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Sample records for activins

  1. Activins and activin antagonists in hepatocellular carcinoma

    Institute of Scientific and Technical Information of China (English)

    Alev Deli; Emanuel Kreidl; Stefan Santifaller; Barbara Trotter; Katja Seir; Walter Berger; Rolf Schulte-Hermann; Chantal Rodgarkia-Dara; Michael Grusch

    2008-01-01

    In many parts of the world hepatocellular carcinoma (HCC) is among the leading causes of cancer-related mortality but the underlying molecular pathology is still insufficiently understood. There is increasing evidence that activins, which are members of the transforming growth factor β (TGFβ) superfamily of growth and differentiation factors, could play important roles in liver carcinogenesis. Activins are disulphide-linked homo-or heterodimers formed from four different β subunits termed βA, βB, βC, and βE, respectively. Activin A, the dimer of two βA subunits, is critically involved in the regulation of cell growth, apoptosis, and tissue architecture in the liver, while the hepatic function of other activins is largely unexplored so far. Negative regulators of activin signals include antagonists in the extracellular space like the binding proteins follistatin and FLRG, and at the cell membrane antagonistic co-receptors like Cripto or BAMBI. Additionally, in the intracellular space inhibitory Smads can modulate and control activin activity. Accumulating data suggest that deregulation of activin signals contributes to pathologic conditions such as chronic inflammation, fibrosis and development of cancer. The current article reviews the alterations in components of the activin signaling pathway that have been observed in HCC and discusses their potential significance for liver tumorigenesis.

  2. Cripto forms a complex with activin and type II activin receptors and can block activin signaling

    Science.gov (United States)

    Gray, Peter C.; Harrison, Craig A.; Vale, Wylie

    2003-01-01

    Activin, nodal, Vg1, and growth and differentiation factor 1 are members of the transforming growth factor β superfamily and signal via the activin type II (ActRII/IIB) and type I (ALK4) serine/threonine kinase receptors. Unlike activins, however, signaling by nodal, Vg1, and growth and differentiation factor 1 requires a coreceptor from the epidermal growth factor-Cripto-FRL1-Cryptic protein family such as Cripto. Cripto has important roles during development and oncogenesis and binds nodal or related ligands and ALK4 to facilitate assembly of type I and type II receptor signaling complexes. Because Cripto mediates signaling via activin receptors and binds directly to ALK4, we tested whether transfection with Cripto would affect the ability of activin to signal and/or interact with its receptors. Here we show that Cripto can form a complex with activin and ActRII/IIB. We were unable to detect activin binding to Cripto in the absence of ActRII/IIB, indicating that unlike nodal, activin requires type II receptors to bind Cripto. If cotransfected with ActRII/IIB and ALK4, Cripto inhibited crosslinking of activin to ALK4 and the association of ALK4 with ActRII/IIB. In addition, Cripto blocked activin signaling when transfected into either HepG2 cells or 293T cells. We have also shown that under conditions in which Cripto facilitates nodal signaling, it antagonizes activin. Inhibition of activin signaling provides an additional example of a Cripto effect on the regulation of signaling by transforming growth factor-β superfamily members. Because activin is a potent inhibitor of cell growth in multiple cell types, these results provide a mechanism that may partially explain the oncogenic action of Cripto. PMID:12682303

  3. Activin receptor subunits in normal and dysfunctional adult human testis

    DEFF Research Database (Denmark)

    Dias, V; Meachem, S; Rajpert-De Meyts, E

    2008-01-01

    The cellular sites of activin action and its regulation in the normal and dysfunctional adult human testis are unknown.......The cellular sites of activin action and its regulation in the normal and dysfunctional adult human testis are unknown....

  4. Uterine-embryonic interaction in pit : activin, follistatin, and activin receptor II in uterus and embryo during early gestation

    NARCIS (Netherlands)

    Pavert, van de S.A.; Boerjan, M.L.; Stroband, H.W.J.; Taverne, M.A.M.; Hurk, van der R.

    2001-01-01

    The mRNA expression patterns of activin A and follistatin in the uterus and embryo, the mRNA expression of the activin receptor II in the embryo, and the localization in the uterus of the immunoreactive activin A and the receptor II proteins in the uterus were examined at gestation days 7-12 after o

  5. Xnrs and activin regulate distinct genes during Xenopus development: activin regulates cell division.

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    Joana M Ramis

    Full Text Available BACKGROUND: The mesoderm of the amphibian embryo is formed through an inductive interaction in which vegetal cells of the blastula-staged embryo act on overlying equatorial cells. Candidate mesoderm-inducing factors include members of the transforming growth factor type beta family such as Vg1, activin B, the nodal-related proteins and derrière. METHODOLOGY AND PRINCIPLE FINDINGS: Microarray analysis reveals different functions for activin B and the nodal-related proteins during early Xenopus development. Inhibition of nodal-related protein function causes the down-regulation of regionally expressed genes such as chordin, dickkopf and XSox17alpha/beta, while genes that are mis-regulated in the absence of activin B tend to be more widely expressed and, interestingly, include several that are involved in cell cycle regulation. Consistent with the latter observation, cells of the involuting dorsal axial mesoderm, which normally undergo cell cycle arrest, continue to proliferate when the function of activin B is inhibited. CONCLUSIONS/SIGNIFICANCE: These observations reveal distinct functions for these two classes of the TGF-beta family during early Xenopus development, and in doing so identify a new role for activin B during gastrulation.

  6. Serum activin B concentration as predictive biomarker for ectopic pregnancy.

    Science.gov (United States)

    Dhiman, Pooja; Senthilkumar, G P; Rajendiran, Soundravally; Sivaraman, K; Soundararaghavan, S; Kulandhasamy, Maheshwari

    2016-05-01

    We evaluated the diagnostic accuracy of activin B in discriminating tubal ectopic pregnancy (tEP) from intrauterine miscarriages (IUM), and normal viable intrauterine pregnancy (IUP). We included 28 women with tEP, 31 women with IUM, and 29 normal IUP, confirmed both by clinical examination and ultrasonography. Serum activin B concentration was measured at the time of admission using the ELISA kit. The median serum activin B concentration was found to be significantly decreased in both tEP (p=0.004) and IUM (p=0.022) compared to normal IUP. When compared between tEP and IUM, activin B concentrations did not differ significantly. ROC analysis of activin B and free β-hCG demonstrated AUC of 0.722 and 0.805, respectively to discriminate tEP from viable IUP. The model including both activin B and free β-hCG improved the discriminating potential with greater AUC (0.824), and specificity (93%) than individual one. To discriminate tEP from IUM, activin B, free β-hCG and combination of both performed poorly. We conclude that serum activin B concentration is lower in tubal ectopic pregnancy, and can discriminate it from normal pregnancy with moderate accuracy. It also shows improved diagnostic potential along with free β-hCG, but cannot distinguish tEP from IUM reliably.

  7. EXPRESSION OF ACTIVIN DURING THE MANDIBULAR DISTRACTION OSTEOGENESIS IN RABBIT

    Institute of Scientific and Technical Information of China (English)

    李昕; 祁佐良; 王炜; 董佳生; 林晓曦; 戴传昌

    2002-01-01

    Objective To investigate the role of activin on osteogenesis during mandibular distraction.Methods Rabbit mandibular distraction model was used and the new regenerating tissue in the distraction zone were harvested at different time points. lmmunohistochemical technique for activin A was performed in the harvested tissues. Results Positive stain was noted in early phases of distraction. At the end of distraction phase osteoblasts and osteoid in primary mineralization front were strongly stained and osteoblasts and osteocytes in peripheral new bone zone were moderately stained. There were also broad activin A stains in osteoblasts and active osteocytes in early consolidation phase. Conclusion The expression of activin is increased during mandibular distraction. It could play an important role in the process of osteoblastic cells secretion, differentiation to osteocytes and bone formation during mandibular distraction.

  8. Role of Activin A in Immune Responses to Breast Cancer

    Science.gov (United States)

    2013-12-01

    secretion of activin A induced by radiation of breast tumor cells is functionally relevant since it further decreases DC maturation and enhances Treg ...regulatory T cells ( Treg ). Recent data indicate that activin A is expressed by some tumors, including breast cancer. Radiotherapy (RT) has the ability to...convert a tumor into an in situ vaccine, but the concomitant increase in Treg has been suggested to hinder its pro-immunogenic effects. Here we

  9. Mutations in inhibin and activin genes associated with human disease.

    Science.gov (United States)

    Shelling, Andrew N

    2012-08-15

    Inhibins and activins are members of the transforming growth factor (TGFβ) superfamily, that includes the TGFβs, inhibins and activins, bone morphogenetic proteins (BMPs) and growth and differentiation factors (GDFs). The family members are expressed throughout the human body, and are involved in the regulation of a range of important functions. The precise regulation of the TGFβ pathways is critical, and mutations of individual molecules or even minor alterations of signalling will have a significant affect on function, that may lead to development of disease or predisposition to the development of disease. The inhibins and activins regulate aspects of the male and female reproductive system, therefore, it is not surprising that most of the diseases associated with abnormalities of the inhibin and activin genes are focused on reproductive disorders and reproductive cancers. In this review, I highlight the role of genetic variants in the development of conditions such as premature ovarian failure, pre-eclampsia, and various reproductive cancers. Given the recent advances in human genetic research, such as genome wide association studies and next generation sequencing, it is likely that inhibins and activins will be shown to play more important roles in a range of human genetic diseases in the future.

  10. Activins and Inhibins: novel regulators of thymocyte development

    Science.gov (United States)

    Licona-Limón, Paula; Alemán-Muench, German; Chimal-Monroy, Jesus; Macías-Silva, Marina; García-Zepeda, Eduardo A.; Matzuk, Martin M.; Fortoul, Teresa I.; Soldevila, Gloria

    2009-01-01

    Activins and inhibins are members of the transforming growth factor β superfamily that act on different cell types and regulate a broad range of cellular processes including proliferation, differentiation, and apoptosis. Here, we provide the first evidence that activins and inhibins regulate specific checkpoints during thymocyte development. We demonstrate that both activin A and inhibin A promote the DN3-DN4 transition in vitro, although they differentially control the transition to the DP stage. Whereas activin A induces the accumulation of a CD8+CD24hiTCRβlo intermediate subpopulation, inhibin A promotes the differentiation of DN4 to DP. In addition, both activin A and inhibin A appear to promote CD8+SP differentiation Moreover, Inhibin α null mice have delayed in vitro T cell development, showing both a decrease in the DN-DP transition and reduced thymocyte numbers, further supporting a role for inhibins in the control of developmental signals taking place during T cell differentiation in vivo. PMID:19338778

  11. Activin tunes GABAergic neurotransmission and modulates anxiety-like behavior.

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    Zheng, F; Adelsberger, H; Müller, M R; Fritschy, J-M; Werner, S; Alzheimer, C

    2009-03-01

    Activin, a member of the transforming growth factor-beta superfamily, affords neuroprotection in acute brain injury, but its physiological functions in normal adult brain are largely unknown. Using transgenic (tg) mice expressing a dominant-negative activin receptor mutant under the control of the CaMKIIalpha promoter in forebrain neurons, we identified activin as a key regulator of gamma-aminobutyric acid (GABA)ergic synapses and anxiety-like behavior. In the open field, wild-type (wt) and tg mice did not differ in spontaneous locomotion and exploration behavior. However, tg mice visited inner fields significantly more often than wt mice. In the light-dark exploration test, tg mice made more exits, spent significantly more time on a well-lit elevated bar and went farther away from the dark box as compared to wt mice. In addition, the anxiolytic effect of diazepam was abrogated in tg mice. Thus the disruption of activin receptor signaling produced a low-anxiety phenotype that failed to respond to benzodiazepines. In whole-cell recordings from hippocampal pyramidal cells, enhanced spontaneous GABA release, increased GABA tonus, reduced benzodiazepine sensitivity and augmented GABA(B) receptor function emerged as likely substrates of the low-anxiety phenotype. These data provide strong evidence that activin influences pre- and postsynaptic components of GABAergic synapses in a highly synergistic fashion. Given the crucial role of GABAergic neurotransmission in emotional states, anxiety and depression, dysfunctions of activin receptor signaling could be involved in affective disorders: and drugs affecting this pathway might show promise for psychopharmacological treatment.

  12. Activin Receptor Signaling Regulates Prostatic Epithelial Cell Adhesion and Viability

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    Derek P. Simon

    2009-04-01

    Full Text Available Mutational changes coupled with endocrine, paracrine, and/or autocrine signals regulate cell division during carcinogenesis. The hormone signals remain undefined, although the absolute requirement in vitro for fetal serum indicates the necessity for a fetal serum factor(s in cell proliferation. Using prostatic cancer cell (PCC lines as a model of cancer cell proliferation, we have identified the fetal serum component activin A and its signaling through the activin receptor type II (ActRII, as necessary, although not sufficient, for PCC proliferation. Activin A induced Smad2 phosphorylation and PCC proliferation, but only in the presence of fetal bovine serum (FBS. Conversely, activin A antibodies and inhibin A suppressed FBS-induced PCC proliferation confirming activin A as one of multiple serum components required for PCC proliferation. Basic fibroblast growth factor was subsequently shown to synergize activin A-induced PCC proliferation. Inhibition of ActRII signaling using a blocking antibody or antisense-P decreased mature ActRII expression, Smad2 phosphorylation, and the apparent viability of PCCs and neuroblastoma cells grown in FBS. Suppression of ActRII signaling in PCC and neuroblastoma cells did not induce apoptosis as indicated by the ratio of active/inactive caspase 3 but did correlate with increased cell detachment and ADAM-15 expression, a disintegrin whose expression is strongly correlated with prostatic metastasis. These findings indicate that ActRII signaling is required for PCC and neuroblastoma cell viability, with ActRII mediating cell fate via the regulation of cell adhesion. That ActRII signaling governs both cell viability and cell adhesion has important implications for developing therapeutic strategies to regulate cancer growth and metastasis.

  13. Intertwining of Activin A and TGFβ Signaling: Dual Roles in Cancer Progression and Cancer Cell Invasion

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    Loomans, Holli A. [Department of Cancer Biology, Vanderbilt University Medical Center, Nashville, TN 37232 (United States); Andl, Claudia D., E-mail: claudia.andl@vanderbilt.edu [Department of Cancer Biology, Vanderbilt University Medical Center, Nashville, TN 37232 (United States); Department of Surgery, Vanderbilt University Medical Center, Nashville, TN 37232 (United States); Vanderbilt Ingram Cancer Center, Vanderbilt University Medical Center, Nashville, TN 37232 (United States); Vanderbilt Digestive Disease Center, Vanderbilt University Medical Center, Nashville, TN 37232 (United States); Vanderbilt Epithelial Biology Center, Vanderbilt University Medical Center, Nashville, TN 37232 (United States)

    2014-12-30

    In recent years, a significant amount of research has examined the controversial role of activin A in cancer. Activin A, a member of the transforming growth factor β (TGFβ) superfamily, is best characterized for its function during embryogenesis in mesoderm cell fate differentiation and reproduction. During embryogenesis, TGFβ superfamily ligands, TGFβ, bone morphogenic proteins (BMPs) and activins, act as potent morphogens. Similar to TGFβs and BMPs, activin A is a protein that is highly systemically expressed during early embryogenesis; however, post-natal expression is overall reduced and remains under strict spatiotemporal regulation. Of importance, normal post-natal expression of activin A has been implicated in the migration and invasive properties of various immune cell types, as well as endometrial cells. Aberrant activin A signaling during development results in significant morphological defects and premature mortality. Interestingly, activin A has been found to have both oncogenic and tumor suppressor roles in cancer. Investigations into the role of activin A in prostate and breast cancer has demonstrated tumor suppressive effects, while in lung and head and neck squamous cell carcinoma, it has been consistently shown that activin A expression is correlated with increased proliferation, invasion and poor patient prognosis. Activin A signaling is highly context-dependent, which is demonstrated in studies of epithelial cell tumors and the microenvironment. This review discusses normal activin A signaling in comparison to TGFβ and highlights how its dysregulation contributes to cancer progression and cell invasion.

  14. Inhibition of somatotroph growth and growth hormone biosynthesis by activin in vitro

    DEFF Research Database (Denmark)

    Billestrup, Nils; González-Manchón, C; Potter, E

    1990-01-01

    ]methionine-labeled cells, could be observed after 24 h of activin treatment, and maximal (70%) inhibition of GH biosynthesis was observed after 3 days. Activin inhibited basal as well as GH-releasing factor (GRF)-, glucocorticoid-, and thyroid hormone-stimulated GH biosynthesis. Inhibin, which is known to reverse...... the effect of activin on FSH secretion, did not reverse the effect of activin on GH biosynthesis. Treatment of somatotrophs with activin for 3 days completely inhibited the growth-promoting effect of GRF on somatotrophs. However, no effect of activin on GRF-stimulated expression of the c-fos protooncogene...... was observed. These data demonstrate that activin, in addition to its stimulatory effect on FSH secretion, is able to inhibit both expression of GH and growth of somatotropic cells....

  15. R-Smad competition controls activin receptor output in Drosophila.

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    Aidan J Peterson

    Full Text Available Animals use TGF-β superfamily signal transduction pathways during development and tissue maintenance. The superfamily has traditionally been divided into TGF-β/Activin and BMP branches based on relationships between ligands, receptors, and R-Smads. Several previous reports have shown that, in cell culture systems, "BMP-specific" Smads can be phosphorylated in response to TGF-β/Activin pathway activation. Using Drosophila cell culture as well as in vivo assays, we find that Baboon, the Drosophila TGF-β/Activin-specific Type I receptor, can phosphorylate Mad, the BMP-specific R-Smad, in addition to its normal substrate, dSmad2. The Baboon-Mad activation appears direct because it occurs in the absence of canonical BMP Type I receptors. Wing phenotypes generated by Baboon gain-of-function require Mad, and are partially suppressed by over-expression of dSmad2. In the larval wing disc, activated Baboon cell-autonomously causes C-terminal Mad phosphorylation, but only when endogenous dSmad2 protein is depleted. The Baboon-Mad relationship is thus controlled by dSmad2 levels. Elevated P-Mad is seen in several tissues of dSmad2 protein-null mutant larvae, and these levels are normalized in dSmad2; baboon double mutants, indicating that the cross-talk reaction and Smad competition occur with endogenous levels of signaling components in vivo. In addition, we find that high levels of Activin signaling cause substantial turnover in dSmad2 protein, providing a potential cross-pathway signal-switching mechanism. We propose that the dual activity of TGF-β/Activin receptors is an ancient feature, and we discuss several ways this activity can modulate TGF-β signaling output.

  16. Activin B is produced early in antral follicular development and suppresses thecal androgen production

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    Young, J M; Henderson, S; Souza, C; Ludlow, H; Groome, N; McNeilly, A S

    2012-01-01

    Little is known about the role of activin B during folliculogenesis. This study investigated the expression levels of activin/inhibin subunits (βA, βB, and α), steroid enzyme, and gonadotrophin receptors in theca (TC) and granulosa cells (GC) by QPCR and activin A and B and inhibin A protein levels in follicular fluid (FF) of developing sheep follicles during estrus and anestrus. The effect of activin B on androgen production from primary TC cultures in vitro was also assessed. During folliculogenesis, in anestrus and estrus, FF activin B concentrations and thecal and GC activin βB mRNA levels decreased as follicle diameter increased from 1–3 to >6 mm regardless of estrogenic status. Estrogenic preovulatory follicles had reduced concentrations of FF activins B and A, and TC and GCs expressed higher levels of activin βA mRNA at 3–4 mm, and TCs more inhibin α mRNA at >4 mm stages of development compared with nonestrogenic follicles. Activin B decreased androstenedione production from primary TCs in vitro, an effect blocked by inhibin A. Thus, sheep follicles 1–3 mm in diameter contained high FF levels of activin B, which decreased as the follicle size increased, and, like activin A, suppressed thecal androgen production in vitro, an effect blocked by inhibin. Furthermore, the theca of large estrogenic follicles expressed high levels of inhibin α and activin βA mRNA suggesting local thecal derived inhibin A production. This would inhibit the negative effects of thecal activins B and A ensuring maximum androgen production for enhanced estradiol production by the preovulatory follicle(s). PMID:22450673

  17. Activin is a potent growth suppressor of epithelial ovarian cancer cells.

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    Ramachandran, Anassuya; Marshall, Elaine S; Love, Donald R; Baguley, Bruce C; Shelling, Andrew N

    2009-11-28

    Although activin is a major cytokine produced by the ovary, its role in epithelial ovarian cancer is poorly defined. Here, we demonstrate a novel role for activin as a growth inhibitor of some (8/16) epithelial ovarian cancer cell lines. Unresponsive cell lines displayed transcriptional downregulation of the activin receptors ACTRIIA and ACTRIB, suggesting resistance to activin signalling. In response to activin, growth inhibited cell lines demonstrated activation of the canonical SMAD2/3/4, transcriptional induction of the CDK inhibitor p15INK4B, suppression of C-MYC levels and a G1 phase cell cycle arrest. Thus, activin is a potent inhibitor of proliferation of some epithelial ovarian cancer cell lines and its role in the pathogenesis of this disease needs to be re-evaluated.

  18. Activin A Levels Are Associated With Abnormal Glucose Regulation in Patients With Myocardial Infarction

    Science.gov (United States)

    Andersen, Geir Ø.; Ueland, Thor; Knudsen, Eva C.; Scholz, Hanne; Yndestad, Arne; Sahraoui, Afaf; Smith, Camilla; Lekva, Tove; Otterdal, Kari; Halvorsen, Bente; Seljeflot, Ingebjørg; Aukrust, Pål

    2011-01-01

    OBJECTIVE On the basis of the role of activin A in inflammation, atherogenesis, and glucose homeostasis, we investigated whether activin A could be related to glucometabolic abnormalities in patients with acute myocardial infarction (MI). RESEARCH DESIGN AND METHODS Activin A measurement and oral glucose tolerance tests (OGTTs) were performed in patients (n = 115) with acute MI, without previously known diabetes, and repeated after 3 months. Release of activin A and potential anti-inflammatory effects of activin A were measured in human endothelial cells. Activin A effects on insulin secretion and inflammation were tested in human pancreatic islet cells. RESULTS 1) In patients with acute MI, serum levels of activin A were significantly higher in those with abnormal glucose regulation (AGR) compared with those with normal glucose regulation. Activin A levels were associated with the presence of AGR 3 months later (adjusted odds ratio 5.1 [95% CI 1.73–15.17], P = 0.003). 2) In endothelial cells, glucose enhanced the release of activin A, whereas activin A attenuated the release of interleukin (IL)-8 and enhanced the mRNA levels of the antioxidant metallothionein. 3) In islet cells, activin A attenuated the suppressive effect of inflammatory cytokines on insulin release, counteracted the ability of these inflammatory cytokines to induce mRNA expression of IL-8, and induced the expression of transforming growth factor-β. CONCLUSIONS We found a significant association between activin A and newly detected AGR in patients with acute MI. Our in vitro findings suggest that this association represents a counteracting mechanism to protect against inflammation, hyperglycemia, and oxidative stress. PMID:21464440

  19. Acute Modulation of Synaptic Plasticity of Pyramidal Neurons by Activin in Adult Hippocampus

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    Yoshitaka eHasegawa

    2014-06-01

    Full Text Available Activin A is known as a neuroprotective factor produced upon acute excitotoxic injury of the hippocampus (in pathological states. We attempt to reveal the role of activin as a neuromodulator in the adult male hippocampus under physiological conditions (in healthy states, which remains largely unknown. We showed endogenous/basal expression of activin in the hippocampal neurons. Localization of activin receptors in dendritic spines (= postsynapses was demonstrated by immunoelectron microscopy. The incubation of hippocampal acute slices with activin A (10 ng/mL, 0.4 nM for 2 h altered the density and morphology of spines in CA1 pyramidal neurons. The total spine density increased by 1.2-fold upon activin treatments. Activin selectively increased the density of large-head spines, without affecting middle-head and small-head spines. Blocking of Erk/MAPK, PKA or PKC prevented the activin-induced spinogenesis by reducing the density of large-head spines, independent of Smad-induced gene transcription which usually takes more than several hours. Incubation of acute slices with activin for 2 h induced the moderate early long-term potentiation (moderate LTP upon weak theta burst stimuli. This moderate LTP induction was blocked by follistatin, MAPK inhibitor (PD98059 and inhibitor of NR2B subunit of NMDA receptors (Ro25-6981. It should be noted that the weak theta burst stimuli alone cannot induce moderate LTP. These results suggest that MAPK-induced phosphorylation of NMDA receptors (including NR2B may play an important role for activin-induced moderate LTP. Taken together, the current results reveal interesting physiological roles of endogenous activin as a synaptic modulator in the adult hippocampus.

  20. Activin A inhibits BMP-signaling by binding ACVR2A and ACVR2B

    DEFF Research Database (Denmark)

    Olsen, Oddrun Elise; Wader, Karin Fahl; Hella, Hanne;

    2015-01-01

    BACKGROUND: Activins are members of the TGF-β family of ligands that have multiple biological functions in embryonic stem cells as well as in differentiated tissue. Serum levels of activin A were found to be elevated in pathological conditions such as cachexia, osteoporosis and cancer. Signaling...

  1. Developmentally regulated SMAD2 and SMAD3 utilization directs activin signalling outcomes

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    Itman, Catherine; Small, Chris; Griswold, Michael; Nagaraja, Ankur K.; Matzuk, Martin M.; Brown, Chester; Jans, David A.; Loveland, Kate L.

    2010-01-01

    Activin is required for testis development. Activin signals via the phosphorylation and nuclear accumulation of SMAD2 and SMAD3. We present novel findings of developmentally regulated activin signalling leading to specific transcriptional outcomes in testicular Sertoli cells. In immature, proliferating, Sertoli cells, activin A induces nuclear accumulation of SMAD3, but not SMAD2, although both proteins become phosphorylated. In post-mitotic differentiating cells, both SMAD proteins accumulate in the nucleus. Furthermore, immature Sertoli cells are sensitive to activin dosage; higher concentrations induce maximal SMAD3 nuclear accumulation and a small increase in nuclear SMAD2. Microarray analysis identified distinct transcriptional outcomes correlating with differential SMAD utilization and new activin target genes, including Gja1 and Serpina5, which are essential for Sertoli cell development and male fertility. In transgenic mice with altered activin bioactivity that display fertility phenotypes, Gja1 and Serpina5 are significantly altered. Thus, differential SMAD utilization in response to activin features during Sertoli cell maturation. PMID:19517569

  2. Activin B promotes epithelial wound healing in vivo through RhoA-JNK signaling pathway.

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    Min Zhang

    Full Text Available BACKGROUND: Activin B has been reported to promote the proliferation and migration of keratinocytes in vitro via the RhoA-JNK signaling pathway, whereas its in vivo role and mechanism in wound healing process has not yet been elucidated. PRINCIPAL FINDINGS: In this study, we explored the potential mechanism by which activin B induces epithelial wound healing in mice. Recombinant lentiviral plasmids, with RhoA (N19 and RhoA (L63 were used to infect wounded KM mice. The wound healing process was monitored after different treatments. Activin B-induced cell proliferation on the wounded skin was visualized by electron microscopy and analyzed by 5'-bromodeoxyuridine (BrdU incorporation assay. Protein expression of p-JNK or p-cJun was determined by immunohistochemical staining and immunoblotting analysis. Activin B efficiently stimulated the proliferation of keratinocytes and hair follicle cells at the wound area and promoted wound closure. RhoA positively regulated activin B-induced wound healing by up-regulating the expression of p-JNK and p-cJun. Moreover, suppression of RhoA activation delayed activin B-induced wound healing, while JNK inhibition recapitulated phenotypes of RhoA inhibition on wound healing. CONCLUSION: These results demonstrate that activin B promotes epithelial wound closure in vivo through the RhoA-Rock-JNK-cJun signaling pathway, providing novel insight into the essential role of activin B in the therapy of wound repair.

  3. Gram-Negative Bacterial Lipopolysaccharide Stimulates Activin A Secretion from Human Amniotic Epithelial Cells

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    Yumiko Abe

    2013-01-01

    Full Text Available Activin A is involved in inflammation. The present study was performed to clarify if lipopolysaccharide, a component of Gram-negative bacteria, stimulates activin A secretion from human amniotic epithelial cells and to determine if activin A plays a role in amnionitis. Fetal membranes were obtained during elective cesarean sections performed in full-term pregnancies of patients without systemic disease, signs of premature delivery, or fetal complications. Amniotic epithelial cells were isolated by trypsinization. The activin A concentrations in the culture media were measured by enzyme-linked immunosorbent assay, and cell proliferation was assessed by 5-bromo-2′-deoxyuridine incorporation. Amniotic epithelial cells secreted activin A in a cell density-dependent manner, and lipopolysaccharide (10 μg/mL enhanced the secretion at each cell density. Lipopolysaccharide (10–50 μg/mL also stimulated activin A secretion in a dose-dependent manner. Contrary to the effect of activin A secretion, lipopolysaccharide inhibited cell proliferation in amniotic epithelial cells. The present study suggests that lipopolysaccharide stimulation of activin A secretion may be a mechanism in the pathogenesis of amnionitis.

  4. Activin B mediated induction of Pdx1 in human embryonic stem cell derived embryoid bodies

    DEFF Research Database (Denmark)

    Frandsen, Ulrik; Pørneki, Ann Dorte Storm; Floridon, Charlotte

    2007-01-01

    Human embryonic stem cells (hESCs) have the potential to provide alternative sources for pancreatic islet grafts. In the present study we have investigated the influence of Activin A and Activin B on the expression of the pancreas marker gene Pdx1 in hESCs differentiated as embryoid bodies (EBs...

  5. Increased activin bioavailability enhances hepatic insulin sensitivity while inducing hepatic steatosis in male mice.

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    Ungerleider, Nathan A; Bonomi, Lara M; Brown, Melissa L; Schneyer, Alan L

    2013-06-01

    The development of insulin resistance is tightly linked to fatty liver disease and is considered a major health concern worldwide, although their mechanistic relationship remains controversial. Activin has emerging roles in nutrient homeostasis, but its metabolic effects on hepatocytes remain unknown. In this study, we investigated the effects of increased endogenous activin bioactivity on hepatic nutrient homeostasis by creating mice with inactivating mutations that deplete the circulating activin antagonists follistatin-like-3 (FSTL3) or the follistatin 315 isoform (FST315; FST288-only mice). We investigated liver histology and lipid content, hepatic insulin sensitivity, and metabolic gene expression including the HepG2 cell and primary hepatocyte response to activin treatment. Both FSTL3-knockout and FST288-only mice had extensive hepatic steatosis and elevated hepatic triglyceride content. Unexpectedly, insulin signaling, as assessed by phospho-Akt (a.k.a. protein kinase B), was enhanced in both mouse models. Pretreatment of HepG2 cells with activin A increased their response to subsequent insulin challenge. Gene expression analysis suggests that increased lipid uptake, enhanced de novo lipid synthesis, decreased lipolysis, and/or enhanced glucose uptake contribute to increased hepatic triglyceride content in these models. However, activin treatment recapitulated only some of these gene changes, suggesting that increased activin bioactivity may be only partially responsible for this phenotype. Nevertheless, our results indicate that activin enhances hepatocyte insulin response, which ultimately leads to hepatic steatosis despite the increased insulin sensitivity. Thus, regulation of activin bioactivity is critical for maintaining normal liver lipid homeostasis and response to insulin, whereas activin agonists may be useful for increasing liver insulin sensitivity.

  6. Activin signal promotes cancer progression and is involved in cachexia in a subset of pancreatic cancer.

    Science.gov (United States)

    Togashi, Yosuke; Kogita, Akihiro; Sakamoto, Hiroki; Hayashi, Hidetoshi; Terashima, Masato; de Velasco, Marco A; Sakai, Kazuko; Fujita, Yoshihiko; Tomida, Shuta; Kitano, Masayuki; Okuno, Kiyotaka; Kudo, Masatoshi; Nishio, Kazuto

    2015-01-28

    We previously reported that activin produces a signal with a tumor suppressive role in pancreatic cancer (PC). Here, the association between plasma activin A and survival in patients with advanced PC was investigated. Contrary to our expectations, however, patients with high plasma activin A levels had a significantly shorter survival period than those with low levels (median survival, 314 days vs. 482 days, P = 0.034). The cellular growth of the MIA PaCa-2 cell line was greatly enhanced by activin A via non-SMAD pathways. The cellular growth and colony formation of an INHBA (beta subunit of inhibin)-overexpressed cell line were also enhanced. In a xenograft study, INHBA-overexpressed cells tended to result in a larger tumor volume, compared with a control. The bodyweights of mice inoculated with INHBA-overexpressed cells decreased dramatically, and these mice all died at an early stage, suggesting the occurrence of activin-induced cachexia. Our findings indicated that the activin signal can promote cancer progression in a subset of PC and might be involved in cachexia. The activin signal might be a novel target for the treatment of PC.

  7. Activin A is increased in the nucleus accumbens following a cocaine binge

    Science.gov (United States)

    Wang, Zi-Jun; Martin, Jennifer A.; Gancarz, Amy M.; Adank, Danielle N.; Sim, Fraser J.; Dietz, David M.

    2017-01-01

    Drug addiction is a long-lasting disease characterized by compulsive drug intake mediated in part by neuronal and biological adaptations in key brain areas, such as the nucleus accumbens (NAc). While we previously demonstrated involvement of the activin 2a receptor in drug taking, the role of its ligand, activin A, in cocaine relapse is unknown. Activin A levels in the NAc were assessed via ELISA and immunohistochemistry (in neurons, astrocytes, and microglia) following a cocaine binge paradigm. Cocaine exposure significantly increased the levels of activin A in the NAc of animals that had self-administered cocaine prior to the 14-day withdrawal compared with levels in saline controls. This was accompanied by an increase in the proportion of IBA1+ microglia in the NAc that were immunopositive for activin A. In contrast, the proportions of NeuN+ neurons and GFAP+ astrocytes that were immunopositive for activin A remained unaltered. In conclusion, these data suggest that increased secretion of activin A, particularly from microglia, in the NAc represents a novel potential target for the treatment of cocaine relapse. PMID:28272550

  8. An Activin Receptor IA/Activin-Like Kinase-2 (R206H Mutation in Fibrodysplasia Ossificans Progressiva

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    Rafael Herrera-Esparza

    2013-01-01

    Full Text Available Fibrodysplasia ossificans progressiva (FOP is an exceptionally rare genetic disease that is characterised by congenital malformations of the great toes and progressive heterotopic ossification (HO in specific anatomical areas. This disease is caused by a mutation in activin receptor IA/activin-like kinase-2 (ACVR1/ALK2. A Mexican family with one member affected by FOP was studied. The patient is a 19-year-old female who first presented with symptoms of FOP at 8 years old; she developed spontaneous and painful swelling of the right scapular area accompanied by functional limitation of movement. Mutation analysis was performed in which genomic DNA as PCR amplified using primers flanking exons 4 and 6, and PCR products were digested with Cac8I and HphI restriction enzymes. The most informative results were obtained with the exon 4 flanking primers and the Cac8I restriction enzyme, which generated a 253 bp product that carries the ACVR1 617G>A mutation, which causes an amino acid substitution of histidine for arginine at position 206 of the glycine-serine (GS domain, and its mutation results in the dysregulation of bone morphogenetic protein (BMP signalling that causes FOP.

  9. Tissue absence initiates regeneration through follistatin-mediated inhibition of activin signaling.

    Science.gov (United States)

    Gaviño, Michael A; Wenemoser, Danielle; Wang, Irving E; Reddien, Peter W

    2013-09-10

    Regeneration is widespread, but mechanisms that activate regeneration remain mysterious. Planarians are capable of whole-body regeneration and mount distinct molecular responses to wounds that result in tissue absence and those that do not. A major question is how these distinct responses are activated. We describe a follistatin homolog (Smed-follistatin) required for planarian regeneration. Smed-follistatin inhibition blocks responses to tissue absence but does not prevent normal tissue turnover. Two activin homologs (Smed-activin-1 and Smed-activin-2) are required for the Smed-follistatin phenotype. Finally, Smed-follistatin is wound-induced and expressed at higher levels following injuries that cause tissue absence. These data suggest that Smed-follistatin inhibits Smed-Activin proteins to trigger regeneration specifically following injuries involving tissue absence and identify a mechanism critical for regeneration initiation, a process important across the animal kingdom. DOI:http://dx.doi.org/10.7554/eLife.00247.001.

  10. Activin receptor signaling regulates cocaine-primed behavioral and morphological plasticity.

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    Gancarz, Amy M; Wang, Zi-Jun; Schroeder, Gabrielle L; Damez-Werno, Diane; Braunscheidel, Kevin M; Mueller, Lauren E; Humby, Monica S; Caccamise, Aaron; Martin, Jennifer A; Dietz, Karen C; Neve, Rachael L; Dietz, David M

    2015-07-01

    Activin receptor signaling, including the transcription factor Smad3, was upregulated in the rat nucleus accumbens (NAc) shell following withdrawal from cocaine. Direct genetic and pharmacological manipulations of this pathway bidirectionally altered cocaine seeking while governing morphological plasticity in NAc neurons. Thus, Activin/Smad3 signaling is induced following withdrawal from cocaine, and such regulation may be a key molecular mechanism underlying behavioral and cellular plasticity in the brain following cocaine self-administration.

  11. Activin A-Smad Signaling Mediates Connective Tissue Growth Factor Synthesis in Liver Progenitor Cells.

    Science.gov (United States)

    Ding, Ze-Yang; Jin, Guan-Nan; Wang, Wei; Sun, Yi-Min; Chen, Wei-Xun; Chen, Lin; Liang, Hui-Fang; Datta, Pran K; Zhang, Ming-Zhi; Zhang, Bixiang; Chen, Xiao-Ping

    2016-03-22

    Liver progenitor cells (LPCs) are activated in chronic liver damage and may contribute to liver fibrosis. Our previous investigation reported that LPCs produced connective tissue growth factor (CTGF/CCN2), an inducer of liver fibrosis, yet the regulatory mechanism of the production of CTGF/CCN2 in LPCs remains elusive. In this study, we report that Activin A is an inducer of CTGF/CCN2 in LPCs. Here we show that expression of both Activin A and CTGF/CCN2 were upregulated in the cirrhotic liver, and the expression of Activin A positively correlates with that of CTGF/CCN2 in liver tissues. We go on to show that Activin A induced de novo synthesis of CTGF/CCN2 in LPC cell lines LE/6 and WB-F344. Furthermore, Activin A contributed to autonomous production of CTGF/CCN2 in liver progenitor cells (LPCs) via activation of the Smad signaling pathway. Smad2, 3 and 4 were all required for this induction. Collectively, these results provide evidence for the fibrotic role of LPCs in the liver and suggest that the Activin A-Smad-CTGF/CCN2 signaling in LPCs may be a therapeutic target of liver fibrosis.

  12. Activin A prevents neuron-like PC12 cell apoptosis after oxygen-glucose deprivation

    Institute of Scientific and Technical Information of China (English)

    Guihua Xu; Zhongxin Xu; Jinting He; Hongliang Guo; Chunli Mei; Jiaoqi Wang; Zhongshu Li; Han Chen; Jing Mang; Hong Yang

    2013-01-01

    In this study, PC12 cells were induced to differentiate into neuron-like cells using nerve growth factor, and were subjected to oxygen-glucose deprivation. Cells were treated with 0, 10, 20, 30, 50, 100 ng/mL exogenous Activin A. The 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl tetrazolium bromide assay and Hoechst 33324 staining showed that the survival percentage of PC12 cells significantly decreased and the rate of apoptosis significantly increased after oxygen-glucose deprivation. Exogenous Activin A significantly increased the survival percentage of PC12 cells in a dose-dependent manner. Reverse transcription-PCR results revealed a significant increase in Activin receptor IIA, Smad3 and Smad4 mRNA levels, which are key sites in the Activin A/Smads signaling pathway, in neuron-like cells subjected to oxygen-glucose deprivation, while mRNA expression of the apoptosis-regulation gene caspase-3 decreased. Our experimental findings indicate that exogenous Activin A plays an anti-apoptotic role and protects neurons by means of activating the Activin A/Smads signaling pathway.

  13. Direct Effects of Activin A on the Activation of Mouse Macrophage RAW264.7 Cells

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    Jingyan Ge; Yinan Wang; Ye Feng; Haiyan Liu; Xueling Cui; Fangfang Chen; Guixiang Tai; Zhonghui Liu

    2009-01-01

    Macrophages play critical roles in innate immune and acquired immune via secreting pro-inflammatory mediators, phagocytosing microorganisms and presenting antigens. Activin A, a member of transforming growth factor β (TGF-β) superfamily, is produced by macrophages and microglia cells. In this study, we reported a direct effect of activin A as a pro-inflammatory factor on mouse macrophage cell line RAW264.7 cells. Our data revealed that activin A could not only increase IL-1v and IL-6 production from RAW264.7 cells, but also promote pinocytic and phagocytic activities of RAW264.7 cells. In addition, activin A obviously up-regulated MHC Ⅱ expression on the surface of RAW264.7 cells, whereas did not influence MHC I expression. Activin A also enhanced CD80 expression, which is a marker of activated macrophages, but did not influence RAW264.7 cell proliferation. These data suggest that activin A may regulate primary macrophage-mediated innate and acquired immune response via promoting the activation of rest macrophages. Cellular & Molecular Immunology.

  14. Activin A induces Langerhans cell differentiation in vitro and in human skin explants.

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    Tiziana Musso

    Full Text Available Langerhans cells (LC represent a well characterized subset of dendritic cells located in the epidermis of skin and mucosae. In vivo, they originate from resident and blood-borne precursors in the presence of keratinocyte-derived TGFbeta. In vitro, LC can be generated from monocytes in the presence of GM-CSF, IL-4 and TGFbeta. However, the signals that induce LC during an inflammatory reaction are not fully investigated. Here we report that Activin A, a TGFbeta family member induced by pro-inflammatory cytokines and involved in skin morphogenesis and wound healing, induces the differentiation of human monocytes into LC in the absence of TGFbeta. Activin A-induced LC are Langerin+, Birbeck granules+, E-cadherin+, CLA+ and CCR6+ and possess typical APC functions. In human skin explants, intradermal injection of Activin A increased the number of CD1a+ and Langerin+ cells in both the epidermis and dermis by promoting the differentiation of resident precursor cells. High levels of Activin A were present in the upper epidermal layers and in the dermis of Lichen Planus biopsies in association with a marked infiltration of CD1a+ and Langerin+ cells. This study reports that Activin A induces the differentiation of circulating CD14+ cells into LC. Since Activin A is abundantly produced during inflammatory conditions which are also characterized by increased numbers of LC, we propose that this cytokine represents a new pathway, alternative to TGFbeta, responsible for LC differentiation during inflammatory/autoimmune conditions.

  15. Substantial Increases Occur in Serum Activins and Follistatin during Lung Transplantation.

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    David M de Kretser

    Full Text Available Lung transplantation exposes the donated lung to a period of anoxia. Re-establishing the circulation after ischemia stimulates inflammation causing organ damage. Since our published data established that activin A is a key pro-inflammatory cytokine, we assessed the roles of activin A and B, and their binding protein, follistatin, in patients undergoing lung transplantation.Sera from 46 patients participating in a published study of remote ischemia conditioning in lung transplantation were used. Serum activin A and B, follistatin and 11 other cytokines were measured in samples taken immediately after anaesthesia induction, after remote ischemia conditioning or sham treatment undertaken just prior to allograft reperfusion and during the subsequent 24 hours.Substantial increases in serum activin A, B and follistatin occurred after the baseline sample, taken before anaesthesia induction and peaked immediately after the remote ischemia conditioning/sham treatment. The levels remained elevated 15 minutes after lung transplantation declining thereafter reaching baseline 2 hours post-transplant. Activin B and follistatin concentrations were lower in patients receiving remote ischemia conditioning compared to sham treated patients but the magnitude of the decrease did not correlate with early transplant outcomes.We propose that the increases in the serum activin A, B and follistatin result from a combination of factors; the acute phase response, the reperfusion response and the use of heparin-based anti-coagulants.

  16. Regulation of C-cadherin function during activin induced morphogenesis of Xenopus animal caps.

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    Brieher, W M; Gumbiner, B M

    1994-07-01

    Treatment of Xenopus animal pole tissue with activin results in the induction of mesodermal cell types and a dramatic elongation of the tissue. The morphogenetic movements involved in the elongation appear similar to those in normal gastrulation, which is driven by cell rearrangement and cell intercalations. We have used this system to explore the potential regulation of cell-cell adhesion and cadherin function during morphogenesis. Quantitative blastomere aggregation assays revealed that activin induction reduced the calcium-dependent adhesion between blastomeres. Activin-induced blastomeres formed smaller aggregates, and a greater proportion of the population remained as single cells compared to uninduced blastomeres. The aggregation was mediated by C-cadherin because C-cadherin was present in the blastomeres during the aggregation assay, and monoclonal antibodies against C-cadherin inhibited the calcium-dependent aggregation of blastomeres. E-cadherin was not detectable until after the completion of the assay and, therefore, does not explain the adhesive differences between induced and uninduced blastomeres. L cells stably expressing C-cadherin (LC cells) were used to demonstrate that C-cadherin activity was specifically altered after activin induction. Blastomeres induced with activin bound fewer LC cells than uninduced blastomers. L cells not expressing C-cadherin did not adhere to blastomeres. The changes in C-cadherin-mediated adhesion occurred without detectable changes in the steady-state levels of C-cadherin or the amount of C-cadherin present on the surface of the cell. Immunoprecipitation of C-cadherin and its associated catenins revealed that the ratio of C-cadherin and the catenins was not altered by activin induction. These results demonstrate that activin decreases the adhesive function of existing C-cadherin molecules on the surface of blastomeres and suggest that decreased cadherin mediated cell-cell adhesion is associated with increased

  17. Activin B regulates islet composition and islet mass but not whole body glucose homeostasis or insulin sensitivity

    Science.gov (United States)

    Bonomi, Lara; Brown, Melissa; Ungerleider, Nathan; Muse, Meghan; Matzuk, Martin M.

    2012-01-01

    Based on the phenotype of the activin-like kinase-7 (ALK7)-null mouse, activins A and B have been proposed to play distinct roles in regulating pancreatic islet function and glucose homeostasis, with activin A acting to enhance islet function and insulin release while activin B antagonizes these actions. We therefore hypothesized that islets from activin B-null (BBKO) mice would have enhanced glucose-stimulated insulin secretion. In addition, we hypothesized that this enhanced islet function would translate into increased whole body glucose tolerance. We tested these hypotheses by analyzing glucose homeostasis, insulin secretion, and islet function in BBKO mice. No differences were observed in fasting glucose or insulin levels, glucose tolerance, or insulin sensitivity compared with weight-matched young or older males. Similarly, there were no significant differences in insulin secretion comparing islets from WT or BBKO males at either age. However, BBKO islets were more sensitive to activin A, myostatin (MSTN), and follistatin (FST) treatments, so that activin A and FST inhibited and MSTN enhanced glucose stimulated insulin secretion. While mean islet area and the distribution of islet areas were not different between the genotypes, islet mass, islet number, and the proportion of α-cells/islet were significantly reduced in BBKO islets. These results indicate that activin B does not antagonize activin A to influence whole body glucose homeostasis or β-cell function but does influence islet mass and proportion of α-cells/islet. Therefore, loss of activin B signaling alone does not account for the ALK7-null phenotype, but activin B may have important roles in modulating islet mass, islet number, and the cellular composition of islets. PMID:22739106

  18. Regulation of Muscle Mass by Follistatin and Activins

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    Lee, Se-Jin; Lee, Yun-Sil; Zimmers, Teresa A.; Soleimani, Arshia; Matzuk, Martin M.; Tsuchida, Kunihiro; Cohn, Ronald D.; Barton, Elisabeth R.

    2010-01-01

    Myostatin is a TGF-β family member that normally acts to limit skeletal muscle mass. Follistatin is a myostatin-binding protein that can inhibit myostatin activity in vitro and promote muscle growth in vivo. Mice homozygous for a mutation in the Fst gene have been shown to die immediately after birth but have a reduced amount of muscle tissue, consistent with a role for follistatin in regulating myogenesis. Here, we show that Fst mutant mice exhibit haploinsufficiency, with muscles of Fst heterozygotes having significantly reduced size, a shift toward more oxidative fiber types, an impairment of muscle remodeling in response to cardiotoxin-induced injury, and a reduction in tetanic force production yet a maintenance of specific force. We show that the effect of heterozygous loss of Fst is at least partially retained in a Mstn-null background, implying that follistatin normally acts to inhibit other TGF-β family members in addition to myostatin to regulate muscle size. Finally, we present genetic evidence suggesting that activin A may be one of the ligands that is regulated by follistatin and that functions with myostatin to limit muscle mass. These findings potentially have important implications with respect to the development of therapeutics targeting this signaling pathway to preserve muscle mass and prevent muscle atrophy in a variety of inherited and acquired forms of muscle degeneration. PMID:20810712

  19. Activin Bioactivity Affects Germ Cell Differentiation in the Postnatal Mouse Testis In Vivo1

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    Mithraprabhu, Sridurga; Mendis, Sirisha; Meachem, Sarah J.; Tubino, Laura; Matzuk, Martin M.; Brown, Chester W.; Loveland, Kate L.

    2010-01-01

    The transforming growth factor beta superfamily ligand activin A controls juvenile testis growth by stimulating Sertoli cell proliferation. Testicular levels are highest in the first postnatal week, when Sertoli cells are proliferating and spermatogonial stem cells first form. Levels decrease sharply as Sertoli cell proliferation ceases and spermatogenic differentiation begins. We hypothesized that changing activin levels also affect germ cell maturation. We detected an acute and developmentally regulated impact of activin on Kit mRNA in cocultures of Sertoli cells and germ cells from Day 8, but not Day 4, mice. Both stereological and flow cytometry analyses identified an elevated spermatogonium:Sertoli cell ratio in Day 7 testes from InhbaBK/BK mice, which have decreased bioactive activin, and the germ cell markers Sycp3, Dazl, and Ccnd3 were significantly elevated in InhbaBK/BK mice. The flow cytometry measurements demonstrated that surface KIT protein is significantly higher in Day 7 InhbaBK/BK germ cells than in wild-type littermates. By Day 14, the germ cell:Sertoli cell ratio did not differ between genotypes, but the transition of type A spermatogonia into spermatocytes was altered in InhbaBK/BK testes. We conclude that regulated activin signaling not only controls Sertoli cell proliferation, as previously described, but also influences the in vivo progression of germ cell maturation in the juvenile testis at the onset of spermatogenesis. PMID:20130270

  20. Activin signalling and pre-eclampsia: from genetic risk to pre-symptomatic biomarker.

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    Williamson, Rachel D; O'Keeffe, Gerard W; Kenny, Louise C

    2015-02-01

    Pre-eclampsia is a multi-system condition in pregnancy that is characterised by the onset of hypertension and proteinuria in women after the 20th week and it remains a leading cause of maternal and fetal mortality. Despite this the causative molecular basis of pre-eclampsia remains poorly understood. As a result, an intensive research effort has focused on understanding the molecular mechanisms involved in pre-eclampsia and using this information to identify new pre-symptomatic bio-markers of the condition. Activin A and its receptor, ACVR2A, have been extensively studied in this regard. Activin A is a member of the transforming growth factor (TGF)-β superfamily that has a wide range of biological functions depending on the cellular context. Recent work has shown that polymorphisms in ACVR2A may be a genetic risk factor for pre-eclampsia. Furthermore, both placenta and serum levels of Activin A are significantly increased in pre-eclampsia suggesting that Activin A may be a possible biomarker for the condition. Here we review the latest advances in this field and link these with new molecular data that suggest that the oxidative stress and pro-inflammatory cytokine production seen in pre-eclampsia may result in increased placental Activin A secretion in an attempt to maintain placental function.

  1. Activin A suppressed hepatic oval cell-mediated liver regeneration in vivo%Activin A抑制卵圆细胞介导肝再生的体内研究

    Institute of Scientific and Technical Information of China (English)

    陈琳; 周巧丹; 丁则阳; 张伟; 张必翔; 梁慧芳; 陈孝平

    2012-01-01

    Objective To study the inhibitory effect and mechanism of Activin A on hepatic oval cell-mediated liver regeneratioa Methods 2-acetylaminofluorene/partial hepatectomy (2-AAF/PH) o-val cell-mediated liver regeneration model was established. One μg Activin A (Activin A group) or normal saline (control S group) was infused into portal vein immediately after 70 % partial hepatectomy. Animals were sacrificed at 2nd, 4th, 6th, and 9th day after hepatectomy. Liver regeneration rate was measured. Changes in Pan-CK positive oval cell number and nuclear bromodeoxyuridine labeling were detected by using immunohistochemistry. The expression of phosphorylated smad2, p15, and p21 were detected by using Western blotting. Results As compared with control group, the Pan-CK positive number was significantly reduced in Activin A group at 6th and 9th day after hepatectomy,and Brdu labeling nuclear number was reduced in Activin A group at 4th,6th and 9th day after hepatectomy. The liver regeneration rate in Activin A group was lower than in control group. Higher levels of phosphorylated smad2,p15 and p21 were detected in Activin A group. Conclusion Activin A could suppress o-val cell-mediated liver regeneration through smad pathway-dependent up-regulation of p15 and p21.%目的 研究Activin A对卵圆细胞介导肝再生的抑制作用及其机制.方法 建立2-乙酰胺基芴/部分肝切除卵圆细胞介导肝再生模型,肝切除后立即经门静脉注射1 μg Activin A(Activin A组),或生理盐水(对照组).肝切除后不同时间点取肝脏标本检测卵圆细胞增殖及肝再生率和磷酸化smad2,p15和p21的表达.结果 Activin A组肝脏中卵圆细胞的增殖及肝再生率均明显低于对照组;Activin A组肝脏中smad2的磷酸化水平及p15,p21的表达水平均高于对照组.结论 Activin A可以通过smad信号通路抑制卵圆细胞介导的肝再生.

  2. Emodin prevents hypoxic-ischemic neuronal injury Involvement of the activin A pathway

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    Hongliang Guo; Xiaoran Shen; Ye Xu; Junliang Yuan; Dongming Zhao; Wenli Hu

    2013-01-01

    Emodin, an extract of dried rhizomes and the root of the Rhizoma Polygoni Cuspidati, can protect neurons from hypoxic-ischemic brain damage. This study aimed to verify the underlying mechanism. After PC12 cells had differentiated into neuron-like cells under the induction of mouse nerve growth factor, cells were subjected to oxygen-glucose deprivation and treated with emodin. Results showed that the viability of neuron-like cells cultured under an ischemia-hypoxia environment decreased, while the expression of activin A and caspase-3 in cells increased. Emodin raised the survival rate of oxygen-glucose deprived neuron-like cells, increased activin A expression, and decreased caspase-3 expression. Experimental findings indicate that emodin can inhibit neuronal apoptosis and alleviate the injury of nerve cells after oxygen-glucose deprivation through the activin A pathway.

  3. Inhibin subunits, follistatin and activin receptors in the human teratocarcinoma cell line Tera-2.

    Science.gov (United States)

    de Jong, F H; de Winter, J P; Wesseling, J G; Timmerman, M A; van Genesen, S; van den Eijnden-van Raaij, A J; van Zoelen, E J

    1993-05-14

    The expression of mRNAs for inhibin subunits was studied in the human teratocarcinoma cell line Tera-2 clone 13 before and after differentiation with retinoic acid (RA). Both alpha- and beta B-subunits of inhibin were expressed. Subsequently, inhibin bio- and immunoactivity in the conditioned media of the Tera-2 cells were determined by studying the release of follicle-stimulating hormone from rat pituitary cells, by immunoassay and by immunoprecipitation using inhibin alpha- and beta B-subunit specific antibodies. Strikingly dissimilar high bio- and low immuno-activities were found. The ensuing hypothesis that the high bioactivity might be due to the presence of the activin-binding protein follistatin was confirmed by immunoprecipitation of 34 and 37 kDa labelled proteins, using a polyclonal anti-follistatin antiserum after culture of the Tera-2 cells with [35S]-methionine. Furthermore, expression of activin receptor types II and IIB mRNA was found in the cells. Addition of 5 microM RA to monolayer cultures of Tera-2 cells resulted in differentiation to flat endoderm-like cells and caused a slight suppression of the expression of the mRNA encoding the inhibin alpha- and beta B-subunits. The expression of follistatin and activin receptor type IIB was strongly suppressed, whereas the expression of the activin receptor type II was not affected. We conclude that Tera-2 cells secrete follistatin and express inhibin subunits and activin receptors differentially during RA-induced differentiation. The role of the decreased expression of follistatin and activin receptor IIB mRNA after addition of RA in the mechanism of RA-induced differentiation remains to be elucidated.

  4. Diagnostic accuracy of serum activin A in detection of ectopic pregnancy

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    Mohammad Ali Roghaei

    2012-01-01

    Full Text Available Background: Ectopic pregnancy (EP still remains a main cause of maternal mortalities. This study is designed to evaluate the accuracy of serum Activin A in detection of ectopic pregnancy. Methods : This prospective observational study was conducted from 2009 to 2010 at two main referral university hospitals, Isfahan University of Medical Sciences, Isfahan, Iran. Two hundred subjects who were under 10 week′s pregnancy with clinical presentations of abdominal pain and vaginal bleeding were enrolled. After sampling serum Activin A, patients underwent ultrasonography, titer of B-HCG and surgery (if indicated and were divided into two groups: EP (n = 100 and intrauterine pregnancy (IUP (n = 100. The mean of Activin A was compared between groups and by ROC curve, the optimal cut off with sensitivity, specificity, positive predictive value (PPV and negative predictive value (NPV were determined. Results : The mean age of women with IUP was 25.4 ± 4.3 years (15-40 years compared with 25.9 ± 4.1 years in women in EP group (P = 0.448. Statistical difference was not found between EP versus IUP groups in gestational age (6.32 ± 1.03 vs. 6.85 ± 1.82 weeks, P = 0.124. The mean of serum Activin A in EP group was 0.264 ± 0.0703 ng/ml versus 0.949 ± 0.5283 ng/ml in IUP group (P < 0.05. According to ROC curve (area under the curve = 0.981, P < 0.05, confidence interval: 0.961-1.000, the optimal cut off was estimated as 0.504 ng/ml with sensitivity of 97% and specificity of 93.5%. Conclusion : This study indicated that the mean of serum Activin A is lower in EP compared with IUP. The serum Activin A has a fair accuracy in detecting EP.

  5. Activin A balances Sertoli and germ cell proliferation in the fetal mouse testis.

    Science.gov (United States)

    Mendis, Sirisha H S; Meachem, Sarah J; Sarraj, Mai A; Loveland, Kate L

    2011-02-01

    Activin affects many aspects of cellular development, including those essential for reproductive fitness. This study examined the contribution of activin A to murine fetal testicular development, revealing contrasting outcomes of activin actions on Sertoli cells and gonocytes. Shortly after sex determination, from Embryonic Day 12.5 (E12.5) through to birth (0 dpp), the activin A subunit transcript (Inhba) level rises in testis but not ovary, followed closely by the Inha transcript (encoding the inhibitory inhibin alpha subunit). Activin receptor transcript levels also change, with Acvr1 (encoding ALK2) and Acvr2b (ActRIIB) significantly higher and lower, respectively, at 0 dpp compared with E13.5 and E15.5. Transcripts encoding the signaling mediators Smad1, Smad3, and Smad4 were higher at 0 dpp compared with E13.5 and E15.5, whereas Smad2, Smad5, and Smad7 were lower. Detection of phosphorylated (P-)SMAD2/3 in nearly all testis cell nuclei indicated widespread transforming growth factor beta (TGFB) and/or activin ligand signaling activity. In contrast to wild-type littermates, activin betaA subunit knockout (Inhba(-/-)) mice have significantly smaller testes at birth, attributable to a 50% lower Sertoli cell number and decreased Sertoli cell proliferation from E13.5. Inhba(-/-) testes contained twice the normal gonocyte number at birth, with some appearing to bypass quiescence. Persistence of widespread P-SMAD2/3 in Inhba(-/-) cells indicates other TGFB superfamily ligands are active in fetal testes. Significant differences in Smad and cell cycle regulator transcript levels correlating to Inhba gene dosage correspond to differences in Sertoli and germ cell numbers. In Inhba(-/-) testes, Cdkn1a (encoding p21(cip1)), identified previously in fetal gonocytes, was lower at E13.5, whereas Cdkn1b (encoding p(27kip1) in somatic cells) was lower at birth, and cyclin D2 mRNA and protein were lower at E15.5 and 0 dpp. Thus, activin A dosage contributes to establishing the

  6. Effects of the Activin A–Follistatin System on Myocardial Cell Apoptosis through the Endoplasmic Reticulum Stress Pathway in Heart Failure

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    Liu, Miao; Mao, Cuiying; Li, Jiayu; Han, Fanglei; Yang, Ping

    2017-01-01

    Background: A previous study suggested that activin A inhibited myocardial cell apoptosis. This study thus aimed to explore the effects of the activin A–follistatin system on myocardial cell apoptosis in heart failure (HF) rats in order to determine whether or not the mechanism operates through the endoplasmic reticulum stress (ERS) pathway. Methods: Myocardial infarction (MI) by vascular deprivation was used to induce HF. The enzyme-linked immunosorbent assay was used to detect activin A, follistatin and brain natriuretic peptide (BNP) contents in serum. Immunohistochemical staining for activin A, follistatin, CCAAT-enhancer-binding protein (C/EBP) homologous protein (CHOP) and caspase-3 was performed on the myocardial tissue. The activin A-stimulated apoptosis of H9c2 cells was tested by flow cytometry. Western blot was used to detect the expression levels of activin A, follistatin and ERS-related proteins. Results: It was found that the high expression of activin A could cause activin A–follistatin system imbalance, inducing myocardial cell apoptosis via ERS in vivo. When HF developed to a certain stage, the expression of follistatin was upregulated to antagonize the expression of activin A. Activin A inhibited cardiomyocyte apoptosis with a low concentration and promoted apoptosis with a high concentration in vitro, also via ERS. Conclusion: Activin A–follistatin system participated in ERS-mediated myocardial cell apoptosis in HF. PMID:28208629

  7. Differential regulation of follicle stimulating hormone by activin A and TGFB1 in murine gonadotropes

    Directory of Open Access Journals (Sweden)

    Miller William L

    2005-12-01

    Full Text Available Abstract Background Activins stimulate the synthesis of follicle stimulating hormone (FSH in pituitary gonadotropes, at least in part, by inducing transcription of its beta subunit (Fshb. Evidence from several laboratories studying transformed murine LbetaT2 gonadotropes indicates that activins signal through Smad-dependent and/or Smad-independent pathways, similar to those used by transforming growth factor beta-1 (TGFB1 in other cell types. Therefore, given common intracellular signaling mechanisms of these two ligands, we examined whether TGFBs can also induce transcription of Fshb in LbetaT2 cells as well as in purified primary murine gonadotropes. Methods Murine Fshb promoter-reporter (-1990/+1 mFshb-luc activity was measured in LbetaT2 cells treated with activin A or TGFB1, and in cells transfected with either activin or TGFB receptors. The ability of the ligands to stimulate phosphorylation of Smads 2 and 3 in LbetaT2 cells was measured by western blot analysis, and expression of TGFB type I and II receptors was assessed by reverse transcriptase polymerase chain reaction in both LbetaT2 cells and primary gonadotropes purified from male mice of different ages. Finally, regulation of endogenous murine Fshb mRNA levels by activin A and TGFB1 in purified gonadotropes and whole pituitary cultures was measured using quantitative RT-PCR. Results Activin A dose-dependently stimulated -1990/+1 mFshb-luc activity in LbetaT2 cells, but TGFB1 had no effect at doses up to 5 nM. Similarly, activin A, but not TGFB1, stimulated Smad 2 and 3 phosphorylation in these cells. Constitutively active forms of the activin (Acvr1b-T206D and TGFB (TGFBR1-T204D type I receptors strongly stimulated -1990/+1 mFshb-luc activity, showing that mechanisms down stream of Tgfbr1 seem to be intact in LbetaT2 cells. RT-PCR analysis of LbetaT2 cells and whole adult murine pituitaries indicated that both expressed Tgfbr1 mRNA, but that Tgfbr2 was not detected in LbetaT2 cells

  8. 1α,25-dihydroxyvitamin D3 stimulates activin A production to fine-tune osteoblast-induced mineralization.

    Science.gov (United States)

    Woeckel, V J; van der Eerden, B C J; Schreuders-Koedam, M; Eijken, M; Van Leeuwen, J P T M

    2013-11-01

    In healthy bones, mineralization has to be tightly controlled to avoid pathological phenotypes. In this study, we investigated interactions between 1α,25(OH)2 D3 (1,25D3) and activin A in the regulation of osteoblast induced mineralization. In human osteoblast cultures, we demonstrated that besides stimulation of mineralization, 1,25D3 also induced activin A, a strong inhibitor of mineralization. Simultaneously, follistatin (FST), the natural antagonist of activin A, was down-regulated by1,25D3. This resulted in an increase in activin A activity during 1,25D3 treatment. We also showed that in 1,25D3-treated osteoblasts, mineralization can be further increased when activin A activity was abrogated by adding exogenous FST. This observation implies that, besides stimulation of mineralization, 1,25D3 also controls activin A-mediated inhibition of mineralization. Besides activin A, 1,25D3 also induces osteocalcin (BGLAP), another inhibitor of mineralization. Warfarin, which has been shown to inactivate osteocalcin, increased 1,25D3-induced mineralization. Interaction between these two systems became evident from the synergistic increase in BGLAP expression upon blocking activin activity in 1,25D3-treated cultures. In conclusion, we demonstrate that 1,25D3 stimulation of mineralization by human osteoblasts is suppressed by concomitant induction of inhibitors of mineralization. Mineralization induction by 1,25D3 may actually be controlled via interplay with activin A and osteocalcin. Finally, this complex regulation of mineralization substantiates the significance of tight control of mineralization to prevent excessive mineralization and consequently reduction in bone quality and strength.

  9. An antibody blocking activin type II receptors induces strong skeletal muscle hypertrophy and protects from atrophy.

    Science.gov (United States)

    Lach-Trifilieff, Estelle; Minetti, Giulia C; Sheppard, KellyAnn; Ibebunjo, Chikwendu; Feige, Jerome N; Hartmann, Steffen; Brachat, Sophie; Rivet, Helene; Koelbing, Claudia; Morvan, Frederic; Hatakeyama, Shinji; Glass, David J

    2014-02-01

    The myostatin/activin type II receptor (ActRII) pathway has been identified to be critical in regulating skeletal muscle size. Several other ligands, including GDF11 and the activins, signal through this pathway, suggesting that the ActRII receptors are major regulatory nodes in the regulation of muscle mass. We have developed a novel, human anti-ActRII antibody (bimagrumab, or BYM338) to prevent binding of ligands to the receptors and thus inhibit downstream signaling. BYM338 enhances differentiation of primary human skeletal myoblasts and counteracts the inhibition of differentiation induced by myostatin or activin A. BYM338 prevents myostatin- or activin A-induced atrophy through inhibition of Smad2/3 phosphorylation, thus sparing the myosin heavy chain from degradation. BYM338 dramatically increases skeletal muscle mass in mice, beyond sole inhibition of myostatin, detected by comparing the antibody with a myostatin inhibitor. A mouse version of the antibody induces enhanced muscle hypertrophy in myostatin mutant mice, further confirming a beneficial effect on muscle growth beyond myostatin inhibition alone through blockade of ActRII ligands. BYM338 protects muscles from glucocorticoid-induced atrophy and weakness via prevention of muscle and tetanic force losses. These data highlight the compelling therapeutic potential of BYM338 for the treatment of skeletal muscle atrophy and weakness in multiple settings.

  10. Serum activin A and follistatin levels in gestational diabetes and the association of the Activin A-Follistatin system with anthropometric parameters in offspring.

    Directory of Open Access Journals (Sweden)

    Silvia Näf

    Full Text Available CONTEXT: The Activin A-Follistatin system has emerged as an important regulator of lipid and glucose metabolism with possible repercussions on fetal growth. OBJECTIVE: To analyze circulating activin A, follistatin and follistatin-like-3 (FSTL3 levels and their relationship with glucose metabolism in pregnant women and their influence on fetal growth and neonatal adiposity. DESIGN AND METHODS: A prospective cohort was studied comprising 207 pregnant women, 129 with normal glucose tolerance (NGT and 78 with gestational diabetes mellitus (GDM and their offspring. Activin A, follistatin and FSTL3 levels were measured in maternal serum collected in the early third trimester of pregnancy. Serial fetal ultrasounds were performed during the third trimester to evaluate fetal growth. Neonatal anthropometry was measured to assess neonatal adiposity. RESULTS: Serum follistatin levels were significantly lower in GDM than in NGT pregnant women (8.21±2.32 ng/mL vs 9.22±3.41, P = 0.012 whereas serum FSTL3 and activin A levels were comparable between the two groups. Serum follistatin concentrations were negatively correlated with HOMA-IR and positively with ultrasound growth parameters such as fractional thigh volume estimation in the middle of the third trimester and percent fat mass at birth. Also, in the stepwise multiple linear regression analysis serum follistatin levels were negatively associated with HOMA-IR (β = -0.199, P = 0.008 and the diagnosis of gestational diabetes (β = -0.138, P = 0.049. Likewise, fractional thigh volume estimation in the middle of third trimester and percent fat mass at birth were positively determined by serum follistatin levels (β = 0.214, P = 0.005 and β = 0.231, P = 0.002, respectively. CONCLUSIONS: Circulating follistatin levels are reduced in GDM compared with NGT pregnant women and they are positively associated with fetal growth and neonatal adiposity. These data suggest a role of

  11. Activin and TGF-β effects on brain development and neural stem cells.

    Science.gov (United States)

    Rodríguez-Martínez, Griselda; Velasco, Iván

    2012-11-01

    Transforming Growth Factor-β (TGF-β) family members are ubiquitously expressed, participating in the regulation of many processes in different cell types both in embryonic and adult stages. Several members of this family, including Activins, TGF-β1-3 and Nodal, have been implicated in the development and maintenance of various organs, in which stem cells play important roles. Although TGF-β was initially considered an injury-related cytokine, it became clear that not only TGF-β, but other members of this family, play critical roles in morphogenesis and cell lineage specification. During brain development, Activin and TGF-βs as well as their cognate receptors, are expressed in different patterns. The roles of Activin and TGF-β during CNS development are sometimes contradictory, because these proteins present different actions depending on the cell type and the context. The aim of this review is to summarize current information on the actions of TGF-β members during developing brain, and also on Neural Stem/Progenitor Cells (NSPC). We focus on the TGF-β subgroup, specifically on the effects of TGF-β1 and Activin A. In the first section we describe the main characteristics of the ligands, its receptors as well as the proteins and mechanisms involved in signaling. Next, we discuss the main advances concerning TGF-β1 and Activin actions during brain development and their roles in NSPC fate decision and neuroprotection both in vitro and in vivo. The emerging picture from these studies suggests that these growth factors can be used to manipulate neurogenesis and might help to achieve restoration after brain deterioration.

  12. Regulation of FSHβ induction in LβT2 cells by BMP2 and an Activin A/BMP2 chimera, AB215.

    Science.gov (United States)

    Jung, Jae Woo; Ahn, Chihoon; Shim, Sun Young; Gray, Peter C; Kwiatkowski, Witek; Choe, Senyon

    2014-10-01

    Activins and bone morphogenetic proteins (BMPs) share activin type 2 signaling receptors but utilize different type 1 receptors and Smads. We designed AB215, a potent BMP2-like Activin A/BMP2 chimera incorporating the high-affinity type 2 receptor-binding epitope of Activin A. In this study, we compare the signaling properties of AB215 and BMP2 in HEK293T cells and gonadotroph LβT2 cells in which Activin A and BMP2 synergistically induce FSHβ. In HEK293T cells, AB215 is more potent than BMP2 and competitively blocks Activin A signaling, while BMP2 has a partial blocking activity. Activin A signaling is insensitive to BMP pathway antagonism in HEK293T cells but is strongly inhibited by constitutively active (CA) BMP type 1 receptors. By contrast, the potencies of AB215 and BMP2 are indistinguishable in LβT2 cells and although AB215 blocks Activin A signaling, BMP2 has no inhibitory effect. Unlike HEK293T, Activin A signaling is strongly inhibited by BMP pathway antagonism in LβT2 cells but is largely unaffected by CA BMP type 1 receptors. BMP2 increases phospho-Smad3 levels in LβT2 cells, in both the absence and the presence of Activin A treatment, and augments Activin A-induced FSHβ. AB215 has the opposite effect and sharply decreases basal phospho-Smad3 levels and blocks Smad2 phosphorylation and FSHβ induction resulting from Activin A treatment. These findings together demonstrate that while AB215 activates the BMP pathway, it has opposing effects to those of BMP2 on FSHβ induction in LβT2 cells apparently due to its ability to block Activin A signaling.

  13. Regulation of muscle growth by multiple ligands signaling through activin type II receptors

    Science.gov (United States)

    Lee, Se-Jin; Reed, Lori A.; Davies, Monique V.; Girgenrath, Stefan; Goad, Mary E. P.; Tomkinson, Kathy N.; Wright, Jill F.; Barker, Christopher; Ehrmantraut, Gregory; Holmstrom, James; Trowell, Betty; Gertz, Barry; Jiang, Man-Shiow; Sebald, Suzanne M.; Matzuk, Martin; Li, En; Liang, Li-fang; Quattlebaum, Edwin; Stotish, Ronald L.; Wolfman, Neil M.

    2005-01-01

    Myostatin is a secreted protein that normally functions as a negative regulator of muscle growth. Agents capable of blocking the myostatin signaling pathway could have important applications for treating human muscle degenerative diseases as well as for enhancing livestock production. Here we describe a potent myostatin inhibitor, a soluble form of the activin type IIB receptor (ACVR2B), which can cause dramatic increases in muscle mass (up to 60% in 2 weeks) when injected into wild-type mice. Furthermore, we show that the effect of the soluble receptor is attenuated but not eliminated in Mstn-/- mice, suggesting that at least one other ligand in addition to myostatin normally functions to limit muscle growth. Finally, we provide genetic evidence that these ligands signal through both activin type II receptors, ACVR2 and ACVR2B, to regulate muscle growth in vivo. PMID:16330774

  14. Regulation of C-cadherin function during activin induced morphogenesis of Xenopus animal caps

    OpenAIRE

    1994-01-01

    Treatment of Xenopus animal pole tissue with activin results in the induction of mesodermal cell types and a dramatic elongation of the tissue. The morphogenetic movements involved in the elongation appear similar to those in normal gastrulation, which is driven by cell rearrangement and cell intercalations. We have used this system to explore the potential regulation of cell-cell adhesion and cadherin function during morphogenesis. Quantitative blastomere aggregation assays revealed that act...

  15. ACVR2A promoter polymorphism rs1424954 in the Activin-A signaling pathway in trophoblasts.

    Science.gov (United States)

    Thulluru, H K; Michel, O J; Oudejans, C B M; van Dijk, M

    2015-04-01

    Pre-eclampsia is a pregnancy-specific disorder and characterized by reduced trophoblast invasion and reduced spiral artery remodeling in the first trimester placenta. A polymorphism located in the promoter region of ACVR2A (rs1424954 (A > G)) has previously been shown to be significantly associated with pre-eclampsia. The effects of this variant on ACVR2A expression and its function in the Activin-A signaling pathway were studied by transfections in SGHPL-5 extravillous trophoblasts followed by qRT-PCR. Here we show that the ACVR2A promoter susceptibility variant causes a downregulation of ACVR2A expression. We also provide evidence for transcription of a so-called PROMPT (PROMoter-uPstream-Transcript) in the opposite direction of ACVR2A, containing the polymorphism, and downregulated when the susceptibility allele is carried, which either shares the same promoter as ACVR2A or is a non-coding RNA that is able to enhance ACVR2A transcription. Furthermore, when the effect of the susceptibility variant is mimicked by knockdown of ACVR2A, physiologic concentrations of Activin-A cause a reduction in NODAL mRNA expression in the SGHPL-5 trophoblasts, indicative of a protective effect as reduction in NODAL expression is associated with an increase in trophoblast invasion. However, at pathologic levels of Activin-A, as found in pre-eclampsia, this effect is no longer seen, and we show this is potentially caused by a lack of downregulation of ACVR2B. The combined data suggest a double hit phenomenon in which the first hit, the promoter variant, together with the second hit, pathological levels of Activin-A, lead to high levels of NODAL, associated with reduced trophoblast invasion and observed in pre-eclamptic placentas. Copyright © 2015 Elsevier Ltd. All rights reserved.

  16. Activin and Bmp4 Signaling Converge on Wnt Activation during Odontogenesis.

    Science.gov (United States)

    Kwon, H-J E; Jia, S; Lan, Y; Liu, H; Jiang, R

    2017-09-01

    Previous studies show that both activin and Bmp4 act as crucial mesenchymal odontogenic signals during early tooth development. Remarkably, mice lacking activin-βA ( Inhba(-/-)) and mice with neural crest-specific inactivation of Bmp4 ( Bmp4(ncko/ncko)) both exhibit bud-stage developmental arrest of the mandibular molar tooth germs while their maxillary molar tooth germs completed morphogenesis. In this study, we found that, whereas expression of Inhba and Bmp4 in the developing tooth mesenchyme is independent of each other, Bmp4(ncko/ncko)Inhba(-/-) compound mutant mice exhibit early developmental arrest of all tooth germs. Moreover, genetic inactivation of Osr2, a negative regulator of the odontogenic function of the Bmp4-Msx1 signaling pathway, rescues mandibular molar morphogenesis in Inhba(-/-) embryos. We recently reported that Osr2 and the Bmp4-Msx1 pathway control the bud-to-cap transition of tooth morphogenesis through antagonistic regulation of expression of secreted Wnt antagonists, including Dkk2 and Sfrp2, in the developing tooth mesenchyme. We show here that expression of Dkk2 messenger RNAs was significantly upregulated and expanded into the tooth bud mesenchyme in Inhba(-/-) embryos in comparison with wild-type littermates. Furthermore, in utero treatment with either lithium chloride, an agonist of canonical Wnt signaling, or the DKK inhibitor IIIC3a rescued mandibular molar tooth morphogenesis in Inhba(-/-) embryos. Together with our finding that the developing mandibular molar tooth bud mesenchyme expresses significantly higher levels of Dkk2 than the developing maxillary molar tooth mesenchyme, these data indicate that Bmp4 and activin signaling pathways converge on activation of the Wnt signaling pathway to promote tooth morphogenesis through the bud-to-cap transition and that the differential effects of loss of activin or Bmp4 signaling on maxillary and mandibular molar tooth morphogenesis are mainly due to the differential expression of Wnt

  17. TGFβ/Activin signalling is required for ribosome biogenesis and cell growth in Drosophila salivary glands

    Science.gov (United States)

    Eusebio, Nadia; Correia, Andreia; Marinho, Joana; Casares, Fernando

    2017-01-01

    Signalling by TGFβ superfamily factors plays an important role in tissue growth and cell proliferation. In Drosophila, the activity of the TGFβ/Activin signalling branch has been linked to the regulation of cell growth and proliferation, but the cellular and molecular basis for these functions are not fully understood. In this study, we show that both the RII receptor Punt (Put) and the R-Smad Smad2 are strongly required for cell and tissue growth. Knocking down the expression of Put or Smad2 in salivary glands causes alterations in nucleolar structure and functions. Cells with decreased TGFβ/Activin signalling accumulate intermediate pre-rRNA transcripts containing internal transcribed spacer 1 regions accompanied by the nucleolar retention of ribosomal proteins. Thus, our results show that TGFβ/Activin signalling is required for ribosomal biogenesis, a key aspect of cellular growth control. Importantly, overexpression of Put enhanced cell growth induced by Drosophila Myc, a well-characterized inducer of nucleolar hypertrophy and ribosome biogenesis. PMID:28123053

  18. Role of Activin-A and Myostatin and Their Signaling Pathway in Human Myometrial and Leiomyoma Cell Function

    Science.gov (United States)

    Islam, Md Soriful; Catherino, William H.; Protic, Olga; Janjusevic, Milijana; Gray, Peter Clarke; Giannubilo, Stefano Raffaele; Ciavattini, Andrea; Lamanna, Pasquale; Tranquilli, Andrea Luigi; Petraglia, Felice

    2014-01-01

    Context: Uterine leiomyomas are highly prevalent benign tumors of premenopausal women and the most common indication for hysterectomy. However, the exact etiology of this tumor is not fully understood. Objective: The objective of the study was to evaluate the role of activin-A and myostatin and their signaling pathways in human myometrial and leiomyoma cells. Design: This was a laboratory study. Setting: Myometrial and leiomyoma cells (primary and cell lines) were cultured in vitro. Patients: The study included premenopausal women who were admitted to the hospital for myomectomy or hysterectomy. Interventions: Primary myometrial and leiomyoma cells and/or cell lines were treated with activin-A (4 nM) and myostatin (4 nM) for different days of interval (to measure proliferation rate) or 30 minutes (to measure signaling molecules) or 48 hours to measure proliferating markers, extracellular matrix mRNA, and/or protein expression by real-time PCR, Western blot, and/or immunocytochemistry. Results: We found that activin-A and myostatin significantly reduce cell proliferation in primary myometrial cells but not in leiomyoma cells as measured by a CyQUANT cell proliferation assay kit. Reduced expression of proliferating cell nuclear antigen and Ki-67 were also observed in myometrial cells in response to activin-A and myostatin treatment. Activin-A also significantly increased mRNA expression of fibronectin, collagen1A1, and versican in primary leiomyoma cells. Finally, we found that activin-A and myostatin activate Smad-2/3 signaling but do not affect ERK or p38 signaling in both myometrial and leiomyoma cells. Conclusions: This study results suggest that activin-A and myostatin can exert antiproliferative and/or fibrotic effects on these cell types via Smad-2/3 signaling. PMID:24606069

  19. Overexpression of activin-A and -B in malignant mesothelioma – Attenuated Smad3 signaling responses and ERK activation promote cell migration and invasive growth

    Energy Technology Data Exchange (ETDEWEB)

    Tamminen, Jenni A.; Yin, Miao [Research Programs Unit, Translational Cancer Biology, University of Helsinki (Finland); Transplantation Laboratory, Haartman Institute, University of Helsinki (Finland); Rönty, Mikko [Helsinki University Central Hospital Laboratory, Helsinki (Finland); Department of Pathology, University of Helsinki (Finland); Sutinen, Eva [Helsinki University Central Hospital Laboratory, Helsinki (Finland); Department of Medicine, Division of Pulmonary Medicine, University of Helsinki (Finland); Pasternack, Arja; Ritvos, Olli [Helsinki University Central Hospital Laboratory, Helsinki (Finland); Department of Bacteriology and Immunology, University of Helsinki (Finland); Myllärniemi, Marjukka [Transplantation Laboratory, Haartman Institute, University of Helsinki (Finland); Helsinki University Central Hospital Laboratory, Helsinki (Finland); Department of Medicine, Division of Pulmonary Medicine, University of Helsinki (Finland); Koli, Katri, E-mail: katri.koli@helsinki.fi [Research Programs Unit, Translational Cancer Biology, University of Helsinki (Finland); Transplantation Laboratory, Haartman Institute, University of Helsinki (Finland)

    2015-03-01

    Activin-A and activin-B, members of the TGF-β superfamily, are regulators of reproductive functions, inflammation and wound healing. These dimeric molecules regulate various cellular activities such as proliferation, migration and suvival. Malignant mesothelioma is an asbestos exposure related tumor affecting mainly pleura and it usually has a dismal prognosis. Here, we demonstrate that both activin-A and -B are abundantly expressed in mesothelioma tumor tissue as well as in cultured primary and established mesothelioma cells. Migratory and invasive mesothelioma cells were also found to have attenuated activation of the Smad2/3 pathway in response to activins. Migration and invasive growth of the cells in three-dimentional matrix was prevented by inhibition of activin activity using a soluble activin receptor 2B (sActR2B-Fc). This was associated with decreased ERK activity. Furthermore, migration and invasive growth was significantly inhibited by blocking ERK phosphorylation. Mesothelioma tumors are locally invasive and our results clearly suggest that acivins have a tumor-promoting function in mesothelioma through increasing expression and switching from canonical Smad3 pathway to non-canonical ERK pathway signaling. Blocking activin activity offers a new therapeutic approach for inhibition of mesothelioma invasive growth. - Highlights: • Activin-A and activin-B are highly expressed in mesothelioma. • Mesothelioma cell migration and invasive growth can be blocked with sActR2B. • Activin induced Smad3 activity is attenuated in invasive mesothelioma cells. • Activins induce ERK activity in mesothelioma cells.

  20. Activin A secreted by human mesenchymal stem cells induces neuronal development and neurite outgrowth in an in vitro model of Alzheimer's disease: neurogenesis induced by MSCs via activin A.

    Science.gov (United States)

    Park, Sang Eon; Lee, Jeongmin; Chang, Eun Hyuk; Kim, Jong Hwa; Sung, Ji-Hee; Na, Duk L; Chang, Jong Wook

    2016-08-01

    Alzheimer's disease (AD) is characterized by progressive loss of memory in addition to cortical atrophy. Cortical atrophy in AD brains begins in the parietal and temporal lobes, which are near the subventricular zone (SVZ). The aim of this study was to activate the neurogenesis in the SVZ of AD brains by human mesenchymal stem cells (hMSCs). Neural stem cells (NSCs) were isolated from SVZ of 4-month-old 5XFAD mice. Co-culture of hMSCs with SVZ-derived NSCs from 5XFAD mice induced neuronal development and neurite outgrowth. To examine the inducing factor of neurogenesis, human cytokine array was performed with co-cultured media, and revealed elevated release of activin A from hMSCs. Also, we confirmed that the mRNA levels of activin A and activin receptor in the SVZ of 5XFAD mice were significantly lower than normal mice. Treatment of human recombinant activin A in SVZ-derived NSCs from 5XFAD mice induced neuronal development and neurite outgrowth. These data suggest that use of hMSCs and activin A to recover neurogenesis in future studies of cortical regeneration to treat AD.

  1. Testicular activin and follistatin levels are elevated during the course of experimental autoimmune epididymo–orchitis in mice

    Science.gov (United States)

    Nicolas, Nour; Michel, Vera; Bhushan, Sudhanshu; Wahle, Eva; Hayward, Susan; Ludlow, Helen; de Kretser, David M.; Loveland, Kate L.; Schuppe, Hans-Christian; Meinhardt, Andreas; Hedger, Mark P.; Fijak, Monika

    2017-01-01

    Experimental autoimmune epididymo-orchitis (EAEO) is a model of chronic inflammation, induced by immunisation with testicular antigens, which reproduces the pathology of some types of human infertility. Activins A and B regulate spermatogenesis and steroidogenesis, but are also pro-inflammatory, pro-fibrotic cytokines. Expression of the activins and their endogenous antagonists, inhibin and follistatin, was examined in murine EAEO. Adult untreated and adjuvant-treated control mice showed no pathology. All mice immunised with testis antigens developed EAEO by 50 days, characterised by loss of germ cells, immune cell infiltration and fibrosis in the testis, similar to biopsies from human inflamed testis. An increase of total CD45+ leukocytes, comprising CD3+ T cells, CD4 + CD8− and CD4 + CD25+ T cells, and a novel population of CD4 + CD8+ double positive T cells was also detected in EAEO testes. This was accompanied by increased expression of TNF, MCP-1 and IL-10. Activin A and B and follistatin protein levels were elevated in EAEO testes, with peak activin expression during the active phase of the disease, whereas mRNA expression of the inhibin B subunits (Inha and Inhbb) and activin receptor subunits (Acvr1b and Acvr2b) were downregulated. These data suggest that activin–follistatin regulation may play a role during the development of EAEO. PMID:28205525

  2. Activin/Nodal signaling controls divergent transcriptional networks in human embryonic stem cells and in endoderm progenitors.

    Science.gov (United States)

    Brown, Stephanie; Teo, Adrian; Pauklin, Siim; Hannan, Nicholas; Cho, Candy H-H; Lim, Bing; Vardy, Leah; Dunn, N Ray; Trotter, Matthew; Pedersen, Roger; Vallier, Ludovic

    2011-08-01

    Activin/Nodal signaling is necessary to maintain pluripotency of human embryonic stem cells (hESCs) and to induce their differentiation toward endoderm. However, the mechanisms by which Activin/Nodal signaling achieves these opposite functions remain unclear. To unravel these mechanisms, we examined the transcriptional network controlled in hESCs by Smad2 and Smad3, which represent the direct effectors of Activin/Nodal signaling. These analyses reveal that Smad2/3 participate in the control of the core transcriptional network characterizing pluripotency, which includes Oct-4, Nanog, FoxD3, Dppa4, Tert, Myc, and UTF1. In addition, similar experiments performed on endoderm cells confirm that a broad part of the transcriptional network directing differentiation is downstream of Smad2/3. Therefore, Activin/Nodal signaling appears to control divergent transcriptional networks in hESCs and in endoderm. Importantly, we observed an overlap between the transcriptional network downstream of Nanog and Smad2/3 in hESCs; whereas, functional studies showed that both factors cooperate to control the expression of pluripotency genes. Therefore, the effect of Activin/Nodal signaling on pluripotency and differentiation could be dictated by tissue specific Smad2/3 partners such as Nanog, explaining the mechanisms by which signaling pathways can orchestrate divergent cell fate decisions.

  3. Placental activin A is required for follicular development during the second half of pregnancy in the golden hamster (Mesocricetus auratus).

    Science.gov (United States)

    Furuta, Chie; Arakawa, Sayoko; Shi, Zhanquan; Watanabe, Gen; Taya, Kazuyoshi

    2008-04-01

    Numerous antral follicles develop during the second half of pregnancy in the golden hamster even though LH and FSH are maintained at basal levels. To investigate the possible hormone actions of activin A associated with follicular development, pregnant golden hamsters were placentectomized on day 6 of pregnancy and animals were sacrificed at day 8, 10, 12, or 14 of pregnancy. There was a drastic decrease in the plasma concentrations of activin A from day 10 of pregnancy in the operated group compared to the controls. Positive immunohistochemical staining of inhibin/activin subunits betaA and betaB in the syncytiotrophoblast of the placenta revealed the source of activin A, AB, or B. The number of healthy follicles did not change until day 12 between the operated and the control groups, but decreased in numbers in the operated groups thereafter. The decreased concentrations of inhibin A, B, and estradiol-17beta in the operated groups at day 10 and 12 correlated well with the number of mature follicles in response to hCG treatment. In conclusion, we revealed that activin A secreted from the placenta induces folliculogenesis to maintain the high levels of estradiol-17beta needed to induce uterine dilatation for fetus growth and impending parturition.

  4. Serum levels of activin A and inhibin A are not related to the increased susceptibility to pre-eclampsia in type I diabetic pregnancies

    DEFF Research Database (Denmark)

    Ekbom, Pia; Damm, Peter; Andersson, Anna-Maria;

    2006-01-01

    Activin A and inhibin A have been found to be elevated in women without diabetes subsequently developing pre-eclampsia. The aim was to investigate whether activin A and inhibin A in serum were elevated in type I diabetic women after developing pre-eclampsia and, if so, were they clinically useful...

  5. Serum levels of activin A and inhibin A are not related to the increased susceptibility to pre-eclampsia in type I diabetic pregnancies

    DEFF Research Database (Denmark)

    Ekbom, Pia; Damm, Peter; Andersson, Anna-Maria;

    2006-01-01

    Activin A and inhibin A have been found to be elevated in women without diabetes subsequently developing pre-eclampsia. The aim was to investigate whether activin A and inhibin A in serum were elevated in type I diabetic women after developing pre-eclampsia and, if so, were they clinically useful...... as predictors of pre-eclampsia....

  6. Analysis of activin/TGFB-signaling modulators within the normal and dysfunctional adult human testis reveals evidence of altered signaling capacity in a subset of seminomas

    DEFF Research Database (Denmark)

    Dias, Vinali L; Rajpert-De Meyts, Ewa; McLachlan, Robert

    2009-01-01

    Activin is a pleiotropic growth factor belonging to the transforming growth factor-beta (TGFB) superfamily of signaling molecules. Regulated activin signaling is known to influence several steps in rodent male gamete differentiation. TGFB ligand isoforms, TGFB1-B3, also influence germ cell survival...

  7. Changes in the reproductive function and developmental phenotypes in mice following intramuscular injection of an activin betaA-expressing plasmid.

    Science.gov (United States)

    Kim, Mi-Nyeu; Park, Moon Nyeo; Jung, Hoi Kyung; Cho, Chunghee; Mayo, Kelly E; Cho, Byung-Nam

    2008-12-16

    The TGF-beta family protein activin has numerous reported activities with some uncertainty in the reproductive axis and development. The precise roles of activin in in vivo system were investigated using a transient gain of function model. To this end, an expression plasmid, pCMV-rAct, with the activin betaA cDNA fused to the cytomegalovirus promoter, was introduced into muscle of the female adult mice by direct injection. Activin betaA mRNA was detected in the muscle by RT-PCR and subsequent Southern blot analysis. Activin betaA was also detected, and western blot analysis revealed a relatively high level of serum activin with correspondingly increased FSH. In the pCMV-rAct-injected female mice, estrus stage within the estrous cycle was extended. Moreover, increased numbers of corpora lutea and a thickened granulosa cell layer with a small antrum in tertiary follicles within the ovary were observed. When injected female mice were mated with males of proven fertility, a subset of embryos died in utero, and most of those that survived exhibited increased body weight. Taken together, our data reveal that activin betaA can directly influence the estrous cycle, an integral part of the reproduction in female mice and activin betaA can also influence the embryo development as an endocrine fashion.

  8. Changes in the reproductive function and developmental phenotypes in mice following intramuscular injection of an activin betaA-expressing plasmid

    Directory of Open Access Journals (Sweden)

    Mayo Kelly E

    2008-12-01

    Full Text Available Abstract Background The TGF-beta family protein activin has numerous reported activities with some uncertainty in the reproductive axis and development. The precise roles of activin in in vivo system were investigated using a transient gain of function model. Methods To this end, an expression plasmid, pCMV-rAct, with the activin betaA cDNA fused to the cytomegalovirus promoter, was introduced into muscle of the female adult mice by direct injection. Results Activin betaA mRNA was detected in the muscle by RT-PCR and subsequent Southern blot analysis. Activin betaA was also detected, and western blot analysis revealed a relatively high level of serum activin with correspondingly increased FSH. In the pCMV-rAct-injected female mice, estrus stage within the estrous cycle was extended. Moreover, increased numbers of corpora lutea and a thickened granulosa cell layer with a small antrum in tertiary follicles within the ovary were observed. When injected female mice were mated with males of proven fertility, a subset of embryos died in utero, and most of those that survived exhibited increased body weight. Conclusion Taken together, our data reveal that activin betaA can directly influence the estrous cycle, an integral part of the reproduction in female mice and activin betaA can also influence the embryo development as an endocrine fashion.

  9. A crucial role of activin A-mediated growth hormone suppression in mouse and human heart failure.

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    Noritoshi Fukushima

    Full Text Available Infusion of bone marrow-derived mononuclear cells (BMMNC has been reported to ameliorate cardiac dysfunction after acute myocardial infarction. In this study, we investigated whether infusion of BMMNC is also effective for non-ischemic heart failure model mice and the underlying mechanisms. Intravenous infusion of BMMNC showed transient cardioprotective effects on animal models with dilated cardiomyopathy (DCM without their engraftment in heart, suggesting that BMMNC infusion improves cardiac function via humoral factors rather than their differentiation into cardiomyocytes. Using conditioned media from sorted BMMNC, we found that the cardioprotective effects were mediated by growth hormone (GH secreted from myeloid (Gr-1(+ cells and the effects was partially mediated by signal transducer and activator of transcription 3 in cardiomyocytes. On the other hand, the GH expression in Gr-1(+ cells was significantly downregulated in DCM mice compared with that in healthy control, suggesting that the environmental cue in heart failure might suppress the Gr-1(+ cells function. Activin A was upregulated in the serum of DCM models and induced downregulation of GH levels in Gr-1(+ cells and serum. Furthermore, humoral factors upregulated in heart failure including angiotensin II upregulated activin A in peripheral blood mononuclear cells (PBMNC via activation of NFκB. Similarly, serum activin A levels were also significantly higher in DCM patients with heart failure than in healthy subjects and the GH levels in conditioned medium from PBMNC of DCM patients were lower than that in healthy subjects. Inhibition of activin A increased serum GH levels and improved cardiac function of DCM model mice. These results suggest that activin A causes heart failure by suppressing GH activity and that inhibition of activin A might become a novel strategy for the treatment of heart failure.

  10. Efficient retina formation requires suppression of both Activin and BMP signaling pathways in pluripotent cells

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    Kimberly A. Wong

    2015-03-01

    Full Text Available Retina formation requires the correct spatiotemporal patterning of key regulatory factors. While it is known that repression of several signaling pathways lead to specification of retinal fates, addition of only Noggin, a known BMP antagonist, can convert pluripotent Xenopus laevis animal cap cells to functional retinal cells. The aim of this study is to determine the intracellular molecular events that occur during this conversion. Surprisingly, blocking BMP signaling alone failed to mimic Noggin treatment. Overexpressing Noggin in pluripotent cells resulted in a concentration-dependent suppression of both Smad1 and Smad2 phosphorylation, which act downstream of BMP and Activin signaling, respectively. This caused a decrease in downstream targets: endothelial marker, xk81, and mesodermal marker, xbra. We treated pluripotent cells with dominant-negative receptors or the chemical inhibitors, dorsomorphin and SB431542, which each target either the BMP or Activin signaling pathway. We determined the effect of these treatments on retina formation using the Animal Cap Transplant (ACT assay; in which treated pluripotent cells were transplanted into the eye field of host embryos. We found that inhibition of Activin signaling, in the presence of BMP signaling inhibition, promotes efficient retinal specification in Xenopus tissue, mimicking the affect of adding Noggin alone. In whole embryos, we found that the eye field marker, rax, expanded when adding both dominant-negative Smad1 and Smad2, as did treating the cells with both dorsomorphin and SB431542. Future studies could translate these findings to a mammalian culture assay, in order to more efficiently produce retinal cells in culture.

  11. Neuroprotective Effects of Exogenous Activin A on Oxygen-Glucose Deprivation in PC12 Cells

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    Zhong-Xin Xu

    2011-12-01

    Full Text Available Ischemic cerebrovascular disease is one of the most common causes of death in the World. Exogenous activin A (ActA protects neurons against toxicity and plays a central role in regulating the brain’s response to injury. In the present study, we investigated the mechanisms involved in the neuroprotective effects of ActA in a model of hypoxic-ischemic brain disease. We found that ActA could effectively increase the survival rate of PC12 cells and relieve oxygen-glucose deprivation (OGD damage. To clarify the neuroprotective mechanisms of ActA, the effects of ActA on the ActA/Smad pathway and on the up-regulation of inducible nitric oxide synthase (NOS and superoxide dismutase (SOD were investigated using OGD in PC12 cells. The results showed that ActA could increase the expression of activin receptor IIA (ActRIIA, Smad3 and Smad4 and that 50 ng/mL and 100 ng/mL of ActA could reduce NO levels and increase SOD activity by 78.9% and 79.9%, respectively. These results suggested that the neuroprotective effects of ActA in ischemia could be related to the activation of the ActA/Smad signaling pathway and to its anti-oxidant activities.

  12. RhoA regulates Activin B-induced stress fiber formation and migration of bone marrow-derived mesenchymal stromal cell through distinct signaling.

    Science.gov (United States)

    Wang, Xueer; Tang, Pei; Guo, Fukun; Zhang, Min; Chen, Yinghua; Yan, Yuan; Tian, Zhihui; Xu, Pengcheng; Zhang, Lei; Zhang, Lu; Zhang, Lin

    2017-01-01

    In our previous study, Activin B induced actin stress fiber formation and cell migration in Bone marrow-derived mesenchymal stem cells (BMSCs) in vitro. However, the underlying molecular mechanisms are not well studied. RhoA is recognized to play a critical role in the regulation of actomyosin cytoskeletal organization and cell migration. Pull-down assay was performed to investigate the activity of RhoA. The dominant-negative mutants of RhoA (RhoA(N19)) was used to determine whether RhoA has a role in Activin B-induced cytoskeleton organization and cell migration in BMSCs. Cytoskeleton organization was examined by fluorescence Rhodamine-phalloidin staining, and cell migration by transwell and cell scratching assay. Western blot was carried out to investigate downstream signaling cascade of RhoA. Inhibitor and siRNAs were used to detect the role of downstream signaling in stress fiber formation and/or cell migration. RhoA was activated by Activin B in BMSCs. RhoA(N19) blocked Activin B-induced stress fiber formation and cell migration. ROCK inhibitor blocked Activin B-induced stress fiber formation but enhanced BMSCs migration. Activin B induced phosphorylation of LIMK2 and Cofilin, which was abolished by ROCK inhibition. Both of siRNA LIMK2 and siRNA Cofilin inhibited Activin B-induced stress fiber formation. RhoA regulates Activin B-induced stress fiber formation and migration of BMSCs. A RhoA-ROCK-LIMK2-Cofilin signaling node exists and regulates actin stress fiber formation. RhoA regulates Activin B-induced cell migration independent of ROCK. Better understanding of the molecular mechanisms of BMSCs migration will help optimize therapeutic strategy to target BMSCs at injured tissues. Copyright © 2016 Elsevier B.V. All rights reserved.

  13. Neuroprotective effects of Activin A on endoplasmic reticulum stress-mediated apoptotic and autophagic PC12 cell death

    Directory of Open Access Journals (Sweden)

    Long-xing Xue

    2017-01-01

    Full Text Available Activin A, a member of the transforming growth factor-beta superfamily, plays a neuroprotective role in multiple neurological diseases. Endoplasmic reticulum (ER stress-mediated apoptotic and autophagic cell death is implicated in a wide range of diseases, including cerebral ischemia and neurodegenerative diseases. Thapsigargin was used to induce PC12 cell death, and Activin A was used for intervention. Our results showed that Activin A significantly inhibited morphological changes in thapsigargin-induced apoptotic cells, and the expression of apoptosis-associated proteins [cleaved-caspase-12, C/EBP homologous protein (CHOP and cleaved-caspase-3] and biomarkers of autophagy (Beclin-1 and light chain 3, and downregulated the expression of thapsigargin-induced ER stress-associated proteins [inositol requiring enzyme-1 (IRE1, tumor necrosis factor receptor-associated factor 2 (TRAF2, apoptosis signal-regulating kinase 1 (ASK1, c-Jun N-terminal kinase (JNK and p38]. The inhibition of thapsigargin-induced cell death was concentration-dependent. These findings suggest that administration of Activin A protects PC12 cells against ER stress-mediated apoptotic and autophagic cell death by inhibiting the activation of the IRE1-TRAF2-ASK1-JNK/p38 cascade.

  14. Identification and expression of Smads associated with TGF-beta/activin/nodal signaling pathways in the rainbow trout (Oncorhynuchus mykiss)

    Science.gov (United States)

    The Smad proteins are essential components of the TGF-beta/activin/nodal family signaling pathway. We report the identification and characterization of transcripts representing 3 receptor Smads (Smad2a, Smad2b, Smad3), 2 common Smads (Smad4a, Smad4b) and one inhibitory Smad (Smad7). Phylogenetic an...

  15. An Activin A/BMP2 chimera displays bone healing properties superior to those of BMP2

    Science.gov (United States)

    Yoon, Byung-Hak; Esquivies, Luis; Ahn, Chihoon; Gray, Peter C.; Ye, Sang-kyu; Kwiatkowski, Witek; Choe, Senyon

    2014-01-01

    Recombinant Bone Morphogenetic Protein 2 (rhBMP2) has been used clinically to treat bone fractures in human patients. However, the high doses of rhBMP2 required for a therapeutic response can cause undesirable side effects. Here, we demonstrate that a novel Activin A/BMP2 (AB2) chimera, AB204, promotes osteogenesis and bone healing much more potently and effectively than rhBMP2. Remarkably, 1 month of AB204 treatment completely heals tibial and calvarial defects of critical size in mice at a concentration 10-fold lower than a dose of rhBMP2 that only partially heals the defect. We determine the structure of AB204 to 2.3 Å that reveals a distinct BMP2-like fold in which the Activin A sequence segments confer insensitivity to the BMP2 antagonist Noggin and an affinity for the Activin/BMP type II receptor ActRII that is 100-fold greater than that of BMP2. The structure also led to our identification of a single Activin A-derived amino acid residue which when mutated to the corresponding BMP2 residue resulted in a significant increase in the affinity of AB204 for its type I receptor BMPRIa and a further enhancement in AB204's osteogenic potency. Together, these findings demonstrate that rationally designed AB2 chimeras can provide BMP2 substitutes with enhanced potency for treating non-union bone fractures. PMID:24692083

  16. An activin A/BMP2 chimera, AB204, displays bone-healing properties superior to those of BMP2.

    Science.gov (United States)

    Yoon, Byung-Hak; Esquivies, Luis; Ahn, Chihoon; Gray, Peter C; Ye, Sang-Kyu; Kwiatkowski, Witek; Choe, Senyon

    2014-09-01

    Recombinant bone morphogenetic protein 2 (rhBMP2) has been used clinically to treat bone fractures in human patients. However, the high doses of rhBMP2 required for a therapeutic response can cause undesirable side effects. Here, we demonstrate that a novel Activin A/BMP2 (AB2) chimera, AB204, promotes osteogenesis and bone healing much more potently and effectively than rhBMP2. Remarkably, 1 month of AB204 treatment completely heals tibial and calvarial defects of critical size in mice at a concentration 10-fold lower than a dose of rhBMP2 that only partially heals the defect. We determine the structure of AB204 to 2.3 Å that reveals a distinct BMP2-like fold in which the Activin A sequence segments confer insensitivity to the BMP2 antagonist Noggin and an affinity for the Activin/BMP type II receptor ActRII that is 100-fold greater than that of BMP2. The structure also led to our identification of a single Activin A-derived amino acid residue, which, when mutated to the corresponding BMP2 residue, resulted in a significant increase in the affinity of AB204 for its type I receptor BMPRIa and a further enhancement in AB204's osteogenic potency. Together, these findings demonstrate that rationally designed AB2 chimeras can provide BMP2 substitutes with enhanced potency for treating non-union bone fractures.

  17. Activin Plays a Key Role in the Maintenance of Long-Term Memory and Late-LTP

    Science.gov (United States)

    Ageta, Hiroshi; Ikegami, Shiro; Miura, Masami; Masuda, Masao; Migishima, Rika; Hino, Toshiaki; Takashima, Noriko; Murayama, Akiko; Sugino, Hiromu; Setou, Mitsutoshi; Kida, Satoshi; Yokoyama, Minesuke; Hasegawa, Yoshihisa; Tsuchida, Kunihiro; Aosaki, Toshihiko; Inokuchi, Kaoru

    2010-01-01

    A recent study has revealed that fear memory may be vulnerable following retrieval, and is then reconsolidated in a protein synthesis-dependent manner. However, little is known about the molecular mechanisms of these processes. Activin [beta]A, a member of the TGF-[beta] superfamily, is increased in activated neuronal circuits and regulates…

  18. MiR-125b Regulates Primordial Follicle Assembly by Targeting Activin Receptor Type 2a in Neonatal Mouse Ovary.

    Science.gov (United States)

    Wang, Shufen; Liu, Jiali; Li, Xinqiang; Ji, Xiaowen; Zhang, Jianfang; Wang, Yue; Cui, Sheng

    2016-04-01

    The establishment of the primordial follicle pool is crucial for fertility in mammalian females, and the interruption of overall micro-RNA production byDicer1conditional knockout in the female reproductive system results in infertility. However, there are few reports about the functions of individual micro-RNA in regulating primordial follicle assembly. The present study aimed to investigate the function of miR-125b, which is conserved and preferentially expressed in mammalian ovary during primordial follicle assembly. Detection of miR-125b in the developing mouse ovaries by real-time PCR and in situ hybridization showed that it was highly expressed perinatally and specifically located in the ovarian somatic cells. MiR-125b overexpression blocked the process of primordial follicle assembly in cultured newborn mouse ovaries, while its knockdown promoted this process. Further studies showed that miR-125b regulated the activin/Smad2 signaling in neonatal mouse ovary by directly targeting the 3'-untranslated region of activin receptor type 2a (Acvr2a). Overexpression of miR-125b in neonatal mouse ovary suppressed theAcvr2aprotein level, attenuating activin/Smad2 signaling, while knockdown of miR-125b showed the opposite effects. In addition, recombinant human activin A (rh-ActA) down-regulated miR-125b in the neonatal mouse ovary. Overexpression of miR-125b attenuated the promoting effects of rh-ActA on primordial follicle assembly. Taken together, these data suggest that miR-125b blocks the process of primordial follicle assembly, and miR-125b may play this role by regulating the expression ofAcvr2ain the activin/Smad2 signaling pathway. © 2016 by the Society for the Study of Reproduction, Inc.

  19. A late requirement for Wnt and FGF signaling during activin-induced formation of foregut endoderm from mouse embryonic stem cells

    DEFF Research Database (Denmark)

    Hansson, Mattias; Olesen, Dorthe R; Peterslund, Janny M L

    2009-01-01

    Here we examine how BMP, Wnt, and FGF signaling modulate activin-induced mesendodermal differentiation of mouse ES cells grown under defined conditions in adherent monoculture. We monitor ES cells containing reporter genes for markers of primitive streak (PS) and its progeny and extend previous....... Notably, activin induction of Gsc-GFP(+) cells appears refractory to inhibition of canonical Wnt signaling but shows a dependence on early as well as late FGF signaling. Additionally, we find a late dependence on FGF signaling during induction of Sox17(+) cells by activin while BMP4-induced T expression...... requires FGF signaling in adherent but not aggregate culture. Lastly, we demonstrate that activin-induced definitive endoderm derived from mouse ES cells can incorporate into the developing foregut endoderm in vivo and adopt a mostly anterior foregut character after further culture in vitro....

  20. RAP-011, an activin receptor ligand trap, increases hemoglobin concentration in hepcidin transgenic mice.

    Science.gov (United States)

    Langdon, Jacqueline M; Barkataki, Sangjucta; Berger, Alan E; Cheadle, Chris; Xue, Qian-Li; Sung, Victoria; Roy, Cindy N

    2015-01-01

    Over expression of hepcidin antimicrobial peptide is a common feature of iron-restricted anemia in humans. We investigated the erythroid response to either erythropoietin or RAP-011, a "murinized" ortholog of sotatercept, in C57BL/6 mice and in hepcidin antimicrobial peptide 1 over expressing mice. Sotatercept, a soluble, activin receptor type IIA ligand trap, is currently being evaluated for the treatment of anemias associated with chronic renal disease, myelodysplastic syndrome, β-thalassemia, and Diamond Blackfan anemia and acts by inhibiting signaling downstream of activin and other Transforming Growth Factor-β superfamily members. We found that erythropoietin and RAP-011 increased hemoglobin concentration in C57BL/6 mice and in hepcidin antimicrobial peptide 1 over expressing mice. While erythropoietin treatment depleted splenic iron stores in C57BL/6 mice, RAP-011 treatment did not deplete splenic iron stores in mice of either genotype. Bone marrow erythroid progenitors from erythropoietin-treated mice exhibited iron-restricted erythropoiesis, as indicated by increased median fluorescence intensity of transferrin receptor immunostaining by flow cytometry. In contrast, RAP-011-treated mice did not exhibit the same degree of iron-restricted erythropoiesis. In conclusion, we have demonstrated that RAP-011 can improve hemoglobin concentration in hepcidin antimicrobial peptide 1 transgenic mice. Our data support the hypothesis that RAP-011 has unique biologic effects which prevent or circumvent depletion of mouse splenic iron stores. RAP-011 may, therefore, be an appropriate therapeutic for trials in human anemias characterized by increased expression of hepcidin antimicrobial peptide and iron-restricted erythropoiesis. © 2014 Wiley Periodicals, Inc.

  1. Activin/Nodal Signaling Supports Retinal Progenitor Specification in a Narrow Time Window during Pluripotent Stem Cell Neuralization

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    Michele Bertacchi

    2015-10-01

    Full Text Available Retinal progenitors are initially found in the anterior neural plate region known as the eye field, whereas neighboring areas undertake telencephalic or hypothalamic development. Eye field cells become specified by switching on a network of eye field transcription factors, but the extracellular cues activating this network remain unclear. In this study, we used chemically defined media to induce in vitro differentiation of mouse embryonic stem cells (ESCs toward eye field fates. Inhibition of Wnt/β-catenin signaling was sufficient to drive ESCs to telencephalic, but not retinal, fates. Instead, retinal progenitors could be generated from competent differentiating mouse ESCs by activation of Activin/Nodal signaling within a narrow temporal window corresponding to the emergence of primitive anterior neural progenitors. Activin also promoted eye field gene expression in differentiating human ESCs. Our results reveal insights into the mechanisms of eye field specification and open new avenues toward the generation of retinal progenitors for translational medicine.

  2. IMMUNOLOCALIZATION OF INHIBIN/ACTIVIN SUBUNITS AND STEROIDOGENIC ENZYMES IN THE TESTES OF AN ADULT AFRICAN ELEPHANT (LOXODONTA AFRICANA).

    Science.gov (United States)

    Li, Qinglin; Lu, Lu; Weng, Qiang; Kawakami, Shigehisa; Saito, Eriko; Yamamoto, Tatsuya; Yamamoto, Yuki; Kaewmanee, Saroch; Nagaoka, Kentaro; Watanabe, Gen; Taya, Kazuyoshi

    2016-06-01

    In this case report, the authors investigated immunolocalization of inhibin α and inhibin/activin βA and βB subunits, as well as steroidogenic enzymes, in the testes of an African elephant. Testes were collected from a reproductively active male African elephant (24 yr old) at autopsy. Histologically, all types of spermatogenic cells including mature-phase spermatozoa were found in the seminiferous tubules. Positive immunostaining for inhibin α and inhibin/activin βA and βB subunits was observed in Sertoli and Leydig cells. In addition, P450scc, 3βHSD, P450c17, and P450arom were also detected in the cytoplasm of Leydig cells. These results suggested that Leydig cells of adult African elephant testes have the ability to synthesize progestin, androgen, and estrogen, whereas both Sertoli and Leydig cells appear as a major source of inhibin secretion in the male African elephant.

  3. Activin/Nodal Signaling Supports Retinal Progenitor Specification in a Narrow Time Window during Pluripotent Stem Cell Neuralization

    Science.gov (United States)

    Bertacchi, Michele; Lupo, Giuseppe; Pandolfini, Luca; Casarosa, Simona; D’Onofrio, Mara; Pedersen, Roger A.; Harris, William A.; Cremisi, Federico

    2015-01-01

    Summary Retinal progenitors are initially found in the anterior neural plate region known as the eye field, whereas neighboring areas undertake telencephalic or hypothalamic development. Eye field cells become specified by switching on a network of eye field transcription factors, but the extracellular cues activating this network remain unclear. In this study, we used chemically defined media to induce in vitro differentiation of mouse embryonic stem cells (ESCs) toward eye field fates. Inhibition of Wnt/β-catenin signaling was sufficient to drive ESCs to telencephalic, but not retinal, fates. Instead, retinal progenitors could be generated from competent differentiating mouse ESCs by activation of Activin/Nodal signaling within a narrow temporal window corresponding to the emergence of primitive anterior neural progenitors. Activin also promoted eye field gene expression in differentiating human ESCs. Our results reveal insights into the mechanisms of eye field specification and open new avenues toward the generation of retinal progenitors for translational medicine. PMID:26388287

  4. Activin type IB receptor signaling in prostate cancer cells promotes lymph node metastasis in a xenograft model

    Energy Technology Data Exchange (ETDEWEB)

    Nomura, Masatoshi, E-mail: nomura@med.kyushu-u.ac.jp [Department of Medicine and Bioregulatory Science, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582 (Japan); Tanaka, Kimitaka; Wang, Lixiang; Goto, Yutaka; Mukasa, Chizu; Ashida, Kenji; Takayanagi, Ryoichi [Department of Medicine and Bioregulatory Science, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582 (Japan)

    2013-01-04

    Highlights: Black-Right-Pointing-Pointer ActRIB signaling induces Snail and S100A4 expressions in prostate cancer cells. Black-Right-Pointing-Pointer The prostate cancer cell lines expressing an active form of ActRIB were established. Black-Right-Pointing-Pointer ActRIB signaling promotes EMT and lymph node metastasis in xenograft model. -- Abstract: Activin, a member of the transforming growth factor-{beta} family, has been known to be a growth and differentiating factor. Despite its pluripotent effects, the roles of activin signaling in prostate cancer pathogenesis are still unclear. In this study, we established several cell lines that express a constitutive active form of activin type IB receptor (ActRIBCA) in human prostate cancer cells, ALVA41 (ALVA-ActRIBCA). There was no apparent change in the proliferation of ALVA-ActRIBCA cells in vitro; however, their migratory ability was significantly enhanced. In a xenograft model, histological analysis revealed that the expression of Snail, a cell-adhesion-suppressing transcription factor, was dramatically increased in ALVA-ActRIBCA tumors, indicating epithelial mesenchymal transition (EMT). Finally, mice bearing ALVA-ActRIBCA cells developed multiple lymph node metastases. In this study, we demonstrated that ActRIBCA signaling can promote cell migration in prostate cancer cells via a network of signaling molecules that work together to trigger the process of EMT, and thereby aid in the aggressiveness and progression of prostate cancers.

  5. Distinct modes of SMAD2 chromatin binding and remodeling shape the transcriptional response to NODAL/Activin signaling

    Science.gov (United States)

    Coda, Davide M; Gaarenstroom, Tessa; East, Philip; Patel, Harshil; Miller, Daniel S J; Lobley, Anna; Matthews, Nik; Stewart, Aengus; Hill, Caroline S

    2017-01-01

    NODAL/Activin signaling orchestrates key processes during embryonic development via SMAD2. How SMAD2 activates programs of gene expression that are modulated over time however, is not known. Here we delineate the sequence of events that occur from SMAD2 binding to transcriptional activation, and the mechanisms underlying them. NODAL/Activin signaling induces dramatic chromatin landscape changes, and a dynamic transcriptional network regulated by SMAD2, acting via multiple mechanisms. Crucially we have discovered two modes of SMAD2 binding. SMAD2 can bind pre-acetylated nucleosome-depleted sites. However, it also binds to unacetylated, closed chromatin, independently of pioneer factors, where it induces nucleosome displacement and histone acetylation. For a subset of genes, this requires SMARCA4. We find that long term modulation of the transcriptional responses requires continued NODAL/Activin signaling. Thus SMAD2 binding does not linearly equate with transcriptional kinetics, and our data suggest that SMAD2 recruits multiple co-factors during sustained signaling to shape the downstream transcriptional program. DOI: http://dx.doi.org/10.7554/eLife.22474.001 PMID:28191871

  6. Activin A induces ovine follicle stimulating hormone beta using -169/-58 bp of its promoter and a simple TATA box

    Directory of Open Access Journals (Sweden)

    Miller William L

    2009-06-01

    Full Text Available Abstract Background Activin A increases production of follicle stimulating hormone (FSH by inducing transcription of its beta subunit (FSHB. This induction has been studied here in LbetaT2 gonadotropes using transient expression of ovine FSHBLuc (-4741 bp of ovine FSHB promoter plus exon/intron 1 linked to Luc. Several sequences between -169/-58 bp of the ovine FSHB proximal promoter are necessary for induction by activin A in LbetaT2 cells, but deletions between -4741/-752 bp decrease induction > 70% suggesting the existence of other important 5' sequences. Induction disappears if a minimal T81 thymidine kinase promoter replaces the ovine FSHB TATA box and 3' exon/intron. The study reported here was designed to determine if sequences outside -169/-58 bp are important for induction of ovine FSHB by activin A. Methods Progressively longer deletions of ovine FSHBLuc were created between -4741/-195 bp. Deletions internal to this region were created also, but replaced with substitute DNA. The ovine FSHB TATA box region (-40/+3 bp was replaced by thymidine kinase and rat prolactin minimal promoters, and substitutions were made in 3' intron/exon sequences. All constructs were tested for basal and activin A-induced expression in LbetaT2 cells. Results Successive 5' deletions progressively lowered fold-induction by activin A from 9.5 to zero, but progressively increased basal expression. Replacing deletions with substitute DNA showed no changes in basal expression or fold-induction. Induction by activin A was supported by the minimal rat prolactin promoter (TATA box but not the thymidine kinase promoter (no TATA box. Replacement mutations in the 3' region did not decrease induction by activin A. Conclusion The data show that specific ovine FSHB sequences 5' to -175 bp or 3' of the transcription start site are not required for induction by activin A. A minimal TATA box promoter supports induction by activin A, but the sequence between the TATA box and

  7. The Notch ligand Delta-like 1 integrates inputs from TGFbeta/Activin and Wnt pathways

    Energy Technology Data Exchange (ETDEWEB)

    Bordonaro, Michael, E-mail: mbordonaro@tcmedc.org; Tewari, Shruti, E-mail: stewari@tcmedc.org; Atamna, Wafa, E-mail: watamna@tcmedc.org; Lazarova, Darina L., E-mail: dlazarova@tcmedc.org

    2011-06-10

    Unlike the well-characterized nuclear function of the Notch intracellular domain, it has been difficult to identify a nuclear role for the ligands of Notch. Here we provide evidence for the nuclear function of the Notch ligand Delta-like 1 in colon cancer (CC) cells exposed to butyrate. We demonstrate that the intracellular domain of Delta-like 1 (Dll1icd) augments the activity of Wnt signaling-dependent reporters and that of the promoter of the connective tissue growth factor (CTGF) gene. Data suggest that Dll1icd upregulates CTGF promoter activity through both direct and indirect mechanisms. The direct mechanism is supported by co-immunoprecipitation of endogenous Smad2/3 proteins and Dll1 and by chromatin immunoprecipitation analyses that revealed the occupancy of Dll1icd on CTGF promoter sequences containing a Smad binding element. The indirect upregulation of CTGF expression by Dll1 is likely due to the ability of Dll1icd to increase Wnt signaling, a pathway that targets CTGF. CTGF expression is induced in butyrate-treated CC cells and results from clonal growth assays support a role for CTGF in the cell growth-suppressive role of butyrate. In conclusion, integration of the Notch, Wnt, and TGFbeta/Activin signaling pathways is in part mediated by the interactions of Dll1 with Smad2/3 and Tcf4.

  8. Activation of FGFR(IIIc) isoforms promotes activin-induced mesendoderm development in mouse embryonic stem cells and reduces Sox17 coexpression in EpCAM+ cells.

    Science.gov (United States)

    Peterslund, Janny M L; Serup, Palle

    2011-05-01

    Activin induces the formation of definitive endoderm from mouse ES cells dependent on active fibroblast growth factor (Fgf) signaling. Here we report that Fgf4 is dispensable for activin A-induced differentiation of mouse ES cells into endoderm. We find that Fgf4(-/-) cells readily differentiate into definitive endoderm without exogenous administration of Fgf4. Additionally, we investigate the spatio-temporal dynamics of Fgf receptor (FGFR) isoform distribution in activin A-treated ES cell cultures and find that FGFR(III)c isoforms are expressed in DE as well as non-DE populations, whereas FGFR2(III)b and FGFR4 are found specifically enriched in the DE fraction. Ligands that preferentially activate the FGFR(III)c isoforms induce mesendoderm markers T and Gsc, but reduce expression of the DE marker Sox17 in activin-induced EpCAM(+) cells. In contrast, ligands specifically activating FGFR(III)b isoforms have no effect on either population. Activation of FGFR(III)c isoforms results in a strong mitogenic effect on activin A-induced ES cell progeny early in the differentiation period whereas activation of FGFR(III)b isoforms has only a moderate mitogenic effect confined to the late differentiation period. We conclude that FGFR(III)c-isoform activation selectively drives the differentiation of mES cells toward mesendoderm and that Fgf4 is dispensable for the differentiation into definitive endoderm.

  9. Effect of physical training on liver expression of activin A and follistatin in a nonalcoholic fatty liver disease model in rats

    Energy Technology Data Exchange (ETDEWEB)

    Silva, R.N. [Departamento de Fisioterapia, Universidade Federal de São Carlos, São Carlos, SP (Brazil); Bueno, P.G. [Departamento de Ciências Fisiológicas, Universidade Federal de São Carlos, São Carlos, SP (Brazil); Avó, L.R.S. [Departamento de Medicina, Universidade Federal de São Carlos, São Carlos, SP (Brazil); Nonaka, K.O.; Selistre-Araújo, H.S. [Departamento de Ciências Fisiológicas, Universidade Federal de São Carlos, São Carlos, SP (Brazil); Leal, A.M.O. [Departamento de Medicina, Universidade Federal de São Carlos, São Carlos, SP (Brazil)

    2014-07-25

    Nonalcoholic fatty liver disease (NAFLD) is characterized by fat accumulation in the liver and is associated with obesity and insulin resistance. Activin A is a member of the transforming growth factor beta (TGF)-β superfamily and inhibits hepatocyte growth. Follistatin antagonizes the biological actions of activin. Exercise is an important therapeutic strategy to reduce the metabolic effects of obesity. We evaluated the pattern of activin A and follistatin liver expression in obese rats subjected to swimming exercise. Control rats (C) and high-fat (HF) diet-fed rats were randomly assigned to a swimming training group (C-Swim and HF-Swim) or a sedentary group (C-Sed and HF-Sed). Activin βA subunit mRNA expression was significantly higher in HF-Swim than in HF-Sed rats. Follistatin mRNA expression was significantly lower in C-Swim and HF-Swim than in either C-Sed or HF-Sed animals. There was no evidence of steatosis or inflammation in C rats. In contrast, in HF animals the severity of steatosis ranged from grade 1 to grade 3. The extent of liver parenchyma damage was less in HF-Swim animals, with the severity of steatosis ranging from grade 0 to grade 1. These data showed that exercise may reduce the deleterious effects of a high-fat diet on the liver, suggesting that the local expression of activin-follistatin may be involved.

  10. Effect of physical training on liver expression of activin A and follistatin in a nonalcoholic fatty liver disease model in rats.

    Science.gov (United States)

    Silva, R N; Bueno, P G; Avó, L R S; Nonaka, K O; Selistre-Araújo, H S; Leal, A M O

    2014-09-01

    Nonalcoholic fatty liver disease (NAFLD) is characterized by fat accumulation in the liver and is associated with obesity and insulin resistance. Activin A is a member of the transforming growth factor beta (TGF)-β superfamily and inhibits hepatocyte growth. Follistatin antagonizes the biological actions of activin. Exercise is an important therapeutic strategy to reduce the metabolic effects of obesity. We evaluated the pattern of activin A and follistatin liver expression in obese rats subjected to swimming exercise. Control rats (C) and high-fat (HF) diet-fed rats were randomly assigned to a swimming training group (C-Swim and HF-Swim) or a sedentary group (C-Sed and HF-Sed). Activin βA subunit mRNA expression was significantly higher in HF-Swim than in HF-Sed rats. Follistatin mRNA expression was significantly lower in C-Swim and HF-Swim than in either C-Sed or HF-Sed animals. There was no evidence of steatosis or inflammation in C rats. In contrast, in HF animals the severity of steatosis ranged from grade 1 to grade 3. The extent of liver parenchyma damage was less in HF-Swim animals, with the severity of steatosis ranging from grade 0 to grade 1. These data showed that exercise may reduce the deleterious effects of a high-fat diet on the liver, suggesting that the local expression of activin-follistatin may be involved.

  11. Effect of physical training on liver expression of activin A and follistatin in a nonalcoholic fatty liver disease model in rats

    Directory of Open Access Journals (Sweden)

    R.N. Silva

    2014-09-01

    Full Text Available Nonalcoholic fatty liver disease (NAFLD is characterized by fat accumulation in the liver and is associated with obesity and insulin resistance. Activin A is a member of the transforming growth factor beta (TGF-β superfamily and inhibits hepatocyte growth. Follistatin antagonizes the biological actions of activin. Exercise is an important therapeutic strategy to reduce the metabolic effects of obesity. We evaluated the pattern of activin A and follistatin liver expression in obese rats subjected to swimming exercise. Control rats (C and high-fat (HF diet-fed rats were randomly assigned to a swimming training group (C-Swim and HF-Swim or a sedentary group (C-Sed and HF-Sed. Activin βA subunit mRNA expression was significantly higher in HF-Swim than in HF-Sed rats. Follistatin mRNA expression was significantly lower in C-Swim and HF-Swim than in either C-Sed or HF-Sed animals. There was no evidence of steatosis or inflammation in C rats. In contrast, in HF animals the severity of steatosis ranged from grade 1 to grade 3. The extent of liver parenchyma damage was less in HF-Swim animals, with the severity of steatosis ranging from grade 0 to grade 1. These data showed that exercise may reduce the deleterious effects of a high-fat diet on the liver, suggesting that the local expression of activin-follistatin may be involved.

  12. Regulation of activin receptor-interacting protein 2 expression in mouse hepatoma Hepal-6 cells and its relationship with collagen type Ⅳ

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    AIM: To investigate the regulation of activin receptor-interacting protein 2 (ARIP2) expression and its possible relationships with collagen type Ⅳ (collagen Ⅳ) in mouse hepatoma cell line Hepal-6 cells.METHODS: The ARIP2 mRNA expression kinetics in Hepal-6 cells was detected by RT-PCR, and its regulation factors were analyzed by treatment with signal transduction activators such as phorbol 12-myristate 13-acetate (PMA), forskolin and A23187. After pcDNA3-ARIP2 was transfected into Hepal-6 cells, the effects of ARIP2 overexpression on activin type Ⅱ receptor (ActRII)and collagen Ⅳ expression were evaluated.RESULTS: The expression levels of ARIP2 mRNA in Hapel-6 cells were elevated in time-dependent manner 12 h after treatment with activin A and endotoxin LPS, but not changed evidently in the early stage of stimulation (2 or 4 h). TheARIP2 mRNA expression was increased after stimulated with signal transduction activators such as PMA and forskolin in Hepal-6 cells, whereas decreased after treatment with A23187 (25.3% ± 5.7% vS 48.1% ± 3.6%, P < 0.01). ARIP2 overexpression could remarkably suppress the expression of ActRIIA mRNA in dose-dependent manner, but has no effect on ActRIIB in Hepal-6 cells induced by activin A. Furthermore, we have found that overexpression of ARIP2 could inhibit collagen Ⅳ mRNA and protein expressions induced by activin A in Hapel-6 cells.CONCLUSION: These findings suggest that ARIP2 expression can be influenced by various factors. ARIP2 may participate in the negative feedback regulation of signal transduction in the late stage by affecting the expression of ActRIIA and play an important role in regulation of development of liver fibrosis induced by activin.

  13. Activin Enhances α- to β-Cell Transdifferentiation as a Source For β-Cells In Male FSTL3 Knockout Mice.

    Science.gov (United States)

    Brown, Melissa L; Andrzejewski, Danielle; Burnside, Amy; Schneyer, Alan L

    2016-03-01

    Diabetes results from inadequate β-cell number and/or function to control serum glucose concentrations so that replacement of lost β-cells could become a viable therapy for diabetes. In addition to embryonic stem cell sources for new β-cells, evidence for transdifferentiation/reprogramming of non-β-cells to functional β-cells is accumulating. In addition, de-differentiation of β-cells observed in diabetes and their subsequent conversion to α-cells raises the possibility that adult islet cell fate is malleable and controlled by local hormonal and/or environmental cues. We previously demonstrated that inactivation of the activin antagonist, follistatin-like 3 (FSTL3) resulted in β-cell expansion and improved glucose homeostasis in the absence of β-cell proliferation. We recently reported that activin directly suppressed expression of critical α-cell genes while increasing expression of β-cell genes, supporting the hypothesis that activin is one of the local hormones controlling islet cell fate and that increased activin signaling accelerates α- to β-cell transdifferentiation. We tested this hypothesis using Gluc-Cre/yellow fluorescent protein (YFP) α-cell lineage tracing technology combined with FSTL3 knockout (KO) mice to label α-cells with YFP. Flow cytometry was used to quantify unlabeled and labeled α- and β-cells. We found that Ins+/YFP+ cells were significantly increased in FSTL3 KO mice compared with wild type littermates. Labeled Ins+/YFP+ cells increased significantly with age in FSTL3 KO mice but not wild type littermates. Sorting results were substantiated by counting fluorescently labeled cells in pancreatic sections. Activin treatment of isolated islets significantly increased the number of YFP+/Ins+ cells. These results suggest that α- to β-cell transdifferentiation is influenced by activin signaling and may contribute substantially to β-cell mass.

  14. Uterine activin receptor-like kinase 5 is crucial for blastocyst implantation and placental development

    Science.gov (United States)

    Peng, Jia; Monsivais, Diana; You, Ran; Zhong, Hua; Pangas, Stephanie A.; Matzuk, Martin M.

    2015-01-01

    Members of the transforming growth factor β (TGF-β) superfamily are key regulators in most developmental and physiological processes. However, the in vivo roles of TGF-β signaling in female reproduction remain uncertain. Activin receptor-like kinase 5 (ALK5) is the major type 1 receptor for the TGF-β subfamily. Absence of ALK5 leads to early embryonic lethality because of severe defects in vascular development. In this study, we conditionally ablated uterine ALK5 using progesterone receptor-cre mice to define the physiological roles of ALK5 in female reproduction. Despite normal ovarian functions and artificial decidualization in conditional knockout (cKO) mice, absence of uterine ALK5 resulted in substantially reduced female reproduction due to abnormalities observed at different stages of pregnancy, including implantation defects, disorganization of trophoblast cells, fewer uterine natural killer (uNK) cells, and impairment of spiral artery remodeling. In our microarray analysis, genes encoding proteins involved in cytokine–cytokine receptor interactions and NK cell-mediated cytotoxicity were down-regulated in cKO decidua compared with control decidua. Flow cytometry confirmed a 10-fold decrease in uNK cells in cKO versus control decidua. According to these data, we hypothesize that TGF-β acts on decidual cells via ALK5 to induce expression of other growth factors and cytokines, which are key regulators in luminal epithelium proliferation, trophoblast development, and uNK maturation during pregnancy. Our findings not only generate a mouse model to study TGF-β signaling in female reproduction but also shed light on the pathogenesis of many pregnancy complications in human, such as recurrent spontaneous abortion, preeclampsia, and intrauterine growth restriction. PMID:26305969

  15. Activin and GDF11 collaborate in feedback control of neuroepithelial stem cell proliferation and fate

    Science.gov (United States)

    Gokoffski, Kimberly K.; Wu, Hsiao-Huei; Beites, Crestina L.; Kim, Joon; Kim, Euiseok J.; Matzuk, Martin M.; Johnson, Jane E.; Lander, Arthur D.; Calof, Anne L.

    2011-01-01

    Studies of the olfactory epithelium model system have demonstrated that production of neurons is regulated by negative feedback. Previously, we showed that a locally produced signal, the TGFβ superfamily ligand GDF11, regulates the genesis of olfactory receptor neurons by inhibiting proliferation of the immediate neuronal precursors (INPs) that give rise to them. GDF11 is antagonized by follistatin (FST), which is also produced locally. Here, we show that Fst–/– mice exhibit dramatically decreased neurogenesis, a phenotype that can only be partially explained by increased GDF11 activity. Instead, a second FST-binding factor, activin βB (ACTβB), inhibits neurogenesis by a distinct mechanism: whereas GDF11 inhibits expansion of INPs, ACTβB inhibits expansion of stem and early progenitor cells. We present data supporting the concept that these latter cells, previously considered two distinct types, constitute a dynamic stem/progenitor population in which individual cells alternate expression of Sox2 and/or Ascl1. In addition, we demonstrate that interplay between ACTβB and GDF11 determines whether stem/progenitor cells adopt a glial versus neuronal fate. Altogether, the data indicate that the transition between stem cells and committed progenitors is neither sharp nor irreversible and that GDF11, ACTβB and FST are crucial components of a circuit that controls both total cell number and the ratio of neuronal versus glial cells in this system. Thus, our findings demonstrate a close connection between the signals involved in the control of tissue size and those that regulate the proportions of different cell types. PMID:21852401

  16. Activin-Like Kinase 2 Functions in Peri-implantation Uterine Signaling in Mice and Humans

    Science.gov (United States)

    Clementi, Caterina; Tripurani, Swamy K.; Large, Michael J.; Edson, Mark A.; Creighton, Chad J.; Hawkins, Shannon M.; Kovanci, Ertug; Kaartinen, Vesa; Lydon, John P.; Pangas, Stephanie A.; DeMayo, Francesco J.; Matzuk, Martin M.

    2013-01-01

    Implantation of a blastocyst in the uterus is a multistep process tightly controlled by an intricate regulatory network of interconnected ovarian, uterine, and embryonic factors. Bone morphogenetic protein (BMP) ligands and receptors are expressed in the uterus of pregnant mice, and BMP2 has been shown to be a key regulator of implantation. In this study, we investigated the roles of the BMP type 1 receptor, activin-like kinase 2 (ALK2), during mouse pregnancy by producing mice carrying a conditional ablation of Alk2 in the uterus (Alk2 cKO mice). In the absence of ALK2, embryos demonstrate delayed invasion into the uterine epithelium and stroma, and upon implantation, stromal cells fail to undergo uterine decidualization, resulting in sterility. Mechanistically, microarray analysis revealed that CCAAT/enhancer-binding protein β (Cebpb) expression is suppressed during decidualization in Alk2 cKO females. These findings and the similar phenotypes of Cebpb cKO and Alk2 cKO mice lead to the hypothesis that BMPs act upstream of CEBPB in the stroma to regulate decidualization. To test this hypothesis, we knocked down ALK2 in human uterine stromal cells (hESC) and discovered that ablation of ALK2 alters hESC decidualization and suppresses CEBPB mRNA and protein levels. Chromatin immunoprecipitation (ChIP) analysis of decidualizing hESC confirmed that BMP signaling proteins, SMAD1/5, directly regulate expression of CEBPB by binding a distinct regulatory sequence in the 3′ UTR of this gene; CEBPB, in turn, regulates the expression of progesterone receptor (PGR). Our work clarifies the conserved mechanisms through which BMPs regulate peri-implantation in rodents and primates and, for the first time, uncovers a linear pathway of BMP signaling through ALK2 to regulate CEBPB and, subsequently, PGR during decidualization. PMID:24244176

  17. The glycoprotein-hormones activin A and inhibin A interfere with dendritic cell maturation

    Directory of Open Access Journals (Sweden)

    Reichardt Holger M

    2008-05-01

    Full Text Available Abstract Background Pregnancy represents an exclusive situation in which the immune and the endocrine system cooperate to prevent rejection of the embryo by the maternal immune system. While immature dendritic cells (iDC in the early pregnancy decidua presumably contribute to the establishment of peripheral tolerance, glycoprotein-hormones of the transforming growth factor beta (TGF-beta family including activin A (ActA and inhibin A (InA are candidates that could direct the differentiation of DCs into a tolerance-inducing phenotype. Methods To test this hypothesis we generated iDCs from peripheral-blood-monocytes and exposed them to TGF-beta1, ActA, as well as InA and Dexamethasone (Dex as controls. Results Both glycoprotein-hormones prevented up-regulation of HLA-DR during cytokine-induced DC maturation similar to Dex but did not influence the expression of CD 40, CD 83 and CD 86. Visualization of the F-actin cytoskeleton confirmed that the DCs retained a partially immature phenotype under these conditions. The T-cell stimulatory capacity of DCs was reduced after ActA and InA exposure while the secretion of cytokines and chemokines was unaffected. Conclusion These findings suggest that ActA and InA interfere with selected aspects of DC maturation and may thereby help preventing activation of allogenic T-cells by the embryo. Thus, we have identified two novel members of the TGF-beta superfamily that could promote the generation of tolerance-inducing DCs.

  18. Uterine activin receptor-like kinase 5 is crucial for blastocyst implantation and placental development.

    Science.gov (United States)

    Peng, Jia; Monsivais, Diana; You, Ran; Zhong, Hua; Pangas, Stephanie A; Matzuk, Martin M

    2015-09-08

    Members of the transforming growth factor β (TGF-β) superfamily are key regulators in most developmental and physiological processes. However, the in vivo roles of TGF-β signaling in female reproduction remain uncertain. Activin receptor-like kinase 5 (ALK5) is the major type 1 receptor for the TGF-β subfamily. Absence of ALK5 leads to early embryonic lethality because of severe defects in vascular development. In this study, we conditionally ablated uterine ALK5 using progesterone receptor-cre mice to define the physiological roles of ALK5 in female reproduction. Despite normal ovarian functions and artificial decidualization in conditional knockout (cKO) mice, absence of uterine ALK5 resulted in substantially reduced female reproduction due to abnormalities observed at different stages of pregnancy, including implantation defects, disorganization of trophoblast cells, fewer uterine natural killer (uNK) cells, and impairment of spiral artery remodeling. In our microarray analysis, genes encoding proteins involved in cytokine-cytokine receptor interactions and NK cell-mediated cytotoxicity were down-regulated in cKO decidua compared with control decidua. Flow cytometry confirmed a 10-fold decrease in uNK cells in cKO versus control decidua. According to these data, we hypothesize that TGF-β acts on decidual cells via ALK5 to induce expression of other growth factors and cytokines, which are key regulators in luminal epithelium proliferation, trophoblast development, and uNK maturation during pregnancy. Our findings not only generate a mouse model to study TGF-β signaling in female reproduction but also shed light on the pathogenesis of many pregnancy complications in human, such as recurrent spontaneous abortion, preeclampsia, and intrauterine growth restriction.

  19. Uterine Activin-Like Kinase 4 Regulates Trophoblast Development During Mouse Placentation.

    Science.gov (United States)

    Peng, Jia; Fullerton, Paul T; Monsivais, Diana; Clementi, Caterina; Su, Gloria H; Matzuk, Martin M

    2015-12-01

    The placenta is the first organ to develop after fertilization. It forms an interface between the maternal uterus and growing fetus to allow nutrient uptake, waste elimination, and gas exchange for a successful pregnancy in both mice and humans. In the past 2 decades, in vivo and in vitro approaches have been used to show that several members of the TGF-β superfamily regulate embryo implantation and placental development. Nodal, a TGF-β superfamily ligand, is essential for mesendoderm formation and left-right axis patterning during embryogenesis, and Nodal null mutants exhibit abnormal placental organization with expansion of trophoblast giant cells and a decrease of spongiotrophoblast and labyrinth. To better understand the importance of Nodal signaling in the uterus, we established a mouse model to conditionally ablate activin-like kinase 4 (ALK4; the Nodal type 1 receptor) using Cre recombinase driven by the progesterone receptor promoter sequences (Pgr-Cre). Alk4 conditional knockout females are subfertile due to placental abnormalities and fetal loss in pregnancy, with a placental disorganization phenotype similar to what is observed in Nodal null mice. Thus, Nodal likely functions as an indirect regulator of placental development by binding to type 1 and type 2 receptors on maternal decidual cells to stimulate expression of unknown regulators of placental development. Our findings not only describe the generation of a mouse model that enables study of Nodal signaling in placentation but also provides insights into the pathogenesis of pregnancy complications in humans, including spontaneous abortion, preeclampsia, intrauterine growth restriction, and preterm birth.

  20. Activin-like kinase 2 functions in peri-implantation uterine signaling in mice and humans.

    Science.gov (United States)

    Clementi, Caterina; Tripurani, Swamy K; Large, Michael J; Edson, Mark A; Creighton, Chad J; Hawkins, Shannon M; Kovanci, Ertug; Kaartinen, Vesa; Lydon, John P; Pangas, Stephanie A; DeMayo, Francesco J; Matzuk, Martin M

    2013-11-01

    Implantation of a blastocyst in the uterus is a multistep process tightly controlled by an intricate regulatory network of interconnected ovarian, uterine, and embryonic factors. Bone morphogenetic protein (BMP) ligands and receptors are expressed in the uterus of pregnant mice, and BMP2 has been shown to be a key regulator of implantation. In this study, we investigated the roles of the BMP type 1 receptor, activin-like kinase 2 (ALK2), during mouse pregnancy by producing mice carrying a conditional ablation of Alk2 in the uterus (Alk2 cKO mice). In the absence of ALK2, embryos demonstrate delayed invasion into the uterine epithelium and stroma, and upon implantation, stromal cells fail to undergo uterine decidualization, resulting in sterility. Mechanistically, microarray analysis revealed that CCAAT/enhancer-binding protein β (Cebpb) expression is suppressed during decidualization in Alk2 cKO females. These findings and the similar phenotypes of Cebpb cKO and Alk2 cKO mice lead to the hypothesis that BMPs act upstream of CEBPB in the stroma to regulate decidualization. To test this hypothesis, we knocked down ALK2 in human uterine stromal cells (hESC) and discovered that ablation of ALK2 alters hESC decidualization and suppresses CEBPB mRNA and protein levels. Chromatin immunoprecipitation (ChIP) analysis of decidualizing hESC confirmed that BMP signaling proteins, SMAD1/5, directly regulate expression of CEBPB by binding a distinct regulatory sequence in the 3' UTR of this gene; CEBPB, in turn, regulates the expression of progesterone receptor (PGR). Our work clarifies the conserved mechanisms through which BMPs regulate peri-implantation in rodents and primates and, for the first time, uncovers a linear pathway of BMP signaling through ALK2 to regulate CEBPB and, subsequently, PGR during decidualization.

  1. Activin-like kinase 2 functions in peri-implantation uterine signaling in mice and humans.

    Directory of Open Access Journals (Sweden)

    Caterina Clementi

    2013-11-01

    Full Text Available Implantation of a blastocyst in the uterus is a multistep process tightly controlled by an intricate regulatory network of interconnected ovarian, uterine, and embryonic factors. Bone morphogenetic protein (BMP ligands and receptors are expressed in the uterus of pregnant mice, and BMP2 has been shown to be a key regulator of implantation. In this study, we investigated the roles of the BMP type 1 receptor, activin-like kinase 2 (ALK2, during mouse pregnancy by producing mice carrying a conditional ablation of Alk2 in the uterus (Alk2 cKO mice. In the absence of ALK2, embryos demonstrate delayed invasion into the uterine epithelium and stroma, and upon implantation, stromal cells fail to undergo uterine decidualization, resulting in sterility. Mechanistically, microarray analysis revealed that CCAAT/enhancer-binding protein β (Cebpb expression is suppressed during decidualization in Alk2 cKO females. These findings and the similar phenotypes of Cebpb cKO and Alk2 cKO mice lead to the hypothesis that BMPs act upstream of CEBPB in the stroma to regulate decidualization. To test this hypothesis, we knocked down ALK2 in human uterine stromal cells (hESC and discovered that ablation of ALK2 alters hESC decidualization and suppresses CEBPB mRNA and protein levels. Chromatin immunoprecipitation (ChIP analysis of decidualizing hESC confirmed that BMP signaling proteins, SMAD1/5, directly regulate expression of CEBPB by binding a distinct regulatory sequence in the 3' UTR of this gene; CEBPB, in turn, regulates the expression of progesterone receptor (PGR. Our work clarifies the conserved mechanisms through which BMPs regulate peri-implantation in rodents and primates and, for the first time, uncovers a linear pathway of BMP signaling through ALK2 to regulate CEBPB and, subsequently, PGR during decidualization.

  2. Inhibition of activin receptor type IIB increases strength and lifespan in myotubularin-deficient mice.

    Science.gov (United States)

    Lawlor, Michael W; Read, Benjamin P; Edelstein, Rachel; Yang, Nicole; Pierson, Christopher R; Stein, Matthew J; Wermer-Colan, Ariana; Buj-Bello, Anna; Lachey, Jennifer L; Seehra, Jasbir S; Beggs, Alan H

    2011-02-01

    X-linked myotubular myopathy (XLMTM) is a congenital disorder caused by deficiency of the lipid phosphatase, myotubularin. Patients with XLMTM often have severe perinatal weakness that requires mechanical ventilation to prevent death from respiratory failure. Muscle biopsy specimens from patients with XLMTM exhibit small myofibers with central nuclei and central aggregations of organelles in many cells. It was postulated that therapeutically increasing muscle fiber size would cause symptomatic improvement in myotubularin deficiency. Recent studies have elucidated an important role for the activin-receptor type IIB (ActRIIB) in regulation of muscle growth and have demonstrated that ActRIIB inhibition results in significant muscle hypertrophy. To evaluate whether promoting muscle hypertrophy can attenuate symptoms resulting from myotubularin deficiency, the effect of ActRIIB-mFC treatment was determined in myotubularin-deficient (Mtm1δ4) mice. Compared with wild-type mice, untreated Mtm1δ4 mice have decreased body weight, skeletal muscle hypotrophy, and reduced survival. Treatment of Mtm1δ4 mice with ActRIIB-mFC produced a 17% extension of lifespan, with transient increases in weight, forelimb grip strength, and myofiber size. Pathologic analysis of Mtm1δ4 mice during treatment revealed that ActRIIB-mFC produced marked hypertrophy restricted to type 2b myofibers, which suggests that oxidative fibers in Mtm1δ4 animals are incapable of a hypertrophic response in this setting. These results support ActRIIB-mFC as an effective treatment for the weakness observed in myotubularin deficiency. Copyright © 2011 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

  3. Transforming Growth Factor β/Activin Signaling Functions as a Sugar-Sensing Feedback Loop to Regulate Digestive Enzyme Expression

    Directory of Open Access Journals (Sweden)

    Wen-bin Alfred Chng

    2014-10-01

    Full Text Available Organisms need to assess their nutritional state and adapt their digestive capacity to the demands for various nutrients. Modulation of digestive enzyme production represents a rational step to regulate nutriment uptake. However, the role of digestion in nutrient homeostasis has been largely neglected. In this study, we analyzed the mechanism underlying glucose repression of digestive enzymes in the adult Drosophila midgut. We demonstrate that glucose represses the expression of many carbohydrases and lipases. Our data reveal that the consumption of nutritious sugars stimulates the secretion of the transforming growth factor β (TGF-β ligand, Dawdle, from the fat body. Dawdle then acts via circulation to activate TGF-β/Activin signaling in the midgut, culminating in the repression of digestive enzymes that are highly expressed during starvation. Thus, our study not only identifies a mechanism that couples sugar sensing with digestive enzyme expression but points to an important role of TGF-β/Activin signaling in sugar metabolism.

  4. DNA Damage Drives an Activin A-Dependent Induction of COX-2 in Premalignant Cells and Lesions

    Science.gov (United States)

    Fordyce, Colleen; Fessenden, Tim; Pickering, Curtis; Jung, Jason; Singla, Veena; Berman, Hal; Tlsty, Thea

    2010-01-01

    COX-2 catalyzes the rate-limiting step in the synthesis of prostaglandins. Its overexpression induces numerous tumor-promoting phenotypes and is associated with cancer metastasis and poor clinical outcome. Although COX-2 inhibitors are promising chemotherapeutic and chemopreventative agents for cancer, the risk of significant cardiovascular and gastrointestinal complications currently outweighs their potential benefits. Systemic complications of COX-2 inhibition could be avoided by specifically decreasing COX-2 expression in epithelial cells. To that end, we have investigated the signal transduction pathway regulating COX-2 expression in response to DNA damage in breast epithelial cells. In variant human mammary epithelial cells that have silenced p16 (vHMEC), double strand DNA damage or telomere malfunction results in a p53-and activin A-dependent induction of COX-2 and continued proliferation. In contrast, telomere malfunction in HMEC with an intact p16/Rb pathway induces cell cycle arrest. Importantly, in ductal carcinoma in situ (DCIS) lesions, high COX-2 expression is associated with high γH2AX, TRF2, activin A and telomere malfunction. These data demonstrate that DNA damage and telomere malfunction can have both cell autonomous and cell non-autonomous consequences and provides a novel mechanism for the propagation of tumorigenesis. PMID:20028875

  5. DNA damage drives an activin a-dependent induction of cyclooxygenase-2 in premalignant cells and lesions.

    Science.gov (United States)

    Fordyce, Colleen; Fessenden, Tim; Pickering, Curtis; Jung, Jason; Singla, Veena; Berman, Hal; Tlsty, Thea

    2010-02-01

    Cyclooxygenase-2 (COX-2) catalyzes the rate-limiting step in the synthesis of prostaglandins. Its overexpression induces numerous tumor-promoting phenotypes and is associated with cancer metastasis and poor clinical outcome. Although COX-2 inhibitors are promising chemotherapeutic and chemopreventative agents for cancer, the risk of significant cardiovascular and gastrointestinal complications currently outweighs their potential benefits. Systemic complications of COX-2 inhibition could be avoided by specifically decreasing COX-2 expression in epithelial cells. To that end, we have investigated the signal transduction pathway regulating the COX-2 expression in response to DNA damage in breast epithelial cells. In variant human mammary epithelial cells that have silenced p16 (vHMEC), double-strand DNA damage or telomere malfunction results in a p53- and activin A-dependent induction of COX-2 and continued proliferation. In contrast, telomere malfunction in HMEC with an intact p16/Rb pathway induces cell cycle arrest. Importantly, in ductal carcinoma in situ lesions, high COX-2 expression is associated with high gammaH2AX, TRF2, activin A, and telomere malfunction. These data show that DNA damage and telomere malfunction can have both cell-autonomous and cell-nonautonomous consequences and can provide a novel mechanism for the propagation of tumorigenesis.

  6. FGF signaling via MAPK is required early and improves Activin A-induced definitive endoderm formation from human embryonic stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Sui, Lina, E-mail: linasui@vub.ac.be [Cell Differentiation Unit, Diabetes Research Center, Vrije Universiteit Brussel (VUB), Laarbeeklaan 103, 1090 Brussels (Belgium); Mfopou, Josue K. [Cell Differentiation Unit, Diabetes Research Center, Vrije Universiteit Brussel (VUB), Laarbeeklaan 103, 1090 Brussels (Belgium); Geens, Mieke; Sermon, Karen [Department of Embryology and Genetics, Vrije Universiteit Brussel (VUB), Laarbeeklaan 103, 1090 Brussels (Belgium); Bouwens, Luc [Cell Differentiation Unit, Diabetes Research Center, Vrije Universiteit Brussel (VUB), Laarbeeklaan 103, 1090 Brussels (Belgium)

    2012-09-28

    Highlights: Black-Right-Pointing-Pointer Deep study the FGF signaling role during DE specification in the context of hESCs. Black-Right-Pointing-Pointer DE differentiation from hESCs has an early dependence on FGF signaling. Black-Right-Pointing-Pointer A serum-free DE protocol is developed based on the findings. Black-Right-Pointing-Pointer The DE cells showed potential to differentiate into pancreatic progenitor cells. -- Abstract: Considering their unlimited proliferation and pluripotency properties, human embryonic stem cells (hESCs) constitute a promising resource applicable for cell replacement therapy. To facilitate this clinical translation, it is critical to study and understand the early stage of hESCs differentiation wherein germ layers are defined. In this study, we examined the role of FGF signaling in Activin A-induced definitive endoderm (DE) differentiation in the absence of supplemented animal serum. We found that activated FGF/MAPK signaling is required at the early time point of Activin A-induced DE formation. In addition, FGF activation increased the number of DE cells compared to Activin A alone. These DE cells could further differentiate into PDX1 and NKX6.1 positive pancreatic progenitors in vitro. We conclude that Activin A combined with FGF/MAPK signaling efficiently induce DE cells in the absence of serum. These findings improve our understanding of human endoderm formation, and constitute a step forward in the generation of clinical grade hESCs progenies for cell therapy.

  7. Enhanced differentiation of human embryonic stem cells to mesenchymal progenitors by inhibition of TGF-beta/Activin/Nodal signaling using SB-431542

    DEFF Research Database (Denmark)

    Mahmood, Amer; Harkness, Linda; Schrøder, Henrik Daa

    2010-01-01

    progenitor cells. We demonstrate that inhibition of TGF-beta/Activin/Nodal signaling during embryoid bodies (EB) formation using SB-431542 (SB) in serum free medium, markedly up-regulated paraxial mesodermal markers (TBX6, TBX5), and several myogenic developmental markers including early myogenic...

  8. TAK-1/p38/nNFκB signaling inhibits myoblast differentiation by increasing levels of Activin A

    Directory of Open Access Journals (Sweden)

    Trendelenburg Anne

    2012-02-01

    Full Text Available Abstract Background Skeletal-muscle differentiation is required for the regeneration of myofibers after injury. The differentiation capacity of satellite cells is impaired in settings of old age, which is at least one factor in the onset of sarcopenia, the age-related loss of skeletal-muscle mass and major cause of frailty. One important cause of impaired regeneration is increased levels of transforming growth factor (TGF-β accompanied by reduced Notch signaling. Pro-inflammatory cytokines are also upregulated in aging, which led us hypothesize that they might potentially contribute to impaired regeneration in sarcopenia. Thus, in this study, we further analyzed the muscle differentiation-inhibition pathway mediated by pro-inflammatory cytokines in human skeletal muscle cells (HuSKMCs. Methods We studied the modulation of HuSKMC differentiation by the pro-inflammatory cytokines interleukin (IL-1α and tumor necrosis factor (TNF-α The grade of differentiation was determined by either imaging (fusion index or creatine kinase (CK activity, a marker of muscle differentiation. Secretion of TGF-β proteins during differentiation was assessed by using a TGF-β-responsive reporter-gene assay and further identified by means of pharmacological and genetic inhibitors. In addition, signaling events were monitored by western blotting and reverse transcription PCR, both in HuSKMC cultures and in samples from a rat sarcopenia study. Results The pro-inflammatory cytokines IL-1α and TNF-α block differentiation of human myoblasts into myotubes. This anti-differentiation effect requires activation of TGF-β-activated kinase (TAK-1. Using pharmacological and genetic inhibitors, the TAK-1 pathway could be traced to p38 and NFκB. Surprisingly, the anti-differentiation effect of the cytokines required the transcriptional upregulation of Activin A, which in turn acted through its established signaling pathway: ActRII/ALK/SMAD. Inhibition of Activin A signaling was

  9. Activin signaling in microsatellite stable colon cancers is disrupted by a combination of genetic and epigenetic mechanisms.

    Directory of Open Access Journals (Sweden)

    Barbara Jung

    Full Text Available BACKGROUND: Activin receptor 2 (ACVR2 is commonly mutated in microsatellite unstable (MSI colon cancers, leading to protein loss, signaling disruption, and larger tumors. Here, we examined activin signaling disruption in microsatellite stable (MSS colon cancers. METHODS: Fifty-one population-based MSS colon cancers were assessed for ACVR1, ACVR2 and pSMAD2 protein. Consensus mutation-prone portions of ACVR2 were sequenced in primary cancers and all exons in colon cancer cell lines. Loss of heterozygosity (LOH was evaluated for ACVR2 and ACVR1, and ACVR2 promoter methylation by methylation-specific PCR and bisulfite sequencing and chromosomal instability (CIN phenotype via fluorescent LOH analysis of 3 duplicate markers. ACVR2 promoter methylation and ACVR2 expression were assessed in colon cancer cell lines via qPCR and IP-Western blots. Re-expression of ACVR2 after demethylation with 5-aza-2'-deoxycytidine (5-Aza was determined. An additional 26 MSS colon cancers were assessed for ACVR2 loss and its mechanism, and ACVR2 loss in all tested cancers correlated with clinicopathological criteria. RESULTS: Of 51 MSS colon tumors, 7 (14% lost ACVR2, 2 (4% ACVR1, and 5 (10% pSMAD2 expression. No somatic ACVR2 mutations were detected. Loss of ACVR2 expression was associated with LOH at ACVR2 (p<0.001 and ACVR2 promoter hypermethylation (p<0.05. ACVR2 LOH, but not promoter hypermethylation, correlated with CIN status. In colon cancer cell lines with fully methylated ACVR2 promoter, loss of ACVR2 mRNA and protein expression was restored with 5-Aza treatment. Loss of ACVR2 was associated with an increase in primary colon cancer volume (p<0.05. CONCLUSIONS: Only a small percentage of MSS colon cancers lose expression of activin signaling members. ACVR2 loss occurs through LOH and ACVR2 promoter hypermethylation, revealing distinct mechanisms for ACVR2 inactivation in both MSI and MSS subtypes of colon cancer.

  10. 激活素A促进鸡胚背根神经节神经突起生长的作用机制%Effect mechanism of activin A in neurite outgrowth of embryonic dorsal root ganglia of the chicken

    Institute of Scientific and Technical Information of China (English)

    方琳; 王轶楠; 刘海岩; 李晨光; 李继茹; 柳忠辉

    2012-01-01

    采用半定量PCR检测激活素II型受体(ActRII)、钙基因相关肽(cGRP)、血管活性肠肽(VIP)mRNA表达,液相一质谱联用仪检测5-羟色胺释放水平。结果显示,5μg/L激活素A能明显促进鸡胚背根神经节神经突起生长,同时可见激活素A促进ActRIIA、CGRPmRNA表达及神经递质5-羟色胺释放,但对ActRIIB及VIPmRNA表达无影响。结果表明,ActivinA可能通过ActivinA—ActRIIA途径发挥作用,其促进鸡胚背根神经节神经突起生长与其上调CGRP表达及5-HT释放有关。%To investigate the effect mechanism of activin A in neurite outgrowth of embryonic dorsal root ganglia (DRG) of the chiken. ActRII,CGRP and VIP mRNA expressions were analyzed by semi-quantitative RT-PCR, and the secretion of serotonin (5-HT) was examined by liquid chromatography-tandem mass spectrometry (LC/MS). Activin A significantly induced neurite outgrowth of DRG and up-regulated the mRNA expressions of the activin receptor type IIA (ActRIIA) and CGRP in DRG. In addition,activin A stimulated 5-HT release from DRG. Activin A might stimulate DRG neurite outgrowth via activin A-ActRIIA pathway and promoting CGRP expression and 5-HT secretion.

  11. Endoglin-mediated suppression of prostate cancer invasion is regulated by activin and bone morphogenetic protein type II receptors.

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    Michael J Breen

    Full Text Available Mortality from prostate cancer (PCa is due to the formation of metastatic disease. Understanding how that process is regulated is therefore critical. We previously demonstrated that endoglin, a type III transforming growth factor β (TGFβ superfamily receptor, suppresses human PCa cell invasion and metastasis. Endoglin-mediated suppression of invasion was also shown by us to be dependent upon the type I TGFβ receptor, activin receptor-like kinase 2 (ALK2, and the downstream effector, Smad1. In this study we demonstrate for the first time that two type II TGFβ receptors are required for endoglin-mediated suppression of invasion: activin A receptor type IIA (ActRIIA and bone morphogenetic protein receptor type II (BMPRII. Downstream signaling through these receptors is predominantly mediated by Smad1. ActRIIA stimulates Smad1 activation in a kinase-dependent manner, and this is required for suppression of invasion. In contrast BMPRII regulates Smad1 in a biphasic manner, promoting Smad1 signaling through its kinase domain but suppressing it through its cytoplasmic tail. BMPRII's Smad1-regulatory effects are dependent upon its expression level. Further, its ability to suppress invasion is independent of either kinase function or tail domain. We demonstrate that ActRIIA and BMPRII physically interact, and that each also interacts with endoglin. The current findings demonstrate that both BMPRII and ActRIIA are necessary for endoglin-mediated suppression of human PCa cell invasion, that they have differential effects on Smad1 signaling, that they make separate contributions to regulation of invasion, and that they functionally and physically interact.

  12. Stimulation of circus movement by activin, bFGF and TGF-beta 2 in isolated animal cap cells of Xenopus laevis.

    Science.gov (United States)

    Minoura, I; Nakamura, H; Tashiro, K; Shiokawa, K

    1995-01-01

    Lobopodium is a hyaline cytoplasmic protrusion which rotates circumferencially around a cell. This movement is called circus movement, which is seen in dissociated cells of amphibian embryos. Relative abundance of the lobopodia-forming cells changes temporally and spatially within Xenopus embryos, reflecting stage-dependent difference of morphogenetic movements. The lobopodia-forming activity of dissociated animal cap cells was stimulated strongly by activin and bFGF, and weakly by TGF-beta 2. In addition, activin A was found to stimulate cellular attachment to the substratum when the cultivation lasted long. Thus, mesoderm-inducing growth factors stimulate lobopodia formation and cellular movements which may be necessary for gastrulation and neurulation in Xenopus early embryos.

  13. La fonction de la voie de signalisation du récepteur Activin de type IIB dans le muscle squelettique adulte.

    OpenAIRE

    Relizani, Karima

    2014-01-01

    Myostatin, a growth factor of the TGF-β family that signals through the activin receptor-IIB (ActRIIB), has been identified as an important negative regulator of skeletal muscle growth. However, its effect on muscle energy metabolism and energy dependent muscle function remains largely unexplored. I here investigated the consequence of impaired ActRIIB signaling for muscle metabolism in two experimental models, i) the constitutive myostatin knockout mice and ii) following pharmacological admi...

  14. Up-regulation of JAM-1 in AR42J cells treated with activin A and betacellulin and the diabetic regenerating islets.

    Science.gov (United States)

    Yoshikumi, Yukako; Ohno, Hideki; Suzuki, Junko; Isshiki, Masashi; Morishita, Yasuyuki; Ohnishi, Hirohide; Yasuda, Hiroshi; Omata, Masao; Fujita, Toshiro; Mashima, Hirosato

    2008-08-01

    Pancreatic AR42J cells demonstrate the pluripotency in precursor cells of the gut endoderm and also provide an excellent model system to study the differentiation of the pancreas. Using the mRNA differential display technique, we identified junctional adhesion molecule-1 (JAM-1), a component of the tight junction, was highly up-regulated during the differentiation of AR42J cells, although junctions were not formed. The expression level of JAM-1 showed an up-regulation in the mRNA level after 3 hours and in the protein level after 24 hours in [activin A + betacellulin]-treated AR42J cells. The expressions of its signaling molecules, PAR-3 and atypical PKC lambda, also increased after the addition of activin A + betacellulin. When JAM-1 was over-expressed in [activin A + betacellulin]-treated AR42J cells, tagged-JAM-1 was observed in cytoplasm as vesicular structures and JAM-1 was colocalized with Rab3B and Rab13, members of the Rab family expressed at tight junctions. In streptozotocin-induced regenerating islets, the expression of JAM-1 was also up-regulated in the mRNA level and the protein level. JAM-1 might therefore play an important role in the differentiation of AR42J cells and the regeneration of pancreatic islets.

  15. 牙鲆(Paralichthys olivaceus) Activin 基因两种β亚基的启动子克隆及生物信息学分析%Bioinformatic Characterization of Promoters of Two Activin-βSubunit Genes in Paralichthys olivaceus

    Institute of Scientific and Technical Information of China (English)

    刘蒙蒙; 王晶; 高金宁; 马丽曼; 张全启

    2015-01-01

    Activin is a member of the transforming growth factor-β (TGF-β) family and regulates sex hormones. It was originally discovered in pig ovarian follicular fluid. Activin contains two β subunits and plays a vital role in the hypothalamus-pituitary-gonad axis (HPG). It regulates the secretion of pituitary gonadotropin, the production of steroid hormones and the maturation of oocyte in ovary. Paralichthys olivaceus is a type of important commercial fish species that has advantageous traits in aquaculture such as the fast growth rate. Better understanding of its reproduction mechanism is essential for the guidance of the breeding of P. olivaceus. In this study, we analyzed the expression and regulation of Activin gene related to the reproductive endocrinology of P. olivaceus. Our data should provide important information for future studies on biological functions of Activin and for the practice in the culture of P. olivaceus. We used the genome walking method to obtain the partial sequence of promoters located in the upstream of Activin βA and βB genes of P. olivaceus, and predicted the binding sites of transcriptional regulation elements using the bioinformatical method. The promoters of these two genes were 2.7 kb and 2.4 kb in length respectively. The results showed that the TATA box of Activin βB was located at 31 bp in the upstream region of the transcription initiation site, however this structure was not found in Activin βA. We found in the two promoters a number of binding sites of the transcription factors including Sp1, Oct-1, C/EBP, CREB, GATA-1, c-Jun, HNF-3, HNF-1, and USF. Moreover, we also found multiple transcription binding sites of endocrine-related factors such as Pit-1, ER, PR, GR, RAR, and RXR. However, the binding sites of MyoD, myogenin and SRY were only found in Activin βA. In conclusion, the bioinformatical analysis suggested that the basic and hormone-inducing expression of both Activin βA and βB was regulated by a variety of factors

  16. Maternal circulating levels of activin A, inhibin A, sFlt-1 and endoglin at parturition in normal pregnancy and pre-eclampsia.

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    Aparna Reddy

    Full Text Available BACKGROUND: Maternal circulating levels of anti-angiogenic factors such as soluble fms-like tyrosine kinase-1 (sFlt-1, endoglin (sEng and placental proteins like activin A and inhibin A are increased before the onset of pre-eclampsia. There is evidence for oxidative stress in pre eclampsia. Recently it was shown that placental oxygen concentration is related to sFlt-1 and inhibin A. In addition it is reported that oxidative stress markers are increased in placental tissue delivered after labour. Therefore, the objective of this study is to investigate if these proteins are altered in maternal circulation of labouring pre-eclampsia and normal pregnancies. METHODOLOGY: To assess the effects of labour, samples were taken from 10 normal pregnant (NP and 10 pre-eclamptic (PE women pre-labour, full dilation, placental delivery and 24 h. To assess the effects of placental delivery, plasma samples were taken from 10NP and 10PE women undergoing elective Caesarean section, pre-delivery, placental delivery and 10 min, 60 min and 24 h post delivery. SFlt-1 and sEng and activin A and inhibin A were measured using commercial and in house ELISA's respectively. RESULTS: The levels of sFlt-1 and sEng were significantly higher in PE compared to NP women in both groups. In labour, sFlt-1 levels increased significantly at full dilatation in PE women, before declining by 24 hr. However there was no significant rise in sEng levels in labour. Activin A and inhibin A levels declined rapidly with placental delivery in NP and PE pregnancies. There was a significant rise in activin A levels during labour in PE compared to pre labour, but inhibin levels did not increase. CONCLUSION: Labour in pre-eclamptic women increases the levels of sFlt-1 and activin A. This pilot data suggests that increase in the maternal levels of these factors in labour could predict and/or contribute to the maternal syndrome postpartum.

  17. Molecular characterization of Activin Receptor Type IIA and its expression during gonadal maturation and growth stages in rohu carp.

    Science.gov (United States)

    Patnaik, Siddhi; Mohanty, Mausumee; Bit, Amrita; Sahoo, Lakshman; Das, Sachidananda; Jayasankar, Pallipuram; Das, Paramananda

    2017-01-01

    Activin receptor type IIA (ActRIIA), a transmembrane serine/threonine kinase receptor is an important regulator of physiological traits, viz., reproduction and body growth in vertebrates including teleosts. However, existing knowledge of its role in regulating fish physiology is limited. To address this, we have cloned and characterized the ActRIIA cDNA of Labeo rohita (rohu), an economically important fish species of the Indian subcontinent. Comparative expression profiling of the receptor gene at various reproductive and growth stages supports to its role in promoting oocyte maturation, spermatogenesis and skeletal muscle development via interaction with multiple ligands of transforming growth factor-β (TGF-β) family. The full-length cDNA of rohu ActRIIA was found to be of 1587bp length encoding 528 amino acids. The three-dimensional structure of the intracellular kinase domain of rohu ActRIIA has also been predicted. Phylogenetic relationship studies showed that the gene is evolutionarily conserved across the vertebrate lineage implicating that the functioning of the receptor is more or less similar in vertebrates. Taken together, these findings could be an initial step towards the use of ActRIIA as a potential candidate gene marker for understanding the complex regulatory mechanism of fish reproduction and growth.

  18. Tbx3 fosters pancreatic cancer growth by increased angiogenesis and activin/nodal-dependent induction of stemness

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    Lukas Perkhofer

    2016-09-01

    Full Text Available Cell fate decisions and pluripotency, but also malignancy depend on networks of key transcriptional regulators. The T-box transcription factor TBX3 has been implicated in the regulation of embryonic stem cell self-renewal and cardiogenesis. We have recently discovered that forced TBX3 expression in embryonic stem cells promotes mesendoderm specification directly by activating key lineage specification factors and indirectly by enhancing paracrine NODAL signalling. Interestingly, aberrant TBX3 expression is associated with breast cancer and melanoma formation. In other cancers, loss of TBX3 expression is associated with a more aggressive phenotype e.g. in gastric and cervical cancer. The precise function of TBX3 in pancreatic ductal adenocarcinoma remains to be determined. In the current study we provide conclusive evidence for TBX3 overexpression in pancreatic cancer samples as compared to healthy tissue. While proliferation remains unaltered, forced TBX3 expression strongly increases migration and invasion, but also angiogenesis in vitro and in vivo. Finally, we describe the TBX3-dependency of cancer stem cells that perpetuate themselves through an autocrine TBX3–ACTIVIN/NODAL signalling loop to sustain stemness. Thus, TBX3 is a new key player among pluripotency-related genes driving cancer formation.

  19. Modified activin receptor IIB ligand trap mitigates ineffective erythropoiesis and disease complications in murine β-thalassemia.

    Science.gov (United States)

    Suragani, Rajasekhar N V S; Cawley, Sharon M; Li, Robert; Wallner, Samantha; Alexander, Mark J; Mulivor, Aaron W; Gardenghi, Sara; Rivella, Stefano; Grinberg, Asya V; Pearsall, R Scott; Kumar, Ravindra

    2014-06-19

    In β-thalassemia, unequal production of α- and β-globin chains in erythroid precursors causes apoptosis and inhibition of late-stage erythroid differentiation, leading to anemia, ineffective erythropoiesis (IE), and dysregulated iron homeostasis. Here we used a murine model of β-thalassemia intermedia (Hbb(th1/th1) mice) to investigate effects of a modified activin receptor type IIB (ActRIIB) ligand trap (RAP-536) that inhibits Smad2/3 signaling. In Hbb(th1/th1) mice, treatment with RAP-536 reduced overactivation of Smad2/3 in splenic erythroid precursors. In addition, treatment of Hbb(th1/th1) mice with RAP-536 reduced α-globin aggregates in peripheral red cells, decreased the elevated reactive oxygen species present in erythroid precursors and peripheral red cells, and alleviated anemia by promoting differentiation of late-stage erythroid precursors and reducing hemolysis. Notably, RAP-536 treatment mitigated disease complications of IE, including iron overload, splenomegaly, and bone pathology, while reducing erythropoietin levels, improving erythrocyte morphology, and extending erythrocyte life span. These results implicate signaling by the transforming growth factor-β superfamily in late-stage erythropoiesis and reveal potential of a modified ActRIIB ligand trap as a novel therapeutic agent for thalassemia syndrome and other red cell disorders characterized by IE.

  20. Administration of soluble activin receptor 2B increases bone and muscle mass in a mouse model of osteogenesis imperfecta

    Institute of Scientific and Technical Information of China (English)

    Douglas J DiGirolamo; Vandana Singhal; Xiaoli Chang; Se-Jin Lee; Emily L Germain-Lee

    2015-01-01

    Osteogenesis imperfecta (OI) comprises a group of heritable connective tissue disorders generally defined by recurrent fractures, low bone mass, short stature and skeletal fragility. Beyond the skeletal complications of OI, many patients also report intolerance to physical activity, fatigue and muscle weakness. Indeed, recent studies have demonstrated that skeletal muscle is also negatively affected by OI, both directly and indirectly. Given the well-established interdependence of bone and skeletal muscle in both physiology and pathophysiology and the observations of skeletal muscle pathology in patients with OI, we investigated the therapeutic potential of simultaneous anabolic targeting of both bone and skeletal muscle using a soluble activin receptor 2B (ACVR2B) in a mouse model of type III OI (oim). Treatment of 12-week-old oim mice with ACVR2B for 4 weeks resulted in significant increases in both bone and muscle that were similar to those observed in healthy, wild-type littermates. This proof of concept study provides encouraging evidence for a holistic approach to treating the deleterious consequences of OI in the musculoskeletal system.

  1. Blockade of the activin receptor IIb activates functional brown adipogenesis and thermogenesis by inducing mitochondrial oxidative metabolism.

    Science.gov (United States)

    Fournier, Brigitte; Murray, Ben; Gutzwiller, Sabine; Marcaletti, Stefan; Marcellin, David; Bergling, Sebastian; Brachat, Sophie; Persohn, Elke; Pierrel, Eliane; Bombard, Florian; Hatakeyama, Shinji; Trendelenburg, Anne-Ulrike; Morvan, Frederic; Richardson, Brian; Glass, David J; Lach-Trifilieff, Estelle; Feige, Jerome N

    2012-07-01

    Brown adipose tissue (BAT) is a key tissue for energy expenditure via fat and glucose oxidation for thermogenesis. In this study, we demonstrate that the myostatin/activin receptor IIB (ActRIIB) pathway, which serves as an important negative regulator of muscle growth, is also a negative regulator of brown adipocyte differentiation. In parallel to the anticipated hypertrophy of skeletal muscle, the pharmacological inhibition of ActRIIB in mice, using a neutralizing antibody, increases the amount of BAT without directly affecting white adipose tissue. Mechanistically, inhibition of ActRIIB inhibits Smad3 signaling and activates the expression of myoglobin and PGC-1 coregulators in brown adipocytes. Consequently, ActRIIB blockade in brown adipose tissue enhances mitochondrial function and uncoupled respiration, translating into beneficial functional consequences, including enhanced cold tolerance and increased energy expenditure. Importantly, ActRIIB inhibition enhanced energy expenditure only at ambient temperature or in the cold and not at thermoneutrality, where nonshivering thermogenesis is minimal, strongly suggesting that brown fat activation plays a prominent role in the metabolic actions of ActRIIB inhibition.

  2. Administration of soluble activin receptor 2B increases bone and muscle mass in a mouse model of osteogenesis imperfecta

    Science.gov (United States)

    DiGirolamo, Douglas J.; Singhal, Vandana; Chang, Xiaoli; Lee, Se-Jin; Germain-Lee, Emily L.

    2015-01-01

    Osteogenesis imperfecta (OI) comprises a group of heritable connective tissue disorders generally defined by recurrent fractures, low bone mass, short stature and skeletal fragility. Beyond the skeletal complications of OI, many patients also report intolerance to physical activity, fatigue and muscle weakness. Indeed, recent studies have demonstrated that skeletal muscle is also negatively affected by OI, both directly and indirectly. Given the well-established interdependence of bone and skeletal muscle in both physiology and pathophysiology and the observations of skeletal muscle pathology in patients with OI, we investigated the therapeutic potential of simultaneous anabolic targeting of both bone and skeletal muscle using a soluble activin receptor 2B (ACVR2B) in a mouse model of type III OI (oim). Treatment of 12-week-old oim mice with ACVR2B for 4 weeks resulted in significant increases in both bone and muscle that were similar to those observed in healthy, wild-type littermates. This proof of concept study provides encouraging evidence for a holistic approach to treating the deleterious consequences of OI in the musculoskeletal system. PMID:26161291

  3. Differential expression of mRNAs encoding the putative inhibin co-receptor (betaglycan) and activin type-I and type-II receptors in preovulatory and prehierarchical follicles of the laying hen ovary.

    Science.gov (United States)

    Lovell, T M; Knight, P G; Gladwell, R T

    2006-02-01

    Ovarian follicle development is primarily regulated by an interplay between the pituitary gonadotrophins, LH and FSH, and ovary-derived steroids. Increasing evidence implicates regulatory roles of transforming growth factor-beta (TGFbeta) superfamily members, including inhibins and activins. The aim of this study was to identify the expression of mRNAs encoding key receptors of the inhibin/activin system in ovarian follicles ranging from 4 mm in diameter to the dominant F1 follicle (approximately 40 mm). Ovaries were collected (n = 16) from mid-sequence hens maintained on a long-day photoschedule (16 h of light:8 h of darkness). All follicles removed were dissected into individual granulosa and thecal layers. RNA was extracted and cDNA synthesized. Real-time quantitative PCR was used to quantify the expression of mRNA encoding betaglycan, activin receptor (ActR) subtypes (type-I, -IIA and -IIB) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH); receptor expression data were normalized to GAPDH expression. Detectable levels of ActRI, -IIA and -IIB and the inhibin co-receptor (betaglycan) expression were found in all granulosa and thecal layers analysed. Granulosa ActRI mRNA peaked (P II activin receptors and betaglycan on granulosa and thecal cells are consistent with a local auto/paracrine role of inhibins and activins in modulating ovarian follicle development, selection and progression in the domestic fowl.

  4. Activin suppresses LPS-induced Toll-like receptor, cytokine and inducible nitric oxide synthase expression in normal human melanocytes by inhibiting NF-κB and MAPK pathway activation.

    Science.gov (United States)

    Kim, Young Il; Park, Seung-Won; Kang, In Jung; Shin, Min Kyung; Lee, Mu-Hyoung

    2015-10-01

    Activins are dimeric growth and differentiation factors that belong to the transforming growth factor (TGF)-β superfamily of structurally related signaling proteins. In the present study, we examined the mechanisms through which activin regulates the lipopolysaccharide (LPS)-induced transcription of Toll-like receptors (TLRs), cytokines and inducible nitric oxide synthase (iNOS) in human melanocytes, as well as the involvement of nuclear factor (NF)-κB and mitogen-activated protein kinase (MAPK) signaling. Cell proliferation was analyzed by cell viability assay, mRNA expression was detected by RT-qPCR, and protein expression was measured by western blot analysis. LPS increased the mRNA expression of TLRs (TLR1-10) and cytokines [interleukin (IL)-1β, IL-6, IL-8 and TNF-α], as well as the mRNA and protein expression of iNOS. Activin decreased the LPS-induced TLR and cytokine mRNA expression, as well as the LPS-induced iNOS mRNA and protein expression. In addition, activin suppressed NF-κB p65 activation and blocked inhibitor of NF-κB (IκBα) degradation in LPS-stimulated melanocytes, and reduced LPS-induced p38 MAPK and MEK/ERK activation. On the whole, our results demonstrated that activin inhibited TLR and cytokine expression in LPS-activated normal human melanocytes and suppressed LPS-induced iNOS gene expression. Moreover, the anti-inflammatory effects of activin were shown to be mediated through the suppression of NF-κB and MAPK signaling, resulting in reduced TLR and iNOS expression, and in the inhibition of inflammatory cytokine expression.

  5. Gonadotropin-induced changes in oviducal mRNA expression levels of sex steroid hormone receptors and activin-related signaling factors in the alligator

    Science.gov (United States)

    Moore, Brandon C.; Forouhar, Sara; Kohno, Satomi; Botteri, Nicole L.; Hamlin, Heather J.; Guillette, Louis J.

    2011-01-01

    Oviducts respond to hormonal cues from ovaries with tissue proliferation and differentiation in preparation of transporting and fostering gametes. These responses produce oviducal microenvironments conducive to reproductive success. Here we investigated changes in circulating plasma sex steroid hormones concentrations and ovarian and oviducal mRNA expression to an in vivo gonadotropin (FSH) challenge in sexually immature, five-month-old alligators. Further, we investigated differences in these observed responses between alligators hatched from eggs collected at a heavily-polluted (Lake Apopka, FL) and minimally-polluted (Lake Woodruff, FL) site. In oviducts, we measured mRNA expression of estrogen, progesterone, and androgen receptors and also beta A and B subunits which homo- or heterodimerize to produce the transforming growth factor activin. In comparison, minimal inhibin alpha subunit mRNA expression suggests that these oviducts produce a primarily activin-dominated signaling milieu. Ovaries responded to a five-day FSH challenge with increased expression of steroidogenic enzyme mRNA which was concomitant with increased circulating sex steroid hormone concentrations. Oviducts in the FSH-challenged Lake Woodruff alligators increased mRNA expression of progesterone and androgen receptors, proliferating cell nuclear antigen, and the activin signaling antagonist follistatin. In contrast, Lake Apopka alligators displayed a diminished increase in ovarian CYP19A1 aromatase expression and no increase in oviducal AR expression, as compared to those observed in Lake Woodruff alligators. These results demonstrate that five-month-old female alligators display an endocrine-responsive ovarian-oviducal axis and environmental pollution exposure may alter these physiological responses. PMID:22154572

  6. Activin-A and Myostatin Response and Steroid Regulation in Human Myometrium: Disruption of Their Signalling in Uterine Fibroid

    Science.gov (United States)

    Bloise, Enrrico; Gray, Peter C.; Carrarelli, Patrizia; Islam, Md. Soriful; De Pascalis, Flavio; Severi, Filiberto Maria; Vale, Wylie; Castellucci, Mario; Petraglia, Felice

    2011-01-01

    Context: Investigation of activin-A (A) and myostatin (M) in human myometrium (HM) and leiomyoma (HL) will explain their involvement in human myometrial pathophysiology. Objective: We aimed to investigate A and M response and steroid regulation in HM. We also evaluated A and M expression and response in HL. Design: Tissues were analyzed and cultured. Patients: Patients included fertile (in proliferative phase) and menopausal women undergoing hysterectomy. Interventions: HM explant cultures were treated with A and M (for Smad-7 mRNA quantification) or estrogen and progesterone (for A and M mRNA quantification). A and M expression levels were also evaluated in menopausal (physiological absence of steroids) HM specimens. A and M and their receptors were evaluated in HL (n = 8, diameter 5–8 cm) compared with their matched HM. HL explants cultures were treated with A and M (for Smad7 mRNA quantification), and, to explain the absence of response, the levels of follistatin, follistatin-related gene (FLRG), and Cripto were evaluated. Results: A and M increased Smad7 expression in HM explants. A and M mRNAs were both reduced after estradiol treatment, unchanged after progesterone treatment, but were higher in menopausal than fertile (in proliferative phase) specimens. A, M, and FLRG were expressed at higher levels in HL compared with adjacent HM, whereas the receptors, follistatin, and Smad7 mRNAs resulted unchanged. Cripto mRNA was expressed only in HL. Conclusions: A and M act on human HM and are regulated by steroids. In HL there is an increase of A, M, FLRG, and Cripto expression. PMID:21177794

  7. Effects of gonadotrophins, growth hormone, and activin A on enzymatically isolated follicle growth, oocyte chromatin organization, and steroid secretion.

    Science.gov (United States)

    Ola, Safiriyu Idowu; Ai, Jun-Shu; Liu, Jing-He; Wang, Qiang; Wang, Zhen-Bo; Chen, Da-Yuan; Sun, Qing-Yuan

    2008-01-01

    So far, standard follicle culture systems can produce blastocyst from less than 40% of the in vitro matured oocytes compared to over 70% in the in vivo counterpart. Because the capacity for embryonic development is strictly associated with the terminal stage of oocyte growth, the nuclear maturity status of the in vitro grown oocyte was the subject of this study. Mouse early preantral follicles (100-130 microm) and early antral follicles (170-200 microm) isolated enzymatically were cultured for 12 and 4 days, respectively, in a collagen-free dish. The serum-based media were supplemented with either 100 mIU/ml FSH (FSH only); 100 mIU/ml FSH + 10 mIU/ml LH (FSH-LH); 100 mIU/ml FSH + 1 mIU/ml GH (FSH-GH) or 100 mIU/ml FSH + 100 ng/ml activin A (FSH-AA). Follicle survival was highest in follicle stimulating hormone (FSH)-AA group in both cultured preantral (91.8%) and antral follicles (82.7%). Survival rates in the other groups ranged between 48% (FSH only, preantral follicle culture) and 78.7% (FSH only, antral follicle culture). Estradiol and progesterone were undetectable in medium lacking gonadotrophins while AA supplementation in synergy with FSH caused increased estradiol secretion and a simultaneously lowered progesterone secretion. Chromatin configuration of oocytes from surviving follicles at the end of culture revealed that there were twice more developmentally incompetent non-surrounded nucleolus (NSN) oocytes (>65%) than the competent surrounded nucleolus (SN) oocytes (produce optimum proportion of developmentally competent oocytes.

  8. Heterozygous disruption of activin receptor-like kinase 1 is associated with increased arterial pressure in mice

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    María González-Núñez

    2015-11-01

    Full Text Available The activin receptor-like kinase 1 (ALK-1 is a type I cell-surface receptor for the transforming growth factor-β (TGF-β family of proteins. Hypertension is related to TGF-β1, because increased TGF-β1 expression is correlated with an elevation in arterial pressure (AP and TGF-β expression is upregulated by the renin-angiotensin-aldosterone system. The purpose of this study was to assess the role of ALK-1 in regulation of AP using Alk1 haploinsufficient mice (Alk1+/−. We observed that systolic and diastolic AP were significantly higher in Alk1+/− than in Alk1+/+ mice, and all functional and structural cardiac parameters (echocardiography and electrocardiography were similar in both groups. Alk1+/− mice showed alterations in the circadian rhythm of AP, with higher AP than Alk1+/+ mice during most of the light period. Higher AP in Alk1+/− mice is not a result of a reduction in the NO-dependent vasodilator response or of overactivation of the peripheral renin-angiotensin system. However, intracerebroventricular administration of losartan had a hypotensive effect in Alk1+/− and not in Alk1+/+ mice. Alk1+/− mice showed a greater hypotensive response to the β-adrenergic antagonist atenolol and higher concentrations of epinephrine and norepinephrine in plasma than Alk1+/+ mice. The number of brain cholinergic neurons in the anterior basal forebrain was reduced in Alk1+/− mice. Thus, we concluded that the ALK-1 receptor is involved in the control of AP, and the high AP of Alk1+/− mice is explained mainly by the sympathetic overactivation shown by these animals, which is probably related to the decreased number of cholinergic neurons.

  9. Novel Protein Interactions with Endoglin and Activin Receptor-like Kinase 1: Potential Role in Vascular Networks*

    Science.gov (United States)

    Xu, Guoxiong; Barrios-Rodiles, Miriam; Jerkic, Mirjana; Turinsky, Andrei L.; Nadon, Robert; Vera, Sonia; Voulgaraki, Despina; Wrana, Jeffrey L.; Toporsian, Mourad; Letarte, Michelle

    2014-01-01

    Endoglin and activin receptor-like kinase 1 are specialized transforming growth factor-beta (TGF-β) superfamily receptors, primarily expressed in endothelial cells. Mutations in the corresponding ENG or ACVRL1 genes lead to hereditary hemorrhagic telangiectasia (HHT1 and HHT2 respectively). To discover proteins interacting with endoglin, ACVRL1 and TGF-β receptor type 2 and involved in TGF-β signaling, we applied LUMIER, a high-throughput mammalian interactome mapping technology. Using stringent criteria, we identified 181 novel unique and shared interactions with ACVRL1, TGF-β receptor type 2, and endoglin, defining potential novel important vascular networks. In particular, the regulatory subunit B-beta of the protein phosphatase PP2A (PPP2R2B) interacted with all three receptors. Interestingly, the PPP2R2B gene lies in an interval in linkage disequilibrium with HHT3, for which the gene remains unidentified. We show that PPP2R2B protein interacts with the ACVRL1/TGFBR2/endoglin complex and recruits PP2A to nitric oxide synthase 3 (NOS3). Endoglin overexpression in endothelial cells inhibits the association of PPP2R2B with NOS3, whereas endoglin-deficient cells show enhanced PP2A-NOS3 interaction and lower levels of endogenous NOS3 Serine 1177 phosphorylation. Our data suggest that endoglin regulates NOS3 activation status by regulating PPP2R2B access to NOS3, and that PPP2R2B might be the HHT3 gene. Furthermore, endoglin and ACVRL1 contribute to several novel networks, including TGF-β dependent and independent ones, critical for vascular function and potentially defective in HHT. PMID:24319055

  10. Exogenous FGF10 can rescue an eye-open at birth phenotype of Fgf10-null mice by activating activin and TGFalpha-EGFR signaling.

    Science.gov (United States)

    Tao, Hirotaka; Ono, Katsuhiko; Kurose, Hitomi; Noji, Sumihare; Ohuchi, Hideyo

    2006-06-01

    Mutant mice deficient in the fibroblast growth factor 10 (Fgf10) gene exhibit an eye-open phenotype at birth. It has previously been shown that FGF10 has a dual role in proliferation and migration during the early and later stages of eyelid development, respectively. To verify the role of FGF10 during eyelid closure, explant culture of Fgf10-null eyelid anlagen was performed, by which it was examined whether or not exogenous FGF10 could rescue the expression of activin betaB and transforming growth factor alpha, known to be required for eyelid closure. We found that the expression of these genes was markedly induced while that of Shh or Ptch1, Ptch2 was not. We also observed the distribution of filamentous actin (F-actin) after FGF10 application in the mutant eyelid explant, finding that the FGF10 protein induced F-actin accumulation. We further examined filopodia of the eyelid leading edge cells, finding the length of the filopodia was significantly reduced in the mutant. These results verify that FGF10 promotes eyelid closure through activating activin and TGFalpha-EGFR signaling.

  11. The syndrome of central hypothyroidism and macroorchidism: IGSF1 controls TRHR and FSHB expression by differential modulation of pituitary TGFβ and Activin pathways

    Science.gov (United States)

    García, Marta; Barrio, Raquel; García-Lavandeira, Montserrat; Garcia-Rendueles, Angela R.; Escudero, Adela; Díaz-Rodríguez, Esther; Gorbenko Del Blanco, Darya; Fernández, Ana; de Rijke, Yolanda B.; Vallespín, Elena; Nevado, Julián; Lapunzina, Pablo; Matre, Vilborg; Hinkle, Patricia M.; Hokken-Koelega, Anita C. S.; de Miguel, María P.; Cameselle-Teijeiro, José Manuel; Nistal, Manuel; Alvarez, Clara V.; Moreno, José C.

    2017-01-01

    IGSF1 (Immunoglobulin Superfamily 1) gene defects cause central hypothyroidism and macroorchidism. However, the pathogenic mechanisms of the disease remain unclear. Based on a patient with a full deletion of IGSF1 clinically followed from neonate to adulthood, we investigated a common pituitary origin for hypothyroidism and macroorchidism, and the role of IGSF1 as regulator of pituitary hormone secretion. The patient showed congenital central hypothyroidism with reduced TSH biopotency, over-secretion of FSH at neonatal minipuberty and macroorchidism from 3 years of age. His markedly elevated inhibin B was unable to inhibit FSH secretion, indicating a status of pituitary inhibin B resistance. We show here that IGSF1 is expressed both in thyrotropes and gonadotropes of the pituitary and in Leydig and germ cells in the testes, but at very low levels in Sertoli cells. Furthermore, IGSF1 stimulates transcription of the thyrotropin-releasing hormone receptor (TRHR) by negative modulation of the TGFβ1-Smad signaling pathway, and enhances the synthesis and biopotency of TSH, the hormone secreted by thyrotropes. By contrast, IGSF1 strongly down-regulates the activin-Smad pathway, leading to reduced expression of FSHB, the hormone secreted by gonadotropes. In conclusion, two relevant molecular mechanisms linked to central hypothyroidism and macroorchidism in IGSF1 deficiency are identified, revealing IGSF1 as an important regulator of TGFβ/Activin pathways in the pituitary. PMID:28262687

  12. Hanging drop cultures of human testis and testis cancer samples: a model used to investigate activin treatment effects in a preserved niche

    Science.gov (United States)

    Jørgensen, A; Young, J; Nielsen, J E; Joensen, U N; Toft, B G; Rajpert-De Meyts, E; Loveland, K L

    2014-01-01

    Background: Testicular germ cell tumours of young adults, seminoma or non-seminomas, are preceded by a pre-invasive precursor, carcinoma in situ (CIS), understood to arise through differentiation arrest of embryonic germ cells. Knowledge about the malignant transformation of germ cells is currently limited by the lack of experimental models. The aim of this study was to establish an experimental tissue culture model to maintain normal and malignant germ cells within their niche and allow investigation of treatment effects. Methods: Human testis and testis cancer specimens from orchidectomies were cultured in ‘hanging drops' and effects of activin A and follistatin treatment were investigated in seminoma cultures. Results: Testis fragments with normal spermatogenesis or CIS cells were cultured for 14 days with sustained proliferation of germ cells and CIS cells and without increased apoptosis. Seminoma cultures survived 7 days, with proliferating cells detectable during the first 5 days. Activin A treatment significantly reduced KIT transcript and protein levels in seminoma cultures, thereby demonstrating a specific treatment response. Conclusions: Hanging drop cultures of human testis and testis cancer samples can be employed to delineate mechanisms governing growth of normal, CIS and tumorigenic germ cells retained within their niche. PMID:24781282

  13. Angiomodulin is required for cardiogenesis of embryonic stem cells and is maintained by a feedback loop network of p63 and Activin-A.

    Science.gov (United States)

    Wolchinsky, Zohar; Shivtiel, Shoham; Kouwenhoven, Evelyn Nathalie; Putin, Daria; Sprecher, Eli; Zhou, Huiqing; Rouleau, Matthieu; Aberdam, Daniel

    2014-01-01

    The transcription factor p63, member of the p53 gene family, encodes for two main isoforms, TAp63 and ΔNp63 with distinct functions on epithelial homeostasis and cancer. Recently, we discovered that TAp63 is essential for in vitro cardiogenesis and heart development in vivo. TAp63 is expressed by embryonic endoderm and acts on cardiac progenitors by a cell-non-autonomous manner. In the present study, we search for cardiogenic secreted factors that could be regulated by TAp63 and, by ChIP-seq analysis, identified Angiomodulin (AGM), also named IGFBP7 or IGFBP-rP1. We demonstrate that AGM is necessary for cardiac commitment of embryonic stem cells (ESCs) and its regulation depends on TAp63 isoform. TAp63 directly activates both AGM and Activin-A during ESC cardiogenesis while these secreted factors modulate TAp63 gene expression by a feedback loop mechanism. The molecular circuitry controlled by TAp63 on AGM/Activin-A signaling pathway and thus on cardiogenesis emphasizes the importance of p63 during early cardiac development.

  14. Angiomodulin is required for cardiogenesis of embryonic stem cells and is maintained by a feedback loop network of p63 and Activin-A

    Directory of Open Access Journals (Sweden)

    Zohar Wolchinsky

    2014-01-01

    Full Text Available The transcription factor p63, member of the p53 gene family, encodes for two main isoforms, TAp63 and ΔNp63 with distinct functions on epithelial homeostasis and cancer. Recently, we discovered that TAp63 is essential for in vitro cardiogenesis and heart development in vivo. TAp63 is expressed by embryonic endoderm and acts on cardiac progenitors by a cell-non-autonomous manner. In the present study, we search for cardiogenic secreted factors that could be regulated by TAp63 and, by ChIP-seq analysis, identified Angiomodulin (AGM, also named IGFBP7 or IGFBP-rP1. We demonstrate that AGM is necessary for cardiac commitment of embryonic stem cells (ESCs and its regulation depends on TAp63 isoform. TAp63 directly activates both AGM and Activin-A during ESC cardiogenesis while these secreted factors modulate TAp63 gene expression by a feedback loop mechanism. The molecular circuitry controlled by TAp63 on AGM/Activin-A signaling pathway and thus on cardiogenesis emphasizes the importance of p63 during early cardiac development.

  15. Sotatercept, a soluble activin receptor type 2A IgG-Fc fusion protein for the treatment of anemia and bone loss.

    Science.gov (United States)

    Raje, Noopur; Vallet, Sonia

    2010-10-01

    Sotatercept (ACE-011), under development by Acceleron Pharma Inc in collaboration with Celgene Corp, is a chimeric protein containing the extracellular domain of the activin receptor 2A (ACVR2A) fused to the Fc domain of human IgG1. Sotatercept contains the binding site of ACVR2A and interferes with downstream signaling cascades, in particular the SMAD pathway, by sequestering activin. The murine counterpart of sotatercept, referred to as RAP-011, has been extensively evaluated in preclinical studies, in particular in models of cancer- and osteoporosis-related bone loss, and the developing companies envisage that sotatercept may also have potential for the treatment of cancer and cancer-related bone loss. In a phase I clinical trial in postmenopausal females, sotatercept increased hematocrit levels, and, in a phase II trial in patients with multiple myeloma, a trend toward improvement in osteolytic lesions as well as antitumor activity was observed. At the time of publication, phase II trials in patients with anemia were ongoing. Future clinical development will rely on an evaluation of the benefits and complications of sotatercept administration, focusing in particular on suppression of ovarian function and increases in hematocrit levels without a consequent risk of hypertension and thrombosis.

  16. Hormonal and photoperiodic modulation of testicular mRNAs coding for inhibin/activin subunits and follistatin in Clethrionomys glareolus, Schreber.

    Science.gov (United States)

    Tähkä, K M; Kaipia, A; Toppari, J; Tähkä, S; Tuuri, T; Tuohimaa, P

    1998-07-01

    Photoperiodic and hormonal modulation of mRNAs for testicular inhibin/activin subunits and follistatin were studied in a seasonally breeding rodent, the bank vole (Clethrionomys glareolus). Photoperiod-induced testicular regression had no effect on the relatively low steady-state levels of follistatin mRNA. Inhibin alpha (I alpha) and beta B (I beta B) mRNA levels were significantly higher in regressed than in active gonads, but inhibin beta A was undetectable. The effect of gonadotropin administration on testicular weight and mRNA concentrations differed between the sexually active and quiescent voles. Neither FSH (1.2 U/kg; s.c. for 5 days) nor hCG (600 IU/kg; s.c. for 5 days) affected testicular weight in sexually active voles, whereas both gonadotropins significantly increased testicular weight in photo-regressed individuals. FSH had no effect on I alpha or I beta B mRNA concentrations in the active testes, whereas excessive hCG challenge induced a decrease in the steady-state levels of these mRNAs. FSH induced an increase in I alpha mRNA concentrations in the regressed gonad, whereas both gonadotropins concomitantly down-regulated I beta B mRNA levels. In conclusion, the high expression of I alpha and I beta B mRNA in the regressed testis imply autocrine and paracrine roles for inhibin/activin in the quiescent gonad of seasonal breeders. Inhibin alpha-subunit expression is at least partly under the control of FSH in the bank vole testis.

  17. [Molecular cloning of the DNA sequence of activin beta A subunit gene mature peptides from panda and related species and its application in the research of phylogeny and taxonomy].

    Science.gov (United States)

    Wang, Xiao-Jing; Wang, Xiao-Xing; Wang, Ya-Jun; Wang, Xi-Zhong; He, Guang-Xin; Chen, Hong-Wei; Fei, Li-Song

    2002-09-01

    Activin, which is included in the transforming growth factor-beta (TGF beta) superfamily of proteins and receptors, is known to have broad-ranging effects in the creatures. The mature peptide of beta A subunit of this gene, one of the most highly conserved sequence, can elevate the basal secretion of follicle-stimulating hormone (FSH) in the pituitary and FSH is pivotal to organism's reproduction. Reproduction block is one of the main reasons which cause giant panda to extinct. The sequence of Activin beta A subunit gene mature peptides has been successfully amplified from giant panda, red panda and malayan sun bear's genomic DNA by using polymerase chain reaction (PCR) with a pair of degenerate primers. The PCR products were cloned into the vector pBlueScript+ of Esherichia coli. Sequence analysis of Activin beta A subunit gene mature peptides shows that the length of this gene segment is the same (359 bp) and there is no intron in all three species. The sequence encodes a peptide of 119 amino acid residues. The homology comparison demonstrates 93.9% DNA homology and 99% homology in amino acid among these three species. Both GenBank blast search result and restriction enzyme map reveal that the sequences of Activin beta A subunit gene mature peptides of different species are highly conserved during the evolution process. Phylogeny analysis is performed with PHYLIP software package. A consistent phylogeny tree has been drawn with three different methods. The software analysis outcome accords with the academic view that giant panda has a closer relationship to the malayan sun bear than the red panda. Giant panda should be grouped into the bear family (Uersidae) with the malayan sun bear. As to the red panda, it would be better that this animal be grouped into the unique family (red panda family) because of great difference between the red panda and the bears (Uersidae).

  18. Effects of c-Jun N-terminal kinase on Activin A/Smads signaling in PC12 cell suffered from oxygen-glucose deprivation.

    Science.gov (United States)

    Wang, J Q; Xu, Z H; Liang, W Z; He, J T; Cui, Y; Liu, H Y; Xue, L X; Shi, W; Shao, Y K; Mang, J; Xu, Z X

    2016-02-29

    Activin A (Act A), a member of transforming growth factor-β (TGF-β) superfamily, is an early gene in response to cerebral ischemia. Growing evidences confirm the neuroprotective effect of Act A in ischemic injury through Act A/Smads signal activation. In this process, regulation networks are involved in modulating the outcomes of Smads signaling. Among these regulators, crosstalk between c-Jun N-terminal kinase (JNK) and Smads signaling has been found in the TGF-β induced epithelial-mesenchymal transition. However, in neural ischemia, the speculative regulation between JNK and Act A/Smads signaling pathways has not been clarified. To explore this issue, an Oxygen Glucose Deprivation (OGD) model was introduced to nerve-like PC12 cells. We found that JNK signal activation occurred at the early time of OGD injury (1 h). Act A administration suppressed JNK phosphorylation. In addition, JNK inhibition could elevate the strength of Smads signaling and attenuate neural apoptosis after OGD injury. Our results indicated a negative regulation effect of JNK on Smads signaling in ischemic injury. Taken together, JNK, as a critical site for neural apoptosis and negative regulator for Act A/Smads signaling, was presumed to be a molecular therapeutic target for ischemia.

  19. Dual Inhibition of Activin/Nodal/TGF-β and BMP Signaling Pathways by SB431542 and Dorsomorphin Induces Neuronal Differentiation of Human Adipose Derived Stem Cells

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    Vedavathi Madhu

    2016-01-01

    Full Text Available Damage to the nervous system can cause devastating diseases or musculoskeletal dysfunctions and transplantation of progenitor stem cells can be an excellent treatment option in this regard. Preclinical studies demonstrate that untreated stem cells, unlike stem cells activated to differentiate into neuronal lineage, do not survive in the neuronal tissues. Conventional methods of inducing neuronal differentiation of stem cells are complex and expensive. We therefore sought to determine if a simple, one-step, and cost effective method, previously reported to induce neuronal differentiation of embryonic stem cells and induced-pluripotent stem cells, can be applied to adult stem cells. Indeed, dual inhibition of activin/nodal/TGF-β and BMP pathways using SB431542 and dorsomorphin, respectively, induced neuronal differentiation of human adipose derived stem cells (hADSCs as evidenced by formation of neurite extensions, protein expression of neuron-specific gamma enolase, and mRNA expression of neuron-specific transcription factors Sox1 and Pax6 and matured neuronal marker NF200. This process correlated with enhanced phosphorylation of p38, Erk1/2, PI3K, and Akt1/3. Additionally, in vitro subcutaneous implants of SB431542 and dorsomorphin treated hADSCs displayed significantly higher expression of active-axonal-growth-specific marker GAP43. Our data offers novel insights into cell-based therapies for the nervous system repair.

  20. In vivo amelioration of endogenous antitumor autoantibodies via low-dose P4N through the LTA4H/activin A/BAFF pathway.

    Science.gov (United States)

    Lin, Yu-Ling; Tsai, Nu-Man; Hsieh, Cheng-Hao; Ho, Shu-Yi; Chang, Jung; Wu, Hsin-Yi; Hsu, Ming-Hua; Chang, Chia-Ching; Liao, Kuang-Wen; Jackson, Tiffany L B; Mold, David E; Huang, Ru Chih C

    2016-11-29

    Cancer progression is associated with the development of antitumor autoantibodies in patients' sera. Although passive treatment with antitumor antibodies has exhibited remarkable therapeutic efficacy, inhibitory effects on tumor progression by endogenous antitumor autoantibodies (EAAs) have been limited. In this study, we show that P4N, a derivative of the plant lignan nordihydroguaiaretic acid (NDGA), enhanced the production of EAAs and inhibited tumor growth at low noncytotoxic concentrations via its immunoregulatory activity. Intratumoral injection of P4N improved the quantity and quality of EAAs, and passive transfer of P4N-induced EAAs dramatically suppressed lung metastasis formation and prolonged the survival of mice inoculated with metastatic CT26 tumor cells. P4N-induced EAAs specifically recognized two surface antigens, 78-kDa glucose-regulated protein (GRP78) and F1F0 ATP synthase, on the plasma membrane of cancer cells. Additionally, P4N treatment led to B-cell proliferation, differentiation to plasma cells, and high titers of autoantibody production. By serial induction of autocrine and paracrine signals in monocytes, P4N increased B-cell proliferation and antibody production via the leukotriene A4 hydrolase (LTA4H)/activin A/B-cell activating factor (BAFF) pathway. This mechanism provides a useful platform for studying and seeking a novel immunomodulator that can be applied in targeting therapy by improving the quantity and quality of the EAAs.

  1. BDNF Reduces Toxic Extrasynaptic NMDA Receptor Signaling via Synaptic NMDA Receptors and Nuclear-Calcium-Induced Transcription of inhba/Activin A

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    David Lau

    2015-08-01

    Full Text Available The health of neurons is critically dependent on the relative signaling intensities of survival-promoting synaptic and death-inducing extrasynaptic NMDA receptors. Here, we show that BDNF is a regulator of this balance and promotes neuroprotection by reducing toxic NMDA receptor signaling. BDNF acts by initiating synaptic NMDA-receptor/nuclear-calcium-driven adaptogenomics, leading to increased expression of inhibin β-A (inhba. Inhibin β-A (its homodimer is known as activin A in turn reduces neurotoxic extrasynaptic NMDA-receptor-mediated calcium influx, thereby shielding neurons against mitochondrial dysfunction, a major cause of excitotoxicity. Thus, BDNF induces acquired neuroprotection by enhancing synaptic activity and lowering extrasynaptic NMDA receptor death signaling through a nuclear calcium-inhibin β-A pathway. This process, which confers protection against ischemic brain damage in a mouse stroke model, may be compromised in Huntington’s disease, Alzheimer’s disease, or aging-related neurodegenerative conditions that are associated with reduced BDNF levels and/or enhanced extrasynaptic NMDA receptor signaling.

  2. Activin receptor-like kinase 7 suppresses lipolysis to accumulate fat in obesity through downregulation of peroxisome proliferator-activated receptor γ and C/EBPα.

    Science.gov (United States)

    Yogosawa, Satomi; Mizutani, Shin; Ogawa, Yoshihiro; Izumi, Tetsuro

    2013-01-01

    We previously identified a quantitative trait locus for adiposity, non-insulin-dependent diabetes 5 (Nidd5), on mouse chromosome 2. In the current study, we identified the actual genetic alteration at Nidd5 as a nonsense mutation of the Acvr1c gene encoding activin receptor-like kinase 7 (ALK7), one of the type I transforming growth factor-β receptors, which results in a COOH-terminal deletion of the kinase domain. We further showed that the ALK7 dysfunction causes increased lipolysis in adipocytes and leads to decreased fat accumulation. Conversely, ALK7 activation inhibits lipolysis by suppressing the expression of adipose lipases. ALK7 and activated Smads repress those lipases by downregulating peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT/enhancer binding protein (C/EBP) α. Although PPARγ and C/EBPα act as adipogenic transcription factors during adipocyte differentiation, they are lipolytic in sum in differentiated adipocytes and are downregulated by ALK7 in obesity to accumulate fat. Under the obese state, ALK7 deficiency improves glucose tolerance and insulin sensitivity by preferentially increasing fat combustion in mice. These findings have uncovered a net lipolytic function of PPARγ and C/EBPα in differentiated adipocytes and point to the ALK7-signaling pathway that is activated in obesity as a potential target of medical intervention.

  3. Activin Receptor-Like Kinase 7 Suppresses Lipolysis to Accumulate Fat in Obesity Through Downregulation of Peroxisome Proliferator–Activated Receptor γ and C/EBPα

    Science.gov (United States)

    Yogosawa, Satomi; Mizutani, Shin; Ogawa, Yoshihiro; Izumi, Tetsuro

    2013-01-01

    We previously identified a quantitative trait locus for adiposity, non-insulin-dependent diabetes 5 (Nidd5), on mouse chromosome 2. In the current study, we identified the actual genetic alteration at Nidd5 as a nonsense mutation of the Acvr1c gene encoding activin receptor-like kinase 7 (ALK7), one of the type I transforming growth factor-β receptors, which results in a COOH-terminal deletion of the kinase domain. We further showed that the ALK7 dysfunction causes increased lipolysis in adipocytes and leads to decreased fat accumulation. Conversely, ALK7 activation inhibits lipolysis by suppressing the expression of adipose lipases. ALK7 and activated Smads repress those lipases by downregulating peroxisome proliferator–activated receptor γ (PPARγ) and CCAAT/enhancer binding protein (C/EBP) α. Although PPARγ and C/EBPα act as adipogenic transcription factors during adipocyte differentiation, they are lipolytic in sum in differentiated adipocytes and are downregulated by ALK7 in obesity to accumulate fat. Under the obese state, ALK7 deficiency improves glucose tolerance and insulin sensitivity by preferentially increasing fat combustion in mice. These findings have uncovered a net lipolytic function of PPARγ and C/EBPα in differentiated adipocytes and point to the ALK7-signaling pathway that is activated in obesity as a potential target of medical intervention. PMID:22933117

  4. 激活素受体相互作用蛋白1及2在小鼠巨噬细胞中的表达%Expression of activin receptor-interacting proteins 1, 2 in mouse macrophages

    Institute of Scientific and Technical Information of China (English)

    崔雪玲; 葛敬岩; 李晨光; 孙洋; 牛立慢; 刘海岩; 柳忠辉; 王轶楠

    2012-01-01

    Objective To investigate the expression and role of activin receptor-interacting proteins 1, 2 (ARIP1, 2) in mouse macrophages. Methods The expression of ARIP1, 2 was determined by immunocytochemical staining. RAW264. 7 cells were co-transfected with plasmids CAGA-lux and CMV-gal, plus plasmids pcDNA3-ARIPl, pcDNA3-ARIP2 or empty plasmid pcDNA3, respectively, then stimulated with activin A, and determined for transcription activity of report gene. The expression of ActR II A mRNA was determined by RT-PCR. Results Immunocytochemical staining proved that ARIP1,2 were expressed in RAW264. 7 cells. The overexpressions of ARIP1, 2 inhibited the transcription of specific gene induced by activin A. The overexpression of ARIP2 inhibited, while that of ARIP1 showed no significant effect on the expression of ActR II A mRNA in RAW264. 7 cells. Conclusion ARIP1, 2 were coexpressed in mouse macrophages, which down-regulated the signal transduction of activin by different action modes.%目的 探讨激活素受体相互作用蛋白(Activin receptor-interacting protein,ARIP)1及2(ARIP1,2)在小鼠巨噬细胞RAW264.7中的表达及作用.方法 采用免疫细胞化学染色法检测RAW264.7细胞中ARIP1,2的表达;将质粒CAGA-lux、CMV-gal分别与表达质粒pcDNA3-ARIP1、pcDNA3-ARIP2或空质粒pcDNA3共转染RAW264.7细胞,应用激活素A刺激,检测细胞内报告基因转录荧光素酶的活性;实时定量RT-PCR检测ActRⅡA mRNA的表达.结果 免疫细胞化学染色结果显示,小鼠巨噬细胞系RAW264.7细胞表达ARIP1,2;RAW264.7细胞过表达ARIP1,2均能抑制Activin A诱导的特异基因转录;过表达ARIP2可抑制RAW264.7细胞ActRⅡA mRNA的表达,而过表达ARIP1对ActRⅡA mRNA的表达无明显影响.结论 ARIP1,2在小鼠巨噬细胞中共表达,并具有下调激活素信号传导的作用,但其作用方式不同.

  5. Crosstalk between the Smad and the Mitogen-Activated Protein Kinase Pathways is Essential for Erythroid Differentiation of Erythroleukemia Cells Induced by TGF-β, Activin, Hydroxyurea and Butyrate.

    Science.gov (United States)

    Akel, Salem; Bertolette, Daniel; Ruscetti, Francis W

    2013-04-22

    The role of crosstalk between the Smad and the MAPK signaling pathways in activin-, transforming growth factor-β (TGF-β)-, hydroxyurea (HU) - and butyrate-dependent erythroid differentiation of K562 leukemic cells was studied. Treatment with all four inducers caused transient phosphorylation of Smad2/3 and MAPK proteins including ERK, p38 and JNK. Use of specific inhibitors of p38, ERK and JNK MAPK proteins, and TGF-β type I receptor indicated that differentiation induced by each of these agents involves activation of Smad2/3 and p38 MAPK, and inhibition of ERK MAPK. Also, treatment of cells with an inhibitor of protein serine/threonine phosphatase, okadaic acid (OA), induced phosphorylation of Smad2/3, and p38 MAPK, coincident with its induction of erythroid differentiation. Specific inhibition of TGF-β type I receptor kinase activity not only abolished TGF-β/activin effects but also prevented Smad2/3 activation and erythroid differentiation induced by OA, HU and butyrate. The TGF-β type I receptor kinase inhibitor blocked OA-induced differentiation but not p38 MAPK phosphorylation demonstrating that signals from both pathways are needed. As previously observed, addition of ERK1/2 MAPK inhibitors upregulated Smad2/3 phosphorylation and enhanced differentiation, but these effects were dependent on signals from the TGF-β type I receptor. These data indicate that activation of both Smad2/3 and p38 MAPK signaling pathways is a prerequisite to induce erythroid differentiation of erythroleukemia cells by activin, TGF-β, HU, OA and butyrate.

  6. bFGF and Activin A function to promote survival and proliferation of single iPS cells in conditioned half-exchange mTeSR1 medium.

    Science.gov (United States)

    Guo, Xiaoling; Lian, Ruiling; Guo, Yonglong; Liu, Qing; Ji, Qingshan; Chen, Jiansu

    2015-07-01

    Human induced pluripotent stem (iPS) cells can be well maintained by clonal growth. The pluripotent growth of single iPS cells is limited by low survival. To facilitate robust single iPS cells cultured in vitro, half-exchange mTeSR1 medium (HM), whole-exchange medium (WM) and iPS cell-derived conditioned medium (iPS-CM) culture were used. The effects of bFGF and Activin A on the growth of single iPS cells were explored. The dissociation and propagation of single iPS cells also included Accutase enzymatic isolation, Rho-associated protein kinase (ROCK) inhibitor Y27632 protection and high-density single-cell seeding (1 × 10(6) cells/well). CCK-8 assays demonstrated that the viability of clonal iPS cells in mTeSR1 medium and single iPS cells in HM, iPS-CM or WM supplemented with 100 ng/ml bFGF and 10 ng/ml Activin A was significantly higher than that in WM. Annexin v and propidium iodide (PI) assay, Calcein AM and EthD-III double staining also confirmed the similar results. ELISA assays showed that the levels of bFGF and Activin A of single iPS cells in HM and iPS-CM were higher than single iPS cells in WM. Meanwhile, Reverse Transcription-Polymerase Chain Reaction (RT-PCR), quantitative Polymerase Chain Reaction (qPCR), Western Blotting (WB), Immunofluorescence (IF) and karyotype analysis revealed that HM culture was able to maintain undifferentiated markers of Nanog, Klf4, Sox2, Oct4, and did not affect the karyotype of iPS cells. Undifferentiated single iPS cells in HM displayed homogenized growth. These findings demonstrate that bFGF and Activin A are important for the survival and growth of single iPS cells. HM culture system combined Accutase, Y27632 and high-density single-cell seeding can facilitate short-term growth of single iPS cells in vitro.

  7. Complete reversal of muscle wasting in experimental cancer cachexia: Additive effects of activin type II receptor inhibition and β-2 agonist.

    Science.gov (United States)

    Toledo, Míriam; Busquets, Sílvia; Penna, Fabio; Zhou, Xiaolan; Marmonti, Enrica; Betancourt, Angelica; Massa, David; López-Soriano, Francisco J; Han, H Q; Argilés, Josep M

    2016-04-15

    Formoterol is a highly potent β2-adrenoceptor-selective agonist, which is a muscle growth promoter in many animal species. Myostatin/activin inhibition reverses skeletal muscle loss and prolongs survival of tumor-bearing animals. The aim of this investigation was to evaluate the effects of a combination of the soluble myostatin receptor ActRIIB (sActRIIB) and the β2-agonist formoterol in the cachectic Lewis lung carcinoma model. The combination of formoterol and sActRIIB was extremely effective in reversing muscle wasting associated with experimental cancer cachexia in mice. Muscle weights from tumor-bearing animals were completely recovered following treatment and this was also reflected in the measured grip strength. This combination increased food intake in both control and tumor-bearing animals. The double treatment also prolonged survival significantly without affecting the weight and growth of the primary tumor. In addition, it significantly reduced the number of metastasis. Concerning the mechanisms for the preservation of muscle mass during cachexia, the effects of formoterol and sActRIIB seemed to be additive, since formoterol reduced the rate of protein degradation (as measured in vitro as tyrosine release, using incubated isolated individual muscles) while sActRIIB only affected protein synthesis (as measured in vivo using tritiated phenylalanine). Formoterol also increased the rate of protein synthesis and this seemed to be favored by the presence of sActRIIB. Combining formoterol and sActRIIB seemed to be a very promising treatment for experimental cancer cachexia. Further studies in human patients are necessary and may lead to a highly effective treatment option for muscle wasting associated with cancer.

  8. Endoglin and activin receptor-like kinase 1 heterozygous mice have a distinct pulmonary and hepatic angiogenic profile and response to anti-VEGF treatment.

    Science.gov (United States)

    Ardelean, Daniela S; Jerkic, Mirjana; Yin, Melissa; Peter, Madonna; Ngan, Bo; Kerbel, Robert S; Foster, F Stuart; Letarte, Michelle

    2014-01-01

    Hereditary hemorrhagic telangiectasia (HHT) is a vascular dysplasia associated with dysregulated angiogenesis and arteriovascular malformations. The disease is caused by mutations in endoglin (ENG; HHT1) or activin receptor-like kinase 1 (ALK1; HHT2) genes, coding for transforming growth factor β (TGF-β) superfamily receptors. Vascular endothelial growth factor (VEGF) has been implicated in HHT and beneficial effects of anti-VEGF treatment were recently reported in HHT patients. To investigate the systemic angiogenic phenotype of Endoglin and Alk1 mutant mice and their response to anti-VEGF therapy, we assessed microvessel density (MVD) in multiple organs after treatment with an antibody to mouse VEGF or vehicle. Lungs were the only organ showing an angiogenic defect, with reduced peripheral MVD and secondary right ventricular hypertrophy (RVH), yet distinctly associated with a fourfold increase in thrombospondin-1 (TSP-1) in Eng (+/-) versus a rise in angiopoietin-2 (Ang-2) in Alk1 (+/-) mice. Anti-VEGF treatment did reduce lung VEGF levels but interestingly, led to an increase in peripheral pulmonary MVD and attenuation of RVH; it also normalized TSP-1 and Ang-2 expression. Hepatic MVD, unaffected in mutant mice, was reduced by anti-VEGF therapy in heterozygous and wild type mice, indicating a liver-specific effect of treatment. Contrast-enhanced micro-ultrasound demonstrated a reduction in hepatic microvascular perfusion after anti-VEGF treatment only in Eng (+/-) mice. Our findings indicate that the mechanisms responsible for the angiogenic imbalance and the response to anti-VEGF therapy differ between Eng and Alk1 heterozygous mice and raise the need for systemic monitoring of anti-angiogenic therapy effects in HHT patients.

  9. The Expression of Human Cytomegalovirus MicroRNA MiR-UL148D during Latent Infection in Primary Myeloid Cells Inhibits Activin A-triggered Secretion of IL-6.

    Science.gov (United States)

    Lau, Betty; Poole, Emma; Krishna, Benjamin; Sellart, Immaculada; Wills, Mark R; Murphy, Eain; Sinclair, John

    2016-08-05

    The successful establishment and maintenance of human cytomegalovirus (HCMV) latency is dependent on the expression of a subset of viral genes. Whilst the exact spectrum and functions of these genes are far from clear, inroads have been made for protein-coding genes. In contrast, little is known about the expression of non-coding RNAs. Here we show that HCMV encoded miRNAs are expressed de novo during latent infection of primary myeloid cells. Furthermore, we demonstrate that miR-UL148D, one of the most highly expressed viral miRNAs during latent infection, directly targets the cellular receptor ACVR1B of the activin signalling axis. Consistent with this, we observed upregulation of ACVR1B expression during latent infection with a miR-UL148D deletion virus (ΔmiR-UL148D). Importantly, we observed that monocytes latently infected with ΔmiR-UL148D are more responsive to activin A stimulation, as demonstrated by their increased secretion of IL-6. Collectively, our data indicates miR-UL148D inhibits ACVR1B expression in latently infected cells to limit proinflammatory cytokine secretion, perhaps as an immune evasion strategy or to postpone cytokine-induced reactivation until conditions are more favourable. This is the first demonstration of an HCMV miRNA function during latency in primary myeloid cells, implicating that small RNA species may contribute significantly to latent infection.

  10. Multiple roles of Activin/Nodal, bone morphogenetic protein, fibroblast growth factor and Wnt/β-catenin signalling in the anterior neural patterning of adherent human embryonic stem cell cultures

    Science.gov (United States)

    Lupo, Giuseppe; Novorol, Claire; Smith, Joseph R.; Vallier, Ludovic; Miranda, Elena; Alexander, Morgan; Biagioni, Stefano; Pedersen, Roger A.; Harris, William A.

    2013-01-01

    Several studies have successfully produced a variety of neural cell types from human embryonic stem cells (hESCs), but there has been limited systematic analysis of how different regional identities are established using well-defined differentiation conditions. We have used adherent, chemically defined cultures to analyse the roles of Activin/Nodal, bone morphogenetic protein (BMP), fibroblast growth factor (FGF) and Wnt/β-catenin signalling in neural induction, anteroposterior patterning and eye field specification in hESCs. We show that either BMP inhibition or activation of FGF signalling is required for effective neural induction, but these two pathways have distinct outcomes on rostrocaudal patterning. While BMP inhibition leads to specification of forebrain/midbrain positional identities, FGF-dependent neural induction is associated with strong posteriorization towards hindbrain/spinal cord fates. We also demonstrate that Wnt/β-catenin signalling is activated during neural induction and promotes acquisition of neural fates posterior to forebrain. Therefore, inhibition of this pathway is needed for efficient forebrain specification. Finally, we provide evidence that the levels of Activin/Nodal and BMP signalling have a marked influence on further forebrain patterning and that constitutive inhibition of these pathways represses expression of eye field genes. These results show that the key mechanisms controlling neural patterning in model vertebrate species are preserved in adherent, chemically defined hESC cultures and reveal new insights into the signals regulating eye field specification. PMID:23576785

  11. Characterization of the human Activin-A receptor type II-like kinase 1 (ACVRL1 promoter and its regulation by Sp1

    Directory of Open Access Journals (Sweden)

    Botella Luisa M

    2010-06-01

    Full Text Available Abstract Background Activin receptor-like kinase 1 (ALK1 is a Transforming Growth Factor-β (TGF-β receptor type I, mainly expressed in endothelial cells that plays a pivotal role in vascular remodelling and angiogenesis. Mutations in the ALK1 gene (ACVRL1 give rise to Hereditary Haemorrhagic Telangiectasia, a dominant autosomal vascular dysplasia caused by a haploinsufficiency mechanism. In spite of its patho-physiological relevance, little is known about the transcriptional regulation of ACVRL1. Here, we have studied the different origins of ACVRL1 transcription, we have analyzed in silico its 5'-proximal promoter sequence and we have characterized the role of Sp1 in the transcriptional regulation of ACVRL1. Results We have performed a 5'Rapid Amplification of cDNA Ends (5'RACE of ACVRL1 transcripts, finding two new transcriptional origins, upstream of the one previously described, that give rise to a new exon undiscovered to date. The 5'-proximal promoter region of ACVRL1 (-1,035/+210 was analyzed in silico, finding that it lacks TATA/CAAT boxes, but contains a remarkably high number of GC-rich Sp1 consensus sites. In cells lacking Sp1, ACVRL1 promoter reporters did not present any significant transcriptional activity, whereas increasing concentrations of Sp1 triggered a dose-dependent stimulation of its transcription. Moreover, silencing Sp1 in HEK293T cells resulted in a marked decrease of ACVRL1 transcriptional activity. Chromatin immunoprecipitation assays demonstrated multiple Sp1 binding sites along the proximal promoter region of ACVRL1 in endothelial cells. Furthermore, demethylation of CpG islands, led to an increase in ACVRL1 transcription, whereas in vitro hypermethylation resulted in the abolishment of Sp1-dependent transcriptional activation of ACVRL1. Conclusions Our results describe two new transcriptional start sites in ACVRL1 gene, and indicate that Sp1 is a key regulator of ACVRL1 transcription, providing new insights into

  12. The crucial role of Activin A on the formation of primordial germ cell-like cells from skin-derived stem cells in vitro.

    Science.gov (United States)

    Sun, Rui; Sun, Yuan-Chao; Ge, Wei; Tan, Hui; Cheng, Shun-Feng; Yin, Shen; Sun, Xiao-Feng; Li, Lan; Dyce, Paul; Li, Julang; Yang, Xiao; Shi, Qing-Hua; Shen, Wei

    2015-01-01

    Primordial germ cells (PGCs) are founder cells of the germ cell lineage, and can be differentiated from stem cells in an induced system in vitro. However, the induction conditions need to be optimized in order to improve the differentiation efficiency. Activin A (ActA) is a member of the TGF-β super family and plays an important role in oogenesis and folliculogenesis. In the present study, we found that ActA promoted PGC-like cells (PGCLCs) formation from mouse skin-derived stem cells (SDSCs) in both embryoid body-like structure (EBLS) differentiation and the co-culture stage in a dose dependent manner. ActA treatment (100 ng/ml) during EBLS differentiation stage and further co-cultured for 6 days without ActA significantly increased PGCLCs from 53.2% to 82.8%, and as well as EBLS differentiation without ActA followed by co-cultured with 100 ng/ml ActA for 4 to 12 days with the percentage of PGCLCs increasing markedly in vitro. Moreover, mice treated with ActA at 100 ng/kg body weight from embryonic day (E) 5.5-12.5 led to more PGCs formation. However, the stimulating effects of ActA were interrupted by Smad3 RNAi, and in an in vitro cultured Smad3(-/-) mouse skin cells scenario. SMAD3 is thus likely a key effecter molecule in the ActA signaling pathway. In addition, we found that the expression of some epiblast cell markers, Fgf5, Dnmt3a, Dnmt3b and Wnt3, was increased in EBLSs cultured for 4 days or PGCLCs co-cultured for 12 days with ActA treatment. Interestingly, at 16 days of differentiation, the percentage of PGCLCs was decreased in the presence of ActA, but the expression of meiosis-relative genes, such as Stra8, Dmc1, Sycp3 and Sycp1, was increased. In conclusion, our data here demonstrated that ActA can promote PGCLC formation from SDSCs in vitro, at early stages of differentiation, and affect meiotic initiation of PGCLCs in later stages.

  13. Expressions of Placenta Inhibin-A, Activin-A and Inhibin-B Genes in Preeclamptic Pregnancy%子痫前期胎盘抑制素A、胎盘激活素A和胎盘抑制素B基因的表达

    Institute of Scientific and Technical Information of China (English)

    董旭东; 吴云萍; 江江; 陈桂仙

    2011-01-01

    Objective: To investigate the mRNA expression and the significance of inhibin-A,activin-A and inhibin-B subunites in the placenta from preeclamptic patients.Methods: The mRNA levels of inhibin-A, activin-A and inhibin-B subunites in the placenta from 44 pre-eclamptic patients and 30 normal pregnancies were measured by RT-PCR.Results: The mRNA levels of inhibin-A α-βA subunite and activin-A βA-βA subunite in the placentas from pre-eclamptic patients were significantly higher than those in the placenta from normal pregnancies (P < 0.05 ), while the mRNA expression of inhibin-B α-βB subunit between the preeclamptic and normal pregnancy groups were no significant difference( P>0.05).Conclutions: The mRNA levels of inhibin-A α-βA subunite and activin-A βA-βA subunite increased significantly in the placenta from pre-eclamptic patients.The levels of inhibin-A and activin-A can reflect the function of trophoblastic cells, so they could be useful markers to monitor the function of placenta, and involve in the pathophysiologic process of preeclampsia.%目的:探讨子痫前期患者胎盘中抑制素A(inhibin-A)、激活素A(activin-A)和抑制素B(inhibin-B)亚单位基因的表达水平及其意义.方法:采用逆转录聚合酶链反应(RT-PCR)技术定量测定44例子痫前期患者和30例正常妊娠妇女胎盘中inhibin-A、activin-A和inhibin-B亚单位基因mRNA的表达.结果:子痫前期组胎盘中inhibin-A a-βA亚单位和activin-A βA-βA亚单位基因mRNA表达水平明显高于对照组,差异有统计学意义(P0.05).结论:子痫前期患者胎盘中的inhibin-A a-βA亚单位和activin-A βA-βA亚单位mRNA表达水平升高,inhibin-A/activin-A水平可反映滋养细胞的功能,是监测胎盘功能有效指标,并参与子痫前期的病理生理过程.

  14. Diagnostic value of serum activin A and follistatin levels indifferent types of endometriosis%激活素A和卵泡抑素在子宫内膜异位症中的诊断价值

    Institute of Scientific and Technical Information of China (English)

    徐小艳; 洛若愚; 邢辉

    2015-01-01

    目的:探讨人激活素 A 和卵泡抑素在腹膜型,卵巢型及深部浸润型子宫内膜异位症中的临床诊断价值。方法子宫内膜异位症患者139例,分成腹膜型内异症组28例,卵巢型内异症组61例,深部浸润型内异症组50例,月经规律的健康女性(对照组)75例,均采集外周静脉血,采用酶联免疫测定试剂盒分析血清中激活素A 和卵泡抑素。结果卵巢内异症组激活素 A 水平[(0.22±0.01)ng/ml]明显高于对照组[(0.17±0.01)ng/ml]、腹膜型内异症组[(0.19±0.01)ng/ml]及深部浸润型内异症组[(0.16±0.02)ng/ml],差异有统计学意义;子宫内膜异位症患者卵泡抑素与对照组比较,差异无统计学意义;深部浸润型内异症组卵泡抑素水平[(1.50±0.17)ng/ml]低于腹膜型内异症组[(2.24±0.42)ng/ml]和卵巢内异症组[(2.34±0.32)ng/ml],差异有统计学意义;激活素 A和卵泡抑素诊断卵巢内异症的 ROC 曲线下面积分别为0.700(95% CI 为0.605~0.794)和0.620(95% CI 为0.510~0.730),二者联合应用不能提高子宫内膜异位症的诊断特异度和灵敏度。结论激活素 A 和卵泡抑素水平在腹膜型内异症和深部浸润型内异症中无明显改变,对诊断卵巢型内异症的准确率有限。%Objective To explore the clinical value of activin A and follistatin levels in women with peritoneal, ovarian and deep inltrating endometriosis. Methods One hundred and thirty﹣nine patients with endometriosis were divided into three groups:peritoneal endometriosis (n =28),ovarian endometrioma (n =61 ),and deep inltrating endometriosis (n =50).Seventy﹣five health women were included as the control group.Peripheral vein blood samples were collected for activin A and follistatin assessment by enzyme immunoassay kits. Results The ovarian endometrioma group [(0.22 ±0.01)ng/ml]had serum

  15. Circulating irisin levels are lower in patients with either stable coronary artery disease (CAD) or myocardial infarction (MI) versus healthy controls, whereas follistatin and activin A levels are higher and can discriminate MI from CAD with similar to CK-MB accuracy.

    Science.gov (United States)

    Anastasilakis, Athanasios D; Koulaxis, Dimitrios; Kefala, Nikoleta; Polyzos, Stergios A; Upadhyay, Jagriti; Pagkalidou, Eirini; Economou, Fotios; Anastasilakis, Chrysostomos D; Mantzoros, Christos S

    2017-08-01

    Several myokines are produced by cardiac muscle. We investigated changes in myokine levels at the time of acute myocardial infarction (MI) and following reperfusion in relation to controls. Patients with MI (MI Group, n=31) treated with percutaneous coronary intervention (PCI) were compared to patients with stable coronary artery disease (CAD) subjected to scheduled PCI (CAD Group, n=40) and controls with symptoms mimicking CAD without stenosis in angiography (Control Group, n=43). The number and degree of stenosis were recorded. Irisin, follistatin, follistatin-like 3, activin A and B, ALT, AST, CK and CK-MB were measured at baseline and 6 or 24h after the intervention. MI and CAD patients had lower irisin than controls (pCAD patients and controls (all p≤0.001). None of the myokines changed following reperfusion. Circulating irisin was associated with the degree of stenosis in all patients (p=0.05). Irisin was not inferior to CK-MB in predicting MI while folistatin and activin A could discriminate MI from CAD patients with similar to CK-MB accuracy. None of these myokines was altered following PCI in contrast to CK-MB. Irisin levels are lower in MI and CAD implying that their production may depend on myocadial blood supply. Follistatin and activin A are higher in MI than in CAD suggesting increased release due to myocardial necrosis. They can predict MI with accuracy similar to CK-MB and their role in the diagnosis of MI remains to be confirmed by prospective large clinical studies. Copyright © 2017 Elsevier Inc. All rights reserved.

  16. 激活素A对Neuro-2a细胞电压门控钠电流的作用%Effects of activin A on voltage-gated Na+ current of Neuro-2a cells

    Institute of Scientific and Technical Information of China (English)

    孙洋; 柳忠辉; 王轶楠; 刘海岩; 王东辉; 葛敬岩

    2010-01-01

    目的:研究激活素A(activin A)对Neuro-2a细胞电压门控钠电流(INa)的作用及其机制,为激活素A在神经系统疾病中的应用奠定基础.方法:培养小鼠源性Neuro-2a细胞,分为IgG对照组和抗激活素受体ⅡA (ActRⅡA)抗体染色组,免疫组织化学染色观察ActRⅡA在Neuro-2a细胞中是否表达;将Neuro-2a细胞随机分为正常对照组和激活素A组,采用全细胞膜片钳技术检测激活素A对Neuro-2a细胞 INa 的作用;采用RT-PCR检测激活素A对血管活性肠肽(VIP)及诱导型一氧化氮合酶(iNOS) mRNA表达的影响.结果:正常Neuro-2a细胞中可检测到ActRⅡA的表达;与正常对照组比较,激活素A明显增加Neuro-2a细胞INa(P< 0.05);激活素A组Neuro-2a细胞VIP mRNA表达明显高于正常对照组(P< 0.05),而iNOS mRNA表达低于正常对照组(P<0.05).结论:激活素A具有增加Neuro-2a细胞电压门控Na+电流的作用,VIP和iNOS途径可能参与了激活素A调控神经细胞的功能.

  17. Activin Receptor-Like Kinase Receptors ALK5 and ALK1 Are Both Required for TGFβ-Induced Chondrogenic Differentiation of Human Bone Marrow-Derived Mesenchymal Stem Cells

    Science.gov (United States)

    de Kroon, Laurie M. G.; Narcisi, Roberto; Blaney Davidson, Esmeralda N.; Cleary, Mairéad A.; van Beuningen, Henk M.; Koevoet, Wendy J. L. M.; van Osch, Gerjo J. V. M.; van der Kraan, Peter M.

    2015-01-01

    Introduction Bone marrow-derived mesenchymal stem cells (BMSCs) are promising for cartilage regeneration because BMSCs can differentiate into cartilage tissue-producing chondrocytes. Transforming Growth Factor β (TGFβ) is crucial for inducing chondrogenic differentiation of BMSCs and is known to signal via Activin receptor-Like Kinase (ALK) receptors ALK5 and ALK1. Since the specific role of these two TGFβ receptors in chondrogenesis is unknown, we investigated whether ALK5 and ALK1 are expressed in BMSCs and whether both receptors are required for chondrogenic differentiation of BMSCs. Materials & Methods ALK5 and ALK1 gene expression in human BMSCs was determined with RT-qPCR. To induce chondrogenesis, human BMSCs were pellet-cultured in serum-free chondrogenic medium containing TGFβ1. Chondrogenesis was evaluated by aggrecan and collagen type IIα1 RT-qPCR analysis, and histological stainings of proteoglycans and collagen type II. To overexpress constitutively active (ca) receptors, BMSCs were transduced either with caALK5 or caALK1. Expression of ALK5 and ALK1 was downregulated by transducing BMSCs with shRNA against ALK5 or ALK1. Results ALK5 and ALK1 were expressed in in vitro-expanded as well as in pellet-cultured BMSCs from five donors, but mRNA levels of both TGFβ receptors did not clearly associate with chondrogenic induction. TGFβ increased ALK5 and decreased ALK1 gene expression in chondrogenically differentiating BMSC pellets. Neither caALK5 nor caALK1 overexpression induced cartilage matrix formation as efficient as that induced by TGFβ. Moreover, short hairpin-mediated downregulation of either ALK5 or ALK1 resulted in a strong inhibition of TGFβ-induced chondrogenesis. Conclusion ALK5 as well as ALK1 are required for TGFβ-induced chondrogenic differentiation of BMSCs, and TGFβ not only directly induces chondrogenesis, but also modulates ALK5 and ALK1 receptor signaling in BMSCs. These results imply that optimizing cartilage formation by

  18. 老年慢性阻塞性肺疾病患者的血清激活素A和转化生长因子β1与肺功能变化的相关性%Clinic significance and correlations of serum activin A,transforming growth factor beta 1 and pulmonary function of elderly patients with chronic obstructive pulmonary disease

    Institute of Scientific and Technical Information of China (English)

    樊晓曦

    2016-01-01

    Objective To explore the clinic significance and correlations of serum activin A,transforming growth factor β1 and pulmonary function of elderly patients with chronic obstructive pulmonary disease. Methods 95 patients with elderly COPD were collected as the research object from September 2014 to September 2015. Sixty healthy subjects served as controls. The concentrations of ACTA and TGF - β1 were deter-mined using ELISA. The pearson correlation test was performed to analyze the correlation between ACTA,TGF - β1 and pulmonary function in-dex. Results Compared with those in healthy controls,plasma ACTA and TGF - β1 levels in elderly COPD increased significantly( P < 0. 05). The higher of the COPD level,the higher the level of ACTA and TGF - beta 1( P < 0. 05). The higher the level of pulmonary function damage, the higher the level of ACTA and TGF - beta 1. Pearson correlation analysis showed that the serum levels of ACTA,TGF - β1 were negatively cor-related with FEV1%( r = - 0. 687,- 0. 751,P < 0. 05),and they were also negatively correlated with FEV1 / FVC( r = - 0. 613,- 0. 598, P < 0. 05). Conclusion ACTA and TGF - β1 play important roles in the progression of COPD,and have correlation with pulmonary function changes.%目的:探讨老年慢性阻塞性肺疾病(COPD)患者的血清激活素 A(ACTA)和转化生长因子β1(TGF -β1)与肺功能变化的相关性。方法选择于2014年9月至2015年9月收治的老年 COPD 患者95例作为研究对象,选取同期60例健康体检者作为对照组。采用酶联免疫吸附法(ELISA)法检测血清 ACTA 和 TGF -β1水平。采用 Pearson 相关性分析法分析 ACTA 和 TGF -β1水平与各肺功能指标的相关性。结果与健康对照组相比,老年 COPD 患者的 ACTA和 TGF -β1水平显著升高( P <0.05)。COPD 严重程度越高,ACTA 和 TGF -β1水平越高( P <0.05)。肺功能损伤等级越高,ACTA 和 TGF -β1水平随之显著增高( P <0.05

  19. 活化素A和视黄酸诱导骨髓间充质干细胞体外分化为胰岛素分泌细胞%In vitro differentiation of bone marrow mesenchymal stem cells induced by activin A and all-trans retinoic acid into insulin-producing cells

    Institute of Scientific and Technical Information of China (English)

    王启伟; 于瑾; 刘兴茂; 李世崇; 叶玲玲; 刘红; 吴本传; 陈昭烈

    2006-01-01

    human being in the 21st century. Islet transplantation is considered to be the most effective approach to cure type Ⅰ diabetes mellitus. However, lack of donor tissue limits the application of this therapy. However, recent progress of stem cell research shows that stem cell therapy may be a potential means to solve this problem.OBJECTIVE: To take activin A and all-trans retinoic acid (AR) in inducing the differentiation of bone marrow mesenchymal stem cells (MSCs) and explore its possibility DESIGN: A randomized controlled experiment.SETTING: Institute of Biotechnology, Academy of Military Medical SciencesMATERIALS: This experiment was conducted at the Institute of Biotechnology, Academy of Military Medical Sciences from November 2004to June 2005. Six male Sprague-Dawley rats, with body mass of 150-160g, were provided by the Experimental Animal Center of Academy of Military Medical Sciences.METHODS: Femoral bone marrow of the rats was extracted under aseptic condition. Bone marrow mesenchymal stem cells (MSCs) were isolated with density gradient centrifugation. Passaged MSCs were randomly divided into 4 groups: high concentration of glucose (HG), AR, beta-mercaptoethanol (ME) and negative control groups. MSCs were induced to differentiate into IPCs with conditional medium containing high concentration glucose, activin A, RA and ME etc. After induction, phenotypes of differentiated cells were examined by immunocytochemistry and reverse transcription-polymerase chain reaction (RT-PCR).MAIN OUTCOME MEASURES: Expression of insulin and glucagon of differentiated cells were examined by immunocytochemistry. Insulin-1 mR-NA expression of differentiated cells was detected by RT-PCR.RESULTS: After bone marrow mesenchymal stem cells were induced,there were scattered insulin-and glucagon-positive cells in the HG group,many insulin-and glucagon-positive cells in the AR and ME groups, and these cells formed insulin-like structure. The expression of insulin-1mRNA could be observed

  20. Activin receptor subunits in normal and dysfunctional adult human testis

    DEFF Research Database (Denmark)

    Dias, V.; Meachem, S.; Rajpert-De, Meyts E.

    2008-01-01

    , carcinoma in situ (CIS), seminoma, non-seminoma and gonadotropin-deprived human testis. ActRIIA mRNA was localized by in situ hybridization. RESULTS: ALK2, ALK4 and ActRIIB proteins were observed in Sertoli cells, spermatogonia and some spermatocytes within normal and gonadotropin-suppressed adult human...... testis; all three receptor subunits were also detected in CIS, seminoma and non-seminoma cells. ActRIIA immunoreactivity was faint to absent in the normal testis and in CIS and non-seminoma cells, whereas some seminoma cells displayed a strong signal. Also in contrast to the normal testis, a majority...

  1. Role of Activin A in Immune Response to Breast Cancer

    Science.gov (United States)

    2015-12-01

    2014;124:2246–59. 41. Postow MA, Callahan MK, Wolchok JD. The antitumor immunity of ipilimumab: (T-cell) memories to last a lifetime? Clin Cancer Res 2012;18...Lagorce-Pages C, et al. Type, density, and location of immune cells within human colorectal tumors predict clinical outcome. Science 2006;313:1960–4...4] Pagès F, Galon J, Dieu-Nosjean MC, Tartour E, Sautès-Fridman C, Fridman WH. Immune infiltration in human tumors: a prognostic factor that should

  2. Role of Activin A in Immune Response to Breast Cancer

    Science.gov (United States)

    2014-12-01

    arabinomannan and mannin, has immunomodulatory properties (123). This was tested in a Japanese phase III, randomized trial in patients with stage IIB – IVA ...tolerability IMRT 5 days a week for 7 weeks with cetuximab and ipi at week 4 for 3, 21 day courses NCI NCT01711515 Cervical cancer (stage IB– IVA ) One arm: ipi...endothelial cells in inflammation. Nat Rev Immunol (2007) 7:803–15. doi:10.1038/nri2171 41. Ley K, Laudanna C, Cybulsky MI, Nourshargh S. Getting to

  3. Role of Activin A in Immune Response to Breast Cancer

    Science.gov (United States)

    2016-12-01

    in enhanced actA release, consistent with a compensatory mechanism described in development (Carvalho et al., J Cell Sci , 2007). Here we tested the...anti-PD-1-resistant preclinical tumor model with which to identify mechanisms of resis- tance, which should provide novel insights to expand the...enhanced actA release, consistent with a compensatory mech- anism described by Carvalho et al., J Cell Sci , 2007. Here we tested the hypothesis that both

  4. 激活素A在细菌性脑膜炎新生儿脑脊液中的变化及其对预后评估的价值%Change of Activin A in Cerebrospinal Fluid of Neonates with Bacterial Meningitis and Its Value for Prognosis Evaluation

    Institute of Scientific and Technical Information of China (English)

    郝丽红; 王琳; 郭静; 周颖; 巴爽; 林书祥

    2012-01-01

    目的 研究新生儿细菌性脑膜炎(BM)脑脊液(CSF)中激活素A( ACT A)水平的变化及其对预后判断的意义.方法 对2010年3月-2011年6月在本院新生儿病房住院的48例确诊BM患儿,进行3~18个月的随访及回顾性分析,分为有并发症和后遗症组(A组)和无并发症和后遗症组(B组).另收集同期住院的非颅内疾病患儿作为对照组(C组).应用EHSA法动态监测3组患儿CSF中ACT A水平.结果 A组患儿急性期CSF中ACT A水平为(544.39±149.62) ng·L-1,B组CSF中ACT A水平为(480.82±128.24) ng·L-1,二组间差异无统计学意义,但2组均高于C组[(181.06±45.20) ng·L-1](Pa<0.01).治疗1周,A组CSF中ACT A水平为(315.84±86.35) ng·L-1、B组为(338.25±99.43) ng·L-1,2组较治疗前显著下降(Pa<0.05),但2组间差异无统计学意义(P=0.432).治疗2周,A组CSF中ACT A水平为(188.19±43.38) ng·L-1,B组为(203.86±50.73) ng·L-1,2组差异无统计学意义(P=0.281).治疗3周,A组CSF中ACT A水平为(107.65±17.65)ng·L-1,B组为( 169.36±28.90)ng·L-1,A组明显低于B组(P =0.000).治疗4周,A组CSF中ACT A水平为(98.54±28.54) ng·L-1,B组为(181.84 ±35.01)ng·L-1,A组显著低于B组(P=0.000).结论 ACT A参与新生儿BM的发病过程,动态检测CSF中ACT A水平,对评估新生儿BM的预后,可能具有重要价值.%Abstract; Objective To evaluate the changes of activin A( ACT A) level in cerebrospinal fluid(CSF) in neonatal bacterial meningitis and its clinical value for prognosis evaluation. Methods Forty - eight neonatal bacterial meningitis in Tianjin Children's Hospital from Mar. 2010 to Jun. 2011 were visited for 3-18 months and analyzed retrospectively. All the patients were divided into 2 groups: neonatal bacterial meningitis with the complications and (or) sequelae group (group A) ,without the complications and sequelae group (group B) ,and the children without brain disease were selected as control group (group C). The levels of ACT A in cerebrospinal

  5. Effects of tripterygium wilfordii polyglucoside on expression of Activin A and influence of transdifferentiation in renal tubulointerstitium of rats with diabetic nephropathy%雷公藤多苷对糖尿病肾病大鼠肾小管间质激活素A表达及转分化的影响

    Institute of Scientific and Technical Information of China (English)

    赵晨光; 于为民; 任小军; 张巍; 李慧

    2014-01-01

    Objective To explore the effects of tripterygium wilfordii polyglucoside (TWP) on expression of Activin A (Act-A) and influence of transdifferentiation in renal tubulointerstitium of rats with diabetes nephropathy (DN).Methods A total of 40 male Wistar rats were randomly divided into normal control,DN model,irbesartan treatment and TWP treatment groups (n =10 each).The animal model of DN was established by an injection of streptozotocin.After modeling,irbesartan treatment and TWP treatment groups received an intragastric injection of corresponding drugs.The body weights were recorded and the 24 h Pro quantitative levels detected at Weeks 4,8,12 and 16 respectively.After sacrificing at Week 16,serum creatinine (Scr) was detected.The renal tissues were excised and examined after HE and PAS staining.The expressions of Act-A,alpha-smooth muscle actin (α-SMA),collagen type Ⅳ (Co-Ⅳ) and E-cad in renal tubulointerstitium were detected by immunohistochemistry.And the expressions of Act-A,α-SMA,Co-Ⅳand mRNA of E-cad in renal tissue were detected by real-time quantitative fluorescent polymerase chain reaction (PCR).Results Compared with DN model group,24 hPro ((67.1 ± 9.6),(61.8 ± 8.0) vs (158.3 ± 13.3) mg),quantitative Scr ((31.7 ± 4.1),(32.6 ± 6.2) vs (55.3 ± 8.9) μmol/L) and renal hypertrophic index ((9.14 ± 0.65),(7.50 ± 0.34) vs (11.28 ± 0.70) mg/g) of irbesartan treatment and TWP treatment groups decreased obviously at Week 16 (all P < 0.05).However,no statistical significance existed between irbesartan treatment and TWP treatment groups.The body weights of irbesartan treatment and TWP treatment groups increased versus DN model group.Pathological sections showed that lesion degree of renal tubulointerstitium became alleviated in each group.Yet no statistical significance existed between irbesartan treatment and TWP treatment groups.Both immunohistochemistry and real-time quantitative fluorescent PCR showed that the expressions of Act-A,α-SMA and Co

  6. Mutations in endoglin and in activin receptor-like kinase 1 among Danish patients with hereditary haemorrhagic telangiectasia

    DEFF Research Database (Denmark)

    Brusgaard, K; Kjeldsen, A D; Poulsen, L

    2004-01-01

    , a presumed spontaneous mutation was characterized. The method developed proved to be very sensitive for mutation detection in both ENG and ALK1. Genetic screening in HHT families facilitates an early treatment strategy for silent HHT manifestations in first degree relatives....... protocol for mutation scanning of the two loci. Twenty-five Danish HHT families were tested. A total of eight new as well as seven previously reported mutations were identified. A founder mutation was characterized present in seven families and possibly introduced around 350 years ago. In one individual...

  7. 激活素受体样激酶1促进人脐静脉内皮细胞增殖和迁徙%The activin receptor-like kinase I promotes proliferation and migration in HUVECs

    Institute of Scientific and Technical Information of China (English)

    李斌; 唐仕波; 林少芬; 孟晶

    2006-01-01

    目的:研究激活素受体样激酶1(ALK1)对人脐静脉内皮细胞的作用.方法:体外培养人脐静脉内皮细胞(HUVECs),RT-PCR分析ALK1和ALK5在HUVEC激活状态下表达的变化.脂质体转染pcDNA3.1+ALK1到HUVECs,流式细胞仪检测HUVECs增殖的改变,boyden小室检测ALK1对HUVECs迁徙的影响.结果:ALK1在HUVEC安静状态高表达,ALK1能促进HUVECs的增殖和迁徙.结论:ALK1通过促进内皮细胞的增殖和迁徙在血管重塑中发挥作用.

  8. Activin receptor-like kinase receptors ALK5 and ALK1 are both required for TGFβ-induced chondrogenic differentiation of human bone marrow-derived mesenchymal stem cells

    NARCIS (Netherlands)

    L.M.G. De Kroon (Laurie); R. Narcisi (Roberto); E.N. Blaney Davidson (Esmeralda); M.A. Cleary (Mairéad); H.M. van Beuningen (Henk); W.J.L.M. Koevoet (Wendy J.L.M.); G.J.V.M. van Osch (Gerjo); P.M. van der Kraan (Peter)

    2015-01-01

    textabstractIntroduction Bone marrow-derived mesenchymal stem cells (BMSCs) are promising for cartilage regeneration because BMSCs can differentiate into cartilage tissue-producing chondrocytes. Transforming Growth Factor beta; (TGFbeta;) is crucial for inducing chondrogenic differentiation of BMSCs

  9. Experiment list: SRX833419 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available 874189,88.6,9.7,10641 GSM1579362: Total-RNAPII Activin; Homo sapiens; ChIP-Seq source_name=H1 human ESCs || ...cell type=embryonic stem cells || passages/media=40-50/mTeSR || treatment=Activin A 4h || antibody=Total RNA

  10. Keratinocyte-derived follistatin regulates epidermal homeostasis and wound repair

    Science.gov (United States)

    Antsiferova, Maria; Klatte, Jennifer E; Bodó, Enikö; Paus, Ralf; Jorcano, José L; Matzuk, Martin M; Werner, Sabine; Kögel, Heidi

    2014-01-01

    Activin is a growth and differentiation factor that controls development and repair of several tissues and organs. Transgenic mice overexpressing activin in the skin were characterized by strongly enhanced wound healing, but also by excessive scarring. In this study, we explored the consequences of targeted activation of activin in the epidermis and hair follicles by generation of mice lacking the activin antagonist follistatin in keratinocytes. We observed enhanced keratinocyte proliferation in the tail epidermis of these animals. After skin injury, an earlier onset of keratinocyte hyperproliferation at the wound edge was observed in the mutant mice, resulting in an enlarged hyperproliferative epithelium. However, granulation tissue formation and scarring were not affected. These results demonstrate that selective activation of activin in the epidermis enhances reepithelialization without affecting the quality of the healed wound. PMID:19079322

  11. Regulatory roles of bone morphogenetic proteins and glucocorticoids in catecholamine production by rat pheochromocytoma cells.

    Science.gov (United States)

    Kano, Yoshihiro; Otsuka, Fumio; Takeda, Masaya; Suzuki, Jiro; Inagaki, Kenichi; Miyoshi, Tomoko; Miyamoto, Manabu; Otani, Hiroyuki; Ogura, Toshio; Makino, Hirofumi

    2005-12-01

    We here report a new physiological system that governs catecholamine synthesis involving bone morphogenetic proteins (BMPs) and activin in the rat pheochromocytoma cell line, PC12. BMP type I receptors, including activin receptor-like kinase-2 (ALK-2) (also referred to as ActRIA) and ALK-3 (BMPRIA), both type II receptors, ActRII and BMPRII, as well as the ligands BMP-2, -4, and -7 and inhibin/activin subunits were expressed in PC12 cells. PC12 cells predominantly secrete dopamine, whereas noradrenaline and adrenaline production is negligible. BMP-2, -4, -6, and -7 and activin A each suppressed dopamine and cAMP synthesis in a dose-dependent fashion. The BMP ligands also decreased 3,4-dihydroxyphenylalanine decarboxylase mRNA expression, whereas activin suppressed tyrosine hydroxylase expression. BMPs induced both Smad1/5/8 phosphorylation and Tlx2-Luc activation, whereas activin stimulated 3TP-Luc activity and p38 MAPK phosphorylation. ERK signaling was not affected by BMPs or activin. Dexamethasone enhanced catecholamine synthesis, accompanying increases in tyrosine hydroxylase and 3,4-dihydroxyphenylalanine decarboxylase transcription without cAMP accumulation. In the presence of dexamethasone, BMPs and activin failed to reduce dopamine as well as cAMP production. In addition, dexamethasone modulated mitotic suppression of PC12 induced by BMPs in a ligand-dependent manner. Furthermore, intracellular BMP signaling was markedly suppressed by dexamethasone treatment and the expression of ALK-2, ALK-3, and BMPRII was significantly inhibited by dexamethasone. Collectively, the endogenous BMP/activin system plays a key role in the regulation of catecholamine production. Controlling activity of the BMP system may be critical for glucocorticoid-induced catecholamine synthesis by adrenomedullar cells.

  12. Microarrays--analysis of signaling pathways.

    Science.gov (United States)

    Ramachandran, Anassuya; Black, Michael A; Shelling, Andrew N; Love, Donald R

    2008-01-01

    Microarrays provide a powerful means of analyzing the expression level of multiple transcripts in two sample populations. In this study, we have used microarray technology to identify genes that are differentially regulated in response to activin-treated ovarian cancer cells. We find a number of biologically relevant genes that are involved in regulating activin signaling and genes potentially contributing to activin-mediated growth arrest appear to be differentially regulated. Thus, microarrays are an important tool for dissecting gene expression changes in normal physiological processes and disease.

  13. Lefty blocks a subset of TGFbeta signals by antagonizing EGF-CFC coreceptors.

    Directory of Open Access Journals (Sweden)

    Simon K Cheng

    2004-02-01

    Full Text Available Members of the EGF-CFC family play essential roles in embryonic development and have been implicated in tumorigenesis. The TGFbeta signals Nodal and Vg1/GDF1, but not Activin, require EGF-CFC coreceptors to activate Activin receptors. We report that the TGFbeta signaling antagonist Lefty also acts through an EGF-CFC-dependent mechanism. Lefty inhibits Nodal and Vg1 signaling, but not Activin signaling. Lefty genetically interacts with EGF-CFC proteins and competes with Nodal for binding to these coreceptors. Chimeras between Activin and Nodal or Vg1 identify a 14 amino acid region that confers independence from EGF-CFC coreceptors and resistance to Lefty. These results indicate that coreceptors are targets for both TGFbeta agonists and antagonists and suggest that subtle sequence variations in TGFbeta signals result in greater ligand diversity.

  14. Experiment list: SRX833418 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available 706512,85.5,6.3,8565 GSM1579361: Total-RNAPII Wnt3a+Activin; Homo sapiens; ChIP-Seq source_name=H1 human ESC...s || cell type=embryonic stem cells || passages/media=40-50/mTeSR || treatment=Wnt3a+Activin A (WA) 4h || antibody=Total

  15. Effect of Activin A-follistatin on steroidogenesis in frozen-thawed human fetal ovary in vitro%激活素A和卵泡抑素对冻融人胎儿卵巢雌二醇分泌的影响

    Institute of Scientific and Technical Information of China (English)

    池海虹; 周颖; 祝怀平; 金畅; 叶碧绿

    2008-01-01

    目的:研究组织培养条件下冻融人胎儿卵巢雌二醇(E2)的分泌及激活素A(Act A)和卵泡抑素(FS)对E分泌的影响.方法:冻融人胎儿卵巢组织体外培养,分别加入基础培养液(对照组,即MEM组),基础培养液+100 ng/ml Act A(Act A组)、基础培养液+100 ng/ml Fs(Fs组)和基础培养液+100 ng/mlAct A+100 ng/ml FS(A+F组),每组4块组织,培养8 d.收集培养2 d、4 d、6 d、8 d各组培养液,测定比较其E,值.结果:培养4 d、6 d,8 d卵巢组织分泌的E.值均高于2 d的E,值(均尸<0.05),随培养时间延长,E.分泌量先增加后减少,在培养后6 d可达较高分泌水平;Act A组E2分泌量明显高于对照组、Fs组和A+F组(均尸<0.01).结论:冻融人胎儿卵巢在组织培养条件下能分泌E2,Act A能促进体外培养胎儿卵巢E2的分泌,其作用可能受到FS的调节.

  16. Targeting senescent cells enhances adipogenesis and metabolic function in old age.

    Science.gov (United States)

    Xu, Ming; Palmer, Allyson K; Ding, Husheng; Weivoda, Megan M; Pirtskhalava, Tamar; White, Thomas A; Sepe, Anna; Johnson, Kurt O; Stout, Michael B; Giorgadze, Nino; Jensen, Michael D; LeBrasseur, Nathan K; Tchkonia, Tamar; Kirkland, James L

    2015-12-19

    Senescent cells accumulate in fat with aging. We previously found genetic clearance of senescent cells from progeroid INK-ATTAC mice prevents lipodystrophy. Here we show that primary human senescent fat progenitors secrete activin A and directly inhibit adipogenesis in non-senescent progenitors. Blocking activin A partially restored lipid accumulation and expression of key adipogenic markers in differentiating progenitors exposed to senescent cells. Mouse fat tissue activin A increased with aging. Clearing senescent cells from 18-month-old naturally-aged INK-ATTAC mice reduced circulating activin A, blunted fat loss, and enhanced adipogenic transcription factor expression within 3 weeks. JAK inhibitor suppressed senescent cell activin A production and blunted senescent cell-mediated inhibition of adipogenesis. Eight weeks-treatment with ruxolitinib, an FDA-approved JAK1/2 inhibitor, reduced circulating activin A, preserved fat mass, reduced lipotoxicity, and increased insulin sensitivity in 22-month-old mice. Our study indicates targeting senescent cells or their products may alleviate age-related dysfunction of progenitors, adipose tissue, and metabolism.

  17. Genetic evidence that SMAD2 is not required for gonadal tumor development in inhibin-deficient mice

    Directory of Open Access Journals (Sweden)

    Weinstein Michael B

    2010-06-01

    Full Text Available Abstract Background Inhibin is a tumor-suppressor and activin antagonist. Inhibin-deficient mice develop gonadal tumors and a cachexia wasting syndrome due to enhanced activin signaling. Because activins signal through SMAD2 and SMAD3 in vitro and loss of SMAD3 attenuates ovarian tumor development in inhibin-deficient females, we sought to determine the role of SMAD2 in the development of ovarian tumors originating from the granulosa cell lineage. Methods Using an inhibin α null mouse model and a conditional knockout strategy, double conditional knockout mice of Smad2 and inhibin alpha were generated in the current study. The survival rate and development of gonadal tumors and the accompanying cachexia wasting syndrome were monitored. Results Nearly identical to the controls, the Smad2 and inhibin alpha double knockout mice succumbed to weight loss, aggressive tumor progression, and death. Furthermore, elevated activin levels and activin-induced pathologies in the liver and stomach characteristic of inhibin deficiency were also observed in these mice. Our results indicate that SMAD2 ablation does not protect inhibin-deficient females from the development of ovarian tumors or the cachexia wasting syndrome. Conclusions SMAD2 is not required for mediating tumorigenic signals of activin in ovarian tumor development caused by loss of inhibin.

  18. Efficient programming of human eye conjunctiva-derived induced pluripotent stem (ECiPS) cells into definitive endoderm-like cells.

    Science.gov (United States)

    Massumi, Mohammad; Hoveizi, Elham; Baktash, Parvaneh; Hooti, Abdollah; Ghazizadeh, Leili; Nadri, Samad; Pourasgari, Farzaneh; Hajarizadeh, Athena; Soleimani, Masoud; Nabiuni, Mohammad; Khorramizadeh, Mohammad R

    2014-03-10

    Due to pluripotency of induced pluripotent stem (iPS) cells, and the lack of immunological incompatibility and ethical issues, iPS cells have been considered as an invaluable cell source for future cell replacement therapy. This study was aimed first at establishment of novel iPS cells, ECiPS, which directly reprogrammed from human Eye Conjunctiva-derived Mesenchymal Stem Cells (EC-MSCs); second, comparing the inductive effects of Wnt3a/Activin A biomolecules to IDE1 small molecule in derivation of definitive endoderm (DE) from the ECiPS cells. To that end, first, the EC-MSCs were transduced by SOKM-expressing lentiviruses and characterized for endogenous expression of embryonic markers Then the established ECiPS cells were induced to DE formation by Wnt3a/Activin A or IDE1. Quantification of GSC, Sox17 and Foxa2 expression, as DE-specific markers, in both mRNA and protein levels revealed that induction of ECiPS cells by either Wnt3a/Activin A or IDE1 could enhance the expression level of the genes; however the levels of increase were higher in Wnt3a/Activin A induced ECiPS-EBs than IDE1 induced cells. Furthermore, the flow cytometry analyses showed no synergistic effect between Activin A and Wnt3a to derive DE-like cells from ECiPS cells. The comparative findings suggest that although both Wnt3a/Activin A signaling and IDE1 molecule could be used for differentiation of iPS into DE cells, the DE-inducing effect of Wnt3a/Activin A was statistically higher than IDE1.

  19. Glyphosate Vedotin for Treatment of Bone Metastatic Castration-Resistant Prostate Cancer

    Science.gov (United States)

    2015-07-01

    15, demonstrating PSA production via stimulation with DHT and dihydroxyepiandosterone at passage 5 but moving on to androgen insensitivity at passage...10, follistatin, activin-A, and DHT . Int J Oncol 14: 1185-1195. PMID: 10339677 90. Dunning WF (1963) Prostate Cancer in the Rat. Natl Cancer Inst

  20. Stress signaling from human mammary epithelial cells contributes to phenotypes of mammographic density.

    Science.gov (United States)

    DeFilippis, Rosa Anna; Fordyce, Colleen; Patten, Kelley; Chang, Hang; Zhao, Jianxin; Fontenay, Gerald V; Kerlikowske, Karla; Parvin, Bahram; Tlsty, Thea D

    2014-09-15

    Telomere malfunction and other types of DNA damage induce an activin A-dependent stress response in mortal nontumorigenic human mammary epithelial cells that subsequently induces desmoplastic-like phenotypes in neighboring fibroblasts. Some characteristics of this fibroblast/stromal response, such as reduced adipocytes and increased extracellular matrix content, are observed not only in tumor tissues but also in disease-free breast tissues at high risk for developing cancer, especially high mammographic density tissues. We found that these phenotypes are induced by repression of the fatty acid translocase CD36, which is seen in desmoplastic and disease-free high mammographic density tissues. In this study, we show that epithelial cells from high mammographic density tissues have more DNA damage signaling, shorter telomeres, increased activin A secretion and an altered DNA damage response compared with epithelial cells from low mammographic density tissues. Strikingly, both telomere malfunction and activin A expression in epithelial cells can repress CD36 expression in adjacent fibroblasts. These results provide new insights into how high mammographic density arises and why it is associated with breast cancer risk, with implications for the definition of novel invention targets (e.g., activin A and CD36) to prevent breast cancer.

  1. Partial promoter substitutions generating transcriptional sentinels of diverse signaling pathways in embryonic stem cells and mice

    DEFF Research Database (Denmark)

    Serup, Palle; Gustavsen, Carsten; Klein, Tino

    2012-01-01

    extracellular signals. We thereby created an allelic series of embryonic stem cells and mice, each containing a signal-responsive sentinel with different fluorescent reporters that respond with sensitivity and specificity to retinoic acids, bone morphogenic proteins, activin A, Wnts or Notch, and that can...

  2. A novel member of the transmembrane serine/threonine kinase receptor family is specifically expressed in the gonads and in mesenchymal cells adjacent to the mullerian duct

    NARCIS (Netherlands)

    W.M. Baarends (Willy); M.J. van Helmond (Marjolein); M. Verhoef-Post (Miriam); P.J.C.M. van der Schoot; J.W. Hoogerbrugge (Jos); J.P. de Winter (Johan); J.Th.J. Uilenbroek (Jan); B. Karels (Bas)

    1994-01-01

    textabstractThe activin and TGF-beta type II receptors are members of a separate subfamily of transmembrane receptors with intrinsic protein kinase activity, which also includes the recently cloned TGF-beta type I receptor. We have isolated and characterized a cDNA clon

  3. Involvement of Ovarian Estradiol Biosynthesis and Pituitary FSH Expression in the Mechanism of Human Chorionic Gonadotropin Stimulation of Uterine Growth in Immature Female Rats

    Directory of Open Access Journals (Sweden)

    Sandrine Rafert

    2016-01-01

    Full Text Available In a previous work, we showed that human Chorionic Gonadotropin (hCG alone is able to stimulate uterine ponderal growth in immature female rats. In the present paper, we provide new information indicating that a single 100 ng hCG injection increased ovarian androgen availability and aromatase activity. These changes are consistent with an increase in ovarian estradiol secretion. Ovarian activin βA and activin βB subunit RNAs expression were also increased following hCG injection. Most interestingly, this treatment also led to an increase in FSHβ subunit mRNA expression in the pituitary, which might be due to hCG-stimulated ovarian activin secretion. We have not been able so far to follow the kinetics of the plasma concentrations of activin and FSH in hCG-treated animals compared to control animals. This has to be explored in the future to provide a more complete assessment of our model.

  4. Partial promoter substitutions generating transcriptional sentinels of diverse signaling pathways in embryonic stem cells and mice

    DEFF Research Database (Denmark)

    Serup, Palle; Gustavsen, Carsten; Klein, Tino;

    2012-01-01

    extracellular signals. We thereby created an allelic series of embryonic stem cells and mice, each containing a signal-responsive sentinel with different fluorescent reporters that respond with sensitivity and specificity to retinoic acids, bone morphogenic proteins, activin A, Wnts or Notch, and that can...

  5. Isolation and differentiation of chondrocytic cells derived from human embryonic stem cells using dlk1/FA1 as a novel surface marker

    DEFF Research Database (Denmark)

    Harkness, Linda; Taipaleenmaki, Hanna; Mahmood, Amer

    2009-01-01

    was not expressed by undifferentiated hESC, but expressed during in vitro embryoid bodies (hEBs) formation upon down-regulation of undifferentiated markers e.g. Oct 3/4. Similarly, dlk1/FA1 was expressed in chondrocytic cells during in vivo teratoma formation. Interestingly, treatment of hEBs with Activin B...

  6. A Simplified Method for Three-Dimensional (3-D Ovarian Tissue Culture Yielding Oocytes Competent to Produce Full-Term Offspring in Mice.

    Directory of Open Access Journals (Sweden)

    Carolyn M Higuchi

    Full Text Available In vitro growth of follicles is a promising technology to generate large quantities of competent oocytes from immature follicles and could expand the potential of assisted reproductive technologies (ART. Isolated follicle culture is currently the primary method used to develop and mature follicles in vitro. However, this procedure typically requires complicated, time-consuming procedures, as well as destruction of the normal ovarian microenvironment. Here we describe a simplified 3-D ovarian culture system that can be used to mature multilayered secondary follicles into antral follicles, generating developmentally competent oocytes in vitro. Ovaries recovered from mice at 14 days of age were cut into 8 pieces and placed onto a thick Matrigel drop (3-D culture for 10 days of culture. As a control, ovarian pieces were cultured on a membrane filter without any Matrigel drop (Membrane culture. We also evaluated the effect of activin A treatment on follicle growth within the ovarian pieces with or without Matrigel support. Thus we tested four different culture conditions: C (Membrane/activin-, A (Membrane/activin+, M (Matrigel/activin-, and M+A (Matrigel/activin+. We found that the cultured follicles and oocytes steadily increased in size regardless of the culture condition used. However, antral cavity formation occurred only in the follicles grown in the 3-D culture system (M, M+A. Following ovarian tissue culture, full-grown GV oocytes were isolated from the larger follicles to evaluate their developmental competence by subjecting them to in vitro maturation (IVM and in vitro fertilization (IVF. Maturation and fertilization rates were higher using oocytes grown in 3-D culture (M, M+A than with those grown in membrane culture (C, A. In particular, activin A treatment further improved 3-D culture (M+A success. Following IVF, two-cell embryos were transferred to recipients to generate full-term offspring. In summary, this simple and easy 3-D ovarian

  7. The nephrogenic potential of the transcription factors osr1, osr2, hnf1b, lhx1 and pax8 assessed in Xenopus animal caps

    Directory of Open Access Journals (Sweden)

    Ryffel Gerhart U

    2011-01-01

    Full Text Available Abstract Background The three distinct types of kidneys, pronephros, mesonephros and metanephros, develop consecutively in vertebrates. The earliest form of embryonic kidney, the pronephros, is derived from intermediate mesoderm and the first expressed genes localized in the pronephros anlage are the transcription factors osr1, osr2, hnf1b, lhx1 and pax8, here referred to as the early nephrogenic transcription factors. However, the pathway inducing nephrogenesis and the network of theses factors are poorly understood. Treatment of the undifferentiated animal pole explant (animal cap of Xenopus with activin A and retinoic acid induces pronephros formation providing a powerful tool to analyze key molecular events in nephrogenesis. Results We have investigated the expression kinetics of the early nephrogenic transcription factors in activin A and retinoic acid treated animal caps and their potential to induce pronephric differentiation. In treated animal caps, expression of osr1, osr2, hnf1b and lhx1 are induced early, whereas pax8 expression occurs later implying an indirect activation. Activin A alone is able to induce osr2 and lhx1 after three hours treatment in animal caps while retinoic acid fails to induce any of these nephrogenic transcription factors. The early expression of the five transcription factors and their interference with pronephros development when overexpressed in embryos suggest that these factors potentially induce nephrogenesis upon expression in animal caps. But no pronephros development is achieved by either overexpression of OSR1, by HNF1B injection with activin A treatment, or the combined application of LHX1 and PAX8, although they influenced the expression of several early nephrogenic transcription factors in some cases. In an additional approach we could show that HNF1B induces several genes important in nephrogenesis and regulates lhx1 expression by an HNF1 binding site in the lhx1 promoter. Conclusions The early

  8. The chronic kidney disease - Mineral bone disorder (CKD-MBD): Advances in pathophysiology.

    Science.gov (United States)

    Hruska, Keith A; Sugatani, Toshifumi; Agapova, Olga; Fang, Yifu

    2017-01-21

    The causes of excess cardiovascular mortality associated with chronic kidney disease (CKD) have been attributed in part to the CKD-mineral bone disorder syndrome (CKD-MBD), wherein, novel cardiovascular risk factors have been identified. New advances in the causes of the CKD-MBD are discussed in this review. They demonstrate that repair and disease processes in the kidneys release factors to the circulation that cause the systemic complications of CKD. The discovery of WNT inhibitors, especially Dickkopf 1 (Dkk1), produced during renal repair as participating in the pathogenesis of the vascular and skeletal components of the CKD-MBD implied that additional pathogenic factors are critical. This lead to the discovery that activin A is a second renal repair factor circulating in increased levels during CKD. Activin A derives from peritubular myofibroblasts of diseased kidneys, wherein it stimulates fibrosis, and decreases tubular klotho expression. Activin A binds to the type 2 activin A receptor, ActRIIA, which is variably affected by CKD in the vasculature. In diabetic/atherosclerotic aortas, specifically in vascular smooth muscle cells (VSMC), ActRIIA signaling is inhibited and contributes to CKD induced VSMC dedifferentiation, osteogenic transition and neointimal atherosclerotic calcification. In nondiabetic/nonatherosclerotic aortas, CKD increases VSMC ActRIIA signaling, and vascular fibroblast signaling causing the latter to undergo osteogenic transition and stimulate vascular calcification. In both vascular situations, a ligand trap for ActRIIA prevented vascular calcification. In the skeleton, activin A is responsible for CKD stimulation of osteoclastogenesis and bone remodeling increasing bone turnover. These studies demonstrate that circulating renal repair and injury factors are causal of the CKD-MBD and CKD associated cardiovascular disease.

  9. New insights into implication of the SLIT/ROBO pathway in the prehierarchical follicle development of hen ovary.

    Science.gov (United States)

    Qin, N; Fan, X C; Zhang, Y Y; Xu, X X; Tyasi, T L; Jing, Y; Mu, F; Wei, M L; Xu, R F

    2015-09-01

    The SLIT/Roundabout (ROBO) pathway is involved in follicle development of mammalian ovary, and 2 secreted hormones activin A and inhibin A have potential roles in modulation of the SLIT/ROBO system, but the related actions remain poorly understood in bird. The aims of the present study were to examine the spatial and temporal expression of the SLIT ligand genes (SLIT1, SLIT2, and SLIT3) and their receptor ROBO1, ROBO2, ROBO3, and ROBO4 genes in various-sized prehierarchical follicles during hen ovary development and the effects of activin A and inhibin A on the expression of these genes in the cultured hen follicles. Our result demonstrated that the transcripts of the 3 SLIT genes were highly expressed in the developing follicles and expression patterns of the SLIT transcripts were different from those of ROBO genes detected by real-time quantitative reverse transcriptase PCR. Both SLIT and ROBO transcripts were predominantly expressed in oocytes and granulosa cells from the prehierarchichal follicles examined by in situ hybridization. The localization for SLIT and ROBO proteins was revealed by immunohistochemistry similar to the spatial distribution of their transcript. In cultured follicles (4 to 8 mm in diameter), the expression levels of SLIT and ROBO members are hormonally regulated by activin A (10 ng/mL) and/or inhibin A (20 ng/mL) after treatment for 24 h. However, the expression of only SLIT2, SLIT3, and ROBO3 mRNA presented a directly opposite response to activin A and inhibin A hormones. These results indicate that SLIT/ROBO pathway is implicated in the prehierarchical follicular development of the hen ovary by an intrafollicular autocrine and/or paracrine action, and is influenced by activin A and inhibin A hormones.

  10. Induction of dorsal mesoderm by soluble, mature Vg1 protein.

    Science.gov (United States)

    Kessler, D S; Melton, D A

    1995-07-01

    Mesoderm induction during Xenopus development has been extensively studied, and two members of the transforming growth factor-beta family, activin beta B and Vg1, have emerged as candidates for a natural inducer of dorsal mesoderm. Heretofore, analysis of Vg1 activity has relied on injection of hybrid Vg1 mRNAs, which have not been shown to direct efficient secretion of ligand and, therefore, the mechanism of mesoderm induction by processed Vg1 protein is unclear. This report describes injection of Xenopus oocytes with a chimeric activin-Vg1 mRNA, encoding the pro-region of activin beta B fused to the mature region of Vg1, resulting in the processing and secretion of mature Vg1. Treatment of animal pole explants with mature Vg1 protein resulted in differentiation of dorsal, but not ventral, mesodermal tissues and dose-dependent activation of both dorsal and ventrolateral mesodermal markers. At high doses, mature Vg1 induced formation of 'embryoids' with a rudimentary axial pattern, head structures including eyes and a functional neuromuscular system. Furthermore, truncated forms of the activin and FGF receptors, which block mesoderm induction in the intact embryo, fully inhibited mature Vg1 activity. To examine the mechanism of inhibition, we have performed receptor-binding assays with radiolabeled Vg1. Finally, follistatin, a specific inhibitor of activin beta B which is shown not to block endogenous dorsal mesoderm induction, failed to inhibit Vg1. The results support a role for endogenous Vg1 in dorsal mesoderm induction during Xenopus development.

  11. Sodium butyrate and dexamethasone promote exocrine pancreatic gene expression in mouse embryonic stem cells

    Institute of Scientific and Technical Information of China (English)

    Meng REN; Li YAN; Chang-zhen SHANG; Jun CAO; Fang-ping LI; Jingyi LI; Hua CHENG; Jun MIN

    2009-01-01

    Aim: The feasibility of inducing endocrine pancreatic differentiation of embryonic stem (ES) cells has been well documented. How-ever, whether ES cells possess the potential for exocrine pancreatic differentiation requires further exploration. Here, we investigated whether sodium butyrate and glucocorticoids were conducive to the exocrine pancreatic differentiation of ES cells. Methods: E14 mouse ES cells were cultured in suspension to form embryoid bodies (EBs). These EBs were cultured in differentiating medium containing varying concentrations of sodium butyrate. The effects of activinA and dexamethasone (Dex) on exocrine differen-tiation were also explored. Finally, the combination of sodium butyrate, activinA, and Dex was used to promote the differentiation of exocrine pancreatic cells. Specific exocrine pancreatic gene expression was detected by reverse transcription polymerase chain reac-tion (RT-PCR) and amylase expression was examined by immunofluorescence staining. Flow cytometry analysis was also performed to determine the percentage of amylase-positive cells after the treatment with activinA, sodium butyrate, and Dex. Results: Exposure of ES cells to 1 mmol/L sodium butyrate for 5 days promoted exocrine pancreatic gene expression. Further combi-nation with Dex and other pancreatic-inducing factors, such as activinA, significantly enhanced the mRNA and protein levels of exocrine pancreatic markers. Additionally, flow cytometry revealed that approximately 17% of the final differentiated cells were amylase-positive. Conclusion: These data indicate that the exocrine pancreatic differentiation of ES cells can be induced by activinA, sodium butyrate, and Dex, providing a potential tool for studying pancreatic differentiation and pancreas-related diseases.

  12. Analysis of C-cadherin regulation during tissue morphogenesis with an activating antibody.

    Science.gov (United States)

    Zhong, Y; Brieher, W M; Gumbiner, B M

    1999-01-25

    The regulation of cadherin-mediated adhesion at the cell surface underlies several morphogenetic processes. To investigate the role of cadherin regulation in morphogenesis and to begin to analyze the molecular mechanisms of cadherin regulation, we have screened for monoclonal antibodies (mAbs) that allow us to manipulate the adhesive state of the cadherin molecule. Xenopus C-cadherin is regulated during convergent extension movements of gastrulation. Treatment of animal pole tissue explants (animal caps) with the mesoderm-inducing factor activin induces tissue elongation and decreases the strength of C-cadherin-mediated adhesion between blastomeres (Brieher, W.M., and B.M. Gumbiner. 1994. J. Cell Biol. 126:519-527). We have generated a mAb to C-cadherin, AA5, that restores strong adhesion to activin-treated blastomeres. This C-cadherin activating antibody strongly inhibits the elongation of animal caps in response to activin without affecting mesodermal gene expression. Thus, the activin-induced decrease in C-cadherin adhesive activity appears to be required for animal cap elongation. Regulation of C-cadherin and its activation by mAb AA5 involve changes in the state of C-cadherin that encompass more than changes in its homophilic binding site. Although mAb AA5 elicited a small enhancement in the functional activity of the soluble C-cadherin ectodomain (CEC1-5), it was not able to restore cell adhesion activity to mutant C-cadherin lacking its cytoplasmic tail. Furthermore, activin treatment regulates the adhesion of Xenopus blastomeres to surfaces coated with two other anti-C-cadherin mAbs, even though these antibodies probably do not mediate adhesion through a normal homophilic binding mechanism. Moreover, mAb AA5 restores strong adhesion to these antibodies. mAb AA5 only activates adhesion of blastomeres to immobilized CEC1-5 when it binds to C-cadherin on the cell surface. It does not work when added to CEC1-5 on the substrate. Together these findings suggest

  13. Differential expression of follistatin and FLRG in human breast proliferative disorders

    Directory of Open Access Journals (Sweden)

    Amaral Vania F

    2009-09-01

    Full Text Available Abstract Background Activins are growth factors acting on cell growth and differentiation. Activins are expressed in high grade breast tumors and they display an antiproliferative effect inducing G0/G1 cell cycle arrest in breast cancer cell lines. Follistatin and follistatin- related gene (FLRG bind and neutralize activins. In order to establish if these activin binding proteins are involved in breast tumor progression, the present study evaluated follistatin and FLRG pattern of mRNA and protein expression in normal human breast tissue and in different breast proliferative diseases. Methods Paraffin embedded specimens of normal breast (NB - n = 8; florid hyperplasia without atypia (FH - n = 17; fibroadenoma (FIB - n = 17; ductal carcinoma in situ (DCIS - n = 10 and infiltrating ductal carcinoma (IDC - n = 15 were processed for follistatin and FLRG immunohistochemistry and in situ hybridization. The area and intensity of chromogen epithelial and stromal staining were analyzed semi-quantitatively. Results Follistatin and FLRG were expressed both in normal tissue and in all the breast diseases investigated. Follistatin staining was detected in the epithelial cytoplasm and nucleus in normal, benign and malignant breast tissue, with a stronger staining intensity in the peri-alveolar stromal cells of FIB at both mRNA and protein levels. Conversely, FLRG area and intensity of mRNA and protein staining were higher both in the cytoplasm and in the nucleus of IDC epithelial cells when compared to NB, while no significant changes in the stromal intensity were observed in all the proliferative diseases analyzed. Conclusion The present findings suggest a role for follistatin in breast benign disease, particularly in FIB, where its expression was increased in stromal cells. The up regulation of FLRG in IDC suggests a role for this protein in the progression of breast malignancy. As activin displays an anti-proliferative effect in human mammary cells, the

  14. Cell-extrinsic consequences of epithelial stress: activation of protumorigenic tissue phenotypes.

    Science.gov (United States)

    Fordyce, Colleen A; Patten, Kelley T; Fessenden, Tim B; DeFilippis, RosaAnna; Hwang, E Shelley; Zhao, Jianxin; Tlsty, Thea D

    2012-12-07

    Tumors are characterized by alterations in the epithelial and stromal compartments, which both contribute to tumor promotion. However, where, when, and how the tumor stroma develops is still poorly understood. We previously demonstrated that DNA damage or telomere malfunction induces an activin A-dependent epithelial stress response that activates cell-intrinsic and cell-extrinsic consequences in mortal, nontumorigenic human mammary epithelial cells (HMECs and vHMECs). Here we show that this epithelial stress response also induces protumorigenic phenotypes in neighboring primary fibroblasts, recapitulating many of the characteristics associated with formation of the tumor stroma (for example, desmoplasia). The contribution of extrinsic and intrinsic DNA damage to acquisition of desmoplastic phenotypes was investigated in primary human mammary fibroblasts (HMFs) co-cultured with vHMECs with telomere malfunction (TRF2-vHMEC) or in HMFs directly treated with DNA-damaging agents, respectively. Fibroblast reprogramming was assessed by monitoring increases in levels of selected protumorigenic molecules with quantitative polymerase chain reaction, enzyme-linked immunosorbent assay, and immunocytochemistry. Dependence of the induced phenotypes on activin A was evaluated by addition of exogenous activin A or activin A silencing. In vitro findings were validated in vivo, in preinvasive ductal carcinoma in situ (DCIS) lesions by using immunohistochemistry and telomere-specific fluorescent in situ hybridization. HMFs either cocultured with TRF2-vHMEC or directly exposed to exogenous activin A or PGE2 show increased expression of cytokines and growth factors, deposition of extracellular matrix (ECM) proteins, and a shift toward aerobic glycolysis. In turn, these "activated" fibroblasts secrete factors that promote epithelial cell motility. Interestingly, cell-intrinsic DNA damage in HMFs induces some, but not all, of the molecules induced as a consequence of cell-extrinsic DNA

  15. EVEN-SKIPPED HOMEOBOX 1 controls human ES cell differentiation by directly repressing GOOSECOID expression

    DEFF Research Database (Denmark)

    Kalisz, Mark; Winzi, Maria Karin; Bisgaard, Hanne Cathrine;

    2012-01-01

    TGFß signaling patterns the primitive streak, yet little is known about transcriptional effectors that mediate the cell fate choices during streak-like development in mammalian embryos and in embryonic stem (ES) cells. Here we demonstrate that cross-antagonistic actions of EVEN-SKIPPED HOMEOBOX 1...... (EVX1) and GOOSECOID (GSC) regulate cell fate decisions in streak-like progenitors derived from human ES cells exposed to BMP4 and/or activin. We found that EVX1 repressed GSC expression and promoted formation of posterior streak-like progeny in response to BMP4, and conversely that GSC repressed EVX1...... expression and was required for development of anterior streak-like progeny in response to activin. Chromatin immunoprecipitation assays showed that EVX1 bound to the GSC 5'-flanking region in BMP4 treated human ES cells, and band shift assays identified two EVX1 binding sites in the GSC 5'-region...

  16. Cripto/GRP78 modulation of the TGF-β pathway in development and oncogenesis

    Science.gov (United States)

    Gray, Peter C.; Vale, Wylie

    2013-01-01

    Cripto is a small, GPI-anchored signaling protein that regulates cellular survival, proliferation, differentiation and migration during normal developmental processes and tumorigenesis. Cripto functions as an obligatory co-receptor for the TGF-β ligands Nodal, GDF1 and GDF3 but attenuates signaling of others such as activin-A, activin-B and TGF-β1. Soluble, secreted forms of Cripto also activate Src, ras/raf/MAPK and PI3K/Akt pathways via a mechanism that remains largely obscure. This review describes the biological roles and signaling mechanisms of Cripto, highlighting our identification of Glucose Regulated Protein 78 (GRP78) as a cell surface receptor/co-factor required for Cripto signaling via both TGF-β and Src/MAPK/PI3K pathways. We discuss emerging evidence indicating that Cripto/GRP78 signaling regulates normal somatic stem cells and their tumorigenic counterparts. PMID:22306319

  17. Distinct gene expression signatures in human embryonic stem cells differentiated towards definitive endoderm at single-cell level

    DEFF Research Database (Denmark)

    Norrman, Karin; Strömbeck, Anna; Semb, Henrik

    2013-01-01

    of anterior definitive endoderm (DE). Here, we differentiated human embryonic stem cells towards DE using three different activin A based treatments. Differentiation efficiencies were evaluated by gene expression profiling over time at cell population level. A panel of key markers was used to study DE...... for the three activin A based protocols applied. Our data provide novel insights in DE gene expression at the cellular level of in vitro differentiated human embryonic stem cells, and illustrate the power of using single-cell gene expression profiling to study differentiation heterogeneity and to characterize......Characterization of directed differentiation of pluripotent stem cells towards therapeutically relevant cell types, including pancreatic beta-cells and hepatocytes, depends on molecular markers and assays that resolve the signature of individual cells. Pancreas and liver both have a common origin...

  18. Molecular Pathways: Cachexia Signaling-A Targeted Approach to Cancer Treatment.

    Science.gov (United States)

    Miyamoto, Yuji; Hanna, Diana L; Zhang, Wu; Baba, Hideo; Lenz, Heinz-Josef

    2016-08-15

    Cancer cachexia is a multifactorial syndrome characterized by an ongoing loss of skeletal muscle mass, which negatively affects quality of life and portends a poor prognosis. Numerous molecular substrates and mechanisms underlie the dysregulation of skeletal muscle synthesis and degradation observed in cancer cachexia, including proinflammatory cytokines (TNFα, IL1, and IL6), and the NF-κB, IGF1/AKT/mTOR, and myostatin/activin-SMAD pathways. Recent preclinical and clinical studies have demonstrated that anti-cachexia drugs (such as MABp1 and soluble receptor antagonist of myostatin/activin) not only prevent muscle wasting but also may prolong overall survival. In this review, we focus on the significance of cachexia signaling in patients with cancer and highlight promising drugs targeting tumor cachexia in clinical development. Clin Cancer Res; 22(16); 3999-4004. ©2016 AACR.

  19. Expression of betaglycan, an inhibin coreceptor, in normal human ovaries and ovarian sex cord-stromal tumors and its regulation in cultured human granulosa-luteal cells.

    Science.gov (United States)

    Liu, Jianqi; Kuulasmaa, Tiina; Kosma, Veli-Matti; Bützow, Ralf; Vänttinen, Teemu; Hydén-Granskog, Christel; Voutilainen, Raimo

    2003-10-01

    Activins and inhibins are often antagonistic in the regulation of ovarian function. TGFbeta type III receptor, betaglycan, has been identified as a coreceptor to enhance the binding of inhibins to activin type II receptor and thus to prevent the binding of activins to their receptor. In this study we characterized the expression and regulation pattern of betaglycan gene in normal ovaries and sex cord-stromal tumors and in cultured human granulosa-luteal cells from women undergoing in vitro fertilization. Expression of betaglycan mRNA was detected by RT-PCR or Northern blotting in normal ovarian granulosa, thecal, and stroma cells as well as in granulosa-luteal cells. Immunohistochemical analysis revealed positive staining for betaglycan in antral and preovulatory follicular granulosa and thecal cells and in corpora lutea of normal ovaries. Furthermore, betaglycan expression was detected in the vast majority of granulosa cell tumors, thecomas, and fibromas, with weaker staining in granulosa cell tumors compared with fibrothecomas. In cultured granulosa-luteal cells, FSH and LH treatment increased dose-dependently the accumulation of betaglycan mRNA, as did the protein kinase A activator dibutyryl cAMP and the protein kinase C inhibitor staurosporine. In contrast, the protein kinase C activator 12-O-tetradecanoyl phorbol 13-acetate had no significant effect on betaglycan mRNA levels. Treatment with prostaglandin E(2) and with its receptor EP2 subtype agonist butaprost increased betaglycan mRNA accumulation and progesterone secretion dose- and time-dependently. In summary, betaglycan gene is expressed in normal human ovarian steroidogenic cells and sex cord-stromal ovarian tumors. The accumulation of its mRNA in cultured granulosa-luteal cells is up-regulated by gonadotropins and prostaglandin E(2), probably via the protein kinase A pathway. The specific expression and regulation pattern of betaglycan gene may be related to the functional antagonism of inhibins to

  20. DMSO efficiently down regulates pluripotency genes in human embryonic stem cells during definitive endoderm derivation and increases the proficiency of hepatic differentiation.

    Directory of Open Access Journals (Sweden)

    Katherine Czysz

    Full Text Available BACKGROUND: Definitive endoderm (DE is one of the three germ layers which during in vivo vertebrate development gives rise to a variety of organs including liver, lungs, thyroid and pancreas; consequently efficient in vitro initiation of stem cell differentiation to DE cells is a prerequisite for successful cellular specification to subsequent DE-derived cell types [1, 2]. In this study we present a novel approach to rapidly and efficiently down regulate pluripotency genes during initiation of differentiation to DE cells by addition of dimethyl sulfoxide (DMSO to Activin A-based culture medium and report its effects on the downstream differentiation to hepatocyte-like cells. MATERIALS AND METHODS: Human embryonic stem cells (hESC were differentiated to DE using standard methods in medium supplemented with 100ng/ml of Activin A and compared to cultures where DE specification was additionally enhanced with different concentrations of DMSO. DE cells were subsequently primed to generate hepatic-like cells to investigate whether the addition of DMSO during formation of DE improved subsequent expression of hepatic markers. A combination of flow cytometry, real-time quantitative reverse PCR and immunofluorescence was applied throughout the differentiation process to monitor expression of pluripotency (POUF5/OCT4 & NANOG, definitive endoderm (SOX17, CXCR4 & GATA4 and hepatic (AFP & ALB genes to generate differentiation stage-specific signatures. RESULTS: Addition of DMSO to the Activin A-based medium during DE specification resulted in rapid down regulation of the pluripotency genes OCT4 and NANOG, accompanied by an increase expression of the DE genes SOX17, CXCR4 and GATA4. Importantly, the expression level of ALB in DMSO-treated cells was also higher than in cells which were differentiated to the DE stage via standard Activin A treatment.

  1. Modulators of erythropoiesis: emerging therapies for hemoglobinopathies and disorders of red cell production.

    Science.gov (United States)

    Breda, Laura; Rivella, Stefano

    2014-04-01

    Use of new compound such as inhibitors of JAK2 or transforming growth factor β-like molecules might soon revolutionize the treatment of β-thalassemia and related disorders. However, this situation requires careful optimization, noting the potential for off-target immune suppression for JAK2 inhibitors and the lack of mechanistic insights for the use of the ligand trap soluble molecules that sequester ligands of activin receptor IIA and B.

  2. Does Skeletal Muscle Mass Influence Breast Cancer? Evaluating Mammary Tumorigenesis and Progression in Genetically Hyper-Muscular Mice

    Science.gov (United States)

    2007-07-01

    preserve muscle in the end-stages of cancer, cancer cachexia . Up to 25% of breast cancer deaths may be attributed to muscle wasting from the complex... cachexia . 15. SUBJECT TERMS Breast cancer, skeletal muscle, myostatin, MPA, DMBA, Activin receptor, cachexia . 16. SECURITY CLASSIFICATION OF: 17...progress, we turned to another question relating skeletal muscle and cancer—pathological muscle wasting in cancer cachexia . (6) (7) (8) Cancer cachexia

  3. Population based model of human embryonic stem cell (hESC differentiation during endoderm induction.

    Directory of Open Access Journals (Sweden)

    Keith Task

    Full Text Available The mechanisms by which human embryonic stem cells (hESC differentiate to endodermal lineage have not been extensively studied. Mathematical models can aid in the identification of mechanistic information. In this work we use a population-based modeling approach to understand the mechanism of endoderm induction in hESC, performed experimentally with exposure to Activin A and Activin A supplemented with growth factors (basic fibroblast growth factor (FGF2 and bone morphogenetic protein 4 (BMP4. The differentiating cell population is analyzed daily for cellular growth, cell death, and expression of the endoderm proteins Sox17 and CXCR4. The stochastic model starts with a population of undifferentiated cells, wherefrom it evolves in time by assigning each cell a propensity to proliferate, die and differentiate using certain user defined rules. Twelve alternate mechanisms which might describe the observed dynamics were simulated, and an ensemble parameter estimation was performed on each mechanism. A comparison of the quality of agreement of experimental data with simulations for several competing mechanisms led to the identification of one which adequately describes the observed dynamics under both induction conditions. The results indicate that hESC commitment to endoderm occurs through an intermediate mesendoderm germ layer which further differentiates into mesoderm and endoderm, and that during induction proliferation of the endoderm germ layer is promoted. Furthermore, our model suggests that CXCR4 is expressed in mesendoderm and endoderm, but is not expressed in mesoderm. Comparison between the two induction conditions indicates that supplementing FGF2 and BMP4 to Activin A enhances the kinetics of differentiation than Activin A alone. This mechanistic information can aid in the derivation of functional, mature cells from their progenitors. While applied to initial endoderm commitment of hESC, the model is general enough to be applicable

  4. Population based model of human embryonic stem cell (hESC) differentiation during endoderm induction.

    Science.gov (United States)

    Task, Keith; Jaramillo, Maria; Banerjee, Ipsita

    2012-01-01

    The mechanisms by which human embryonic stem cells (hESC) differentiate to endodermal lineage have not been extensively studied. Mathematical models can aid in the identification of mechanistic information. In this work we use a population-based modeling approach to understand the mechanism of endoderm induction in hESC, performed experimentally with exposure to Activin A and Activin A supplemented with growth factors (basic fibroblast growth factor (FGF2) and bone morphogenetic protein 4 (BMP4)). The differentiating cell population is analyzed daily for cellular growth, cell death, and expression of the endoderm proteins Sox17 and CXCR4. The stochastic model starts with a population of undifferentiated cells, wherefrom it evolves in time by assigning each cell a propensity to proliferate, die and differentiate using certain user defined rules. Twelve alternate mechanisms which might describe the observed dynamics were simulated, and an ensemble parameter estimation was performed on each mechanism. A comparison of the quality of agreement of experimental data with simulations for several competing mechanisms led to the identification of one which adequately describes the observed dynamics under both induction conditions. The results indicate that hESC commitment to endoderm occurs through an intermediate mesendoderm germ layer which further differentiates into mesoderm and endoderm, and that during induction proliferation of the endoderm germ layer is promoted. Furthermore, our model suggests that CXCR4 is expressed in mesendoderm and endoderm, but is not expressed in mesoderm. Comparison between the two induction conditions indicates that supplementing FGF2 and BMP4 to Activin A enhances the kinetics of differentiation than Activin A alone. This mechanistic information can aid in the derivation of functional, mature cells from their progenitors. While applied to initial endoderm commitment of hESC, the model is general enough to be applicable either to a system of

  5. Role of PCSK5 expression in mouse ovarian follicle development: identification of the inhibin α- and β-subunits as candidate substrates.

    Directory of Open Access Journals (Sweden)

    Monica Antenos

    Full Text Available Inhibin and activin are essential dimeric glycoproteins belonging to the transforming growth factor-beta (TGFβ superfamily. Inhibin is a heterodimer of α- and β-subunits, whereas activin is a homodimer of β-subunits. Production of inhibin is regulated during the reproductive cycle and requires the processing of pro-ligands to produce mature hormone. Furin is a subtilisin-like proprotein convertase (proconvertase that activates precursor proteins by cleavage at basic sites during their transit through the secretory pathway and/or at the cell surface. We hypothesized that furin-like proconvertases are central regulators of inhibin α- and β-subunit processing within the ovary. We analyzed the expression of the proconvertases furin, PCSK5, PCSK6, and PCSK7 in the developing mouse ovary by real-time quantitative RT-PCR. The data showed that proconvertase enzymes are temporally expressed in ovarian cells. With the transition from two-layer secondary to pre-antral follicle, only PCSK5 mRNA was significantly elevated. Activin A selectively enhanced expression of PCSK5 mRNA and decreased expression of furin and PCSK6 in cultured two-layer secondary follicles. Inhibition of proconvertase enzyme activity by dec-RVKR-chloromethylketone (CMK, a highly specific and potent competitive inhibitor of subtilisin-like proconvertases, significantly impeded both inhibin α- and β-subunit maturation in murine granulosa cells. Overexpression of PC5/6 in furin-deficient cells led to increased inhibin α- and β(B-subunit maturation. Our data support the role of proconvertase PCSK5 in the processing of ovarian inhibin subunits during folliculogenesis and suggest that this enzyme may be an important regulator of inhibin and activin bioavailability.

  6. Domain Modeling: NP_001607.1 [SAHG[Archive

    Lifescience Database Archive (English)

    Full Text Available NP_001607.1 chr2 Crystal structure of Activin receptor type II kinase domain from h...uman p2qlua_ chr2/NP_001607.1/NP_001607.1_holo_192-485.pdb blast 198K,199A,206V,217A,247L,267T,268A,269F,270H,271E,273G,329L 322D,324K,326K __A 0 ...

  7. Molecular Signaling Networks That Choreograph Epimorphic Fin Regeneration in Zebrafish – A Mini-Review

    OpenAIRE

    Tal, Tamara L; Franzosa, Jill A.; Tanguay, Robert L.

    2009-01-01

    This short review provides a current synopsis of caudal fin regeneration in zebrafish with an emphasis on the molecular signaling networks that dictate epimorphic regeneration. At the outset, the fundamentals of caudal fin architecture and the stages of epimorphic regeneration are described. This is followed by a detailed look at the main networks implicated in fin regeneration, namely the Wnt, fibroblast growth factor, activin-βA, retinoic acid and hedgehog signaling pathways. Throughout thi...

  8. Inhibins Tune the Thymocyte Selection Process by Regulating Thymic Stromal Cell Differentiation

    Directory of Open Access Journals (Sweden)

    Ebzadrel Carbajal-Franco

    2015-01-01

    Full Text Available Inhibins and Activins are members of the TGF-β superfamily that regulate the differentiation of several cell types. These ligands were initially identified as hormones that regulate the hypothalamus-pituitary-gonadal axis; however, increasing evidence has demonstrated that they are key regulators in the immune system. We have previously demonstrated that Inhibins are the main Activin ligands expressed in the murine thymus and that they regulate thymocyte differentiation, promoting the DN3-DN4 transition and the selection of SP thymocytes. As Inhibins are mainly produced by thymic stromal cells, which also express Activin receptors and Smad proteins, we hypothesized that Inhibins might play a role in stromal cell differentiation and function. Here, we demonstrate that, in the absence of Inhibins, thymic conventional dendritic cells display reduced levels of MHC Class II (MHCII and CD86. In addition, the ratio between cTECs and mTECs was affected, indicating that mTEC differentiation was favoured and cTEC diminished in the absence of Inhibins. These changes appeared to impact thymocyte selection leading to a decreased selection of CD4SP thymocytes and increased generation of natural regulatory T cells. These findings demonstrate that Inhibins tune the T cell selection process by regulating both thymocyte and stromal cell differentiation.

  9. Designer TGFβ superfamily ligands with diversified functionality.

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    George P Allendorph

    Full Text Available Transforming Growth Factor--beta (TGFβ superfamily ligands, including Activins, Growth and Differentiation Factors (GDFs, and Bone Morphogenetic Proteins (BMPs, are excellent targets for protein-based therapeutics because of their pervasiveness in numerous developmental and cellular processes. We developed a strategy termed RASCH (Random Assembly of Segmental Chimera and Heteromer, to engineer chemically-refoldable TGFβ superfamily ligands with unique signaling properties. One of these engineered ligands, AB208, created from Activin-βA and BMP-2 sequences, exhibits the refolding characteristics of BMP-2 while possessing Activin-like signaling attributes. Further, we find several additional ligands, AB204, AB211, and AB215, which initiate the intracellular Smad1-mediated signaling pathways more strongly than BMP-2 but show no sensitivity to the natural BMP antagonist Noggin unlike natural BMP-2. In another design, incorporation of a short N-terminal segment from BMP-2 was sufficient to enable chemical refolding of BMP-9, without which was never produced nor refolded. Our studies show that the RASCH strategy enables us to expand the functional repertoire of TGFβ superfamily ligands through development of novel chimeric TGFβ ligands with diverse biological and clinical values.

  10. Hepcidin expression in the liver of rats fed a magnesium-deficient diet.

    Science.gov (United States)

    Ishizaki, Natsumi; Kotani, Megumi; Funaba, Masayuki; Matsui, Tohru

    2011-10-01

    Mg deficiency accelerates Fe accumulation in the liver, which may induce various metabolic disturbances. In the present study, we examined the gene expression of Hepcidin, a peptide hormone produced in the liver to regulate intestinal Fe absorption negatively, in Mg-deficient rats. Although liver Fe concentration was significantly higher in rats fed an Mg-deficient diet for 4 weeks than in rats fed a control diet, Hepcidin expression in the liver was comparable between the dietary groups. Previous studies revealed that Fe overload up-regulated Hepcidin expression through transcriptional activation by Fe-induced bone morphogenetic protein (Bmp) 6, a growth/differentiation factor belonging to the transforming growth factor-β family, in the liver. Mg deficiency up-regulated the expression of Bmp6 but did not affect the expression of inhibition of DNA binding 1, a sensitive Bmp-responsive gene. In addition, the expression of Bmp receptors such as activin receptor-like kinase 2 (Alk2), activin receptor type IIA (Actr2a), activin receptor type IIB (Actr2b) and Bmp type II receptor (Bmpr2) was lower in the liver of Mg-deficient rats than in that of control rats. The present study indicates that accumulation of hepatic Fe by Mg deficiency is a stimulant inducing Bmp6 expression but not Hepcidin expression by blunting Bmp signalling possibly resulting from down-regulation of the receptor expression. Unresponsive Hepcidin expression may have a role in Mg deficiency-induced changes related to increased liver Fe.

  11. SOX9: a stem cell transcriptional regulator of secreted niche signaling factors.

    Science.gov (United States)

    Kadaja, Meelis; Keyes, Brice E; Lin, Mingyan; Pasolli, H Amalia; Genander, Maria; Polak, Lisa; Stokes, Nicole; Zheng, Deyou; Fuchs, Elaine

    2014-02-15

    Hair follicles (HFs) undergo cyclical periods of growth, which are fueled by stem cells (SCs) at the base of the resting follicle. HF-SC formation occurs during HF development and requires transcription factor SOX9. Whether and how SOX9 functions in HF-SC maintenance remain unknown. By conditionally targeting Sox9 in adult HF-SCs, we show that SOX9 is essential for maintaining them. SOX9-deficient HF-SCs still transition from quiescence to proliferation and launch the subsequent hair cycle. However, once activated, bulge HF-SCs begin to differentiate into epidermal cells, which naturally lack SOX9. In addition, as HF-SC numbers dwindle, outer root sheath production is not sustained, and HF downgrowth arrests prematurely. Probing the mechanism, we used RNA sequencing (RNA-seq) to identify SOX9-dependent transcriptional changes and chromatin immunoprecipitation (ChIP) and deep sequencing (ChIP-seq) to identify SOX9-bound genes in HF-SCs. Intriguingly, a large cohort of SOX9-sensitive targets encode extracellular factors, most notably enhancers of Activin/pSMAD2 signaling. Moreover, compromising Activin signaling recapitulates SOX9-dependent defects, and Activin partially rescues them. Overall, our findings reveal roles for SOX9 in regulating adult HF-SC maintenance and suppressing epidermal differentiation in the niche. In addition, our studies expose a role for SCs in coordinating their own behavior in part through non-cell-autonomous signaling within the niche.

  12. Aldosterone breakthrough caused by chronic blockage of angiotensin II type 1 receptors in human adrenocortical cells: possible involvement of bone morphogenetic protein-6 actions.

    Science.gov (United States)

    Otani, Hiroyuki; Otsuka, Fumio; Inagaki, Kenichi; Suzuki, Jiro; Miyoshi, Tomoko; Kano, Yoshihiro; Goto, Junko; Ogura, Toshio; Makino, Hirofumi

    2008-06-01

    Circulating aldosterone concentrations occasionally increase after initial suppression with angiotensin II (Ang II) converting enzyme inhibitors or Ang II type 1 receptor blockers (ARBs), a phenomenon referred to as aldosterone breakthrough. However, the underlying mechanism causing the aldosterone breakthrough remains unknown. Here we investigated whether aldosterone breakthrough occurs in human adrenocortical H295R cells in vitro. We recently reported that bone morphogenetic protein (BMP)-6, which is expressed in adrenocortical cells, enhances Ang II- but not potassium-induced aldosterone production in human adrenocortical cells. Accordingly, we examined the roles of BMP-6 in aldosterone breakthrough induced by long-term treatment with ARB. Ang II stimulated aldosterone production by adrenocortical cells. This Ang II stimulation was blocked by an ARB, candesartan. Interestingly, the candesartan effects on Ang II-induced aldosterone synthesis and CYP11B2 expression were attenuated in a course of candesartan treatment for 15 d. The impairment of candesartan effects on Ang II-induced aldosterone production was also observed in Ang II- or candesartan-pretreated cells. Levels of Ang II type 1 receptor mRNA were not changed by chronic candesartan treatment. However, BMP-6 enhancement of Ang II-induced ERK1/2 signaling was resistant to candesartan. The BMP-6-induced Smad1, -5, and -8 phosphorylation, and BRE-Luc activity was augmented in the presence of Ang II and candesartan in the chronic phase. Chronic Ang II exposure decreased cellular expression levels of BMP-6 and its receptors activin receptor-like kinase-2 and activin type II receptor mRNAs. Cotreatment with candesartan reversed the inhibitory effects of Ang II on the expression levels of these mRNAs. The breakthrough phenomenon was attenuated by neutralization of endogenous BMP-6 and activin receptor-like kinase-2. Collectively, these data suggest that changes in BMP-6 availability and response may be involved

  13. [Immunotherapy for the treatment of acute appendicitis in children].

    Science.gov (United States)

    Bulanova, A A; Akhanzaripov, Z A

    1994-08-01

    The immune status was studied during the development of the disease in 182 children who were operated on for acute appendicitis. T lymphocytes and their subpopulations circulating in the blood, as well as B lymphocytes, immunoglobulins A, M, G, and immune complexes were determined. The character of changes of these values before the operation and in various postoperative periods were determined. The effect of complex treatment, including T-activin, on the clinical and immunological parameters in children with acute appendicitis was appraised. Analysis of the results showed that a transitory immunodepressive state forms in children with the disease, which is more marked in the destructive form, with normalization of the main values of cell-mediated and humoral immunity by the 7th day after appendectomy. In a complicated course of acute appendicitis the state of immunodeficiency is torpid in character and does not return to normal values even after clinical recovery, i.e. before discharge from the clinic. Inclusion of the immunostimulating agent T-activin into the complex treatment of patients with appendicitis ensures a more rapid involution of the main clinical manifestations of the disease. The therapeutic effect was most pronounced in destructive appendicitis: after 3 days of treatment the pain syndrome was encountered twice less frequently and intestinal paresis more than twice less frequently in these patients, and the term of hospital stay (8.8 +/- 0.4 days) was less shorter than for children of the control group (12.2 +/- 1.9 days) who did not receive T-activin in the therapeutic complex.

  14. Gene regulatory network interactions in sea urchin endomesoderm induction.

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    Aditya J Sethi

    2009-02-01

    Full Text Available A major goal of contemporary studies of embryonic development is to understand large sets of regulatory changes that accompany the phenomenon of embryonic induction. The highly resolved sea urchin pregastrular endomesoderm-gene regulatory network (EM-GRN provides a unique framework to study the global regulatory interactions underlying endomesoderm induction. Vegetal micromeres of the sea urchin embryo constitute a classic endomesoderm signaling center, whose potential to induce archenteron formation from presumptive ectoderm was demonstrated almost a century ago. In this work, we ectopically activate the primary mesenchyme cell-GRN (PMC-GRN that operates in micromere progeny by misexpressing the micromere determinant Pmar1 and identify the responding EM-GRN that is induced in animal blastomeres. Using localized loss-of -function analyses in conjunction with expression of endo16, the molecular definition of micromere-dependent endomesoderm specification, we show that the TGFbeta cytokine, ActivinB, is an essential component of this induction in blastomeres that emit this signal, as well as in cells that respond to it. We report that normal pregastrular endomesoderm specification requires activation of the Pmar1-inducible subset of the EM-GRN by the same cytokine, strongly suggesting that early micromere-mediated endomesoderm specification, which regulates timely gastrulation in the sea urchin embryo, is also ActivinB dependent. This study unexpectedly uncovers the existence of an additional uncharacterized micromere signal to endomesoderm progenitors, significantly revising existing models. In one of the first network-level characterizations of an intercellular inductive phenomenon, we describe an important in vivo model of the requirement of ActivinB signaling in the earliest steps of embryonic endomesoderm progenitor specification.

  15. Differentiation of murine embryonic stem and induced pluripotent stem cells to renal lineage in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Morizane, Ryuji [Department of Internal Medicine, Keio University School of Medicine, Tokyo (Japan); Monkawa, Toshiaki, E-mail: monkawa@sc.itc.keio.ac.jp [Department of Internal Medicine, Keio University School of Medicine, Tokyo (Japan); Itoh, Hiroshi [Department of Internal Medicine, Keio University School of Medicine, Tokyo (Japan)

    2009-12-25

    Embryonic stem (ES) cells which have the unlimited proliferative capacity and extensive differentiation potency can be an attractive source for kidney regeneration therapies. Recent breakthroughs in the generation of induced pluripotent stem (iPS) cells have provided with another potential source for the artificially-generated kidney. The purpose of this study is to know how to differentiate mouse ES and iPS cells into renal lineage. We used iPS cells from mouse fibroblasts by transfection of four transcription factors, namely Oct4, Sox2, c-Myc and Klf4. Real-time PCR showed that renal lineage markers were expressed in both ES and iPS cells after the induction of differentiation. It also showed that a tubular specific marker, KSP progressively increased to day 18, although the differentiation of iPS cells was slower than ES cells. The results indicated that renal lineage cells can be differentiated from both murine ES and iPS cells. Several inducing factors were tested whether they influenced on cell differentiation. In ES cells, both of GDNF and BMP7 enhanced the differentiation to metanephric mesenchyme, and Activin enhanced the differentiation of ES cells to tubular cells. Activin also enhanced the differentiation of iPS cells to tubular cells, although the enhancement was lower than in ES cells. ES and iPS cells have a potential to differentiate to renal lineage cells, and they will be an attractive resource of kidney regeneration therapy. This differentiation is enhanced by Activin in both ES and iPS cells.

  16. Maternal Nodal inversely affects NODAL and STOX1 expression in the fetal placenta

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    Hari Krishna Thulluru

    2013-08-01

    Full Text Available Nodal, a secreted signaling protein from the TGFβ-super family plays a vital role during early embryonic development. Recently, it was found that maternal decidua-specific Nodal knockout mice show intrauterine growth restriction (IUGR and preterm birth. As the chromosomal location of NODAL is in the same linkage area as the susceptibility gene STOX1, associated with the familial form of early-onset, IUGR-complicated pre-eclampsia, their potential maternal-fetal interaction was investigated. Pre-eclamptic mothers with children who carried the STOX1 susceptibility allele themselves all carried the NODAL H165R SNP, which causes a 50% reduced activity. Surprisingly, in decidua Nodal knockout mice the fetal placenta showed up-regulation of STOX1 and NODAL expression. Conditioned media of human first trimester decidua and a human endometrial stromal cell line (T-HESC treated with siRNAs against NODAL or carrying the H165R SNP were also able to induce NODAL and STOX1 expression when added to SGHPL-5 first trimester extravillous trophoblast cells. Finally, a human TGFß-BMP-Signaling-Pathway PCR-Array on decidua and the T-HESC cell line with Nodal knockdown revealed upregulation of Activin-A, which was confirmed in conditioned media by ELISA. We show that maternal decidua Nodal knockdown gives upregulation of NODAL and STOX1 mRNA expression in fetal extravillous trophoblast cells, potentially via upregulation of Activin-A in the maternal decidua. As both Activin-A and Nodal have been implicated in pre-eclampsia, being increased in serum of pre-eclamptic women and upregulated in pre-eclamptic placentas respectively, this interaction at the maternal-fetal interface might play a substantial role in the development of pre-eclampsia.

  17. Induction and selection of Sox17-expressing endoderm cells generated from murine embryonic stem cells.

    Science.gov (United States)

    Schroeder, Insa S; Sulzbacher, Sabine; Nolden, Tobias; Fuchs, Joerg; Czarnota, Judith; Meisterfeld, Ronny; Himmelbauer, Heinz; Wobus, Anna M

    2012-01-01

    Embryonic stem (ES) cells offer a valuable source for generating insulin-producing cells. However, current differentiation protocols often result in heterogeneous cell populations of various developmental stages. Here we show the activin A-induced differentiation of mouse ES cells carrying a homologous dsRed-IRES-puromycin knock-in within the Sox17 locus into the endoderm lineage. Sox17-expressing cells were selected by fluorescence-assisted cell sorting (FACS) and characterized at the transcript and protein level. Treatment of ES cells with high concentrations of activin A for 10 days resulted in up to 19% Sox17-positive cells selected by FACS. Isolated Sox17-positive cells were characterized by defini- tive endoderm-specific Sox17/Cxcr4/Foxa2 transcripts, but lacked pluripotency-associated Oct4 mRNA and protein. The Sox17-expressing cells showed downregulation of extraembryonic endoderm (Sox7, Afp, Sdf1)-, mesoderm (Foxf1, Meox1)- and ectoderm (Pax6, NeuroD6)-specific transcripts. The presence of Hnf4α, Hes1 and Pdx1 mRNA demonstrated the expression of primitive gut/foregut cell-specific markers. Ngn3, Nkx6.1 and Nkx2.2 transcripts in Sox17-positive cells were determined as properties of pancreatic endocrine progenitors. Immunocytochemistry of activin A-induced Sox17-positive embryoid bodies revealed coexpression of Cxcr4 and Foxa2. Moreover, the histochemical demonstration of E-cadherin-, Cxcr4-, Sox9-, Hnf1β- and Ngn3-positive epithelial-like structures underlined the potential of Sox17-positive cells to further differentiate into the pancreatic lineage. By reducing the heterogeneity of the ES cell progeny, Sox17-expressing cells are a suitable model to evaluate the effects of growth and differentiation factors and of culture conditions to delineate the differentiation process for the generation of pancreatic cells in vitro.

  18. Pentoxifylline Attenuates Proteinuria in Anti-Thy1 Glomerulonephritis via Downregulation of Nuclear Factor-κB and Smad2/3 Signaling

    Science.gov (United States)

    Chen, Yung-Ming; Chiang, Wen-Chih; Yang, Yalin; Lai, Chun-Fu; Wu, Kwan-Dun; Lin, Shuei-Liong

    2015-01-01

    Anti-Thy1 glomerulonephritis is a rat nephritis model closely simulating human mesangial proliferative glomerulonephritis. It affects primarily the mesangium, yet displays substantial proteinuria during the course. This study investigated the molecular signals underlying proteinuria in this disease and the modulation of which by the known antiproteinuric agent, pentoxifylline. Male Wistar rats were randomly divided into a control group and nephritic groups with or without treatment with IMD-0354 (an IκB kinase inhibitor), SB431542 (an activin receptor–like kinase inhibitor) or pentoxifylline. Kidney sections were prepared for histological examinations. Glomeruli were isolated for mRNA and protein analysis. Urine samples were collected for protein and nephrin quantitation. One day after nephritis induction, proteinuria developed together with ultrastructural changes of the podocyte and downregulation of podocyte mRNA and protein expression. These were associated with upregulation of tumor necrosis factor (TNF)-α and transforming growth factor (TGF)-β/activins mRNAs and activation of nuclear factor (NF)-κB p65 and Smad2/3. IMD-0354 attenuated proteinuria on d 1, whereas SB431542 decreased proteinuria on d 3 and 5, in association with partial restoration of downregulated podocyte mRNA and protein expression. Pentoxifylline attenuated proteinuria and nephrinuria through the course, plus inhibition of p-NF-κB p65 (d 1) and p-Smad2/3 (d 5) and partial reversal of downregulated podocyte mRNA and protein. Our data show that the pathogenesis of proteinuria in anti-Thy1 glomerulonephritis involves TNF-α and TGF-β/activin pathways, and the evolution of this process can be attenuated by pentoxifylline via downregulation of NF-κB and Smad signals and restoration of the podocyte component of the glomerular filtration barrier. PMID:25879629

  19. Follistatin N terminus differentially regulates muscle size and fat in vivo.

    Science.gov (United States)

    Zheng, Hui; Qiao, Chunping; Tang, Ruhang; Li, Jianbin; Bulaklak, Karen; Huang, Zhenhua; Zhao, Chunxia; Dai, Yi; Li, Juan; Xiao, Xiao

    2017-09-15

    Delivery of follistatin (FST) represents a promising strategy for both muscular dystrophies and diabetes, as FST is a robust antagonist of myostatin and activin, which are critical regulators of skeletal muscle and adipose tissues. FST is a multi-domain protein, and deciphering the function of different domains will facilitate novel designs for FST-based therapy. Our study aims to investigate the role of the N-terminal domain (ND) of FST in regulating muscle and fat mass in vivo. Different FST constructs were created and packaged into the adeno-associated viral vector (AAV). Overexpression of wild-type FST in normal mice greatly increased muscle mass while decreasing fat accumulation, whereas overexpression of an N terminus mutant or N terminus-deleted FST had no effect on muscle mass but moderately decreased fat mass. In contrast, FST-I-I containing the complete N terminus and double domain I without domain II and III had no effect on fat but increased skeletal muscle mass. The effects of different constructs on differentiated C2C12 myotubes were consistent with the in vivo finding. We hypothesized that ND was critical for myostatin blockade, mediating the increase in muscle mass, and was less pivotal for activin binding, which accounts for the decrease in the fat tissue. An in vitro TGF-beta1-responsive reporter assay revealed that FST-I-I and N terminus-mutated or -deleted FST showed differential responses to blockade of activin and myostatin. Our study provided direct in vivo evidence for a role of the ND of FST, shedding light on future potential molecular designs for FST-based gene therapy.

  20. Narrative review: environmental and genetic basis of orofacial clefts.

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    César Rivera

    2013-04-01

    Full Text Available Complex genetic process guides the embryonic head development. Alterations in these processes result in structural abnormalities. An example of these are the cleft lip and palate. The major pathways involved in the fissures are families: the Fibroblast Growth Factor (FGF family, the Hedgehog (HH family, the Wingless (WNT family and the Transforming Growth Factor beta (TGF-β family, which includes the Bone Morphogenetic Proteins (BMPs and Activins. In this review, we discuss some of the celular/molecular processes and environmental factors involved in the development of the orofacial complex, ending with therapeutic possibilities and potential clinical relevance to the accumulated evidence.

  1. Cloning and analysing of 5‘ flanking region of Xenopus organizer gene noggin

    Institute of Scientific and Technical Information of China (English)

    TAOQINHUA; JINGYANG; 等

    1999-01-01

    Xenopus organizer specific gene noggin possesses nearly all the characterestic properties of the action of organizer to specify the embryonic body acis.To analyze how the maternal inherited factors control its expression pattern,we cloned the 5' regulatory region of noggin gene.The 1.5 kb upstream sequense could direct reporter gene to express in vivo and data from deletion analysis indicated that a 229 base pair fragmet is essential for activating noggin expression.We further demonstrated that the response elements within this regulatory region were indeed under the control of growth factor activin and Wnt signaling pathway components.

  2. Gonadotrope-specific expression and regulation of ovine follicle stimulating hormone Beta: transgenic and adenoviral approaches using primary murine gonadotropes.

    Directory of Open Access Journals (Sweden)

    Jingjing Jia

    Full Text Available The beta subunit of follicle stimulating hormone (FSHB is expressed specifically in pituitary gonadotropes in vertebrates. Transgenic mouse studies have shown that enhancers in the proximal promoter between -172/-1 bp of the ovine FSHB gene are required for gonadotrope expression of ovine FSHB. These enhancers are associated with regulation by activins and gonadotropin releasing hormone (GnRH. Additional distal promoter sequence between -4741/-750 bp is also required for expression. New transgenic studies presented here focus on this distal region and narrow it to 1116 bp between -1866/-750 bp. In addition, adenoviral constructs were produced to identify these critical distal sequences using purified primary mouse gonadotropes as an in vitro model system. The adenoviral constructs contained -2871 bp, -750 bp or -232 bp of the ovine FSHB promoter. They all showed gonadotrope-specific regulation since they were induced only in purified primary gonadotropes by activin A (50 ng/ml and inhibited by GnRH (100 nM in the presence of activin (except -232FSHBLuc. However, basal expression of all three viral constructs (in the presence of follistatin to block cellular induction by activin was relatively high in pituitary non-gonadotropes as well as gonadotropes. Thus, gonadotrope-specific regulation associated with the proximal promoter was observed as expected, but the model was blind to distal promoter elements between -2871/-750 necessary for gonadotrope-specific expression of ovine FSHB in vivo. The new adenoviral-based in vitro technique did detect, however, a novel GnRH response element between -750 bp and -232 bp of the ovine FSHB promoter. We conclude that adenoviral-based studies in primary gonadotropes can adequately recognize regulatory elements on the ovine FSHB promoter associated with gonadotrope-specific regulation/expression, but that more physiologically based techniques, such as transgenic studies, will be needed to identify sequences

  3. [Expression of TGFbeta family factors and FGF2 in mouse and human embryonic stem cells maintained in different culture systems].

    Science.gov (United States)

    Lifantseva, N V; Kol'tsova, A M; Polianskaia, G G; Gordeeva, O F

    2013-01-01

    Mouse and human embryonic stem cells are in different states of pluripotency (naive/ground and primed states). Mechanisms of signaling regulation in cells with ground and primed states of pluripotency are considerably different. In order to understand the contribution of endogenous and exogenous factors in the maintenance of a metastable state of the cells in different phases ofpluripotency, we examined the expression of TGFbeta family factors (ActivinA, Nodal, Leftyl, TGFbeta1, GDF3, BMP4) and FGF2 initiating the appropriate signaling pathways in mouse and human embryonic stem cells (mESCs, hESCs) and supporting feeder cells. Quantitative real-time PCR analysis of gene expression showed that the expression patterns of endogenous factors studied were considerably different in mESCs and hESCs. The most significant differences were found in the levels of endogenous expression of TGFbeta1, BMP4 and ActivinA. The sources of exogenous factors ActivnA, TGFbeta1, and FGF2 for hESCs are feeder cells (mouse and human embryonic fibroblasts) expressing high levels of these factors, as well as low levels of BMP4. Thus, our data demonstrated that the in vitro maintenance of metastable state of undifferentiated pluripotent cells is achieved in mESCs and hESCs using different schemes of the regulations of ActivinA/Nodal/Lefty/Smad2/3BMP/Smad1/5/8 endogenous branches of TGFbeta signaling. The requirement for exogenous stimulation or inhibition of these signaling pathways is due to different patterns of endogenous expression of TGFbeta family factors and FGF2 in the mESCs and hESCs. For the hESCs, enhanced activity of ActivinA/Nodal/Lefty/Smad2/3 signaling by exogenous factor stimulation is necessary to mitigate the effects of BMP/Smadl/5/8 signaling pathways that promote cell differentiation into the extraembryonic structures. Significant differences in endogenous FGF2 expression in the cells in the ground and primary states of pluripotency demonstrate diverse involvement of this

  4. miR-27 negatively regulates pluripotency-associated genes in human embryonal carcinoma cells.

    Directory of Open Access Journals (Sweden)

    Heiko Fuchs

    Full Text Available Human embryonic stem cells and human embryonal carcinoma cells have been studied extensively with respect to the transcription factors (OCT4, SOX2 and NANOG, epigenetic modulators and associated signalling pathways that either promote self-renewal or induce differentiation in these cells. The ACTIVIN/NODAL axis (SMAD2/3 of the TGFß signalling pathway coupled with FGF signalling maintains self-renewal in these cells, whilst the BMP (SMAD1,5,8 axis promotes differentiation. Here we show that miR-27, a somatic-enriched miRNA, is activated upon RNAi-mediated suppression of OCT4 function in human embryonic stem cells. We further demonstrate that miR-27 negatively regulates the expression of the pluripotency-associated ACTIVIN/NODAL axis (SMAD2/3 of the TGFß signalling pathway by targeting ACVR2A, TGFßR1 and SMAD2. Additionally, we have identified a number of pluripotency-associated genes such as NANOG, LIN28, POLR3G and NR5A2 as novel miR-27 targets. Transcriptome analysis revealed that miR-27 over-expression in human embryonal carcinoma cells leads indeed to a significant up-regulation of genes involved in developmental pathways such as TGFß- and WNT-signalling.

  5. Connective Tissue Growth Factor Is Required for Normal Follicle Development and Ovulation

    Science.gov (United States)

    Nagashima, Takashi; Kim, Jaeyeon; Li, Qinglei; Lydon, John P.; DeMayo, Francesco J.; Lyons, Karen M.

    2011-01-01

    Connective tissue growth factor (CTGF) is a cysteine-rich protein the synthesis and secretion of which are hypothesized to be selectively regulated by activins and other members of the TGF-β superfamily. To investigate the in vivo roles of CTGF in female reproduction, we generated Ctgf ovarian and uterine conditional knockout (cKO) mice. Ctgf cKO mice exhibit severe subfertility and multiple reproductive defects including disrupted follicle development, decreased ovulation rates, increased numbers of corpus luteum, and smaller but functionally normal uterine horns. Steroidogenesis is disrupted in the Ctgf cKO mice, leading to increased levels of serum progesterone. We show that disrupted follicle development is accompanied by a significant increase in granulosa cell apoptosis. Moreover, despite normal cumulus expansion, Ctgf cKO mice exhibit a significant decrease in oocytes ovulated, likely due to impaired ovulatory process. During analyses of mRNA expression, we discovered that Ctgf cKO granulosa cells show gene expression changes similar to our previously reported granulosa cell-specific knockouts of activin and Smad4, the common TGF-β family intracellular signaling protein. We also discovered a significant down-regulation of Adamts1, a progesterone-regulated gene that is critical for the remodeling of extracellular matrix surrounding granulosa cells of preovulatory follicles. These findings demonstrate that CTGF is a downstream mediator in TGF-β and progesterone signaling cascades and is necessary for normal follicle development and ovulation. PMID:21868453

  6. Novel use of rituximab in a case of Riedel's thyroiditis refractory to glucocorticoids and tamoxifen.

    Science.gov (United States)

    Soh, Shui-Boon; Pham, Alan; O'Hehir, Robyn E; Cherk, Martin; Topliss, Duncan J

    2013-09-01

    A 42-year-old woman presented with a rapidly enlarging right-sided thyroid mass and underwent hemithyroidectomy. Riedel's thyroiditis was only diagnosed upon surgical decompression of the right carotid artery 2 years later. She became more symptomatic as Riedel's thyroiditis progressed, and mediastinal fibrosclerosis developed over the next 12 months. Oral prednisolone failed to improve her condition, and she was commenced on tamoxifen. Despite initial improvement, her symptoms recurred 2 years later, mainly arising from compression of the trachea and esophagus at the thoracic inlet. Fluorodeoxyglucose positron emission tomographic scan showed locally advanced active invasive fibrosclerosis in the neck and mediastinum. An elevated activin-A level of 218 pg/mL was consistent with active inflammation. IgG subtypes (including IgG4) were normal. Two courses of iv methylprednisolone were given but only produced transient improvement. Subsequently, the patient received 3 doses of i.v. rituximab at monthly intervals and had prompt sustained symptomatic improvement. Activin-A level decreased to 122 pg/mL 10 months after rituximab therapy. Fluorodeoxyglucose positron emission tomographic scan 6 weeks after therapy showed reduction in inflammation. A further scan at 10 months demonstrated ongoing response to rituximab. This is a case of refractory Riedel's thyroiditis with symptomatic, biochemical, and radiological improvement that has persisted 14 months after rituximab. The likelihood and duration of response to rituximab in Riedel's thyroiditis requires further study.

  7. FGF, Insulin, and SMAD Signaling Cooperate for Avian Primordial Germ Cell Self-Renewal

    Directory of Open Access Journals (Sweden)

    Jemima Whyte

    2015-12-01

    Full Text Available Precise self-renewal of the germ cell lineage is fundamental to fertility and reproductive success. The early precursors for the germ lineage, primordial germ cells (PGCs, survive and proliferate in several embryonic locations during their migration to the embryonic gonad. By elucidating the active signaling pathways in migratory PGCs in vivo, we were able to create culture conditions that recapitulate this embryonic germ cell environment. In defined medium conditions without feeder cells, the growth factors FGF2, insulin, and Activin A, signaling through their cognate-signaling pathways, were sufficient for self-renewal of germline-competent PGCs. Forced expression of constitutively active MEK1, AKT, and SMAD3 proteins could replace their respective upstream growth factors. Unexpectedly, we found that BMP4 could replace Activin A in non-clonal growth conditions. These defined medium conditions identify the key molecular pathways required for PGC self-renewal and will facilitate efforts in biobanking of chicken genetic resources and genome editing.

  8. Investigational drugs in phase I and phase II clinical trials for thalassemia.

    Science.gov (United States)

    Motta, Irene; Scaramellini, Natalia; Cappellini, Maria Domenica

    2017-07-01

    Regular transfusion and iron chelation are the current treatment of severe forms of thalassemia. As a consequence of this demanding supportive treatment, there are several unmet therapeutic needs. Due to a deeper understanding in the pathophysiology of thalassemia, new therapeutic strategies have been developed that are now in pre-clinical and clinical trials. Areas covered: Activin receptor ligand traps (luspatercept and sotatercept), drugs targeting ineffective erythropoiesis, showed encouraging results in Phase I and II clinical trials. A phase III clinical trial is currently ongoing. Ruxolitinib, a Jak2 inhibitor, has been tested to limit stress erythropoiesis in a phase II clinical trial. In addition, improvement in iron chelation has been developed. Moreover, several trials of gene therapy are currently active in different countries with different lentiviral vectors. Expert opinion: The most promising molecules are the activin receptor ligand traps. Together with gene therapy these could be an alternative to bone marrow transplant, aiming towards a curative strategy. The main limit to gene therapy seems to be the conditioning regimen, thus an in vivo gene therapy would be more suitable. At pre-clinical level gene editing is showing extremely encouraging results.

  9. dSno facilitates baboon signaling in the Drosophila brain by switching the affinity of Medea away from Mad and toward dSmad2.

    Science.gov (United States)

    Takaesu, Norma T; Hyman-Walsh, Cathy; Ye, Ying; Wisotzkey, Robert G; Stinchfield, Michael J; O'connor, Michael B; Wotton, David; Newfeld, Stuart J

    2006-11-01

    A screen for modifiers of Dpp adult phenotypes led to the identification of the Drosophila homolog of the Sno oncogene (dSno). The dSno locus is large, transcriptionally complex and contains a recent retrotransposon insertion that may be essential for dSno function, an intriguing possibility from the perspective of developmental evolution. dSno is highly transcribed in the embryonic central nervous system and transcripts are most abundant in third instar larvae. dSno mutant larvae have proliferation defects in the optic lobe of the brain very similar to those seen in baboon (Activin type I receptor) and dSmad2 mutants. This suggests that dSno is a mediator of Baboon signaling. dSno binds to Medea and Medea/dSno complexes have enhanced affinity for dSmad2. Alternatively, Medea/dSno complexes have reduced affinity for Mad such that, in the presence of dSno, Dpp signaling is antagonized. We propose that dSno functions as a switch in optic lobe development, shunting Medea from the Dpp pathway to the Activin pathway to ensure proper proliferation. Pathway switching in target cells is a previously unreported mechanism for regulating TGFbeta signaling and a novel function for Sno/Ski family proteins.

  10. Follistatin is critical for mouse uterine receptivity and decidualization.

    Science.gov (United States)

    Fullerton, Paul T; Monsivais, Diana; Kommagani, Ramakrishna; Matzuk, Martin M

    2017-06-13

    Embryo implantation remains a significant challenge for assisted reproductive technology, with implantation failure occurring in ∼50% of in vitro fertilization attempts. Understanding the molecular mechanisms underlying uterine receptivity will enable the development of new interventions and biomarkers. TGFβ family signaling in the uterus is critical for establishing and maintaining pregnancy. Follistatin (FST) regulates TGFβ family signaling by selectively binding TGFβ family ligands and sequestering them. In humans, FST is up-regulated in the decidua during early pregnancy, and women with recurrent miscarriage have lower endometrial expression of FST during the luteal phase. Because global knockout of Fst is perinatal lethal in mice, we generated a conditional knockout (cKO) of Fst in the uterus using progesterone receptor-cre to study the roles of uterine Fst during pregnancy. Uterine Fst-cKO mice demonstrate severe fertility defects and deliver only 2% of the number of pups delivered by control females. In Fst-cKO mice, the uterine luminal epithelium does not respond properly to estrogen and progesterone signals and remains unreceptive to embryo attachment by continuing to proliferate and failing to differentiate. The uterine stroma of Fst-cKO mice also responds poorly to artificial decidualization, with lower levels of proliferation and differentiation. In the absence of uterine FST, activin B expression and signaling are up-regulated, and bone morphogenetic protein (BMP) signals are impaired. Our findings support a model in which repression of activin signaling by FST enables uterine receptivity by preserving critical BMP signaling.

  11. MicroRNA-498 Inhibition Enhances the Differentiation of Human Adipose-Derived Mesenchymal Stem Cells into Podocyte-Like Cells.

    Science.gov (United States)

    Zhang, Lina; Li, Kanghua; Yan, Xi; Liang, Xiaolei; Wang, Shihua; Han, Qin; Zhao, Robert Chunhua

    2015-12-15

    Podocyte depletion is a key event in the progression of end-stage kidney disease (ESKD) resulting in nephrotic proteinuria and renal failure, but the treatment options are limited to dialysis and renal transplantation. So there is an urgent need for renal regenerative therapies. Generation of podocytes from human stem cells is regarded as a promising therapeutic strategy to repair or regenerate the damaged kidneys; however, the reliable induction system remains a challenge. In this study, we established a two-stage induction protocol for podocyte generation from human adipose-derived mesenchymal stem cells (hAD-MSCs). We initially established a condition that induces hAD-MSCs toward intermediate mesoderm cells with activin A and high concentration of retinoic acid (RA). Subsequently, by using the combination of activin A and low concentration of RA and BMP7, we generated podocyte-like cells expressing multiple podocyte-specific markers and able to integrate into a developing nephron of embryonic kidney explant culture and ameliorate proteinuria and kidney injure in adriamycin-treated mice. Furthermore, we identified that miRNA-498 inhibitor has potential to improve the differentiation of hAD-MSCs into podocyte-like cells and established a robust induction protocol. Thereby, our study advocated an efficient method for the induction of kidney podocyte-like (iPod) cells from hAD-MSCs and provided an ideal candidate for regenerative therapies of the kidney.

  12. Gene expression analysis in gonads and brain of catfish Clarias batrachus after the exposure of malathion.

    Science.gov (United States)

    Prathibha, Y; Murugananthkumar, R; Rajakumar, A; Laldinsangi, C; Sudhakumari, C C; Mamta, S K; Dutta-Gupta, A; Senthilkumaran, B

    2014-04-01

    Pesticides like malathion have the potential to disrupt development and reproduction of aquatic organisms including fishes. To investigate the likely consequences of malathion exposure at low doses in juvenile catfish, Clarias batrachus, we studied the expression pattern of genes encoding certain transcription factors, activin A, sex steroid or orphan nuclear receptors and steroidogenic enzymes which are known to be involved in gonadal development along with histological changes. To compare further, we also analyzed certain brain specific genes related to gonadal axis. Fifty days post hatch catfish fingerlings were exposed continuously to 1 and 10 µg/L of malathion for 21 days. Results from these experiments indicated that transcript levels of various genes were altered by the treatments, which may further affect the gonadal development either directly or indirectly through brain. Histological analysis revealed slow progression of spermatogenesis in testis, while in ovary, the oil droplet oocytes were found to be higher after treatment (10 µg/L). Our findings revealed that the exposure of malathion, even at low doses, hinder or modulate early gonadal development differentially by targeting gene expression pattern of transcription factors, activin A, sex steroid or orphan nuclear receptors and steroidogenic enzymes with an evidence on histological changes. Further, some of the genes showed differential expression at the level of brain in male and female sex after the exposure of malathion.

  13. [Regulation of in vitro and in vivo differentiation of mouse embryonic stem cells, embryonic germ cells, and teratocarcinoma cells by TGFb family signaling factors].

    Science.gov (United States)

    Gordeeva, O F; Nikonova, T M; Lifantseva, N V

    2009-01-01

    The activity of specific signaling and transcription factors determines the cell fate in normal development and in tumor transformation. The transcriptional profiles of gene-components of different branches of TGFbeta family signaling pathways were studied in experimental models of initial stages of three-dimensional in vitro differentiation of embryonic stem cells, embryonic germ cells and teratocarcinoma cells and in teratomas and teratocarcinomas developed after their transplantation into immunodeficient Nude mice. Gene profile analysis of studied cell systems have revealed that expression patterns of ActivinA, Nodal, Lefty1, Lefty2, TGF TGFbeta1, BMP4, and GDF were identical in pluripotent stem cells whereas the mRNAs of all examined genes with the exception of Inhibin betaA/ActivinA were detected in the teratocarcinoma cells. These results indicate that differential activity of signaling pathways of the TGFbeta family factors regulates pluripotent state maintenance and pluripotent stem cell differentiation into the progenitors of three germ layers and extraembryonic structures and that normal expression pattern of TGFbeta family factors is rearranged in embryonic teratocarcinoma cells during tumor growth in vitro and in vivo.

  14. A miR-590/Acvr2a/Rad51b Axis Regulates DNA Damage Repair during mESC Proliferation

    Directory of Open Access Journals (Sweden)

    Qidong Liu

    2014-12-01

    Full Text Available Embryonic stem cells (ESCs enable rapid proliferation that also causes DNA damage. To maintain genomic stabilization during rapid proliferation, ESCs must have an efficient system to repress genotoxic stress. Here, we show that withdrawal of leukemia inhibitory factor (LIF, which maintains the self-renewal capability of mouse ESCs (mESCs, significantly inhibits the cell proliferation and DNA damage of mESCs and upregulates the expression of miR-590. miR-590 promotes single-strand break (SSB and double-strand break (DSB damage repair, thus slowing proliferation of mESCs without influencing stemness. miR-590 directly targets Activin receptor type 2a (Acvr2a to mediate Activin signaling. We identified the homologous recombination-mediated repair (HRR gene, Rad51b, as a downstream molecule of the miR-590/Acvr2a pathway regulating the SSB and DSB damage repair and cell cycle. Our study shows that a miR-590/Acvr2a/Rad51b signaling axis ensures the stabilization of mESCs by balancing DNA damage repair and rapid proliferation during self-renewal.

  15. An interplay between extracellular signalling and the dynamics of the exit from pluripotency drives cell fate decisions in mouse ES cells

    Directory of Open Access Journals (Sweden)

    David A. Turner

    2014-06-01

    Full Text Available Embryonic Stem cells derived from the epiblast tissue of the mammalian blastocyst retain the capability to differentiate into any adult cell type and are able to self-renew indefinitely under appropriate culture conditions. Despite the large amount of knowledge that we have accumulated to date about the regulation and control of self-renewal, efficient directed differentiation into specific tissues remains elusive. In this work, we have analysed in a systematic manner the interaction between the dynamics of loss of pluripotency and Activin/Nodal, BMP4 and Wnt signalling in fate assignment during the early stages of differentiation of mouse ES cells in culture. During the initial period of differentiation, cells exit from pluripotency and enter an Epi-like state. Following this transient stage, and under the influence of Activin/Nodal and BMP signalling, cells face a fate choice between differentiating into neuroectoderm and contributing to Primitive Streak fates. We find that Wnt signalling does not suppress neural development as previously thought and that it aids both fates in a context dependent manner. Our results suggest that as cells exit pluripotency they are endowed with a primary neuroectodermal fate and that the potency to become endomesodermal rises with time. We suggest that this situation translates into a “race for fates” in which the neuroectodermal fate has an advantage.

  16. Comparative proteomic profiling of human osteoblast-derived extracellular matrices identifies proteins involved in mesenchymal stromal cell osteogenic differentiation and mineralization.

    Science.gov (United States)

    Baroncelli, Marta; van der Eerden, Bram C; Kan, Yik-Yang; Alves, Rodrigo D; Demmers, Jeroen A; van de Peppel, Jeroen; van Leeuwen, Johannes P

    2018-01-01

    The extracellular matrix (ECM) is a dynamic component of tissue architecture that physically supports cells and actively influences their behavior. In the context of bone regeneration, cell-secreted ECMs have become of interest as they reproduce tissue-architecture and modulate the promising properties of mesenchymal stem cells (MSCs). We have previously created an in vitro model of human osteoblast-derived devitalized ECM that was osteopromotive for MSCs. The aim of this study was to identify ECM regulatory proteins able to modulate MSC differentiation to broaden the spectrum of MSC clinical applications. To this end, we created two additional models of devitalized ECMs with different mineralization phenotypes. Our results showed that the ECM derived from osteoblast-differentiated MSCs had increased osteogenic potential compared to ECM derived from undifferentiated MSCs and non-ECM cultures. Proteomic analysis revealed that structural ECM proteins and ribosomal proteins were upregulated in the ECM from undifferentiated MSCs. A similar response profile was obtained by treating osteoblast-differentiating MSCs with Activin-A. Extracellular proteins were upregulated in Activin-A ECM, whereas mitochondrial and membrane proteins were downregulated. In summary, this study illustrates that the composition of different MSC-secreted ECMs is important to regulate the osteogenic differentiation of MSCs. These models of devitalized ECMs could be used to modulate MSC properties to regulate bone quality. © 2017 Wiley Periodicals, Inc.

  17. Multiple Signaling Pathways Control Tbx6 Expression during Xenopus Myogenesis

    Institute of Scientific and Technical Information of China (English)

    Pan-Feng FANG; Rui-Ying HU; Xing-Yue HE; Xiao-Yan DING

    2004-01-01

    Tbx6 is critical for somite specification and myogenesis initiation.It has been shown that Activin/Nodal,VegT/Nodal,FGF,and BMP signaling pathways are involved early in specifying mesoderm or later in patterning mesoderm,and Xnot plays roles in setting up the boundary between notochord and paraxial mesoderm.In this study,we introduce the dominant negative form of above genes into embryos to evaluate if they are responsible for regulating Tbx6 expression.The results show that: (1)Activin/Nodal and VegT/Nodal signals are necessary for both initiation and maintenance of Tbx6 expression,and Nodal is sufficient to induce ectopic Tbx6 expression;(2) FGF signal is necessary for the initiation and maintenance of Tbx6,but it is not sufficient to induce Tbx6 expression;(3) BMP is also necessary for the expression of Tbx6,and the induction of Tbx6 expression by BMP is dose dependent;(4) Xnot has no effect on the expression of Tbx6.Our results suggest that several signaling pathways are involved in regulating Tbx6expression,and pave the route to reveal the molecular mechanism of initiating myogenesis.

  18. L6E9 Myoblasts Are Deficient of Myostatin and Additional TGF- Members Are Candidates to Developmentally Control Their Fiber Formation

    Directory of Open Access Journals (Sweden)

    Stefania Rossi

    2010-01-01

    Full Text Available This work provides evidence that the robust myoblast differentiation observed in L6E9 cells is causally linked to deficiency of myostatin, which, conversely, has been found to be expressed in C2C12 cells. However, despite the absence of endogenous myostatin, L6E9 myoblasts expressed functional Activin receptors type II (ActRIIs and follistatin as well as the highly related TGF- members Activins and GDF11, suggesting that in this cell line the regulation of fiber size might be under the control of multiple regulators regardless of myostatin. In line with this hypothesis, delivery of a dominant-negative ActRIIb form or the increase of follistatin, as obtained via Trichostatin treatment or stable transfection of a short human follistatin form, enhanced the L6E9 cell differentiation and further increased the size of myotubes, suggesting that L6E9 myoblasts provide a spontaneous myostatin knock-out in vitro model to study TGF- ligands involved in developmental regulation of fiber size.

  19. Cholera Toxin Promotes Th17 Cell Differentiation by Modulating Expression of Polarizing Cytokines and the Antigen-Presenting Potential of Dendritic Cells.

    Directory of Open Access Journals (Sweden)

    Jung-Ok Kang

    Full Text Available Cholera toxin (CT, an exotoxin produced by Vibrio cholera, acts as a mucosal adjuvant. In a previous study, we showed that CT skews differentiation of CD4 T cells to IL-17-producing Th17 cells. Here, we found that intranasal administration of CT induced migration of migratory dendritic cell (DC populations, CD103+ DCs and CD11bhi DCs, to the lung draining mediastinal lymph nodes (medLN. Among those DC subsets, CD11bhi DCs that were relatively immature had a major role in Th17 cell differentiation after administration of CT. CT-treated BMDCs showed reduced expression of MHC class II and CD86, similar to CD11bhi DCs in medLN, and these BMDCs promoted Th17 cell differentiation more potently than other BMDCs expressing higher levels of MHC class II and CD86. By analyzing the expression of activation markers such as CD25 and CD69, proliferation and IL-2 production, we determined that CT-treated BMDCs showed diminished antigen-presenting potential to CD4+ T cells compared with normal BMDCs. We also found that CT-stimulated BMDCs promote activin A expression as well as IL-6 and IL-1β, and activin A had a synergic role with TGF-β1 in CT-mediated Th17 cell differentiation. Taken together, our results suggest that CT-stimulated DCs promote Th17 cell differentiation by not only modulating antigen-presenting potential but also inducing Th polarizing cytokines.

  20. Cholera Toxin Promotes Th17 Cell Differentiation by Modulating Expression of Polarizing Cytokines and the Antigen-Presenting Potential of Dendritic Cells

    Science.gov (United States)

    Kang, Jung-Ok; Lee, Jee-Boong

    2016-01-01

    Cholera toxin (CT), an exotoxin produced by Vibrio cholera, acts as a mucosal adjuvant. In a previous study, we showed that CT skews differentiation of CD4 T cells to IL-17-producing Th17 cells. Here, we found that intranasal administration of CT induced migration of migratory dendritic cell (DC) populations, CD103+ DCs and CD11bhi DCs, to the lung draining mediastinal lymph nodes (medLN). Among those DC subsets, CD11bhi DCs that were relatively immature had a major role in Th17 cell differentiation after administration of CT. CT-treated BMDCs showed reduced expression of MHC class II and CD86, similar to CD11bhi DCs in medLN, and these BMDCs promoted Th17 cell differentiation more potently than other BMDCs expressing higher levels of MHC class II and CD86. By analyzing the expression of activation markers such as CD25 and CD69, proliferation and IL-2 production, we determined that CT-treated BMDCs showed diminished antigen-presenting potential to CD4+ T cells compared with normal BMDCs. We also found that CT-stimulated BMDCs promote activin A expression as well as IL-6 and IL-1β, and activin A had a synergic role with TGF-β1 in CT-mediated Th17 cell differentiation. Taken together, our results suggest that CT-stimulated DCs promote Th17 cell differentiation by not only modulating antigen-presenting potential but also inducing Th polarizing cytokines. PMID:27271559

  1. Characterization of the nutritional endoderm in the direct developing frog Eleutherodactylus coqui.

    Science.gov (United States)

    Karadge, Uma; Elinson, Richard P

    2013-11-01

    Unlike Xenopus laevis, Eleutherodactylus coqui develops without a tadpole. The yolk-rich vegetal region of the embryo forms a transient nutritive tissue, the nutritional endoderm (NE). The definitive endoderm (DE) in E. coqui comes from cells closer to the animal pole in contrast to its vegetal origin in X. laevis. RNA important for initiating the endoderm specification network is absent in presumptive NE cells, raising the question whether signaling occurs in them. We explored the nature of NE and asked how differences between NE and DE cells arise. We identified differences between NE and DE that first become evident at gastrula, when NE cells become multinucleated. Nuclear β-catenin, an essential cofactor of sox 17, important for endoderm formation in X. laevis, is present in NE and DE at gastrula but remains in NE long after it is not seen in DE. We cloned E. coqui homologs of TGFβs activin b and derriere and provide evidence for their maternal expression. We also detected activin b and derriere RNAs in NE at gastrula and show that NE possesses some mesoderm-inducing activity, but it is delayed with respect to DE. Our findings indicate that altered development of NE begins at gastrula. RNAs important for mesendoderm induction and some mesoderm-inducing activity are present in NE.

  2. Chronic alcohol ingestion exacerbates skeletal muscle myopathy in HIV-1 transgenic rats

    Directory of Open Access Journals (Sweden)

    Bratina Margaux A

    2011-08-01

    Full Text Available Abstract Background Separately, chronic alcohol ingestion and HIV-1 infection are associated with severe skeletal muscle derangements, including atrophy and wasting, weakness, and fatigue. One prospective cohort study reported that 41% of HIV-infected patients met the criteria for alcoholism, however; few reports exist on the co-morbid effects of these two disease processes on skeletal muscle homeostasis. Thus, we analyzed the atrophic effects of chronic alcohol ingestion in HIV-1 transgenic rats and identified alterations to several catabolic and anabolic factors. Findings Relative plantaris mass, total protein content, and fiber cross-sectional area were reduced in each experimental group compared to healthy, control-fed rats. Alcohol abuse further reduced plantaris fiber area in HIV-1 transgenic rats. Consistent with previous reports, gene levels of myostatin and its receptor activin IIB were not increased in HIV-1 transgenic rat muscle. However, myostatin and activin IIB were induced in healthy and HIV-1 transgenic rats fed alcohol for 12 weeks. Catabolic signaling factors such as TGFβ1, TNFα, and phospho-p38/total-p38 were increased in all groups compared to controls. There was no effect on IL-6, leukemia inhibitory factor (LIF, cardiotrophin-1 (CT-1, or ciliary neurotrophic factor (CNTF in control-fed, transgenic rats. However, the co-morbidity of chronic alcohol abuse and HIV-1-related protein expression decreased expression of the two anabolic factors, CT-1 and CNTF. Conclusions Consistent with previous reports, alcohol abuse accentuated skeletal muscle atrophy in an animal model of HIV/AIDS. While some catabolic pathways known to drive alcoholic or HIV-1-associated myopathies were also elevated in this co-morbid model (e.g., TGFβ1, consistent expression patterns were not apparent. Thus, specific alterations to signaling mechanisms such as the induction of the myostatin/activin IIB system or reductions in growth factor signaling via

  3. Effect of the probiotic Lactobacillus rhamnosus on the expression of genes involved in European eel spermatogenesis.

    Science.gov (United States)

    Vílchez, M Carmen; Santangeli, Stefania; Maradonna, Francesca; Gioacchini, Giorgia; Verdenelli, Cristina; Gallego, Victor; Peñaranda, David S; Tveiten, Helge; Pérez, Luz; Carnevali, Oliana; Asturiano, Juan F

    2015-11-01

    Positive effects of probiotics on fish reproduction have been reported in several species. In the present study, 40 male European eels were weekly treated with recombinant hCG for 9 weeks and with three different concentrations (10(3), 10(5), and 10(6) CFU/mL) of probiotic Lactobacillus rhamnosus IMC 501 (Sinbyotec, Italy). The probiotics were daily added to the water from the sixth week of the hCG treatment. Males from the treated and control groups were sacrificed after 1, 2, and 3 weeks of probiotic treatment (seventh-ninth weeks of hCG treatment); at this point, sperm and testis samples were also collected. Sperm volume was estimated, and motility was analyzed by computer-assisted sperm analysis software. Alternations in transcription of specific genes involved in reproductive process such as activin, androgen receptors α and β (arα and arβ), progesterone receptor 1 (pr1), bone morphogenetic protein 15 (bmp15), and FSH receptor (fshr) were analyzed in the testis. After 2 weeks of probiotic treatment, sperm production and sperm motility parameters (percentage of motile cells and percentage of straight-swimming spermatozoa) were increased in the European eel treated with 10(5) CFU/mL compared to controls or to the other probiotic doses. These changes were associated with increases in messenger RNA expression of activin, arα, arβ, pr1, and fshr. Conversely, after 3 weeks, activin and pr1 expression decreased. No significant changes were observed on bmp15 expression throughout the duration of the treatment with 10(5) CFU/mL dose. The lowest and highest probiotic dose (10(3) and 10(6) CFU/mL, respectively) inhibited the transcription of all genes along all the experiment, except for arα and arβ after 1 week of probiotic treatment when compared to controls. The changes observed by transcriptomic analysis and the sperm parameters suggest that a treatment with L rhamnosus at 10(5) CFU/mL for 2 weeks could improve spermatogenesis process in Anguilla

  4. TGF-β Signaling Transduetion and Carcinoma%TGF-β信号转导与肿瘤

    Institute of Scientific and Technical Information of China (English)

    王守立; 杨光华; 步宏

    2004-01-01

    转化生长因子β(transforming growth factors—β,TGF—β)是一类多功能的多肽类生长因子,主要包括TGF-βs、激活素(activin)/抑制素(inhibins)、骨形态形成蛋白(bone morphogenetic proteins,BMPs)等三大类40多个成员,其中研究较多的是TGF-βs,包括TGF—β1至TGF—β5。近年来,大量文献报道TGF—β信号传导通路的异常与肿瘤的发生、发展、转移等密切相关,现就TGF—β信号传导通路中主要成分与肿瘤的关系做如下综述.

  5. Follistatin288 Regulates Germ Cell Cyst Breakdown and Primordial Follicle Assembly in the Mouse Ovary.

    Science.gov (United States)

    Wang, Zhengpin; Niu, Wanbao; Wang, Yijing; Teng, Zhen; Wen, Jia; Xia, Guoliang; Wang, Chao

    2015-01-01

    In mammals, the primordial follicle pool represents the entire reproductive potential of a female. The transforming growth factor-β (TGF-β) family member activin (ACT) contributes to folliculogenesis, although the exact mechanism is not known. The role of FST288, the strongest ACT-neutralizing isoform of follistatin (FST), during cyst breakdown and primordial follicle formation in the fetal mice ovary was assessed using an in vitro culture system. FST was continuously expressed in the oocytes as well as the cuboidal granulosa cells of growing follicles in perinatal mouse ovaries. Treatment with FST288 delayed germ cell nest breakdown, particularly near the periphery of the ovary, and dramatically decreased the percentage of primordial follicles. In addition, there was a dramatic decrease in proliferation of granulosa cells and somatic cell expression of Notch signaling was impaired. In conclusion, FST288 impacts germ cell nest breakdown and primordial follicle assembly by inhibiting somatic cell proliferation.

  6. Treating cancer cachexia to treat cancer

    Directory of Open Access Journals (Sweden)

    Lee Se-Jin

    2011-01-01

    Full Text Available Abstract Skeletal muscle wasting is a major component of cachectic states found in a variety of disease settings, including cancer. As increasing caloric intake often provides little benefit in combating muscle loss in cachectic patients, a major research focus has been to develop strategies stimulating muscle anabolic pathways - in an attempt to fight the catabolic pathways induced during cachexia. Two recent papers have reported the beneficial effects of blocking the myostatin/activin signalling pathway in mouse models of cancer cachexia. We discuss the implications of their findings both with respect to the role that this signalling pathway may play in the aetiology of cachexia and with respect to the prospects for targeting this pathway as a therapeutic strategy in patients with cachexia.

  7. Coco regulates dorsoventral specification of germ layers via inhibition of TGFβ signalling

    Science.gov (United States)

    Bates, Thomas J. D.; Vonica, Alin; Heasman, Janet; Brivanlou, Ali H.; Bell, Esther

    2013-01-01

    One of the earliest steps in embryonic development is the specification of the germ layers, the subdivision of the blastula embryo into endoderm, mesoderm and ectoderm. Maternally expressed members of the Transforming Growth Factor β (TGFβ) family influence all three germ layers; the ligands are required to induce endoderm and mesoderm, whereas inhibitors are required for formation of the ectoderm. Here, we demonstrate a vital role for maternal Coco, a secreted antagonist of TGFβ signalling, in this process. We show that Coco is required to prevent Activin and Nodal signals in the dorsal marginal side of the embryo from invading the prospective ectoderm, thereby restricting endoderm- and mesoderm-inducing signals to the vegetal and marginal zones of the pre-gastrula Xenopus laevis embryo. PMID:24026124

  8. Coco is a dual activity modulator of TGFβ signaling

    Science.gov (United States)

    Deglincerti, Alessia; Haremaki, Tomomi; Warmflash, Aryeh; Sorre, Benoit; Brivanlou, Ali H.

    2015-01-01

    The TGFβ signaling pathway is a crucial regulator of developmental processes and disease. The activity of TGFβ ligands is modulated by various families of soluble inhibitors that interfere with the interactions between ligands and receptors. In an unbiased, genome-wide RNAi screen to identify genes involved in ligand-dependent signaling, we unexpectedly identified the BMP/Activin/Nodal inhibitor Coco as an enhancer of TGFβ1 signaling. Coco synergizes with TGFβ1 in both cell culture and Xenopus explants. Molecularly, Coco binds to TGFβ1 and enhances TGFβ1 binding to its receptor Alk5. Thus, Coco acts as both an inhibitor and an enhancer of signaling depending on the ligand it binds. This finding raises the need for a global reconsideration of the molecular mechanisms regulating TGFβ signaling. PMID:26116664

  9. Changes in Blood Components in Aphtha Patients with Excess Heat.

    Science.gov (United States)

    Qin, Lu; Li, Yan; Jiao, Yifeng; Fu, Danqing; Ye, Li; Ji, Jinjun; Xie, Guanqun; Fan, Yongsheng; Xu, Li

    2016-01-01

    "Superior heat" is a popularization expression in TCM heat syndrome and has no counterpart in the modern medical system concept. Oral ulcer is considered to be a kind of clinical manifestation of "superior heat." Aphtha is a common and frequently occurring disease, which can be divided into excess heat and Yin deficiency. The aphtha of excess heat manifests the syndromes of acute occurrence, severe local symptoms, obvious swelling and pain, red tongue, yellow coating, and fast-powerful pulse. In this study, we found that there was an abnormal immune regulation in aphtha patients induced by excess heat. There are changes in the blood components, including abnormal serum protein expression (IL-4, MMP-19, MMP-9, and Activin A) and a higher percentage of CD4(+)CD25(+)Treg cells in the peripheral blood lymphocytes of the EXP group. Changes in the blood environment may be an important factor in the occurrence of aphtha caused by excess heat.

  10. Identification of maverick, a novel member of the TGF-beta superfamily in Drosophila.

    Science.gov (United States)

    Nguyen, M; Parker, L; Arora, K

    2000-07-01

    The transforming growth factor-beta (TGF-beta) superfamily of structurally related ligands regulates essential signaling pathways that control many aspects of cell behavior in organisms across the phylogenetic spectrum. Here we report the identification of maverick (mav), a gene that encodes a new member of the TGF-beta superfamily in Drosophila. Phylogenetic analysis and sequence comparison suggest that Mav cannot be easily assigned to any one sub-family, since it is equally related to BMP, activin and TGF-beta ligands. mav maps to the fourth chromosome and is expressed throughout development. In situ hybridization experiments reveal the presence of maternally derived mav transcript in precellular blastoderm embryos. Later in development, mav is expressed in a dynamic pattern in the developing gut, both in endodermal and visceral mesodermal cells. In adult females, high levels of mav mRNA are present in late stage egg chambers.

  11. Cachexia.

    Science.gov (United States)

    Graul, A I; Stringer, M; Sorbera, L

    2016-09-01

    Cachexia is a multiorgan, multifactorial and often irreversible wasting syndrome associated with cancer and other serious, chronic illnesses including AIDS, chronic heart failure, chronic kidney disease and chronic obstructive pulmonary disease. Treatment of the patient with cachexia is currently targeted to correcting the two underlying features of the condition: anorexia and metabolic disturbances. Greater understanding of the mechanisms behind cachexia and muscle wasting have led to new therapeutic possibilities, however. Several classes of drugs are under active development for cachexia including drugs acting on hormone receptors or cytokine receptors, myostatin/activin pathway antagonists, beta-adrenoceptor agonists and cannabinoids. This review will cover the pathophysiology, epidemiology, diagnosis, treatment, drug candidates under active development and targets for therapeutic intervention of cachexia.

  12. Isolation and Characterization of Node/Notochord-like Cells from Mouse Embryonic Stem Cells

    DEFF Research Database (Denmark)

    Winzi, Maria Karin; Maddox-Hyttel, Poul; Dale, J Kim

    2011-01-01

    The homeobox gene Noto is expressed in the node and its derivative the notochord. Here we use a targeted Noto-GFP reporter to isolate and characterize node/notochord-like cells derived from mouse embryonic stem cells. We find very few Noto-expressing cells after spontaneous differentiation. However......, the number of Noto-expressing cells was increased when using Activin A to induce a Foxa2- and Brachyury-expressing progenitor population, whose further differentiation into Noto-expressing cells was improved by simultaneous inhibition of BMP, Wnt, and retinoic acid signaling. Noto-GFP(+) cells expressed...... the node/notochord markers Noto, Foxa2, Shh, Noggin, Chordin, Foxj1, and Brachyury; showed a vacuolarization characteristic of notochord cells; and can integrate into midline structures when grafted into Hensen's node of gastrulating chicken embryos. The ability to generate node/notochord-like cells...

  13. Generation of integration free induced pluripotent stem cells from fibrodysplasia ossificans progressiva (FOP) patients from urine samples.

    Science.gov (United States)

    Hildebrand, Laura; Rossbach, Bella; Kühnen, Peter; Gossen, Manfred; Kurtz, Andreas; Reinke, Petra; Seemann, Petra; Stachelscheid, Harald

    2016-01-01

    Fibrodysplasia ossificans progressiva (FOP) is an extremely rare, autosomal dominant transmitted genetic disease. Patients experience progressive bone formation replacing tendons, ligaments, muscle and soft tissue. Cause of FOP are gain-of-function mutations in the Bone Morphogenetic Protein (BMP) receptor Activin A receptor type 1 (ACVR1) (Kaplan et al., 2008). The most common mutation is R206H, which leads to the substitution of codon 206 from arginine to histidine (Shore et al., 2006). Here, we describe the derivation and characterization of two hiPSC lines from two FOP patients, both carrying the mutation R206H. Cells were isolated from urine and reprogrammed using integration free Sendai virus vectors under defined conditions.

  14. Follistatin288 Regulates Germ Cell Cyst Breakdown and Primordial Follicle Assembly in the Mouse Ovary.

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    Zhengpin Wang

    Full Text Available In mammals, the primordial follicle pool represents the entire reproductive potential of a female. The transforming growth factor-β (TGF-β family member activin (ACT contributes to folliculogenesis, although the exact mechanism is not known. The role of FST288, the strongest ACT-neutralizing isoform of follistatin (FST, during cyst breakdown and primordial follicle formation in the fetal mice ovary was assessed using an in vitro culture system. FST was continuously expressed in the oocytes as well as the cuboidal granulosa cells of growing follicles in perinatal mouse ovaries. Treatment with FST288 delayed germ cell nest breakdown, particularly near the periphery of the ovary, and dramatically decreased the percentage of primordial follicles. In addition, there was a dramatic decrease in proliferation of granulosa cells and somatic cell expression of Notch signaling was impaired. In conclusion, FST288 impacts germ cell nest breakdown and primordial follicle assembly by inhibiting somatic cell proliferation.

  15. Hepatic differentiation of porcine embryonic stem cells for translational research of hepatocyte transplantation.

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    Park, K M; Hussein, K H; Ghim, J H; Ahn, C; Cha, S H; Lee, G S; Hong, S H; Yang, S; Woo, H M

    2015-04-01

    Porcine embryonic stem cells (ES) are considered attractive preclinical research tools for human liver diseases. Although several studies previously reported generation of porcine ES, none of these studies has described hepatic differentiation from porcine ES. The aim of this study was to generate hepatocytes from porcine ES and analyze their characteristics. We optimized conditions for definitive endoderm induction and developed a 4-step hepatic differentiation protocol. A brief serum-free condition with activin A efficiently induced definitive endoderm differentiation from porcine ES. The porcine ES-derived hepatocyte-like cells highly expressed hepatic markers including albumin and α-fetoprotein, and displayed liver characteristics such as glycogen storage, lipid production, and low-density lipoprotein uptake. For the first time, we describe a highly efficient protocol for hepatic differentiation from porcine ES. Our findings provide valuable information for translational liver research using porcine models, including hepatic regeneration and transplant studies, drug screening, and toxicology.

  16. TAp63 is important for cardiac differentiation of embryonic stem cells and heart development.

    Science.gov (United States)

    Rouleau, Matthieu; Medawar, Alain; Hamon, Laurent; Shivtiel, Shoham; Wolchinsky, Zohar; Zhou, Huiqing; De Rosa, Laura; Candi, Eleonora; de la Forest Divonne, Stéphanie; Mikkola, Marja L; van Bokhoven, Hans; Missero, Caterina; Melino, Gerry; Pucéat, Michel; Aberdam, Daniel

    2011-11-01

    p63, a member of the p53 family, is essential for skin morphogenesis and epithelial stem cell maintenance. Here, we report an unexpected role of TAp63 in cardiogenesis. p63 null mice exhibit severe defects in embryonic cardiac development, including dilation of both ventricles, a defect in trabeculation and abnormal septation. This was accompanied by myofibrillar disarray, mitochondrial disorganization, and reduction in spontaneous calcium spikes. By the use of embryonic stem cells (ESCs), we show that TAp63 deficiency prevents expression of pivotal cardiac genes and production of cardiomyocytes. TAp63 is expressed by endodermal cells. Coculture of p63-knockdown ESCs with wild-type ESCs, supplementation with Activin A, or overexpression of GATA-6 rescue cardiogenesis. Therefore, TAp63 acts in a non-cell-autonomous manner by modulating expression of endodermal factors. Our findings uncover a critical role for p63 in cardiogenesis that could be related to human heart disease.

  17. Molecular signaling networks that choreograph epimorphic fin regeneration in zebrafish - a mini-review.

    Science.gov (United States)

    Tal, Tamara L; Franzosa, Jill A; Tanguay, Robert L

    2010-01-01

    This short review provides a current synopsis of caudal fin regeneration in zebrafish with an emphasis on the molecular signaling networks that dictate epimorphic regeneration. At the outset, the fundamentals of caudal fin architecture and the stages of epimorphic regeneration are described. This is followed by a detailed look at the main networks implicated in fin regeneration, namely the Wnt, fibroblast growth factor, activin-betaA, retinoic acid and hedgehog signaling pathways. Throughout this mini-review, these molecular networks are examined through the lens of wound healing, blastema formation or regenerative outgrowth, three of the main stages of epimorphic regeneration. Next, the emerging role of noncoding RNAs as regulators of regeneration and mechanisms of regenerative termination are discussed. Finally, the implications for future research and the broader field of regenerative medicine are examined.

  18. Isolation and characterization of node/notochord-like cells from mouse embryonic stem cells.

    Science.gov (United States)

    Winzi, Maria K; Hyttel, Poul; Dale, Jacqueline Kim; Serup, Palle

    2011-11-01

    The homeobox gene Noto is expressed in the node and its derivative the notochord. Here we use a targeted Noto-GFP reporter to isolate and characterize node/notochord-like cells derived from mouse embryonic stem cells. We find very few Noto-expressing cells after spontaneous differentiation. However, the number of Noto-expressing cells was increased when using Activin A to induce a Foxa2- and Brachyury-expressing progenitor population, whose further differentiation into Noto-expressing cells was improved by simultaneous inhibition of BMP, Wnt, and retinoic acid signaling. Noto-GFP(+) cells expressed the node/notochord markers Noto, Foxa2, Shh, Noggin, Chordin, Foxj1, and Brachyury; showed a vacuolarization characteristic of notochord cells; and can integrate into midline structures when grafted into Hensen's node of gastrulating chicken embryos. The ability to generate node/notochord-like cells in vitro will aid the biochemical characterization of these developmentally important structures.

  19. The daf-4 gene encodes a bone morphogenetic protein receptor controlling C. elegans dauer larva development.

    Science.gov (United States)

    Estevez, M; Attisano, L; Wrana, J L; Albert, P S; Massagué, J; Riddle, D L

    1993-10-14

    The bone morphogenetic protein (BMP) family is a conserved group of signalling molecules within the transforming growth factor-beta (TGF-beta) superfamily. This group, including the Drosophila decapentaplegic (dpp) protein and the mammalian BMPs, mediates cellular interactions and tissue differentiation during development. Here we show that a homologue of human BMPs controls a developmental switch in the life cycle of the free-living soil nematode Caenorhabditis elegans. Starvation and overcrowding induce C. elegans to form a developmentally arrested, third-stage dauer larva. The daf-4 gene, which acts to inhibit dauer larva formation and promote growth, encodes a receptor protein kinase similar to the daf-1, activin and TGF-beta receptor serine/threonine kinases. When expressed in monkey COS cells, the daf-4 receptor binds human BMP-2 and BMP-4. The daf-4 receptor is the first to be identified for any growth factor in the BMP family.

  20. Chordin is required for the Spemann organizer transplantation phenomenon in Xenopus embryos.

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    Oelgeschläger, Michael; Kuroda, Hiroki; Reversade, Bruno; De Robertis, E M

    2003-02-01

    We analyzed the Chordin requirement in Xenopus development. Targeting of both chordin Xenopus laevis pseudoalleles with morpholino antisense oligomers (Chd-MO) markedly decreased Chordin production. Embryos developed with moderately reduced dorsoanterior structures and expanded ventroposterior tissues, phenocopying the zebrafish chordino mutant. A strong requirement for Chordin in dorsal development was revealed by experimental manipulations. First, dorsalization by lithium chloride treatment was completely blocked by Chd-MO. Second, Chd-MO inhibited elongation and muscle differentiation in Activin-treated animal caps. Third, Chd-MO completely blocked the induction of the central nervous system (CNS), somites, and notochord by organizer tissue transplanted to the ventral side of host embryos. Unexpectedly, transplantations into the dorsal side revealed a cell-autonomous requirement of Chordin for neural plate differentiation.

  1. Partial promoter substitutions generating transcriptional sentinels of diverse signaling pathways in embryonic stem cells and mice

    Science.gov (United States)

    Serup, Palle; Gustavsen, Carsten; Klein, Tino; Potter, Leah A.; Lin, Robert; Mullapudi, Nandita; Wandzioch, Ewa; Hines, Angela; Davis, Ashley; Bruun, Christine; Engberg, Nina; Petersen, Dorthe R.; Peterslund, Janny M. L.; MacDonald, Raymond J.; Grapin-Botton, Anne; Magnuson, Mark A.; Zaret, Kenneth S.

    2012-01-01

    SUMMARY Extracellular signals in development, physiology, homeostasis and disease often act by regulating transcription. Herein we describe a general method and specific resources for determining where and when such signaling occurs in live animals and for systematically comparing the timing and extent of different signals in different cellular contexts. We used recombinase-mediated cassette exchange (RMCE) to test the effect of successively deleting conserved genomic regions of the ubiquitously active Rosa26 promoter and substituting the deleted regions for regulatory sequences that respond to diverse extracellular signals. We thereby created an allelic series of embryonic stem cells and mice, each containing a signal-responsive sentinel with different fluorescent reporters that respond with sensitivity and specificity to retinoic acids, bone morphogenic proteins, activin A, Wnts or Notch, and that can be adapted to any pathway that acts via DNA elements. PMID:22888097

  2. The expression pattern of Xenopus Mox-2 implies a role in initial mesodermal differentiation.

    Science.gov (United States)

    Candia, A F; Wright, C V

    1995-07-01

    We have isolated a Xenopus homolog of the murine Mox-2 gene. As is the case for the mouse homolog, mesoderm specific expression of Xenopus Mox-2 (X. Mox-2) expression begins during gastrulation. Using whole mount in situ hybridization, we show that X. Mox-2 is expressed in undifferentiated dorsal, lateral and ventral mesoderm in the posterior of neurula/tailbud embryos, with expression more anteriorly detected in the dermatomes. In the tailbud tadpole, X. Mox-2 is expressed in tissues of the tailbud itself that represent a site of continued gastrulation-like processes resulting in mesoderm formation. X. Mox-2 is not expressed in the marginal zone of blastula, nor in the dorsal lip of gastrula, nor midline tissues (i.e. prospective notochord). Treatments that affect mesodermal patterning during embryonic development, including LiCl and ultraviolet light, and injection of mRNAs encoding BMP-4, or dominant negative activin and FGF receptors, produce changes in X. Mox-2 expression consistent with the types of tissues affected by these manipulations. X. Mox-2 expression is induced more in animal caps treated with FGF than those treated with activin. Together with the fact that X. Mox-2 activation in animal caps requires protein synthesis, our data suggest that X. Mox-2 is involved in initial mesodermal differentiation, downstream of molecules affecting mesoderm induction and determination such as Brachyury and goosecoid, and upstream of factors controlling terminal differentiation such as MyoD and myf5. X. Mox-2, therefore, is another useful marker for understanding the formation of mesoderm in amphibian development.

  3. Recombinant myostatin reduces highly expressed microRNAs in differentiating C2C12 cells

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    Zachary A. Graham

    2017-03-01

    Full Text Available Myostatin is small glycopeptide that is produced and secreted by skeletal muscle. It is a potent negative regulator of muscle growth that has been associated with conditions of frailty. In C2C12 cells, myostatin limits cell differentiation. Myostatin acts through activin receptor IIB, activin receptor-like kinase (ALK and Smad transcription factors. microRNAs (miRNA are short, 22 base pair nucleotides that bind to the 3′ UTR of target mRNA to repress translation or reduce mRNA stability. In the present study, expression in differentiating C2C12 cells of the myomiRs miR-1 and 133a were down-regulated following treatment with 1 µg of recombinant myostatin at 1 d post-induction of differentiation while all myomiRs (miR-1, 133a/b and 206 were upregulated by SB431542, a potent ALK4/5/7 inhibitor which reduces Smad2 signaling, at 1 d and all, with the exception of miR-206, were upregulated by SB431542 at 3 d. The expression of the muscle-enriched miR-486 was greater following treatment with SB431542 but not altered by myostatin. Other highly expressed miRNAs in skeletal muscle, miR-23a/b and 145, were altered only at 1 d post-induction of differentiation. miR-27b responded differently to treatments at 1 d, where it was upregulated, as compared to 3 d, where it was downregulated. Neither myostatin nor SB431542 altered cell size or cell morphology. The data indicate that myostatin represses myomiR expression in differentiating C2C12 cells and that inhibition of Smad signaling with SB431542 can result in large changes in highly expressed miRNAs in differentiating myoblasts.

  4. The interactions between the stimulatory effect of follicle-stimulating hormone and the inhibitory effect of estrogen on mouse primordial folliculogenesis.

    Science.gov (United States)

    Lei, Lei; Jin, Shiying; Mayo, Kelly E; Woodruff, Teresa K

    2010-01-01

    The murine primordial follicle pool develops largely within 3 days after birth through germline nest breakdown and enclosure of oocytes within pregranulosa cells. The mechanisms that trigger primordial follicle formation likely are influenced by a transition from the maternal to fetal hormonal milieu at the time of birth. High levels of maternal estrogen maintain intact germline nest in fetal ovary, and decrease of estrogen after birth is permissive of follicle formation. In the present study, we measured an increase in neonatal serum follicle-stimulating hormone (FSH), which corresponded to falling estradiol (E(2)) levels during the critical window of primordial follicle formation (Postnatal Days 1-3). To determine whether fetal hormones contribute in an active manner to primordial follicle formation, mouse fetal ovaries (17.5 days postcoitus) were cultured in vitro at two concentrations of E(2) (meant to reflect maternal and fetal levels of E(2)) and FSH for 6 days. High levels of E(2) (10(-6) M) inhibited germline nest breakdown, and this effect was significantly reduced when fetal ovaries were cultured in the low E(2) concentration (10(-10) M). FSH facilitated germline nest breakdown and primordial follicle formation under both high and low E(2) culture conditions. Low E(2) was identified as being more permissive for the effects of FSH on primordial follicle formation by stimulating the up-regulation of Fshr and activin beta A subunit (Inhba) expression, pregranulosa cell proliferation, and oocyte survival. The decrease of E(2) plus the presence of FSH after birth are critical for primordial follicle formation and the expression of oocyte-specific transcription factors (Figla and Nobox) in that inappropriate exposure to FSH or E(2) during follicle formation resulted in premature or delayed primordial folliculogenesis. In conclusion, with the drop of E(2) level after birth, FSH promotes primordial follicle formation in mice by stimulating local activin signaling

  5. The Xenopus receptor tyrosine kinase Xror2 modulates morphogenetic movements of the axial mesoderm and neuroectoderm via Wnt signaling.

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    Hikasa, Hiroki; Shibata, Mikihito; Hiratani, Ichiro; Taira, Masanori

    2002-11-01

    The Spemann organizer plays a central role in neural induction, patterning of the neuroectoderm and mesoderm, and morphogenetic movements during early embryogenesis. By seeking genes whose expression is activated by the organizer-specific LIM homeobox gene Xlim-1 in Xenopus animal caps, we isolated the receptor tyrosine kinase Xror2. Xror2 is expressed initially in the dorsal marginal zone, then in the notochord and the neuroectoderm posterior to the midbrain-hindbrain boundary. mRNA injection experiments revealed that overexpression of Xror2 inhibits convergent extension of the dorsal mesoderm and neuroectoderm in whole embryos, as well as the elongation of animal caps treated with activin, whereas it does not appear to affect cell differentiation of neural tissue and notochord. Interestingly, mutant constructs in which the kinase domain was point-mutated or deleted (named Xror2-TM) also inhibited convergent extension, and did not counteract the wild-type, suggesting that the ectodomain of Xror2 per se has activities that may be modulated by the intracellular domain. In relation to Wnt signaling for planar cell polarity, we observed: (1) the Frizzled-like domain in the ectodomain is required for the activity of wild-type Xror2 and Xror2-TM; (2) co-expression of Xror2 with Xwnt11, Xfz7, or both, synergistically inhibits convergent extension in embryos; (3) inhibition of elongation by Xror2 in activin-treated animal caps is reversed by co-expression of a dominant negative form of Cdc42 that has been suggested to mediate the planar cell polarity pathway of Wnt; and (4) the ectodomain of Xror2 interacts with Xwnts in co-immunoprecipitation experiments. These results suggest that Xror2 cooperates with Wnts to regulate convergent extension of the axial mesoderm and neuroectoderm by modulating the planar cell polarity pathway of Wnt.

  6. Biomarkers of brain injury in the premature infant

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    Martha V. Douglas-Escobar

    2013-01-01

    Full Text Available The term encephalopathy of prematurity encompasses not only the acute brain injury (such as intraventricular hemorrhage but also complex disturbance on the infant’s subsequent brain development. In premature infants, the most frequent recognized source of brain injury is intraventricular hemorrhage (IVH and periventricular leukomalacia (PVL. Furthermore 20-25% infants with birth weigh less than 1,500 g will have IVH and that proportion increases to 45% if the birth weight is less than 500-750 g. In addition, nearly 60% of very low birth weight newborns will have hypoxic-ischemic injury. Therefore permanent lifetime neurodevelopmental disabilities are frequent in premature infants. Innovative approach to prevent or decrease brain injury in preterm infants requires discovery of biomarkers able to discriminate infants at risk for injury, monitor the progression of the injury and assess efficacy of neuroprotective clinical trials. In this article, we will review biomarkers studied in premature infants with IVH, Post-hemorrhagic ventricular dilation (PHVD and PVL including: S100b, Activin A, erythropoietin, chemokine CCL 18, GFAP and NFL will also be examined. Some of the most promising biomarkers for IVH are S100β and Activin. The concentrations of TGF-β1, MMP-9 and PAI-1 in cerebrospinal fluid could be used to discriminate patients that will require shunt after post-hemorrhagic ventricular dilation. Neonatal brain injury is frequent in premature infants admitted to the neonatal intensive care and we hope to contribute to the awareness and interest in clinical validation of established as well as novel neonatal brain injury biomarkers.

  7. Biomarkers of brain injury in the premature infant.

    Science.gov (United States)

    Douglas-Escobar, Martha; Weiss, Michael D

    2012-01-01

    The term "encephalopathy of prematurity" encompasses not only the acute brain injury [such as intraventricular hemorrhage (IVH)] but also complex disturbance on the infant's subsequent brain development. In premature infants, the most frequent recognized source of brain injury is IVH and periventricular leukomalacia (PVL). Furthermore 20-25% infants with birth weigh less than 1,500 g will have IVH and that proportion increases to 45% if the birth weight is less than 500-750 g. In addition, nearly 60% of very low birth weight newborns will have hypoxic-ischemic injury. Therefore permanent lifetime neurodevelopmental disabilities are frequent in premature infants. Innovative approach to prevent or decrease brain injury in preterm infants requires discovery of biomarkers able to discriminate infants at risk for injury, monitor the progression of the injury, and assess efficacy of neuroprotective clinical trials. In this article, we will review biomarkers studied in premature infants with IVH, Post-hemorrhagic ventricular dilation (PHVD), and PVL including: S100b, Activin A, erythropoietin, chemokine CCL 18, GFAP, and NFL will also be examined. Some of the most promising biomarkers for IVH are S100β and Activin. The concentrations of TGF-β1, MMP-9, and PAI-1 in cerebrospinal fluid could be used to discriminate patients that will require shunt after PHVD. Neonatal brain injury is frequent in premature infants admitted to the neonatal intensive care and we hope to contribute to the awareness and interest in clinical validation of established as well as novel neonatal brain injury biomarkers.

  8. The TGF-β pathway mediates doxorubicin effects on cardiac endothelial cells.

    Science.gov (United States)

    Sun, Zuyue; Schriewer, Jill; Tang, Mingxin; Marlin, Jerry; Taylor, Frederick; Shohet, Ralph V; Konorev, Eugene A

    2016-01-01

    Elevated ALK4/5 ligands including TGF-β and activins have been linked to cardiovascular remodeling and heart failure. Doxorubicin (Dox) is commonly used as a model of cardiomyopathy, a condition that often precedes cardiovascular remodeling and heart failure. In 7-8-week-old C57Bl/6 male mice treated with Dox we found decreased capillary density, increased levels of ALK4/5 ligand and Smad2/3 transcripts, and increased expression of Smad2/3 transcriptional targets. Human cardiac microvascular endothelial cells (HCMVEC) treated with Dox also showed increased levels of ALK4/5 ligands, Smad2/3 transcriptional targets, a decrease in proliferation and suppression of vascular network formation in a HCMVEC and human cardiac fibroblasts co-culture assay. Our hypothesis is that the deleterious effects of Dox on endothelial cells are mediated in part by the activation of the TGF-β pathway. We used the inhibitor of ALK4/5 kinases SB431542 (SB) in concert with Dox to ascertain the role of TGF-β pathway activation in doxorubicin induced endothelial cell defects. SB prevented the suppression of HCMVEC proliferation in the presence of TGF-β2 and activin A, and alleviated the inhibition of HCMVEC proliferation by Dox. SB also prevented the suppression of vascular network formation in co-cultures of HCMVEC and human cardiac fibroblasts treated with Dox. Our results show that the inhibition of the TGF-β pathway alleviates the detrimental effects of Dox on endothelial cells in vitro.

  9. Female Infertility and Disrupted Angiogenesis Are Actions of Specific Follistatin Isoforms

    Science.gov (United States)

    Lin, Shyr-Yeu; Craythorn, Rebecca G.; O’Connor, Anne E.; Matzuk, Martin M.; Girling, Jane E.; Morrison, John R.; de Kretser, David M.

    2008-01-01

    The circulating and tissue-bound forms of follistatin (FST315 and FST288, respectively) modulate the actions of activins. FST knockout (KO/null) mice, lacking both isoforms, die perinatally with defects in lung, skin, and the musculoskeletal system. Using constructs of the human FST gene engineered to enable expression of each isoform under the control of natural regulatory elements, transgenic mouse lines were created and crossed with FST null mice to attempt to rescue the neonatal lethality. FST288 expression alone did not rescue the neonatal lethality, but mice expressing FST315 on the KO background survived to adulthood with normal lung and skin morphology and partial reversal of the musculoskeletal defects noted in FST KO mice. The FST315 rescue mice displayed a short period of neonatal growth retardation, impaired tail growth, and female infertility. The latter may be due to failure of corpus luteum formation, a decline in the ovarian follicular population, and an augmented uterine inflammatory response to mating. Failure of corpus luteum formation and impaired tail growth indicate abnormal vascularization and suggest that FST288 is required for the promotion of angiogenesis. The augmented uterine inflammatory response may result from the failure of FST315 to modulate the proinflammatory actions of activin A in the uterus or may result from the altered steroid milieu associated with the ovarian abnormalities. Although we cannot definitively conclude that the remaining defects are due to the absence of a particular isoform or due to variable expression of each, these models have demonstrated novel physiological processes that are influenced by FST. PMID:17932109

  10. Different Concentrations of FGF Ligands, FGF2 or FGF8 Determine Distinct States of WNT-Induced Presomitic Mesoderm.

    Science.gov (United States)

    Sudheer, Smita; Liu, Jinhua; Marks, Matthias; Koch, Frederic; Anurin, Anna; Scholze, Manuela; Senft, Anna Dorothea; Wittler, Lars; Macura, Karol; Grote, Phillip; Herrmann, Bernhard G

    2016-07-01

    Presomitic mesoderm (PSM) cells are the precursors of the somites, which flank both sides of the neural tube and give rise to the musculo-skeletal system shaping the vertebrate body. WNT and FGF signaling control the formation of both the PSM and the somites and show a graded distribution with highest levels in the posterior PSM. We have used reporters for the mesoderm/PSM control genes T, Tbx6, and Msgn1 to investigate the differentiation of mouse ESCs from the naïve state via EpiSCs to PSM cells. Here we show that the activation of WNT signaling by CHIR99021 (CH) in combination with FGF ligand induces embryo-like PSM at high efficiency. By varying the FGF ligand concentration, the state of PSM cells formed can be altered. High FGF concentration supports posterior PSM formation, whereas low FGF generates anterior/differentiating PSM, in line with in vivo data. Furthermore, the level of Msgn1 expression depends on the FGF ligand concentration. We also show that Activin/Nodal signaling inhibits CH-mediated PSM induction in EpiSCs, without affecting T-expression. Inversely, Activin/Nodal inhibition enhances PSM induction by WNT/high FGF signaling. The ability to generate PSM cells of either posterior or anterior PSM identity with high efficiency in vitro will promote the investigation of the gene regulatory networks controlling the formation of nascent PSM cells and their switch to differentiating/somitic paraxial mesoderm. Stem Cells 2016;34:1790-1800.

  11. miR-200c and GATA binding protein 4 regulate human embryonic stem cell renewal and differentiation

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    Hsiao-Ning Huang

    2014-03-01

    Full Text Available Human embryonic stem cells (hESCs are functionally unique for their self-renewal ability and pluripotency, but the molecular mechanisms giving rise to these properties are not fully understood. hESCs can differentiate into embryoid bodies (EBs containing ectoderm, mesoderm, and endoderm. In the miR-200 family, miR-200c was especially enriched in undifferentiated hESCs and significantly downregulated in EBs. The knockdown of the miR-200c in hESCs downregulated Nanog expression, upregulated GATA binding protein 4 (GATA4 expression, and induced hESC apoptosis. The knockdown of GATA4 rescued hESC apoptosis induced by downregulation of miR-200c. miR-200c directly targeted the 3′-untranslated region of GATA4. Interestingly, the downregulation of GATA4 significantly inhibited EB formation in hESCs. Overexpression of miR-200c inhibited EB formation and repressed the expression of ectoderm, endoderm, and mesoderm markers, which could partially be rescued by ectopic expression of GATA4. Fibroblast growth factor (FGF and activin A/nodal can sustain hESC renewal in the absence of feeder layer. Inhibition of transforming growth factor-β (TGF-β/activin A/nodal signaling by SB431542 treatment downregulated the expression of miR-200c. Overexpression of miR-200c partially rescued the expression of Nanog/phospho-Smad2 that was downregulated by SB431542 treatment. Our observations have uncovered novel functions of miR-200c and GATA4 in regulating hESC renewal and differentiation.

  12. Dynamic, large-scale profiling of transcription factor activity from live cells in 3D culture.

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    Michael S Weiss

    Full Text Available BACKGROUND: Extracellular activation of signal transduction pathways and their downstream target transcription factors (TFs are critical regulators of cellular processes and tissue development. The intracellular signaling network is complex, and techniques that quantify the activities of numerous pathways and connect their activities to the resulting phenotype would identify the signals and mechanisms regulating tissue development. The ability to investigate tissue development should capture the dynamic pathway activity and requires an environment that supports cellular organization into structures that mimic in vivo phenotypes. Taken together, our objective was to develop cellular arrays for dynamic, large-scale quantification of TF activity as cells organized into spherical structures within 3D culture. METHODOLOGY/PRINCIPAL FINDINGS: TF-specific and normalization reporter constructs were delivered in parallel to a cellular array containing a well-established breast cancer cell line cultured in Matrigel. Bioluminescence imaging provided a rapid, non-invasive, and sensitive method to quantify luciferase levels, and was applied repeatedly on each sample to monitor dynamic activity. Arrays measuring 28 TFs identified up to 19 active, with 13 factors changing significantly over time. Stimulation of cells with β-estradiol or activin A resulted in differential TF activity profiles evolving from initial stimulation of the ligand. Many TFs changed as expected based on previous reports, yet arrays were able to replicate these results in a single experiment. Additionally, arrays identified TFs that had not previously been linked with activin A. CONCLUSIONS/SIGNIFICANCE: This system provides a method for large-scale, non-invasive, and dynamic quantification of signaling pathway activity as cells organize into structures. The arrays may find utility for investigating mechanisms regulating normal and abnormal tissue growth, biomaterial design, or as a

  13. In-vitro study of the effect of anti-hypertensive drugs on placental hormones and angiogenic proteins synthesis in pre-eclampsia.

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    Subrata Gangooly

    Full Text Available INTRODUCTION: Antihypertensive drugs lower the maternal blood pressure in pre-eclampsia (PE by direct or central vasodilatory mechanisms but little is known about the direct effects of these drugs on placental functions. OBJECTIVE: The aim of our study is to evaluate the effect of labetolol, hydralazine, α-methyldopa and pravastatin on the synthesis of placental hormonal and angiogenic proteins know to be altered in PE. DESIGN: Placental villous explants from late onset PE (n = 3 and normotensive controls (n = 6 were cultured for 3 days at 10 and 20% oxygen (O2 with variable doses anti-hypertensive drugs. The levels of activin A, inhibin A, human Chorionic Gonadotrophin (hCG, soluble fms-like tyrosine kinase-1 (sFlt-1 and soluble endoglin (sEng were measured in explant culture media on day 1, 2 and 3 using standard immunoassays. Data at day 1 and day 3 were compared. RESULTS: Spontaneous secretion of sEndoglin and sFlt-1 were higher (p < 0.05 in villous explants from PE pregnancies compared to controls. There was a significant time dependent decrease in the secretion of sFlt-1 and sEndoglin in PE cases, which was seen only for sFlt-1 in controls. In both PE cases and controls the placental protein secretions were not affected by varying doses of anti-hypertensive drugs or the different O2 concentration cultures, except for Activin, A which was significantly (p < 0.05 higher in controls at 10% O2. INTERPRETATION: Our findings suggest that the changes previously observed in maternal serum hormones and angiogenic proteins level after anti-hypertensive treatment in PE could be due to a systemic effect of the drugs on maternal blood pressure and circulation rather than a direct effect of these drugs on placental biosynthesis and/or secretion.

  14. Follistatin concentrations in maternal and fetal fluids during the oestrous cycle, gestation and parturition in Merino sheep.

    Science.gov (United States)

    McFarlane, J R; Xia, Y; O'Shea, T; Hayward, S; O'Connor, A E; De Kretser, D M

    2002-08-01

    The aim of this study was to investigate the changes in follistatin, an activin binding protein, during the oestrous cycle, gestation and parturition in ewes using a radioimmunoassay for total follistatin, which uses dissociating reagents to remove the interference of activin. Follistatin concentrations remained unchanged (2.7 +/- 0.2 ng ml(-1)) during the oestrous cycle and decreased as pregnancy progressed. Follistatin concentrations in allantoic fluid also decreased during gestation, whereas in amniotic fluid follistatin concentrations reached a peak at day 75 of gestation (9.8 ng ml(-1)) and had decreased to 4.4 ng ml(-1) at day 140. Follistatin concentrations in fetal blood (7.0 +/- 0.5 ng ml(-1)) did not change from day 50 to day 140 of gestation but were significantly higher than in matched maternal samples (3.1 +/- 0.3 ng ml(-1)). Circulating follistatin in ewes was significantly increased on the day of parturition (5.6 +/- 0.6 ng ml(-1)) compared with the days before parturition (2.7 +/- 0.4 ng ml(-1)), but had decreased by day 2 after birth. Blood samples from newborn lambs showed that plasma follistatin concentration (13.4 +/- 2.3 ng ml(-1)) was significantly higher than that of the mothers and remained high for at least 7 days after birth. These data support previous studies of the human menstrual cycle indicating that follistatin is not an endocrine signal from the ovary; however, in contrast to human pregnancies, follistatin concentrations in sheep decreased and become high only after or during parturition. This difference observed between species may reflect different physiological effects of follistatin or may be the result of measurement of different isoforms.

  15. Induction of cardiac muscle differentiation in isolated animal pole explants of Xenopus laevis embryos.

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    Logan, M; Mohun, T

    1993-07-01

    We have isolated a cDNA fragment encoding a portion of the myosin heavy chain alpha-isoform (XMHC alpha) in the amphibian, Xenopus laevis. The XMHC alpha transcript is highly enriched in adult heart RNA and is expressed exclusively in embryonic heart tissue. It therefore provides a tissue-specific marker for cardiac muscle differentiation during early embryogenesis. Using an RNAase protection assay, we can detect the onset of cardiac muscle differentiation in an anterior, ventral region of tailbud embryos, many hours before the appearance of a beating heart. Whole-mount in situ RNA hybridisation indicates that expression of the XMHC alpha gene is restricted to the developing heart primordium. XMHC alpha gene expression can also be induced in isolated animal pole explants of blastulae by treatment with the growth factor, activin A. Induction is dose-dependent, requiring high doses of the growth factor compared with that required for myotomal (skeletal) muscle differentiation. In contrast, no XMHC alpha transcripts are detected in explants incubated with basic FGF, despite the induction of myotomal muscle differentiation. Activin-induced explants show a similar temporal pattern of XMHC alpha gene expression to that found in normal embryogenesis. Furthermore, cells expressing this gene appear clustered in one or two foci within fused explant aggregates, which often show regular, spontaneous contractions after several days in culture. These results show that terminal differentiation of cardiac muscle can occur in growth factor-induced explants and may be distinguished from skeletal muscle differentiation by the dose and nature of the inducing factor.

  16. Over-expression of Follistatin-like 3 attenuates fat accumulation and improves insulin sensitivity in mice.

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    Brandt, Claus; Hansen, Rasmus Hvass; Hansen, Jakob Bondo; Olsen, Caroline Holkmann; Galle, Pia; Mandrup-Poulsen, Thomas; Gehl, Julie; Pedersen, Bente Klarlund; Hojman, Pernille

    2015-02-01

    Follistatin-like 3 (fstl3), a natural inhibitor of members of the TGF-β family, increases during resistance training in human plasma. Fstl3 primarily binds myostatin and activin A, and thereby inhibits their functions. We hypothesize that blocking myostatin and activin A signalling through systemic fstl3 over-expression protects against diet-induced obesity and insulin resistance. Fstl3 was over-expressed by DNA electrotransfer in tibialis anterior, quadriceps and gastrocnemius muscles in female C57BL/C mice, and the mice were subsequently randomized to chow or high-fat feeding. Body weight, food intake, fat accumulation by MR scanning, and glucose, insulin and glucagon tolerance were evaluated, as was the response in body weight and metabolic parameters to 24h fasting. Effects of fstl3 on pancreatic insulin and glucagon content, and pancreatic islet morphology were determined. Fstl3 over-expression reduced fat accumulation during high-fat feeding by 16%, and liver fat by 50%, as determined by MRI. No changes in body weight were observed, while the weight of the transfected muscles increased by 10%. No transcriptional changes were found in the subcutaneous adipose tissue. Fstl3 mice displayed improved insulin sensitivity and muscle insulin signalling. In contrast, glucose tolerance was impaired in high-fat fed fstl3 mice, which was explained by increased hepatic glucagon sensitivity and glucose output, as well as a decrease in the pancreatic insulin/glucagon ratio. Accordingly, fstl3 transfection improved counter-regulation to 24h fasting. Fstl3 over-expression regulates insulin and glucagon sensitivities through increased muscular insulin action, as well as increased hepatic glucagon sensitivity and pancreatic glucagon content. Copyright © 2015 Elsevier Inc. All rights reserved.

  17. Follistatin is a novel biomarker for lung adenocarcinoma in humans.

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    Fangfang Chen

    Full Text Available Follistatin (FST, a single chain glycoprotein, is originally isolated from follicular fluid of ovary. Previous studies have revealed that serum FST served as a biomarker for pregnancy and ovarian mucinous tumor. However, whether FST can serve as a biomarker for diagnosis in lung adenocarcinoma of humans remains unclear.The study population consisted of 80 patients with lung adenocarcinoma, 40 patients with ovarian adenocarcinoma and 80 healthy subjects. Serum FST levels in patients and healthy subjects were measured using ELISA. The results showed that the positive ratio of serum FST levels was 51.3% (41/80, which was comparable to the sensitivity of FST in 40 patients with ovarian adenocarcinoma (60%, 24/40 using the 95th confidence interval for the healthy subject group as the cut-off value. FST expressions in lung adenocarcinoma were examined by immunohistochemical staining, we found that lung adenocarcinoma could produce FST and there was positive correlation between the level of FST expression and the differential degree of lung adenocarcinoma. Furthermore, the results showed that primary cultured lung adenocarcinoma cells could secrete FST, while cells derived from non-tumor lung tissues almost did not produce FST. In addition, the results of CCK8 assay and flow cytometry showed that using anti-FST monoclonal antibody to neutralize endogenous FST significantly augmented activin A-induced lung adenocarcinoma cells apoptosis.These data indicate that lung adenocarcinoma cells can secret FST into serum, which may be beneficial to the survival of adenocarcinoma cells by neutralizing activin A action. Thus, FST can serve as a promising biomarker for diagnosis of lung adenocarcinoma and a useful biotherapy target for lung adenocarcinoma.

  18. Differentiation of intestinal absorptive cells derived from mouse embryonic bodies can be promoted by inducing the differentiation of definitive endoderm in vivo%小鼠拟胚体中定型内胚层的比例对小肠吸收细胞分化的促进作用

    Institute of Scientific and Technical Information of China (English)

    邓娜; 于涛; 石柳; 兰绍阳; 周慧敏; 陈浩; 陈其奎

    2011-01-01

    AIM: To investigate the effect of inducing the differentiation of definitive endoderm derived from mouse embryonic bodies (Ebs) cultured bythe hanging drop method in promoting the differentiation of intestinal absorptive cells in vivo.METHODS: The differentiation of definitive endoderm during Ebs formation derived from mouse ES-E14TG2a embryonic stem cells (ESC) and the role of Activin A in promoting its differentiation were monitored by detecting its markers by RT-PCR and fluorescence-activated cell sorting. Subsequently, the Ebs with high proportion of definitive endoderm were hypodermi-cally engrafted into the back of NOD/SCID mice to form grafts. The markers for small intestinal absorptive cells, including SI, LPH, and Fabp2, were detected in these grafts by quantitative RT-PCR and immunohistochemistry.RESULTS: The marker genes for definitive endoderm were more highly expressed in the 5-day Ebs than in other stages of Ebs (Gsc: 0.9809 ± 0.1001 νs 0.5435 ± 0.0821,0.5525 ± 0.0786,0.2234 ± 0.0425; Tm4sf2: 0.9231 ± 0.1121 vs 0.0017 ± 0.0007, 0.0176 ± 0.0058, 0.6542 ± 0.0742; Gpcl: 0.8639 ± 0.1098 vs 0.5882 ± 0.1027,0.7112 ± 0.0956, 0.4239 ± 0.0874, all P < 0.05). The percentage of definitive endoderm cells in the 5-day Ebs induced with 50 μg/L Activin A (SF-A group) was significantly higher than that in controls (all P < 0.05). SI and LPH mRNA expression in the grafts from the SF-A group was significantly higher than that in control groups (all P < 0.05). Immu-nohistochemical analysis revealed that Fabp2 was expressed in some immature cells without specific structure or adenoid structures in the grafts from the SF-A group.CONCLUSION: The differentiation of definitive endoderm derived from mouse ESC could be induced with 50 ug/L Activin A in Ebs cultured by the hanging drop method. Increasing the proportion of definitive endoderm in Ebs promotes the differentiation of intestinal absorptive cells in vivo.%目的:探讨悬浮状态下小鼠拟胚体中

  19. Fibrodysplasia Ossificans Progressiva: Clinical and Genetic Aspects

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    Pignolo Robert J

    2011-12-01

    Full Text Available Abstract Fibrodysplasia ossificans progressiva (FOP is a severely disabling heritable disorder of connective tissue characterized by congenital malformations of the great toes and progressive heterotopic ossification that forms qualitatively normal bone in characteristic extraskeletal sites. The worldwide prevalence is approximately 1/2,000,000. There is no ethnic, racial, gender, or geographic predilection to FOP. Children who have FOP appear normal at birth except for congenital malformations of the great toes. During the first decade of life, sporadic episodes of painful soft tissue swellings (flare-ups occur which are often precipitated by soft tissue injury, intramuscular injections, viral infection, muscular stretching, falls or fatigue. These flare-ups transform skeletal muscles, tendons, ligaments, fascia, and aponeuroses into heterotopic bone, rendering movement impossible. Patients with atypical forms of FOP have been described. They either present with the classic features of FOP plus one or more atypical features [FOP plus], or present with major variations in one or both of the two classic defining features of FOP [FOP variants]. Classic FOP is caused by a recurrent activating mutation (617G>A; R206H in the gene ACVR1/ALK2 encoding Activin A receptor type I/Activin-like kinase 2, a bone morphogenetic protein (BMP type I receptor. Atypical FOP patients also have heterozygous ACVR1 missense mutations in conserved amino acids. The diagnosis of FOP is made by clinical evaluation. Confirmatory genetic testing is available. Differential diagnosis includes progressive osseous heteroplasia, osteosarcoma, lymphedema, soft tissue sarcoma, desmoid tumors, aggressive juvenile fibromatosis, and non-hereditary (acquired heterotopic ossification. Although most cases of FOP are sporadic (noninherited mutations, a small number of inherited FOP cases show germline transmission in an autosomal dominant pattern. At present, there is no definitive

  20. Human Embryonic Stem Cells Form Functional Thyroid Follicles

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    Latif, Rauf; Davies, Terry F.

    2015-01-01

    Objective: The molecular events that lead to human thyroid cell speciation remain incompletely characterized. It has been shown that overexpression of the regulatory transcription factors Pax8 and Nkx2-1 (ttf-1) directs murine embryonic stem (mES) cells to differentiate into thyroid follicular cells by initiating a transcriptional regulatory network. Such cells subsequently organized into three-dimensional follicular structures in the presence of extracellular matrix. In the current study, human embryonic stem (hES) cells were studied with the aim of recapitulating this scenario and producing functional human thyroid cell lines. Methods: Reporter gene tagged pEZ-lentiviral vectors were used to express human PAX8-eGFP and NKX2-1-mCherry in the H9 hES cell line followed by differentiation into thyroid cells directed by Activin A and thyrotropin (TSH). Results: Both transcription factors were expressed efficiently in hES cells expressing either PAX8, NKX2-1, or in combination in the hES cells, which had low endogenous expression of these transcription factors. Further differentiation of the double transfected cells showed the expression of thyroid-specific genes, including thyroglobulin (TG), thyroid peroxidase (TPO), the sodium/iodide symporter (NIS), and the TSH receptor (TSHR) as assessed by reverse transcription polymerase chain reaction and immunostaining. Most notably, the Activin/TSH-induced differentiation approach resulted in thyroid follicle formation and abundant TG protein expression within the follicular lumens. On stimulation with TSH, these hES-derived follicles were also capable of dose-dependent cAMP generation and radioiodine uptake, indicating functional thyroid epithelial cells. Conclusion: The induced expression of PAX8 and NKX2-1 in hES cells was followed by differentiation into thyroid epithelial cells and their commitment to form functional three-dimensional neo-follicular structures. The data provide proof of principal that hES cells can be

  1. Thyroid Follicle Formation and Thyroglobulin Expression in Multipotent Endodermal Stem Cells

    Science.gov (United States)

    Latif, Rauf; Davies, Terry F.

    2013-01-01

    Objective The aim of this study was to assess the impact of transcriptional induction on thyroid follicular cell (TFC) differentiation from endodermally matured embryonic stem (ES) cells. The thyroid transcription factors—NKx2 homeobox 1 (NKx2-1, formerly called TTF-1) and Paired box gene 8 (Pax8)—are known to associate biochemically and synergistically in the activation of thyroid functional genes including the sodium/iodide symporter (NIS), thyrotropin (TSH) receptor (TSHR), thyroglobulin (Tg), and thyroid peroxidase (TPO) genes. In this study, we investigated the ability of ectopically expressed Pax8 and NKx2-1 to further the induction and differentiation of murine ES cells into potential TFCs. Methods ES cells were stably transfected with either the Pax8 gene, the NKx2-1 gene, or both genes to study the induction of NIS, TSHR, Tg, and TPO genes as assessed using both quantitative reverse-transcription polymerase chain reaction (qRT-PCR) and protein expression. The derived cells were cultured with or without the presence of activin A to allow their differentiation into multipotent endodermal cells. Results The four thyroid-specific genes NIS, TSHR, Tg, and TPO were all significantly activated by expressing both transcription factors within the same ES cell. In contrast, significant but much lower transcriptional activity of the TSHR, Tg, and TPO genes was detected in cells expressing just NKx2-1, and only the NIS and TSHR genes responded to Pax8 alone. No Tg protein expression could be detected prior to their development into endodermal derivatives. However, after further differentiation of postembryoid body ES cells with activin A and TSH into endodermal cell lines, those cells with dual transfection of Pax8 and NKx2-1 demonstrated greatly enhanced expression of the NIS, TSHR, Tg, and TPO genes to such a degree that it was similar to that found in control thyroid cells. Furthermore, these same cells formed three-dimensional neofollicles in vitro and

  2. Influence of the photoperiod on TGF-β1 and p15 expression in hamster Leydig cells.

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    Gonzalez, Candela R; Calandra, Ricardo S; Gonzalez-Calvar, Silvia I

    2012-07-01

    Adult hamsters exposed to short photoperiods show a marked atrophy of their internal reproductive organs, including a reduction in size, though not number of Leydig cells. Transforming growth factor-β1 (TGF-β1) is involved in the regulation of growth and proliferation of different cell types. The aim of the present study was to examine the influence of photoperiod on the protein and gene expression of TGF-β1 and its receptors as well as gene expression of p15. The effect of TGF-β1 on the expression of p15 in purified Leydig cells from regressed and non-regressed hamster testes was also tested. Protein and gene expression of TGF-β1 was detected in both regressed and non-regressed testes. In contrast to the activin receptor-like kinase 1 (ALK-1), the TGF-β1, the activin receptor-like kinase 5 (ALK-5) and the co-receptor endoglin all showed a greater basal expression in regressed than non-regressed hamster testes. Melatonin induced the TGF-β1 mRNA expression in purified Leydig cells from non-regressed testes. The p15 mRNA level was greater in regressed than non-regressed testes. A high dose of TGF-β1 during a short incubation period increased the p15 mRNA level in Leydig cells from non-regressed testes. ALK-5 and mitogen-activated protein kinase (MAPK) p38 might have played a role in this process. In regressed hamster testes, the p15 mRNA level increased due to a low dose of TGF-β1 after short incubation periods and to a high dose after longer incubation periods; in both instances, ALK-5, ERK 1/2 and p38 were involved. Collectively, these results suggest that the alterations in p15 expression, mediated by MAPK, are involved in the shift between the active and inactive states in hamster Leydig cells.

  3. Long-Term Expandable SOX9+ Chondrogenic Ectomesenchymal Cells from Human Pluripotent Stem Cells

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    Katsutsugu Umeda

    2015-04-01

    Full Text Available Here we report the successful generation and long-term expansion of SOX9-expressing CD271+PDGFRα+CD73+ chondrogenic ectomesenchymal cells from the PAX3/SOX10/FOXD3-expressing MIXL1−CD271hiPDGFRαloCD73− neural crest-like progeny of human pluripotent stem cells in a chemically defined medium supplemented with Nodal/Activin/transforming growth factorβ (TGFβ inhibitor and fibroblast growth factor (FGF. When “primed” with TGFβ, such cells efficiently formed translucent cartilage particles, which were completely mineralized in 12 weeks in immunocompromized mice. The ectomesenchymal cells were expandable without loss of chondrogenic potential for at least 16 passages. They maintained normal karyotype for at least 10 passages and expressed genes representing embryonic progenitors (SOX4/12, LIN28A/B, cranial mesenchyme (ALX1/3/4, and chondroprogenitors (SOX9, COL2A1 of neural crest origin (SOX8/9, NGFR, NES. Ectomesenchyme is a source of many craniofacial bone and cartilage structures. The method we describe for obtaining a large quantity of human ectomesenchymal cells will help to model craniofacial disorders in vitro and potentially provide cells for the repair of craniofacial damage.

  4. TGF-β superfamily gene expression and induction of the Runx1 transcription factor in adult neurogenic regions after brain injury.

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    Trevor T Logan

    Full Text Available Traumatic brain injury (TBI increases neurogenesis in the forebrain subventricular zone (SVZ and the hippocampal dentate gyrus (DG. Transforming growth factor-β (TGF-β superfamily cytokines are important regulators of adult neurogenesis, but their involvement in the regulation of this process after brain injury is unclear. We subjected adult mice to controlled cortical impact (CCI injury, and isolated RNA from the SVZ and DG at different post-injury time points. qPCR array analysis showed that cortical injury caused significant alterations in the mRNA expression of components and targets of the TGF-β, BMP, and activin signaling pathways in the SVZ and DG after injury, suggesting that these pathways could regulate post-injury neurogenesis. In both neurogenic regions, the injury also induced expression of Runt-related transcription factor-1 (Runx1, which can interact with intracellular TGF-β Smad signaling pathways. CCI injury strongly induced Runx1 expression in activated and proliferating microglial cells throughout the neurogenic regions. Runx1 protein was also expressed in a subset of Nestin- and GFAP-expressing putative neural stem or progenitor cells in the DG and SVZ after injury. In the DG only, these Runx1+ progenitors proliferated. Our data suggest potential roles for Runx1 in the processes of microglial cell activation and proliferation and in neural stem cell proliferation after TBI.

  5. Differential regulation of gonadotropins (FSH and LH) and growth hormone (GH) by neuroendocrine, endocrine, and paracrine factors in the zebrafish--an in vitro approach.

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    Lin, Sze-Wah; Ge, Wei

    2009-01-15

    Recently, zebrafish has quickly risen as a model species for functional analysis of the brain-pituitary-gonad axis. However, one of the hurdles for such work in this popular model organism is the small size of its pituitary gland, which makes it difficult to investigate the regulation of pituitary hormone expression and secretion in vitro. To provide a solution to this problem and demonstrate the value of zebrafish in reproductive endocrinology, the present study was undertaken to establish a primary pituitary cell culture followed by investigating the regulation of FSHbeta (fshb), LHbeta (lhb), and GH (gh) expression by a variety of neuroendocrine, endocrine, and paracrine factors. All the factors examined influenced the expression of fshb, lhb, and ghin vitro except epidermal growth factor (EGF) despite the expression of its receptor egfr in the pituitary. Acting in a similar manner, gonadal steroids (estradiol and testosterone) stimulated both fshb and lhb, but had no effect on gh. In contrast, all other factors tested (gonadotropin-releasing hormone, GnRH; pituitary adenylate cyclase-activating polypeptide, PACAP; activin/follistatin, and insulin-like growth factor I, IGF-I) exhibited distinct effects on the expression of the three target genes studied, suggesting roles for these factors in the differential regulation of two gonadotropins and growth hormone and therefore the gonadotrophic and somatotrophic axes.

  6. Efficient feeder-free episomal reprogramming with small molecules.

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    Junying Yu

    Full Text Available Genetic reprogramming of human somatic cells to induced pluripotent stem cells (iPSCs could offer replenishable cell sources for transplantation therapies. To fulfill their promises, human iPSCs will ideally be free of exogenous DNA (footprint-free, and be derived and cultured in chemically defined media free of feeder cells. Currently, methods are available to enable efficient derivation of footprint-free human iPSCs. However, each of these methods has its limitations. We have previously derived footprint-free human iPSCs by employing episomal vectors for transgene delivery, but the process was inefficient and required feeder cells. Here, we have greatly improved the episomal reprogramming efficiency using a cocktail containing MEK inhibitor PD0325901, GSK3β inhibitor CHIR99021, TGF-β/Activin/Nodal receptor inhibitor A-83-01, ROCK inhibitor HA-100 and human leukemia inhibitory factor. Moreover, we have successfully established a feeder-free reprogramming condition using chemically defined medium with bFGF and N2B27 supplements and chemically defined human ESC medium mTeSR1 for the derivation of footprint-free human iPSCs. These improvements enabled the routine derivation of footprint-free human iPSCs from skin fibroblasts, adipose tissue-derived cells and cord blood cells. This technology will likely be valuable for the production of clinical-grade human iPSCs.

  7. Effects of S-adenosylmethionine decarboxylase, polyamines, amino acids, and weak bases (amines and ammonia) on development and ribosomal RNA synthesis in Xenopus embryos.

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    Shiokawa, Koichiro; Aso, Mai; Kondo, Takeshi; Takai, Jun-Ichi; Yoshida, Junki; Mishina, Takamichi; Fuchimukai, Kota; Ogasawara, Tsukasa; Kariya, Taro; Tashiro, Kosuke; Igarashi, Kazuei

    2010-02-01

    We have been studying control mechanisms of gene expression in early embryogenesis in a South African clawed toad Xenopus laevis, especially during the period of midblastula transition (MBT), or the transition from the phase of active cell division (cleavage stage) to the phase of extensive morphogenesis (post-blastular stages). We first found that ribosomal RNA synthesis is initiated shortly after MBT in Xenopus embryos and those weak bases, such as amines and ammonium ion, selectively inhibit the initiation and subsequent activation of rRNA synthesis. We then found that rapidly labeled heterogeneous mRNA-like RNA is synthesized in embryos at pre-MBT stage. We then performed cloning and expression studies of several genes, such as those for activin receptors, follistatin and aldolases, and then reached the studies of S-adenosylmethionine decarboxylase (SAMDC), a key enzyme in polyamine metabolism. Here, we cloned a Xenopus SAMDC cDNA and performed experiments to overexpress the in vitro-synthesized SAMDC mRNA in Xenopus early embryos, and found that the maternally preset program of apoptosis occurs in cleavage stage embryos, which is executed when embryos reach the stage of MBT. In the present article, we first summarize results on SAMDC and the maternal program of apoptosis, and then describe our studies on small-molecular-weight substances like polyamines, amino acids, and amines in Xenopus embryos. Finally, we summarize our studies on weak bases, especially on ammonium ion, as the specific inhibitor of ribosomal RNA synthesis in Xenopus embryonic cells.

  8. Fibrocyte-like cells from intrauterine growth restriction placentas have a reduced ability to stimulate angiogenesis.

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    Riddell, Meghan R; Winkler-Lowen, Bonnie; Jiang, Yanyan; Guilbert, Larry J; Davidge, Sandra T

    2013-09-01

    Intrauterine growth restriction (IUGR) is a common complication of pregnancy whereby the fetus fails to achieve its genetic growth potential. Malformation of the placental vasculature is observed in IUGR and may be due to the development of the placenta in a chronically hypoxic environment. Recently, we identified that the predominant stromal cells in the angiogenic zones of the placenta are fibrocyte-like cells. The conditioned medium from fibrocyte-like cells (FcCM) has been shown to stimulate angiogenesis in vitro. Thus, we hypothesized that FcCM from IUGR cells would have a reduced ability to stimulate angiogenesis and that chronic hypoxia would decrease the ability of both normal and IUGR fibrocyte-like cells to stimulate angiogenesis. IUGR FcCM had a reduced ability to stimulate endothelial tubule-like structure formation and an increased ability to stimulate endothelial migration compared with normal FcCM. However, normal and IUGR FcCM produced in chronic hypoxia did not alter endothelial proliferation, migration, or tubule-like structure formation. IUGR FcCM was found to have reduced levels of the pro-angiogenic cytokine IL-8 and increased levels of the anti-angiogenic factors activin-A and pigment epithelium-derived growth factor. Thus, alterations in the ability of IUGR fibrocyte-like cells to stimulate angiogenesis may contribute to the development of vascular malformation in IUGR, but in vitro these changes cannot be attributed to a chronically hypoxic environment.

  9. Inhibition of myostatin does not ameliorate disease features of severe spinal muscular atrophy mice

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    Sumner, Charlotte J.; Wee, Claribel D.; Warsing, Leigh C.; Choe, Dong W.; Ng, Andrew S.; Lutz, Cathleen; Wagner, Kathryn R.

    2009-01-01

    There is currently no treatment for the inherited motor neuron disease, spinal muscular atrophy (SMA). Severe SMA causes lower motor neuron loss, impaired myofiber development, profound muscle weakness and early mortality. Myostatin is a transforming growth factor-β family member that inhibits muscle growth. Loss or blockade of myostatin signaling increases muscle mass and improves muscle strength in mouse models of primary muscle disease and in the motor neuron disease, amyotrophic lateral sclerosis. In this study, we evaluated the effects of blocking myostatin signaling in severe SMA mice (hSMN2/delta7SMN/mSmn−/−) by two independent strategies: (i) transgenic overexpression of the myostatin inhibitor follistatin and (ii) post-natal administration of a soluble activin receptor IIB (ActRIIB-Fc). SMA mice overexpressing follistatin showed little increase in muscle mass and no improvement in motor function or survival. SMA mice treated with ActRIIB-Fc showed minimal improvement in motor function, and no extension of survival compared with vehicle-treated mice. Together these results suggest that inhibition of myostatin may not be a promising therapeutic strategy in severe forms of SMA. PMID:19477958

  10. Stem cell-dependent formation of a functional anterior regeneration pole in planarians requires Zic and Forkhead transcription factors.

    Science.gov (United States)

    Vogg, Matthias C; Owlarn, Suthira; Pérez Rico, Yuvia A; Xie, Jianlei; Suzuki, Yoko; Gentile, Luca; Wu, Wei; Bartscherer, Kerstin

    2014-06-15

    Planarians can regenerate their head within days. This process depends on the direction of adult stem cells to wound sites and the orchestration of their progenitors to commit to appropriate lineages and to arrange into patterned tissues. We identified a zinc finger transcription factor, Smed-ZicA, as a downstream target of Smed-FoxD, a Forkhead transcription factor required for head regeneration. Smed-zicA and Smed-FoxD are co-expressed with the Wnt inhibitor notum and the Activin inhibitor follistatin in a cluster of cells at the anterior-most tip of the regenerating head - the anterior regeneration pole - and in surrounding stem cell progeny. Depletion of Smed-zicA and Smed-FoxD by RNAi abolishes notum and follistatin expression at the pole and inhibits head formation downstream of initial polarity decisions. We suggest a model in which ZicA and FoxD transcription factors synergize to control the formation of Notum- and Follistatin-producing anterior pole cells. Pole formation might constitute an early step in regeneration, resulting in a signaling center that orchestrates cellular events in the growing tissue.

  11. Nicalin and its binding partner Nomo are novel Nodal signaling antagonists.

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    Haffner, Christof; Frauli, Mélanie; Topp, Stephanie; Irmler, Martin; Hofmann, Kay; Regula, Jörg T; Bally-Cuif, Laure; Haass, Christian

    2004-08-04

    Nodals are signaling factors of the transforming growth factor-beta (TGFbeta) superfamily with a key role in vertebrate development. They control a variety of cell fate decisions required for the establishment of the embryonic body plan. We have identified two highly conserved transmembrane proteins, Nicalin and Nomo (Nodal modulator, previously known as pM5), as novel antagonists of Nodal signaling. Nicalin is distantly related to Nicastrin, a component of the Alzheimer's disease-associated gamma-secretase, and forms a complex with Nomo. Ectopic expression of both proteins in zebrafish embryos causes cyclopia, a phenotype that can arise from a defect in mesendoderm patterning mediated by the Nodal signaling pathway. Accordingly, downregulation of Nomo resulted in an increase in anterior axial mesendoderm and the development of an enlarged hatching gland. Inhibition of Nodal signaling by ectopic expression of Lefty was rescued by reducing Nomo levels. Furthermore, Nodal- as well as Activin-induced signaling was inhibited by Nicalin and Nomo in a cell-based reporter assay. Our data demonstrate that the Nicalin/Nomo complex antagonizes Nodal signaling during mesendodermal patterning in zebrafish.

  12. Prediction of spontaneous preterm delivery from maternal factors, obstetric history and placental perfusion and function at 11-13 weeks.

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    Beta, Jarek; Akolekar, Ranjit; Ventura, Walter; Syngelaki, Argyro; Nicolaides, Kypros H

    2011-01-01

    To develop a model for prediction of spontaneous delivery before 34 weeks based on maternal factors, placental perfusion and function at 11-13 weeks' gestation. Two groups of studies: first, screening study of maternal characteristics, serum pregnancy-associated plasma protein-A (PAPP-A), free β-human chorionic gonadotrophin (β-hCG) and uterine artery pulsatility index (PI). Second, case-control studies of maternal serum or plasma concentration of placental growth factor (PlGF), placental protein 13 (PP13), a disintegrin and metalloprotease 12 (ADAM12), inhibin-A and activin-A. Regression analysis was used to develop a model for the prediction of spontaneous early delivery. Spontaneous early delivery occurred in 365 (1.1%) of the 34 025 pregnancies. A model based on maternal factors could detect 38.2% of the preterm deliveries in women with previous pregnancies at or beyond 16 weeks and 18.4% in those without, at a false positive rate (FPR) of 10%. In the preterm delivery group, compared with unaffected pregnancies there were no significant differences in the markers of placental perfusion or function, except for PAPP-A which was reduced. Patient-specific risk of preterm delivery is provided by maternal factors and obstetric history. Placental perfusion and function at 11-13 weeks are not altered in pregnancies resulting in spontaneous early delivery. Copyright © 2011 John Wiley & Sons, Ltd.

  13. Gene expression analysis of a murine model with pulmonary vascular remodeling compared to end-stage IPAH lungs

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    Shimodaira Kayoko

    2012-11-01

    Full Text Available Abstract Background Idiopathic pulmonary arterial hypertension (IPAH continues to be one of the most serious intractable diseases that might start with activation of several triggers representing the genetic susceptibility of a patient. To elucidate what essentially contributes to the onset and progression of IPAH, we investigated factors playing an important role in IPAH by searching discrepant or controversial expression patterns between our murine model and those previously published for human IPAH. We employed the mouse model, which induced muscularization of pulmonary artery leading to hypertension by repeated intratracheal injection of Stachybotrys chartarum, a member of nonpathogenic and ubiquitous fungus in our envelopment. Methods Microarray assays with ontology and pathway analyses were performed with the lungs of mice. A comparison was made of the expression patterns of biological pathways between our model and those published for IPAH. Results Some pathways in our model showed the same expression patterns in IPAH, which included bone morphogenetic protein (BMP signaling with down-regulation of BMP receptor type 2, activin-like kinase type 1, and endoglin. On the other hand, both Wnt/planar cell polarity (PCP signaling and its downstream Rho/ROCK signaling were found alone to be activated in IPAH and not in our model. Conclusions Activation of Wnt/PCP signaling, in upstream positions of the pathway, found alone in lungs from end stage IPAH may play essential roles in the pathogenesis of the disease.

  14. Antero-posterior tissue polarity links mesoderm convergent extension to axial patterning.

    Science.gov (United States)

    Ninomiya, Hiromasa; Elinson, Richard P; Winklbauer, Rudolf

    2004-07-15

    Remodelling its shape, or morphogenesis, is a fundamental property of living tissue. It underlies much of embryonic development and numerous pathologies. Convergent extension (CE) of the axial mesoderm of vertebrates is an intensively studied model for morphogenetic processes that rely on cell rearrangement. It involves the intercalation of polarized cells perpendicular to the antero-posterior (AP) axis, which narrows and lengthens the tissue. Several genes have been identified that regulate cell behaviour underlying CE in zebrafish and Xenopus. Many of these are homologues of genes that control epithelial planar cell polarity in Drosophila. However, elongation of axial mesoderm must be also coordinated with the pattern of AP tissue specification to generate a normal larval morphology. At present, the long-range control that orients CE with respect to embryonic axes is not understood. Here we show that the chordamesoderm of Xenopus possesses an intrinsic AP polarity that is necessary for CE, functions in parallel to Wnt/planar cell polarity signalling, and determines the direction of tissue elongation. The mechanism that establishes AP polarity involves graded activin-like signalling and directly links mesoderm AP patterning to CE.

  15. The neurotrophin-receptor-related protein NRH1 is essential for convergent extension movements.

    Science.gov (United States)

    Sasai, Noriaki; Nakazawa, Yoko; Haraguchi, Tomoko; Sasai, Yoshiki

    2004-08-01

    Early spherical Xenopus laevis embryos are transformed into a streamlined shape through convergent extension movements. Here we report that a p75(NTR)-related transmembrane protein, NRH1, has an essential function in the regulation of these movements. NRH1 was expressed in marginal zone tissues of the gastrula and in the posterior ectoderm of the neurula. Attenuation of the NRH1 function inhibited convergent extension movements in the embryo and in activin-treated animal caps. NRH1 activated downstream effectors of the Wnt/planar cell polarity pathway: small GTPases and the cascade of MKK7-JNK. Furthermore, gain- and loss-of-function phenotypes of NRH1 were rescued by co-injection of dominant-negative and constitutively active forms of these downstream effectors, respectively, suggesting that NRH1 functions as a positive modulator of planar cell polarity signalling. Interestingly, NRH1 does not require Dishevelled (Xdsh) for the activation of these downstream effectors or translocation of Xdsh to the membrane, suggesting that NRH1 signalling interacts with planar cell polarity signalling downstream of Xdsh. This demonstrates an essential role for p75(NTR)-related signalling in early embryonic morphogenesis.

  16. Impact of 900 MHz electromagnetic field exposure on main male reproductive hormone levels: a Rattus norvegicus model.

    Science.gov (United States)

    Sepehrimanesh, Masood; Saeb, Mehdi; Nazifi, Saeed; Kazemipour, Nasrin; Jelodar, Gholamali; Saeb, Saeedeh

    2014-09-01

    This work analyzes the effects of radiofrequency-electromagnetic field (RF-EMF) exposure on the reproductive system of male rats, assessed by measuring circulating levels of FSH, LH, inhibin B, activin B, prolactin, and testosterone. Twenty adult male Sprague-Dawley rats (180 ± 10 g) were exposed to 900 MHz RF-EMF in four equal separated groups. The duration of exposure was 1, 2, and 4 h/day over a period of 30 days and sham-exposed animals were kept under the same environmental conditions as the exposed group except with no RF-EMF exposure. Before the exposure, at 15 and 30 days of exposure, determination of the abovementioned hormone levels was performed using ELISA. At the end of the experiment, FSH and LH values of the long time exposure (LTE) group were significantly higher than the sham-exposed group (p exposure (p exposure (p exposure and it may possibly affect reproductive functions. However, testosterone and inhibin B concentrations as a fertility marker and spermatogenesis were decreased significantly.

  17. Impact of 900 MHz electromagnetic field exposure on main male reproductive hormone levels: a Rattus norvegicus model

    Science.gov (United States)

    Sepehrimanesh, Masood; Saeb, Mehdi; Nazifi, Saeed; Kazemipour, Nasrin; Jelodar, Gholamali; Saeb, Saeedeh

    2014-09-01

    This work analyzes the effects of radiofrequency-electromagnetic field (RF-EMF) exposure on the reproductive system of male rats, assessed by measuring circulating levels of FSH, LH, inhibin B, activin B, prolactin, and testosterone. Twenty adult male Sprague-Dawley rats (180 ± 10 g) were exposed to 900 MHz RF-EMF in four equal separated groups. The duration of exposure was 1, 2, and 4 h/day over a period of 30 days and sham-exposed animals were kept under the same environmental conditions as the exposed group except with no RF-EMF exposure. Before the exposure, at 15 and 30 days of exposure, determination of the abovementioned hormone levels was performed using ELISA. At the end of the experiment, FSH and LH values of the long time exposure (LTE) group were significantly higher than the sham-exposed group ( p exposure ( p exposure ( p exposure and it may possibly affect reproductive functions. However, testosterone and inhibin B concentrations as a fertility marker and spermatogenesis were decreased significantly.

  18. Wnt/BMP信号通路激活高效诱导人多能干细胞分化为血管平滑肌细胞%Generation of vascular smooth muscle cells from human pluripotent stem cells via the activation of Wnt and BMP signaling

    Institute of Scientific and Technical Information of China (English)

    李艳辉; 罗丹; 高颖

    2016-01-01

    目的 探讨体外采用无血清化学成分确定培养基诱导人多能干细胞(human pluripotent stem cell,hPSC)分化为血管平滑肌细胞的优化方案.方法 hPSC(H1/H9胚胎干细胞及诱导多能干细胞)采用无血清、化学成分确定的培养基进行单层培养,并依据诱导分化所使用的生长因子不同分为两组:A组采用骨形态蛋白4(BMP4)+激活素A(Activin A);B组采用BMP4+ Activin A +Wnt信号通路激活剂CHIR99021.在两组干细胞诱导分化后的第9天,流式分选获取的CD31+、CD34+血管前体细胞,并经PDGF-BB定向诱导分化为血管平滑肌细胞.采用免疫荧光染色及实时定量荧光聚合酶链式反应(real-time QPCR)对分化获取的细胞进行表型鉴定.结果 流式分选结果显示,B组诱导方案应用于3种多能干细胞时能获得38.8%~ 51.4%的CD31+、CD34+血管前体细胞,显著高于A组诱导方案的11.7%~22.5% (P <0.05).两种诱导方案获得的血管前体细胞经定向诱导分化后,免疫荧光染色及real-time QPCR检测显示所有分化细胞均具有SM22-α、SMA-α、Calponin等肌源性标志的表达.结论 在采用无血清化学成分确定培养基培养条件下,同时激活BMP及Wnt信号通路可稳定、高效地将hPSC细胞诱导分化为血管平滑肌细胞,从而有望为细胞移植治疗提供“临床应用级别”的种子细胞.%Objective To establish an optimized protocol for rapid and efficient differentiation of human pluripotent stem cell into vascular smooth muscle progenitor cells with serum-free and chemically defined medium.Methods Monolayer H1/ H9 embryo stem cells(ESC) and induced pluripotent stem cells(iPSC) were cultured in serum-free and chemically defined medium,and divided into two groups according to the supplemented growth factors in the first two days.A group:added bone morphogenetic protein 4 (BMP4) and Activin A;B group:supplemented BMP4,Activin A and CHIR99021.CD31 +,CD34 + vascular progenitor

  19. The structure of myostatin:follistatin 288: insights into receptor utilization and heparin binding

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    Cash, Jennifer N.; Rejon, Carlis A.; McPherron, Alexandra C.; Bernard, Daniel J.; Thompson, Thomas B.; (UCIN); (McGill); (NIH)

    2009-09-29

    Myostatin is a member of the transforming growth factor-{beta} (TGF-{beta}) family and a strong negative regulator of muscle growth. Here, we present the crystal structure of myostatin in complex with the antagonist follistatin 288 (Fst288). We find that the prehelix region of myostatin very closely resembles that of TGF-{beta} class members and that this region alone can be swapped into activin A to confer signalling through the non-canonical type I receptor Alk5. Furthermore, the N-terminal domain of Fst288 undergoes conformational rearrangements to bind myostatin and likely acts as a site of specificity for the antagonist. In addition, a unique continuous electropositive surface is created when myostatin binds Fst288, which significantly increases the affinity for heparin. This translates into stronger interactions with the cell surface and enhanced myostatin degradation in the presence of either Fst288 or Fst315. Overall, we have identified several characteristics unique to myostatin that will be paramount to the rational design of myostatin inhibitors that could be used in the treatment of muscle-wasting disorders.

  20. Secreted Stress-Induced Phosphoprotein 1 Activates the ALK2-SMAD Signaling Pathways and Promotes Cell Proliferation of Ovarian Cancer Cells

    Directory of Open Access Journals (Sweden)

    Chia-Lung Tsai

    2012-08-01

    Full Text Available Stress-induced phosphoprotein 1 (STIP1, a cochaperone that organizes other chaperones, heat shock proteins (HSPs, was recently shown to be secreted by human ovarian cancer cells. In neuronal tissues, binding to prion protein was required for STIP1 to activate the ERK (extracellular-regulated MAP kinase signaling pathways. However, we report that STIP1 binding to a bone morphogenetic protein (BMP receptor, ALK2 (activin A receptor, type II-like kinase 2, was necessary and sufficient to stimulate proliferation of ovarian cancer cells. The binding of STIP1 to ALK2 activated the SMAD signaling pathway, leading to transcriptional activation of ID3 (inhibitor of DNA binding 3, promoting cell proliferation. In conclusion, ovarian-cancer-tissue-secreted STIP1 stimulates cancer cell proliferation by binding to ALK2 and activating the SMAD-ID3 signaling pathways. Although animal studies are needed to confirm these mechanisms in vivo, our results may pave the way for developing novel therapeutic strategies for ovarian cancer.

  1. Establishment of trophoblast stem cells under defined culture conditions in mice.

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    Yasuhide Ohinata

    Full Text Available The inner cell mass (ICM and trophoblast cell lineages duet early embryonic development in mammals. After implantation, the ICM forms the embryo proper as well as some extraembryonic tissues, whereas the trophoectoderm (TE exclusively forms the fetal portion of the placenta and the trophoblast giant cells. Although embryonic stem (ES cells can be derived from ICM in cultures of mouse blastocysts in the presence of LIF and/or combinations of small-molecule chemical compounds, and the undifferentiated pluripotent state can be stably maintained without use of serum and feeder cells, defined culture conditions for derivation and maintenance of undifferentiated trophoblast stem (TS cells have not been established. Here, we report that addition of FGF2, activin A, XAV939, and Y27632 are necessary and sufficient for derivation of TS cells from both of E3.5 blastocysts and E6.5 early postimplantation extraembryonic ectoderm. Moreover, the undifferentiated TS cell state can be stably maintained in chemically defined culture conditions. Cells derived in this manner expressed TS cell marker genes, including Eomes, Elf5, Cdx2, Klf5, Cdh1, Esrrb, Sox2, and Tcfap2c; differentiated into all trophoblast subtypes (trophoblast giant cells, spongiotrophoblast, and labyrinthine trophoblast in vitro; and exclusively contributed to trophoblast lineages in chimeric animals. This delineation of minimal requirements for derivation and self-renewal provides a defined platform for precise description and dissection of the molecular state of TS cells.

  2. Induced Pluripotent Stem Cells to Model Human Fibrodysplasia Ossificans Progressiva

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    Jie Cai

    2015-12-01

    Full Text Available Fibrodysplasia ossificans progressiva (FOP is a rare disease characterized by progressive ossification of soft tissues, for which there is no effective treatment. Mutations in the bone morphogenetic protein (BMP type I receptor activin receptor-like kinase 2 (ACVR1/ALK2 are the main cause of FOP. We generated human induced pluripotent stem cells (hiPSCs from FOP patients with the ALK2 R206H mutation. The mutant ALK2 gene changed differentiation efficiencies of hiPSCs into FOP bone-forming progenitors: endothelial cells (ECs and pericytes. ECs from FOP hiPSCs showed reduced expression of vascular endothelial growth factor receptor 2 and could transform into mesenchymal cells through endothelial-mesenchymal transition. Increased mineralization of pericytes from FOP hiPSCs could be partly inhibited by the ALK2 kinase inhibitor LDN-212854. Thus, differentiated FOP hiPSCs recapitulate some aspects of the disease phenotype in vitro, and they could be instrumental in further elucidating underlying mechanisms of FOP and development of therapeutic drug candidates.

  3. Fatal rupture of pulmonary arteriovenous malformation in hereditary haemorrhagic telangiectasis and severe PAH

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    D. Montani

    2009-03-01

    Full Text Available 29-yr-old patient with hereditary haemorrhagic telangiectasia was referred to the present authors’ centre with progressive exertional dyspnoea. Pulmonary arterial hypertension (PAH was suspected on Doppler echocardiography and confirmed by right heart catheterisation demonstrating severe PAH. Genetic analysis found an activin receptor-like kinase-1 gene missense mutation. Chest radiography and computed tomodensitometry of the chest revealed a pulmonary arteriovenous malformation with a 5-mm diameter feeding artery in the right lower lobe. Embolisation of the arteriovenous malformation was discussed, but was considered a very high-risk procedure that could aggravate PAH and was therefore not performed. Haemodynamics were improved by dual endothelin receptor antagonist and inhaled iloprost but the patient subsequently died suddenly of a rupture of the arteriovenous malformation into the pleural cavity. Severe PAH is generally considered a contraindication to performing pulmonary arteriovenous malformation embolisation because of the risk of worsening of PAH. However, given the significant risk of rupture, paradoxical embolism and haemoptysis, and the lack of data regarding the evolution of pulmonary pressure after embolisation in PAH, pulmonary arteriovenous malformation embolisation should not be absolutely contraindicated and might be considered in patients with stable PAH.

  4. Histone deacetylase inhibitor valproic acid promotes the differentiation of human induced pluripotent stem cells into hepatocyte-like cells.

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    Yuki Kondo

    Full Text Available In this study, we aimed to elucidate the effects and mechanism of action of valproic acid on hepatic differentiation from human induced pluripotent stem cell-derived hepatic progenitor cells. Human induced pluripotent stem cells were differentiated into endodermal cells in the presence of activin A and then into hepatic progenitor cells using dimethyl sulfoxide. Hepatic progenitor cells were matured in the presence of hepatocyte growth factor, oncostatin M, and dexamethasone with valproic acid that was added during the maturation process. After 25 days of differentiation, cells expressed hepatic marker genes and drug-metabolizing enzymes and exhibited drug-metabolizing enzyme activities. These expression levels and activities were increased by treatment with valproic acid, the timing and duration of which were important parameters to promote differentiation from human induced pluripotent stem cell-derived hepatic progenitor cells into hepatocytes. Valproic acid inhibited histone deacetylase activity during differentiation of human induced pluripotent stem cells, and other histone deacetylase inhibitors also enhanced differentiation into hepatocytes. In conclusion, histone deacetylase inhibitors such as valproic acid can be used to promote hepatic differentiation from human induced pluripotent stem cell-derived hepatic progenitor cells.

  5. Tissue inhibitor of metalloproteinase-3 is up-regulated by transforming growth factor-beta1 in vitro and expressed in fibroblastic foci in vivo in idiopathic pulmonary fibrosis.

    Science.gov (United States)

    García-Alvarez, Jorge; Ramirez, Remedios; Checa, Marco; Nuttall, Robert K; Sampieri, Clara L; Edwards, Dylan R; Selman, Moisés; Pardo, Annie

    2006-05-01

    Idiopathic pulmonary fibrosis (IPF) is characterized by fibroblast expansion and extracellular matrix accumulation. However, the mechanisms involved in matrix remodeling have not been elucidated. In this study, the authors aimed to evaluate the expression of the tissue inhibitors of matrix metalloproteinases (TIMPs) in human fibroblasts and whole tissues from IPF and normal lungs. They also determined the role of mitogen-activated protein kinase (MAPK) in TIMP3 expression. TIMP1, TIMP2, and TIMP3 were highly expressed in lung fibroblasts. Transforming growth factor (TGF)-beta1, a profibrotic mediator, induced strong up-regulation of TIMP3 at the mRNA and protein levels. The authors examined whether the MAPK pathway was involved in TGF-beta1-induced TIMP3 expression. TGF-beta1 induced the phosphorylation of p38 and extracellular signal-regulated kinase (ERK)1/2. Biochemical blockade of p38 by SB203580, but not of the ERK MAPK pathway, inhibited the effect of this factor. The effect was also blocked by the tyrosine kinase inhibitor genistein and by antagonizing TGF-beta1 receptor type I (activin-linked kinase [ALK5]). In IPF tissues TIMP3 gene expression was significantly increased and the protein was localized to fibroblastic foci and extracellular matrix. Our findings suggest that TGF-beta1-induced TIMP3 may be an important mediator in lung fibrogenesis.

  6. In vitro differentiation potential of human haematopoietic CD34(+) cells towards pancreatic β-cells.

    Science.gov (United States)

    Sunitha, Manne Mudhu; Srikanth, Lokanathan; Santhosh Kumar, Pasupuleti; Chandrasekhar, Chodimella; Sarma, Potukuchi Venkata Gurunadha Krishna

    2016-10-01

    Haematopoietic stem cells (HSCs) possess multipotent ability to differentiate into various types of cells on providing appropriate niche. In the present study, the differentiating potential of human HSCs into β-cells of islets of langerhans was explored. Human HSCs were apheretically isolated from a donor and cultured. Phenotypic characterization of CD34 glycoprotein in the growing monolayer HSCs was confirmed by immunocytochemistry and flow cytometry techniques. HSCs were induced by selection with beta cell differentiating medium (BDM), which consists of epidermal growth factor (EGF), fibroblast growth factor (FGF), transferrin, Triiodo-l-Tyronine, nicotinamide and activin A. Distinct morphological changes of differentiated cells were observed on staining with dithizone (DTZ) and expression of PDX1, insulin and synaptophysin was confirmed by immunocytochemistry. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis revealed distinct expression of specific β-cell markers, pancreatic and duodenal homeobox-1 (PDX1), glucose transporter-2 (GLUT-2), synaptophysin (SYP) and insulin (INS) in these differentiated cells compared to HSCs. Further, these cells exhibited elevated expression of INS gene at 10 mM glucose upon inducing with different glucose concentrations. The prominent feature of the obtained β-cells was the presence of glucose sensors, which was determined by glucokinase activity and high glucokinase activity compared with CD34(+) stem cells. These findings illustrate the differentiation of CD34(+) HSCs into β-cells of islets of langerhans.

  7. Generation of insulin-producing cells from gnotobiotic porcine skin-derived stem cells

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    Yang, Ji Hoon; Lee, Sung Ho; Heo, Young Tae [Department of Bioscience and Biotechnology, Bio-Organ Research Center, Konkuk University, Seoul 143-701 (Korea, Republic of); Uhm, Sang Jun [Department of Animal Biotechnology, Bio-Organ Research Center, Konkuk University, Seoul 143-701 (Korea, Republic of); Lee, Hoon Taek, E-mail: htl3675@konkuk.ac.kr [Department of Animal Biotechnology, Bio-Organ Research Center, Konkuk University, Seoul 143-701 (Korea, Republic of)

    2010-07-09

    A major problem in the treatment of type 1 diabetes mellitus is the limited availability of alternative sources of insulin-producing cells for islet transplantation. In this study, we investigated the effect of bone morphogenetic protein 4 (BMP-4) treatments of gnotobiotic porcine skin-derived stem cells (gSDSCs) on their reprogramming and subsequent differentiation into insulin-producing cells (IPCs). We isolated SDSCs from the ear skin of a gnotobiotic pig. During the proliferation period, the cells expressed stem-cell markers Oct-4, Sox-2, and CD90; nestin expression also increased significantly. The cells could differentiate into IPCs after treatments with activin-A, glucagon-like peptide-1 (GLP-1), and nicotinamide. After 15 days in the differentiation medium, controlled gSDSCs began expressing endocrine progenitor genes and proteins (Ngn3, Neuro-D, PDX-1, NKX2.2, NKX6.1, and insulin). The IPCs showed increased insulin synthesis after glucose stimulation. The results indicate that stem cells derived from the skin of gnotobiotic pigs can differentiate into IPCs under the appropriate conditions in vitro. Our three-stage induction protocol could be applied without genetic modification to source IPCs from stem cells in the skin of patients with diabetes for autologous transplantation.

  8. A Multi-Lineage Screen Reveals mTORC1 Inhibition Enhances Human Pluripotent Stem Cell Mesendoderm and Blood Progenitor Production

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    Emanuel Joseph Paul Nazareth

    2016-05-01

    Full Text Available Human pluripotent stem cells (hPSCs exist in heterogeneous micro-environments with multiple subpopulations, convoluting fate-regulation analysis. We patterned hPSCs into engineered micro-environments and screened responses to 400 small-molecule kinase inhibitors, measuring yield and purity outputs of undifferentiated, neuroectoderm, mesendoderm, and extra-embryonic populations. Enrichment analysis revealed mammalian target of rapamycin (mTOR inhibition as a strong inducer of mesendoderm. Dose responses of mTOR inhibitors such as rapamycin synergized with Bone Morphogenetic protein 4 (BMP4 and activin A to enhance the yield and purity of BRACHYURY-expressing cells. Mechanistically, small interfering RNA knockdown of RAPTOR, a component of mTOR complex 1, phenocopied the mesendoderm-enhancing effects of rapamycin. Functional analysis during mesoderm and endoderm differentiation revealed that mTOR inhibition increased the output of hemogenic endothelial cells 3-fold, with a concomitant enhancement of blood colony-forming cells. These data demonstrate the power of our multi-lineage screening approach and identify mTOR signaling as a node in hPSC differentiation to mesendoderm and its derivatives.

  9. Growth factors and myometrium: biological effects in uterine fibroid and possible clinical implications

    Science.gov (United States)

    Ciarmela, Pasquapina; Islam, Md. Soriful; Reis, Fernando M.; Gray, Peter C.; Bloise, Enrrico; Petraglia, Felice; Vale, Wylie; Castellucci, Mario

    2011-01-01

    BACKGROUND Growth factors are proteins secreted by a number of cell types that are capable of modulating cellular growth, proliferation and cellular differentiation. It is well accepted that uterine cellular events such as proliferation and differentiation are regulated by sex steroids and their actions in target tissues are mediated by local production of growth factors acting through paracrine and/or autocrine mechanisms. Myometrial mass is ultimately modified in pregnancy as well as in tumour conditions such as leiomyoma and leiomyosarcoma. Leiomyomas, also known as fibroids, are benign tumours of the uterus, considered to be one of the most frequent causes of infertility in reproductive years in women. METHODS For this review, we searched the database MEDLINE and Google Scholar for articles with content related to growth factors acting on myometrium; the findings are hereby reviewed and discussed. RESULTS Different growth factors such as epidermal growth factor (EGF), transforming growth factor-α (TGF-α), heparin-binding EGF (HB-EGF), acidic fibroblast growth factor (aFGF), basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), insulin-like growth factor (IGF), platelet-derived growth factor (PDGF) and TGF-β perform actions in myometrium and in leiomyomas. In addition to these growth factors, activin and myostatin have been recently identified in myometrium and leiomyoma. CONCLUSIONS Growth factors play an important role in the mechanisms involved in myometrial patho-physiology. PMID:21788281

  10. Cripto Binds Transforming Growth Factor β (TGF-β) and Inhibits TGF-β Signaling▿

    Science.gov (United States)

    Gray, Peter C.; Shani, Gidi; Aung, Kevin; Kelber, Jonathan; Vale, Wylie

    2006-01-01

    Cripto is a developmental oncoprotein and a member of the epidermal growth factor-Cripto, FRL-1, Cryptic family of extracellular signaling molecules. In addition to having essential functions during embryogenesis, Cripto is highly expressed in tumors and promotes tumorigenesis. During development, Cripto acts as an obligate coreceptor for transforming growth factor β (TGF-β) ligands, including nodals, growth and differentiation factor 1 (GDF1), and GDF3. As an oncogene, Cripto is thought to promote tumor growth via mechanisms including activation of mitogenic signaling pathways and antagonism of activin signaling. Here, we provide evidence supporting a novel mechanism in which Cripto inhibits the tumor suppressor function of TGF-β. Cripto bound TGF-β and reduced the association of TGF-β with its type I receptor, TβRI. Consistent with its ability to block receptor assembly, Cripto suppressed TGF-β signaling in multiple cell types and diminished the cytostatic effects of TGF-β in mammary epithelial cells. Furthermore, targeted disruption of Cripto expression by use of small inhibitory RNA enhanced TGF-β signaling, indicating that endogenous Cripto plays a role in restraining TGF-β responses. PMID:17030617

  11. TGF-β superfamily members from the helminth Fasciola hepatica show intrinsic effects on viability and development.

    Science.gov (United States)

    Japa, Ornampai; Hodgkinson, Jane E; Emes, Richard D; Flynn, Robin J

    2015-03-11

    The helminth Fasciola hepatica causes fasciolosis throughout the world, a major disease of livestock and an emerging zoonotic disease in humans. Sustainable control mechanisms such as vaccination are urgently required. To discover potential vaccine targets we undertook a genome screen to identify members of the transforming growth factor (TGF) family of proteins. Herein we describe the discovery of three ligands belonging to this superfamily and the cloning and characterisation of an activin/TGF like molecule we term FhTLM. FhTLM has a limited expression pattern both temporally across the parasite stages but also spatially within the worm. Furthermore, a recombinant form of this protein is able to enhance the rate (or magnitude) of multiple developmental processes of the parasite indicating a conserved role for this protein superfamily in the developmental biology of a major trematode parasite. Our study demonstrates for the first time the existence of this protein superfamily within F. hepatica and assigns a function to one of the three identified ligands. Moreover further exploration of this superfamily may yield future targets for diagnostic or vaccination purposes due to its stage restricted expression and functional role.

  12. Fibromodulin: a master regulator of myostatin controlling progression of satellite cells through a myogenic program.

    Science.gov (United States)

    Lee, Eun Ju; Jan, Arif Tasleem; Baig, Mohammad Hassan; Ashraf, Jalaluddin Mohammad; Nahm, Sang-Soep; Kim, Yong-Woon; Park, So-Young; Choi, Inho

    2016-08-01

    Differentiation of muscle satellite cells (MSCs) involves interaction of the proteins present in the extracellular matrix (ECM) with MSCs to regulate their activity, and therefore phenotype. Herein, we report fibromodulin (FMOD), a member of the proteoglycan family participating in the assembly of ECM, as a novel regulator of myostatin (MSTN) during myoblast differentiation. In addition to having a pronounced effect on the expression of myogenic marker genes [myogenin (MYOG) and myosin light chain 2 (MYL2)], FMOD was found to maintain the transcriptional activity of MSTN Moreover, coimmunoprecipitation and in silico studies performed to investigate the interaction of FMOD helped confirm that it antagonizes MSTN function by distorting its folding and preventing its binding to activin receptor type IIB. Furthermore, in vivo studies revealed that FMOD plays an active role in healing by increasing satellite cell recruitment to sites of injury. Together, these findings disclose a hitherto unrecognized regulatory role for FMOD in MSCs and highlight new mechanisms whereby FMOD circumvents the inhibitory effects of MSTN and triggers myoblast differentiation. These findings offer a basis for the design of novel MSTN inhibitors that promote muscle regeneration after injury or for the development of pharmaceutical agents for the treatment of different muscle atrophies.-Lee, E. J., Jan, A. T., Baig, M. H., Ashraf, J. M., Nahm, S.-S., Kim, Y.-W., Park, S.-Y., Choi, I. Fibromodulin: a master regulator of myostatin controlling progression of satellite cells through a myogenic program.

  13. Tumor inoculation site affects the development of cancer cachexia and muscle wasting.

    Science.gov (United States)

    Matsuyama, Tatsuzo; Ishikawa, Takeshi; Okayama, Tetsuya; Oka, Kaname; Adachi, Satoko; Mizushima, Katsura; Kimura, Reiko; Okajima, Manabu; Sakai, Hiromi; Sakamoto, Naoyuki; Katada, Kazuhiro; Kamada, Kazuhiro; Uchiyama, Kazuhiko; Handa, Osamu; Takagi, Tomohisa; Kokura, Satoshi; Naito, Yuji; Itoh, Yoshito

    2015-12-01

    The phenotype and severity of cancer cachexia differ among tumor types and metastatic site in individual patients. In this study, we evaluated if differences in tumor microenvironment would affect the development of cancer cachexia in a murine model, and demonstrated that body weight, adipose tissue and gastrocnemius muscle decreased in tumor-bearing mice. Interestingly, a reduction in heart weight was observed in the intraperitoneal tumor group but not in the subcutaneous group. We evaluated 23 circulating cytokines and members of the TGF-β family, and found that levels of IL-6, TNF-α and activin A increased in both groups of tumor-bearing mice. Eotaxin and G-CSF levels in the intraperitoneal tumor group were higher than in the subcutaneous group. Atrogin 1 and MuRF1 mRNA expressions in the gastrocnemius muscle increased significantly in both groups of tumor-bearing mice, however, in the myocardium, expression of these mRNAs increased in the intraperitoneal group but not in subcutaneous group. Based on these results, we believe that differences in microenvironment where tumor cells develop can affect the progression and phenotype of cancer cachexia through alterations in various circulating factors derived from the tumor microenvironment.

  14. TGF-β: An Important Mediator of Allergic Disease and a Molecule with Dual Activity in Cancer Development

    Directory of Open Access Journals (Sweden)

    Belen Tirado-Rodriguez

    2014-01-01

    Full Text Available The transforming growth factor-β (TGF-β superfamily is a family of structurally related proteins that includes TGF-β, activins/inhibins, and bone morphogenic proteins (BMPs. Members of the TGF-β superfamily regulate cellular functions such as proliferation, apoptosis, differentiation, and migration and thus play key roles in organismal development. TGF-β is involved in several human diseases, including autoimmune disorders and vascular diseases. Activation of the TGF-β receptor induces phosphorylation of serine/threonine residues and triggers phosphorylation of intracellular effectors (Smads. Once activated, Smad proteins translocate to the nucleus and induce transcription of their target genes, regulating various processes and cellular functions. Recently, there has been an attempt to correlate the effect of TGF-β with various pathological entities such as allergic diseases and cancer, yielding a new area of research known as “allergooncology," which investigates the mechanisms by which allergic diseases may influence the progression of certain cancers. This knowledge could generate new therapeutic strategies aimed at correcting the pathologies in which TGF-β is involved. Here, we review recent studies that suggest an important role for TGF-β in both allergic disease and cancer progression.

  15. Anti-angiogenic therapeutic strategies in hereditary hemorrhagic telangiectasia

    Directory of Open Access Journals (Sweden)

    Daniela S. Ardelean

    2015-02-01

    Full Text Available Hereditary hemorrhagic telangiectasia (HHT is an autosomal dominant vascular dysplastic disorder, characterized by recurrent nosebleeds (epistaxis, multiple telangiectases and arteriovenous malformations (AVMs in major organs. Mutations in Endoglin (ENG or CD105 and Activin receptor-like kinase 1 (ACVRL1 or ALK1 genes of the TGF-β superfamily receptors are responsible for HHT1 and HHT2 respectively and account for the majority of HHT cases. Haploinsufficiency in ENG and ALK1 is recognized at the underlying cause of HHT. However, the mechanisms responsible for the predisposition to and generation of AVMs, the hallmark of this disease, are poorly understood. Recent data suggest that dysregulated angiogenesis contributes to the pathogenesis of HHT and that the vascular endothelial growth factor, VEGF, may be implicated in this disease, by modulating the angiogenic balance in the affected tissues. Hence, anti-angiogenic therapies that target the abnormal vessels and restore the angiogenic balance are candidates for treatment of HHT. Here we review the experimental evidence for dysregulated angiogenesis in HHT, the anti-angiogenic therapeutic strategies used in animal models and some patients with HHT and the potential benefit of the anti-angiogenic treatment for ameliorating this severe, progressive vascular disease.

  16. Uterine ALK3 is essential during the window of implantation

    Science.gov (United States)

    Monsivais, Diana; Clementi, Caterina; Peng, Jia; Titus, Mary M.; Barrish, James P.; Creighton, Chad J.; Lydon, John P.; DeMayo, Francesco J.; Matzuk, Martin M.

    2016-01-01

    The window of implantation is defined by the inhibition of uterine epithelial proliferation, structural epithelial cell remodeling, and attenuated estrogen (E2) response. These changes occur via paracrine signaling between the uterine epithelium and stroma. Because implantation defects are a major cause of infertility in women, identifying these signaling pathways will improve infertility interventions. Bone morphogenetic proteins (BMPs) are TGF-β family members that regulate the postimplantation and midgestation stages of pregnancy. In this study, we discovered that signaling via activin-like kinase 3 (ALK3/BMPR1A), a BMP type 1 receptor, is necessary for blastocyst attachment. Conditional knockout (cKO) of ALK3 in the uterus was obtained by producing Alk3flox/flox-Pgr-cre–positive females. Alk3 cKO mice are sterile and have defects in the luminal uterine epithelium, including increased microvilli density and maintenance of apical cell polarity. Moreover, Alk3 cKO mice exhibit an elevated uterine E2 response and unopposed epithelial cell proliferation during the window of implantation. We determined that dual transcriptional regulation of Kruppel-like factor 15 (Klf15), by both the transforming growth factor β (TGF-β) transcription factor SMAD family member 4 (SMAD4) and progesterone receptor (PR), is necessary to inhibit uterine epithelial cell proliferation, a key step for embryo implantation. Our findings present a convergence of BMP and steroid hormone signaling pathways in the regulation of uterine receptivity. PMID:26721398

  17. Loss of inhibin alpha uncouples oocyte-granulosa dynamics and disrupts postnatal folliculogenesis

    Science.gov (United States)

    Myers, Michelle; Middlebrook, Brooke S.; Matzuk, Martin M.; Pangas, Stephanie A.

    2009-01-01

    Targeted disruption of the inhibin α gene (Inha−/−) in mice results in an ovarian phenotype of granulosa cell tumors that renders the animals infertile. Little is known about the reproductive defects prior to tumor development. Here, we report novel data on early follicle dynamics in Inha−/− mice, which demonstrate that inhibin α has important consequences upon follicle development. Morphological changes in both germ and somatic cells were evident in postnatal day 12 ovaries, with Inha−/− mice exhibiting numerous multilayered follicles that were far more advanced than those observed in age-matched controls. These changes were accompanied by alterations in follicle dynamics such that Inha−/− ovaries had fewer follicles in the resting pool and more committed in the growth phase. Absence of inhibin α resulted in advanced follicular maturation as marked by premature loss of anti-Müllerian hormone (AMH) in secondary follicles. Additionally, gene expression analysis revealed changes in factors known to be vital for oocyte and follicle development. Together, these data provide key evidence to suggest that regulation of the inhibin/activin system is essential for early folliculogenesis in the prepubertal mouse ovary. PMID:19666016

  18. Redundant Roles of SMAD2 and SMAD3 in Ovarian Granulosa Cells In Vivo ▿

    Science.gov (United States)

    Li, Qinglei; Pangas, Stephanie A.; Jorgez, Carolina J.; Graff, Jonathan M.; Weinstein, Michael; Matzuk, Martin M.

    2008-01-01

    Transforming growth factor β (TGF-β) superfamily members are critical in maintaining cell growth and differentiation in the ovary. Although signaling of activins, TGF-βs, growth differentiation factor 9, and nodal converge preferentially to SMAD2 and SMAD3, the in vivo functions and redundancy of these SMADs in the ovary and female reproduction remain largely unidentified. To circumvent the deleterious phenotypic aspects of ubiquitous deletion of Smad2 and Smad3, a conditional knockout strategy was formulated to selectively inactivate Smad2, Smad3, or both Smad2 and Smad3 in ovarian granulosa cells. While granulosa cell ablation of individual Smad2 or Smad3 caused insignificant changes in female fertility, deletion of both Smad2 and Smad3 led to dramatically reduced female fertility and fecundity. These defects were associated with the disruption of multiple ovarian processes, including follicular development, ovulation, and cumulus cell expansion. Furthermore, the impaired expansion of cumulus cells may be partially associated with altered cumulus expansion-related transcripts that are regulated by SMAD2/3 signaling. Our results indicate that SMAD2 and SMAD3 function redundantly in vivo to maintain normal female fertility and further support the involvement of an intraovarian SMAD2/3 pathway in mediating oocyte-produced signals essential for coordinating key events of the ovulatory process. PMID:18809571

  19. Stem cells decreased neuronal cell death after hypoxic stress in primary fetal rat neurons in vitro.

    Science.gov (United States)

    Sakai, Tetsuro; Xu, Yan

    2012-01-01

    To explore stem cell-mediated neuronal protection through extracellular signaling pathways by transplanted stem cells, we sought to identify potential candidate molecules responsible for neuronal protection using an in vitro coculture system. Primary fetal rat hippocampal neurons underwent hypoxia (≤1% oxygen) for 96 h nad then were returned to a normoxic condition. The study group then received rat umbilical cord matrix-derived stem cells, while the control group received fresh media only. The experimental group showed decreased neuronal apoptosis compared to the control group [44.5 ± 1.6% vs. 71.0 ± 4.2% (mean ± SD, p = 0.0005) on day 5] and higher neuronal survival (4.9 ± 1.2 cells/100× field vs. 2.2 ± 0.3, p = 0.02 on day 5). Among 90 proteins evaluated using a protein array, stem cell coculture media showed increased protein secretion of TIMP-1 (5.61-fold), TIMP-2 (4.88), CNTF-Rα (3.42), activin A (2.20), fractalkine (2.04), CCR4 (2.02), and decreased secretion in MIP-2 (0.30-fold), AMPK α1 (0.43), TROY (0.48), and TIMP-3 (0.50). This study demonstrated that coculturing stem cells with primary neurons in vitro decreased neuronal cell death after hypoxia with significantly altered protein secretion. The results suggest that stem cells may offer neuronal protection through extracellular signaling.

  20. Growth differentiation factor 8 suppresses cell proliferation by up-regulating CTGF expression in human granulosa cells.

    Science.gov (United States)

    Chang, Hsun-Ming; Pan, Hui-Hui; Cheng, Jung-Chien; Zhu, Yi-Min; Leung, Peter C K

    2016-02-15

    Connective tissue growth factor (CTGF) is a matricellular protein that plays a critical role in the development of ovarian follicles. Growth differentiation factor 8 (GDF8) is mainly, but not exclusively, expressed in the mammalian musculoskeletal system and is a potent negative regulator of skeletal muscle growth. The aim of this study was to investigate the effects of GDF8 and CTGF on the regulation of cell proliferation in human granulosa cells and to examine its underlying molecular determinants. Using dual inhibition approaches (inhibitors and small interfering RNAs), we have demonstrated that GDF8 induces the up-regulation of CTGF expression through the activin receptor-like kinase (ALK)4/5-mediated SMAD2/3-dependent signaling pathways. In addition, the increase in CTGF expression contributes to the GDF8-induced suppressive effect on granulosa cell proliferation. Our findings suggest that GDF8 and CTGF may play critical roles in the regulation of proliferative events in human granulosa cells. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  1. Inactivation of Smad4 leads to impaired ocular development and cataract formation

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Ying, E-mail: yingliu@doheny.org [Department of Ophthalmology and Doheny Eye Institute, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033 (United States); Sun Yet-sen University, Zhongshan Ophthalmic Center, State Key Ophthalmic Laboratory, Guangzhou 510060 (China); Kawai, Kirio; Khashabi, Shabnam [Department of Ophthalmology and Doheny Eye Institute, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033 (United States); Deng, Chuxia [Laboratory of Biochemistry and Metabolism, NIDDK, National Institutes of Health, Bethesda, MD 20892 (United States); Liu, Yi-Hsin; Yiu, Samuel [Department of Ophthalmology and Doheny Eye Institute, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033 (United States)

    2010-10-01

    Research highlights: {yields} Inactivation of Smad4 caused disruption in the development of the anterior segment. {yields} Inactivation of Smad4 failed to disrupt early lens development. {yields} Smad4 controlled lens cell cycle and cell death processes. {yields} Smad4 may regulate actin stress fiber assembly and eyelid epithelial movement. -- Abstract: Purpose: Signaling by members of the TGF{beta} superfamily of molecules is essential for embryonic development and homeostasis. Smad4, a key intracellular mediator in TGF{beta} signaling, forms transcriptional activator complexes with Activin-, BMP-, and TGF{beta}-restricted Smad proteins. However, the functional role of Smad4 in controlling different visual system compartments has not been fully investigated. Methods: Using the Pax6 promoter-driven Cre transgenic, smad4 was conditionally inactivated in the lens, cornea and ectoderm of the eyelids. Standard histological and molecular analytical approaches were employed to reveal morphological and cellular changes. Results: Inactivation of Smad4 in the lens led to microphthalmia and cataract formation in addition to the persistent adhesion of the retina to the lens and the iris to the cornea. Inactivation of Smad4 from the ectoderm of the eyelid and cornea caused disruption to eyelid fusion and proper development of the corneal epithelium and corneal stroma. Conclusions: Smad4 is required for the development and maintenance of the lens in addition to the proper development of the cornea, eyelids, and retina.

  2. Derivation and transcriptional profiling analysis of pluripotent stem cell lines from rat blastocysts

    Institute of Scientific and Technical Information of China (English)

    Chunliang Li; Ying Yang; Junjie Gu; Yu Ma; Ying Jin

    2009-01-01

    Embryonic stem (ES) cells are derived from blastocyst-stage embryos. Their unique properties of self-renewal and pluripotency make them an attractive tool for basic research and a potential cell resource for therapy. ES cells of mouse and human have been successfully generated and applied in a wide range of research. However, no genuine ES cell lines have been obtained from rat to date. In this study, we identified pluripotent cells in early rat embryos using specific antibodies against markers of pluripotent stem cells. Subsequently, by modifying the culture medium for rat blastocysts, we derived pluripotent rat ES-llke cell lines, which expressed pluripotency markers and formed embryoid bodies (EBs) in vitro. Importantly, these rat ES-like cells were able to produce teratomas. Both EBs and teratomas contained tissues from all three embryonic germ layers, in addition, from the rat ES-like cells, we derived a rat primitive endoderm (PrE) cell line. Furthermore, we conducted transcriptional profiling of the rat ES-like cells and identified the unique molecular signature of the rat pluripotent stem cells. Our analysis demonstrates that multiple signaling pathways, including the BMP, Activin and roTOR pathways, may be involved in keeping the rat ES-like cells in an undifferentiated state. The cell lines and information obtained in this study will accelerate our understanding of the molecular regulation underlying pluripotency and guide us in the appropriate manipulation of ES cells from a particular species.

  3. Down-regulation of phosphoglucomutase 3 mediates sulforaphane-induced cell death in LNCaP prostate cancer cells

    Directory of Open Access Journals (Sweden)

    Lee Hyo-Jeong

    2010-12-01

    Full Text Available Abstract Background Sulforaphane (SFN is an isothiocyanate found in cruciferous vegetables that exerts anti-oxidant, anti-inflammatory, anti-cancer and radio-sensitizing activities. Nonetheless, the mechanism responsible for SFN-induced cell death is not fully understood. In the present study, anti-cancer mechanism of SFN was elucidated in LNCaP prostate cancer cells. Results SFN exerted cytotoxicity and increased TUNEL positive cells in a concentration-dependent manner in LNCaP cells. Proteomics study revealed that levels of nine proteins including tubulin β-2, phosphoglucomutase-3 (PGM3, melanoma-derived leucine zipper containing extra-nuclear factor, activin A type I receptor precursor, smoothelin-A, KIA0073, hypothetical protein LOC57691 and two unnamed proteins were changed over 8 folds in SFN treated LNCaP cells compared to untreated control. We have further confirmed that SFN reduced PGM3 expression with western blotting and showed that PGM3 siRNA enhanced cytotoxicity demonstrated by cell morphology and TUNEL assays in LNCaP cells. Conclusion Taken together, these findings suggest that PGM3 plays a role in mediating SFN-induced cell death in LNCaP cells, and is a potential molecular therapeutic target for prostate cancer.

  4. A Modified Method of Insulin Producing Cells’ Generation from Bone Marrow-Derived Mesenchymal Stem Cells

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    Paweł Czubak

    2014-01-01

    Full Text Available Type 1 diabetes mellitus is a result of autoimmune destruction of pancreatic insulin producing β-cells and so far it can be cured only by insulin injection, by pancreas transplantation, or by pancreatic islet cells’ transplantation. The methods are, however, imperfect and have a lot of disadvantages. Therefore new solutions are needed. The best one would be the use of differentiated mesenchymal stem cells (MSCs. In the present study, we investigated the potential of the bone marrow-derived MSCs line for in vitro differentiation into insulin producing cells (IPSs. We applied an 18-day protocol to differentiate MSCs. Differentiating cells formed cell clusters some of which resembled pancreatic islet-like cells. Using dithizone we confirmed the presence of insulin in the cells. What is more, the expression of proinsulin C-peptide in differentiated IPCs was analyzed by flow cytometry. For the first time, we investigated the influence of growth factors’ concentration on IPCs differentiation efficiency. We have found that an increase in the concentration of growth factors up to 60 ng/mL of β-FGF/EGF and 30 ng/mL of activin A/β-cellulin increases the percentage of IPCs. Further increase of growth factors does not show any increase of the percentage of differentiated cells. Our findings suggest that the presented protocol can be adapted for differentiation of insulin producing cells from stem cells.

  5. Research on Potential Biomarkers in Hereditary Haemorrhagic Telangiectasia

    Directory of Open Access Journals (Sweden)

    Luisa Maria Botella

    2015-03-01

    Full Text Available Hereditary Hemorrhagic Telangiectasia (HHT is a genetically heterogeneous disorder, involving mutations in two predominant genes known as Endoglin (ENG; HHT1 and Activin receptor like kinase 1 (ACVRL1/ALK1; HHT2, as well as in some less frequent genes, such as MADH4/SMAD4 (JP-HHT or BMP9/GDF2 (HHT5. The diagnosis of HHT patients currently remains at the clinical level, according to the Curaçao criteria, whereas the molecular diagnosis is used to confirm or rule out suspected HHT cases, especially when a well characterized index case is present in the family or in an isolated population. Unfortunately, many suspected patients do not present a clear HHT diagnosis or do not show pathogenic mutations in HHT genes, prompting the need to investigate additional biomarkers of the disease. Here, several HHT biomarkers and novel methodological approaches developed during the last years will be reviewed. On one hand, products detected in plasma or serum samples: soluble proteins (VEGF, TGF-β1, soluble endoglin, angiopoietin-2 and microRNA variants (miR-27a, miR-205, miR-210. On the other hand, differential HHT gene expression fingerprinting, Next Generation Sequencing (NGS of a panel of genes involved in HHT, and infrared spectroscopy combined with Artificial Neural Network (ANN patterns will also be reviewed. All these biomarkers might help to improve and refine HHT diagnosis by distinguishing from the non-HHT population.

  6. A mouse model of hereditary hemorrhagic telangiectasia generated by transmammary-delivered immunoblocking of BMP9 and BMP10

    Science.gov (United States)

    Ruiz, Santiago; Zhao, Haitian; Chandakkar, Pallavi; Chatterjee, Prodyot K.; Papoin, Julien; Blanc, Lionel; Metz, Christine N.; Campagne, Fabien; Marambaud, Philippe

    2016-01-01

    Hereditary hemorrhagic telangiectasia (HHT) is a potentially life-threatening genetic vascular disorder caused by loss-of-function mutations in the genes encoding activin receptor-like kinase 1 (ALK1), endoglin, Smad4, and bone morphogenetic protein 9 (BMP9). Injections of mouse neonates with BMP9/10 blocking antibodies lead to HHT-like vascular defects in the postnatal retinal angiogenesis model. Mothers and their newborns share the same immunity through the transfer of maternal antibodies during lactation. Here, we investigated whether the transmammary delivery route could improve the ease and consistency of administering anti-BMP9/10 antibodies in the postnatal retinal angiogenesis model. We found that anti-BMP9/10 antibodies, when intraperitoneally injected into lactating dams, are efficiently transferred into the blood circulation of lactationally-exposed neonatal pups. Strikingly, pups receiving anti-BMP9/10 antibodies via lactation displayed consistent and robust vascular pathology in the retina, which included hypervascularization and defects in arteriovenous specification, as well as the presence of multiple and massive arteriovenous malformations. Furthermore, RNA-Seq analyses of neonatal retinas identified an increase in the key pro-angiogenic factor, angiopoietin-2, as the most significant change in gene expression triggered by the transmammary delivery of anti-BMP9/10 antibodies. Transmammary-delivered BMP9/10 immunoblocking in the mouse neonatal retina is therefore a practical, noninvasive, reliable, and robust model of HHT vascular pathology. PMID:27874028

  7. Pharmacologic Options for the Treatment of Sarcopenia.

    Science.gov (United States)

    Morley, John E

    2016-04-01

    Sarcopenia is now clinically defined as a loss of muscle mass coupled with functional deterioration (either walking speed or distance or grip strength). Based on the FRAX studies suggesting that the questions without bone mineral density can be used to screen for osteoporosis, there is now a valid simple questionnaire to screen for sarcopenia, i.e., the SARC-F. Numerous factors have been implicated in the pathophysiology of sarcopenia. These include genetic factors, mitochondrial defects, decreased anabolic hormones (e.g., testosterone, vitamin D, growth hormone and insulin growth hormone-1), inflammatory cytokine excess, insulin resistance, decreased protein intake and activity, poor blood flow to muscle and deficiency of growth derived factor-11. Over the last decade, there has been a remarkable increase in our understanding of the molecular biology of muscle, resulting in a marked increase in potential future targets for the treatment of sarcopenia. At present, resistance exercise, protein supplementation, and vitamin D have been established as the basic treatment of sarcopenia. High-dose testosterone increases muscle power and function, but has a number of potentially limiting side effects. Other drugs in clinical development include selective androgen receptor molecules, ghrelin agonists, myostatin antibodies, activin IIR antagonists, angiotensin converting enzyme inhibitors, beta antagonists, and fast skeletal muscle troponin activators. As sarcopenia is a major predictor of frailty, hip fracture, disability, and mortality in older persons, the development of drugs to treat it is eagerly awaited.

  8. 5'UTR mutations of ENG cause hereditary hemorrhagic telangiectasia.

    Science.gov (United States)

    Damjanovich, Kristy; Langa, Carmen; Blanco, Francisco J; McDonald, Jamie; Botella, Luisa M; Bernabeu, Carmelo; Wooderchak-Donahue, Whitney; Stevenson, David A; Bayrak-Toydemir, Pinar

    2011-12-22

    Hereditary hemorrhagic telangiectasia (HHT) is a vascular disorder characterized by epistaxis, arteriovenous malformations, and telangiectases. The majority of the patients have a mutation in the coding region of the activin A receptor type II-like 1 (ACVRL1) or Endoglin (ENG) gene. However, in approximately 15% of cases, sequencing analysis and deletion/duplication testing fail to identify mutations in the coding regions of these genes. Knowing its vital role in transcription and translation control, we were prompted to investigate the 5'untranslated region (UTR) of ENG. We sequenced the 5'UTR of ENG for 154 HHT patients without mutations in ENG or ACVRL1 coding regions. We found a mutation (c.-127C > T), which is predicted to affect translation initiation and alter the reading frame of endoglin. This mutation was found in a family with linkage to the ENG, as well as in three other patients, one of which had an affected sibling with the same mutation. In vitro expression studies showed that a construct with the c.-127C > T mutation alters the translation and decreases the level of the endoglin protein. In addition, a c.-9G > A mutation was found in three patients, one of whom was homozygous for this mutation. Expression studies showed decreased protein levels suggesting that the c.-9G > A is a hypomorphic mutation. Our results emphasize the need for the inclusion of the 5'UTR region of ENG in clinical testing for HHT.

  9. Hereditary hemorrhagic telangiectasia: two distinct ENG deletions in one family.

    Science.gov (United States)

    Wooderchak, W; Gedge, F; McDonald, M; Krautscheid, P; Wang, X; Malkiewicz, J; Bukjiok, C J; Lewis, T; Bayrak-Toydemir, P

    2010-11-01

    Hereditary hemorrhagic telangiectasia (HHT) is an autosomal dominant disorder characterized by aberrant vascular development. Mutations in endoglin (ENG) or activin A receptor type II-like 1 (ACVRL1) account for around 90% of HHT patients, 10% of those are large deletions or duplications. We report here the first observation of two distinct, large ENG deletions segregating in one pedigree. An ENG exon 4-7 deletion was observed in a patient with HHT. This deletion was identified in several affected family members. However, some affected family members had an ENG exon 3 deletion instead. These deletions were detected by multiplex ligation-dependent probe amplification and confirmed by mRNA sequencing and an oligo-CGH array. Linkage analysis revealed that one individual with the exon 3 deletion inherited the same chromosome from his mother who has the exon 4-7 deletion. This finding has important clinical implications because it shows that targeted family-specific mutation analysis for exon deletions could have led to the misdiagnosis of some affected family members. © 2010 John Wiley & Sons A/S.

  10. Clinical features and mutations in the ENG, ACVRL1, and SMAD4 genes in Korean patients with hereditary hemorrhagic telangiectasia.

    Science.gov (United States)

    Lee, Seung-Tae; Kim, Jee-Ah; Jang, Shin-Yi; Kim, Duk-Kyung; Do, Young Soo; Suh, Gee Young; Kim, Jong-Won; Ki, Chang-Seok

    2009-02-01

    Hereditary hemorrhagic telangiectasia (HHT) is an inherited disorder that is characterized by abnormal communication between the arteries and veins in the skin, mucosa, and various organs. HHT has been reported to show significant phenotypic variability and genetic heterogeneity with wide ethnic and geographic variations. Although mutations in the endoglin (ENG) and activin A receptor type II-like 1 (ACVRL1) genes have been known to cause HHT for more than 10 yr, little is known about the clinical features or genetic background of Korean patients with HHT. In addition, mutations in mothers against decapentaplegic homolog 4 (SMAD4) are also seen in patients with the combined syndrome of juvenile polyposis and HHT. This study examined five Korean patients with the typical manifestations of HHT such as frequent epistaxis and pulmonary arteriovenous malformations. Direct sequencing of the ENG and ACVRL1 genes revealed one known mutation, ENG c.277C>T, in one patient and two novel mutations, ENG c.992-1G>C and ACVRL1 c.81dupT in two patients, respectively. The remaining two patients with negative results were screened for SMAD4 mutations as well as gross deletions of ENG and ACVRL1 using multiple ligation-dependent probe amplification, but none was detected. Despite the small number of patients investigated, we firstly report Korean patients with genetically confirmed HHT, and show the genetic and allelic heterogeneity underlying HHT.

  11. Differentiation of murine embryonic stem cells to thyrocytes requires insulin and insulin-like growth factor-1.

    Science.gov (United States)

    Arufe, Maria C; Lu, Min; Lin, Reigh-Yi

    2009-04-03

    The mechanisms controlling thyrocyte development during embryonic stem (ES) cell differentiation have only been partially elucidated, although previous studies have suggested the participation of thyroid stimulating hormone (TSH) in these processes. To further define the role of TSH in this context, we have studied a murine ES cell line in which green fluorescent protein (GFP) cDNA is targeted to the TSH receptor (TSHR) gene, linking the expression of GFP to the transcription of the endogenous TSHR gene. We demonstrate that, in the initial stages of embryoid body formation, activin A and TSH induce the differentiation of definitive endoderm and thyrocyte progenitors expressing Sox17, Foxa2, and TSHR. These thyrocyte progenitors are then converted into cellular aggregates that, in the presence of insulin and IGF-1, further differentiate into mature thyroglobulin-expressing thyrocytes. Our data suggest that, despite the fact that TSH is important for the induction and specification of thyrocytes from ES cells, insulin and IGF-1 are crucial for thyrocyte maturation. Our method provides a powerful in vitro differentiation model for studying the mechanisms of early thyrocyte lineage development.

  12. A Small Molecule that Promotes Cardiac Differentiation of Human Pluripotent Stem Cells under Defined, Cytokine- and Xeno-free Conditions

    Directory of Open Access Journals (Sweden)

    Itsunari Minami

    2012-11-01

    Full Text Available Human pluripotent stem cells (hPSCs, including embryonic stem cells and induced pluripotent stem cells, are potentially useful in regenerative therapies for heart disease. For medical applications, clinical-grade cardiac cells must be produced from hPSCs in a defined, cost-effective manner. Cell-based screening led to the discovery of KY02111, a small molecule that promotes differentiation of hPSCs to cardiomyocytes. Although the direct target of KY02111 remains unknown, results of the present study suggest that KY02111 promotes differentiation by inhibiting WNT signaling in hPSCs but in a manner that is distinct from that of previously studied WNT inhibitors. Combined use of KY02111 and WNT signaling modulators produced robust cardiac differentiation of hPSCs in a xeno-free, defined medium, devoid of serum and any kind of recombinant cytokines and hormones, such as BMP4, Activin A, or insulin. The methodology has potential as a means for the practical production of human cardiomyocytes for regeneration therapies.

  13. Emerging therapies for the treatment of osteoporosis

    Directory of Open Access Journals (Sweden)

    Garima Bhutani

    2013-01-01

    Full Text Available Osteoporosis is a chronic disease of the osseous system characterized by decreased bone strength and increased fracture risk. It is due to an imbalance in the dynamic ongoing processes of bone formation and bone resorption. Currently available osteoporosis therapies like bisphosphonates, selective estrogen receptor modulators (SERMs, and denosumab are anti-resorptive agents. Parathyroid hormone analogs like teriparatide are the only anabolic agents currently approved for osteoporosis treatment. The side-effects and limited efficacy of the presently available therapies has encouraged extensive research into the pathophysiology of the disease and newer drug targets for its treatment. The novel anti-resorptive agents being developed are newer SERMs, osteoprotegerin, c-src (cellular-sarcoma kinase inhibitors, αVβ3 integrin antagonists, cathepsin K inhibitors, chloride channel inhibitors, and nitrates. Upcoming anabolic agents include calcilytics, antibodies against sclerostin and Dickkopf-1, statins, matrix extracellular phosphoglycoprotein fragments activin inhibitiors, and endo-cannabinoid agonists. Many of these new drugs are still in development. This article provides an insight into the emerging drugs for the treatment of osteoporosis.

  14. Efficient Differentiation of Mouse Embryonic Stem Cells into Insulin-Producing Cells

    Directory of Open Access Journals (Sweden)

    Szu-Hsiu Liu

    2012-01-01

    Full Text Available Embryonic stem (ES cells are a potential source of a variety of differentiated cells for cell therapy, drug discovery, and toxicology screening. Here, we present an efficacy strategy for the differentiation of mouse ES cells into insulin-producing cells (IPCs by a two-step differentiation protocol comprising of (i the formation of definitive endoderm in monolayer culture by activin A, and (ii this monolayer endoderm being induced to differentiate into IPCs by nicotinamide, insulin, and laminin. Differentiated cells can be obtained within approximately 7 days. The differentiation IPCs combined application of RT-PCR, ELISA, and immunofluorescence to characterize phenotypic and functional properties. In our study, we demonstrated that IPCs produced pancreatic transcription factors, endocrine progenitor marker, definitive endoderm, pancreatic β-cell markers, and Langerhans α and δ cells. The IPCs released insulin in a manner that was dose dependent upon the amount of glucose added. These techniques may be able to be applied to human ES cells, which would have very important ramifications for treating human disease.

  15. CNS repair and axon regeneration: Using genetic variation to determine mechanisms.

    Science.gov (United States)

    Tedeschi, Andrea; Omura, Takao; Costigan, Michael

    2017-01-01

    The importance of genetic diversity in biological investigation has been recognized since the pioneering studies of Gregor Johann Mendel and Charles Darwin. Research in this area has been greatly informed recently by the publication of genomes from multiple species. Genes regulate and create every part and process in a living organism, react with the environment to create each living form and morph and mutate to determine the history and future of each species. The regenerative capacity of neurons differs profoundly between animal lineages and within the mammalian central and peripheral nervous systems. Here, we discuss research that suggests that genetic background contributes to the ability of injured axons to regenerate in the mammalian central nervous system (CNS), by controlling the regulation of specific signaling cascades. We detail the methods used to identify these pathways, which include among others Activin signaling and other TGF-β superfamily members. We discuss the potential of altering these pathways in patients with CNS damage and outline strategies to promote regeneration and repair by combinatorial manipulation of neuron-intrinsic and extrinsic determinants. Copyright © 2016 Elsevier Inc. All rights reserved.

  16. Treatment of renal anemia: Erythropoiesis stimulating agents and beyond

    Directory of Open Access Journals (Sweden)

    Patrick Biggar

    2017-09-01

    Full Text Available Anemia, complicating the course of chronic kidney disease, is a significant parameter, whether interpreted as subjective impairment or an objective prognostic marker. Renal anemia is predominantly due to relative erythropoietin (EPO deficiency. EPO inhibits apoptosis of erythrocyte precursors. Studies using EPO substitution have shown that increasing hemoglobin (Hb levels up to 10-11 g/dL is associated with clinical improvement. However, it has not been unequivocally proven that further intensification of erythropoiesis stimulating agent (ESA therapy actually leads to a comprehensive benefit for the patient, especially as ESAs are potentially associated with increased cerebro-cardiovascular events. Recently, new developments offer interesting options not only via stimulating erythropoeisis but also by employing additional mechanisms. The inhibition of activin, a member of the transforming growth factor superfamily, has the potential to correct anemia by stimulating liberation of mature erythrocyte forms and also to mitigate disturbed mineral and bone metabolism as well. Hypoxia-inducible factor prolyl hydroxylase inhibitors also show pleiotropic effects, which are at the focus of present research and have the potential of reducing mortality. However, conventional ESAs offer an extensive body of safety evidence, against which the newer substances should be measured. Carbamylated EPO is devoid of Hb augmenting effects whilst exerting promising tissue protective properties. Additionally, the role of hepcidin antagonists is discussed. An innovative new hemodialysis blood tube system, reducing blood contact with air, conveys a totally different and innocuous option to improve renal anemia by reducing mechanical hemolysis.

  17. Society for Reproductive Biology Founders' Lecture 2009. Preparing fertile soil: the importance of endometrial receptivity.

    Science.gov (United States)

    Salamonsen, Lois A; Nie, Guiying; Hannan, Natalie J; Dimitriadis, Evdokia

    2009-01-01

    The human endometrium is receptive for implantation of a blastocyst for only 4-5 days in each menstrual cycle. Failure of implantation is a major reason for infertility in women and the inability to achieve endometrial receptivity is responsible for much of the failure of reproductive technologies. Endometrial receptivity requires changes in the uterine luminal and glandular cells, particularly in terms of their secretory capacity and altered expression of adhesion molecules. In parallel with these changes, decidualisation (differentiation) of the endometrial stroma is initiated in women during the receptive phase, regardless of the presence of a blastocyst. Increased leucocyte numbers are also important. The microenvironments provided by the endometrium during the receptive phase and that support implantation are highly complex and constantly changing as implantation progresses. The present review provides a comprehensive overview of the cellular and molecular events of human implantation. It also summarises work from our laboratories emphasising the functional importance of proprotein convertase 6, along with key cytokines (interleukin-11, leukaemia inhibitory factor, activin A) and chemokines (including CX3CL1 and CCL14), during implantation. Of particular importance is how these mediators contribute to receptivity and how they are disturbed in infertile women. Factors that are critical for uterine receptivity may also be manipulated to provide new contraceptive strategies for women.

  18. ALK7 Gene Polymorphism is Associated with Metabolic Syndrome Risk and Cardiovascular Remodeling

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Wenchao; Wang, Hui; Zhang, Wei [Key Laboratory of Cardiovascular Remodeling and Function Research Chinese Ministry of Education and Chinese Ministry of Public Health, Qilu Hospital of Shandong University, Jinan (China); Lv, Ruijuan [Department of Emergency, Qilu Hospital of Shandong University, Jinan (China); Wang, Zhihao [Key Laboratory of Cardiovascular Remodeling and Function Research Chinese Ministry of Education and Chinese Ministry of Public Health, Qilu Hospital of Shandong University, Jinan (China); Department of Geriatrics, Qilu Hospital of Shandong University, Jinan (China); Shang, Yuanyuan; Zhang, Yun; Zhong, Ming [Key Laboratory of Cardiovascular Remodeling and Function Research Chinese Ministry of Education and Chinese Ministry of Public Health, Qilu Hospital of Shandong University, Jinan (China); Chen, Yuguo; Tang, Mengxiong, E-mail: tangmengxiongsdu8@163.com [Key Laboratory of Cardiovascular Remodeling and Function Research Chinese Ministry of Education and Chinese Ministry of Public Health, Qilu Hospital of Shandong University, Jinan (China); Department of Emergency, Qilu Hospital of Shandong University, Jinan (China)

    2013-08-15

    Activin receptor-like kinase 7 (ALK7) is a type I receptor for the TGF-β superfamily and has recently been demonstrated to play an important role in the maintenance of metabolic homeostasis. To investigate the association of the ALK7 gene polymorphism with metabolic syndrome (MetS) and cardiovascular remodeling in MetS patients. The single nucleotide polymorphism rs13010956 in the ALK7 gene was genotyped in 351 Chinese subjects undergoing carotid and cardiac ultrasonography. The associations of the ALK7 gene polymorphism with the MetS phenotype, MetS parameters, and cardiovascular ultrasonic features were analyzed. The rs13010956 polymorphism in the ALK7 gene was found to be significantly associated with the MetS phenotype in females (p < 0.05) and was also significantly associated with blood pressure in the total (p < 0.05) and female populations (p < 0.01). Further analysis revealed that rs13010956 was associated with mean intima-media thickness of the carotid arteries in females (p < 0.05). After control for body mass index, blood pressure, fasting blood glucose, and triglycerides, rs13010956 was also found to be significantly associated with left ventricular mass index in the total (p < 0.05) and female populations (p < 0.05). Our findings suggested that the ALK7 gene polymorphism rs13010956 was significantly associated with MetS risk in females and may be involved in cardiovascular remodeling in MetS patients.

  19. Toward a better understanding of the interaction between TGF-β family members and their ALK receptors

    KAUST Repository

    Romano, Valentina

    2012-02-22

    Transforming growth factor-beta (TGF-β) proteins are a family of structurally related extracellular proteins that trigger their signaling functions through interaction with the extracellular domains of their cognate serine/threonine kinase receptors. The specificity of TGF-β/receptor binding is complex and gives rise to multiple functional roles. Additionally, it is not completely understood at the atomic level. Here, we use the most reliable computational methods currently available to study systems involving activin-like kinase (ALK) receptors ALK4 and ALK7 and their multiple TGF-β ligands. We built models for all these proteins and their complexes for which experimental structures are not available. By analyzing the surfaces of interaction in six different TGF-β/ALK complexes we could infer which are the structural distinctive features of the ligand-receptor binding mode. Furthermore, this study allowed us to rationalize why binding of the growth factors GDF3 and Nodal to the ALK4 receptor requires the Cripto co-factor, whilst binding to the ALK7 receptor does not. © Springer-Verlag 2012.

  20. Directed pancreatic acinar differentiation of mouse embryonic stem cells via embryonic signalling molecules and exocrine transcription factors.

    Directory of Open Access Journals (Sweden)

    Fabien Delaspre

    Full Text Available Pluripotent embryonic stem cells (ESC are a promising cellular system for generating an unlimited source of tissue for the treatment of chronic diseases and valuable in vitro differentiation models for drug testing. Our aim was to direct differentiation of mouse ESC into pancreatic acinar cells, which play key roles in pancreatitis and pancreatic cancer. To that end, ESC were first differentiated as embryoid bodies and sequentially incubated with activin A, inhibitors of Sonic hedgehog (Shh and bone morphogenetic protein (BMP pathways, fibroblast growth factors (FGF and retinoic acid (RA in order to achieve a stepwise increase in the expression of mRNA transcripts encoding for endodermal and pancreatic progenitor markers. Subsequent plating in Matrigel® and concomitant modulation of FGF, glucocorticoid, and folllistatin signalling pathways involved in exocrine differentiation resulted in a significant increase of mRNAs encoding secretory enzymes and in the number of cells co-expressing their protein products. Also, pancreatic endocrine marker expression was down-regulated and accompanied by a significant reduction in the number of hormone-expressing cells with a limited presence of hepatic marker expressing-cells. These findings suggest a selective activation of the acinar differentiation program. The newly differentiated cells were able to release α-amylase and this feature was greatly improved by lentiviral-mediated expression of Rbpjl and Ptf1a, two transcription factors involved in the maximal production of digestive enzymes. This study provides a novel method to produce functional pancreatic exocrine cells from ESC.

  1. Transforming growth factor-beta induces nerve growth factor expression in pancreatic stellate cells by activation of the ALK-5 pathway.

    Science.gov (United States)

    Haas, Stephan L; Fitzner, Brit; Jaster, Robert; Wiercinska, Eliza; Gaitantzi, Haristi; Jesnowski, Ralf; Jesenowski, Ralf; Löhr, J-Matthias; Singer, Manfred V; Dooley, Steven; Breitkopf, Katja

    2009-10-01

    Nerve growth factor (NGF), a survival factor for neurons enforces pain by sensitizing nociceptors. Also in the pancreas, NGF was associated with pain and it can stimulate the proliferation of pancreatic cancer cells. Hepatic stellate cells (HSC) respond to NGF with apoptosis. Transforming growth factor (TGF)-beta, one of the strongest pro-fibrogenic activators of pancreatic stellate cells (PSC) induced NGF and its two receptors in an immortalized human cell line (ihPSC) and primary rat PSC (prPSC) as determined by RT-PCR, western blot, and immunofluorescence. In contrast to HSC, PSC expressed both NGF receptors, although p75(NTR) expression was weak in prPSC. In contrast to ihPSC TGF-beta activated both Smad signaling cascades in prPSC. NGF secretion was diminished by the activin-like kinase (ALK)-5 inhibitor SB431542, indicating the predominant role of ALK5 in activating the NGF system in PSC. While NGF did not affect proliferation or survival of PSC it induced expression of Inhibitor of Differentiation-1. We conclude that under conditions of upregulated TGF-beta, like fibrosis, NGF levels will also increase in PSC which might contribute to pancreatic wound healing responses.

  2. Effect of vitamin B12 on cleft palate induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin and dexamethasone in mice.

    Science.gov (United States)

    Zhao, Shu-Fan; Chai, Mao-Zhou; Wu, Min; He, Yong-Hong; Meng, Tian; Shi, Bing

    2014-03-01

    The purpose of this study was to investigate the effect of vitamin B12 on palatal development by co-administration of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and dexamethasone (DEX). We examined the morphological and histological features of the palatal shelf and expression levels of key signaling molecules (transforming growth factor-β3 (TGF-β3) and TGF-β type I receptor (activin receptor-like kinase 5, ALK5)) during palatogenesis among a control group (Group A), TCDD+DEX exposed group (Group B), and TCDD+DEX+vitamin B12 exposed group (Group C). While we failed to find that vitamin B12 decreased the incidence of cleft palate induced by TCDD+DEX treatment, the expression levels of key signaling molecules (TGF-β3 and ALK5) during palatogenesis were significantly modulated. In TCDD+DEX exposed and TCDD+DEX+vitamin B12 exposed groups, palatal shelves could not contact in the midline due to their small sizes. Our results suggest that vitamin B12 may inhibit the expression of some cleft palate inducers such as TGF-β3 and ALK5 in DEX+TCDD exposed mice, which may be beneficial against palatogenesis to some degree, even though we were unable to observe a protective role of vitamin B12 in morphological and histological alterations of palatal shelves induced by DEX and TCDD.

  3. A pilot study of angiogenin in heart failure with preserved ejection fraction: a novel potential biomarker for diagnosis and prognosis?

    Science.gov (United States)

    Jiang, Hong; Zhang, Lei; Yu, Ying; Liu, Ming; Jin, Xuejuan; Zhang, Peipei; Yu, Peng; Zhang, Shuning; Zhu, Hongmin; Chen, Ruizhen; Zou, Yunzeng; Ge, Junbo

    2014-11-01

    Characteristics of heart failure with preserved ejection fraction (HFPEF) have not yet been fully understood. The objectives of this pilot study are to detect protein expression profile in the sera of HFPEF patients, and to identify potential biomarkers for the disease. Five hundred and seven proteins were detected in the sera of healthy volunteers and patients with either HFPEF or hypertension using antibody microarrays (three in each group). The results showed that the serum concentrations of 17 proteins (e.g. angiogenin, activin A and artemin) differed considerably between HFPEF and non-HFPEF patients (hypertensive patients and healthy controls), while a protein expression pattern distinct from that in non-HFPEF patients was associated with HFPEF patients. The up-regulation of angiogenin in both HFPEF patients with LVEF ≥50% (P = 0.004) and a subset of HFPEF patients with LVEF = 41-49% (P HFPEF patients and 16 healthy controls. Meanwhile, angiogenin distinguished HFPEF patients from controls with a mean area under the receiver operating characteristic curve of 0.88 (P HFPEF patients were positively correlated with Lg(N-terminal pro-B-type natriuretic peptide, NT-proBNP) (P HFPEF.

  4. Uterine ALK3 is essential during the window of implantation.

    Science.gov (United States)

    Monsivais, Diana; Clementi, Caterina; Peng, Jia; Titus, Mary M; Barrish, James P; Creighton, Chad J; Lydon, John P; DeMayo, Francesco J; Matzuk, Martin M

    2016-01-19

    The window of implantation is defined by the inhibition of uterine epithelial proliferation, structural epithelial cell remodeling, and attenuated estrogen (E2) response. These changes occur via paracrine signaling between the uterine epithelium and stroma. Because implantation defects are a major cause of infertility in women, identifying these signaling pathways will improve infertility interventions. Bone morphogenetic proteins (BMPs) are TGF-β family members that regulate the postimplantation and midgestation stages of pregnancy. In this study, we discovered that signaling via activin-like kinase 3 (ALK3/BMPR1A), a BMP type 1 receptor, is necessary for blastocyst attachment. Conditional knockout (cKO) of ALK3 in the uterus was obtained by producing Alk3(flox) (/flox)-Pgr-cre-positive females. Alk3 cKO mice are sterile and have defects in the luminal uterine epithelium, including increased microvilli density and maintenance of apical cell polarity. Moreover, Alk3 cKO mice exhibit an elevated uterine E2 response and unopposed epithelial cell proliferation during the window of implantation. We determined that dual transcriptional regulation of Kruppel-like factor 15 (Klf15), by both the transforming growth factor β (TGF-β) transcription factor SMAD family member 4 (SMAD4) and progesterone receptor (PR), is necessary to inhibit uterine epithelial cell proliferation, a key step for embryo implantation. Our findings present a convergence of BMP and steroid hormone signaling pathways in the regulation of uterine receptivity.

  5. microRNA-21 mediates stretch-induced osteogenic differentiation in human periodontal ligament stem cells.

    Science.gov (United States)

    Wei, Fulan; Liu, Dongxu; Feng, Cheng; Zhang, Fan; Yang, Shuangyan; Hu, Yijun; Ding, Gang; Wang, Songlin

    2015-02-01

    microRNAs (miRNAs) are short 20- to 22-nucleotide noncoding RNAs that negatively regulate the expression of target genes at the post-transcriptional level. The expression of specific miRNAs and their roles in the osteogenic differentiation of human periodontal ligament stem cells (PDLSCs) exposed to mechanical stretch remain unclear. Here, we found that stretch induced both osteogenic differentiation and the differential expression of miR-21 in PDLSCs. Furthermore, we identified activin receptor type IIB (ACVR2B) as a target gene of miR-21. Luciferase reporter assays showed that miR-21 interacts directly with the 3'-untranslated repeat sequence of ACVR2B mRNA. Mechanical stretch suppressed ACVR2B protein levels in PDLSCs, and this suppressive effect was modulated when endogenous miR-21 levels were either enhanced or inhibited. Both stretch and the expression of miR-21 altered endogenous ACVR2B protein levels and thus the osteogenic differentiation of PDLSCs. In addition, gain- and loss of function of ACVR2B mediated the osteogenic differentiation of PDLSCs. This study demonstrates that miR-21 is a mechanosensitive gene that plays an important role in the osteogenic differentiation of PDLSCs exposed to stretch.

  6. Human Umbilical Cord Blood-Derived Serum for Culturing the Supportive Feeder Cells of Human Pluripotent Stem Cell Lines

    Directory of Open Access Journals (Sweden)

    Ruttachuk Rungsiwiwut

    2016-01-01

    Full Text Available Although human pluripotent stem cells (hPSCs can proliferate robustly on the feeder-free culture system, genetic instability of hPSCs has been reported in such environment. Alternatively, feeder cells enable hPSCs to maintain their pluripotency. The feeder cells are usually grown in a culture medium containing fetal bovine serum (FBS prior to coculture with hPSCs. The use of FBS might limit the clinical application of hPSCs. Recently, human cord blood-derived serum (hUCS showed a positive effect on culture of mesenchymal stem cells. It is interesting to test whether hUCS can be used for culture of feeder cells of hPSCs. This study was aimed to replace FBS with hUCS for culturing the human foreskin fibroblasts (HFFs prior to feeder cell preparation. The results showed that HFFs cultured in hUCS-containing medium (HFF-hUCS displayed fibroblastic features, high proliferation rates, short population doubling times, and normal karyotypes after prolonged culture. Inactivated HFF-hUCS expressed important genes, including Activin A, FGF2, and TGFβ1, which have been implicated in the maintenance of hPSC pluripotency. Moreover, hPSC lines maintained pluripotency, differentiation capacities, and karyotypic stability after being cocultured for extended period with inactivated HFF-hUCS. Therefore, the results demonstrated the benefit of hUCS for hPSCs culture system.

  7. FK506 activates BMPR2, rescues endothelial dysfunction, and reverses pulmonary hypertension

    Science.gov (United States)

    Spiekerkoetter, Edda; Tian, Xuefei; Cai, Jie; Hopper, Rachel K.; Sudheendra, Deepti; Li, Caiyun G.; El-Bizri, Nesrine; Sawada, Hirofumi; Haghighat, Roxanna; Chan, Roshelle; Haghighat, Leila; de Jesus Perez, Vinicio; Wang, Lingli; Reddy, Sushma; Zhao, Mingming; Bernstein, Daniel; Solow-Cordero, David E.; Beachy, Philip A.; Wandless, Thomas J.; ten Dijke, Peter; Rabinovitch, Marlene

    2013-01-01

    Dysfunctional bone morphogenetic protein receptor-2 (BMPR2) signaling is implicated in the pathogenesis of pulmonary arterial hypertension (PAH). We used a transcriptional high-throughput luciferase reporter assay to screen 3,756 FDA-approved drugs and bioactive compounds for induction of BMPR2 signaling. The best response was achieved with FK506 (tacrolimus), via a dual mechanism of action as a calcineurin inhibitor that also binds FK-binding protein-12 (FKBP12), a repressor of BMP signaling. FK506 released FKBP12 from type I receptors activin receptor-like kinase 1 (ALK1), ALK2, and ALK3 and activated downstream SMAD1/5 and MAPK signaling and ID1 gene regulation in a manner superior to the calcineurin inhibitor cyclosporine and the FKBP12 ligand rapamycin. In pulmonary artery endothelial cells (ECs) from patients with idiopathic PAH, low-dose FK506 reversed dysfunctional BMPR2 signaling. In mice with conditional Bmpr2 deletion in ECs, low-dose FK506 prevented exaggerated chronic hypoxic PAH associated with induction of EC targets of BMP signaling, such as apelin. Low-dose FK506 also reversed severe PAH in rats with medial hypertrophy following monocrotaline and in rats with neointima formation following VEGF receptor blockade and chronic hypoxia. Our studies indicate that low-dose FK506 could be useful in the treatment of PAH. PMID:23867624

  8. Kinase/phosphatase overexpression reveals pathways regulating hippocampal neuron morphology.

    Science.gov (United States)

    Buchser, William J; Slepak, Tatiana I; Gutierrez-Arenas, Omar; Bixby, John L; Lemmon, Vance P

    2010-07-01

    Development and regeneration of the nervous system requires the precise formation of axons and dendrites. Kinases and phosphatases are pervasive regulators of cellular function and have been implicated in controlling axodendritic development and regeneration. We undertook a gain-of-function analysis to determine the functions of kinases and phosphatases in the regulation of neuron morphology. Over 300 kinases and 124 esterases and phosphatases were studied by high-content analysis of rat hippocampal neurons. Proteins previously implicated in neurite growth, such as ERK1, GSK3, EphA8, FGFR, PI3K, PKC, p38, and PP1a, were confirmed to have effects in our functional assays. We also identified novel positive and negative neurite growth regulators. These include neuronal-developmentally regulated kinases such as the activin receptor, interferon regulatory factor 6 (IRF6) and neural leucine-rich repeat 1 (LRRN1). The protein kinase N2 (PKN2) and choline kinase alpha (CHKA) kinases, and the phosphatases PPEF2 and SMPD1, have little or no established functions in neuronal function, but were sufficient to promote neurite growth. In addition, pathway analysis revealed that members of signaling pathways involved in cancer progression and axis formation enhanced neurite outgrowth, whereas cytokine-related pathways significantly inhibited neurite formation.

  9. Strategies for exploring TGF-β signaling in Drosophila.

    Science.gov (United States)

    Peterson, Aidan J; O'Connor, Michael B

    2014-06-15

    The TGF-β pathway is an evolutionarily conserved signal transduction module that mediates diverse biological processes in animals. In Drosophila, both the BMP and Activin branches are required for viability. Studies rooted in classical and molecular genetic approaches continue to uncover new developmental roles for TGF-β signaling. We present an overview of the secreted ligands, transmembrane receptors and cellular Smad transducer proteins that compose the core pathway in Drosophila. An assortment of tools have been developed to conduct tissue-specific loss- and gain-of-function experiments for these pathway components. We discuss the deployment of these reagents, with an emphasis on appropriate usage and limitations of the available tools. Throughout, we note reagents that are in need of further improvement or development, and signaling features requiring further study. A general theme is that comparison of phenotypes for ligands, receptors, and Smads can be used to map tissue interactions, and to separate canonical and non-canonical signaling activities. Core TGF-β signaling components are subject to multiple layers of regulation, and are coupled to context-specific inputs and outputs. In addition to fleshing out how TGF-β signaling serves the fruit fly, we anticipate that future studies will uncover new regulatory nodes and modes and will continue to advance paradigms for how TGF-β signaling regulates general developmental processes.

  10. Roles for transforming growth factor beta superfamily proteins in early folliculogenesis.

    Science.gov (United States)

    Trombly, Daniel J; Woodruff, Teresa K; Mayo, Kelly E

    2009-01-01

    Primordial follicle formation and the subsequent transition of follicles to the primary and secondary stages encompass the early events during folliculogenesis in mammals. These processes establish the ovarian follicle pool and prime follicles for entry into subsequent growth phases during the reproductive cycle. Perturbations during follicle formation can affect the size of the primordial follicle pool significantly, and alterations in follicle transition can cause follicles to arrest at immature stages or result in premature depletion of the follicle reserve. Determining the molecular events that regulate primordial follicle formation and early follicle growth may lead to the development of new fertility treatments. Over the last decade, many of the growth factors and signaling proteins that mediate the early stages of folliculogenesis have been identified using mouse genetic models, in vivo injection studies, and ex vivo organ culture approaches. These studies reveal important roles for the transforming growth factor beta (TGF-beta) superfamily of proteins in the ovary. This article reviews these roles for TGF-beta family proteins and focuses in particular on work from our laboratories on the functions of activin in early folliculogenesis.

  11. Evaluation of Electrical Impedance as a Biomarker of Myostatin Inhibition in Wild Type and Muscular Dystrophy Mice.

    Directory of Open Access Journals (Sweden)

    Benjamin Sanchez

    Full Text Available Non-invasive and effort independent biomarkers are needed to better assess the effects of drug therapy on healthy muscle and that affected by muscular dystrophy (mdx. Here we evaluated the use of multi-frequency electrical impedance for this purpose with comparison to force and histological parameters.Eight wild-type (wt and 10 mdx mice were treated weekly with RAP-031 activin type IIB receptor at a dose of 10 mg kg-1 twice weekly for 16 weeks; the investigators were blinded to treatment and disease status. At the completion of treatment, impedance measurements, in situ force measurements, and histology analyses were performed.As compared to untreated animals, RAP-031 wt and mdx treated mice had greater body mass (18% and 17%, p 70 Hz, but not in the mdx animals. In contrast, maximum force normalized by muscle mass was unchanged in the wt animals and lower in the mdx animals by 21% (p < 0.01. Similarly, myofiber size was only non-significantly higher in treated versus untreated animals (8% p = 0.44 and 12% p = 0.31 for wt and mdx animals, respectively.Our findings demonstrate electrical impedance of muscle reproduce the functional and histological changes associated with myostatin pathway inhibition and do not reflect differences in muscle size or volume. This technique deserves further study in both animal and human therapeutic trials.

  12. Cinnabar-Induced Subchronic Renal Injury Is Associated with Increased Apoptosis in Rats

    Directory of Open Access Journals (Sweden)

    Ying Wang

    2015-01-01

    Full Text Available The aim of this study was to explore the role of apoptosis in cinnabar-induced renal injury in rats. To test this role, rats were dosed orally with cinnabar (1 g/kg/day for 8 weeks or 12 weeks, and the control rats were treated with 5% carboxymethylcellulose solution. Levels of urinary mercury (UHg, renal mercury (RHg, serum creatinine (SCr, and urine kidney injury molecule 1 (KIM-1 were assessed, and renal pathology was analyzed. Apoptotic cells were identified and the apoptotic index was calculated. A rat antibody array was used to analyze expression of cytokines associated with apoptosis. Results from these analyses showed that UHg, RHg, and urine KIM-1, but not SCr, levels were significantly increased in cinnabar-treated rats. Renal pathological changes in cinnabar-treated rats included vacuolization of tubular cells, formation of protein casts, infiltration of inflammatory cells, and increase in the number of apoptotic tubular cells. In comparison to the control group, expression of FasL, Fas, TNF-α, TRAIL, activin A, and adiponectin was upregulated in the cinnabar-treated group. Collectively, our results suggest that prolonged use of cinnabar results in kidney damage due to accumulation of mercury and that the underlying mechanism involves apoptosis of tubular cells via a death receptor-mediated pathway.

  13. Functional high-resolution time-course expression analysis of human embryonic stem cells undergoing cardiac induction

    Directory of Open Access Journals (Sweden)

    Ilaria Piccini

    2016-12-01

    Full Text Available Cardiac induction of human embryonic stem cells (hESCs is a process bearing increasing medical relevance, yet it is poorly understood from a developmental biology perspective. Anticipated technological progress in deriving stably expandable cardiac precursor cells or in advancing cardiac subtype specification protocols will likely require deeper insights into this fascinating system. Recent improvements in controlling hESC differentiation now enable a near-homogeneous induction of the cardiac lineage. This is based on an optimized initial stimulation of mesoderm-inducing signaling pathways such as Activin and/or FGF, BMP, and WNT, followed by WNT inhibition as a secondary requirement. Here, we describe a comprehensive data set based on varying hESC differentiation conditions in a systematic manner and recording high-resolution differentiation time-courses analyzed by genome-wide expression profiling (GEO accession number GSE67154. As a baseline, hESCs were differentiated into cardiomyocytes under optimal conditions. Moreover, in additional time-series, individual signaling factors were withdrawn from the initial stimulation cocktail to reveal their specific roles via comparison to the standard condition. Hence, this data set presents a rich resource for hypothesis generation in studying human cardiac induction, as we reveal numbers of known as well as uncharacterized genes prominently marking distinct intermediate stages in the process. These data will also be useful for identifying putative cardiac master regulators in the human system as well as for characterizing expandable cardiac stem cells.

  14. Cancer Stem Cells, EMT, and Developmental Pathway Activation in Pancreatic Tumors

    Directory of Open Access Journals (Sweden)

    Maarten F. Bijlsma

    2012-10-01

    Full Text Available Pancreatic cancer is a disease with remarkably poor patient survival rates. The frequent presence of metastases and profound chemoresistance pose a severe problem for the treatment of these tumors. Moreover, cross-talk between the tumor and the local micro-environment contributes to tumorigenicity, metastasis and chemoresistance. Compared to bulk tumor cells, cancer stem cells (CSC have reduced sensitivity to chemotherapy. CSC are tumor cells with stem-like features that possess the ability to self-renew, but can also give rise to more differentiated progeny. CSC can be identified based on increased in vitro spheroid- or colony formation, enhanced in vivo tumor initiating potential, or expression of cell surface markers. Since CSC are thought to be required for the maintenance of a tumor cell population, these cells could possibly serve as a therapeutic target. There appears to be a causal relationship between CSC and epithelial-to-mesenchymal transition (EMT in pancreatic tumors. The occurrence of EMT in pancreatic cancer cells is often accompanied by re-activation of developmental pathways, such as the Hedgehog, WNT, NOTCH, and Nodal/Activin pathways. Therapeutics based on CSC markers, EMT, developmental pathways, or tumor micro-environment could potentially be used to target pancreatic CSC. This may lead to a reduction of tumor growth, metastatic events, and chemoresistance in pancreatic cancer.

  15. Cancer Stem Cells, EMT, and Developmental Pathway Activation in Pancreatic Tumors

    Energy Technology Data Exchange (ETDEWEB)

    Hindriksen, Sanne; Bijlsma, Maarten F., E-mail: m.f.bijlsma@amc.uva.nl [Laboratory for Experimental Oncology and Radiobiology, Academic Medical Centre, Meibergdreef 9, 1105AZ Amsterdam (Netherlands)

    2012-10-12

    Pancreatic cancer is a disease with remarkably poor patient survival rates. The frequent presence of metastases and profound chemoresistance pose a severe problem for the treatment of these tumors. Moreover, cross-talk between the tumor and the local micro-environment contributes to tumorigenicity, metastasis and chemoresistance. Compared to bulk tumor cells, cancer stem cells (CSC) have reduced sensitivity to chemotherapy. CSC are tumor cells with stem-like features that possess the ability to self-renew, but can also give rise to more differentiated progeny. CSC can be identified based on increased in vitro spheroid- or colony formation, enhanced in vivo tumor initiating potential, or expression of cell surface markers. Since CSC are thought to be required for the maintenance of a tumor cell population, these cells could possibly serve as a therapeutic target. There appears to be a causal relationship between CSC and epithelial-to-mesenchymal transition (EMT) in pancreatic tumors. The occurrence of EMT in pancreatic cancer cells is often accompanied by re-activation of developmental pathways, such as the Hedgehog, WNT, NOTCH, and Nodal/Activin pathways. Therapeutics based on CSC markers, EMT, developmental pathways, or tumor micro-environment could potentially be used to target pancreatic CSC. This may lead to a reduction of tumor growth, metastatic events, and chemoresistance in pancreatic cancer.

  16. Brachyury and SMAD signalling collaboratively orchestrate distinct mesoderm and endoderm gene regulatory networks in differentiating human embryonic stem cells.

    Science.gov (United States)

    Faial, Tiago; Bernardo, Andreia S; Mendjan, Sasha; Diamanti, Evangelia; Ortmann, Daniel; Gentsch, George E; Mascetti, Victoria L; Trotter, Matthew W B; Smith, James C; Pedersen, Roger A

    2015-06-15

    The transcription factor brachyury (T, BRA) is one of the first markers of gastrulation and lineage specification in vertebrates. Despite its wide use and importance in stem cell and developmental biology, its functional genomic targets in human cells are largely unknown. Here, we use differentiating human embryonic stem cells to study the role of BRA in activin A-induced endoderm and BMP4-induced mesoderm progenitors. We show that BRA has distinct genome-wide binding landscapes in these two cell populations, and that BRA interacts and collaborates with SMAD1 or SMAD2/3 signalling to regulate the expression of its target genes in a cell-specific manner. Importantly, by manipulating the levels of BRA in cells exposed to different signalling environments, we demonstrate that BRA is essential for mesoderm but not for endoderm formation. Together, our data illuminate the function of BRA in the context of human embryonic development and show that the regulatory role of BRA is context dependent. Our study reinforces the importance of analysing the functions of a transcription factor in different cellular and signalling environments.

  17. Origins of pluripotent stem cells.

    Science.gov (United States)

    Roelen, B A J; Chuva De Sousa Lopes, S M

    2011-08-01

    Different types of pluripotent stem cells can be identified and cultured in vitro. Here an overview is presented of the various pluripotent stem cells types. Embryonal carcinoma (EC) cells that have been cultured in vitro provided the groundwork for future pluripotent cell cultures. Conditions established for these cells such as culture on a feeder layer of mouse embryonic fibroblasts and the importance of fetal calf serum were initially also used for the culture of mouse embryonic stem (ES) cells derived from the inner cell masses of blastocysts. Embryonic stem cells derived from human blastocysts were found to require different conditions and are cultured in the presence of activin and basic fibroblast growth factor. Recently pluripotent stem cells have also been derived from mouse peri-implantation epiblasts. Since these epiblast stem cells (EpiSCs) require the same conditions as the human ES cells it has been suggested that human ES cells are more similar to mouse EpiSCs than to mouse ES cells. Pluripotent cell lines have also been derived from migratory primordial germ cells and spermatogonial stem cells. The creation of pluripotent stem cells from adult cells by the introduction of reprogramming transcription factors, so-called induced pluripotent stem (iPS) cells allowed the derivation of patient-specific pluripotent stem cells without the need of creation of a human blastocyst after cloning by somatic cells nuclear transfer. Recently it has become clear however that iPS cells may be quite different to ES cells in terms of epigenetics.

  18. Expression of Smad2 and Smad4 in rhesus monkey endometrium during the menstrual cycle and early pregnancy

    Institute of Scientific and Technical Information of China (English)

    LIN Haiyan; WANG Hongmei; LI Qinglei; WANG Juan; ZHANG Xuan; LIU Donglin; LIU Huitu; ZHU Cheng

    2003-01-01

    Expression of Smad2 and Smad4 mRNAs in the endometrium of rhesus monkey on Days 8, 20 and 28 of the normal menstrual cycle and on Days 12, 18 and 26 of early pregnancy was detected using in situ hybridization. The results showed that Smad2 and Smad4 mRNAs were mainly localized in luminal epithelium and glandular epithelium. The expression of Smad2 mRNA in glandular epithelium was sustained at moderate level on Days 8, 20 and 28 of the menstrual cycle, while the expression of Smad4 gradually increased with the menstrual cycle. Both Smad2 and Smad4 mRNAs in functionalis glandular epithelium were expressed at the highest levels on Day 12 of early pregnancy, while in basalis glandular epithelium the most abundant expression of both Smads occurred on Days 12 and 18 of pregnancy. On Day 26, both Smads mRNAs were expressed at the lowest levels either in functionalis or in basalis. The data suggest that the epithelium is the major compartment where TGF-βs/activins exert their biological effects via Smads, and that Smad4 may play a role in the maintenance of endometrial gland function during secreting period of the menstrual cycle. During lacunar stage of early pregnancy, Smad2 and Smad4 are implicated in the tissue remodeling of endometrial functionalis and basalis, and during early villous stage both Smads are functional primarily in basalis.

  19. The LIM class homeobox gene lim5: implied role in CNS patterning in Xenopus and zebrafish.

    Science.gov (United States)

    Toyama, R; Curtiss, P E; Otani, H; Kimura, M; Dawid, I B; Taira, M

    1995-08-01

    LIM homeobox genes are characterized by encoding proteins in which two cysteine-rich LIM domains are associated with a homeodomain. We report the isolation of a gene, named Xlim-5 in Xenopus and lim5 in the zebrafish, that is highly similar in sequence but quite distinct in expression pattern from the previously described Xlim-1/lim1 gene. In both species studied the lim5 gene is expressed in the entire ectoderm in the early gastrula embryo. The Xlim-5 gene is activated in a cell autonomous manner in ectodermal cells, and this activation is suppressed by the mesoderm inducer activin. During neurulation, expression of the lim5 gene in both the frog and fish embryo is rapidly restricted to an anterior region in the developing neural plate/keel. In the 2-day Xenopus and 24-hr zebrafish embryo, this region becomes more sharply defined, forming a strongly lim5-expressing domain in the diencephalon anterior to the midbrain-forebrain boundary. In addition, regions of less intense lim5 expression are seen in the zebrafish embryo in parts of the telencephalon, in the anterior diencephalon coincident with the postoptic commissure, and in restricted regions of the midbrain, hindbrain, and spinal cord. Expression in ventral forebrain is abolished from the 5-somite stage onward in cyclops mutant fish. These results imply a role for lim5 in the patterning of the nervous system, in particular in the early specification of the diencephalon.

  20. Discovery of 7-methoxy-6-[4-(4-methyl-1,3-thiazol-2-yl)-1H-imidazol-5-yl]-1,3-benzothiazole (TASP0382088): a potent and selective transforming growth factor-β type I receptor inhibitor as a topical drug for alopecia.

    Science.gov (United States)

    Amada, Hideaki; Asanuma, Hajime; Koami, Takeshi; Okada, Atsushi; Endo, Mayumi; Ueda, Yasuji; Naruse, Takumi; Ikeda, Akiko

    2013-01-01

    7-Methoxy-6-[4-(4-methyl-1,3-thiazol-2-yl)-1H-imidazol-5-yl]-1,3-benzothiazole 11 (TASP0382088) was synthesized and evaluated as transforming growth factor-β (TGF-β) type I receptor (also known as activin receptor-like kinase 5 or ALK5) inhibitor. Compound 11, a potent and selective ALK5 inhibitor, exhibited good enzyme inhibitory activity (IC50=4.8 nM) as well as inhibitory activity against TGF-β-induced Smad2/3 phosphorylation at a cellular level (IC50=17 nM). The introduction of a methoxy group to the benzothiazole ring in 1 and the break up of the planarity between the imidazole ring and the thiazole ring improved the solubility in the lotion base of 11. Furthermore, the topical application of 3% 11 lotion significantly inhibited Smad2 phosphorylation in mouse skin at 8 h after application (71% inhibition, compared with vehicle-treated animals).

  1. Structural and Functional Insights into Endoglin Ligand Recognition and Binding

    Science.gov (United States)

    Alt, Aaron; Miguel-Romero, Laura; Donderis, Jordi; Aristorena, Mikel; Blanco, Francisco J.; Round, Adam; Rubio, Vicente; Bernabeu, Carmelo; Marina, Alberto

    2012-01-01

    Endoglin, a type I membrane glycoprotein expressed as a disulfide-linked homodimer on human vascular endothelial cells, is a component of the transforming growth factor (TGF)-β receptor complex and is implicated in a dominant vascular dysplasia known as hereditary hemorrhagic telangiectasia as well as in preeclampsia. It interacts with the type I TGF-β signaling receptor activin receptor-like kinase (ALK)1 and modulates cellular responses to Bone Morphogenetic Protein (BMP)-9 and BMP-10. Structurally, besides carrying a zona pellucida (ZP) domain, endoglin contains at its N-terminal extracellular region a domain of unknown function and without homology to any other known protein, therefore called the orphan domain (OD). In this study, we have determined the recognition and binding ability of full length ALK1, endoglin and constructs encompassing the OD to BMP-9 using combined methods, consisting of surface plasmon resonance and cellular assays. ALK1 and endoglin ectodomains bind, independently of their glycosylation state and without cooperativity, to different sites of BMP-9. The OD comprising residues 22 to 337 was identified among the present constructs as the minimal active endoglin domain needed for partner recognition. These studies also pinpointed to Cys350 as being responsible for the dimerization of endoglin. In contrast to the complete endoglin ectodomain, the OD is a monomer and its small angle X-ray scattering characterization revealed a compact conformation in solution into which a de novo model was fitted. PMID:22347366

  2. Structural and functional insights into endoglin ligand recognition and binding.

    Directory of Open Access Journals (Sweden)

    Aaron Alt

    Full Text Available Endoglin, a type I membrane glycoprotein expressed as a disulfide-linked homodimer on human vascular endothelial cells, is a component of the transforming growth factor (TGF-β receptor complex and is implicated in a dominant vascular dysplasia known as hereditary hemorrhagic telangiectasia as well as in preeclampsia. It interacts with the type I TGF-β signaling receptor activin receptor-like kinase (ALK1 and modulates cellular responses to Bone Morphogenetic Protein (BMP-9 and BMP-10. Structurally, besides carrying a zona pellucida (ZP domain, endoglin contains at its N-terminal extracellular region a domain of unknown function and without homology to any other known protein, therefore called the orphan domain (OD. In this study, we have determined the recognition and binding ability of full length ALK1, endoglin and constructs encompassing the OD to BMP-9 using combined methods, consisting of surface plasmon resonance and cellular assays. ALK1 and endoglin ectodomains bind, independently of their glycosylation state and without cooperativity, to different sites of BMP-9. The OD comprising residues 22 to 337 was identified among the present constructs as the minimal active endoglin domain needed for partner recognition. These studies also pinpointed to Cys350 as being responsible for the dimerization of endoglin. In contrast to the complete endoglin ectodomain, the OD is a monomer and its small angle X-ray scattering characterization revealed a compact conformation in solution into which a de novo model was fitted.

  3. Sarcopenia--The search for emerging biomarkers.

    Science.gov (United States)

    Kalinkovich, Alexander; Livshits, Gregory

    2015-07-01

    Sarcopenia, an age-related decline in skeletal muscle mass and function, dramatically affects the life quality of elder people. In view of increasing life expectancy, sarcopenia renders a heavy burden on the health care system. However, although there is a consensus that sarcopenia is a multifactorial syndrome, its etiology, underlying mechanisms, and even definition remain poorly delineated, thus, preventing development of a precise treatment strategy. The main aim of our review is to critically analyze potential sarcopenia biomarkers in light of the molecular mechanisms of their involvement in sarcopenia pathogenesis. Normal muscle mass and function maintenance are proposed to be dependent on the dynamic balance between the positive regulators of muscle growth such as bone morphogenetic proteins (BMPs), brain-derived neurotrophic factor (BDNF), follistatin (FST) and irisin, and negative regulators including TGFβ, myostatin, activins A and B, and growth and differentiation factor-15 (GDF-15). We hypothesize that the shift in this balance to muscle growth inhibitors, along with increased expression of the C- terminal agrin fragment (CAF) associated with age-dependent neuromuscular junction (NMJ) dysfunction, as well as skeletal muscle-specific troponin T (sTnT), a key component of contractile machinery, is a main mechanism underlying sarcopenia pathogenesis. Thus, this review proposes and emphasizes that these molecules are the emerging sarcopenia biomarkers.

  4. Mutation study of Spanish patients with Hereditary Hemorrhagic Telangiectasia

    Directory of Open Access Journals (Sweden)

    Blanco Francisco J

    2008-08-01

    Full Text Available Abstract Background Hereditary Hemorrhagic Telangiectasia (HHT is an autosomal dominant and age-dependent vascular disorder characterised mainly by mutations in the Endoglin (ENG or activin receptor-like kinase-1 (ALK1, ACVRL1 genes. Methods Here, we have identified 22 ALK1 mutations and 15 ENG mutations, many of which had not previously been reported, in independent Spanish families afflicted with HHT. Results We identified mutations in thirty-seven unrelated families. A detailed analysis of clinical symptoms was recorded for each patient analyzed, with a higher significant presence of pulmonary arteriovenous malformations (PAVM in HHT1 patients over HHT2. Twenty-two mutations in ALK1 and fifteen in ENG genes were identified. Many of them, almost half, represented new mutations in ALK1 and in ENG. Missense mutations in ENG and ALK1 were localized in a tridimensional protein structure model. Conclusion Overall, ALK1 mutations (HHT2 were predominant over ENG mutations (HHT1 in our Spanish population, in agreement with previous data from our country and other Mediterranean countries (France, Italy, but different to Northern Europe or North America. There was a significant increase of PAVM associated with HHT1 over HHT2 in these families.

  5. Reproductive hormones in breast cancer bone metastasis: The role of inhibins

    Directory of Open Access Journals (Sweden)

    Caroline Wilson

    2016-09-01

    Full Text Available The spread of breast cancer cells to bone and survival in this new metastatic environment is influenced not only by the genetic signature of the cells, but also multiple host cells and soluble factors produced locally (paracrine or from distant sites (endocrine. Disrupting this metastatic process has been evaluated in clinical trials of the bone targeted agents bisphosphonates and denosumab and have shown that these agents reduce the recurrence of breast cancer in postmenopausal women only, suggesting the efficacy of the drugs are influenced by levels of reproductive endocrine hormones. The molecular mechanism driving this differential effect has not been definitively identified, however, there is evidence that both reproductive hormones and bisphosphonates can affect similar paracrine factors and cellular components of the bone metastatic niche. This review focuses on how the ovarian endocrine hormone, inhibin, interacts with the paracrine factors activin and follistatin, abundant in the primary tumour and bone microenvironment, with subsequent effects on tumour cell survival. Inhibin also affects the cellular components of the bone microenvironment primarily the osteoblastic niche. Recent evidence has shown that bisphosphonates also alter this niche, which may represent a common mechanism by which inhibin and bisphosphonates interact to influence disease outcomes in early breast cancer. Further research is needed to fully elucidate these molecular mechanisms to enable understanding and future development of alternative bone targeted treatments with anti-tumour efficacy in premenopausal women.

  6. Endocrine and ovarian responses in water buffalo cows immunized against inhibin and subjected to the Ovsynch protocol

    Institute of Scientific and Technical Information of China (English)

    Abdalla Bahareldin-Ali; QIN Guang-sheng; GUO Ri-hong; Anastasia Tsigkou; TAN Zheng-zhun; HUANG Jian; LI Hui; SHI Zhen-dan

    2015-01-01

    The aim of this study was to investigate the feasibility of stimulating ovarian fol icle development in order to improve fertility in water buffalo cows by immunization against inhibin. The experiment was carried out in early summer (May) and included 24 multi-parity crossbred Murrah-Swamp buffaloes that were divided into immunized (n=11) and control (n=13) groups. Each immunized cow was administered with a 2-mL immunogen of mineral oil adjuvant containing 2 mg of recombinant inhibinα-subunit fusion protein. The controls were treated with the adjuvant only. Al animals received Ovsynch protocol treatment, starting on the day of the antigen administration, and they were artiifcial y inseminated upon behavioral estrus. As a result, al of the immunized buffaloes generated antibodies against inhibin during the experimental period and had higher plasma concentrations of fol icle-stimulating hormone (FSH), activin, and estradiol (E2) related to estrous expression. A higher proportion of immunized animals expressed estrus behavior than did the controls (72%vs. 30%, P0.05). These results demonstrate that immunization against inhibin, coupled with the treatment with the Ovsynch protocol, can constitute a new technique to increase fertility in water buffalo cows.

  7. Glucocorticoid Insensitivity in Virally Infected Airway Epithelial Cells Is Dependent on Transforming Growth Factor-β Activity

    Science.gov (United States)

    Radwan, Asmaa; Keenan, Christine R.; Langenbach, Shenna Y.; Li, Meina; Londrigan, Sarah L.; Gualano, Rosa C.; Stewart, Alastair G.

    2017-01-01

    Asthma and chronic obstructive pulmonary disease (COPD) exacerbations are commonly associated with respiratory syncytial virus (RSV), rhinovirus (RV) and influenza A virus (IAV) infection. The ensuing airway inflammation is resistant to the anti-inflammatory actions of glucocorticoids (GCs). Viral infection elicits transforming growth factor-β (TGF-β) activity, a growth factor we have previously shown to impair GC action in human airway epithelial cells through the activation of activin-like kinase 5 (ALK5), the type 1 receptor of TGF-β. In the current study, we examine the contribution of TGF-β activity to the GC-resistance caused by viral infection. We demonstrate that viral infection of human bronchial epithelial cells with RSV, RV or IAV impairs GC anti-inflammatory action. Poly(I:C), a synthetic analog of double-stranded RNA, also impairs GC activity. Both viral infection and poly(I:C) increase TGF-β expression and activity. Importantly, the GC impairment was attenuated by the selective ALK5 (TGFβRI) inhibitor, SB431542 and prevented by the therapeutic agent, tranilast, which reduced TGF-β activity associated with viral infection. This study shows for the first time that viral-induced glucocorticoid-insensitivity is partially mediated by activation of endogenous TGF-β. PMID:28046097

  8. Novel therapies for osteoporosis.

    Science.gov (United States)

    Makras, Polyzois; Delaroudis, Sideris; Anastasilakis, Athanasios D

    2015-10-01

    Since the identification of osteoporosis as a major health issue in aging populations and the subsequent development of the first treatment modalities for its management, considerable progress has been made in our understanding of the mechanisms controlling bone turnover and disease pathophysiology, thus enabling the pinpointing of new targets for intervention. This progress, along with advances in biotechnology, has rendered possible the development of ever more sophisticated treatments employing novel mechanisms of action. Denosumab, a monoclonal antibody against RANKL, approved for the treatment of postmenopausal and male osteoporosis, significantly and continuously increases bone mineral density (BMD) and maintains a low risk of vertebral, non-vertebral, and hip fractures for up to 8 years. Currently available combinations of estrogens with selective estrogen receptor modulators moderately increase BMD without causing the extra-skeletal adverse effects of each compound alone. The cathepsin K inhibitor odanacatib has recently been shown to decrease vertebral, non-vertebral, and hip fracture rates and is nearing approval. Romosozumab, an anti-sclerosin antibody, and abaloparatide, a PTH-related peptide analog, are at present in advanced stages of clinical evaluation, so far demonstrating efficaciousness together with a favorable safety profile. Several other agents are currently in earlier clinical and preclinical phases of development, including dickkopf-1 antagonists, activin A antagonists, β-arrestin analogs, calcilytics, and Src tyrosine kinase inhibitors.

  9. Regulation of embryonic stem cell self-renewal and differentiation by TGF-β family signaling

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    Embryonic stem (ES) cells are characterized by their ability to indefinitely self-renew and potential to differentiate into all the cell lineages of the body. ES cells are considered to have potential applications in regenerative medicine. In particular, the emergence of an ES cell analogue-induced pluripotent stem (iPS) cells via somatic cell reprogramming by co-expressing a limited number of critical stemness-related transcriptional factors has solved the problem of obtaining patient-specific pluripotent cells, encouraging researchers to develop more specific and functional cell lineages from ES or iPS cells for broad therapeutic applications. ES cell fate choice is delicately controlled by a core transcriptional network, epigenetic modification profiles and complex signaling cascades both intrinsically and extrinsically. Of these signals, transforming growth factor β (TGF-β) family members, including TGF-β, bone morphogenetic protein (BMP), Activin and Nodal, have been reported to influence cell self-renewal and a broad spectrum of lineage differentiation in ES cells, in accordance with the key roles of TGF-β family signaling in early embryo development. In this review, the roles of TGF-β family signals in coordinating ES cell fate determination are summarized.

  10. Effect of vitamin B12 on cleft palate induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin and dexamethasone in mice*

    Science.gov (United States)

    Zhao, Shu-fan; Chai, Mao-zhou; Wu, Min; He, Yong-hong; Meng, Tian; Shi, Bing

    2014-01-01

    The purpose of this study was to investigate the effect of vitamin B12 on palatal development by co-administration of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and dexamethasone (DEX). We examined the morphological and histological features of the palatal shelf and expression levels of key signaling molecules (transforming growth factor-β3 (TGF-β3) and TGF-β type I receptor (activin receptor-like kinase 5, ALK5)) during palatogenesis among a control group (Group A), TCDD+DEX exposed group (Group B), and TCDD+DEX+vitamin B12 exposed group (Group C). While we failed to find that vitamin B12 decreased the incidence of cleft palate induced by TCDD+DEX treatment, the expression levels of key signaling molecules (TGF-β3 and ALK5) during palatogenesis were significantly modulated. In TCDD+DEX exposed and TCDD+DEX+vitamin B12 exposed groups, palatal shelves could not contact in the midline due to their small sizes. Our results suggest that vitamin B12 may inhibit the expression of some cleft palate inducers such as TGF-β3 and ALK5 in DEX+TCDD exposed mice, which may be beneficial against palatogenesis to some degree, even though we were unable to observe a protective role of vitamin B12 in morphological and histological alterations of palatal shelves induced by DEX and TCDD. PMID:24599693

  11. A single-cell and feeder-free culture system for monkey embryonic stem cells.

    Science.gov (United States)

    Ono, Takashi; Suzuki, Yutaka; Kato, Yosuke; Fujita, Risako; Araki, Toshihiro; Yamashita, Tomoko; Kato, Hidemasa; Torii, Ryuzo; Sato, Naoya

    2014-01-01

    Primate pluripotent stem cells (PSCs), including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), hold great potential for research and application in regenerative medicine and drug discovery. To maximize primate PSC potential, a practical system is required for generating desired functional cells and reproducible differentiation techniques. Much progress regarding their culture systems has been reported to date; however, better methods would still be required for their practical use, particularly in industrial and clinical fields. Here we report a new single-cell and feeder-free culture system for primate PSCs, the key feature of which is an originally formulated serum-free medium containing FGF and activin. In this culture system, cynomolgus monkey ESCs can be passaged many times by single-cell dissociation with traditional trypsin treatment and can be propagated with a high proliferation rate as a monolayer without any feeder cells; further, typical PSC properties and genomic stability can be retained. In addition, it has been demonstrated that monkey ESCs maintained in the culture system can be used for various experiments such as in vitro differentiation and gene manipulation. Thus, compared with the conventional culture system, monkey ESCs grown in the aforementioned culture system can serve as a cell source with the following practical advantages: simple, stable, and easy cell maintenance; gene manipulation; cryopreservation; and desired differentiation. We propose that this culture system can serve as a reliable platform to prepare primate PSCs useful for future research and application.

  12. RAP-011 improves erythropoiesis in zebrafish model of Diamond-Blackfan anemia through antagonizing lefty1.

    Science.gov (United States)

    Ear, Jason; Huang, Haigen; Wilson, Tianna; Tehrani, Zahra; Lindgren, Anne; Sung, Victoria; Laadem, Abderrahmane; Daniel, Thomas O; Chopra, Rajesh; Lin, Shuo

    2015-08-13

    Diamond-Blackfan Anemia (DBA) is a bone marrow failure disorder characterized by low red blood cell count. Mutations in ribosomal protein genes have been identified in approximately half of all DBA cases. Corticosteriod therapy and bone marrow transplantation are common treatment options for patients; however, significant risks and complications are associated with these treatment options. Therefore, novel therapeutic approaches are needed for treating DBA. Sotatercept (ACE-011, and its murine ortholog RAP-011) acts as an activin receptor type IIA ligand trap, increasing hemoglobin and hematocrit in pharmacologic models, in healthy volunteers, and in patients with β-thalassemia, by expanding late-stage erythroblasts through a mechanism distinct from erythropoietin. Here, we evaluated the effects of RAP-011 in zebrafish models of RPL11 ribosome deficiency. Treatment with RAP-011 dramatically restored hemoglobin levels caused by ribosome stress. In zebrafish embryos, RAP-011 likely stimulates erythropoietic activity by sequestering lefty1 from erythroid cells. These findings identify lefty1 as a signaling component in the development of erythroid cells and rationalize the use of sotatercept in DBA patients. © 2015 by The American Society of Hematology.

  13. RAP-011 augments callus formation in closed fractures in rats.

    Science.gov (United States)

    Morse, Alyson; Cheng, Tegan L; Peacock, Lauren; Mikulec, Kathy; Little, David G; Schindeler, Aaron

    2016-02-01

    ACE-011 is a bone anabolic agent generated by fusing the extracellular domain of the Activin Type 2A receptor (ActRIIA) to an IgG-Fc. The orthopedic utility of ACE-011 was investigated using a murine analogue, RAP-011. Initially, a rat closed fracture model was tested using bi-weekly (biw) 10 mg/kg RAP-011. RAP-011 significantly increased callus length and callus bone volume (BV, +43% at 6w, p RAP-011(p)] and "sustained" [RAP-011(s)] regimens of biw 0.6mg/kg × 2, 0.35mg/kg × 3 and 0.18mg/kg × 2, 0.1mg/kg × 7 respectively, compared with PTH(1-34) (25 μg/kg/d) and vehicle controls. RAP-011 treatments gave modest increases in callus length and callus BV at 6w (p RAP-011 is effective in promoting bone formation during repair, but optimizing callus bone quality will require further investigation. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

  14. Research progress in the genetics of hyperuricaemia and gout.

    Science.gov (United States)

    Min, Zheng; Junwu, Ma

    2016-04-01

    Gout is one of the most common inflammatory arthritis caused by hyperuricaemia, which is affected by both genetic factors and environmental factors. Early researches show that a few of rare monogenic mutations, such as PRPS1 and HPRT1 mutations, lead to abnormal purine anabolism and then cause hyperuricaemia and gout. In recent years, genome-wide association studies (GWAS) have identified dozens of susceptibility loci and/or candidate genes associated with hyperuricemia and gout. Loss-of-function mutations in SLC2A9, SLC22A11, and SLC22A12 cause hereditary hypouricaemia, while their overexpression may increase the reabsorption of uric acid. In contrast, loss-of-function mutations in ABCG2, SLC17A1, and SLC17A3 cause urate underexcretion of renal and intestinal. These variations leading to blood uric acid excretion disorder (excess reabsorption and underexcretion) are the main genetic factors affecting hyperuicemia and gout. Moreover, to some degree, inhibins-activins growth factor system, transcription factors, cytoskeleton and gene-environment interaction can also affect the level of blood uric acid. In addition, two risk genes, RFX3 and KCNQ1, which might impair immune response and lead to functional deficiency of beta cell were recently discovered to influence hyperuiceamia and gout in Han Chinese. This paper systematically reviews genetic studies on hyperuricaemia and gout to improve our understanding of pathogenesis of hyperuricaemia and gout.

  15. Deficient signaling via Alk2 (Acvr1 leads to bicuspid aortic valve development.

    Directory of Open Access Journals (Sweden)

    Penny S Thomas

    Full Text Available Bicuspid aortic valve (BAV is the most common congenital cardiac anomaly in humans. Despite recent advances, the molecular basis of BAV development is poorly understood. Previously it has been shown that mutations in the Notch1 gene lead to BAV and valve calcification both in human and mice, and mice deficient in Gata5 or its downstream target Nos3 have been shown to display BAVs. Here we show that tissue-specific deletion of the gene encoding Activin Receptor Type I (Alk2 or Acvr1 in the cushion mesenchyme results in formation of aortic valve defects including BAV. These defects are largely due to a failure of normal development of the embryonic aortic valve leaflet precursor cushions in the outflow tract resulting in either a fused right- and non-coronary leaflet, or the presence of only a very small, rudimentary non-coronary leaflet. The surviving adult mutant mice display aortic stenosis with high frequency and occasional aortic valve insufficiency. The thickened aortic valve leaflets in such animals do not show changes in Bmp signaling activity, while Map kinase pathways are activated. Although dysfunction correlated with some pro-osteogenic differences in gene expression, neither calcification nor inflammation were detected in aortic valves of Alk2 mutants with stenosis. We conclude that signaling via Alk2 is required for appropriate aortic valve development in utero, and that defects in this process lead to indirect secondary complications later in life.

  16. Generation of monoclonal antibodies specific for cell surface molecules expressed on early mouse endoderm.

    Science.gov (United States)

    Gadue, Paul; Gouon-Evans, Valerie; Cheng, Xin; Wandzioch, Ewa; Zaret, Kenneth S; Grompe, Markus; Streeter, Philip R; Keller, Gordon M

    2009-09-01

    The development of functional cell populations such as hepatocytes and pancreatic beta cells from embryonic stem cell (ESC) is dependent on the efficient induction of definitive endoderm early in the differentiation process. To monitor definitive endoderm formation in mouse ESC differentiation cultures in a quantitative fashion, we generated a reporter cell line that expresses human CD25 from the Foxa3 locus and human CD4 from the Foxa2 locus. Induction of these reporter ESCs with high concentrations of activin A led to the development of a CD25-Foxa3+CD4-Foxa2+ population within 4-5 days of culture. Isolation and characterization of this population showed that it consists predominantly of definitive endoderm that is able to undergo hepatic specification under the appropriate conditions. To develop reagents that can be used for studies on endoderm development from unmanipulated ESCs, from induced pluripotent stem cells, and from the mouse embryo, we generated monoclonal antibodies against the CD25-Foxa3+CD4-Foxa2+ population. With this approach, we identified two antibodies that react specifically with endoderm from ESC cultures and from the early embryo. The specificity of these antibodies enables one to quantitatively monitor endoderm development in ESC differentiation cultures, to study endoderm formation in the embryo, and to isolate pure populations of culture- or embryo-derived endodermal cells.

  17. Antihepatic Fibrosis Effect of Active Components Isolated from Green Asparagus (Asparagus officinalis L.) Involves the Inactivation of Hepatic Stellate Cells.

    Science.gov (United States)

    Zhong, Chunge; Jiang, Chunyu; Xia, Xichun; Mu, Teng; Wei, Lige; Lou, Yuntian; Zhang, Xiaoshu; Zhao, Yuqing; Bi, Xiuli

    2015-07-08

    Green asparagus (Asparagus officinalis L.) is a vegetable with numerous nutritional properties. In the current study, a total of 23 compounds were isolated from green asparagus, and 9 of these compounds were obtained from this genus for the first time. Preliminary data showed that the ethyl acetate (EtOAc)-extracted fraction of green asparagus exerted a stronger inhibitory effect on the growth of t-HSC/Cl-6 cells, giving an IC50 value of 45.52 μg/mL. The biological activities of the different compounds isolated from the EtOAc-extracted fraction with respect to antihepatic fibrosis were investigated further. Four compounds, C3, C4, C10, and C12, exhibited profound inhibitory effect on the activation of t-HSC/Cl-6 cells induced by TNF-α. The activation t-HSC/Cl-6 cells, which led to the production of fibrotic matrix (TGF-β1, activin C) and accumulation of TNF-α, was dramatically decreased by these compounds. The mechanisms by which these compounds inhibited the activation of hepatic stellate cells appeared to be associated with the inactivation of TGF-β1/Smad signaling and c-Jun N-terminal kinases, as well as the ERK phosphorylation cascade.

  18. Elevation of myostatin and FOXOs in prolonged muscular impairment induced by eccentric contractions in rat medial gastrocnemius muscle.

    Science.gov (United States)

    Ochi, Eisuke; Hirose, Tatsuro; Hiranuma, Kenji; Min, Seok-Ki; Ishii, Naokata; Nakazato, Koichi

    2010-02-01

    This study aimed to investigate torque deficit and activation of protein synthesis and/or protein degradation signaling pathways during the early and recovery phase after high- and low-velocity eccentric contractions (ECs). Male Wistar rats (n = 36) were randomly divided into fast angular velocity ECs group (FAST; 180 degrees/s; n = 12), slow ECs group (SLOW; 30 degrees/s; n = 12), and control group (control; n = 12). ECs comprised four sets of five forced dorsiflexions combined with electrical stimulation of the plantar flexors. Isometric tetanic torque was measured before and after ECs. Tissue contents of Akt(P) (P, phosphorylated), mammalian target of rapamycin (mTOR)(P), 70-kDa ribosomal protein S6 kinase (P70S6k), P70S6k(P), forkhead transcription factor 1 of the O class (FOXO1), FOXO1(P), FOXO3, FOXO3(P), myostatin, and activin receptor type IIB (ActRIIB) were measured. The isometric tetanic torque after ECs was significantly lower in FAST than in SLOW (days 1, 3, and 5, P muscular function and activation of protein synthesis and/or protein degradation signaling pathways.

  19. A novel role for fibronectin type I domain in the regulation of human hematopoietic cell adhesiveness through binding to follistatin domains of FLRG and follistatin.

    Science.gov (United States)

    Maguer-Satta, Véronique; Forissier, Stéphanie; Bartholin, Laurent; Martel, Sylvie; Jeanpierre, Sandrine; Bachelard, Elodie; Rimokh, Ruth

    2006-02-15

    FLRG and follistatin belong to the family of follistatin proteins involved in the regulation of various biological effects, such as hematopoiesis, mediated by their binding to activin and BMP, both members of the TGFbeta family. To further characterize the function of FLRG, we searched for other possible functional partners using a yeast two-hybrid screen. We identified human fibronectin as a new partner for both FLRG and follistatin. We also demonstrated that their physical interaction is mediated by type I motifs of fibronectin and follistatin domains. We then analyzed the biological consequences of these protein interactions on the regulation of hematopoiesis. For the first time, we associated a biological effect with the regulation of human hematopoietic cell adhesiveness of both the type I motifs of fibronectin and the follistatin domains of FLRG and follistatin. Indeed, we observed a significant and specific dose-dependent increase of cell adhesion to fibronectin in the presence of FLRG or follistatin, using either a human hematopoietic cell line or primary cells. In particular, we observed a significantly increased adhesion of immature hematopoietic precursors (CFC, LTC-IC). Altogether these results highlight a new mechanism by which FLRG and follistatin regulate human hematopoiesis.

  20. Cloning, characterization, and tissue expression pattern of mouse Nma/BAMBI during odontogenesis.

    Science.gov (United States)

    Knight, C; Simmons, D; Gu, T T; Gluhak-Heinrich, J; Pavlin, D; Zeichner-David, M; MacDougall, M

    2001-10-01

    Degenerate oligonucleotides to consensus serine kinase functional domains previously identified a novel, partial rabbit tooth cDNA (Zeichner-David et al., 1992) that was used in this study to identify a full-length mouse clone. A 1390-base-pair cDNA clone was isolated encoding a putative 260-amino-acid open reading frame containing a hydrophobic 25-amino-acid potential transmembrane domain. This clone shares some homology with the TGF-beta type I receptor family, but lacks the intracellular kinase domain. DNA database analysis revealed that this clone has 86% identity to a newly isolated human gene termed non-metastatic gene A and 80% identity to a Xenopus cDNA clone termed BMP and activin membrane bound inhibitor. Here we report the mouse Nma/BAMBI cDNA sequence, the tissue expression pattern, and confirmed expression in dental cell lines. This study demonstrates that Nma/BAMBI is a highly conserved protein across species and is expressed at high levels during odontogenesis.

  1. BAMBI Promotes C2C12 Myogenic Differentiation by Enhancing Wnt/β-Catenin Signaling

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    Qiangling Zhang

    2015-08-01

    Full Text Available Bone morphogenic protein and activin membrane-bound inhibitor (BAMBI is regarded as an essential regulator of cell proliferation and differentiation that represses transforming growth factor-β and enhances Wnt/β-catenin signaling in various cell types. However, its role in skeletal muscle remains largely unknown. In the current study, we found that the expression level of BAMBI peaked in the early differentiation phase of the C2C12 rodent myoblast cell line. Knockdown of BAMBI via siRNA inhibited C2C12 differentiation, indicated by repressed MyoD, MyoG, and MyHC expression as well as reductions in the differentiation and fusion indices. BAMBI knockdown reduced the activity of Wnt/β-catenin signaling, as characterized by the decreased nuclear translocation of β-catenin and the lowered transcription of Axin2, which is a well-documented target gene of the Wnt/β-catenin signaling pathway. Furthermore, treatment with LiCl, an activator of Wnt/β-catenin signaling, rescued the reduction in C2C12 differentiation caused by BAMBI siRNA. Taken together, our data suggest that BAMBI is required for normal C2C12 differentiation, and that its role in myogenesis is mediated by the Wnt/β-catenin pathway.

  2. Creating frog heart as an organ: in vitro-induced heart functions as a circulatory organ in vivo.

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    Kinoshita, Masayoshi; Ariizumi, Takashi; Yuasa, Shinsuke; Miyoshi, Shunichirou; Komazaki, Shinji; Fukuda, Keiichi; Asashima, Makoto

    2010-01-01

    Cardiomyocytes have been induced from various pluripotent cells, such as embryonic stem cells and myeloid stem cells; however, the generation of cardiac tissues beyond two-dimensional cell-sheets has not been reported. Creating higher order, three-dimensional structures that are unique to heart is the long-awaited next step in realizing cardiac regenerative medicine. We have previously shown that cardiomyocytes can be induced in vitro from undifferentiated cells (animal caps) excised from Xenopus embryos. Cardiomyocytes were induced by first dissociating the animal caps and then reaggregating them following treatment with activin. Here, we describe an interesting method for creating a complete ectopic heart in vivo, involving the introduction of in vitro-created tissue during early embryogenesis. Thus, animal cap reaggregates were transplanted into the abdomen of late-neurula-stage embryos, resulting in two-chambered hearts being formed. The dual-heart larvae matured into adult animals with transplanted hearts intact. Involvement of transplanted hearts in systemic circulation was demonstrated. Moreover, the ectopic hearts possessed higher order structures such as atrium and ventricle, and were morphologically, histologically, and electrophysiologically identical to original hearts. This system should facilitate the study of heart organogenesis and may promote a shift from tissue to organ engineering for clinical applications.

  3. Quantitative and semiquantitative immunoassay of growth factors and cytokines in the conditioned medium of STO and CF-1 mouse feeder cells.

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    Talbot, Neil C; Sparks, Wendy O; Powell, Anne M; Kahl, Stanislaw; Caperna, Thomas J

    2012-01-01

    Feeder cells of irradiated mouse fibroblasts are commonly used for, and are generally necessary for, the in vitro maintenance and growth of many fastidious cell types, particularly embryonic stem cells or induced pluripotent stem cells. Quantitative and semiquantitative immunoassays of conditioned media were performed to identify some of the soluble cytokines, chemokines, protein hormones, and cell matrix/adhesion molecules that are elaborated from two commonly used feeder cells, STO and CF-1. Among those quantitatively assayed, the most abundant cytokine proteins expressed by the feeder cells were activin A, hepatocyte growth factor (HGF), insulin-like growth factor 1, insulin-like growth factor 2, insulin-like growth factor binding protein (IGFBP)-6, macrophage colony-stimulating factor (a.k.a. CSF-1), and pigment epithelium-derived factor (a.k.a. serine protease inhibitor, clade F, member 1). CF-1 cells expressed ten times more activin A than STO cells and also produced larger amounts of interleukin-6 and IGFBP-2, IGFBP-3, IGFBP-4, and IGFBP-5. Conversely, STO cell produced almost ten times more HGF and five times more stem cell factor (a.k.a. c-kit ligand) than CF-1 cells. Assayed semiquantitatively, relatively large amounts of chemokines were produced by both feeder cells including fractalkine (CX3CL1), interferon-inducible protein 10 (a.k.a. CXCL10 and cytokine-responsive gene-2, CRG-2), monocyte chemotactic protein (MCP)-1 (a.k.a. CCL2 and junctional epithelium chemokine (JE), MCP-5/CCL12), keratinocyte-derived chemokine (a.k.a. CXCL1 and growth-related oncogene alpha, GROα), nephroblastoma overexpressed gene (CCN3, IGFBP-9), stromal cell-derived factor 1 (CXCL12), and serpin E1 (PAI-1). In contrast to one another, STO produced more CXCL16 than CF-1 cells, and CF-1 cell produced more MCP-5 (CCL12), macrophage inflammatory protein (MIP)-1α (CCL3), MIP-1β (CCL4), pentraxin-3 (TSG-14), and platelet factor-4 (CXCL4) than STO cells. Soluble adhesion molecule

  4. Endothelins Inhibit Osmotic Swelling of Rat Retinal Glial and Bipolar Cells by Activation of Growth Factor Signaling.

    Science.gov (United States)

    Vogler, Stefanie; Grosche, Antje; Pannicke, Thomas; Wiedemann, Peter; Reichenbach, Andreas; Bringmann, Andreas

    2016-10-01

    Water accumulation in retinal glial (Müller) and neuronal cells resulting in cellular swelling contributes to the development of retinal edema and neurodegeneration. Here, we show that endothelin-1 (ET-1) dose-dependently inhibits the hypoosmotic swelling of Müller cells in freshly isolated retinal slices of control and diabetic rats, with a maximal inhibition at 100 nM. Osmotic Müller cell swelling was also inhibited by ET-2. The effect of ET-1 was mediated by activation of ETA and ETB receptors resulting in transactivation of metabotropic glutamate receptors, purinergic P2Y1, and adenosine A1 receptors. ET-1 (but not ET-2) also inhibited the osmotic swelling of bipolar cells in retinal slices, but failed to inhibit the swelling of freshly isolated bipolar cells. The inhibitory effect of ET-1 on the bipolar cell swelling in retinal slices was abrogated by inhibitors of the FGF receptor kinase (PD173074) and of TGF-β1 superfamily activin receptor-like kinase receptors (SB431542), respectively. Both Müller and bipolar cells displayed immunoreactivities of ETA and ETB receptor proteins. The data may suggest that neuroprotective effects of ETs in the retina are in part mediated by prevention of the cytotoxic swelling of retinal glial and bipolar cells. ET-1 acts directly on Müller cells, while the inhibitory effect of ET-1 on bipolar cell swelling is indirectly mediated, via stimulation of the release of growth factors like bFGF and TGF-β1 from Müller cells.

  5. Muscle-bone interactions: From experimental models to the clinic? A critical update.

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    Laurent, Michaël R; Dubois, Vanessa; Claessens, Frank; Verschueren, Sabine M P; Vanderschueren, Dirk; Gielen, Evelien; Jardí, Ferran

    2016-09-01

    Bone is a biomechanical tissue shaped by forces from muscles and gravitation. Simultaneous bone and muscle decay and dysfunction (osteosarcopenia or sarco-osteoporosis) is seen in ageing, numerous clinical situations including after stroke or paralysis, in neuromuscular dystrophies, glucocorticoid excess, or in association with vitamin D, growth hormone/insulin like growth factor or sex steroid deficiency, as well as in spaceflight. Physical exercise may be beneficial in these situations, but further work is still needed to translate acceptable and effective biomechanical interventions like vibration therapy from animal models to humans. Novel antiresorptive and anabolic therapies are emerging for osteoporosis as well as drugs for sarcopenia, cancer cachexia or muscle wasting disorders, including antibodies against myostatin or activin receptor type IIA and IIB (e.g. bimagrumab). Ideally, increasing muscle mass would increase muscle strength and restore bone loss from disuse. However, the classical view that muscle is unidirectionally dominant over bone via mechanical loading is overly simplistic. Indeed, recent studies indicate a role for neuronal regulation of not only muscle but also bone metabolism, bone signaling pathways like receptor activator of nuclear factor kappa-B ligand (RANKL) implicated in muscle biology, myokines affecting bone and possible bone-to-muscle communication. Moreover, pharmacological strategies inducing isolated myocyte hypertrophy may not translate into increased muscle power because tendons, connective tissue, neurons and energy metabolism need to adapt as well. We aim here to critically review key musculoskeletal molecular pathways involved in mechanoregulation and their effect on the bone-muscle unit as a whole, as well as preclinical and emerging clinical evidence regarding the effects of sarcopenia therapies on osteoporosis and vice versa.

  6. In situ guided tissue regeneration in musculoskeletal diseases and aging : Implementing pathology into tailored tissue engineering strategies.

    Science.gov (United States)

    Jakob, Franz; Ebert, Regina; Rudert, Maximilian; Nöth, Ulrich; Walles, Heike; Docheva, Denitsa; Schieker, Matthias; Meinel, Lorenz; Groll, Jürgen

    2012-03-01

    In situ guided tissue regeneration, also addressed as in situ tissue engineering or endogenous regeneration, has a great potential for population-wide "minimal invasive" applications. During the last two decades, tissue engineering has been developed with remarkable in vitro and preclinical success but still the number of applications in clinical routine is extremely small. Moreover, the vision of population-wide applications of ex vivo tissue engineered constructs based on cells, growth and differentiation factors and scaffolds, must probably be deemed unrealistic for economic and regulation-related issues. Hence, the progress made in this respect will be mostly applicable to a fraction of post-traumatic or post-surgery situations such as big tissue defects due to tumor manifestation. Minimally invasive procedures would probably qualify for a broader application and ideally would only require off the shelf standardized products without cells. Such products should mimic the microenvironment of regenerating tissues and make use of the endogenous tissue regeneration capacities. Functionally, the chemotaxis of regenerative cells, their amplification as a transient amplifying pool and their concerted differentiation and remodeling should be addressed. This is especially important because the main target populations for such applications are the elderly and diseased. The quality of regenerative cells is impaired in such organisms and high levels of inhibitors also interfere with regeneration and healing. In metabolic bone diseases like osteoporosis, it is already known that antagonists for inhibitors such as activin and sclerostin enhance bone formation. Implementing such strategies into applications for in situ guided tissue regeneration should greatly enhance the efficacy of tailored procedures in the future.

  7. Measurement of dimeric inhibin using a modified two-site immunoradiometric assay specific for oxidized (Met O) inhibin.

    Science.gov (United States)

    Knight, P G; Muttukrishna, S

    1994-06-01

    Several years ago we developed a novel two-site immunoradiometric assay (IRMA) for dimeric inhibin. However, relative to the purified 32 kDa bovine inhibin standard used at that time, the immunopotencies of crude inhibin-containing samples were much less than their biopotencies estimated by pituitary cell bioassay. In attempting to improve assay performance and resolve this discrepancy we recently discovered that introduction of a preassay oxidation step to the IRMA results in a dramatic increase in the immunopotencies of inhibin-containing test samples (e.g.: bovine, human, porcine follicular fluid (FF)) and of a new (purified in 1993) 32 kDa bovine inhibin standard. However, the oxidation step did not affect the immunopotency of our original standard (purified in 1987), indicating that this material had undergone spontaneous oxidation during long-term storage, thus accounting for its higher immunopotency in our original IRMA and providing an explanation for the discrepancy between immunoactivity and bioactivity referred to above. These findings, together with other observations on the behaviour of oxidized and non-oxidized samples of inhibin, related peptide fragments and inhibin-containing samples in the IRMA and alpha subunit radioimmunoassay (RIA), indicate that the anti-beta A82-114 monoclonal antibody (E4) used as tracer in the IRMA binds selectively to the oxidized (Met O89,91,108) form of the peptide. This property of the antibody can be exploited to advantage by incorporating simple modifications to existing inhibin/activin immunoassays to ensure that all samples and standards are fully oxidized before antibody addition.(ABSTRACT TRUNCATED AT 250 WORDS)

  8. Effects of cellular origin on differentiation of human induced pluripotent stem cell-derived endothelial cells.

    Science.gov (United States)

    Hu, Shijun; Zhao, Ming-Tao; Jahanbani, Fereshteh; Shao, Ning-Yi; Lee, Won Hee; Chen, Haodong; Snyder, Michael P; Wu, Joseph C

    2016-06-02

    Human induced pluripotent stem cells (iPSCs) can be derived from various types of somatic cells by transient overexpression of 4 Yamanaka factors (OCT4, SOX2, C-MYC, and KLF4). Patient-specific iPSC derivatives (e.g., neuronal, cardiac, hepatic, muscular, and endothelial cells [ECs]) hold great promise in drug discovery and regenerative medicine. In this study, we aimed to evaluate whether the cellular origin can affect the differentiation, in vivo behavior, and single-cell gene expression signatures of human iPSC-derived ECs. We derived human iPSCs from 3 types of somatic cells of the same individuals: fibroblasts (FB-iPSCs), ECs (EC-iPSCs), and cardiac progenitor cells (CPC-iPSCs). We then differentiated them into ECs by sequential administration of Activin, BMP4, bFGF, and VEGF. EC-iPSCs at early passage (10 < P < 20) showed higher EC differentiation propensity and gene expression of EC-specific markers (PECAM1 and NOS3) than FB-iPSCs and CPC-iPSCs. In vivo transplanted EC-iPSC-ECs were recovered with a higher percentage of CD31(+) population and expressed higher EC-specific gene expression markers (PECAM1, KDR, and ICAM) as revealed by microfluidic single-cell quantitative PCR (qPCR). In vitro EC-iPSC-ECs maintained a higher CD31(+) population than FB-iPSC-ECs and CPC-iPSC-ECs with long-term culturing and passaging. These results indicate that cellular origin may influence lineage differentiation propensity of human iPSCs; hence, the somatic memory carried by early passage iPSCs should be carefully considered before clinical translation.

  9. Effects of cellular origin on differentiation of human induced pluripotent stem cell–derived endothelial cells

    Science.gov (United States)

    Zhao, Ming-Tao; Jahanbani, Fereshteh; Lee, Won Hee; Snyder, Michael P.

    2016-01-01

    Human induced pluripotent stem cells (iPSCs) can be derived from various types of somatic cells by transient overexpression of 4 Yamanaka factors (OCT4, SOX2, C-MYC, and KLF4). Patient-specific iPSC derivatives (e.g., neuronal, cardiac, hepatic, muscular, and endothelial cells [ECs]) hold great promise in drug discovery and regenerative medicine. In this study, we aimed to evaluate whether the cellular origin can affect the differentiation, in vivo behavior, and single-cell gene expression signatures of human iPSC–derived ECs. We derived human iPSCs from 3 types of somatic cells of the same individuals: fibroblasts (FB-iPSCs), ECs (EC-iPSCs), and cardiac progenitor cells (CPC-iPSCs). We then differentiated them into ECs by sequential administration of Activin, BMP4, bFGF, and VEGF. EC-iPSCs at early passage (10 < P < 20) showed higher EC differentiation propensity and gene expression of EC-specific markers (PECAM1 and NOS3) than FB-iPSCs and CPC-iPSCs. In vivo transplanted EC-iPSC–ECs were recovered with a higher percentage of CD31+ population and expressed higher EC-specific gene expression markers (PECAM1, KDR, and ICAM) as revealed by microfluidic single-cell quantitative PCR (qPCR). In vitro EC-iPSC–ECs maintained a higher CD31+ population than FB-iPSC–ECs and CPC-iPSC–ECs with long-term culturing and passaging. These results indicate that cellular origin may influence lineage differentiation propensity of human iPSCs; hence, the somatic memory carried by early passage iPSCs should be carefully considered before clinical translation. PMID:27398408

  10. A Key Role for Inhibins in Dendritic Cell Maturation and Function.

    Science.gov (United States)

    Olguín-Alor, Roxana; de la Fuente-Granada, Marisol; Bonifaz, Laura C; Antonio-Herrera, Laura; García-Zepeda, Eduardo A; Soldevila, Gloria

    2016-01-01

    Inhibins are members of the TGFβ superfamily, which regulate many cellular processes including differentiation, proliferation, survival and apoptosis. Although initially described as hormones regulating the hypothalamus-pituitary-gonadal axis, based on their ability to antagonize Activins, our group has recently reported that they play a role in thymocyte differentiation and survival, as well as in thymic stromal cell maturation and nTreg generation. Here, we used Inhibin knock out mice (Inhα-/-) to investigate the role of Inhibins in peripheral dendritic cell maturation and function. We first demonstrated that LPS treated Inhα+/+ bone marrow derived dendritic cells (BMDC) were capable to produce significant levels of Inhibin A. Interestingly, Inhα-/- BMDC showed reduced MHCII and CD86 upregulation and increased PD-L1 expression in response to LPS compared to Inhα+/+, which correlated with reduced ability to induce proliferation of allogeneic T cells. The "semi-mature" phenotype displayed by Inhα-/- mBMDC correlated with increased levels of IL-10 and slightly decreased IL-6 production after LPS stimulation. In addition, Inhα-/- mBMDC showed impaired migration towards CCL19 and CCL21, assessed by in vitro chemotaxis and in vivo competitive homing experiments, despite their normal CCR7 expression. Furthermore, in vivo LPS-induced DC maturation was also diminished in Inhα-/- mice, specially within the LC (CD207+ CD11b+ CD103-) subpopulation. Finally, analysis of delayed type hypersensitivity responses in Inhα-/- mice, showed reduced ear swelling as a result of reduced cellular infiltration in the skin, correlating with impaired homing of CD207+ DCs to the draining lymph nodes. In summary, our data demonstrate for the first time that Inhibins play a key role in peripheral DC maturation and function, regulating the balance between immunity and tolerance.

  11. The GnRH receptor and the response of gonadotrope cells to GnRH pulse frequency code. A story of an atypical adaptation of cell function relying on a lack of receptor homologous desensitization.

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    Christian Bleux

    2010-01-01

    Full Text Available Brain control of the reproductive system is mediated through hypothalamic gonadotropin-releasing hormone (GnRH which activates specific receptors (GnRHR present at the surface of the pituitary gonadotropes to trigger secretion of the two gonadotropins LH and FSH. A unique feature of this system is the high dependence on the secretion mode of GnRH, which is basically pulsatile but undergoes considerable fluctuations in pulse frequency pattern in response to endogenous or external factors. How the physiological fluctuations of GnRH secretion that orchestrate normal reproduction are decoded by the gonadotrope cell machinery to ultimately control gonadotropin release and/or subunit gene transcription has been the subject of intensive studies during the past decades. Surprisingly, the mammalian GnRHR is unique among G protein-coupled receptor family as it lacks the carboxy-terminal tail usually involved in classical endocytotic process. Accordingly, it does not desensitize properly and internalizes very poorly. Both this atypical intrinsic property and post-receptor events may thus contribute to decode the GnRH signal. This includes the participation of a network of signaling pathways that differently respond to GnRH together with a growing amount of genes differentially sensitive to pulse frequency. Among these are two pairs of genes, the transcription factors EGR-1 and NAB, and the regulatory factors activin and follistatin, that function as intracellular autoregulatory feedback loops controlling respectively LHbeta and FSHbeta gene expression and hence, LH and FSH synthesis. Pituitary gonadotropes thus represent a unique model of cells functionally adapted to respond to a considerably fluctuating neuroendocrine stimulation, from short individual pulses to sustained GnRH as observed at the proestrus of ovarian cycle. Altogether, the data emphasize the adaptative reciprocal complementarity of hypothalamic GnRH neurones and pituitary gonadotropes to

  12. Levetiracetam attenuates hippocampal expression of synaptic plasticity-related immediate early and late response genes in amygdala-kindled rats

    Science.gov (United States)

    2010-01-01

    Background The amygdala-kindled rat is a model for human temporal lobe epilepsy and activity-dependent synaptic plasticity. Hippocampal RNA isolated from amygdala-kindled rats at different kindling stages was analyzed to identify kindling-induced genes. Furthermore, effects of the anti-epileptic drug levetiracetam on kindling-induced gene expression were examined. Results Cyclooxygenase-2 (Cox-2), Protocadherin-8 (Pcdh8) and TGF-beta-inducible early response gene-1 (TIEG1) were identified and verified as differentially expressed transcripts in the hippocampus of kindled rats by in situ hybridization and quantitative RT-PCR. In addition, we identified a panel of 16 additional transcripts which included Arc, Egr3/Pilot, Homer1a, Ania-3, MMP9, Narp, c-fos, NGF, BDNF, NT-3, Synaptopodin, Pim1 kinase, TNF-α, RGS2, Egr2/krox-20 and β-A activin that were differentially expressed in the hippocampus of amygdala-kindled rats. The list consists of many synaptic plasticity-related immediate early genes (IEGs) as well as some late response genes encoding transcription factors, neurotrophic factors and proteins that are known to regulate synaptic remodelling. In the hippocampus, induction of IEG expression was dependent on the afterdischarge (AD) duration. Levetiracetam, 40 mg/kg, suppressed the development of kindling measured as severity of seizures and AD duration. In addition, single animal profiling also showed that levetiracetam attenuated the observed kindling-induced IEG expression; an effect that paralleled the anti-epileptic effect of the drug on AD duration. Conclusions The present study provides mRNA expression data that suggest that levetiracetam attenuates expression of genes known to regulate synaptic remodelling. In the kindled rat, levetiracetam does so by shortening the AD duration thereby reducing the seizure-induced changes in mRNA expression in the hippocampus. PMID:20105316

  13. Lin28 proteins are required for germ layer specification in Xenopus.

    Science.gov (United States)

    Faas, Laura; Warrander, Fiona C; Maguire, Richard; Ramsbottom, Simon A; Quinn, Diana; Genever, Paul; Isaacs, Harry V

    2013-03-01

    Lin28 family proteins share a unique structure, with both zinc knuckle and cold shock RNA-binding domains, and were originally identified as regulators of developmental timing in Caenorhabditis elegans. They have since been implicated as regulators of pluripotency in mammalian stem cells in culture. Using Xenopus tropicalis, we have undertaken the first analysis of the effects on the early development of a vertebrate embryo resulting from global inhibition of the Lin28 family. The Xenopus genome contains two Lin28-related genes, lin28a and lin28b. lin28a is expressed zygotically, whereas lin28b is expressed both zygotically and maternally. Both lin28a and lin28b are expressed in pluripotent cells of the Xenopus embryo and are enriched in cells that respond to mesoderm-inducing signals. The development of axial and paraxial mesoderm is severely abnormal in lin28 knockdown (morphant) embryos. In culture, the ability of pluripotent cells from the embryo to respond to the FGF and activin/nodal-like mesoderm-inducing pathways is compromised following inhibition of lin28 function. Furthermore, there are complex effects on the temporal regulation of, and the responses to, mesoderm-inducing signals in lin28 morphant embryos. We provide evidence that Xenopus lin28 proteins play a key role in choreographing the responses of pluripotent cells in the early embryo to the signals that regulate germ layer specification, and that this early function is probably independent of the recognised role of Lin28 proteins in negatively regulating let-7 miRNA biogenesis.

  14. A Combination of Culture Conditions and Gene Expression Analysis Can Be Used to Investigate and Predict hES Cell Differentiation Potential towards Male Gonadal Cells.

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    Kristín Rós Kjartansdóttir

    Full Text Available Human embryonic stem cell differentiation towards various cell types belonging to ecto-, endo- and mesodermal cell lineages has been demonstrated, with high efficiency rates using standardized differentiation protocols. However, germ cell differentiation from human embryonic stem cells has been very inefficient so far. Even though the influence of various growth factors has been evaluated, the gene expression of different cell lines in relation to their differentiation potential has not yet been extensively examined. In this study, the potential of three male human embryonic stem cell lines to differentiate towards male gonadal cells was explored by analysing their gene expression profiles. The human embryonic stem cell lines were cultured for 14 days as monolayers on supporting human foreskin fibroblasts or as spheres in suspension, and were differentiated using BMP7, or spontaneous differentiation by omitting exogenous FGF2. TLDA analysis revealed that in the undifferentiated state, these cell lines have diverse mRNA profiles and exhibit significantly different potentials for differentiation towards the cell types present in the male gonads. This potential was associated with important factors directing the fate of the male primordial germ cells in vivo to form gonocytes, such as SOX17 or genes involved in the NODAL/ACTIVIN pathway, for example. Stimulation with BMP7 in suspension culture resulted in up-regulation of cytoplasmic SOX9 protein expression in all three lines. The observation that human embryonic stem cells differentiate towards germ and somatic cells after spontaneous and BMP7-induced stimulation in suspension emphasizes the important role of somatic cells in germ cell differentiation in vitro.

  15. GnRH-induced Ca2+ signaling patterns and gonadotropin secretion in pituitary gonadotrophs. Functional adaptations to both ordinary and extraordinary physiological demands

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    María Luisa eDurán-Pastén

    2013-09-01

    Full Text Available Pituitary gonadotrophs are a small fraction of the anterior pituitary population, yet they synthesize gonadotropins: luteinizing (LH and follicle stimulating (FSH, essential for gametogenesis and steroidogenesis. LH is secreted via a regulated pathway while FSH release is mostly constitutive and controlled by synthesis. Although gonadotrophs fire action potentials spontaneously, the intracellular Ca2+ rises produced do not influence secretion, which is mainly driven by Gonadotropin Releasing Hormone (GnRH, a decapeptide synthesized in the hypothalamus and released in a pulsatile manner into the hypophyseal portal circulation. GnRH binding to G protein coupled receptors triggers Ca2+ mobilization from InsP3-sensitive intracellular pools, generating the global Ca2+ elevations necessary for secretion. Ca2+ signaling responses to increasing [GnRH] vary in stereotyped fashion from subthreshold to baseline spiking (oscillatory, to biphasic (spike-oscillatory or spike-plateau. This progression varies somewhat in gonadotrophs from different species and biological preparations. Both baseline spiking and biphasic GnRH-induced Ca2+ signals control LH/FSH synthesis and exocytosis. Estradiol and testosterone regulate gonadotropin secretion through feedback mechanisms, while FSH synthesis and release are influenced by activin, inhibin and follistatin. Adaptation to physiological events like the estrous cycle, involves changes in GnRH sensitivity and LH/FSH synthesis: in proestrus, estradiol feedback regulation abruptly changes from negative to positive, causing the pre-ovulatory LH surge. Similarly, when testosterone levels drop after orquiectomy the lack of negative feedback on pituitary and hypothalamus boosts both GnRH and LH secretion, gonadotrophs GnRH sensitivity increases and Ca2+ signaling patterns change. In addition, gonadotrophs proliferate and grow. These plastic changes denote a more vigorous functional adaptation in response to an extraordinary

  16. Transforming growth factor-beta type 1 receptor (ALK5) and Smad proteins mediate TIMP-1 and collagen synthesis in experimental intestinal fibrosis.

    Science.gov (United States)

    Medina, Carlos; Santos-Martinez, Maria Jose; Santana, Alfredo; Paz-Cabrera, Maria Cristina; Johnston, Michael J; Mourelle, Marisabel; Salas, Antonio; Guarner, Francisco

    2011-08-01

    Transforming growth factor β (TGF-β) is known to play a key role in intestinal fibrosis; however, the underlying mechanisms are not well understood. TGF-β signal transduction is through TGF-β receptors, including the TGF-β type 1 receptor. Most cell types contain a TGF-β type 1 receptor form known as activin receptor-like kinase 5 (ALK5), which propagates the signal to the nucleus through the phosphorylation of Smad2 and Smad3 proteins. Therefore, we assessed the effect of the disruption of TGF-β/ALK5/Smad signalling by an ALK5 inhibitor (SD-208) in two experimental animal models of intestinal fibrosis: anaerobic bacteria- and trinitrobenzensulphonic acid-induced colitis. In addition, isolated myofibroblasts were pretreated with SD-208 and exposed to recombinant TGF-β1. Finally, myofibroblasts were transfected with ALK5, Smad2, and Smad3-specific siRNA. Up-regulation of ALK5 and TIMP-1, phosphorylation of Smad2 and Smad3 proteins, and increased intestinal wall collagen deposition were found in both experimental animal models. These effects were decreased by SD-208. TGF-β1 treatment also induced phosphorylation of Smad2 and Smad3 and up-regulation of ALK5 protein, TIMP-1, and α2 type 1 collagen gene expression in isolated myofibroblasts. Again these effects were inhibited by SD-208. Also, ALK5, Smad2, and Smad3 siRNA abolished the induction of TIMP-1 and α2 type 1 collagen. Our findings provide evidence that the TGF-β/ALK5/Smad pathway participates in the pathogenesis of experimental intestinal fibrosis. These data show promise for the development of an effective therapeutic intervention in this condition.

  17. Role of oxygen tension and genetic background during the epigenetic conversion of mouse fibroblasts into insulin secreting cells

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    Alessandro Zenobi

    2015-07-01

    Full Text Available Epigenetic cell conversion overcomes the stability of a mature cell phenotype transforming a somatic cell in an unlimited source of autologous cells of a different type. It is based on the exposure to a demethylating agent followed by an induction protocol. In our work we exposed mouse dermal fibroblasts to the demethylating agent 5-azacytidine. Cell differentiation was directed toward the endocrine pancreatic lineage with a sequential combination of Activin A, Retinoic Acid, B27 supplement, ITS and bFGF. The overall duration of the process was 10 days. Aim of this work was to evaluate the role of oxygen during differentiation of dermal fibroblasts derived from two different mouse strains, NOD and C57 BL/6J. During differentiation, both cell lines were cultured either in the standard in vitro culture 20% oxygen concentration or in the lower and more physiological 5% of oxygen. Our results show that C57 BL/6J cells are able to differentiate into insulin secreting cells in both oxygen tensions with a higher amount of insulin release in low oxygen conditions. On the other hand, cells of NOD mice, which are physiologically predisposed to the onset of diabetes, differentiate in 20% of oxygen but not in low oxygen and they died after three days of culture. However, if these cells are moved to 5% of oxygen after their differentiation in high oxygen they remain viable for up to four days. Furthermore, their capacity to release insulin remains unchanged for 24 hours. Results suggest that genetic background has a profound effect on the role of oxygen during the in vitro differentiation process, possibly reflecting the different susceptibility to the disease of the strains used in the experiment.Supported by EFSD and Carraresi Foundation

  18. Action mechanism of inhibin α-subunit on the development of Sertoli cells and first wave of spermatogenesis in mice.

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    Kailai Cai

    Full Text Available Inhibin is an important marker of Sertoli cell (SC activity in animals with impaired spermatogenesis. However, the precise relationship between inhibin and SC activity is unknown. To investigate this relationship, we partially silenced both the transcription and translation of the gene for the α-subunit of inhibin, Inha, using recombinant pshRNA vectors developed with RNAi-Ready pSIREN-RetroQ-ZsGreen Vector (Clontech Laboratories, Mountain View, Calif. We found that Inha silencing suppresses the cell-cycle regulators Cyclin D1 and Cyclin E and up-regulates the cell-cycle inhibitor P21 (as detected by Western blot analysis, thereby increasing the number of SCs in the G1 phase of the cell cycle and decreasing the amount in the S-phase of the cell cycle (as detected by flow cytometry. Inha silencing also suppressed Pdgfa, Igf1, and Kitl mRNA levels and up-regulated Tgfbrs, Inhba, Inhbb, Cyp11a1, Dhh, and Tjp1 mRNA levels (as indicated by real-time polymerase chain reaction [PCR] analysis. These findings indicate that Inha has the potential to influence the availability of the ligand inhibin and its antagonist activin in the SC in an autocrine manner and inhibit the progression of SC from G1 to S. It may also participate in the development of the blood-testis barrier, Leydig cells, and spermatogenesis through its effect on Dhh, Tjp1, Kitl, and Pdgfa. Real-time PCR and Western blot analyses of Inha, Inhba, and Inhbb mRNA and Inha levels over time show that Inha plays an important role in the formation of round spermatid during the first wave of spermatogenesis in mice.

  19. Combinatory microarray and SuperSAGE analyses identify pairing-dependently transcribed genes in Schistosoma mansoni males, including follistatin.

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    Silke Leutner

    2013-11-01

    Full Text Available BACKGROUND: Schistosomiasis is a disease of world-wide importance and is caused by parasitic flatworms of the genus Schistosoma. These parasites exhibit a unique reproduction biology as the female's sexual maturation depends on a constant pairing-contact to the male. Pairing leads to gonad differentiation in the female, and even gene expression of some gonad-associated genes is controlled by pairing. In contrast, no morphological changes have been observed in males, although first data indicated an effect of pairing also on gene transcription in males. METHODOLOGY/PRINCIPAL FINDINGS: To investigate the influence of pairing on males, we performed a combinatory approach applying SuperSAGE and microarray hybridization, generating the most comprehensive data-set on differential transcription available to date. Of 6,326 sense transcripts detected by both analyses, 29 were significantly differentially transcribed. Besides mutual confirmation, the two methods complemented each other as shown by data comparison and real-time PCR, which revealed a number of genes with consistent regulation across all methods. One of the candidate genes, follistatin of S. mansoni (SmFst was characterized in more detail by in situ hybridization and yeast two-hybrid (Y2H interaction analyses with potential binding partners. CONCLUSIONS/SIGNIFICANCE: Beyond confirming previously hypothesized differences in metabolic processes between pairing-experienced (EM and pairing-unexperienced males (UM, our data indicate that neuronal processes are involved in male-female interaction but also TGFβ-signaling. One candidate revealing significant down-regulation in EM was the TGFβ-pathway controlling molecule follistatin (SmFst. First functional analyses demonstrated SmFst interaction with the S. mansoni TGFβ-receptor agonists inhibin/activin (SmInAct and bone morphogenic protein (SmBMP, and all molecules colocalized in the testes. This indicates a yet unknown role of the TGF

  20. A novel therapeutic approach to treating obesity through modulation of TGFβ signaling.

    Science.gov (United States)

    Koncarevic, Alan; Kajimura, Shingo; Cornwall-Brady, Milton; Andreucci, Amy; Pullen, Abigail; Sako, Dianne; Kumar, Ravindra; Grinberg, Asya V; Liharska, Katia; Ucran, Jeffrey A; Howard, Elizabeth; Spiegelman, Bruce M; Seehra, Jasbir; Lachey, Jennifer

    2012-07-01

    Obesity results from disproportionately high energy intake relative to energy expenditure. Many therapeutic strategies have focused on the intake side of the equation, including pharmaceutical targeting of appetite and digestion. An alternative approach is to increase energy expenditure through physical activity or adaptive thermogenesis. A pharmacological way to increase muscle mass and hence exercise capacity is through inhibition of the activin receptor type IIB (ActRIIB). Muscle mass and strength is regulated, at least in part, by growth factors that signal via ActRIIB. Administration of a soluble ActRIIB protein comprised of a form of the extracellular domain of ActRIIB fused to a human Fc (ActRIIB-Fc) results in a substantial muscle mass increase in normal mice. However, ActRIIB is also present on and mediates the action of growth factors in adipose tissue, although the function of this system is poorly understood. In the current study, we report the effect of ActRIIB-Fc to suppress diet-induced obesity and linked metabolic dysfunctions in mice fed a high-fat diet. ActRIIB-Fc induced a brown fat-like thermogenic gene program in epididymal white fat, as shown by robustly increased expression of the thermogenic genes uncoupling protein 1 and peroxisomal proliferator-activated receptor-γ coactivator 1α. Finally, we identified multiple ligands capable of reducing thermogenesis that represent likely target ligands for the ActRIIB-Fc effects on the white fat depots. These data demonstrate that novel therapeutic ActRIIB-Fc improves obesity and obesity-linked metabolic disease by both increasing skeletal muscle mass and by inducing a gene program of thermogenesis in the white adipose tissues.

  1. Functional and splicing defect analysis of 23 ACVRL1 mutations in a cohort of patients affected by Hereditary Hemorrhagic Telangiectasia.

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    Ferdos Alaa El Din

    Full Text Available Hereditary Hemorrhagic Telangiectasia syndrome (HHT or Rendu-Osler-Weber (ROW syndrome is an autosomal dominant vascular disorder. Two most common forms of HHT, HHT1 and HHT2, have been linked to mutations in the endoglin (ENG and activin receptor-like kinase 1 (ACVRL1or ALK1 genes respectively. This work was designed to examine the pathogenicity of 23 nucleotide variations in ACVRL1 gene detected in more than 400 patients. Among them, 14 missense mutations and one intronic variant were novels, and 8 missense mutations were previously identified with questionable implication in HHT2. The functionality of missense mutations was analyzed in response to BMP9 (specific ligand of ALK1, the maturation of the protein products and their localization were analyzed by western blot and fluorescence microscopy. The splicing impairment of the intronic and of two missense mutations was examined by minigene assay. Functional analysis showed that 18 out of 22 missense mutations were defective. Splicing analysis revealed that one missense mutation (c.733A>G, p.Ile245Val affects the splicing of the harboring exon 6. Similarly, the intronic mutation outside the consensus splicing sites (c.1048+5G>A in intron 7 was seen pathogenic by splicing study. Both mutations induce a frame shift creating a premature stop codon likely resulting in mRNA degradation by NMD surveillance mechanism. Our results confirm the haploinsufficiency model proposed for HHT2. The affected allele of ACVRL1 induces mRNA degradation or the synthesis of a protein lacking the receptor activity. Furthermore, our data demonstrate that functional and splicing analyses together, represent two robust diagnostic tools to be used by geneticists confronted with novel or conflicted ACVRL1 mutations.

  2. Identification of novel tissue-specific genes by analysis of microarray databases: a human and mouse model.

    Science.gov (United States)

    Song, Yan; Ahn, Jinsoo; Suh, Yeunsu; Davis, Michael E; Lee, Kichoon

    2013-01-01

    Understanding the tissue-specific pattern of gene expression is critical in elucidating the molecular mechanisms of tissue development, gene function, and transcriptional regulations of biological processes. Although tissue-specific gene expression information is available in several databases, follow-up strategies to integrate and use these data are limited. The objective of the current study was to identify and evaluate novel tissue-specific genes in human and mouse tissues by performing comparative microarray database analysis and semi-quantitative PCR analysis. We developed a powerful approach to predict tissue-specific genes by analyzing existing microarray data from the NCBI's Gene Expression Omnibus (GEO) public repository. We investigated and confirmed tissue-specific gene expression in the human and mouse kidney, liver, lung, heart, muscle, and adipose tissue. Applying our novel comparative microarray approach, we confirmed 10 kidney, 11 liver, 11 lung, 11 heart, 8 muscle, and 8 adipose specific genes. The accuracy of this approach was further verified by employing semi-quantitative PCR reaction and by searching for gene function information in existing publications. Three novel tissue-specific genes were discovered by this approach including AMDHD1 (amidohydrolase domain containing 1) in the liver, PRUNE2 (prune homolog 2) in the heart, and ACVR1C (activin A receptor, type IC) in adipose tissue. We further confirmed the tissue-specific expression of these 3 novel genes by real-time PCR. Among them, ACVR1C is adipose tissue-specific and adipocyte-specific in adipose tissue, and can be used as an adipocyte developmental marker. From GEO profiles, we predicted the processes in which AMDHD1 and PRUNE2 may participate. Our approach provides a novel way to identify new sets of tissue-specific genes and to predict functions in which they may be involved.

  3. Primitive cardiac cells from human embryonic stem cells.

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    Hudson, James; Titmarsh, Drew; Hidalgo, Alejandro; Wolvetang, Ernst; Cooper-White, Justin

    2012-06-10

    Pluripotent stem cell-derived cardiomyocytes are currently being investigated for in vitro human heart models and as potential therapeutics for heart failure. In this study, we have developed a differentiation protocol that minimizes the need for specific human embryonic stem cell (hESC) line optimization. We first reduced the heterogeneity that exists within the starting population of bulk cultured hESCs by using cells adapted to single-cell passaging in a 2-dimensional (2D) culture format. Compared with bulk cultures, single-cell cultures comprised larger fractions of TG30(hi)/OCT4(hi) cells, corresponding to an increased expression of pluripotency markers OCT4 and NANOG, and reduced expression of early lineage-specific markers. A 2D temporal differentiation protocol was then developed, aimed at reducing the inherent heterogeneity and variability of embryoid body-based protocols, with induction of primitive streak cells using bone morphogenetic protein 4 and activin A, followed by cardiogenesis via inhibition of Wnt signaling using the small molecules IWP-4 or IWR-1. IWP-4 treatment resulted in a large percentage of cells expressing low amounts of cardiac myosin heavy chain and expression of early cardiac progenitor markers ISL1 and NKX2-5, thus indicating the production of large numbers of immature cardiomyocytes (~65,000/cm(2) or ~1.5 per input hESC). This protocol was shown to be effective in HES3, H9, and, to a lesser, extent, MEL1 hESC lines. In addition, we observed that IWR-1 induced predominantly atrial myosin light chain (MLC2a) expression, whereas IWP-4 induced expression of both atrial (MLC2a) and ventricular (MLC2v) forms. The intrinsic flexibility and scalability of this 2D protocol mean that the output population of primitive cardiomyocytes will be particularly accessible and useful for the investigation of molecular mechanisms driving terminal cardiomyocyte differentiation, and potentially for the future treatment of heart failure.

  4. Novel insights into embryonic stem cell self-renewal revealed through comparative human and mouse systems biology networks.

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    Dowell, Karen G; Simons, Allen K; Bai, Hao; Kell, Braden; Wang, Zack Z; Yun, Kyuson; Hibbs, Matthew A

    2014-05-01

    Embryonic stem cells (ESCs), characterized by their ability to both self-renew and differentiate into multiple cell lineages, are a powerful model for biomedical research and developmental biology. Human and mouse ESCs share many features, yet have distinctive aspects, including fundamental differences in the signaling pathways and cell cycle controls that support self-renewal. Here, we explore the molecular basis of human ESC self-renewal using Bayesian network machine learning to integrate cell-type-specific, high-throughput data for gene function discovery. We integrated high-throughput ESC data from 83 human studies (~1.8 million data points collected under 1,100 conditions) and 62 mouse studies (~2.4 million data points collected under 1,085 conditions) into separate human and mouse predictive networks focused on ESC self-renewal to analyze shared and distinct functional relationships among protein-coding gene orthologs. Computational evaluations show that these networks are highly accurate, literature validation confirms their biological relevance, and reverse transcriptase polymerase chain reaction (RT-PCR) validation supports our predictions. Our results reflect the importance of key regulatory genes known to be strongly associated with self-renewal and pluripotency in both species (e.g., POU5F1, SOX2, and NANOG), identify metabolic differences between species (e.g., threonine metabolism), clarify differences between human and mouse ESC developmental signaling pathways (e.g., leukemia inhibitory factor (LIF)-activated JAK/STAT in mouse; NODAL/ACTIVIN-A-activated fibroblast growth factor in human), and reveal many novel genes and pathways predicted to be functionally associated with self-renewal in each species. These interactive networks are available online at www.StemSight.org for stem cell researchers to develop new hypotheses, discover potential mechanisms involving sparsely annotated genes, and prioritize genes of interest for experimental validation

  5. A Smad3 transgenic reporter reveals TGF-beta control of zebrafish spinal cord development.

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    Casari, Alessandro; Schiavone, Marco; Facchinello, Nicola; Vettori, Andrea; Meyer, Dirk; Tiso, Natascia; Moro, Enrico; Argenton, Francesco

    2014-12-01

    TGF-beta (TGFβ) family mediated Smad signaling is involved in mesoderm and endoderm specifications, left-right asymmetry formation and neural tube development. The TGFβ1/2/3 and Activin/Nodal signal transduction cascades culminate with activation of SMAD2 and/or SMAD3 transcription factors and their overactivation are involved in different pathologies with an inflammatory and/or uncontrolled cell proliferation basis, such as cancer and fibrosis. We have developed a transgenic zebrafish reporter line responsive to Smad3 activity. Through chemical, genetic and molecular approaches we have seen that this transgenic line consistently reproduces in vivo Smad3-mediated TGFβ signaling. Reporter fluorescence is activated in phospho-Smad3 positive cells and is responsive to both Smad3 isoforms, Smad3a and 3b. Moreover, Alk4 and Alk5 inhibitors strongly repress the reporter activity. In the CNS, Smad3 reporter activity is particularly high in the subpallium, tegumentum, cerebellar plate, medulla oblongata and the retina proliferative zone. In the spinal cord, the reporter is activated at the ventricular zone, where neuronal progenitor cells are located. Colocalization methods show in vivo that TGFβ signaling is particularly active in neuroD+ precursors. Using neuronal transgenic lines, we observed that TGFβ chemical inhibition leads to a decrease of differentiating cells and an increase of proliferation. Similarly, smad3a and 3b knock-down alter neural differentiation showing that both paralogues play a positive role in neural differentiation. EdU proliferation assay and pH3 staining confirmed that Smad3 is mainly active in post-mitotic, non-proliferating cells. In summary, we demonstrate that the Smad3 reporter line allows us to follow in vivo Smad3 transcriptional activity and that Smad3, by controlling neural differentiation, promotes the progenitor to precursor switch allowing neural progenitors to exit cell cycle and differentiate.

  6. Defining the Earliest Transcriptional Steps of Chondrogenic Progenitor Specification during the Formation of the Digits in the Embryonic Limb

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    Diaz-Mendoza, Manuel J.; Garcia-Porrero, Juan A.; Hurle, Juan M.

    2011-01-01

    The characterization of genes involved in the formation of cartilage is of key importance to improve cell-based cartilage regenerative therapies. Here, we have developed a suitable experimental model to identify precocious chondrogenic events in vivo by inducing an ectopic digit in the developing embryo. In this model, only 12 hr after the implantation of a Tgfβ bead, in the absence of increased cell proliferation, cartilage forms in undifferentiated interdigital mesoderm and in the course of development, becomes a structurally and morphologically normal digit. Systematic quantitative PCR expression analysis, together with other experimental approaches allowed us to establish 3 successive periods preceding the formation of cartilage. The “pre-condensation stage”, occurring within the first 3 hr of treatment, is characterized by the activation of connective tissue identity transcriptional factors (such as Sox9 and Scleraxis) and secreted factors (such as Activin A and the matricellular proteins CCN-1 and CCN-2) and the downregulation of the galectin CG-8. Next, the “condensation stage” is characterized by intense activation of Smad 1/5/8 BMP-signaling and increased expression of extracellular matrix components. During this period, the CCN matricellular proteins promote the expression of extracellular matrix and cell adhesion components. The third period, designated the “pre-cartilage period”, precedes the formation of molecularly identifiable cartilage by 2–3 hr and is characterized by the intensification of Sox 9 gene expression, along with the stimulation of other pro-chondrogenic transcription factors, such as HifIa. In summary, this work establishes a temporal hierarchy in the regulation of pro-chondrogenic genes preceding cartilage differentiation and provides new insights into the relative roles of secreted factors and cytoskeletal regulators that direct the first steps of this process in vivo. PMID:21931747

  7. Defining the earliest transcriptional steps of chondrogenic progenitor specification during the formation of the digits in the embryonic limb.

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    Carlos I Lorda-Diez

    Full Text Available The characterization of genes involved in the formation of cartilage is of key importance to improve cell-based cartilage regenerative therapies. Here, we have developed a suitable experimental model to identify precocious chondrogenic events in vivo by inducing an ectopic digit in the developing embryo. In this model, only 12 hr after the implantation of a Tgfβ bead, in the absence of increased cell proliferation, cartilage forms in undifferentiated interdigital mesoderm and in the course of development, becomes a structurally and morphologically normal digit. Systematic quantitative PCR expression analysis, together with other experimental approaches allowed us to establish 3 successive periods preceding the formation of cartilage. The "pre-condensation stage", occurring within the first 3 hr of treatment, is characterized by the activation of connective tissue identity transcriptional factors (such as Sox9 and Scleraxis and secreted factors (such as Activin A and the matricellular proteins CCN-1 and CCN-2 and the downregulation of the galectin CG-8. Next, the "condensation stage" is characterized by intense activation of Smad 1/5/8 BMP-signaling and increased expression of extracellular matrix components. During this period, the CCN matricellular proteins promote the expression of extracellular matrix and cell adhesion components. The third period, designated the "pre-cartilage period", precedes the formation of molecularly identifiable cartilage by 2-3 hr and is characterized by the intensification of Sox 9 gene expression, along with the stimulation of other pro-chondrogenic transcription factors, such as HifIa. In summary, this work establishes a temporal hierarchy in the regulation of pro-chondrogenic genes preceding cartilage differentiation and provides new insights into the relative roles of secreted factors and cytoskeletal regulators that direct the first steps of this process in vivo.

  8. Myostatin inhibition in muscle, but not adipose tissue, decreases fat mass and improves insulin sensitivity.

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    Tingqing Guo

    Full Text Available Myostatin (Mstn is a secreted growth factor expressed in skeletal muscle and adipose tissue that negatively regulates skeletal muscle mass. Mstn(-/- mice have a dramatic increase in muscle mass, reduction in fat mass, and resistance to diet-induced and genetic obesity. To determine how Mstn deletion causes reduced adiposity and resistance to obesity, we analyzed substrate utilization and insulin sensitivity in Mstn(-/- mice fed a standard chow. Despite reduced lipid oxidation in skeletal muscle, Mstn(-/- mice had no change in the rate of whole body lipid oxidation. In contrast, Mstn(-/- mice had increased glucose utilization and insulin sensitivity as measured by indirect calorimetry, glucose and insulin tolerance tests, and hyperinsulinemic-euglycemic clamp. To determine whether these metabolic effects were due primarily to the loss of myostatin signaling in muscle or adipose tissue, we compared two transgenic mouse lines carrying a dominant negative activin IIB receptor expressed specifically in adipocytes or skeletal muscle. We found that inhibition of myostatin signaling in adipose tissue had no effect on body composition, weight gain, or glucose and insulin tolerance in mice fed a standard diet or a high-fat diet. In contrast, inhibition of myostatin signaling in skeletal muscle, like Mstn deletion, resulted in increased lean mass, decreased fat mass, improved glucose metabolism on standard and high-fat diets, and resistance to diet-induced obesity. Our results demonstrate that Mstn(-/- mice have an increase in insulin sensitivity and glucose uptake, and that the reduction in adipose tissue mass in Mstn(-/- mice is an indirect result of metabolic changes in skeletal muscle. These data suggest that increasing muscle mass by administration of myostatin antagonists may be a promising therapeutic target for treating patients with obesity or diabetes.

  9. Simple suspension culture system of human iPS cells maintaining their pluripotency for cardiac cell sheet engineering.

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    Haraguchi, Yuji; Matsuura, Katsuhisa; Shimizu, Tatsuya; Yamato, Masayuki; Okano, Teruo

    2015-12-01

    In this study, a simple three-dimensional (3D) suspension culture method for the expansion and cardiac differentiation of human induced pluripotent stem cells (hiPSCs) is reported. The culture methods were easily adapted from two-dimensional (2D) to 3D culture without any additional manipulations. When hiPSCs were directly applied to 3D culture from 2D in a single-cell suspension, only a few aggregated cells were observed. However, after 3 days, culture of the small hiPSC aggregates in a spinner flask at the optimal agitation rate created aggregates which were capable of cell passages from the single-cell suspension. Cell numbers increased to approximately 10-fold after 12 days of culture. The undifferentiated state of expanded hiPSCs was confirmed by flow cytometry, immunocytochemistry and quantitative RT-PCR, and the hiPSCs differentiated into three germ layers. When the hiPSCs were subsequently cultured in a flask using cardiac differentiation medium, expression of cardiac cell-specific genes and beating cardiomyocytes were observed. Furthermore, the culture of hiPSCs on Matrigel-coated dishes with serum-free medium containing activin A, BMP4 and FGF-2 enabled it to generate robust spontaneous beating cardiomyocytes and these cells expressed several cardiac cell-related genes, including HCN4, MLC-2a and MLC-2v. This suggests that the expanded hiPSCs might maintain the potential to differentiate into several types of cardiomyocytes, including pacemakers. Moreover, when cardiac cell sheets were fabricated using differentiated cardiomyocytes, they beat spontaneously and synchronously, indicating electrically communicative tissue. This simple culture system might enable the generation of sufficient amounts of beating cardiomyocytes for use in cardiac regenerative medicine and tissue engineering.

  10. Variation in Telangiectasia Predisposing Genes Is Associated With Overall Radiation Toxicity

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    Tanteles, George A. [Department of Genetics, University of Leicester, Leicester (United Kingdom); Department of Cancer Studies and Molecular Medicine, University Hospitals of Leicester, Leicester Royal Infirmary, Leicester (United Kingdom); Murray, Robert J.S. [Department of Genetics, University of Leicester, Leicester (United Kingdom); Mills, Jamie [Department of Cancer Studies and Molecular Medicine, University Hospitals of Leicester, Leicester Royal Infirmary, Leicester (United Kingdom); Barwell, Julian [Department of Genetics, University of Leicester, Leicester (United Kingdom); Department of Cancer Studies and Molecular Medicine, University Hospitals of Leicester, Leicester Royal Infirmary, Leicester (United Kingdom); Chakraborti, Prabir [Department of Clinical Oncology, Derby Hospitals NHS Foundation Trust, Derby (United Kingdom); Chan, Steve [Department of Clinical Oncology, Nottingham University Hospitals NHS Trust, Nottingham (United Kingdom); Cheung, Kwok-Leung [Division of Breast Surgery, University of Nottingham, Nottingham (United Kingdom); Ennis, Dawn [Department of Clinical Oncology, Derby Hospitals NHS Foundation Trust, Derby (United Kingdom); Khurshid, Nazish [Department of Genetics, University of Leicester, Leicester (United Kingdom); Lambert, Kelly [Department of Breast Surgery, University Hospitals of Leicester, Glenfield Hospital, Leicester (United Kingdom); Machhar, Rohan; Meisuria, Mitul [Department of Genetics, University of Leicester, Leicester (United Kingdom); Osman, Ahmed; Peat, Irene [Department of Cancer Studies and Molecular Medicine, University Hospitals of Leicester, Leicester Royal Infirmary, Leicester (United Kingdom); Sahota, Harjinder [Department of Genetics, University of Leicester, Leicester (United Kingdom); Woodings, Pamela [Department of Clinical Oncology, Derby Hospitals NHS Foundation Trust, Derby (United Kingdom); Talbot, Christopher J., E-mail: cjt14@le.ac.uk [Department of Genetics, University of Leicester, Leicester (United Kingdom); and others

    2012-11-15

    Purpose: In patients receiving radiotherapy for breast cancer where the heart is within the radiation field, cutaneous telangiectasiae could be a marker of potential radiation-induced heart disease. We hypothesized that single nucleotide polymorphisms (SNPs) in genes known to cause heritable telangiectasia-associated disorders could predispose to such late, normal tissue vascular damage. Methods and Materials: The relationship between cutaneous telangiectasia as a late normal tissue radiation injury phenotype in 633 breast cancer patients treated with radiotherapy was examined. Patients were clinically assessed for the presence of cutaneous telangiectasia and genotyped at nine SNPs in three candidate genes. Candidate SNPs were within the endoglin (ENG) and activin A receptor, type II-like 1 (ACVRL1) genes, mutations in which cause hereditary hemorrhagic telangiectasia and the ataxia-telangiectasia mutated (ATM) gene associated with ataxia-telangiectasia. Results: A total of 121 (19.1%) patients exhibited a degree of cutaneous telangiectasiae on clinical examination. Regression was used to examine the associations between the presence of telangiectasiae in patients who underwent breast-conserving surgery, controlling for the effects of boost and known brassiere size (n=388), and individual geno- or haplotypes. Inheritance of ACVRL1 SNPs marginally contributed to the risk of cutaneous telangiectasiae. Haplotypic analysis revealed a stronger association between inheritance of a ATM haplotype and the presence of cutaneous telangiectasiae, fibrosis and overall toxicity. No significant association was observed between telangiectasiae and the coinheritance of the candidate ENG SNPs. Conclusions: Genetic variation in the ATM gene influences reaction to radiotherapy through both vascular damage and increased fibrosis. The predisposing variation in the ATM gene will need to be better defined to optimize it as a predictive marker for assessing radiotherapy late effects.

  11. Thalassemia 2016: Modern medicine battles an ancient disease.

    Science.gov (United States)

    Rund, Deborah

    2016-01-01

    Thalassemia was first clinically described nearly a century ago and treatment of this widespread genetic disease has greatly advanced during this period. DNA-based diagnosis elucidated the molecular basis of the disease and clarified the variable clinical picture. It also paved the way for modern methods of carrier identification and prevention via DNA-based prenatal diagnosis. Every aspect of supportive care, including safer blood supply, more regular transfusions, specific monitoring of iron overload, parenteral and oral chelation, and other therapies, has prolonged life and improved the quality of life of these patients. Significant advances have also been made in allogenic bone marrow transplantation, the only curative therapy. Recently, there has been a rejuvenated interest in studying thalassemia at the basic science level, leading to the discovery of previously unknown mechanisms leading to anemia and enabling the development of novel therapies. These will potentially improve the treatment of, and possibly cure the disease. Pathways involving activin receptors, heat shock proteins, JAK2 inhibitors and macrophage targeted therapy, among others, are being studied or are currently in clinical trials for treating thalassemia. Novel types of genetic therapies are in use or under investigation. In addition to the challenges of treating each individual patient, the longer survival of thalassemia patients has raised considerations regarding worldwide control of thalassemia, since prevention is not universally implemented. This review will trace a number of the original medical milestones of thalassemia diagnosis and treatment, as well as some of the most recent developments which may lead to innovative therapeutic modalities. © 2015 Wiley Periodicals, Inc.

  12. Cloning of a novel signaling molecule, AMSH-2, that potentiates transforming growth factor β signaling

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    Pandey Akhilesh

    2004-01-01

    Full Text Available Abstract Background Transforming growth factor-βs (TGF-βs, bone morphogenetic proteins (BMPs and activins are important regulators of developmental cell growth and differentiation. Signaling by these factors is mediated chiefly by the Smad family of latent transcription factors. Results There are a large number of uncharacterized cDNA clones that code for novel proteins with homology to known signaling molecules. We have identified a novel molecule from the HUGE database that is related to a previously known molecule, AMSH (associated molecule with the SH3 domain of STAM, an adapter shown to be involved in BMP signaling. Both of these molecules contain a coiled-coil domain located within the amino-terminus region and a JAB (Domain in Jun kinase activation domain binding protein and proteasomal subunits domain at the carboxy-terminus. We show that this novel molecule, which we have designated AMSH-2, is widely expressed and its overexpression potentiates activation of TGF-β-dependent promoters. Coimmunoprecipitation studies indicated that Smad7 and Smad2, but not Smad3 or 4, interact with AMSH-2. We show that overexpression of AMSH-2 decreases the inhibitory effect of Smad7 on TGF-β signaling. Finally, we demonstrate that knocking down AMSH-2 expression by RNA interference decreases the activation of 3TP-lux reporter in response to TGF-β. Conclusions This report implicates AMSH and AMSH-2 as a novel family of molecules that positively regulate the TGF-β signaling pathway. Our results suggest that this effect could be partially explained by AMSH-2 mediated decrease of the action of Smad7 on TGF-β signaling pathway.

  13. Supplementation of Magnolol Attenuates Skeletal Muscle Atrophy in Bladder Cancer-Bearing Mice Undergoing Chemotherapy via Suppression of FoxO3 Activation and Induction of IGF-1.

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    Meng-Chuan Chen

    Full Text Available Skeletal muscle atrophy, the most prominent phenotypic feature of cancer cachexia, is often observed in cancer patients undergoing chemotherapy. Magnolol (M extracted from Magnolia officinalis exhibits several pharmacological effects including anti-inflammatory and anticancer activities. In this study, we investigated whether magnolol supplementation protects against the development of cachexia symptoms in bladder cancer-bearing mice undergoing chemotherapy. Combined treatment of magnolol with chemotherapeutic drugs, such as gemcitabine and cisplatin (TGCM or gemcitabine (TGM, markedly attenuates the body weight loss and skeletal muscle atrophy compared with conventional chemotherapy (TGC. The antiatrophic effect of magnolol may be associated with inhibition of myostatin and activin A formation, as well as FoxO3 transcriptional activity resulting from Akt activation, thereby suppressing ubiquitin ligases MuRF-1 and MAFbx/atrogin-1 expression, as well as proteasomal enzyme activity. Notably, magnolol-induced insulin-like growth factor 1 (IGF-1 production and related protein synthesis may also contribute to its protective effects. The decreased food intake, and intestinal injury and dysfunction observed in the mice of TGC group were significantly improved in the TGCM and TGM groups. Moreover, the increased inflammatory responses evidenced by elevation of proinflammatory cytokine formation and NF-κB activation occurred in the atrophying muscle of TGC group were markedly inhibited in mice of combined treatment with magnolol. In summary, these findings support that magnolol is a promising chemopreventive supplement for preventing chemotherapy-induced skeletal muscle atrophy associated with cancer cachexia by suppressing muscle protein degradation, and inflammatory responses, as well as increasing IGF-1-mediated protein synthesis.

  14. Acute inhibition of myostatin-family proteins preserves skeletal muscle in mouse models of cancer cachexia

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    Benny Klimek, Margaret E.; Aydogdu, Tufan [Department of Cell Biology and Anatomy, University of Miami Miller School of Medicine, Miami, FL (United States); Link, Majik J.; Pons, Marianne [Molecular Oncology Program, Division of Surgical Oncology, DeWitt Daughtry Family Department of Surgery, University of Miami Miller School of Medicine, Miami, FL (United States); Koniaris, Leonidas G. [Department of Cell Biology and Anatomy, University of Miami Miller School of Medicine, Miami, FL (United States); Molecular Oncology Program, Division of Surgical Oncology, DeWitt Daughtry Family Department of Surgery, University of Miami Miller School of Medicine, Miami, FL (United States); Molecular Oncology and Experimental Therapeutics Program, Sylvester Comprehensive Cancer Center, University of Miami Miller School of Medicine, Miami, FL (United States); Zimmers, Teresa A., E-mail: tzimmers@med.miami.edu [Department of Cell Biology and Anatomy, University of Miami Miller School of Medicine, Miami, FL (United States); Molecular Oncology Program, Division of Surgical Oncology, DeWitt Daughtry Family Department of Surgery, University of Miami Miller School of Medicine, Miami, FL (United States); Molecular Oncology and Experimental Therapeutics Program, Sylvester Comprehensive Cancer Center, University of Miami Miller School of Medicine, Miami, FL (United States)

    2010-01-15

    Cachexia, progressive loss of fat and muscle mass despite adequate nutrition, is a devastating complication of cancer associated with poor quality of life and increased mortality. Myostatin is a potent tonic muscle growth inhibitor. We tested how myostatin inhibition might influence cancer cachexia using genetic and pharmacological approaches. First, hypermuscular myostatin null mice were injected with Lewis lung carcinoma or B16F10 melanoma cells. Myostatin null mice were more sensitive to tumor-induced cachexia, losing more absolute mass and proportionately more muscle mass than wild-type mice. Because myostatin null mice lack expression from development, however, we also sought to manipulate myostatin acutely. The histone deacetylase inhibitor Trichostatin A has been shown to increase muscle mass in normal and dystrophic mice by inducing the myostatin inhibitor, follistatin. Although Trichostatin A administration induced muscle growth in normal mice, it failed to preserve muscle in colon-26 cancer cachexia. Finally we sought to inhibit myostatin and related ligands by administration of the Activin receptor extracellular domain/Fc fusion protein, ACVR2B-Fc. Systemic administration of ACVR2B-Fc potently inhibited muscle wasting and protected adipose stores in both colon-26 and Lewis lung carcinoma cachexia, without affecting tumor growth. Enhanced cachexia in myostatin knockouts indicates that host-derived myostatin is not the sole mediator of muscle wasting in cancer. More importantly, skeletal muscle preservation with ACVR2B-Fc establishes that targeting myostatin-family ligands using ACVR2B-Fc or related molecules is an important and potent therapeutic avenue in cancer cachexia.

  15. Directed differentiation of human pluripotent stem cells into intestinal tissue in vitro.

    Science.gov (United States)

    Spence, Jason R; Mayhew, Christopher N; Rankin, Scott A; Kuhar, Matthew F; Vallance, Jefferson E; Tolle, Kathryn; Hoskins, Elizabeth E; Kalinichenko, Vladimir V; Wells, Susanne I; Zorn, Aaron M; Shroyer, Noah F; Wells, James M

    2011-02-03

    Studies in embryonic development have guided successful efforts to direct the differentiation of human embryonic and induced pluripotent stem cells (PSCs) into specific organ cell types in vitro. For example, human PSCs have been differentiated into monolayer cultures of liver hepatocytes and pancreatic endocrine cells that have therapeutic efficacy in animal models of liver disease and diabetes, respectively. However, the generation of complex three-dimensional organ tissues in vitro remains a major challenge for translational studies. Here we establish a robust and efficient process to direct the differentiation of human PSCs into intestinal tissue in vitro using a temporal series of growth factor manipulations to mimic embryonic intestinal development. This involved activin-induced definitive endoderm formation, FGF/Wnt-induced posterior endoderm pattering, hindgut specification and morphogenesis, and a pro-intestinal culture system to promote intestinal growth, morphogenesis and cytodifferentiation. The resulting three-dimensional intestinal 'organoids' consisted of a polarized, columnar epithelium that was patterned into villus-like structures and crypt-like proliferative zones that expressed intestinal stem cell markers. The epithelium contained functional enterocytes, as well as goblet, Paneth and enteroendocrine cells. Using this culture system as a model to study human intestinal development, we identified that the combined activity of WNT3A and FGF4 is required for hindgut specification whereas FGF4 alone is sufficient to promote hindgut morphogenesis. Our data indicate that human intestinal stem cells form de novo during development. We also determined that NEUROG3, a pro-endocrine transcription factor that is mutated in enteric anendocrinosis, is both necessary and sufficient for human enteroendocrine cell development in vitro. PSC-derived human intestinal tissue should allow for unprecedented studies of human intestinal development and disease.

  16. Directed Differentiation of Human Embryonic Stem Cells into Prostate Organoids In Vitro and its Perturbation by Low-Dose Bisphenol A Exposure.

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    Esther L Calderon-Gierszal

    Full Text Available Studies using rodent and adult human prostate stem-progenitor cell models suggest that developmental exposure to the endocrine disruptor Bisphenol-A (BPA can predispose to prostate carcinogenesis with aging. Unknown at present is whether the embryonic human prostate is equally susceptible to BPA during its natural developmental window. To address this unmet need, we herein report the construction of a pioneer in vitro human prostate developmental model to study the effects of BPA. The directed differentiation of human embryonic stem cells (hESC into prostatic organoids in a spatial system was accomplished with precise temporal control of growth factors and steroids. Activin-induced definitive endoderm was driven to prostate specification by combined exposure to WNT10B and FGF10. Matrigel culture for 20-30 days in medium containing R-Spondin-1, Noggin, EGF, retinoic acid and testosterone was sufficient for mature prostate organoid development. Immunofluorescence and gene expression analysis confirmed that organoids exhibited cytodifferentiation and functional properties of the human prostate. Exposure to 1 nM or 10 nM BPA throughout differentiation culture disturbed early morphogenesis in a dose-dependent manner with 1 nM BPA increasing and 10 nM BPA reducing the number of branched structures formed. While differentiation of branched structures to mature organoids seemed largely unaffected by BPA exposure, the stem-like cell population increased, appearing as focal stem cell nests that have not properly entered lineage commitment rather than the rare isolated stem cells found in normally differentiated structures. These findings provide the first direct evidence that low-dose BPA exposure targets hESC and perturbs morphogenesis as the embryonic cells differentiate towards human prostate organoids, suggesting that the developing human prostate may be susceptible to disruption by in utero BPA exposures.

  17. Induction of Skin-Derived Precursor Cells from Human Induced Pluripotent Stem Cells

    Science.gov (United States)

    Sugiyama-Nakagiri, Yoriko; Fujimura, Tsutomu; Moriwaki, Shigeru

    2016-01-01

    The generation of full thickness human skin from dissociated cells is an attractive approach not only for treating skin diseases, but also for treating many systemic disorders. However, it is currently not possible to obtain an unlimited number of skin dermal cells. The goal of this study was to develop a procedure to produce skin dermal stem cells from induced pluripotent stem cells (iPSCs). Skin-derived precursor cells (SKPs) were isolated as adult dermal precursors that could differentiate into both neural and mesodermal progenies and could reconstitute the dermis. Thus, we attempted to generate SKPs from iPSCs that could reconstitute the skin dermis. Human iPSCs were initially cultured with recombinant noggin and SB431542, an inhibitor of activin/nodal and TGFβ signaling, to induce neural crest progenitor cells. Those cells were then treated with SKP medium that included CHIR99021, a WNT signal activator. The induction efficacy from neural crest progenitor cells to SKPs was more than 97%. No other modifiers tested were able to induce those cells. Those human iPSC-derived SKPs (hiPSC-SKPs) showed a similar gene expression signature to SKPs isolated from human skin dermis. Human iPSC-SKPs differentiated into neural and mesodermal progenies, including adipocytes, skeletogenic cell types and Schwann cells. Moreover, they could be induced to follicular type keratinization when co-cultured with human epidermal keratinocytes. We here provide a new efficient protocol to create human skin dermal stem cells from hiPSCs that could contribute to the treatment of various skin disorders. PMID:27992514

  18. The kielin/chordin-like protein (KCP) attenuates high-fat diet-induced obesity and metabolic syndrome in mice.

    Science.gov (United States)

    Soofi, Abdul; Wolf, Katherine I; Emont, Margo P; Qi, Nathan; Martinez-Santibanez, Gabriel; Grimley, Edward; Ostwani, Wesam; Dressler, Gregory R

    2017-06-02

    Obesity and its associated complications such as insulin resistance and non-alcoholic fatty liver disease are reaching epidemic proportions. In mice, the TGF-β superfamily is implicated in the regulation of white and brown adipose tissue differentiation. The kielin/chordin-like protein (KCP) is a secreted regulator of the TGF-β superfamily pathways that can inhibit both TGF-β and activin signals while enhancing bone morphogenetic protein (BMP) signaling. However, KCP's effects on metabolism and obesity have not been studied in animal models. Therefore, we examined the effects of KCP loss or gain of function in mice that were maintained on either a regular or a high-fat diet. KCP loss sensitized the mice to obesity and associated complications such as glucose intolerance and adipose tissue inflammation and fibrosis. In contrast, transgenic mice that expressed KCP in the kidney, liver, and adipose tissues were resistant to developing high-fat diet-induced obesity and had significantly reduced white adipose tissue. Moreover, KCP overexpression shifted the pattern of SMAD signaling in vivo, increasing the levels of phospho (P)-SMAD1 and decreasing P-SMAD3. Adipocytes in culture showed a cell-autonomous effect in response to added TGF-β1 or BMP7. Metabolic profiling indicated increased energy expenditure in KCP-overexpressing mice and reduced expenditure in the KCP mutants with no effect on food intake or activity. These findings demonstrate that shifting the TGF-β superfamily signaling with a secreted protein can alter the physiology and thermogenic properties of adipose tissue to reduce obesity even when mice are fed a high-fat diet. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  19. Stable expression and characterization of N-terminal tagged recombinant human bone morphogenetic protein 15

    Science.gov (United States)

    Li, Qinglei; Rajanahally, Saneal; Edson, Mark A.; Matzuk, Martin M.

    2009-01-01

    Oocyte-derived growth factors are critically involved in multiple ovarian processes via paracrine actions. Although recombinant proteins have been applied to dissect the physiological functions of these factors, variation of activities among different protein preparations remains an issue. To further elucidate the roles of one of these growth factors, bone morphogenetic protein 15 (BMP15), in mediating oocyte-regulated molecular and cellular events and to explore its potential clinical application, we engineered the human BMP15 sequence to efficiently produce bioactive recombinant human BMP15 (rhBMP15). The proteolytic cleavage site of the hBMP15 precursor was optimized to facilitate the production of the mature protein, and a FLAG-tag was placed at the N-terminus of the mature region to ease purification and avoid potential interference of the tag with the cystine knot structure. The rhBMP15 protein was purified using anti-FLAG M2 affinity gel. Our results demonstrated that the N-terminal tagged rhBMP15 was efficiently processed in HEK-293 cells. Furthermore, the purified rhBMP15 could activate SMAD1/5/8 and induce the transcription of genes encoding cumulus expansion-related transcripts (Ptx3, Has2, Tnfaip6 and Ptgs2), inhibitory SMADs (Smad6 and Smad7), BMP antagonists (Grem1 and Fst), activin/inhibin βA (Inhba) and βB (Inhbb) subunits, etc. Thus, our rhBMP15 containing a genetically modified cleavage sequence and an N-terminal FLAG-tag can be efficiently produced, processed and secreted in a mammalian expression system. The purified rhBMP15 is also biologically active and very stable, and can induce the expression of a variety of mouse granulosa cell genes. PMID:19651638

  20. Transforming growth factor β receptor type 1 is essential for female reproductive tract integrity and function.

    Directory of Open Access Journals (Sweden)

    Qinglei Li

    2011-10-01

    Full Text Available The transforming growth factor β (TGFβ superfamily proteins are principle regulators of numerous biological functions. Although recent studies have gained tremendous insights into this growth factor family in female reproduction, the functions of the receptors in vivo remain poorly defined. TGFβ type 1 receptor (TGFBR1, also known as activin receptor-like kinase 5, is the major type 1 receptor for TGFβ ligands. Tgfbr1 null mice die embryonically, precluding functional characterization of TGFBR1 postnatally. To study TGFBR1-mediated signaling in female reproduction, we generated a mouse model with conditional knockout (cKO of Tgfbr1 in the female reproductive tract using anti-Müllerian hormone receptor type 2 promoter-driven Cre recombinase. We found that Tgfbr1 cKO females are sterile. However, unlike its role in growth differentiation factor 9 (GDF9 signaling in vitro, TGFBR1 seems to be dispensable for GDF9 signaling in vivo. Strikingly, we discovered that the Tgfbr1 cKO females develop oviductal diverticula, which impair embryo development and transit of embryos to the uterus. Molecular analysis further demonstrated the dysregulation of several cell differentiation and migration genes (e.g., Krt12, Ace2, and MyoR that are potentially associated with female reproductive tract development. Moreover, defective smooth muscle development was also revealed in the uteri of the Tgfbr1 cKO mice. Thus, TGFBR1 is required for female reproductive tract integrity and function, and disruption of TGFBR1-mediated signaling leads to catastrophic structural and functional consequences in the oviduct and uterus.

  1. Transforming Growth Factor β Receptor Type 1 Is Essential for Female Reproductive Tract Integrity and Function

    Science.gov (United States)

    Li, Qinglei; Agno, Julio E.; Edson, Mark A.; Nagaraja, Ankur K.; Nagashima, Takashi; Matzuk, Martin M.

    2011-01-01

    The transforming growth factor β (TGFβ) superfamily proteins are principle regulators of numerous biological functions. Although recent studies have gained tremendous insights into this growth factor family in female reproduction, the functions of the receptors in vivo remain poorly defined. TGFβ type 1 receptor (TGFBR1), also known as activin receptor-like kinase 5, is the major type 1 receptor for TGFβ ligands. Tgfbr1 null mice die embryonically, precluding functional characterization of TGFBR1 postnatally. To study TGFBR1–mediated signaling in female reproduction, we generated a mouse model with conditional knockout (cKO) of Tgfbr1 in the female reproductive tract using anti-Müllerian hormone receptor type 2 promoter-driven Cre recombinase. We found that Tgfbr1 cKO females are sterile. However, unlike its role in growth differentiation factor 9 (GDF9) signaling in vitro, TGFBR1 seems to be dispensable for GDF9 signaling in vivo. Strikingly, we discovered that the Tgfbr1 cKO females develop oviductal diverticula, which impair embryo development and transit of embryos to the uterus. Molecular analysis further demonstrated the dysregulation of several cell differentiation and migration genes (e.g., Krt12, Ace2, and MyoR) that are potentially associated with female reproductive tract development. Moreover, defective smooth muscle development was also revealed in the uteri of the Tgfbr1 cKO mice. Thus, TGFBR1 is required for female reproductive tract integrity and function, and disruption of TGFBR1–mediated signaling leads to catastrophic structural and functional consequences in the oviduct and uterus. PMID:22028666

  2. Sex-related differences in gene expression in human skeletal muscle.

    Directory of Open Access Journals (Sweden)

    Stephen Welle

    Full Text Available There is sexual dimorphism of skeletal muscle, the most obvious feature being the larger muscle mass of men. The molecular basis for this difference has not been clearly defined. To identify genes that might contribute to the relatively greater muscularity of men, we compared skeletal muscle gene expression profiles of 15 normal men and 15 normal women by using comprehensive oligonucleotide microarrays. Although there were sex-related differences in expression of several hundred genes, very few of the differentially expressed genes have functions that are obvious candidates for explaining the larger muscle mass of men. The men tended to have higher expression of genes encoding mitochondrial proteins, ribosomal proteins, and a few translation initiation factors. The women had >2-fold greater expression than the men (P<0.0001 of two genes that encode proteins in growth factor pathways known to be important in regulating muscle mass: growth factor receptor-bound 10 (GRB10 and activin A receptor IIB (ACVR2B. GRB10 encodes a protein that inhibits insulin-like growth factor-1 (IGF-1 signaling. ACVR2B encodes a myostatin receptor. Quantitative RT-PCR confirmed higher expression of GRB10 and ACVR2B genes in these women. In an independent microarray study of 10 men and 9 women with facioscapulohumeral dystrophy, women had higher expression of GRB10 (2.7-fold, P<0.001 and ACVR2B (1.7-fold, P<0.03. If these sex-related differences in mRNA expression lead to reduced IGF-1 activity and increased myostatin activity, they could contribute to the sex difference in muscle size.

  3. Genetics and genomics of pulmonary arterial hypertension.

    Science.gov (United States)

    Soubrier, Florent; Chung, Wendy K; Machado, Rajiv; Grünig, Ekkehard; Aldred, Micheala; Geraci, Mark; Loyd, James E; Elliott, C Gregory; Trembath, Richard C; Newman, John H; Humbert, Marc

    2013-12-24

    Major discoveries have been obtained within the last decade in the field of hereditary predisposition to pulmonary arterial hypertension (PAH). Among them, the identification of bone morphogenetic protein receptor type 2 (BMPR2) as the major predisposing gene and activin A receptor type II-like kinase-1 (ACVRL1, also known as ALK1) as the major gene when PAH is associated with hereditary hemorrhagic telangiectasia. The mutation detection rate for the known genes is approximately 75% in familial PAH, but the mutation shortfall remains unexplained even after careful molecular investigation of these genes. To identify additional genetic variants predisposing to PAH, investigators harnessed the power of next-generation sequencing to successfully identify additional genes that will be described in this report. Furthermore, common genetic predisposing factors for PAH can be identified by genome-wide association studies and are detailed in this paper. The careful study of families and routine genetic diagnosis facilitated natural history studies based on large registries of PAH patients to be set up in different countries. These longitudinal or cross-sectional studies permitted the clinical characterization of PAH in mutation carriers to be accurately described. The availability of molecular genetic diagnosis has opened up a new field for patient care, including genetic counseling for a severe disease, taking into account that the major predisposing gene has a highly variable penetrance between families. Molecular information can be drawn from the genomic study of affected tissues in PAH, in particular, pulmonary vascular tissues and cells, to gain insight into the mechanisms leading to the development of the disease. High-throughput genomic techniques, on the basis of next-generation sequencing, now allow the accurate quantification and analysis of ribonucleic acid, species, including micro-ribonucleic acids, and allow for a genome-wide investigation of epigenetic or

  4. A novel ENG mutation causing impaired co-translational processing of endoglin associated with hereditary hemorrhagic telangiectasia.

    Science.gov (United States)

    Suzuki, Atsuo; Nakashima, Daisuke; Miyawaki, Yuhri; Fujita, Junko; Maki, Asuka; Fujimori, Yuta; Takagi, Akira; Murate, Takashi; Teranishi, Masaaki; Matsushita, Tadashi; Saito, Hidehiko; Kojima, Tetsuhito

    2012-05-01

    Hereditary hemorrhagic telangiectasia (HHT) is an inherited autosomal dominant vascular dysplasia caused by mutations in mainly the endoglin gene (ENG) or activin-like kinase receptor 1 (ALK1) gene (ACVRL1). We investigated the molecular basis of HHT in a Japanese patient, and identified a novel missense mutation in ENG (c.38T>A, p.Leu13Gln) located in the signal peptide's hydrophobic core, but not in ACVRL1. In experiments in COS-1 cells, the Leu13Gln (L13Q) mutant endoglin appeared to be expressed as a precursor form, probably due to impaired protein processing. Flow cytometry analyses of the COS-1 cells transiently expressing recombinant endoglins revealed that the wild-type endoglin was detected on the cell surface, but the L13Q mutant was not. We also analyzed expression patterns of the recombinant endoglins by immunofluorescent staining, and found that the wild-type co-localized with the endoplasmic reticulum (ER), but the L13Q mutant did not. These results implied that the L13Q mutant endoglin fails to insert into the ER, probably due to destruction of the hydrophobic core structure in the signal peptide to be recognized by signal recognition particles. Thus, the Leu13 in the signal peptide of endoglin might be essential for correct protein processing through the ER and cell-surface expression. Taken together, the novel c.38T>A mutation in ENG would impair co-translational processing of the endoglin, and could be responsible for HHT in this patient. Copyright © 2011 Elsevier Ltd. All rights reserved.

  5. Cloned, CD117 selected human amniotic fluid stem cells are capable of modulating the immune response.

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    Emily C Moorefield

    Full Text Available Amniotic fluid stem (AFS cells are broadly multipotent, can be expanded extensively in culture, are not tumorigenic and can be readily cryopreserved for cell banking. Mesenchymal stem cells (MSC show immunomodulatory activity and secrete a wide spectrum of cytokines and chemokines that suppress inflammatory responses, block mixed lymphocyte reactions (MLR and other immune reactions, and have proven therapeutic against conditions such as graft-versus-host disease. AFS cells resemble MSCs in many respects including surface marker expression and differentiation potential. We therefore hypothesized that AFS cells may exhibit similar immunomodulatory capabilities. We present data to demonstrate that direct contact with AFS cells inhibits lymphocyte activation. In addition, we show that cell-free supernatants derived from AFS cells primed with total blood monocytes or IL-1β, a cytokine released by monocytes and essential in mediation of the inflammatory response, also inhibited lymphocyte activation. Further investigation of AFS cell-free supernatants by protein array revealed secretion of multiple factors in common with MSCs that are known to be involved in immune regulation including growth related oncogene (GRO and monocyte chemotactic protein (MCP family members as well as interleukin-6 (IL-6. AFS cells activated by PBMCs released several additional cytokines as compared to BM-MSCs, including macrophage inflammatory protein-3α (MIP-3α, MIP-1α and Activin. AFS cells also released higher levels of MCP-1 and lower levels of MCP-2 compared to BM-MSCs in response to IL-1β activation. This suggests that there may be some AFS-specific mechanisms of inhibition of lymphocyte activation. Our results indicate that AFS cells are able to suppress inflammatory responses in vitro and that soluble factors are an essential component in the communication between lymphocytes and AFS cells. Their extensive self-renewal capacity, possibility for banking and

  6. Epigenetic Regulation of GDF2 Suppresses Anoikis in Ovarian and Breast Epithelia

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    Archana Varadaraj

    2015-11-01

    Full Text Available Anoikis, a cell death mechanism triggered upon cell-matrix detachment, is regarded as a physiological suppressor of metastasis that can be regulated by a diverse array of signals. The protein encoded by GDF2 is BMP9 and is a member of the bone morphogenetic protein family and the transforming growth factor (TGF β superfamily with emerging yet controversial roles in carcinogenesis. In an attempt to identify the function of growth and differentiation factor 2 (GDF2 in epithelial systems, we examined the signaling machinery that is involved and cell fate decisions in response to GDF2 in ovarian and breast epithelia. We find that GDF2 can robustly activate the SMAD1/5 signaling axis by increasing complex formation between the type I receptor serine threonine kinases activin receptor-like kinase (ALK 3 and ALK6 and the type II receptor serine threonine kinase BMPRII. This activation is independent of cross talk with the SMAD2-transforming growth factor β pathway. By activating SMAD1/5, epithelial cells regulate anchorage-independent growth by increasing anoikis sensitivity that is dependent on GDF2’s ability to sustain the activation of SMAD1/5 via ALK3 and ALK6. Consistent with a role for GDF2 in promoting anoikis susceptibility, the analysis of cell lines and patient data suggests epigenetic silencing of GDF2 in cancer cell lines and increased promoter methylation in patients. These findings collectively indicate an antimetastatic role for GDF2 in ovarian and breast cancer. The work also implicates loss of GDF2 via promoter methylation-mediated downregulation in promotion of carcinogenesis with significant relevance for the use of epigenetic drugs currently in clinical trials.

  7. Bone morphogenetic proteins: from structure to clinical use

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    Granjeiro J.M.

    2005-01-01

    Full Text Available Bone morphogenetic proteins (BMPs are multi-functional growth factors belonging to the transforming growth factor ß superfamily. Family members are expressed during limb development, endochondral ossification, early fracture, and cartilage repair. The activity of BMPs was first identified in the 1960s but the proteins responsible for bone induction were unknown until the purification and cloning of human BMPs in the 1980s. To date, about 15 BMP family members have been identified and characterized. The signal triggered by BMPs is transduced through serine/threonine kinase receptors, type I and II subtypes. Three type I receptors have been shown to bind BMP ligands, namely: type IA and IB BMP receptors and type IA activin receptors. BMPs seem to be involved in the regulation of cell proliferation, survival, differentiation and apoptosis, but their hallmark is their ability to induce bone, cartilage, ligament, and tendon formation at both heterotopic and orthotopic sites. This suggests that, in the future, they may play a major role in the treatment of bone diseases. Several animal studies have illustrated the potential of BMPs to enhance spinal fusion, repair critical-size defects, accelerate union, and heal articular cartilage lesions. Difficulties in producing and purifying BMPs from bone tissue have prompted the attempts made by several laboratories, including ours, to express these proteins in the recombinant form in heterologous systems. This review focuses on BMP structure, molecular mechanisms of action and significance and potential applications in medical, dental and veterinary practice for the treatment of cartilage and bone-related diseases.

  8. Regulators of Tfh cell differentiation

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    Gajendra Motiram Jogdand

    2016-11-01

    Full Text Available The follicular helper T (Tfh cells help is critical for activation of B cells, antibody class switching and germinal center formation. The Tfh cells are characterized by the expression of CXCR5, ICOS, PD-1, Bcl-6, and IL-21. They are involved in clearing infections and are adversely linked with autoimmune diseases and also have a role in viral replication as well as clearance. Tfh cells are generated from naïve CD4 T cells with sequential steps involving cytokine signaling (IL-21, IL-6, IL-12, activin A, migration and positioning in the germinal center by CXCR5, surface receptors (ICOS/ICOSL, SAP/SLAM as well as transcription factor (Bcl-6, c-Maf, STAT3 signaling and repressor miR155. On the other hand Tfh generation is negatively regulated at specific steps of Tfh generation by specific cytokine (IL-2, IL-7, surface receptor (PD-1, CTLA-4, transcription factors Blimp-1, STAT5, T-bet, KLF-2 signaling and repressor miR 146a. Interestingly, miR 17-92 and FOXO1 acts as a positive as well as a negative regulator of Tfh differentiation depending on the time of expression and disease specificity. Tfh cells are also generated from the conversion of other effector T cells as exemplified by Th1 cells converting into Tfh during viral infection. The mechanistic details of effector T cells conversion into Tfh are yet to be clear. To manipulate Tfh cells for therapeutic implication and or for effective vaccination strategies, it is important to know positive and negative regulators of Tfh generation. Hence, in this review we have highlighted and interlinked molecular signaling from cytokines, surface receptors, transcription factors, ubiquitin Ligase and miRNA as positive and negative regulators for Tfh differentiation.

  9. Construction and Activity Assay of Transcription Activator-like Effector Nuclease (TALEN) Plasmids for ALK4 Gene Knock-out%针对ALK4基因的TALEN质粒构建与活性鉴定

    Institute of Scientific and Technical Information of China (English)

    曾凡才; 顾洪; 王轲; 周红

    2014-01-01

    旨在利用类转录激活因子效应物核酸酶(Transcription activator-like effector nuclease,TALEN)技术构建具有活性的敲除类激活素激酶受体4(Activin receptor-like kinase 4,ALK4)的TALEN质粒.利用TALEN在线设计工具,根据TALEN设计原则和ALK4剪接异构体的共同序列确定基因敲除的靶位点、TALEN识别序列和用于活性验证的限制性酶切位点.利用质粒文库试剂盒快速构建TALEN质粒,并通过酶切、测序和BLAST比对加以验证.应用脂质体转染法将构建质粒导入HEK293T细胞,通过共转染的pEGFP-N1质粒判断转染效率.利用嘌呤霉素进行阳性筛选后提取基因组DNA,PCR扩增靶序列,HhaⅠ酶切纯化后的PCR产物.结果显示,来自转染TALEN质粒细胞基因组的PCR产物的酶切效率明显下降,提示部分细胞的ALK4基因发生了突变.首次成功构建了在HEK293T细胞中有活性的TALEN质粒.

  10. Losartan prevents acquired epilepsy via TGF-β signaling suppression.

    Science.gov (United States)

    Bar-Klein, Guy; Cacheaux, Luisa P; Kamintsky, Lyn; Prager, Ofer; Weissberg, Itai; Schoknecht, Karl; Cheng, Paul; Kim, Soo Young; Wood, Lydia; Heinemann, Uwe; Kaufer, Daniela; Friedman, Alon

    2014-06-01

    Acquired epilepsy is frequently associated with structural lesions after trauma, stroke, and infections. Although seizures are often difficult to treat, there is no clinically applicable strategy to prevent the development of epilepsy in patients at risk. We have recently shown that vascular injury is associated with activation of albumin-mediated transforming growth factor β (TGF-β) signaling, and followed by local inflammatory response and epileptiform activity ex vivo. Here we investigated albumin-mediated TGF-β signaling and tested the efficacy of blocking the TGF-β pathway in preventing epilepsy. We addressed the role of TGF-β signaling in epileptogenesis in 2 different rat models of vascular injury, combining in vitro and in vivo biochemical assays, gene expression, and magnetic resonance and direct optical imaging for blood-brain barrier permeability and vascular reactivity. Long-term electrocorticographic recordings were acquired in freely behaving animals. We demonstrate that serum-derived albumin preferentially induces activation of the activin receptor-like kinase 5 pathway of TGF-β receptor I in astrocytes. We further show that the angiotensin II type 1 receptor antagonist, losartan, previously identified as a blocker of peripheral TGF-β signaling, effectively blocks albumin-induced TGF-β activation in the brain. Most importantly, losartan prevents the development of delayed recurrent spontaneous seizures, an effect that persists weeks after drug withdrawal. TGF-β signaling, activated in astrocytes by serum-derived albumin, is involved in epileptogenesis. We propose losartan, a drug approved by the US Food and Drug Administration, as an efficient antiepileptogenic therapy for epilepsy associated with vascular injury. © 2014 American Neurological Association.

  11. TGF-β1 exerts opposing effects on grass carp leukocytes: implication in teleost immunity, receptor signaling and potential self-regulatory mechanisms.

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    Mu Yang

    Full Text Available In fish immunity, the regulatory role of transforming growth factor-β1 (TGF-β1 has not been fully characterized. Here we examined the immunoregulatory effects of TGF-β1 in grass carp peripheral blood leukocytes (PBL and head kidney leukocytes (HKL. It is interesting that TGF-β1 consistently stimulated the cell viability and the mRNA levels of pro-inflammatory cytokines (Tnfα and Ifnγ and T/B cell markers [Cd4-like (Cd4l, Cd8α, Cd8β and Igμ] in PBL, which contrasted with its inhibitory tone in HKL. Further studies showed that grass carp TGF-β1 type I receptor, activin receptor-like kinase 5 (ALK5, was indispensable for the immunoregulatory effects of TGF-β1 in PBL and HKL. Notably, TGF-β1 persistently attenuated ALK5 expression, whereas immunoneutralization of endogenous grass carp TGF-β1 could increase ALK5 mRNA and protein levels. It is consistent with the observation that TGF-β1 decreased the number of ALK5(+ leukocytes in PBL and HKL, revealing a negative regulation of TGF-β1 signaling at the receptor level. Moreover, transient treatment with TGF-β1 for 24 h was sufficient to induce similar cellular responses compared with the continuous treatment. This indicated a possible mechanism by which TGF-β1 triggered the down-regulation of ALK5 mRNA and protein, leading to the desensitization of grass carp leukocytes toward TGF-β1. Accordingly, our data revealed a dual role of TGF-β1 in teleost immunity in which it can serve as a positive or negative control device and provided additional mechanistic insights as to how TGF-β1 controls its signaling in vertebrate leukocytes.

  12. Phorbol ester-treated human acute myeloid leukemia cells secrete G-CSF, GM-CSF and erythroid differentiation factor into serum-free media in primary culture.

    Science.gov (United States)

    Scher, W; Eto, Y; Ejima, D; Den, T; Svet-Moldavsky, I A

    1990-12-10

    Upon treatment with the phorbol ester, tetradecanoylphorbol 13-acetate (PMA), peripheral mononuclear blood cells from patients with acute myeloid leukemia secrete into serum-free cell-conditioned media (PMA-CCM) at least three distinct nondialysable 'hematopoietic' factors: granulocyte-colony-stimulating factor (G-CSF), granulocyte/macrophage-colony-stimulating factor (GM-CSF) and erythroid differentiation factor (EDF, activin A). G-CSF was identified by its stimulation of [3H]thymidine incorporation into a G-CSF-responsive cell line, NSF-60, and the inhibition of its stimulation by a G-CSF-specific monoclonal antibody (MAB). GM-CSF was identified by its stimulation of [3H]thymidine incorporation into a GM-CSF-responsive line, TALL-101, and the inhibition of its stimulation by a GM-CSF-specific MAB. EDF was identified by its ability to stimulate erythroid differentiation in mouse erythroleukemia cell lines, its identical retention times to those of authentic EDF on three successive reverse-phase HPLC columns and characterization of its penultimate N-terminal residue as leucine which is the same as that of authentic EDF. Both authentic EDF and the erythroid-stimulating activity in PMA-CCM were found to act synergistically with a suboptimal inducing concentration of a well-studied inducing agent, dimethyl sulfoxide, in inducing erythroid differentiation. In addition, a fourth activity was observed in PMA-CCM: normal human fetal bone marrow cell-proliferation stimulating activity (FBMC-PSA). FBMC-PSA was identified by its ability to stimulate the growth of granulocytes and macrophages in FBMC suspension cultures, which neither recombinant G-CSF or GM-CSF were found to do.

  13. Emerging roles of BMP9 and BMP10 in hereditary hemorrhagic telangiectasia

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    Emmanuelle eTillet

    2015-01-01

    Full Text Available Rendu-Osler-Weber syndrome, also known as hereditary hemorrhagic telangiectasia (HHT, is an autosomal dominant vascular disorder. Three genes are causally related to HHT: the ENG gene encoding endoglin, a co-receptor of the TGFß family (HHT1, the ACVRL1 gene encoding ALK1 (activin receptor-like kinase 1, a type I receptor of the TGFß family (HHT2, and the SMAD4 gene, encoding a transcription factor critical for this signaling pathway. Bone morphogenetic proteins (BMPs are growth factors of the TGFß family. Among them, BMP9 and BMP10 have been shown to bind directly with high affinity to ALK1 and endoglin, and BMP9 mutations have recently been linked to a vascular-anomaly syndrome that has phenotypic overlap with HHT. BMP9 and BMP10 are both circulating cytokines in blood, and the current working model is that BMP9 and BMP10 maintain a quiescent endothelial state that is dependent on the level of ALK1/endoglin activation on endothelial cells. In accordance with this model, to explain the etiology of HHT we hypothesize that a deficient BMP9/BMP10/ALK1/endoglin pathway may lead to re-activation of angiogenesis or a greater sensitivity to an angiogenic stimulus. Resulting endothelial hyperproliferation and hypermigration may lead to vasodilatation and formation of arteriovenous malformation (AVM. HHT would thus result from a defect in the angiogenic balance. This review will focus on the emerging role played by BMP9 and BMP10 in the development of this disease and the therapeutic approaches that this opens.

  14. The immunosuppressive signature of menstrual blood mesenchymal stem cells entails opposite effects on experimental arthritis and graft versus host diseases.

    Science.gov (United States)

    Luz-Crawford, Patricia; Torres, Maria J; Noël, Daniele; Fernandez, Ainoa; Toupet, Karine; Alcayaga-Miranda, Francisca; Tejedor, Gautier; Jorgensen, Christian; Illanes, Sebastian E; Figueroa, Fernando E; Djouad, Farida; Khoury, Maroun

    2016-02-01

    Recently, a noninvasive and highly proliferative stem cell population from menstrual blood called MenSCs has been identified. Despite their use in clinical studies, their immunomodulatory properties have not yet been investigated. In this context, we studied the immunosuppressive properties of MenSCs in comparison with the well-characterized bone marrow derived-MSCs (BM-MSCs). Using an in vitro proliferation assays, we showed that MenSCs displayed a lower suppressive effect on peripheral blood mononuclear cells and in particular on the proinflammatory CD4(+) IFN-γ(+) and CD8(+) IFNγ(+) cells than BM-MSCs. Moreover, compared to BM-MSCs, MenSCs activated with IFN-γ and IL-1β produced lower amounts of immunosuppressive factors such as IDO, PDL-1, PGE2, and Activin A and exhibited a substantial lower expression level of IFN-γ receptor subunits. In the collagen induced arthritis model, while BM-MSCs administration resulted in a potent therapeutic effect associated with a significant decrease of proinflammatory T cell frequency in the lymph nodes, MenSCs injection did not. In contrast, in the xeno-GVHD model, only MenSCs administration significantly increased the survival of mice. This beneficial effect mediated by MenSCs was associated with a higher capacity to migrate into the intestine and liver and not to their anti-inflammatory capacities. All together our results demonstrate for the first time that the therapeutic potential of MSC in the experimental xeno-GVHD model is independent of their immunosuppressive properties. These findings should be taken into consideration for the development of safe and effective cell therapies.

  15. Human amniotic fluid stem cells support undifferentiated propagation and pluripotency of human embryonic stem cell without b-FGF in a density dependent manner.

    Science.gov (United States)

    Ma, Xiaorong; Li, Huanqi; Xin, Shujia; Ma, Yueting; Ouyang, Tianxiang

    2014-01-01

    Human embryonic stem cells (hESCs) are pluripotent cells which can give rise to almost all adult cell lineages. Culture system of hESCs is complex, requiring exogenous b-FGF and feeder cell layer. Human mesenchymal stem cells (MSCs) not only synthesize soluble cytokines or factors such as b-FGF, but also provide other mechanism which might play positive role on sustaining hESCs propagation and pluripotency. Human amniotic fluid stem (AFS) cells, which share characteristics of both embryonic and adult stem cells, have been regarded as promising cells for regenerative medicine. Taking advantage by AFS cells, we studied the ability of AFS cells in supporting undifferentiated propagation and pluripotency of Chinese population derived X-01 hESCs. Human AF-type amniotic fluid stem cells (hAF-AFSCs) transcribed genes including Activin A, TGF-β1, Noggin and b-FGF, which involved in maintaining pluripotency and self-renewal of hESCs. Compared to mouse embryonic fibroblasts (MEFs), hAF-AFSCs secreted higher concentration of b-FGF which was important in hESCs culture (P FGF supplementation, keeping undifferentiated status. While exogenous b-FGF was obviated, propagation of hESCs with undifferentiated status was dependent on density of hAF-AFSC feeder layer. Lower density of hAF-AFSCs resulted in rapid decline in undifferentiated clone number, while higher ones hindered the growth of colonies. The most appropriate hAF-AFSCs feeder density to maintain the X-01 hESC line without exogenous b-FGF was 15-20×10(4)/well. To the best of our knowledge, this is the first study demonstrating that hAF-AFSCs could support undifferentiated propagation and pluripotency of Chinese population derived hESCs without exogenous b-FGF supplementation.

  16. Drosophila Smad2 Opposes Mad Signaling during Wing Vein Development

    Science.gov (United States)

    Sander, Veronika; Eivers, Edward; Choi, Renee H.; De Robertis, Edward M.

    2010-01-01

    In the vertebrates, the BMP/Smad1 and TGF-β/Smad2 signaling pathways execute antagonistic functions in different contexts of development. The differentiation of specific structures results from the balance between these two pathways. For example, the gastrula organizer/node of the vertebrates requires a region of low Smad1 and high Smad2 signaling. In Drosophila, Mad regulates tissue determination and growth in the wing, but the function of dSmad2 in wing patterning is largely unknown. In this study, we used an RNAi loss-of-function approach to investigate dSmad2 signaling during wing development. RNAi-mediated knockdown of dSmad2 caused formation of extra vein tissue, with phenotypes similar to those seen in Dpp/Mad gain-of-function. Clonal analyses revealed that the normal function of dSmad2 is to inhibit the response of wing intervein cells to the extracellular Dpp morphogen gradient that specifies vein formation, as measured by expression of the activated phospho-Mad protein. The effect of dSmad2 depletion in promoting vein differentiation was dependent on Medea, the co-factor shared by Mad and dSmad2. Furthermore, double RNAi experiments showed that Mad is epistatic to dSmad2. In other words, depletion of Smad2 had no effect in Mad-deficient wings. Our results demonstrate a novel role for dSmad2 in opposing Mad-mediated vein formation in the wing. We propose that the main function of dActivin/dSmad2 in Drosophila wing development is to antagonize Dpp/Mad signaling. Possible molecular mechanisms for the opposition between dSmad2 and Mad signaling are discussed. PMID:20442782

  17. Clinical Outcome 3 Years After Autologous Chondrocyte Implantation Does Not Correlate With the Expression of a Predefined Gene Marker Set in Chondrocytes Prior to Implantation but Is Associated With Critical Signaling Pathways.

    Science.gov (United States)

    Stenberg, Johan; de Windt, Tommy S; Synnergren, Jane; Hynsjö, Lars; van der Lee, Josefine; Saris, Daniel B F; Brittberg, Mats; Peterson, Lars; Lindahl, Anders

    2014-09-01

    There is a need for tools to predict the chondrogenic potency of autologous cells for cartilage repair. To evaluate previously proposed chondrogenic biomarkers and to identify new biomarkers in the chondrocyte transcriptome capable of predicting clinical success or failure after autologous chondrocyte implantation. Controlled laboratory study and case-control study; Level of evidence, 3. Five patients with clinical improvement after autologous chondrocyte implantation and 5 patients with graft failures 3 years after implantation were included. Surplus chondrocytes from the transplantation were frozen for each patient. Each chondrocyte sample was subsequently thawed at the same time point and cultured for 1 cell doubling, prior to RNA purification and global microarray analysis. The expression profiles of a set of predefined marker genes (ie, collagen type II α1 [COL2A1], bone morphogenic protein 2 [BMP2], fibroblast growth factor receptor 3 [FGFR3], aggrecan [ACAN], CD44, and activin receptor-like kinase receptor 1 [ACVRL1]) were also evaluated. No significant difference in expression of the predefined marker set was observed between the success and failure groups. Thirty-nine genes were found to be induced, and 38 genes were found to be repressed between the 2 groups prior to autologous chondrocyte implantation, which have implications for cell-regulating pathways (eg, apoptosis, interleukin signaling, and β-catenin regulation). No expressional differences that predict clinical outcome could be found in the present study, which may have implications for quality control assessments of autologous chondrocyte implantation. The subtle difference in gene expression regulation found between the 2 groups may strengthen the basis for further research, aiming at reliable biomarkers and quality control for tissue engineering in cartilage repair. The present study shows the possible limitations of using gene expression before transplantation to predict the chondrogenic and

  18. FGF7 and cell density are required for final differentiation of pancreatic amylase-positive cells from human ES cells.

    Science.gov (United States)

    Takizawa-Shirasawa, Sakiko; Yoshie, Susumu; Yue, Fengming; Mogi, Akimi; Yokoyama, Tadayuki; Tomotsune, Daihachiro; Sasaki, Katsunori

    2013-12-01

    The major molecular signals of pancreatic exocrine development are largely unknown. We examine the role of fibroblast growth factor 7 (FGF7) in the final induction of pancreatic amylase-containing exocrine cells from induced-pancreatic progenitor cells derived from human embryonic stem (hES) cells. Our protocol consisted in three steps: Step I, differentiation of definitive endoderm (DE) by activin A treatment of hES cell colonies; Step II, differentiation of pancreatic progenitor cells by re-plating of the cells of Step I onto 24-well plates at high density and stimulation with all-trans retinoic acid; Step III, differentiation of pancreatic exocrine cells with a combination of FGF7, glucagon-like peptide 1 and nicotinamide. The expression levels of pancreatic endodermal markers such as Foxa2, Sox17 and gut tube endoderm marker HNF1β were up-regulated in both Step I and II. Moreover, in Step III, the induced cells expressed pancreatic markers such as amylase, carboxypeptidase A and chymotrypsinogen B, which were similar to those in normal human pancreas. From day 8 in Step III, cells immunohistochemically positive for amylase and for carboxypeptidase A, a pancreatic exocrine cell product, were induced by FGF7. Pancreatic progenitor Pdx1-positive cells were localized in proximity to the amylase-positive cells. In the absence of FGF7, few amylase-positive cells were identified. Thus, our three-step culture protocol for human ES cells effectively induces the differentiation of amylase- and carboxypeptidase-A-containing pancreatic exocrine cells.

  19. Luteinizing hormone receptor (lhcgr) as a marker gene for characterizing estrogenic endocrine-disrupting chemicals in zebrafish ovarian follicle cells.

    Science.gov (United States)

    Liu, Ka-Cheuk; Wu, Rudolf S S; Ge, Wei

    2013-10-01

    The adverse effects of endocrine-disrupting chemicals (EDCs) have been well documented; however, the action mechanisms of many EDCs remain elusive and controversial. Furthermore, the highly diversified chemical structures and low environmental concentrations of EDCs present a major challenge to their chemical detection. Clearly, there is an urgent need for simple and reliable bioassays to detect EDCs in the environment and unravel their action mechanisms. We have recently identified luteinizing hormone receptor (lhcgr) as a robust estradiol (E2)-responsive gene in cultured zebrafish ovarian follicle cells. The expression of lhcgr exhibited a distinct biphasic response to E2 over a 24-h time-course treatment, making this a unique system for characterizing estrogenic EDCs. This study was undertaken to validate this platform by testing a wide range of EDCs, including 17α-ethinylestradiol (EE2), diethylstilbestrol (DES), bisphenol A (BPA), genistein (GEN), 1,1,1-trichloro-2-(2-chlorophenyl)-2-(4-chlorophenyl)ethane (o,p'-DDT), vinclozolin (VIN), bis(2-ethylhexyl) phthalate (DEHP), 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), and 2,2',4,4'-tetrabromodiphenyl ether (BDE-47). Diethylstilbestrol (DES), EE2 and o,p'-DDT mimicked E2 and induced a biphasic expression of lhcgr while BPA and GEN stimulated a monophasic expression in the 24-h time-course. In contrast, BDE-47, DEHP and VIN had no effect, whereas TCDD decreased lhcgr expression. Dose-response experiment showed that E2, EE2 and DES had the highest potency, which was followed by GEN, BPA and o,p'-DDT. The effects of estrogenic EDCs were further confirmed by their potentiation of hCG-induced activin βA2 subunit (inhbab) expression. In conclusion, the present study showed that the expression of lhcgr in cultured zebrafish follicle cells and its biphasic response to estrogens provide a unique in vitro platform for screening and categorizing estrogenic substances and deciphering their action mechanisms.

  20. Krwawienia czynnościowe

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    Tomasz Soszka

    2010-08-01

    Full Text Available Dysfunctional uterine bleedings (DUB are all these abnormal uterine bleedings (AUB after exclusion ofanatomical and organic lesions as well as systemic and iatrogenic changes and pregnancy. In the presented paperspecial attention was paid to the diagnostic and therapeutic treatment in an outpatient clinic, including thepatient interaction in the process. State-of the-art knowledge on the menstrual cycle pathophysiology and DUBhas been presented basing on the angiogenesis model. The participation of many biologically active substances(including among others: kinins, endothelins, coagulation and fibrinolytic factors, prostanoids and platelet factorsin breakdown and regenerative processes within functional layers of the endometrium has been described.The regulative role of steroid hormones (estrogens, progesterone, activin-follistatin was taken into consideration.The carefully taken medical history, the proper selection of diagnostic tools followed by the discussionwith the patient on the pharmacological or, if desired, surgical options constitutes the whole treatment processin the case of troublesome DUB. In the paper new diagnostic and therapeutic methods including a wide rangeof scanning techniques, endometrial sampling and endometrial resection as well as ablation techniques havebeen presented. Special attention was paid to outpatient clinic methods (e.g. Pipella, minihysteroscopy, thermalablation, photodynamic ablation and cryoablation. The pharmaceutical treatment depends on the intensity ofbleeding (acute or chronic and consists of estrogens, oral, transdermal and vaginal contraceptives, IUD system,progestin-oestrogen injections, Danazol and GnRH. Besides the hormones, non-steroid anti-inflammatory and antifibrinolytic drugs appeared to be effective inthe treatment of DUB. One has to pay attention to menarche which in some ways reflects the proper local andsystemic haemostatic mechanisms. Basing on these observations one may to decide to

  1. A Key Role for Inhibins in Dendritic Cell Maturation and Function

    Science.gov (United States)

    Olguín-Alor, Roxana; de la Fuente-Granada, Marisol; Bonifaz, Laura C.; Antonio-Herrera, Laura; García-Zepeda, Eduardo A.; Soldevila, Gloria

    2016-01-01

    Inhibins are members of the TGFβ superfamily, which regulate many cellular processes including differentiation, proliferation, survival and apoptosis. Although initially described as hormones regulating the hypothalamus-pituitary-gonadal axis, based on their ability to antagonize Activins, our group has recently reported that they play a role in thymocyte differentiation and survival, as well as in thymic stromal cell maturation and nTreg generation. Here, we used Inhibin knock out mice (Inhα-/-) to investigate the role of Inhibins in peripheral dendritic cell maturation and function. We first demonstrated that LPS treated Inhα+/+ bone marrow derived dendritic cells (BMDC) were capable to produce significant levels of Inhibin A. Interestingly, Inhα-/- BMDC showed reduced MHCII and CD86 upregulation and increased PD-L1 expression in response to LPS compared to Inhα+/+, which correlated with reduced ability to induce proliferation of allogeneic T cells. The “semi-mature” phenotype displayed by Inhα-/- mBMDC correlated with increased levels of IL-10 and slightly decreased IL-6 production after LPS stimulation. In addition, Inhα-/- mBMDC showed impaired migration towards CCL19 and CCL21, assessed by in vitro chemotaxis and in vivo competitive homing experiments, despite their normal CCR7 expression. Furthermore, in vivo LPS-induced DC maturation was also diminished in Inhα-/- mice, specially within the LC (CD207+ CD11b+ CD103-) subpopulation. Finally, analysis of delayed type hypersensitivity responses in Inhα-/- mice, showed reduced ear swelling as a result of reduced cellular infiltration in the skin, correlating with impaired homing of CD207+ DCs to the draining lymph nodes. In summary, our data demonstrate for the first time that Inhibins play a key role in peripheral DC maturation and function, regulating the balance between immunity and tolerance. PMID:27936218

  2. Identification of Pathways Mediating Growth Differentiation Factor5-Induced Tenogenic Differentiation in Human Bone Marrow Stromal Cells.

    Directory of Open Access Journals (Sweden)

    Sik-Loo Tan

    Full Text Available To date, the molecular signalling mechanisms which regulate growth factors-induced MSCs tenogenic differentiation remain largely unknown. Therefore, a study to determine the global gene expression profile of tenogenic differentiation in human bone marrow stromal cells (hMSCs using growth differentiation factor 5 (GDF5 was conducted. Microarray analyses were conducted on hMSCs cultures supplemented with 100 ng/ml of GDF5 and compared to undifferentiated hMSCs and adult tenocytes. Results of QuantiGene® Plex assay support the use and interpretation of the inferred gene expression profiles and pathways information. From the 27,216 genes assessed, 873 genes (3.21% of the overall human transcriptome were significantly altered during the tenogenic differentiation process (corrected p<0.05. The genes identified as potentially associated with tenogenic differentiation were ARHGAP29, CCL2, integrin alpha 8 and neurofilament medium polypeptides. These genes, were mainly associated with cytoskeleton reorganization (stress fibers formation signaling. Pathway analysis demonstrated the potential molecular pathways involved in tenogenic differentiation were: cytoskeleton reorganization related i.e. keratin filament signaling and activin A signaling; cell adhesion related i.e. chemokine and adhesion signaling; and extracellular matrix related i.e. arachidonic acid production signaling. Further investigation using atomic force microscopy and confocal laser scanning microscopy demonstrated apparent cytoskeleton reorganization in GDF5-induced hMSCs suggesting that cytoskeleton reorganization signaling is an important event involved in tenogenic differentiation. Besides, a reduced nucleostemin expression observed suggested a lower cell proliferation rate in hMSCs undergoing tenogenic differentiation. Understanding and elucidating the tenogenic differentiation signalling pathways are important for future optimization of tenogenic hMSCs for functional tendon cell

  3. A novel nodal enhancer dependent on pluripotency factors and smad2/3 signaling conditions a regulatory switch during epiblast maturation.

    Directory of Open Access Journals (Sweden)

    Costis Papanayotou

    2014-06-01

    Full Text Available During early development, modulations in the expression of Nodal, a TGFβ family member, determine the specification of embryonic and extra-embryonic cell identities. Nodal has been extensively studied in the mouse, but aspects of its early expression remain unaccounted for. We identified a conserved hotspot for the binding of pluripotency factors at the Nodal locus and called this sequence "highly bound element" (HBE. Luciferase-based assays, the analysis of fluorescent HBE reporter transgenes, and a conditional mutation of HBE allowed us to establish that HBE behaves as an enhancer, is activated ahead of other Nodal enhancers in the epiblast, and is essential to Nodal expression in embryonic stem cells (ESCs and in the mouse embryo. We also showed that HBE enhancer activity is critically dependent on its interaction with the pluripotency factor Oct4 and on Activin/Nodal signaling. Use of an in vitro model of epiblast maturation, relying on the differentiation of ESCs into epiblast stem cells (EpiSCs, revealed that this process entails a shift in the regulation of Nodal expression from an HBE-driven phase to an ASE-driven phase, ASE being another autoregulatory Nodal enhancer. Deletion of HBE in ESCs or in EpiSCs allowed us to show that HBE, although not necessary for Nodal expression in EpiSCs, is required in differentiating ESCs to activate the differentiation-promoting ASE and therefore controls this regulatory shift. Our findings clarify how early Nodal expression is regulated and suggest how this regulation can promote the specification of extra-embryonic precusors without inducing premature differentiation of epiblast cells. More generally, they open new perspectives on how pluripotency factors achieve their function.

  4. Autonomous isolation, long-term culture and differentiation potential of adult salivary gland-derived stem/progenitor cells.

    Science.gov (United States)

    Baek, Hyunjung; Noh, Yoo Hun; Lee, Joo Hee; Yeon, Soo-In; Jeong, Jaemin; Kwon, Heechung

    2014-09-01

    Salivary gland stem/progenitor cells belong to the endodermal lineage and may serve as good candidates to replace their dysfunctional counterparts. The objective of this study was to isolate large numbers of salivary gland tissue-derived stem cells (SGSCs) from adult rats in order to develop a clinically applicable method that does not involve sorting or stem cell induction by duct ligation. We analysed SGSCs isolated from normal rat salivary glands to determine whether they retained the major characteristics of stem cells, self-renewal and multipotency, especially with respect to the various endodermal cell types. SGSCs expressed high levels of integrin α6β1 and c-kit, which are surface markers of SGSCs. In particular, the integrin α6β1(+) /c-kit(+) salivary gland cells maintained the morphology, proliferation activity and multipotency of stem cells for up to 92 passages in 12 months. Furthermore, we analysed the capacity of SGSCs to differentiate into endoderm lineage cell types, such as acinar-like and insulin-secreting cells. When cultured on growth factor reduced matrigel, the morphology of progenitor cells changed to acinar-like structures and these cells expressed the acinar cell-specific marker, α-amylase, and tight junction markers. Moreover, reverse transcription-polymerase chain reaction (RT-PCR) data showed increased expression of pancreatic cell markers, including insulin, Pdx1, pan polypeptide and neurogenin-3, when these cells formed pancreatic clusters in the presence of activin A, exendin-4 and retinoic acid. These data demonstrate that adult salivary stem/progenitor cells may serve as a potential source for cell therapy in salivary gland hypofunction and diabetes.

  5. Downregulated miR-195 detected in preeclamptic placenta affects trophoblast cell invasion via modulating ActRIIA expression.

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    Yang Bai

    Full Text Available BACKGROUND: Preeclampsia (PE is a pregnancy-specific syndrome manifested by on-set of hypertension and proteinuria after 20 weeks of gestation. Abnormal placenta development has been generally accepted as initial cause of the disorder. Recently, miR-195 was found to be down-regulated in preeclamptic placentas compared with normal pregnant ones, indicating possible association of this small molecule with placental pathology of preeclampsia. By far the function of miR-195 in the development of placenta remains unknown. METHODOLOGY/PRINCIPAL FINDINGS: Bioinformatic assay predicted ActRIIA as one of the targets for miR-195. By using Real-time PCR, Western blotting and Dual Luciferase Assay, we validated that ActRIIA was the direct target of miR-195 in human trophoblast cells. Transwell insert invasion assay showed that miR-195 could promote cell invasion in trophoblast cell line, HTR8/SVneo cells, and the effect could be abrogated by overexpressed ActRIIA. In preeclamptic placenta tissues, pri-miR-195 and mature miR-195 expressions were down-regulated, whereas ActRIIA level appeared to be increased when compared with that in gestational-week-matched normal placentas. CONCLUSIONS/SIGNIFICANCE: This is the first report on the function of miR-195 in human placental trophoblast cells which reveals an invasion-promoting effect of the small RNA via repressing ActRIIA. Aberrant expression of miR-195 may contribute to the occurrence of preeclampsia through interfering with Activin/Nodal signaling mediated by ActRIIA in human placenta.

  6. Porcine induced pluripotent stem cells analogous to naïve and primed embryonic stem cells of the mouse.

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    Telugu, Bhanu Prakash V L; Ezashi, Toshihiko; Roberts, R Michael

    2010-01-01

    Authentic or naïve embryonic stem cells (ESC) have probably never been derived from the inner cell mass (ICM) of pig blastocysts, despite over 25 years of effort. Recently, several groups, including ours, have reported induced pluripotent stem cells (iPSC) from swine by reprogramming somatic cells with a combination of four factors, OCT4 (POU5F1)/SOX2/KLF4/c-MYC delivered by retroviral transduction. The porcine (p) iPSC resembled human (h) ESC and the mouse "Epiblast stem cells" (EpiSC) in their colony morphology and expression of pluripotent genes, and are likely dependent on FGF2/ACTIVIN/NODAL signaling, therefore representing a primed ESC state. These cells are likely to advance swine as a model in biomedical research, since grafts could potentially be matched to the animal that donated the cells for re-programming. The objective of the present work has been to develop naïve piPSC. Employing a combination of seven reprogramming factors assembled on episomal vectors, we successfully reprogrammed porcine embryonic fibroblasts on a modified LIF-medium supplemented with two kinase inhibitors; CHIR99021, which inhibits GSK-3beta, and PD0325901, a MEK inhibitor. The derived piPSC bear a striking resemblance to naïve mESC in colony morphology, are dependent on LIF to maintain an undifferentiated phenotype, and express markers consistent with pluripotency. They exhibit high telomerase activity, a short cell cycle interval, and a normal karyotype, and are able to generate teratomas. Currently, the competence of these lines for contributing to germ-line chimeras is being tested.

  7. Induced pluripotent stem cells from pigs and other ungulate species: an alternative to embryonic stem cells?

    Science.gov (United States)

    Ezashi, T; Telugu, B P V L; Roberts, R M

    2012-08-01

    Robust embryonic stem cell (ESC) lines from livestock species have been difficult to derive and maintain, and unlike mouse ESC, have not contributed to our ability to understand directed differentiation in vitro. Nor have such cells yet provided a simpler means than pronuclear injection to manipulate the genomes of agriculturally important species, such as cattle, sheep and pigs. Induced pluripotent stem cells (iPSC) generated by reprogramming somatic cells, such as fibroblasts, with a set of stemness genes, most usually but not exclusively POU5F1, SOX2, KLF4 and c-MYC, offer an alternative to ESC in these regards, as they exhibit a pluripotent phenotype resembling that of ESC, yet are readily generated in the laboratory. Accordingly, such cells, in association with cloning technologies, may be useful for introducing complex genetic changes into livestock, although this potential has yet to be demonstrated. Porcine iPSC may be especially valuable because the pig is a prime biomedical model for tissue transplantation. In general, iPSC from livestock, like those from humans, are of the epiblast type and depend upon FGF2 and activin/nodal signalling systems to maintain their pluripotency and growth. Recent experiments, in which newly reprogrammed porcine and bovine cells were selected on a LIF-based medium in presence of specific protein kinase inhibitors, have allowed iPSC cells of the naïve type, resembling the more amenable blastocyst-derived mouse ESC and iPSC to be isolated. However, hurdles still remain if such cells are to achieve their biotechnological promise.

  8. Model systems for studying trophoblast differentiation from human pluripotent stem cells.

    Science.gov (United States)

    Ezashi, Toshihiko; Telugu, Bhanu Prakash V L; Roberts, R Michael

    2012-09-01

    This review focuses on a now well-established model for generating cells of the trophoblast (TB) lineage by treating human embryonic stem cells (ESC) and induced pluripotent stem cells (iPSC) with the growth factor BMP4. We first discuss the opposing roles of FGF2 and BMP4 in directing TB formation and the need to exclude the former from the growth medium to minimize the co-induction of mesoderm and endoderm. Under these conditions, there is up-regulation of several transcription factors implicated in TB lineage emergence within 3 h of BMP4 exposure and, over a period of days and especially under a high O(2) gas atmosphere, gradual appearance of cell types carrying markers for more differentiated TB cell types, including extravillous TB and syncytioTB. We describe the potential value of including low molecular weight pharmaceutical agents that block activin A (INHBA) and FGF2 signaling to support BMP4-directed differentiation. We contend that the weight of available evidence supports the contention that BMP4 converts human ESC and iPSC of the so-called epiblast type unidirectionally to TB. We also consider the argument that BMP4 treatment of human ESC in the absence of exogenous FGF2 leads only to the emergence of mesoderm derivatives to be seriously flawed. Instead, we propose that, when signaling networks supporting pluripotency ESC or iPSC become unsustainable and when specification towards extra-embryonic mesoderm and endoderm are rendered inoperative, TB emerges as a major default state to pluripotency.

  9. Multiple myeloma mesenchymal stromal cells: Contribution to myeloma bone disease and therapeutics.

    Science.gov (United States)

    Garcia-Gomez, Antonio; Sanchez-Guijo, Fermin; Del Cañizo, M Consuelo; San Miguel, Jesus F; Garayoa, Mercedes

    2014-07-26

    Multiple myeloma is a hematological malignancy in which clonal plasma cells proliferate and accumulate within the bone marrow. The presence of osteolytic lesions due to increased osteoclast (OC) activity and suppressed osteoblast (OB) function is characteristic of the disease. The bone marrow mesenchymal stromal cells (MSCs) play a critical role in multiple myeloma pathophysiology, greatly promoting the growth, survival, drug resistance and migration of myeloma cells. Here, we specifically discuss on the relative contribution of MSCs to the pathophysiology of osteolytic lesions in light of the current knowledge of the biology of myeloma bone disease (MBD), together with the reported genomic, functional and gene expression differences between MSCs derived from myeloma patients (pMSCs) and their healthy counterparts (dMSCs). Being MSCs the progenitors of OBs, pMSCs primarily contribute to the pathogenesis of MBD because of their reduced osteogenic potential consequence of multiple OB inhibitory factors and direct interactions with myeloma cells in the bone marrow. Importantly, pMSCs also readily contribute to MBD by promoting OC formation and activity at various levels (i.e., increasing RANKL to OPG expression, augmenting secretion of activin A, uncoupling ephrinB2-EphB4 signaling, and through augmented production of Wnt5a), thus further contributing to OB/OC uncoupling in osteolytic lesions. In this review, we also look over main signaling pathways involved in the osteogenic differentiation of MSCs and/or OB activity, highlighting amenable therapeutic targets; in parallel, the reported activity of bone-anabolic agents (at preclinical or clinical stage) targeting those signaling pathways is commented.

  10. Construction of functional pancreatic artificial islet tissue composed of fibroblast-modified polylactic- co-glycolic acid membrane and pancreatic stem cells.

    Science.gov (United States)

    Liu, Liping; Tan, Jing; Li, Baoyuan; Xie, Qian; Sun, Junwen; Pu, Hongli; Zhang, Li

    2017-09-01

    Objective To improve the biocompatibility between polylactic- co-glycolic acid membrane and pancreatic stem cells, rat fibroblasts were used to modify the polylactic- co-glycolic acid membrane. Meanwhile, we constructed artificial islet tissue by compound culturing the pancreatic stem cells and the fibroblast-modified polylactic- co-glycolic acid membrane and explored the function of artificial islets in diabetic nude mice. Methods Pancreatic stem cells were cultured on the fibroblast-modified polylactic- co-glycolic acid membrane in dulbecco's modified eagle medium containing activin-A, β-catenin, and exendin-4. The differentiated pancreatic stem cells combined with modified polylactic- co-glycolic acid membrane were implanted subcutaneously in diabetic nude mice. The function of artificial islet tissue was explored by detecting blood levels of glucose and insulin in diabetic nude mice. Moreover, the proliferation and differentiation of pancreatic stem cells on modified polylactic- co-glycolic acid membrane as well as the changes on the tissue structure of artificial islets were investigated by immunofluorescence and haematoxylin and eosin staining. Results The pancreatic stem cells differentiated into islet-like cells and secreted insulin when cultured on fibroblast-modified polylactic- co-glycolic acid membrane. Furthermore, when the artificial islet tissues were implanted into diabetic nude mice, the pancreatic stem cells combined with polylactic- co-glycolic acid membrane modified by fibroblasts proliferated, differentiated, and secreted insulin to reduce blood glucose levels in diabetic nude mice. Conclusion Pancreatic stem cells can be induced to differentiate into islet-like cells in vitro. In vivo, the artificial islet tissue can effectively regulate the blood glucose level in nude mice within a short period. However, as time increased, the structure of the artificial islets was destroyed due to the erosion of blood cells that resulted in the gradual

  11. GDNF-independent ureteric budding: role of PI3K-independent activation of AKT and FOSB/JUN/AP-1 signaling

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    James B. Tee

    2013-07-01

    A significant fraction of mice deficient in either glial cell-derived neurotrophic factor (GDNF or its co-receptors (Gfrα1, Ret, undergoes ureteric bud (UB outgrowth leading to the formation of a rudimentary kidney. Previous studies using the isolated Wolffian duct (WD culture indicate that activation of fibroblast growth factor (FGF receptor signaling, together with suppression of BMP/Activin signaling, is critical for GDNF-independent WD budding (Maeshima et al., 2007. By expression analysis of embryonic kidney from Ret(−/− mice, we found the upregulation of several FGFs, including FGF7. To examine the intracellular pathways, we then analyzed GDNF-dependent and GDNF-independent budding in the isolated WD culture. In both conditions, Akt activation was found to be important; however, whereas this occurred through PI3-kinase in GDNF-dependent budding, in the case of GDNF-independent budding, Akt activation was apparently via a PI3-kinase independent mechanism. Jnk signaling and the AP-1 transcription factor complex were also implicated in GDNF-independent budding. FosB, a binding partner of c-Jun in the formation of AP-1, was the most highly upregulated gene in the ret knockout kidney (in which budding had still occurred, and we found that its siRNA-mediated knockdown in isolated WDs also blocked GDNF-independent budding. Taken together with the finding that inhibition of Jnk signaling does not block Akt activation/phosphorylation in GDNF-independent budding, the data support necessary roles for both FosB/Jun/AP-1 signaling and PI3-kinase-independent activation of Akt in GDNF-independent budding. A model is proposed for signaling events that involve Akt and JNK working to regulate GDNF-independent WD budding.

  12. Gene expression profiling reveals new potential players of gonad differentiation in the chicken embryo.

    Directory of Open Access Journals (Sweden)

    Gwenn-Aël Carré

    of chicken gonad differentiation and provides evidence of the preferential expression of BMPs in the developing ovary and Inhibin/Activin subunits in the developing testis.

  13. Resveratrol Ameliorates the Maturation Process of β-Cell-Like Cells Obtained from an Optimized Differentiation Protocol of Human Embryonic Stem Cells

    Science.gov (United States)

    Pezzolla, Daniela; López-Beas, Javier; Lachaud, Christian C.; Domínguez-Rodríguez, Alejandro; Smani, Tarik; Hmadcha, Abdelkrim; Soria, Bernat

    2015-01-01

    Human embryonic stem cells (hESCs) retain the extraordinary capacity to differentiate into different cell types of an adult organism, including pancreatic β-cells. For this particular lineage, although a lot of effort has been made in the last ten years to achieve an efficient and reproducible differentiation protocol, it was not until recently that this aim was roughly accomplished. Besides, several studies evidenced the impact of resveratrol (RSV) on insulin secretion, even though the mechanism by which this polyphenol potentiates glucose-stimulated insulin secretion (GSIS) is still not clear. The aim of this study was to optimize an efficient differentiation protocol that mimics in vivo pancreatic organogenesis and to investigate whether RSV may improve the final maturation step to obtain functional insulin-secreting cells. Our results indicate that treatment of hESCs (HS-181) with activin-A induced definitive endoderm differentiation as detected by the expression of SOX17 and FOXA2. Addition of retinoic acid (RA), Noggin and Cyclopamine promoted pancreatic differentiation as indicated by the expression of the early pancreatic progenitor markers ISL1, NGN3 and PDX1. Moreover, during maturation in suspension culture, differentiating cells assembled in islet-like clusters, which expressed specific endocrine markers such as PDX1, SST, GCG and INS. Similar results were confirmed with the human induced Pluripotent Stem Cell (hiPSC) line MSUH-001. Finally, differentiation protocols incorporating RSV treatment yielded numerous insulin-positive cells, induced significantly higher PDX1 expression and were able to transiently normalize glycaemia when transplanted in streptozotocin (STZ) induced diabetic mice thus promoting its survival. In conclusion, our strategy allows the efficient differentiation of hESCs into pancreatic endoderm capable of generating β-cell-like cells and demonstrates that RSV improves the maturation process. PMID:25774684

  14. Two different network topologies yield bistability in models of mesoderm and anterior mesendoderm specification in amphibians.

    Science.gov (United States)

    Brown, L E; King, J R; Loose, M

    2014-07-21

    Understanding the Gene Regulatory Networks (GRNs) that underlie development is a major question for systems biology. The establishment of the germ layers is amongst the earliest events of development and has been characterised in numerous model systems. The establishment of the mesoderm is best characterised in the frog Xenopus laevis and has been well studied both experimentally and mathematically. However, the Xenopus network has significant differences from that in mouse and humans, including the presence of multiple copies of two key genes in the network, Mix and Nodal. The axolotl, a urodele amphibian, provides a model with all the benefits of amphibian embryology but crucially only a single Mix and Nodal gene required for the specification of the mesoderm. Remarkably, the number of genes within the network is not the only difference. The interaction between Mix and Brachyury, two transcription factors involved in the establishment of the endoderm and mesoderm respectively, is not conserved. While Mix represses Brachyury in Xenopus, it activates Brachyury in axolotl. Thus, whilst the topology of the networks in the two species differs, both are able to form mesoderm and endoderm in vivo. Based on current knowledge of the structure of the mesendoderm GRN we develop deterministic models that describe the time evolution of transcription factors in a single axolotl cell and compare numerical simulations with previous results from Xenopus. The models are shown to have stable steady states corresponding to mesoderm and anterior mesendoderm, with the in vitro model showing how the concentration of Activin can determine cell fate, while the in vivo model shows that β-catenin concentration can determine cell fate. Moreover, our analysis suggests that additional components must be important in the axolotl network in the specification of the full range of tissues.

  15. ZO-1 regulates Erk, Smad1/5/8, Smad2, and RhoA activities to modulate self-renewal and differentiation of mouse embryonic stem cells.

    Science.gov (United States)

    Xu, Jianliang; Lim, Sophia Beng Hui; Ng, Mei Yong; Ali, Safiah Mohamed; Kausalya, Jaya P; Limviphuvadh, Vachiranee; Maurer-Stroh, Sebastian; Hunziker, Walter

    2012-09-01

    ZO-1/Tjp1 is a cytosolic adaptor that links tight junction (TJ) transmembrane proteins to the actin cytoskeleton and has also been implicated in regulating cell proliferation and differentiation by interacting with transcriptional regulators and signaling proteins. To explore possible roles for ZO-1 in mouse embryonic stem cells (mESCs), we inactivated the ZO-1 locus by homologous recombination. The lack of ZO-1 was found to affect mESC self-renewal and differentiation in the presence of leukemia-inhibiting factor (LIF) and Bmp4 or following removal of the growth factors. Our data suggest that ZO-1 suppresses Stat3 and Smad1/5/8 activities and sustains extracellular-signal-regulated kinase (Erk) activity to promote mESC differentiation. Interestingly, Smad2, critical for human but not mESC self-renewal, was hyperactivated in ZO-1(-/-) mESCs and RhoA protein levels were concomitantly enhanced, suggesting attenuation of the noncanonical transforming growth factor β (Tgfβ)/Activin/Nodal pathway that mediates ubiquitination and degradation of RhoA via the TJ proteins Occludin, Par6, and Smurf1 and activation of the canonical Smad2-dependent pathway. Furthermore, Bmp4-induced differentiation of mESCs in the absence of LIF was suppressed in ZO-1(-/-) mESCs, but differentiation down the neural or cardiac lineages was not disturbed. These findings reveal novel roles for ZO-1 in mESC self-renewal, pluripotency, and differentiation by influencing several signaling networks that regulate these processes. Possible implications for the differing relevance of Smad2 in mESC and human ESC self-renewal and how ZO-1 may connect to the different pathways are discussed.