BMP signaling regulates satellite cell-dependent postnatal muscle growth.

Science.gov (United States)

Stantzou, Amalia; Schirwis, Elija; Swist, Sandra; Alonso-Martin, Sonia; Polydorou, Ioanna; Zarrouki, Faouzi; Mouisel, Etienne; Beley, Cyriaque; Julien, Anaïs; Le Grand, Fabien; Garcia, Luis; Colnot, Céline; Birchmeier, Carmen; Braun, Thomas; Schuelke, Markus; Relaix, Frédéric; Amthor, Helge

2017-08-01

Postnatal growth of skeletal muscle largely depends on the expansion and differentiation of resident stem cells, the so-called satellite cells. Here, we demonstrate that postnatal satellite cells express components of the bone morphogenetic protein (BMP) signaling machinery. Overexpression of noggin in postnatal mice (to antagonize BMP ligands), satellite cell-specific knockout of Alk3 (the gene encoding the BMP transmembrane receptor) or overexpression of inhibitory SMAD6 decreased satellite cell proliferation and accretion during myofiber growth, and ultimately retarded muscle growth. Moreover, reduced BMP signaling diminished the adult satellite cell pool. Abrogation of BMP signaling in satellite cell-derived primary myoblasts strongly diminished cell proliferation and upregulated the expression of cell cycle inhibitors p21 and p57 In conclusion, these results show that BMP signaling defines postnatal muscle development by regulating satellite cell-dependent myofiber growth and the generation of the adult muscle stem cell pool. © 2017. Published by The Company of Biologists Ltd.

Interaction of TGFβ and BMP signaling pathways during chondrogenesis.

Directory of Open Access Journals (Sweden)

Bettina Keller

2011-01-01

Full Text Available TGFβ and BMP signaling pathways exhibit antagonistic activities during the development of many tissues. Although the crosstalk between BMP and TGFβ signaling pathways is well established in bone development, the relationship between these two pathways is less well defined during cartilage development and postnatal homeostasis. We generated hypomorphic mouse models of cartilage-specific loss of BMP and TGFβ signaling to assess the interaction of these pathways in postnatal growth plate homeostasis. We further used the chondrogenic ATDC5 cell line to test effects of BMP and TGFβ signaling on each other's downstream targets. We found that conditional deletion of Smad1 in chondrocytes resulted in a shortening of the growth plate. The addition of Smad5 haploinsufficiency led to a more severe phenotype with shorter prehypertrophic and hypertrophic zones and decreased chondrocyte proliferation. The opposite growth plate phenotype was observed in a transgenic mouse model of decreased chondrocytic TGFβ signaling that was generated by expressing a dominant negative form of the TGFβ receptor I (ΔTβRI in cartilage. Histological analysis demonstrated elongated growth plates with enhanced Ihh expression, as well as an increased proliferation rate with altered production of extracellular matrix components. In contrast, in chondrogenic ATDC5 cells, TGFβ was able to enhance BMP signaling, while BMP2 significantly reduces levels of TGF signaling. In summary, our data demonstrate that during endochondral ossification, BMP and TGFβ signaling can have antagonistic effects on chondrocyte proliferation and differentiation in vivo. We also found evidence of direct interaction between the two signaling pathways in a cell model of chondrogenesis in vitro.

AcvR1-mediated BMP signaling in second heart field is required for arterial pole development: implications for myocardial differentiation and regional identity.

Science.gov (United States)

Thomas, Penny S; Rajderkar, Sudha; Lane, Jamie; Mishina, Yuji; Kaartinen, Vesa

2014-06-15

BMP signaling plays an essential role in second heart field-derived heart and arterial trunk development, including myocardial differentiation, right ventricular growth, and interventricular, outflow tract and aortico-pulmonary septation. It is mediated by a number of different BMP ligands, and receptors, many of which are present simultaneously. The mechanisms by which they regulate morphogenetic events and degree of redundancy amongst them have still to be elucidated. We therefore assessed the role of BMP Type I receptor AcvR1 in anterior second heart field-derived cell development, and compared it with that of BmpR1a. By removing Acvr1 using the driver Mef2c[AHF]-Cre, we show that AcvR1 plays an essential role in arterial pole morphogenesis, identifying defects in outflow tract wall and cushion morphology that preceded a spectrum of septation defects from double outlet right ventricle to common arterial trunk in mutants. Its absence caused dysregulation in gene expression important for myocardial differentiation (Isl1, Fgf8) and regional identity (Tbx2, Tbx3, Tbx20, Tgfb2). Although these defects resemble to some degree those in the equivalent Bmpr1a mutant, a novel gene knock-in model in which Bmpr1a was expressed in the Acvr1 locus only partially restored septation in Acvr1 mutants. These data show that both BmpR1a and AcvR1 are needed for normal heart development, in which they play some non-redundant roles, and refine our understanding of the genetic and morphogenetic processes underlying Bmp-mediated heart development important in human congenital heart disease. Copyright © 2014 Elsevier Inc. All rights reserved.

Dragon enhances BMP signaling and increases transepithelial resistance in kidney epithelial cells.

Science.gov (United States)

Xia, Yin; Babitt, Jodie L; Bouley, Richard; Zhang, Ying; Da Silva, Nicolas; Chen, Shanzhuo; Zhuang, Zhenjie; Samad, Tarek A; Brenner, Gary J; Anderson, Jennifer L; Hong, Charles C; Schneyer, Alan L; Brown, Dennis; Lin, Herbert Y

2010-04-01

The neuronal adhesion protein Dragon acts as a bone morphogenetic protein (BMP) coreceptor that enhances BMP signaling. Given the importance of BMP signaling in nephrogenesis and its putative role in the response to injury in the adult kidney, we studied the localization and function of Dragon in the kidney. We observed that Dragon localized predominantly to the apical surfaces of tubular epithelial cells in the thick ascending limbs, distal convoluted tubules, and collecting ducts of mice. Dragon expression was weak in the proximal tubules and glomeruli. In mouse inner medullary collecting duct (mIMCD3) cells, Dragon generated BMP signals in a ligand-dependent manner, and BMP4 is the predominant endogenous ligand for the Dragon coreceptor. In mIMCD3 cells, BMP4 normally signaled through BMPRII, but Dragon enhanced its signaling through the BMP type II receptor ActRIIA. Dragon and BMP4 increased transepithelial resistance (TER) through the Smad1/5/8 pathway. In epithelial cells isolated from the proximal tubule and intercalated cells of collecting ducts, we observed coexpression of ActRIIA, Dragon, and BMP4 but not BMPRII. Taken together, these results suggest that Dragon may enhance BMP signaling in renal tubular epithelial cells and maintain normal renal physiology.

Orphan nuclear receptor TLX regulates astrogenesis by modulating BMP signaling

Directory of Open Access Journals (Sweden)

Song eQin

2014-04-01

Full Text Available Neural stem cells (NSCs are self-renewing multipotent progenitors that generate both neurons and glia. The precise control of NSC behavior is fundamental to the architecture and function of the central nervous system. We previously demonstrated that the orphan nuclear receptor TLX is required for postnatal NSC activation and neurogenesis in the neurogenic niche. Here, we show that TLX modulates BMP-SMAD signaling to control the timing of postnatal astrogenesis. Genes involved in the BMP signaling pathway, such as Bmp4, Hes1, and Id3, are upregulated in postnatal brains lacking Tlx. Chromatin immunoprecipitation and electrophoretic mobility shift assays reveal that TLX can directly bind the enhancer region of Bmp4. In accordance with elevated BMP signaling, the downstream effectors SMAD1/5/8 are activated by phosphorylation in Tlx mutant mice. Consequently, Tlx mutant brains exhibit an early appearance and increased number of astrocytes with marker expression of glial fibrillary acidic protein (GFAP and S100B. Taken together, these results suggest that TLX tightly controls postnatal astrogenesis through the modulation of BMP-SMAD signaling pathway activity.

Orphan nuclear receptor TLX regulates astrogenesis by modulating BMP signaling.

Science.gov (United States)

Qin, Song; Niu, Wenze; Iqbal, Nida; Smith, Derek K; Zhang, Chun-Li

2014-01-01

Neural stem cells (NSCs) are self-renewing multipotent progenitors that generate both neurons and glia. The precise control of NSC behavior is fundamental to the architecture and function of the central nervous system. We previously demonstrated that the orphan nuclear receptor TLX is required for postnatal NSC activation and neurogenesis in the neurogenic niche. Here, we show that TLX modulates bone morphogenetic protein (BMP)-SMAD signaling to control the timing of postnatal astrogenesis. Genes involved in the BMP signaling pathway, such as Bmp4, Hes1, and Id3, are upregulated in postnatal brains lacking Tlx. Chromatin immunoprecipitation and electrophoretic mobility shift assays reveal that TLX can directly bind the enhancer region of Bmp4. In accordance with elevated BMP signaling, the downstream effectors SMAD1/5/8 are activated by phosphorylation in Tlx mutant mice. Consequently, Tlx mutant brains exhibit an early appearance and increased number of astrocytes with marker expression of glial fibrillary acidic protein (GFAP) and S100B. Taken together, these results suggest that TLX tightly controls postnatal astrogenesis through the modulation of BMP-SMAD signaling pathway activity.

BMP-2 functions independently of SHH signaling and triggers cell condensation and apoptosis in regenerating axolotl limbs

Directory of Open Access Journals (Sweden)

Finnson Kenneth

2010-02-01

Full Text Available Abstract Background Axolotls have the unique ability, among vertebrates, to perfectly regenerate complex body parts, such as limbs, after amputation. In addition, axolotls pattern developing and regenerating autopods from the anterior to posterior axis instead of posterior to anterior like all tetrapods studied to date. Sonic hedgehog is important in establishing this anterior-posterior axis of limbs in all tetrapods including axolotls. Interestingly, its expression is conserved (to the posterior side of limb buds and blastemas in axolotl limbs as in other tetrapods. It has been suggested that BMP-2 may be the secondary mediator of sonic hedgehog, although there is mounting evidence to the contrary in mice. Since BMP-2 expression is on the anterior portion of developing and regenerating limbs prior to digit patterning, opposite to the expression of sonic hedgehog, we examined whether BMP-2 expression was dependent on sonic hedgehog signaling and whether it affects patterning of the autopod during regeneration. Results The expression of BMP-2 and SOX-9 in developing and regenerating axolotl limbs corresponded to the first digits forming in the anterior portion of the autopods. The inhibition of sonic hedgehog signaling with cyclopamine caused hypomorphic limbs (during development and regeneration but did not affect the expression of BMP-2 and SOX-9. Overexpression of BMP-2 in regenerating limbs caused a loss of digits. Overexpression of Noggin (BMP inhibitor in regenerating limbs also resulted in a loss of digits. Histological analysis indicated that the loss due to BMP-2 overexpression was the result of increased cell condensation and apoptosis while the loss caused by Noggin was due to a decrease in cell division. Conclusion The expression of BMP-2 and its target SOX-9 was independent of sonic hedgehog signaling in developing and regenerating limbs. Their expression correlated with chondrogenesis and the appearance of skeletal elements has

Molecular mechanisms of BMP-induced bone formation: Cross-talk between BMP and NF-κB signaling pathways in osteoblastogenesis

Directory of Open Access Journals (Sweden)

Eijiro Jimi

2010-02-01

Full Text Available Osteoblasts are bone-forming cells that differentiate from mesenchymal stem cells. Differentiation processes are coordinately and dynamically controlled in the mesenchymal cells by specific signal transduction pathways. Bone morphogenetic proteins (BMPs, members of the TGF-β superfamily, induce not only bone formation in vivo, but also osteoblast differentiation of mesenchymal cells in vitro. BMP signals are transduced from plasma membrane receptors to the nucleus through both Smad-dependent and -independent pathways, and are regulated by many extracellular and intercellular proteins that interact with BMPs or components of BMP signaling pathways. To understand the molecular mechanisms underlying the role of BMPs in osteoblast differentiation, it is important to elucidate the BMP signaling transduction pathways that are active during osteoblast differentiation. In this review, we summarize the BMP signaling pathways that are known to function in osteoblast development. We also describe our recent findings regarding the molecular mechanisms underlying the cross-talk between BMP/Smad and NF-κB pathways in osteoblast differentiation.

Wnt5a signaling is a substantial constituent in bone morphogenetic protein-2-mediated osteoblastogenesis

International Nuclear Information System (INIS)

Nemoto, Eiji; Ebe, Yukari; Kanaya, Sousuke; Tsuchiya, Masahiro; Nakamura, Takashi; Tamura, Masato; Shimauchi, Hidetoshi

2012-01-01

Highlights: ► Wnt5a is identified in osteoblasts in tibial growth plate and bone marrow. ► Osteoblastic differentiation is associated with increased expression of Wnt5a/Ror2. ► Wnt5a/Ror2 signaling is important for BMP-2-mediated osteoblastic differentiation. ► Wnt5a/Ror2 operates independently of BMP-Smad pathway. -- Abstract: Wnts are secreted glycoproteins that mediate developmental and post-developmental physiology by regulating cellular processes including proliferation, differentiation, and apoptosis through β-catenin-dependent canonical and β-catenin-independent noncanonical pathway. It has been reported that Wnt5a activates noncanonical Wnt signaling through receptor tyrosine kinase-like orphan receptor 2 (Ror2). Although it appears that Wnt5a/Ror2 signaling supports normal bone physiology, the biological significance of noncanonical Wnts in osteogenesis is essentially unknown. In this study, we identified expression of Wnt5a in osteoblasts in the ossification zone of the tibial growth plate as well as bone marrow of the rat tibia as assessed by immunohistochemistry. In addition, we show that osteoblastic differentiation mediated by BMP-2 is associated with increased expression of Wnt5a and Ror2 using cultured pre-osteoblasts, MC3T3-E1 cells. Silencing gene expression of Wnt5a and Ror2 in MC3T3-E1 cells results in suppression of BMP-2-mediated osteoblastic differentiation, suggesting that Wnt5a and Ror2 signaling are of substantial importance for BMP-2-mediated osteoblastic differentiation. BMP-2 stimulation induced phosphorylation of Smad1/5/8 in a similar fashion in both siWnt5a-treated cells and control cells, suggesting that Wnt5a was dispensable for the phosphorylation of Smads by BMP-2. Taken together, our results suggest that Wnt5a/Ror2 signaling appears to be involved in BMP-2-mediated osteoblast differentiation in a Smad independent pathway.

Opposing nodal and BMP signals regulate left-right asymmetry in the sea urchin larva.

Directory of Open Access Journals (Sweden)

Yi-Jyun Luo

Full Text Available Nodal and BMP signals are important for establishing left-right (LR asymmetry in vertebrates. In sea urchins, Nodal signaling prevents the formation of the rudiment on the right side. However, the opposing pathway to Nodal signaling during LR axis establishment is not clear. Here, we revealed that BMP signaling is activated in the left coelomic pouch, specifically in the veg2 lineage, but not in the small micromeres. By perturbing BMP activities, we demonstrated that BMP signaling is required for activating the expression of the left-sided genes and the formation of the left-sided structures. On the other hand, Nodal signals on the right side inhibit BMP signaling and control LR asymmetric separation and apoptosis of the small micromeres. Our findings show that BMP signaling is the positive signal for left-sided development in sea urchins, suggesting that the opposing roles of Nodal and BMP signals in establishing LR asymmetry are conserved in deuterostomes.

BMP and Ihh/PTHrP signaling interact to coordinate chondrocyte proliferation and differentiation.

Science.gov (United States)

Minina, E; Wenzel, H M; Kreschel, C; Karp, S; Gaffield, W; McMahon, A P; Vortkamp, A

2001-11-01

During endochondral ossification, two secreted signals, Indian hedgehog (Ihh) and parathyroid hormone-related protein (PTHrP), have been shown to form a negative feedback loop regulating the onset of hypertrophic differentiation of chondrocytes. Bone morphogenetic proteins (BMPs), another family of secreted factors regulating bone formation, have been implicated as potential interactors of the Ihh/PTHrP feedback loop. To analyze the relationship between the two signaling pathways, we used an organ culture system for limb explants of mouse and chick embryos. We manipulated chondrocyte differentiation by supplementing these cultures either with BMP2, PTHrP and Sonic hedgehog as activators or with Noggin and cyclopamine as inhibitors of the BMP and Ihh/PTHrP signaling systems. Overexpression of Ihh in the cartilage elements of transgenic mice results in an upregulation of PTHrP expression and a delayed onset of hypertrophic differentiation. Noggin treatment of limbs from these mice did not antagonize the effects of Ihh overexpression. Conversely, the promotion of chondrocyte maturation induced by cyclopamine, which blocks Ihh signaling, could not be rescued with BMP2. Thus BMP signaling does not act as a secondary signal of Ihh to induce PTHrP expression or to delay the onset of hypertrophic differentiation. Similar results were obtained using cultures of chick limbs. We further investigated the role of BMP signaling in regulating proliferation and hypertrophic differentiation of chondrocytes and identified three functions of BMP signaling in this process. First we found that maintaining a normal proliferation rate requires BMP and Ihh signaling acting in parallel. We further identified a role for BMP signaling in modulating the expression of IHH: Finally, the application of Noggin to mouse limb explants resulted in advanced differentiation of terminally hypertrophic cells, implicating BMP signaling in delaying the process of hypertrophic differentiation itself. This

BMP-2 Overexpression Augments Vascular Smooth Muscle Cell Motility by Upregulating Myosin Va via Erk Signaling

Directory of Open Access Journals (Sweden)

Ming Zhang

2014-01-01

Full Text Available Background. The disruption of physiologic vascular smooth muscle cell (VSMC migration initiates atherosclerosis development. The biochemical mechanisms leading to dysfunctional VSMC motility remain unknown. Recently, cytokine BMP-2 has been implicated in various vascular physiologic and pathologic processes. However, whether BMP-2 has any effect upon VSMC motility, or by what manner, has never been investigated. Methods. VSMCs were adenovirally transfected to genetically overexpress BMP-2. VSMC motility was detected by modified Boyden chamber assay, confocal time-lapse video assay, and a colony wounding assay. Gene chip array and RT-PCR were employed to identify genes potentially regulated by BMP-2. Western blot and real-time PCR detected the expression of myosin Va and the phosphorylation of extracellular signal-regulated kinases 1/2 (Erk1/2. Immunofluorescence analysis revealed myosin Va expression locale. Intracellular Ca2+ oscillations were recorded. Results. VSMC migration was augmented in VSMCs overexpressing BMP-2 in a dose-dependent manner. siRNA-mediated knockdown of myosin Va inhibited VSMC motility. Both myosin Va mRNA and protein expression significantly increased after BMP-2 administration and were inhibited by Erk1/2 inhibitor U0126. BMP-2 induced Ca2+ oscillations, generated largely by a “cytosolic oscillator”. Conclusion. BMP-2 significantly increased VSMCs migration and myosin Va expression, via the Erk signaling pathway and intracellular Ca2+ oscillations. We provide additional insight into the pathophysiology of atherosclerosis, and inhibition of BMP-2-induced myosin Va expression may represent a potential therapeutic strategy.

SMOC can act as both an antagonist and an expander of BMP signaling.

Science.gov (United States)

Thomas, J Terrig; Eric Dollins, D; Andrykovich, Kristin R; Chu, Tehyen; Stultz, Brian G; Hursh, Deborah A; Moos, Malcolm

2017-03-21

The matricellular protein SMOC (Secreted Modular Calcium binding protein) is conserved phylogenetically from vertebrates to arthropods. We showed previously that SMOC inhibits bone morphogenetic protein (BMP) signaling downstream of its receptor via activation of mitogen-activated protein kinase (MAPK) signaling. In contrast, the most prominent effect of the Drosophila orthologue, pentagone ( pent ), is expanding the range of BMP signaling during wing patterning. Using SMOC deletion constructs we found that SMOC-∆EC, lacking the extracellular calcium binding (EC) domain, inhibited BMP2 signaling, whereas SMOC-EC (EC domain only) enhanced BMP2 signaling. The SMOC-EC domain bound HSPGs with a similar affinity to BMP2 and could expand the range of BMP signaling in an in vitro assay by competition for HSPG-binding. Together with data from studies in vivo we propose a model to explain how these two activities contribute to the function of Pent in Drosophila wing development and SMOC in mammalian joint formation.

Sphingosine 1-phosphate receptor activation enhances BMP-2-induced osteoblast differentiation

International Nuclear Information System (INIS)

Sato, Chieri; Iwasaki, Tsuyoshi; Kitano, Sachie; Tsunemi, Sachi; Sano, Hajime

2012-01-01

Highlights: ► We investigated the role of S1P signaling for osteoblast differentiation. ► Both S1P and FTY enhanced BMP-2-stimulated osteoblast differentiation by C2C12 cells. ► S1P signaling enhanced BMP-2-stimulated Smad and ERK phosphorylation by C2C12 cells. ► MEK/ERK signaling is a pathway underlying S1P signaling for osteoblast differentiation. -- Abstract: We previously demonstrated that sphingosine 1-phosphate (S1P) receptor-mediated signaling induced proliferation and prostaglandin productions by synovial cells from rheumatoid arthritis (RA) patients. In the present study we investigated the role of S1P receptor-mediated signaling for osteoblast differentiation. We investigated osteoblast differentiation using C2C12 myoblasts, a cell line derived from murine satellite cells. Osteoblast differentiation was induced by the treatment of bone morphogenic protein (BMP)-2 in the presence or absence of either S1P or FTY720 (FTY), a high-affinity agonist of S1P receptors. Osteoblast differentiation was determined by osteoblast-specific transcription factor, Runx2 mRNA expression, alkaline phosphatase (ALP) activity and osteocalcin production by the cells. Smad1/5/8 and extracellular signal-regulated kinase (ERK) 1/2 phosphorylation was examined by Western blotting. Osteocalcin production by C2C12 cells were determined by ELISA. Runx2 expression and ALP activity by BMP-2-stimulated C2C12 cells were enhanced by addition of either S1P or FTY. Both S1P and FTY enhanced BMP-2-induced ERK1/2 and Smad1/5/8 phosphorylation. The effect of FTY was stronger than that of S1P. S1P receptor-mediated signaling on osteoblast differentiation was inhibited by addition of mitogen-activated protein kinase/ERK kinase (MEK) 1/2 inhibitor, indicating that the S1P receptor-mediated MEK1/2-ERK1/2 signaling pathway enhanced BMP-2-Smad signaling. These results indicate that S1P receptor-mediated signaling plays a crucial role for osteoblast differentiation.

S113R mutation in SLC33A1 leads to neurodegeneration and augmented BMP signaling in a mouse model

Directory of Open Access Journals (Sweden)

Pingting Liu

2017-01-01

Full Text Available The S113R mutation (c.339T>G (MIM #603690.0001 in SLC33A1 (MIM #603690, an ER membrane acetyl-CoA transporter, has been previously identified in individuals with hereditary spastic paraplegia type 42 (SPG42; MIM #612539. SLC33A1 has also been shown to inhibit the bone morphogenetic protein (BMP signaling pathway in zebrafish. To better understand the function of SLC33A1, we generated and characterized Slc33a1S113R knock-in mice. Homozygous Slc33a1S113R mutant mice were embryonic lethal, whereas heterozygous Slc33a1 mutant mice (Slc33a1wt/mut exhibited behavioral abnormalities and central neurodegeneration, which is consistent with hereditary spastic paraplegia (HSP phenotypes. Importantly, we found an upregulation of BMP signaling in the nervous system and mouse embryonic fibroblasts of Slc33a1wt/mut mice. Using a sciatic nerve crush injury model in vivo and dorsal root ganglion (DRG culture in vitro we showed that injury-induced axonal regeneration in Slc33a1wt/mut mice was accelerated and mediated by upregulated BMP signaling. Exogenous addition of BMP signaling antagonist, noggin, could efficiently alleviate the accelerated injury-induced axonal regrowth. These results indicate that SLC33A1 can negatively regulate BMP signaling in mice, further supporting the notion that upregulation of BMP signaling is a common mechanism of a subset of hereditary spastic paraplegias.

A new molecular logic for BMP-mediated dorsoventral patterning in the leech Helobdella.

Science.gov (United States)

Kuo, Dian-Han; Weisblat, David A

2011-08-09

Bone morphogenetic protein (BMP) signaling is broadly implicated in dorsoventral (DV) patterning of bilaterally symmetric animals [1-3], and its role in axial patterning apparently predates the birth of Bilateria [4-7]. In fly and vertebrate embryos, BMPs and their antagonists (primarily Sog/chordin) diffuse and interact to generate signaling gradients that pattern fields of cells [8-10]. Work in other species reveals diversity in essential facets of this ancient patterning process, however. Here, we report that BMP signaling patterns the DV axis of segmental ectoderm in the leech Helobdella, a clitellate annelid (superphylum Lophotrochozoa) featuring stereotyped developmental cell lineages, but the detailed mechanisms of DV patterning in Helobdella differ markedly from fly and vertebrates. In Helobdella, BMP2/4s are expressed broadly, rather than in dorsal territory, whereas a dorsally expressed BMP5-8 specifies dorsal fate by short-range signaling. A BMP antagonist, gremlin, is upregulated by BMP5-8 in dorsolateral, rather than ventral territory, and yet the BMP-antagonizing activity of gremlin is required for normal ventral cell fates. Gremlin promotes ventral fates without disrupting dorsal fates by selectively inhibiting BMP2/4s, not BMP5-8. Thus, DV patterning in the development of the leech revealed unexpected evolutionary plasticity of the conserved BMP patterning system, presumably reflecting its adaptation to different modes of embryogenesis. Copyright © 2011 Elsevier Ltd. All rights reserved.

Kaempferol inhibits vascular smooth muscle cell migration by modulating BMP-mediated miR-21 expression.

Science.gov (United States)

Kim, Kwangho; Kim, Sunghwan; Moh, Sang Hyun; Kang, Hara

2015-09-01

Bioflavonoids are known to induce cardioprotective effects by inhibiting vascular smooth muscle cell (VSMC) proliferation and migration. Kaempferol has been shown to inhibit VSMC proliferation. However, little is known about the effect of kaempferol on VSMC migration and the underlying molecular mechanisms. Our studies provide the first evidence that kaempferol inhibits VSMC migration by modulating the BMP4 signaling pathway and microRNA expression levels. Kaempferol activates the BMP signaling pathway, induces miR-21 expression and downregulates DOCK4, 5, and 7, leading to inhibition of cell migration. Moreover, kaempferol antagonizes the PDGF-mediated pro-migratory effect. Therefore, our study uncovers a novel regulatory mechanism of VSMC migration by kaempferol and suggests that miRNA modulation by kaempferol is a potential therapy for cardiovascular diseases.

Integrating patterning signals: Wnt/GSK3 regulates the duration of the BMP/Smad1 signal.

Science.gov (United States)

Fuentealba, Luis C; Eivers, Edward; Ikeda, Atsushi; Hurtado, Cecilia; Kuroda, Hiroki; Pera, Edgar M; De Robertis, Edward M

2007-11-30

BMP receptors determine the intensity of BMP signals via Smad1 C-terminal phosphorylations. Here we show that a finely controlled cell biological pathway terminates this activity. The duration of the activated pSmad1(Cter) signal was regulated by sequential Smad1 linker region phosphorylations at conserved MAPK and GSK3 sites required for its polyubiquitinylation and transport to the centrosome. Proteasomal degradation of activated Smad1 and total polyubiquitinated proteins took place in the centrosome. Inhibitors of the Erk, p38, and JNK MAPKs, as well as GSK3 inhibitors, prolonged the duration of a pulse of BMP7. Wnt signaling decreased pSmad1(GSK3) antigen levels and redistributed it from the centrosome to cytoplasmic LRP6 signalosomes. In Xenopus embryos, it was found that Wnts induce epidermis and that this required an active BMP-Smad pathway. Epistatic experiments suggested that the dorsoventral (BMP) and anteroposterior (Wnt/GSK3) patterning gradients are integrated at the level of Smad1 phosphorylations during embryonic pattern formation.

Bmp signaling is at the heart of vertebrate left-right asymmetry

NARCIS (Netherlands)

Verhoeven, M.C.

2009-01-01

Bone morphogenetic protein (Bmp) signaling is vitally important in many aspects of cardiac development. These include cardiac induction and differentiation and establishing the L/R axis. In this thesis, we focus on the role of Bmp signaling in securing proper cardiac asymmetry, by (1) establishing

Characterization of wise protein and its molecular mechanism to interact with both Wnt and BMP signals.

Science.gov (United States)

Lintern, Katherine B; Guidato, Sonia; Rowe, Alison; Saldanha, José W; Itasaki, Nobue

2009-08-21

Cross-talk of BMP and Wnt signaling pathways has been implicated in many aspects of biological events during embryogenesis and in adulthood. A secreted protein Wise and its orthologs (Sostdc1, USAG-1, and Ectodin) have been shown to modulate Wnt signaling and also inhibit BMP signals. Modulation of Wnt signaling activity by Wise is brought about by an interaction with the Wnt co-receptor LRP6, whereas BMP inhibition is by binding to BMP ligands. Here we have investigated the mode of action of Wise on Wnt and BMP signals. It was found that Wise binds LRP6 through one of three loops formed by the cystine knot. The Wise deletion construct lacking the LRP6-interacting loop domain nevertheless binds BMP4 and inhibits BMP signals. Moreover, BMP4 does not interfere with Wise-LRP6 binding, suggesting separate domains for the physical interaction. Functional assays also show that the ability of Wise to block Wnt1 activity through LRP6 is not impeded by BMP4. In contrast, the ability of Wise to inhibit BMP4 is prevented by additional LRP6, implying a preference of Wise in binding LRP6 over BMP4. In addition to the interaction of Wise with BMP4 and LRP6, the molecular characteristics of Wise, such as glycosylation and association with heparan sulfate proteoglycans on the cell surface, are suggested. This study helps to understand the multiple functions of Wise at the molecular level and suggests a possible role for Wise in balancing Wnt and BMP signals.

Sphingosine 1-phosphate receptor activation enhances BMP-2-induced osteoblast differentiation

Energy Technology Data Exchange (ETDEWEB)

Sato, Chieri [Division of Rheumatology, Department of Internal Medicine, Hyogo College of Medicine, 1-1 Mukogawa-cho, Nishinomiya, Hyogo 663-8501 (Japan); Iwasaki, Tsuyoshi, E-mail: tsuyo-i@huhs.ac.jp [Division of Pharmacotherapy, Department of Pharmacy, School of Pharmacy, Hyogo University of Health Sciences, 1-3-6 Minatojima, Chuo-ku, Kobe 650-8530 (Japan); Kitano, Sachie; Tsunemi, Sachi; Sano, Hajime [Division of Rheumatology, Department of Internal Medicine, Hyogo College of Medicine, 1-1 Mukogawa-cho, Nishinomiya, Hyogo 663-8501 (Japan)

2012-06-22

Highlights: Black-Right-Pointing-Pointer We investigated the role of S1P signaling for osteoblast differentiation. Black-Right-Pointing-Pointer Both S1P and FTY enhanced BMP-2-stimulated osteoblast differentiation by C2C12 cells. Black-Right-Pointing-Pointer S1P signaling enhanced BMP-2-stimulated Smad and ERK phosphorylation by C2C12 cells. Black-Right-Pointing-Pointer MEK/ERK signaling is a pathway underlying S1P signaling for osteoblast differentiation. -- Abstract: We previously demonstrated that sphingosine 1-phosphate (S1P) receptor-mediated signaling induced proliferation and prostaglandin productions by synovial cells from rheumatoid arthritis (RA) patients. In the present study we investigated the role of S1P receptor-mediated signaling for osteoblast differentiation. We investigated osteoblast differentiation using C2C12 myoblasts, a cell line derived from murine satellite cells. Osteoblast differentiation was induced by the treatment of bone morphogenic protein (BMP)-2 in the presence or absence of either S1P or FTY720 (FTY), a high-affinity agonist of S1P receptors. Osteoblast differentiation was determined by osteoblast-specific transcription factor, Runx2 mRNA expression, alkaline phosphatase (ALP) activity and osteocalcin production by the cells. Smad1/5/8 and extracellular signal-regulated kinase (ERK) 1/2 phosphorylation was examined by Western blotting. Osteocalcin production by C2C12 cells were determined by ELISA. Runx2 expression and ALP activity by BMP-2-stimulated C2C12 cells were enhanced by addition of either S1P or FTY. Both S1P and FTY enhanced BMP-2-induced ERK1/2 and Smad1/5/8 phosphorylation. The effect of FTY was stronger than that of S1P. S1P receptor-mediated signaling on osteoblast differentiation was inhibited by addition of mitogen-activated protein kinase/ERK kinase (MEK) 1/2 inhibitor, indicating that the S1P receptor-mediated MEK1/2-ERK1/2 signaling pathway enhanced BMP-2-Smad signaling. These results indicate that S1P

Morphogen and community effects determine cell fates in response to BMP4 signaling in human embryonic stem cells.

Science.gov (United States)

Nemashkalo, Anastasiia; Ruzo, Albert; Heemskerk, Idse; Warmflash, Aryeh

2017-09-01

Paracrine signals maintain developmental states and create cell fate patterns in vivo and influence differentiation outcomes in human embryonic stem cells (hESCs) in vitro Systematic investigation of morphogen signaling is hampered by the difficulty of disentangling endogenous signaling from experimentally applied ligands. Here, we grow hESCs in micropatterned colonies of 1-8 cells ('µColonies') to quantitatively investigate paracrine signaling and the response to external stimuli. We examine BMP4-mediated differentiation in µColonies and standard culture conditions and find that in µColonies, above a threshold concentration, BMP4 gives rise to only a single cell fate, contrary to its role as a morphogen in other developmental systems. Under standard culture conditions BMP4 acts as a morphogen but this requires secondary signals and particular cell densities. We find that a 'community effect' enforces a common fate within µColonies, both in the state of pluripotency and when cells are differentiated, and that this effect allows a more precise response to external signals. Using live cell imaging to correlate signaling histories with cell fates, we demonstrate that interactions between neighbors result in sustained, homogenous signaling necessary for differentiation. © 2017. Published by The Company of Biologists Ltd.

The Gyc76C Receptor Guanylyl Cyclase and the Foraging cGMP-Dependent Kinase Regulate Extracellular Matrix Organization and BMP Signaling in the Developing Wing of Drosophila melanogaster.

Science.gov (United States)

Schleede, Justin; Blair, Seth S

2015-10-01

The developing crossveins of the wing of Drosophila melanogaster are specified by long-range BMP signaling and are especially sensitive to loss of extracellular modulators of BMP signaling such as the Chordin homolog Short gastrulation (Sog). However, the role of the extracellular matrix in BMP signaling and Sog activity in the crossveins has been poorly explored. Using a genetic mosaic screen for mutations that disrupt BMP signaling and posterior crossvein development, we identify Gyc76C, a member of the receptor guanylyl cyclase family that includes mammalian natriuretic peptide receptors. We show that Gyc76C and the soluble cGMP-dependent kinase Foraging, likely linked by cGMP, are necessary for normal refinement and maintenance of long-range BMP signaling in the posterior crossvein. This does not occur through cell-autonomous crosstalk between cGMP and BMP signal transduction, but likely through altered extracellular activity of Sog. We identify a novel pathway leading from Gyc76C to the organization of the wing extracellular matrix by matrix metalloproteinases, and show that both the extracellular matrix and BMP signaling effects are largely mediated by changes in the activity of matrix metalloproteinases. We discuss parallels and differences between this pathway and other examples of cGMP activity in both Drosophila melanogaster and mammalian cells and tissues.

Bmp indicator mice reveal dynamic regulation of transcriptional response.

Directory of Open Access Journals (Sweden)

Anna L Javier

Full Text Available Cellular responses to Bmp ligands are regulated at multiple levels, both extracellularly and intracellularly. Therefore, the presence of these growth factors is not an accurate indicator of Bmp signaling activity. While a common approach to detect Bmp signaling activity is to determine the presence of phosphorylated forms of Smad1, 5 and 8 by immunostaining, this approach is time consuming and not quantitative. In order to provide a simpler readout system to examine the presence of Bmp signaling in developing animals, we developed BRE-gal mouse embryonic stem cells and a transgenic mouse line that specifically respond to Bmp ligand stimulation. Our reporter identifies specific transcriptional responses that are mediated by Smad1 and Smad4 with the Schnurri transcription factor complex binding to a conserved Bmp-Responsive Element (BRE, originally identified among Drosophila, Xenopus and human Bmp targets. Our BRE-gal mES cells specifically respond to Bmp ligands at concentrations as low as 5 ng/ml; and BRE-gal reporter mice, derived from the BRE-gal mES cells, show dynamic activity in many cellular sites, including extraembryonic structures and mammary glands, thereby making this a useful scientific tool.

Activation of PKA/CREB Signaling is Involved in BMP9-Induced Osteogenic Differentiation of Mesenchymal Stem Cells

Directory of Open Access Journals (Sweden)

Hongyu Zhang

2015-09-01

Full Text Available Background/Aims: BMP9 is highly capable of promoting osteogenic differentiation of mesenchymal stem cells (MSCs although the molecular mechanism involved is largely unknown. Here, we explored the detail role of PKA/CREB signaling in BMP9-induced osteogenic differentiation. Methods: Activation status of PKA/CREB signaling is assessed by nonradioactive assay and Western blot. Using PKA inhibitors and a dominant negative protein of CREB (A-CREB, we investigated the effect of PKA/CREB signaling on BMP9-induced osteogenic differentiation. Results: We found that BMP9 promotes PKA activity and enhances CREB phosphorylation in MSCs. BMP9 is shown to down-regulate protein kinase A inhibitor γ (PKIγ expression. We demonstrated that PKA inhibitors suppress BMP9-induced early osteogenic marker alkaline phosphatase (ALP activity in MSCs as well as late osteogenic markers osteopontin (OPN, osteocalcin (OCN and matrix mineralization. We found that PKA inhibitor reduces BMP9-induced Runx2 activation and p38 phosphorylation in MSCs. Lastly, interference of CREB function by A-CREB decreased BMP9-induced osteogenic differentiation as well. Conclusion: Our results revealed that BMP9 may activate PKA/CREB signaling in MSCs through suppression of PKIγ expression. It is noteworthy that inhibition of PKA/CREB signaling may impair BMP9-induced osteogenic differentiation of MSCs, implying that activation of PKA/CREB signaling is required for BMP9 osteoinductive activity.

Drosophila Nociceptive Sensitization Requires BMP Signaling via the Canonical SMAD Pathway.

Science.gov (United States)

Follansbee, Taylor L; Gjelsvik, Kayla J; Brann, Courtney L; McParland, Aidan L; Longhurst, Colin A; Galko, Michael J; Ganter, Geoffrey K

2017-08-30

Nociceptive sensitization is a common feature in chronic pain, but its basic cellular mechanisms are only partially understood. The present study used the Drosophila melanogaster model system and a candidate gene approach to identify novel components required for modulation of an injury-induced nociceptive sensitization pathway presumably downstream of Hedgehog. This study demonstrates that RNAi silencing of a member of the Bone Morphogenetic Protein (BMP) signaling pathway, Decapentaplegic (Dpp), specifically in the Class IV multidendritic nociceptive neuron, significantly attenuated ultraviolet injury-induced sensitization. Furthermore, overexpression of Dpp in Class IV neurons was sufficient to induce thermal hypersensitivity in the absence of injury. The requirement of various BMP receptors and members of the SMAD signal transduction pathway in nociceptive sensitization was also demonstrated. The effects of BMP signaling were shown to be largely specific to the sensitization pathway and not associated with changes in nociception in the absence of injury or with changes in dendritic morphology. Thus, the results demonstrate that Dpp and its pathway play a crucial and novel role in nociceptive sensitization. Because the BMP family is so strongly conserved between vertebrates and invertebrates, it seems likely that the components analyzed in this study represent potential therapeutic targets for the treatment of chronic pain in humans. SIGNIFICANCE STATEMENT This report provides a genetic analysis of primary nociceptive neuron mechanisms that promote sensitization in response to injury. Drosophila melanogaster larvae whose primary nociceptive neurons were reduced in levels of specific components of the BMP signaling pathway, were injured and then tested for nocifensive responses to a normally subnoxious stimulus. Results suggest that nociceptive neurons use the BMP2/4 ligand, along with identified receptors and intracellular transducers to transition to a

Patterning of the dorsal-ventral axis in echinoderms: insights into the evolution of the BMP-chordin signaling network.

Directory of Open Access Journals (Sweden)

François Lapraz

2009-11-01

Full Text Available Formation of the dorsal-ventral axis of the sea urchin embryo relies on cell interactions initiated by the TGFbeta Nodal. Intriguingly, although nodal expression is restricted to the ventral side of the embryo, Nodal function is required for specification of both the ventral and the dorsal territories and is able to restore both ventral and dorsal regions in nodal morpholino injected embryos. The molecular basis for the long-range organizing activity of Nodal is not understood. In this paper, we provide evidence that the long-range organizing activity of Nodal is assured by a relay molecule synthesized in the ventral ectoderm, then translocated to the opposite side of the embryo. We identified this relay molecule as BMP2/4 based on the following arguments. First, blocking BMP2/4 function eliminated the long-range organizing activity of an activated Nodal receptor in an axis rescue assay. Second, we demonstrate that BMP2/4 and the corresponding type I receptor Alk3/6 functions are both essential for specification of the dorsal region of the embryo. Third, using anti-phospho-Smad1/5/8 immunostaining, we show that, despite its ventral transcription, the BMP2/4 ligand triggers receptor mediated signaling exclusively on the dorsal side of the embryo, one of the most extreme cases of BMP translocation described so far. We further report that the pattern of pSmad1/5/8 is graded along the dorsal-ventral axis and that two BMP2/4 target genes are expressed in nested patterns centered on the region with highest levels of pSmad1/5/8, strongly suggesting that BMP2/4 is acting as a morphogen. We also describe the very unusual ventral co-expression of chordin and bmp2/4 downstream of Nodal and demonstrate that Chordin is largely responsible for the spatial restriction of BMP2/4 signaling to the dorsal side. Thus, unlike in most organisms, in the sea urchin, a single ventral signaling centre is responsible for induction of ventral and dorsal cell fates. Finally

Interaction of Wnt Signaling with BMP/Smad Signaling during the Transition from Cell Proliferation to Myogenic Differentiation in Mouse Myoblast-Derived Cells

Directory of Open Access Journals (Sweden)

Kumiko Terada

2013-01-01

Full Text Available Background. Wnt signaling is involved in muscle formation through β-catenin-dependent or -independent pathways, but interactions with other signaling pathways including transforming growth factor β/Smad have not been precisely elucidated. Results. As Wnt4 stimulates myogenic differentiation by antagonizing myostatin (GDF8 activity, we examined the role of Wnt4 signaling during muscle differentiation in the C2C12 myoblast cell line. Among several extrinsic signaling molecules examined in a microarray analysis of C2C12 cells during the transition from cell proliferation to differentiation after mitogen deprivation, bone morphogenetic protein 4 (BMP4 expression was prominently increased. Wnt4 overexpression had similar effects on BMP4 expression. BMP4 was able to inhibit muscle differentiation when added to the culture medium. BMP4 and noggin had no effects on the cellular localization of β-catenin induced by Wnt3a; however, the BMP4-induced phosphorylation of Smad1/5/8 was enhanced by Wnt4, but not by Wnt3a. The BMP antagonist noggin effectively stimulated muscle differentiation through binding to endogenous BMPs, and the effect of noggin was enhanced by the presence of Wnt3a and Wnt4. Conclusion. These results suggest that BMP/Smad pathways are modified through Wnt signaling during the transition from progenitor cell proliferation to myogenic differentiation, although Wnt/β-catenin signaling is not modified with BMP/Smad signaling.

The Gyc76C Receptor Guanylyl Cyclase and the Foraging cGMP-Dependent Kinase Regulate Extracellular Matrix Organization and BMP Signaling in the Developing Wing of Drosophila melanogaster.

Directory of Open Access Journals (Sweden)

Justin Schleede

2015-10-01

Full Text Available The developing crossveins of the wing of Drosophila melanogaster are specified by long-range BMP signaling and are especially sensitive to loss of extracellular modulators of BMP signaling such as the Chordin homolog Short gastrulation (Sog. However, the role of the extracellular matrix in BMP signaling and Sog activity in the crossveins has been poorly explored. Using a genetic mosaic screen for mutations that disrupt BMP signaling and posterior crossvein development, we identify Gyc76C, a member of the receptor guanylyl cyclase family that includes mammalian natriuretic peptide receptors. We show that Gyc76C and the soluble cGMP-dependent kinase Foraging, likely linked by cGMP, are necessary for normal refinement and maintenance of long-range BMP signaling in the posterior crossvein. This does not occur through cell-autonomous crosstalk between cGMP and BMP signal transduction, but likely through altered extracellular activity of Sog. We identify a novel pathway leading from Gyc76C to the organization of the wing extracellular matrix by matrix metalloproteinases, and show that both the extracellular matrix and BMP signaling effects are largely mediated by changes in the activity of matrix metalloproteinases. We discuss parallels and differences between this pathway and other examples of cGMP activity in both Drosophila melanogaster and mammalian cells and tissues.

Structural Basis of the Human Endoglin-BMP9 Interaction: Insights into BMP Signaling and HHT1

Directory of Open Access Journals (Sweden)

Takako Saito

2017-05-01

Full Text Available Endoglin (ENG/CD105 is an essential endothelial cell co-receptor of the transforming growth factor β (TGF-β superfamily, mutated in hereditary hemorrhagic telangiectasia type 1 (HHT1 and involved in tumor angiogenesis and preeclampsia. Here, we present crystal structures of the ectodomain of human ENG and its complex with the ligand bone morphogenetic protein 9 (BMP9. BMP9 interacts with a hydrophobic surface of the N-terminal orphan domain of ENG, which adopts a new duplicated fold generated by circular permutation. The interface involves residues mutated in HHT1 and overlaps with the epitope of tumor-suppressing anti-ENG monoclonal TRC105. The structure of the C-terminal zona pellucida module suggests how two copies of ENG embrace homodimeric BMP9, whose binding is compatible with ligand recognition by type I but not type II receptors. These findings shed light on the molecular basis of the BMP signaling cascade, with implications for future therapeutic interventions in this fundamental pathway.

Dual role of BMP signaling in the regulation of Drosophila intestinal stem cell self-renewal.

Science.gov (United States)

Tian, Aiguo; Jiang, Jin

2017-10-02

Many adult organs including Drosophila adult midguts rely on resident stem cells to replenish damaged cells during tissue homeostasis and regeneration. Previous studies have shown that, upon injury, intestinal stem cells (ISCs) in the midguts can increase proliferation and lineage differentiation to meet the demand for tissue repair. Our recent study has demonstrated that, in response to certain injury, midguts can expand ISC population size as an additional regenerative mechanism. We found that injury elicited by bleomycin feeding or bacterial infection increased the production of two BMP ligands (Dpp and Gbb) in enterocytes (ECs), leading to elevated BMP signaling in progenitor cells that drove an expansion of ISCs by promoting their symmetric self-renewing division. Interestingly, we also found that BMP signaling in ECs inhibits the production of Dpp and Gbb, and that this negative feedback mechanism is required to reset ISC pool size to the homeostatic state. Our findings suggest that BMP signaling exerts two opposing influences on stem cell activity depending on where it acts: BMP signaling in progenitor cells promotes ISC self-renewal while BMP signaling in ECs restricts ISC self-renewal by preventing excessive production of BMP ligands. Our results further suggest that transient expansion of ISC population in conjunction with increasing ISC proliferation provides a more effective strategy for tissue regeneration.

Timing is everything: Reiterative Wnt, BMP and RA signaling regulate developmental competence during endoderm organogenesis.

Science.gov (United States)

Rankin, Scott A; McCracken, Kyle W; Luedeke, David M; Han, Lu; Wells, James M; Shannon, John M; Zorn, Aaron M

2018-02-01

A small number of signaling pathways are used repeatedly during organogenesis, and they can have drastically different effects on the same population of cells depending on the embryonic stage. How cellular competence changes over developmental time is not well understood. Here we used Xenopus, mouse, and human pluripotent stem cells to investigate how the temporal sequence of Wnt, BMP, and retinoic acid (RA) signals regulates endoderm developmental competence and organ induction, focusing on respiratory fate. While Nkx2-1+ lung fate is not induced until late somitogenesis stages, here we show that lung competence is restricted by the gastrula stage as a result of Wnt and BMP-dependent anterior-posterior (A-P) patterning. These early Wnt and BMP signals make posterior endoderm refractory to subsequent RA/Wnt/BMP-dependent lung induction. We further mapped how RA modulates the response to Wnt and BMP in a temporal specific manner. In the gastrula RA promotes posterior identity, however in early somite stages of development RA regulates respiratory versus pharyngeal potential in anterior endoderm and midgut versus hindgut potential in posterior endoderm. Together our data suggest a dynamic and conserved response of vertebrate endoderm during organogenesis, wherein early Wnt/BMP/RA impacts how cells respond to later Wnt/BMP/RA signals, illustrating how reiterative combinatorial signaling can regulate both developmental competence and subsequent fate specification. Copyright © 2017 Elsevier Inc. All rights reserved.

Dkk1 haploinsufficiency requires expression of Bmp2 for bone anabolic activity

Science.gov (United States)

Intini, Giuseppe; Nyman, Jeffry S.

2015-01-01

Bone fractures remain a serious health burden and prevention and enhanced healing of fractures has been obtained by augmenting either BMP or Wnt signaling. However, whether BMP and Wnt signaling are both required or are self-sufficient for anabolic and fracture healing activities has never been fully elucidated. Mice haploinsufficient for Dkk1 (Dkk1+/−) exhibit a high bone mass phenotype due to an up-regulation of canonical Wnt signaling while mice lacking Bmp2 expression in the limbs (Bmp2c/c;Prx1::cre) succumb to spontaneous fracture and are unable to initiate fracture healing; combined, these mice offer an opportunity to examine the requirement for activated BMP signaling on the anabolic and fracture healing activity of Wnts. When Dkk1+/− mice were crossed with Bmp2c/c;Prx1::cre mice, the offspring bearing both genetic alterations were unable to increase bone mass and heal fractures, indicating that increased canonical Wnt signaling is unable to exploit its activity in absence of Bmp2. Thus, our data suggest that BMP signaling is required for Wnt-mediated anabolic activity and that therapies aimed at preventing fractures and fostering fracture repair may need to target both pathways for maximal efficacy. PMID:25603465

Bone morphogenetic protein 9 (BMP9) and BMP10 enhance tumor necrosis factor-α-induced monocyte recruitment to the vascular endothelium mainly via activin receptor-like kinase 2.

Science.gov (United States)

Mitrofan, Claudia-Gabriela; Appleby, Sarah L; Nash, Gerard B; Mallat, Ziad; Chilvers, Edwin R; Upton, Paul D; Morrell, Nicholas W

2017-08-18

Bone morphogenetic proteins 9 and 10 (BMP9/BMP10) are circulating cytokines with important roles in endothelial homeostasis. The aim of this study was to investigate the roles of BMP9 and BMP10 in mediating monocyte-endothelial interactions using an in vitro flow adhesion assay. Herein, we report that whereas BMP9/BMP10 alone had no effect on monocyte recruitment, at higher concentrations both cytokines synergized with tumor necrosis factor-α (TNFα) to increase recruitment to the vascular endothelium. The BMP9/BMP10-mediated increase in monocyte recruitment in the presence of TNFα was associated with up-regulated expression levels of E-selectin, vascular cell adhesion molecule (VCAM-1), and intercellular adhesion molecule 1 (ICAM-1) on endothelial cells. Using siRNAs to type I and II BMP receptors and the signaling intermediaries (Smads), we demonstrated a key role for ALK2 in the BMP9/BMP10-induced surface expression of E-selectin, and both ALK1 and ALK2 in the up-regulation of VCAM-1 and ICAM-1. The type II receptors, BMPR-II and ACTR-IIA were both required for this response, as was Smad1/5. The up-regulation of cell surface adhesion molecules by BMP9/10 in the presence of TNFα was inhibited by LDN193189, which inhibits ALK2 but not ALK1. Furthermore, LDN193189 inhibited monocyte recruitment induced by TNFα and BMP9/10. BMP9/10 increased basal IκBα protein expression, but did not alter p65/RelA levels. Our findings suggest that higher concentrations of BMP9/BMP10 synergize with TNFα to induce the up-regulation of endothelial selectins and adhesion molecules, ultimately resulting in increased monocyte recruitment to the vascular endothelium. This process is mediated mainly via the ALK2 type I receptor, BMPR-II/ACTR-IIA type II receptors, and downstream Smad1/5 signaling. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

BMP4 signaling is involved in the generation of inner ear sensory epithelia

Directory of Open Access Journals (Sweden)

Wang Yucheng

2005-08-01

Full Text Available Abstract Background The robust expression of BMP4 in the incipient sensory organs of the inner ear suggests possible roles for this signaling protein during induction and development of auditory and vestibular sensory epithelia. Homozygous BMP4-/- animals die before the inner ear's sensory organs develop, which precludes determining the role of BMP4 in these organs with simple gene knockout experiments. Results Here we use a chicken otocyst culture system to perform quantitative studies on the development of inner ear cell types and show that hair cell and supporting cell generation is remarkably reduced when BMP signaling is blocked, either with its antagonist noggin or by using soluble BMP receptors. Conversely, we observed an increase in the number of hair cells when cultured otocysts were treated with exogenous BMP4. BMP4 treatment additionally prompted down-regulation of Pax-2 protein in proliferating sensory epithelial progenitors, leading to reduced progenitor cell proliferation. Conclusion Our results implicate BMP4 in two events during chicken inner ear sensory epithelium formation: first, in inducing the switch from proliferative sensory epithelium progenitors to differentiating epithelial cells and secondly, in promoting the differentiation of hair cells within the developing sensory epithelia.

Zirconium ions up-regulate the BMP/SMAD signaling pathway and promote the proliferation and differentiation of human osteoblasts.

Directory of Open Access Journals (Sweden)

Yongjuan Chen

Full Text Available Zirconium (Zr is an element commonly used in dental and orthopedic implants either as zirconia (ZrO2 or in metal alloys. It can also be incorporated into calcium silicate-based ceramics. However, the effects of in vitro culture of human osteoblasts (HOBs with soluble ionic forms of Zr have not been determined. In this study, primary culture of human osteoblasts was conducted in the presence of medium containing either ZrCl4 or Zirconium (IV oxynitrate (ZrO(NO32 at concentrations of 0, 5, 50 and 500 µM, and osteoblast proliferation, differentiation and calcium deposition were assessed. Incubation of human osteoblast cultures with Zr ions increased the proliferation of human osteoblasts and also gene expression of genetic markers of osteoblast differentiation. In 21 and 28 day cultures, Zr ions at concentrations of 50 and 500 µM increased the deposition of calcium phosphate. In addition, the gene expression of BMP2 and BMP receptors was increased in response to culture with Zr ions and this was associated with increased phosphorylation of SMAD1/5. Moreover, Noggin suppressed osteogenic gene expression in HOBs co-treated with Zr ions. In conclusion, Zr ions appear able to induce both the proliferation and the differentiation of primary human osteoblasts. This is associated with up-regulation of BMP2 expression and activation of BMP signaling suggesting this action is, at least in part, mediated by BMP signaling.

Zirconium Ions Up-Regulate the BMP/SMAD Signaling Pathway and Promote the Proliferation and Differentiation of Human Osteoblasts

Science.gov (United States)

Chen, Yongjuan; Roohani-Esfahani, Seyed-Iman; Lu, ZuFu; Zreiqat, Hala; Dunstan, Colin R.

2015-01-01

Zirconium (Zr) is an element commonly used in dental and orthopedic implants either as zirconia (ZrO2) or in metal alloys. It can also be incorporated into calcium silicate-based ceramics. However, the effects of in vitro culture of human osteoblasts (HOBs) with soluble ionic forms of Zr have not been determined. In this study, primary culture of human osteoblasts was conducted in the presence of medium containing either ZrCl4 or Zirconium (IV) oxynitrate (ZrO(NO3)2) at concentrations of 0, 5, 50 and 500 µM, and osteoblast proliferation, differentiation and calcium deposition were assessed. Incubation of human osteoblast cultures with Zr ions increased the proliferation of human osteoblasts and also gene expression of genetic markers of osteoblast differentiation. In 21 and 28 day cultures, Zr ions at concentrations of 50 and 500 µM increased the deposition of calcium phosphate. In addition, the gene expression of BMP2 and BMP receptors was increased in response to culture with Zr ions and this was associated with increased phosphorylation of SMAD1/5. Moreover, Noggin suppressed osteogenic gene expression in HOBs co-treated with Zr ions. In conclusion, Zr ions appear able to induce both the proliferation and the differentiation of primary human osteoblasts. This is associated with up-regulation of BMP2 expression and activation of BMP signaling suggesting this action is, at least in part, mediated by BMP signaling. PMID:25602473

NOX4 mediates BMP4-induced upregulation of TRPC1 and 6 protein expressions in distal pulmonary arterial smooth muscle cells.

Directory of Open Access Journals (Sweden)

Qian Jiang

Full Text Available Our previous studies demonstrated that bone morphogenetic protein 4 (BMP4 mediated, elevated expression of canonical transient receptor potential (TRPC largely accounts for the enhanced proliferation in pulmonary arterial smooth muscle cells (PASMCs. In the present study, we sought to determine the signaling pathway through which BMP4 up-regulates TRPC expression.We employed recombinant human BMP4 (rhBMP4 to determine the effects of BMP4 on NADPH oxidase 4 (NOX4 and reactive oxygen species (ROS production in rat distal PASMCs. We also designed small interfering RNA targeting NOX4 (siNOX4 and detected whether NOX4 knockdown affects rhBMP4-induced ROS, TRPC1 and 6 expression, cell proliferation and intracellular Ca2+ determination in PASMCs.In rhBMP4 treated rat distal PASMCs, NOX4 expression was (226.73±11.13 %, and the mean ROS level was (123.65±1.62 % of that in untreated control cell. siNOX4 transfection significantly reduced rhBMP4-induced elevation of the mean ROS level in PASMCs. Moreover, siNOX4 transfection markedly reduced rhBMP4-induced elevation of TRPC1 and 6 proteins, basal [Ca2+]i and SOCE. Furthermore, compared with control group (0.21±0.001, the proliferation of rhBMP4 treated cells was significantly enhanced (0.41±0.001 (P<0.01. However, such increase was attenuated by knockdown of NOX4. Moreover, external ROS (H2O2 100 µM, 24 h rescued the effects of NOX4 knockdown, which included the declining of TRPC1 and 6 expression, basal intracellular calcium concentration ([Ca2+]i and store-operated calcium entry (SOCE, suggesting that NOX4 plays as an important mediator in BMP4-induced proliferation and intracellular calcium homeostasis.These results suggest that BMP4 may increase ROS level, enhance TRPC1 and 6 expression and proliferation by up-regulating NOX4 expression in PASMCs.

Bone Morphogenetic Protein 15 (BMP15) Acts as a BMP and Wnt Inhibitor during Early Embryogenesis*

Science.gov (United States)

Di Pasquale, Elisa; Brivanlou, Ali H.

2009-01-01

Bone morphogenetic protein 15 (BMP15) belongs to an unusual subgroup of the transforming growth factor β (TGFβ) superfamily of signaling ligands as it lacks a key cysteine residue in the mature region required for proper intermolecular dimerization. Naturally occurring BMP15 mutation leads to early ovarian failure in humans, and BMP15 has been shown to activate the Smad1/5/8 pathway in that context. Despite its important role in germ cell specification, the embryological function of BMP15 remains unknown. Surprisingly, we find that during early Xenopus embryogenesis BMP15 acts solely as an inhibitor of the Smad1/5/8 pathway and the Wnt pathway. BMP15 gain-of-function leads to embryos with secondary ectopic heads and to direct neural induction in intact explants. BMP15 inhibits BMP4-mediated epidermal induction in dissociated explants. BMP15 strongly inhibits BRE response induced by BMP4 and blocks phosphorylation and activation of Smad1/5/8 MH2-domain. Mechanistically, BMP15 protein specifically interacts with BMP4 protein, suggesting inhibition upstream of receptor binding. Loss-of-function experiments using morpholinos or a naturally occurring human BMP15 dominant-negative mutant (BMP15-Y235C) leads to embryos lacking head. BMP15-Y235C also eliminates the inhibitory activity of BMP15 on BRE (BMP-responsive element). Finally, we show that BMP15 inhibits the canonical branch of the Wnt pathway, upstream of β-catenin. We, thus, demonstrate that BMP15 is necessary and sufficient for the specification of dorso-anterior structures and highlight novel mechanisms of BMP15 function that strongly suggest a reinterpretation of its function in ovaries specially for ovarian failure. PMID:19553676

BMP-2 Induced Expression of Alx3 That Is a Positive Regulator of Osteoblast Differentiation.

Directory of Open Access Journals (Sweden)

Takashi Matsumoto

Full Text Available Bone morphogenetic proteins (BMPs regulate many aspects of skeletal development, including osteoblast and chondrocyte differentiation, cartilage and bone formation, and cranial and limb development. Among them, BMP-2, one of the most potent osteogenic signaling molecules, stimulates osteoblast differentiation, while it inhibits myogenic differentiation in C2C12 cells. To evaluate genes involved in BMP-2-induced osteoblast differentiation, we performed cDNA microarray analyses to compare BMP-2-treated and -untreated C2C12 cells. We focused on Alx3 (aristaless-like homeobox 3 which was clearly induced during osteoblast differentiation. Alx3, a homeobox gene related to the Drosophilaaristaless gene, has been linked to developmental functions in craniofacial structures and limb development. However, little is known about its direct relationship with bone formation. In the present study, we focused on the mechanisms of Alx3 gene expression and function during osteoblast differentiation induced by BMP-2. In C2C12 cells, BMP-2 induced increase of Alx3 gene expression in both time- and dose-dependent manners through the BMP receptors-mediated SMAD signaling pathway. In addition, silencing of Alx3 by siRNA inhibited osteoblast differentiation induced by BMP-2, as showed by the expressions of alkaline phosphatase (Alp, Osteocalcin, and Osterix, while over-expression of Alx3 enhanced osteoblast differentiation induced by BMP-2. These results indicate that Alx3 expression is enhanced by BMP-2 via the BMP receptors mediated-Smad signaling and that Alx3 is a positive regulator of osteoblast differentiation induced by BMP-2.

Oxygen-glucose deprivation preconditioning protects neurons against oxygen-glucose deprivation/reperfusion induced injury via bone morphogenetic protein-7 mediated ERK, p38 and Smad signalling pathways.

Science.gov (United States)

Guan, Junhong; Du, Shaonan; Lv, Tao; Qu, Shengtao; Fu, Qiang; Yuan, Ye

2016-01-01

Bone morphogenetic protein (BMP)-7 mediated neuroprotective effect of cerebral ischemic preconditioning (IPC) has been studied in an ischemic animal model, but the underlying cellular mechanisms have not been clearly clarified. In this study, primary cortical neurons and the SH-SY5Y cell line were used to investigate the role of BMP-7 and its downstream signals in the neuroprotective effects of oxygen-glucose deprivation preconditioning (OGDPC). Immunocytochemistry was used to detect the expression of neurofilament in neurons. MTT and lactate dehydrogenase activity assays were used to measure the cytotoxicity. Western blot was used to detect the protein expression of BMP-7 and downstream signals. BMP inhibitor, mitogen-activated protein kinase inhibitors, Smad inhibitor and siRNA of Smad 1 were used to investigate the role of corresponding signalling pathways in the OGDPC. Results showed that OGDPC-induced overexpression of BMP-7 in primary cortical neurons and SH-SY5Y cells. Both of endogenous and exogenous BMP-7 could replicate the neuroprotective effects seen in OGDPC pretreatment. In addition, extracellular regulated protein kinases, p38 and Smad signalling pathway were found to be involved in the neuroprotective effects mediated by OGDPC via BMP-7. This study primarily reveals the cellular mechanisms of the neuroprotection mediated by OGDPC, and provides evidence for better understanding of this intrinsic factor against ischemia. © 2015 Wiley Publishing Asia Pty Ltd.

Abnormal Activation of BMP Signaling Causes Myopathy in Fbn2 Null Mice.

Directory of Open Access Journals (Sweden)

Gerhard Sengle

2015-06-01

Full Text Available Fibrillins are large extracellular macromolecules that polymerize to form the backbone structure of connective tissue microfibrils. Mutations in the gene for fibrillin-1 cause the Marfan syndrome, while mutations in the gene for fibrillin-2 cause Congenital Contractural Arachnodactyly. Both are autosomal dominant disorders, and both disorders affect musculoskeletal tissues. Here we show that Fbn2 null mice (on a 129/Sv background are born with reduced muscle mass, abnormal muscle histology, and signs of activated BMP signaling in skeletal muscle. A delay in Myosin Heavy Chain 8, a perinatal myosin, was found in Fbn2 null forelimb muscle tissue, consistent with the notion that muscle defects underlie forelimb contractures in these mice. In addition, white fat accumulated in the forelimbs during the early postnatal period. Adult Fbn2 null mice are already known to demonstrate persistent muscle weakness. Here we measured elevated creatine kinase levels in adult Fbn2 null mice, indicating ongoing cycles of muscle injury. On a C57Bl/6 background, Fbn2 null mice showed severe defects in musculature, leading to neonatal death from respiratory failure. These new findings demonstrate that loss of fibrillin-2 results in phenotypes similar to those found in congenital muscular dystrophies and that FBN2 should be considered as a candidate gene for recessive congenital muscular dystrophy. Both in vivo and in vitro evidence associated muscle abnormalities and accumulation of white fat in Fbn2 null mice with abnormally activated BMP signaling. Genetic rescue of reduced muscle mass and accumulation of white fat in Fbn2 null mice was accomplished by deleting a single allele of Bmp7. In contrast to other reports that activated BMP signaling leads to muscle hypertrophy, our findings demonstrate the exquisite sensitivity of BMP signaling to the fibrillin-2 extracellular environment during early postnatal muscle development. New evidence presented here suggests that

Lactoferricin enhances BMP7-stimulated anabolic pathways in intervertebral disc cells.

Science.gov (United States)

Ellman, Michael B; Kim, Jaesung; An, Howard S; Chen, Di; Kc, Ranjan; Li, Xin; Xiao, Guozhi; Yan, Dongyao; Suh, Joon; van Wjnen, Andre J; Wang, James H-C; Kim, Su-Gwan; Im, Hee-Jeong

2013-07-25

Bone-morphogenetic protein-7 (BMP7) is a well-known anabolic and anti-catabolic growth factor on intervertebral disc (IVD) matrix and cell homeostasis. Similarly, Lactoferricin B (LfcinB) has recently been shown to have pro-anabolic, anti-catabolic, anti-oxidative and/or anti-inflammatory effects in bovine disc cells in vitro. In this study, we investigated the potential benefits of using combined peptide therapy with LfcinB and BMP7 for intervertebral disc matrix repair and to understand cellular and signaling mechanisms controlled by these factors. We studied the effects of BMP7 and LfcinB as individual treatments and combined therapy on bovine nucleus pulposus (NP) cells by assessing proteoglycan (PG) accumulation and synthesis, and the gene expression of matrix protein aggrecan and transcription factor SOX-9. We also analyzed the role of Noggin, a BMP antagonist, in IVD tissue and examined its effect after stimulation with LfcinB. To understand the molecular mechanisms by which LfcinB synergizes with BMP7, we investigated the ERK-SP1 axis as a downstream intracellular signaling regulator involved in BMP7 and LfcinB-mediated activities. Treatment of bovine NP cells cultured in alginate with LfcinB plus BMP7 synergistically stimulates PG synthesis and accumulation in part by upregulation of aggrecan gene expression. The synergism results from LfcinB-mediated activation of Sp1 and SMAD signaling pathways by (i) phosphorylation of SMAD 1/5/8; (ii) downregulation of SMAD inhibitory factors [i.e., noggin and SMAD6 (inhibitory SMAD)]; and (iii) upregulation of SMAD4 (universal co-SMAD). These data indicate that LfcinB-suppression of Noggin may eliminate the negative feedback of BMP7, thereby maximizing biological activity of BMP7 and ultimately shifting homeostasis to a pro-anabolic state in disc cells. We propose that combination growth factor therapy using BMP7 and LfcinB may be beneficial for treatment of disc degeneration. Copyright © 2013 Elsevier B.V. All

BMP signalling differentially regulates distinct haematopoietic stem cell types

NARCIS (Netherlands)

M. Crisan (Mihaela); P. Solaimani Kartalaei (Parham); C.S. Vink (Chris); T. Yamada-Inagawa (Tomoko); K. Bollerot (Karine); W.F.J. van IJcken (Wilfred); R. Van Der Linden (Reinier); S.C. de Sousa Lopes (Susana Chuva); R. Monteiro (Rui); C.L. Mummery (Christine); E.A. Dzierzak (Elaine)

2015-01-01

textabstractAdult haematopoiesis is the outcome of distinct haematopoietic stem cell (HSC) subtypes with self-renewable repopulating ability, but with different haematopoietic cell lineage outputs. The molecular basis for this heterogeneity is largely unknown. BMP signalling regulates HSCs as they

BMP Suppresses PTEN Expression via RAS/ERK Signaling

OpenAIRE

Beck, Stayce E.; Carethers, John M.

2007-01-01

Bone morphogenetic protein (BMP), a member of the transforming growth factor β family, classically utilizes the SMAD signaling pathway for its growth suppressive effects, and loss of this signaling cascade may accelerate cell growth. In the colon cancer predisposition syndrome Juvenile Polyposis, as well as in the late progression stages of nonsyndromic colorectal cancers, SMAD4 function is typically abrogated. Here, we utilized the SMAD4-null SW480 colon cancer cell line to examine BMPs effe...

Low-intensity pulsed ultrasound accelerates tooth movement via activation of the BMP-2 signaling pathway.

Directory of Open Access Journals (Sweden)

Hui Xue

Full Text Available The present study was designed to determine the underlying mechanism of low-intensity pulsed ultrasound (LIPUS induced alveolar bone remodeling and the role of BMP-2 expression in a rat orthodontic tooth movement model. Orthodontic appliances were placed between the homonymy upper first molars and the upper central incisors in rats under general anesthesia, followed by daily 20-min LIPUS or sham LIPUS treatment beginning at day 0. Tooth movement distances and molecular changes were evaluated at each observation point. In vitro and in vivo studies were conducted to detect HGF (Hepatocyte growth factor/Runx2/BMP-2 signaling pathways and receptor activator of NFκB ligand (RANKL expression by quantitative real time PCR (qRT-PCR, Western blot and immunohistochemistry. At day 3, LIPUS had no effect on the rat orthodontic tooth movement distance and BMP-2-induced alveolar bone remodeling. However, beginning at day 5 and for the following time points, LIPUS significantly increased orthodontic tooth movement distance and BMP-2 signaling pathway and RANKL expression compared with the control group. The qRT-PCR and Western blot data in vitro and in vivo to study BMP-2 expression were consistent with the immunohistochemistry observations. The present study demonstrates that LIPUS promotes alveolar bone remodeling by stimulating the HGF/Runx2/BMP-2 signaling pathway and RANKL expression in a rat orthodontic tooth movement model, and LIPUS increased BMP-2 expression via Runx2 regulation.

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

Directory of Open Access Journals (Sweden)

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.

Sirenomelia in Bmp7 and Tsg compound mutant mice: requirement for Bmp signaling in the development of ventral posterior mesoderm.

Science.gov (United States)

Zakin, Lise; Reversade, Bruno; Kuroda, Hiroki; Lyons, Karen M; De Robertis, Eddy M

2005-05-01

Sirenomelia or mermaid-like phenotype is one of the principal human congenital malformations that can be traced back to the stage of gastrulation. Sirenomelia is characterized by the fusion of the two hindlimbs into a single one. In the mouse, sirens have been observed in crosses between specific strains and as the consequence of mutations that increase retinoic acid levels. We report that the loss of bone morphogenetic protein 7 (Bmp7) in combination with a half dose or complete loss of twisted gastrulation (Tsg) causes sirenomelia in the mouse. Tsg is a Bmp- and chordin-binding protein that has multiple effects on Bmp metabolism in the extracellular space; Bmp7 is one of many Bmps and is shown here to bind to Tsg. In Xenopus, co-injection of Tsg and Bmp7 morpholino oligonucleotides (MO) has a synergistic effect, greatly inhibiting formation of ventral mesoderm and ventral fin tissue. In the mouse, molecular marker studies indicate that the sirenomelia phenotype is associated with a defect in the formation of ventroposterior mesoderm. These experiments demonstrate that dorsoventral patterning of the mouse posterior mesoderm is regulated by Bmp signaling, as is the case in other vertebrates. Sirens result from a fusion of the hindlimb buds caused by a defect in the formation of ventral mesoderm.

A balance of FGF and BMP signals regulates cell cycle exit and Equarin expression in lens cells

Science.gov (United States)

Jarrin, Miguel; Pandit, Tanushree; Gunhaga, Lena

2012-01-01

In embryonic and adult lenses, a balance of cell proliferation, cell cycle exit, and differentiation is necessary to maintain physical function. The molecular mechanisms regulating the transition of proliferating lens epithelial cells to differentiated primary lens fiber cells are poorly characterized. To investigate this question, we used gain- and loss-of-function analyses to modulate fibroblast growth factor (FGF) and/or bone morphogenetic protein (BMP) signals in chick lens/retina explants. Here we show that FGF activity plays a key role for proliferation independent of BMP signals. Moreover, a balance of FGF and BMP signals regulates cell cycle exit and the expression of Ccdc80 (also called Equarin), which is expressed at sites where differentiation of lens fiber cells occurs. BMP activity promotes cell cycle exit and induces Equarin expression in an FGF-dependent manner. In contrast, FGF activity is required but not sufficient to induce cell cycle exit or Equarin expression. Furthermore, our results show that in the absence of BMP activity, lens cells have increased cell cycle length or are arrested in the cell cycle, which leads to decreased cell cycle exit. Taken together, these findings suggest that proliferation, cell cycle exit, and early differentiation of primary lens fiber cells are regulated by counterbalancing BMP and FGF signals. PMID:22718906

A BMP-mediated transcriptional cascade involving Cash1 and Tlx-3 specifies first-order relay sensory neurons in the developing hindbrain.

Science.gov (United States)

Hornbruch, Amata; Ma, Grace; Ballermann, Mark A; Tumova, Katerina; Liu, Dan; Cairine Logan, C

2005-07-01

The divergent homeobox-containing transcription factor, Tlx-3 (also known as Hox11L2/Rnx), is required for proper formation of first-order relay sensory neurons in the developing vertebrate brainstem. To date, however, the inductive signals and transcriptional regulatory cascade underlying their development are poorly understood. We previously isolated the chick Tlx-3 homologue and showed it is expressed early (i.e. beginning at HH15) in distinct subcomponents of both the trigeminal/solitary and vestibular nuclei. Here we show via in vivo rhombomere inversions that expression of Tlx-3 is under control of local environmental signals. Our RNA in situ analysis shows expression of the BMP-specific receptor, Bmpr-1b, correlates well with Tlx-3. Furthermore, manipulation of the BMP signaling pathway in vivo via electroporation of expression vectors encoding either BMP or NOGGIN coupled with MASH1 gain-of-function experiments demonstrate that a BMP-mediated transcriptional cascade involving Cash1 and Tlx-3 specifies first-order relay sensory neurons in the developing brainstem. Notably, high-level Noggin misexpression results in an increase in newly differentiated Tlx-3+ neurons that correlates with a corresponding increase in the number of Calretinin+ neurons in vestibular nuclei at later developmental stages strongly suggesting that Tlx-3, in addition to being required for proper formation of somatic as well as visceral sensory neurons in the trigeminal and solitary nuclei, respectively, is sufficient for proper formation of special somatic sensory neurons in vestibular nuclei.

BMP4 and BMP7 Suppress StAR and Progesterone Production via ALK3 and SMAD1/5/8-SMAD4 in Human Granulosa-Lutein Cells.

Science.gov (United States)

Zhang, Han; Klausen, Christian; Zhu, Hua; Chang, Hsun-Ming; Leung, Peter C K

2015-11-01

Adequate production of progesterone by the corpus luteum is critical to the successful establishment of pregnancy. In animal models, bone morphogenetic protein (BMP) 4 and BMP7 have been shown to suppress either basal or gonadotropin-induced progesterone production, depending on the species examined. However, the effects of BMP4 and BMP7 on progesterone production in human granulosa cells are unknown. In the present study, we used immortalized (SVOG) and primary human granulosa-lutein cells to investigate the effects of BMP4 and BMP7 on steroidogenic acute regulatory protein (StAR) expression and progesterone production and to examine the underlying molecular mechanism. Treatment of primary and immortalized human granulosa cells with recombinant BMP4 or BMP7 decreased StAR expression and progesterone accumulation. In SVOG cells, the suppressive effects of BMP4 and BMP7 on StAR expression were blocked by pretreatment with inhibitors of activin receptor-like kinase (ALK)2/3/6 (dorsomorphin) or ALK2/3 (DMH1) but not ALK4/5/7 (SB-431542). Moreover, small interfering RNA-mediated depletion of ALK3, but not ALK2 or ALK6, reversed the effects of BMP4 and BMP7 on StAR expression. Likewise, BMP4- and BMP7-induced phosphorylation of SMAD 1/5/8 was reversed by treatment with DMH1 or small interfering RNA targeting ALK3. Knockdown of SMAD4, the essential common SMAD for BMP/TGF-β signaling, abolished the effects of BMP4 and BMP7 on StAR expression. Our results suggest that BMP4 and BMP7 down-regulate StAR and progesterone production via ALK3 and SMAD1/5/8-SMAD4 signaling in human granulosa-lutein cells.

GDF3 is a BMP inhibitor that can activate Nodal signaling only at very high doses

Science.gov (United States)

Levine, Ariel J.; Levine, Zachary J.; Brivanlou, Ali H.

2013-01-01

Within the TGF-β superfamily, there are approximately forty ligands divided into two major branches: the TGF-β/Activin/Nodal ligands and the BMP/GDF ligands. We studied the ligand GDF3 and found that it inhibits signaling by its co-family members, the BMPs; however, GDF3 has been described by others to have Nodal-like activity. Here, we show that GDF3 can activate Nodal signaling, but only at very high doses and only upon mRNA over-expression. In contrast, GDF3 inhibits BMP signaling upon over-expression of GDF3 mRNA, as recombinant protein, and regardless of its dose. We therefore further characterized the mechanism through which GDF3 protein acts as a specific BMP inhibitor and found that the BMP inhibitory activity of GDF3 resides redundantly in the unprocessed, predominant form and in the mature form of the protein. These results confirm and extend the activity that we described for GDF3 and illuminate the experimental basis for the different observations of others. We suggest that GDF3 is either a bi-functional TGF-β ligand, or, more likely, that it is a BMP inhibitor that can artificially activate Nodal signaling under non-physiological conditions. PMID:18823971

Nanostructured hydroxyapatite surfaces-mediated adsorption alters recognition of BMP receptor IA and bioactivity of bone morphogenetic protein-2.

Science.gov (United States)

Huang, Baolin; Yuan, Yuan; Ding, Sai; Li, Jianbo; Ren, Jie; Feng, Bo; Li, Tong; Gu, Yuantong; Liu, Changsheng

2015-11-01

Highly efficient loading of bone morphogenetic protein-2 (BMP-2) onto carriers with desirable performance is still a major challenge in the field of bone regeneration. Till now, the nanoscaled surface-induced changes of the structure and bioactivity of BMP-2 remains poorly understood. Here, the effect of nanoscaled surface on the adsorption and bioactivity of BMP-2 was investigated with a series of hydroxyapatite surfaces (HAPs): HAP crystal-coated surface (HAP), HAP crystal-coated polished surface (HAP-Pol), and sintered HAP crystal-coated surface (HAP-Sin). The adsorption dynamics of recombinant human BMP-2 (rhBMP-2) and the accessibility of the binding epitopes of adsorbed rhBMP-2 for BMP receptors (BMPRs) were examined by a quartz crystal microbalance with dissipation. Moreover, the bioactivity of adsorbed rhBMP-2 and the BMP-induced Smad signaling were investigated with C2C12 model cells. A noticeably high mass-uptake of rhBMP-2 and enhanced recognition of BMPR-IA to adsorbed rhBMP-2 were found on the HAP-Pol surface. For the rhBMP-2-adsorbed HAPs, both ALP activity and Smad signaling increased in the order of HAP-Sinuses of rhBMP-2 in clinical applications and arouse broad interests among researchers in the fields of nano-biotechnology, biomaterials and bone tissue engineering. Copyright © 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Gallic acid inhibits vascular calcification through the blockade of BMP2-Smad1/5/8 signaling pathway.

Science.gov (United States)

Kee, Hae Jin; Cho, Soo-Na; Kim, Gwi Ran; Choi, Sin Young; Ryu, Yuhee; Kim, In Kyeom; Hong, Young Joon; Park, Hyung Wook; Ahn, Youngkeun; Cho, Jeong Gwan; Park, Jong Chun; Jeong, Myung Ho

2014-11-01

Vascular calcification is associated with increased risk of morbidity and mortality in patients with cardiovascular diseases, chronic kidney diseases, and diabetes. Gallic acid, a natural compound found in gallnut and green tea, is known to be antifungal, antioxidant, and anticancer. Here we investigated the effect of gallic acid on vascular smooth muscle cell (VSMC) calcification and the underlying mechanism. Gallic acid inhibited inorganic phosphate-induced osteoblast differentiation markers as well as calcification phenotypes (as determined by calcium deposition, Alizarin Red, and Von Kossa staining). Knockdown of BMP2 or Noggin blocked phosphate-induced calcification. Gallic acid suppressed phosphorylation of Smad1/5/8 protein induced by inorganic phosphate. Taken together, we suggest that gallic acid acts as a novel therapeutic agent of vascular calcification by mediating BMP2-Smad1/5/8 signaling pathway. Copyright © 2014 Elsevier Inc. All rights reserved.

Select polyphenolic fractions from dried plum enhance osteoblast activity through BMP-2 signaling.

Science.gov (United States)

Graef, Jennifer L; Rendina-Ruedy, Elizabeth; Crockett, Erica K; Ouyang, Ping; King, Jarrod B; Cichewicz, Robert H; Lucas, Edralin A; Smith, Brenda J

2018-05-01

Dried plum supplementation has been shown to enhance bone formation while suppressing bone resorption. Evidence from previous studies has demonstrated that these responses can be attributed in part to the fruit's polyphenolic compounds. The purpose of this study was to identify the most bioactive polyphenolic fractions of dried plum with a focus on their osteogenic activity and to investigate their mechanisms of action under normal and inflammatory conditions. Utilizing chromatographic techniques, six fractions of polyphenolic compounds were prepared from a crude extract of dried plum. Initial screening assays revealed that two fractions (DP-FrA and DP-FrB) had the greatest osteogenic potential. Subsequent experiments using primary bone-marrow-derived osteoblast cultures demonstrated these two fractions enhanced extracellular alkaline phosphatase (ALP), an indicator of osteoblast activity, and mineralized nodule formation under normal conditions. Both fractions enhanced bone morphogenetic protein (BMP) signaling, as indicated by increased Bmp2 and Runx2 gene expression and protein levels of phosphorylated Smad1/5. DP-FrB was most effective at up-regulating Tak1 and Smad1, as well as protein levels of phospho-p38. Under inflammatory conditions, TNF-α suppressed ALP and tended to decrease nodule formation (P=.0674). This response coincided with suppressed gene expression of Bmp2 and the up-regulation of Smad6, an inhibitor of BMP signaling. DP-FrA and DP-FrB partially normalized these responses. Our results show that certain fractions of polyphenolic compounds in dried plum up-regulate osteoblast activity by enhancing BMP signaling, and when this pathway is inhibited by TNF-α, the osteogenic response is attenuated. Copyright © 2017 Elsevier Inc. All rights reserved.

BMP6 down-regulates GDNF expression through SMAD1/5 and ERK1/2 signaling pathways in human granulosa-lutein cells.

Science.gov (United States)

Zhang, Xin-Yue; Chang, Hsun-Ming; Taylor, Elizabeth L; Leung, Peter C K; Liu, Rui-Zhi

2018-05-09

Bone morphogenetic protein 6 (BMP6) is a critical regulator of follicular development that is expressed in mammalian oocytes and granulosa cells. Glial cell line-derived neurotrophic factor (GDNF) is an intraovarian neurotrophic factor that plays an essential role in regulating mammalian oocyte maturation. The aim of this study was to investigate the effect of BMP6 on the regulation of GDNF expression and the potential underlying mechanisms. We used an established immortalized human granulosa cell line (SVOG cells) and primary human granulosa-lutein cells as in vitro cell models. Our results showed that BMP6 significantly down-regulated the expression of GDNF in both SVOG and primary human granulosa-lutein cells. Using dual inhibition approaches (kinase receptor inhibitor and small interfering RNA knockdown), our results showed that both ALK2 and ALK3 are involved in BMP6-induced down-regulation of GDNF. In addition, BMP6 induced the phosphorylation of SMAD1/5/8 and ERK1/2 but not AKT or p38. Among three downstream mediators, both SMAD1 and SMAD5 are involved in BMP6-induced down-regulation of GDNF. Moreover, concomitant knockdown of endogenous SMAD4 and inhibition of ERK1/2 activity completely reversed BMP6-induced down-regulation of GDNF, indicating that both SMAD and ERK1/2 signaling pathways are required for the regulatory effect of BMP6 on GDNF expression. Our findings suggest an additional role for an intrafollicular growth factor in regulating follicular function through their paracrine interactions in human granulosa cells.

Reduced BMP signaling results in hindlimb fusion with lethal pelvic/urogenital organ aplasia: a new mouse model of sirenomelia.

Science.gov (United States)

Suzuki, Kentaro; Adachi, Yasuha; Numata, Tomokazu; Nakada, Shoko; Yanagita, Motoko; Nakagata, Naomi; Evans, Sylvia M; Graf, Daniel; Economides, Aris; Haraguchi, Ryuma; Moon, Anne M; Yamada, Gen

2012-01-01

Sirenomelia, also known as mermaid syndrome, is a developmental malformation of the caudal body characterized by leg fusion and associated anomalies of pelvic/urogenital organs including bladder, kidney, rectum and external genitalia. Most affected infants are stillborn, and the few born alive rarely survive beyond the neonatal period. Despite the many clinical studies of sirenomelia in humans, little is known about the pathogenic developmental mechanisms that cause the complex array of phenotypes observed. Here, we provide new evidences that reduced BMP (Bone Morphogenetic Protein) signaling disrupts caudal body formation in mice and phenocopies sirenomelia. Bmp4 is strongly expressed in the developing caudal body structures including the peri-cloacal region and hindlimb field. In order to address the function of Bmp4 in caudal body formation, we utilized a conditional Bmp4 mouse allele (Bmp4(flox/flox)) and the Isl1 (Islet1)-Cre mouse line. Isl1-Cre is expressed in the peri-cloacal region and the developing hindimb field. Isl1Cre;Bmp4(flox/flox) conditional mutant mice displayed sirenomelia phenotypes including hindlimb fusion and pelvic/urogenital organ dysgenesis. Genetic lineage analyses indicate that Isl1-expressing cells contribute to both the aPCM (anterior Peri-Cloacal Mesenchyme) and the hindlimb bud. We show Bmp4 is essential for the aPCM formation independently with Shh signaling. Furthermore, we show Bmp4 is a major BMP ligand for caudal body formation as shown by compound genetic analyses of Bmp4 and Bmp7. Taken together, this study reveals coordinated development of caudal body structures including pelvic/urogenital organs and hindlimb orchestrated by BMP signaling in Isl1-expressing cells. Our study offers new insights into the pathogenesis of sirenomelia.

Reduced BMP signaling results in hindlimb fusion with lethal pelvic/urogenital organ aplasia: a new mouse model of sirenomelia.

Directory of Open Access Journals (Sweden)

Kentaro Suzuki

Full Text Available Sirenomelia, also known as mermaid syndrome, is a developmental malformation of the caudal body characterized by leg fusion and associated anomalies of pelvic/urogenital organs including bladder, kidney, rectum and external genitalia. Most affected infants are stillborn, and the few born alive rarely survive beyond the neonatal period. Despite the many clinical studies of sirenomelia in humans, little is known about the pathogenic developmental mechanisms that cause the complex array of phenotypes observed. Here, we provide new evidences that reduced BMP (Bone Morphogenetic Protein signaling disrupts caudal body formation in mice and phenocopies sirenomelia. Bmp4 is strongly expressed in the developing caudal body structures including the peri-cloacal region and hindlimb field. In order to address the function of Bmp4 in caudal body formation, we utilized a conditional Bmp4 mouse allele (Bmp4(flox/flox and the Isl1 (Islet1-Cre mouse line. Isl1-Cre is expressed in the peri-cloacal region and the developing hindimb field. Isl1Cre;Bmp4(flox/flox conditional mutant mice displayed sirenomelia phenotypes including hindlimb fusion and pelvic/urogenital organ dysgenesis. Genetic lineage analyses indicate that Isl1-expressing cells contribute to both the aPCM (anterior Peri-Cloacal Mesenchyme and the hindlimb bud. We show Bmp4 is essential for the aPCM formation independently with Shh signaling. Furthermore, we show Bmp4 is a major BMP ligand for caudal body formation as shown by compound genetic analyses of Bmp4 and Bmp7. Taken together, this study reveals coordinated development of caudal body structures including pelvic/urogenital organs and hindlimb orchestrated by BMP signaling in Isl1-expressing cells. Our study offers new insights into the pathogenesis of sirenomelia.

Kinesin Khc-73/KIF13B modulates retrograde BMP signaling by influencing endosomal dynamics at the Drosophila neuromuscular junction.

Science.gov (United States)

Liao, Edward H; Gray, Lindsay; Tsurudome, Kazuya; El-Mounzer, Wassim; Elazzouzi, Fatima; Baim, Christopher; Farzin, Sarah; Calderon, Mario R; Kauwe, Grant; Haghighi, A Pejmun

2018-01-01

Retrograde signaling is essential for neuronal growth, function and survival; however, we know little about how signaling endosomes might be directed from synaptic terminals onto retrograde axonal pathways. We have identified Khc-73, a plus-end directed microtubule motor protein, as a regulator of sorting of endosomes in Drosophila larval motor neurons. The number of synaptic boutons and the amount of neurotransmitter release at the Khc-73 mutant larval neuromuscular junction (NMJ) are normal, but we find a significant decrease in the number of presynaptic release sites. This defect in Khc-73 mutant larvae can be genetically enhanced by a partial genetic loss of Bone Morphogenic Protein (BMP) signaling or suppressed by activation of BMP signaling in motoneurons. Consistently, activation of BMP signaling that normally enhances the accumulation of phosphorylated form of BMP transcription factor Mad in the nuclei, can be suppressed by genetic removal of Khc-73. Using a number of assays including live imaging in larval motor neurons, we show that loss of Khc-73 curbs the ability of retrograde-bound endosomes to leave the synaptic area and join the retrograde axonal pathway. Our findings identify Khc-73 as a regulator of endosomal traffic at the synapse and modulator of retrograde BMP signaling in motoneurons.

Cyclic dermal BMP signalling regulates stem cell activation during hair regeneration.

Science.gov (United States)

Plikus, Maksim V; Mayer, Julie Ann; de la Cruz, Damon; Baker, Ruth E; Maini, Philip K; Maxson, Robert; Chuong, Cheng-Ming

2008-01-17

In the age of stem cell engineering it is critical to understand how stem cell activity is regulated during regeneration. Hairs are mini-organs that undergo cyclic regeneration throughout adult life, and are an important model for organ regeneration. Hair stem cells located in the follicle bulge are regulated by the surrounding microenvironment, or niche. The activation of such stem cells is cyclic, involving periodic beta-catenin activity. In the adult mouse, regeneration occurs in waves in a follicle population, implying coordination among adjacent follicles and the extrafollicular environment. Here we show that unexpected periodic expression of bone morphogenetic protein 2 (Bmp2) and Bmp4 in the dermis regulates this process. This BMP cycle is out of phase with the WNT/beta-catenin cycle, thus dividing the conventional telogen into new functional phases: one refractory and the other competent for hair regeneration, characterized by high and low BMP signalling, respectively. Overexpression of noggin, a BMP antagonist, in mouse skin resulted in a markedly shortened refractory phase and faster propagation of the regenerative wave. Transplantation of skin from this mutant onto a wild-type host showed that follicles in donor and host can affect their cycling behaviours mutually, with the outcome depending on the equilibrium of BMP activity in the dermis. Administration of BMP4 protein caused the competent region to become refractory. These results show that BMPs may be the long-sought 'chalone' inhibitors of hair growth postulated by classical experiments. Taken together, results presented in this study provide an example of hierarchical regulation of local organ stem cell homeostasis by the inter-organ macroenvironment. The expression of Bmp2 in subcutaneous adipocytes indicates physiological integration between these two thermo-regulatory organs. Our findings have practical importance for studies using mouse skin as a model for carcinogenesis, intra-cutaneous drug

Overexpression of BMP3 in the developing skeleton alters endochondral bone formation resulting in spontaneous rib fractures.

Science.gov (United States)

Gamer, Laura W; Cox, Karen; Carlo, Joelle M; Rosen, Vicki

2009-09-01

Bone morphogenetic protein-3 (BMP) has been identified as a negative regulator in the skeleton as mice lacking BMP3 have increased bone mass. To further understand how BMP3 mediates bone formation, we created transgenic mice overexpressing BMP3 using the type I collagen promoter. BMP3 transgenic mice displayed spontaneous rib fractures that were first detected at E17.0. The fractures were due to defects in differentiation of the periosteum and late hypertrophic chondrocytes resulting in thinner cortical bone with decreased mineralization. As BMP3 modulates BMP and activin signaling through ActRIIB, we examined the ribs of ActRIIB receptor knockout mice and found they had defects in late chondrogenesis and mineralization similar to BMP3 transgenic mice. These data suggest that BMP3 exerts its effects in the skeleton by altering signaling through ActRIIB in chondrocytes and the periosteum, and this results in defects in bone collar formation and late hypertrophic chondrocyte maturation leading to decreased mineralization and less bone. 2009 Wiley-Liss, Inc.

Histone deacetylases control neurogenesis in embryonic brain by inhibition of BMP2/4 signaling.

Directory of Open Access Journals (Sweden)

Maya Shakèd

Full Text Available BACKGROUND: Histone-modifying enzymes are essential for a wide variety of cellular processes dependent upon changes in gene expression. Histone deacetylases (HDACs lead to the compaction of chromatin and subsequent silencing of gene transcription, and they have recently been implicated in a diversity of functions and dysfunctions in the postnatal and adult brain including ocular dominance plasticity, memory consolidation, drug addiction, and depression. Here we investigate the role of HDACs in the generation of neurons and astrocytes in the embryonic brain. PRINCIPAL FINDINGS: As a variety of HDACs are expressed in differentiating neural progenitor cells, we have taken a pharmacological approach to inhibit multiple family members. Inhibition of class I and II HDACs in developing mouse embryos with trichostatin A resulted in a dramatic reduction in neurogenesis in the ganglionic eminences and a modest increase in neurogenesis in the cortex. An identical effect was observed upon pharmacological inhibition of HDACs in in vitro-differentiating neural precursors derived from the same brain regions. A reduction in neurogenesis in ganglionic eminence-derived neural precursors was accompanied by an increase in the production of immature astrocytes. We show that HDACs control neurogenesis by inhibition of the bone morphogenetic protein BMP2/4 signaling pathway in radial glial cells. HDACs function at the transcriptional level by inhibiting and promoting, respectively, the expression of Bmp2 and Smad7, an intracellular inhibitor of BMP signaling. Inhibition of the BMP2/4 signaling pathway restored normal levels of neurogenesis and astrogliogenesis to both ganglionic eminence- and cortex-derived cultures in which HDACs were inhibited. CONCLUSIONS: Our results demonstrate a transcriptionally-based regulation of BMP2/4 signaling by HDACs both in vivo and in vitro that is critical for neurogenesis in the ganglionic eminences and that modulates cortical

Apc bridges Wnt/{beta}-catenin and BMP signaling during osteoblast differentiation of KS483 cells

Energy Technology Data Exchange (ETDEWEB)

Miclea, Razvan L., E-mail: R.L.Miclea@lumc.nl [Department of Pediatrics, Leiden University Medical Centre (LUMC), Leiden (Netherlands); Horst, Geertje van der, E-mail: G.van_der_Horst@lumc.nl [Department of Urology, LUMC, Leiden (Netherlands); Robanus-Maandag, Els C., E-mail: E.C.Robanus@lumc.nl [Department of Human Genetics, LUMC, Leiden (Netherlands); Loewik, Clemens W.G.M., E-mail: C.W.G.M.Lowik@lumc.nl [Department of Endocrinology and Metabolic Diseases, LUMC, Leiden (Netherlands); Oostdijk, Wilma, E-mail: W.Oostdijk@lumc.nl [Department of Pediatrics, Leiden University Medical Centre (LUMC), Leiden (Netherlands); Wit, Jan M., E-mail: J.M.Wit@lumc.nl [Department of Pediatrics, Leiden University Medical Centre (LUMC), Leiden (Netherlands); Karperien, Marcel, E-mail: H.B.J.Karperien@tnw.utwente.nl [MIRA Institute for Biomedical Technology and Technical Medicine, Department of Tissue Regeneration, University of Twente, Zuidhorst Room ZH 144, Drienerlolaan 5, 7522 NB Enschede (Netherlands)

2011-06-10

The canonical Wnt signaling pathway influences the differentiation of mesenchymal cell lineages in a quantitative and qualitative fashion depending on the dose of {beta}-catenin signaling. Adenomatous polyposis coli (Apc) is the critical intracellular regulator of {beta}-catenin turnover. To better understand the molecular mechanisms underlying the role of Apc in regulating the differentiation capacity of skeletal progenitor cells, we have knocked down Apc in the murine mesenchymal stem cell-like KS483 cells by stable expression of Apc-specific small interfering RNA. In routine culture, KSFrt-Apc{sub si} cells displayed a mesenchymal-like spindle shape morphology, exhibited markedly decreased proliferation and increased apoptosis. Apc knockdown resulted in upregulation of the Wnt/{beta}-catenin and the BMP/Smad signaling pathways, but osteogenic differentiation was completely inhibited. This effect could be rescued by adding high concentrations of BMP-7 to the differentiation medium. Furthermore, KSFrt-Apc{sub si} cells showed no potential to differentiate into chondrocytes or adipocytes. These results demonstrate that Apc is essential for the proliferation, survival and differentiation of KS483 cells. Apc knockdown blocks the osteogenic differentiation of skeletal progenitor cells, a process that can be overruled by high BMP signaling.

BMP signaling in the human fetal ovary is developmentally regulated and promotes primordial germ cell apoptosis.

Science.gov (United States)

Childs, Andrew J; Kinnell, Hazel L; Collins, Craig S; Hogg, Kirsten; Bayne, Rosemary A L; Green, Samira J; McNeilly, Alan S; Anderson, Richard A

2010-08-01

Primordial germ cells (PGCs) are the embryonic precursors of gametes in the adult organism, and their development, differentiation, and survival are regulated by a combination of growth factors collectively known as the germ cell niche. Although many candidate niche components have been identified through studies on mouse PGCs, the growth factor composition of the human PGC niche has not been studied extensively. Here we report a detailed analysis of the expression of components of the bone morphogenetic protein (BMP) signaling apparatus in the human fetal ovary, from postmigratory PGC proliferation to the onset of primordial follicle formation. We find developmentally regulated and reciprocal patterns of expression of BMP2 and BMP4 and identify germ cells to be the exclusive targets of ovarian BMP signaling. By establishing long-term cultures of human fetal ovaries in which PGCs are retained within their physiological niche, we find that BMP4 negatively regulates postmigratory PGC numbers in the human fetal ovary by promoting PGC apoptosis. Finally, we report expression of both muscle segment homeobox (MSX)1 and MSX2 in the human fetal ovary and reveal a selective upregulation of MSX2 expression in human fetal ovary in response to BMP4, suggesting this gene may act as a downstream effector of BMP-induced apoptosis in the ovary, as in other systems. These data reveal for the first time growth factor regulation of human PGC development in a physiologically relevant context and have significant implications for the development of cultures systems for the in vitro maturation of germ cells, and their derivation from pluripotent stem cells.

BMP signaling protects telencephalic fate by repressing eye identity and its Cxcr4-dependent morphogenesis.

Science.gov (United States)

Bielen, Holger; Houart, Corinne

2012-10-16

Depletion of Wnt signaling is a major requirement for the induction of the anterior prosencephalon. However, the molecular events driving the differential regionalization of this area into eye-field and telencephalon fates are still unknown. Here we show that the BMP pathway is active in the anterior neural ectoderm during late blastula to early gastrula stage in zebrafish. Bmp2b mutants and mosaic loss-of-function experiments reveal that BMP acts as a repressor of eye-field fate through inhibition of its key transcription factor Rx3, thereby protecting the future telencephalon from acquiring eye identity. This BMP-driven mechanism initiates the establishment of the telencephalon prior to the involvement of Wnt antagonists from the anterior neural border. Furthermore, we demonstrate that Rx3 and BMP are respectively required to maintain and restrict the chemokine receptor cxcr4a, which in turn contributes to the morphogenetic separation of eye-field and telencephalic cells during early neurulation. Copyright © 2012 Elsevier Inc. All rights reserved.

MiRNA-20a promotes osteogenic differentiation of human mesenchymal stem cells by co-regulating BMP signaling.

Science.gov (United States)

Zhang, Jin-fang; Fu, Wei-ming; He, Ming-liang; Xie, Wei-dong; Lv, Qing; Wan, Gang; Li, Guo; Wang, Hua; Lu, Gang; Hu, Xiang; Jiang, Su; Li, Jian-na; Lin, Marie C M; Zhang, Ya-ou; Kung, Hsiang-fu

2011-01-01

Osteogenic differentiation of mesenchymal stem cells (MSCs) is a complex process, which is regulated by various factors including microRNAs. Our preliminary data showed that the expression of endogenous miR-20a was increased during the course of osteogenic differentiation. Simultaneously, the expression of osteoblast markers and regulators BMP2, BMP4, Runx2, Osx, OCN and OPN was also elevated whereas adipocyte markers PPARγ and osteoblast antagonist, Bambi and Crim1, were downregulated, thereby suggesting that miR-20a plays an important role in regulating osteoblast differentiation. To validate this hypothesis, we tested its effects on osteogenic differentiation by introducing miR-20a mimics and lentiviral-miR20a-expression vectors into hMSCs. We showed that miR-20a promoted osteogenic differentiation by the upregulation of BMP/Runx2 signaling. We performed bioinformatics analysis and predicted that PPARγ, Bambi and Crim1 would be potential targets of miR-20a. PPARγ is a negative regulator of BMP/Runx2 signaling whereas Bambi or Crim1 are antagonists of the BMP pathway. Furthermore, we confirmed that all these molecules were indeed the targets of miR-20a by luciferase reporter, quantitative RT-PCR and western blot assays. Similarly to miR-20a overexpression, the osteogenesis was enhanced by the silence of PPARγ, Bambi or Crim1 by specific siRNAs. Taken together, for the first time, we demonstrated that miR-20a promoted the osteogenesis of hMSCs in a co-regulatory pattern by targeting PPARγ, Bambi and Crim1, the negative regulators of BMP signaling.

The response of early neural genes to FGF signaling or inhibition of BMP indicate the absence of a conserved neural induction module

Directory of Open Access Journals (Sweden)

Rogers Crystal D

2011-12-01

Full Text Available Abstract Background The molecular mechanism that initiates the formation of the vertebrate central nervous system has long been debated. Studies in Xenopus and mouse demonstrate that inhibition of BMP signaling is sufficient to induce neural tissue in explants or ES cells respectively, whereas studies in chick argue that instructive FGF signaling is also required for the expression of neural genes. Although additional signals may be involved in neural induction and patterning, here we focus on the roles of BMP inhibition and FGF8a. Results To address the question of necessity and sufficiency of BMP inhibition and FGF signaling, we compared the temporal expression of the five earliest genes expressed in the neuroectoderm and determined their requirements for induction at the onset of neural plate formation in Xenopus. Our results demonstrate that the onset and peak of expression of the genes vary and that they have different regulatory requirements and are therefore unlikely to share a conserved neural induction regulatory module. Even though all require inhibition of BMP for expression, some also require FGF signaling; expression of the early-onset pan-neural genes sox2 and foxd5α requires FGF signaling while other early genes, sox3, geminin and zicr1 are induced by BMP inhibition alone. Conclusions We demonstrate that BMP inhibition and FGF signaling induce neural genes independently of each other. Together our data indicate that although the spatiotemporal expression patterns of early neural genes are similar, the mechanisms involved in their expression are distinct and there are different signaling requirements for the expression of each gene.

Inhibition of BMP signaling overcomes acquired resistance to cetuximab in oral squamous cell carcinomas.

Science.gov (United States)

Yin, Jinlong; Jung, Ji-Eun; Choi, Sun Il; Kim, Sung Soo; Oh, Young Taek; Kim, Tae-Hoon; Choi, Eunji; Lee, Sun Joo; Kim, Hana; Kim, Eun Ok; Lee, Yu Sun; Chang, Hee Jin; Park, Joo Yong; Kim, Yeejeong; Yun, Tak; Heo, Kyun; Kim, Youn-Jae; Kim, Hyunggee; Kim, Yun-Hee; Park, Jong Bae; Choi, Sung Weon

2018-02-01

Despite expressing high levels of the epidermal growth factor receptor (EGFR), a majority of oral squamous cell carcinoma (OSCC) patients show limited response to cetuximab and ultimately develop drug resistance. However, mechanism underlying cetuximab resistance in OSCC is not clearly understood. Here, using a mouse orthotopic xenograft model of OSCC, we show that bone morphogenic protein-7-phosphorylated Smad-1, -5, -8 (BMP7-p-Smad1/5/8) signaling contributes to cetuximab resistance. Tumor cells isolated from the recurrent cetuximab-resistant xenograft models exhibited low EGFR expression but extremely high levels of p-Smad1/5/8. Treatment with the bone morphogenic protein receptor type 1 (BMPRI) inhibitor, DMH1 significantly reduced cetuximab-resistant OSCC tumor growth, and combined treatment of DMH1 and cetuximab remarkably reduced relapsed tumor growth in vivo. Importantly, p-Smad1/5/8 level was elevated in cetuximab-resistant patients and this correlated with poor prognosis. Collectively, our results indicate that the BMP7-p-Smad1/5/8 signaling is a key pathway to acquired cetuximab resistance, and demonstrate that combination therapy of cetuximab and a BMP signaling inhibitor as potentially a new therapeutic strategy for overcoming acquired resistance to cetuximab in OSCC. Copyright © 2017 Elsevier B.V. All rights reserved.

Regulation of extracellular matrix organization by BMP signaling in Caenorhabditis elegans.

Science.gov (United States)

Schultz, Robbie D; Bennett, Emily E; Ellis, E Ann; Gumienny, Tina L

2014-01-01

In mammals, Bone Morphogenetic Protein (BMP) pathway signaling is important for the growth and homeostasis of extracellular matrix, including basement membrane remodeling, scarring, and bone growth. A conserved BMP member in Caenorhabditis elegans, DBL-1, regulates body length in a dose-sensitive manner. Loss of DBL-1 pathway signaling also results in increased anesthetic sensitivity. However, the physiological basis of these pleiotropic phenotypes is largely unknown. We created a DBL-1 over-expressing strain and show that sensitivity to anesthetics is inversely related to the dose of DBL-1. Using pharmacological, genetic analyses, and a novel dye permeability assay for live, microwave-treated animals, we confirm that DBL-1 is required for the barrier function of the cuticle, a specialized extracellular matrix. We show that DBL-1 signaling is required to prevent animals from forming tail-entangled aggregates in liquid. Stripping lipids off the surface of wild-type animals recapitulates this phenotype. Finally, we find that DBL-1 signaling affects ultrastructure of the nematode cuticle in a dose-dependent manner, as surface lipid content and cuticular organization are disrupted in animals with genetically altered DBL-1 levels. We propose that the lipid layer coating the nematode cuticle normally prevents tail entanglement, and that reduction of this layer by loss of DBL-1 signaling promotes aggregation. This work provides a physiological mechanism that unites the DBL-1 signaling pathway roles of not only body size regulation and drug responsiveness, but also the novel Hoechst 33342 staining and aggregation phenotypes, through barrier function, content, and organization of the cuticle.

Regulation of extracellular matrix organization by BMP signaling in Caenorhabditis elegans.

Directory of Open Access Journals (Sweden)

Robbie D Schultz

Full Text Available In mammals, Bone Morphogenetic Protein (BMP pathway signaling is important for the growth and homeostasis of extracellular matrix, including basement membrane remodeling, scarring, and bone growth. A conserved BMP member in Caenorhabditis elegans, DBL-1, regulates body length in a dose-sensitive manner. Loss of DBL-1 pathway signaling also results in increased anesthetic sensitivity. However, the physiological basis of these pleiotropic phenotypes is largely unknown. We created a DBL-1 over-expressing strain and show that sensitivity to anesthetics is inversely related to the dose of DBL-1. Using pharmacological, genetic analyses, and a novel dye permeability assay for live, microwave-treated animals, we confirm that DBL-1 is required for the barrier function of the cuticle, a specialized extracellular matrix. We show that DBL-1 signaling is required to prevent animals from forming tail-entangled aggregates in liquid. Stripping lipids off the surface of wild-type animals recapitulates this phenotype. Finally, we find that DBL-1 signaling affects ultrastructure of the nematode cuticle in a dose-dependent manner, as surface lipid content and cuticular organization are disrupted in animals with genetically altered DBL-1 levels. We propose that the lipid layer coating the nematode cuticle normally prevents tail entanglement, and that reduction of this layer by loss of DBL-1 signaling promotes aggregation. This work provides a physiological mechanism that unites the DBL-1 signaling pathway roles of not only body size regulation and drug responsiveness, but also the novel Hoechst 33342 staining and aggregation phenotypes, through barrier function, content, and organization of the cuticle.

Production of BMP4 by endothelial cells is crucial for endogenous thymic regeneration

Science.gov (United States)

Wertheimer, Tobias; Velardi, Enrico; Tsai, Jennifer; Cooper, Kirsten; Xiao, Shiyun; Kloss, Christopher C.; Ottmüller, Katja J.; Mokhtari, Zeinab; Brede, Christian; deRoos, Paul; Kinsella, Sinéad; Palikuqi, Brisa; Ginsberg, Michael; Young, Lauren F.; Kreines, Fabiana; Lieberman, Sophia R.; Lazrak, Amina; Guo, Peipei; Malard, Florent; Smith, Odette M.; Shono, Yusuke; Jenq, Robert R.; Hanash, Alan M.; Nolan, Daniel J.; Butler, Jason M.; Beilhack, Andreas; Manley, Nancy R.; Rafii, Shahin; Dudakov, Jarrod A; van den Brink, Marcel RM

2018-01-01

The thymus is extremely sensitive to damage but also has a remarkable ability to repair itself. However, the mechanisms underlying this endogenous regeneration remain poorly understood and this capacity diminishes considerably with age. Here we show that thymic endothelial cells (ECs) comprise a critical pathway of regeneration, via their production of BMP4. ECs increased their production of BMP4 after thymic damage, and abrogating BMP4 signalling or production by either pharmacologic or genetic inhibition impaired thymic repair. EC-derived BMP4 acted on thymic epithelial cells (TECs) to increase their expression of Foxn1, a key transcription factor involved in TEC development, maintenance and regeneration; and its downstream targets such as Dll4, itself a key mediator of thymocyte development and regeneration. These studies demonstrate the importance of the BMP4 pathway in endogenous tissue regeneration and offer a potential clinical approach to enhance T cell immunity. PMID:29330161

crm-1 facilitates BMP signaling to control body size in Caenorhabditis elegans.

Science.gov (United States)

Fung, Wong Yan; Fat, Ko Frankie Chi; Eng, Cheah Kathryn Song; Lau, Chow King

2007-11-01

We have identified in Caenorhabditis elegans a homologue of the vertebrate Crim1, crm-1, which encodes a putative transmembrane protein with multiple cysteine-rich (CR) domains known to have bone morphogenetic proteins (BMPs) binding activity. Using the body morphology of C. elegans as an indicator, we showed that attenuation of crm-1 activity leads to a small body phenotype reminiscent of that of BMP pathway mutants. We showed that the crm-1 loss-of-function phenotype can be rescued by constitutive supply of sma-4 activity. crm-1 can enhance BMP signaling and this activity is dependent on the presence of the DBL-1 ligand and its receptors. crm-1 is expressed in neurons at the ventral nerve cord, where the DBL-1 ligand is produced. However, ectopic expression experiments reveal that crm-1 gene products act outside the DBL-1 producing cells and function non-autonomously to facilitate dbl/sma pathway signaling to control body size.

BMP Sustains Embryonic Stem Cell Self-Renewal through Distinct Functions of Different Krüppel-like Factors.

Science.gov (United States)

Morikawa, Masato; Koinuma, Daizo; Mizutani, Anna; Kawasaki, Natsumi; Holmborn, Katarina; Sundqvist, Anders; Tsutsumi, Shuichi; Watabe, Tetsuro; Aburatani, Hiroyuki; Heldin, Carl-Henrik; Miyazono, Kohei

2016-01-12

Bone morphogenetic protein (BMP) signaling exerts paradoxical roles in pluripotent stem cells (PSCs); it sustains self-renewal of mouse embryonic stem cells (ESCs), while it induces differentiation in other PSCs, including human ESCs. Here, we revisit the roles of BMP-4 using mouse ESCs (mESCs) in naive and primed states. SMAD1 and SMAD5, which transduce BMP signals, recognize enhancer regions together with KLF4 and KLF5 in naive mESCs. KLF4 physically interacts with SMAD1 and suppresses its activity. Consistently, a subpopulation of cells with active BMP-SMAD can be ablated without disturbing the naive state of the culture. Moreover, Smad1/5 double-knockout mESCs stay in the naive state, indicating that the BMP-SMAD pathway is dispensable for it. In contrast, the MEK5-ERK5 pathway mediates BMP-4-induced self-renewal of mESCs by inducing Klf2, a critical factor for the ground state pluripotency. Our study illustrates that BMP exerts its self-renewing effect through distinct functions of different Krüppel-like factors. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Ectopic application of recombinant BMP-2 and BMP-4 can change patterning of developing chick facial primordia.

Science.gov (United States)

Barlow, A J; Francis-West, P H

1997-01-01

The facial primordia initially consist of buds of undifferentiated mesenchyme, which give rise to a variety of tissues including cartilage, muscle and nerve. These must be arranged in a precise spatial order for correct function. The signals that control facial outgrowth and patterning are largely unknown. The bone morphogenetic proteins Bmp-2 and Bmp-4 are expressed in discrete regions at the distal tips of the early facial primordia suggesting possible roles for BMP-2 and BMP-4 during chick facial development. We show that expression of Bmp-4 and Bmp-2 is correlated with the expression of Msx-1 and Msx-2 and that ectopic application of BMP-2 and BMP-4 can activate Msx-1 and Msx-2 gene expression in the developing facial primordia. We correlate this activation of gene expression with changes in skeletal development. For example, activation of Msx-1 gene expression across the distal tip of the mandibular primordium is associated with an extension of Fgf-4 expression in the epithelium and bifurcation of Meckel's cartilage. In the maxillary primordium, extension of the normal domain of Msx-1 gene expression is correlated with extended epithelial expression of shh and bifurcation of the palatine bone. We also show that application of BMP-2 can increase cell proliferation of the mandibular primordia. Our data suggest that BMP-2 and BMP-4 are part of a signalling cascade that controls outgrowth and patterning of the facial primordia.

FoxC2 Enhances BMP7-Mediated Anabolism in Nucleus Pulposus Cells of the Intervertebral Disc

OpenAIRE

Wang, Zheng; Fu, Changfeng; Chen, Yong; Xu, Feng; Wang, Zhenyu; Qu, Zhigang; Liu, Yi

2016-01-01

Bone-morphogenetic protein-7 (BMP-7) is a growth factor that plays a major role in mediating anabolism and anti-catabolism of the intervertebral disc matrix and cell homeostasis. In osteoblasts, Forkhead box protein C2 (FoxC2) is a downstream target of BMPs and promotes cell proliferation and differentiation. However, the role FoxC2 may play in degenerative human intervertebral disc tissue and the relationship between FoxC2 and BMP-7 in nucleus pulposus (NP) cells remain to be elucidated. Thi...

Constitutive activation of BMP signalling abrogates experimental metastasis of OVCA429 cells via reduced cell adhesion

Directory of Open Access Journals (Sweden)

Shepherd Trevor G

2010-02-01

Full Text Available Abstract Background Activation of bone morphogenetic protein (BMP4 signalling in human ovarian cancer cells induces a number of phenotypic changes in vitro, including altered cell morphology, adhesion, motility and invasion, relative to normal human ovarian surface epithelial cells. From these in vitro analyses, we had hypothesized that active BMP signalling promotes the metastatic potential of ovarian cancer. Methods To test this directly, we engineered OVCA429 human ovarian cancer cells possessing doxycycline-inducible expression of a constitutively-active mutant BMP receptor, ALK3QD, and administered these cells to immunocompromised mice. Further characterization was performed in vitro to address the role of activated BMP signalling on the EOC phenotype, with particular emphasis on epithelial-mesenchymal transition (EMT and cell adhesion. Results Unexpectedly, doxycycline-induced ALK3QD expression in OVCA429 cells reduced tumour implantation on peritoneal surfaces and ascites formation when xenografted into immunocompromised mice by intraperitoneal injection. To determine the potential mechanisms controlling this in vivo observation, we followed with several cell culture experiments. Doxycycline-induced ALK3QD expression enhanced the refractile, spindle-shaped morphology of cultured OVCA429 cells eliciting an EMT-like response. Using in vitro wound healing assays, we observed that ALK3QD-expressing cells migrated with long, cytoplasmic projections extending into the wound space. The phenotypic alterations of ALK3QD-expressing cells correlated with changes in specific gene expression patterns of EMT, including increased Snail and Slug and reduced E-cadherin mRNA expression. In addition, ALK3QD signalling reduced β1- and β3-integrin expression, critical molecules involved in ovarian cancer cell adhesion. The combination of reduced E-cadherin and β-integrin expression correlates directly with the reduced EOC cell cohesion in spheroids and

Wnt and BMP signaling crosstalk in regulating dental stem cells: Implications in dental tissue engineering

Directory of Open Access Journals (Sweden)

Fugui Zhang

2016-12-01

Full Text Available Tooth is a complex hard tissue organ and consists of multiple cell types that are regulated by important signaling pathways such as Wnt and BMP signaling. Serious injuries and/or loss of tooth or periodontal tissues may significantly impact aesthetic appearance, essential oral functions and the quality of life. Regenerative dentistry holds great promise in treating oral/dental disorders. The past decade has witnessed a rapid expansion of our understanding of the biological features of dental stem cells, along with the signaling mechanisms governing stem cell self-renewal and differentiation. In this review, we first summarize the biological characteristics of seven types of dental stem cells, including dental pulp stem cells, stem cells from apical papilla, stem cells from human exfoliated deciduous teeth, dental follicle precursor cells, periodontal ligament stem cells, alveolar bone-derived mesenchymal stem cells (MSCs, and MSCs from gingiva. We then focus on how these stem cells are regulated by bone morphogenetic protein (BMP and/or Wnt signaling by examining the interplays between these pathways. Lastly, we analyze the current status of dental tissue engineering strategies that utilize oral/dental stem cells by harnessing the interplays between BMP and Wnt pathways. We also highlight the challenges that must be addressed before the dental stem cells may reach any clinical applications. Thus, we can expect to witness significant progresses to be made in regenerative dentistry in the coming decade.

BMP signaling modulates hepcidin expression in zebrafish embryos independent of hemojuvelin.

Directory of Open Access Journals (Sweden)

Yann Gibert

2011-01-01

Full Text Available Hemojuvelin (Hjv, a member of the repulsive-guidance molecule (RGM family, upregulates transcription of the iron regulatory hormone hepcidin by activating the bone morphogenetic protein (BMP signaling pathway in mammalian cells. Mammalian models have identified furin, neogenin, and matriptase-2 as modifiers of Hjv's function. Using the zebrafish model, we evaluated the effects of hjv and its interacting proteins on hepcidin expression during embryonic development. We found that hjv is strongly expressed in the notochord and somites of the zebrafish embryo and that morpholino knockdown of hjv impaired the development of these structures. Knockdown of hjv or other hjv-related genes, including zebrafish orthologs of furin or neogenin, however, failed to decrease hepcidin expression relative to liver size. In contrast, overexpression of bmp2b or knockdown of matriptase-2 enhanced the intensity and extent of hepcidin expression in zebrafish embryos, but this occurred in an hjv-independent manner. Furthermore, we demonstrated that zebrafish hjv can activate the human hepcidin promoter and enhance BMP responsive gene expression in vitro, but is expressed at low levels in the zebrafish embryonic liver. Taken together, these data support an alternative mechanism for hepcidin regulation during zebrafish embryonic development, which is independent of hjv.

Quinoline compound KM11073 enhances BMP-2-dependent osteogenic differentiation of C2C12 cells via activation of p38 signaling and exhibits in vivo bone forming activity.

Directory of Open Access Journals (Sweden)

Seung-hwa Baek

Full Text Available Recombinant human bone morphogenetic protein (rhBMP-2 has been approved by the FDA for clinical application, but its use is limited due to high cost and a supra-physiological dose for therapeutic efficacy. Therefore, recent studies have focused on the generation of new therapeutic small molecules to induce bone formation or potentiate the osteogenic activity of BMP-2. Here, we show that [4-(7-chloroquinolin-4-yl piperazino][1-phenyl-5-(trifluoromethyl-1H-pyrazol-4-yl]methanone (KM11073 strongly enhances the BMP-2-stimulated induction of alkaline phosphatase (ALP, an early phase biomarker of osteoblast differentiation, in bi-potential mesenchymal progenitor C2C12 cells. The KM11073-mediated ALP induction was inhibited by the BMP antagonist noggin, suggesting that its osteogenic activity occurs via BMP signaling. In addition, a pharmacological inhibition study suggested the involvement of p38 activation in the osteogenic action of KM11073 accompanied by enhanced expression of BMP-2, -6, and -7 mRNA. Furthermore, the in vivo osteogenic activity of KM11073 was confirmed in zebrafish and mouse calvarial bone formation models, suggesting the possibility of its single use for bone formation. In conclusion, the combination of rhBMP-2 with osteogenic small molecules could reduce the use of expensive rhBMP-2, mitigating the undesirable side effects of its supra-physiological dose for therapeutic efficacy. Moreover, due to their inherent physical properties, small molecules could represent the next generation of regenerative medicine.

Emerging roles of BMP9 and BMP10 in hereditary hemorrhagic telangiectasia

Directory of Open Access Journals (Sweden)

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.

Systems biology derived source-sink mechanism of BMP gradient formation.

Science.gov (United States)

Zinski, Joseph; Bu, Ye; Wang, Xu; Dou, Wei; Umulis, David; Mullins, Mary C

2017-08-09

A morphogen gradient of Bone Morphogenetic Protein (BMP) signaling patterns the dorsoventral embryonic axis of vertebrates and invertebrates. The prevailing view in vertebrates for BMP gradient formation is through a counter-gradient of BMP antagonists, often along with ligand shuttling to generate peak signaling levels. To delineate the mechanism in zebrafish, we precisely quantified the BMP activity gradient in wild-type and mutant embryos and combined these data with a mathematical model-based computational screen to test hypotheses for gradient formation. Our analysis ruled out a BMP shuttling mechanism and a bmp transcriptionally-informed gradient mechanism. Surprisingly, rather than supporting a counter-gradient mechanism, our analyses support a fourth model, a source-sink mechanism, which relies on a restricted BMP antagonist distribution acting as a sink that drives BMP flux dorsally and gradient formation. We measured Bmp2 diffusion and found that it supports the source-sink model, suggesting a new mechanism to shape BMP gradients during development.

A computational model for BMP movement in sea urchin embryos.

Science.gov (United States)

van Heijster, Peter; Hardway, Heather; Kaper, Tasso J; Bradham, Cynthia A

2014-12-21

Bone morphogen proteins (BMPs) are distributed along a dorsal-ventral (DV) gradient in many developing embryos. The spatial distribution of this signaling ligand is critical for correct DV axis specification. In various species, BMP expression is spatially localized, and BMP gradient formation relies on BMP transport, which in turn requires interactions with the extracellular proteins Short gastrulation/Chordin (Chd) and Twisted gastrulation (Tsg). These binding interactions promote BMP movement and concomitantly inhibit BMP signaling. The protease Tolloid (Tld) cleaves Chd, which releases BMP from the complex and permits it to bind the BMP receptor and signal. In sea urchin embryos, BMP is produced in the ventral ectoderm, but signals in the dorsal ectoderm. The transport of BMP from the ventral ectoderm to the dorsal ectoderm in sea urchin embryos is not understood. Therefore, using information from a series of experiments, we adapt the mathematical model of Mizutani et al. (2005) and embed it as the reaction part of a one-dimensional reaction-diffusion model. We use it to study aspects of this transport process in sea urchin embryos. We demonstrate that the receptor-bound BMP concentration exhibits dorsally centered peaks of the same type as those observed experimentally when the ternary transport complex (Chd-Tsg-BMP) forms relatively quickly and BMP receptor binding is relatively slow. Similarly, dorsally centered peaks are created when the diffusivities of BMP, Chd, and Chd-Tsg are relatively low and that of Chd-Tsg-BMP is relatively high, and the model dynamics also suggest that Tld is a principal regulator of the system. At the end of this paper, we briefly compare the observed dynamics in the sea urchin model to a version that applies to the fly embryo, and we find that the same conditions can account for BMP transport in the two types of embryos only if Tld levels are reduced in sea urchin compared to fly. Copyright © 2014 Elsevier Ltd. All rights

Smad, PI3K/Akt, and Wnt-dependent signaling pathways are involved in BMP-4-induced ESC self-renewal.

Science.gov (United States)

Lee, Min Young; Lim, Hyun Woo; Lee, Sang Hun; Han, Ho Jae

2009-08-01

It is known that bone morphogenetic protein 4 (BMP-4) has a diverse effect on ESCs. However, its precise mechanism in mouse ESCs is not fully understood. We evaluated the effect of BMP-4 on ESC proliferation and its related signal cascades in this study. BMP-4 significantly increased the level of [(3)H]-thymidine incorporation in time- (> or =8 hours) and dose- (> or =10 ng/ml) dependent manners. Additionally, BMP-4 increased cyclin D1 and decreased p27(kip1) expression values in a time-dependent manner. The increases in BMP-4-induced [(3)H]-thymidine incorporation and cyclin D1 expression were inhibited by the BMP-4 receptor antagonist noggin. BMP-4 increased Wnt1 expression. Wnt1 expression was attenuated by Smad4 small interfering RNA (siRNA), and BMP-4-induced cyclin D1 expression was inhibited by Smad4 and Wnt1 siRNAs. BMP-4 also activated beta-catenin, which was blocked by Smad4 and Wnt1 siRNAs. In addition, BMP-4 induced Akt phosphorylation. BMP-4-induced beta-catenin activation and cyclin D1 expression were attenuated by phosphatidyl inositol 3-kinase (PI3K) siRNA and Akt inhibitor. Additionally, downregulation of Smad4, Wnt1, and PI3K expression by siRNA decreased the levels of pluripotency marker mRNAs of ESCs, including Oct4, Sox2, and FoxD3. Our results suggested that BMP-4-induced [(3)H]-thymidine incorporation was significantly attenuated by Smad4, Wnt1, and PI3K knockdown. In conclusion, BMP-4 contributed to the maintenance of cell proliferation and the pluripotent state by Smad, PI3K/Akt, and Wnt1/beta-catenin in mouse ESCs.

Noggin and Wnt3a enable BMP4-dependent differentiation of telencephalic stem cells into GluR-agonist responsive neurons

DEFF Research Database (Denmark)

Andersson, Therese; Duckworth, Joshua K; Fritz, Nicolas

2011-01-01

levels, that in turn exerted a concentration-dependent inhibition of BMP4-mediated mesenchymal differentiation of NSCs. Instead, BMP4 exposure of NSCs induced neuronal differentiation in mesenchyme-preventing conditions, whereas treatment with recombinant noggin alone did not. Wnt signaling is known...... to be essential for the development of neurons derived from the dorsal telencephalon, and co-stimulation of NSCs with BMP4+Wnt3a resulted in a synergistic effect yielding significantly increased number of mature neurons compared to stimulation with each factor alone. Thus whereas only a subset of BMP4-induced...... neurons derived from telencephalic NSCs, responded to glutamate receptor (GluR) agonists, over 80% of BMP4+Wnt3a-induced neurons responded appropriately to GluR-agonists. Our results increase the understanding of the role for BMP4 in differentiation of telencephalic multipotent progenitors, and reveal...

Twisted gastrulation, a BMP antagonist, exacerbates podocyte injury.

Directory of Open Access Journals (Sweden)

Sachiko Yamada

Full Text Available Podocyte injury is the first step in the progression of glomerulosclerosis. Previous studies have demonstrated the beneficial effect of bone morphogenetic protein 7 (Bmp7 in podocyte injury and the existence of native Bmp signaling in podocytes. Local activity of Bmp7 is controlled by cell-type specific Bmp antagonists, which inhibit the binding of Bmp7 to its receptors. Here we show that the product of Twisted gastrulation (Twsg1, a Bmp antagonist, is the central negative regulator of Bmp function in podocytes and that Twsg1 null mice are resistant to podocyte injury. Twsg1 was the most abundant Bmp antagonist in murine cultured podocytes. The administration of Bmp induced podocyte differentiation through Smad signaling, whereas the simultaneous administration of Twsg1 antagonized the effect. The administration of Bmp also inhibited podocyte proliferation, whereas simultaneous administration of Twsg1 antagonized the effect. Twsg1 was expressed in the glomerular parietal cells (PECs and distal nephron of the healthy kidney, and additionally in damaged glomerular cells in a murine model of podocyte injury. Twsg1 null mice exhibited milder hypoalbuminemia and hyperlipidemia, and milder histological changes while maintaining the expression of podocyte markers during podocyte injury model. Taken together, our results show that Twsg1 plays a critical role in the modulation of protective action of Bmp7 on podocytes, and that inhibition of Twsg1 is a promising means of development of novel treatment for podocyte injury.

Regulation of Msx-1, Msx-2, Bmp-2 and Bmp-4 during foetal and postnatal mammary gland development.

Science.gov (United States)

Phippard, D J; Weber-Hall, S J; Sharpe, P T; Naylor, M S; Jayatalake, H; Maas, R; Woo, I; Roberts-Clark, D; Francis-West, P H; Liu, Y H; Maxson, R; Hill, R E; Dale, T C

1996-09-01

Expression of the Msx-1 and Msx-2 homeobox genes have been shown to be coordinately regulated with the Bmp-2 and Bmp-4 ligands in a variety of developing tissues. Here we report that transcripts from all four genes are developmentally regulated during both foetal and postnatal mammary gland development. The location and time-course of the Bmp and Msx expression point to a role for Msx and Bmp gene products in the control of epithelial-mesenchymal interactions. Expression of Msx-2, but not Msx-1, Bmp-2 or Bmp-4 was decreased following ovariectomy, while expression of the human Msx-2 homologue was regulated by 17beta-oestradiol in the MCF-7 breast cancer cell line. The regulation of Msx-2 expression by oestrogen raises the possibility that hormonal regulation of mammary development is mediated through the control of epithelial-mesenchymal interactions.

Regulation of Gastric Lgr5+ve Cell Homeostasis by Bone Morphogenetic Protein (BMP Signaling and Inflammatory StimuliSummary

Directory of Open Access Journals (Sweden)

Wei Ye

Full Text Available Background & Aims: Gastric Leucine-rich repeat-containing G-protein-coupled receptor 5 (Lgr5 cells exert important functions during injury and homeostasis. Bone morphogenetic protein (BMP signaling regulates gastric inflammation and epithelial homeostasis. We investigated if BMP signaling controls the fate of Lgr5+ve cells during inflammation. Methods: The H+/K+-adenosine triphosphatase β-subunit promoter was used to express the BMP inhibitor noggin (Nog in the stomach (H+/K+-Nog mice. Inhibition of BMP signaling in Lgr5 cells was achieved by crossing Lgr5-EGFP-ires-CreERT2 (Lgr5-Cre mice to mice with floxed alleles of BMP receptor 1A (Lgr5-Cre;Bmpr1aflox/flox mice. Lgr5/GFP+ve cells were isolated using flow cytometry. Lineage tracing studies were conducted by crossing Lgr5-Cre mice to mice that express Nog and tdTomato (Lgr5-Cre;H+/K+-Nog;Rosa26-tdTom. Infection with Helicobacter felis was used to induce inflammation. Morphology of the mucosa was analyzed by H&E staining. Distribution of H+/K+-adenosine triphosphatase-, IF-, Ki67-, CD44-, CD44v9-, and bromodeoxyuridine-positive cells was analyzed by immunostaining. Expression of neck and pit cell mucins was determined by staining with the lectins Griffonia (Bandeiraea simplicifolia lectin II and Ulex europaeus agglutinin 1, respectively. Id1, Bmpr1a, Lgr5, c-Myc, and Cd44 messenger RNAs were measured by quantitative reverse-transcription polymerase chain reaction. Results: Lgr5-Cre;Bmpr1aflox/flox mice showed diminished expression of Bmpr1a in Lgr5/GFP+ve cells. Infection of Lgr5-Cre;Bmpr1aflox/flox mice with H felis led to enhanced inflammation, increased cell proliferation, parietal cell loss, and to the development of metaplasia and dysplasia. Infected Lgr5-Cre;H+/K+-Nog;Rosa26-tdTom mice, but not control mice, showed the presence of tomato+ve glands lining the lesser curvature that stained positively with Griffonia (Bandeiraea simplicifolia lectin II and Ulex europaeus agglutinin 1, and

Regulation of bone morphogenetic protein signalling and cranial osteogenesis by Gpc1 and Gpc3.

Science.gov (United States)

Dwivedi, Prem P; Grose, Randall H; Filmus, Jorge; Hii, Charles S T; Xian, Cory J; Anderson, Peter J; Powell, Barry C

2013-08-01

From birth, the vault of the skull grows at a prodigious rate, driven by the activity of osteoblastic cells at the fibrous joints (sutures) that separate the bony calvarial plates. One in 2500 children is born with a medical condition known as craniosynostosis because of premature bony fusion of the calvarial plates and a cessation of bone growth at the sutures. Bone morphogenetic proteins (BMPs) are potent growth factors that promote bone formation. Previously, we found that Glypican-1 (GPC1) and Glypican-3 (GPC3) are expressed in cranial sutures and are decreased during premature suture fusion in children. Although glypicans are known to regulate BMP signalling, a mechanistic link between GPC1, GPC3 and BMPs and osteogenesis has not yet been investigated. We now report that human primary suture mesenchymal cells coexpress GPC1 and GPC3 on the cell surface and release them into the media. We show that they inhibit BMP2, BMP4 and BMP7 activities, which both physically interact with BMP2 and that immunoblockade of endogenous GPC1 and GPC3 potentiates BMP2 activity. In contrast, increased levels of GPC1 and GPC3 as a result of overexpression or the addition of recombinant protein, inhibit BMP2 signalling and BMP2-mediated osteogenesis. We demonstrate that BMP signalling in suture mesenchymal cells is mediated by both SMAD-dependent and SMAD-independent pathways and that GPC1 and GPC3 inhibit both pathways. GPC3 inhibition of BMP2 activity is independent of attachment of the glypican on the cell surface and post-translational glycanation, and thus appears to be mediated by the core glypican protein. The discovery that GPC1 and GPC3 regulate BMP2-mediated osteogenesis, and that inhibition of endogenous GPC1 and GPC3 potentiates BMP2 responsiveness of human suture mesenchymal cells, indicates how downregulation of glypican expression could lead to the bony suture fusion that characterizes craniosynostosis. Copyright © 2013 Elsevier Inc. All rights reserved.

A targeted glycan-related gene screen reveals heparan sulfate proteoglycan sulfation regulates WNT and BMP trans-synaptic signaling.

Directory of Open Access Journals (Sweden)

Neil Dani

Full Text Available A Drosophila transgenic RNAi screen targeting the glycan genome, including all N/O/GAG-glycan biosynthesis/modification enzymes and glycan-binding lectins, was conducted to discover novel glycan functions in synaptogenesis. As proof-of-product, we characterized functionally paired heparan sulfate (HS 6-O-sulfotransferase (hs6st and sulfatase (sulf1, which bidirectionally control HS proteoglycan (HSPG sulfation. RNAi knockdown of hs6st and sulf1 causes opposite effects on functional synapse development, with decreased (hs6st and increased (sulf1 neurotransmission strength confirmed in null mutants. HSPG co-receptors for WNT and BMP intercellular signaling, Dally-like Protein and Syndecan, are differentially misregulated in the synaptomatrix of these mutants. Consistently, hs6st and sulf1 nulls differentially elevate both WNT (Wingless; Wg and BMP (Glass Bottom Boat; Gbb ligand abundance in the synaptomatrix. Anterograde Wg signaling via Wg receptor dFrizzled2 C-terminus nuclear import and retrograde Gbb signaling via synaptic MAD phosphorylation and nuclear import are differentially activated in hs6st and sulf1 mutants. Consequently, transcriptional control of presynaptic glutamate release machinery and postsynaptic glutamate receptors is bidirectionally altered in hs6st and sulf1 mutants, explaining the bidirectional change in synaptic functional strength. Genetic correction of the altered WNT/BMP signaling restores normal synaptic development in both mutant conditions, proving that altered trans-synaptic signaling causes functional differentiation defects.

Adenoviral Mediated Expression of BMP2 by Bone Marrow Stromal Cells Cultured in 3D Copolymer Scaffolds Enhances Bone Formation.

Science.gov (United States)

Sharma, Sunita; Sapkota, Dipak; Xue, Ying; Sun, Yang; Finne-Wistrand, Anna; Bruland, Ove; Mustafa, Kamal

2016-01-01

Selection of appropriate osteoinductive growth factors, suitable delivery method and proper supportive scaffold are critical for a successful outcome in bone tissue engineering using bone marrow stromal cells (BMSC). This study examined the molecular and functional effect of a combination of adenoviral mediated expression of bone morphogenetic protein-2 (BMP2) in BMSC and recently developed and characterized, biodegradable Poly(L-lactide-co-є-caprolactone){poly(LLA-co-CL)}scaffolds in osteogenic molecular changes and ectopic bone formation by using in vitro and in vivo approaches. Pathway-focused custom PCR array, validation using TaqMan based quantitative RT-PCR (qRT-PCR) and ALP staining showed significant up-regulation of several osteogenic and angiogenic molecules, including ALPL and RUNX2 in ad-BMP2 BMSC group grown in poly(LLA-co-CL) scaffolds both at 3 and 14 days. Micro CT and histological analyses of the subcutaneously implanted scaffolds in NOD/SCID mice revealed significantly increased radiopaque areas, percentage bone volume and formation of vital bone in ad-BMP2 scaffolds as compared to the control groups both at 2 and 8 weeks. The increased bone formation in the ad-BMP2 group in vivo was paralleled at the molecular level with concomitant over-expression of a number of osteogenic and angiogenic genes including ALPL, RUNX2, SPP1, ANGPT1. The increased bone formation in ad-BMP2 explants was not found to be associated with enhanced endochondral activity as evidenced by qRT-PCR (SOX9 and FGF2) and Safranin O staining. Taken together, combination of adenoviral mediated BMP-2 expression in BMSC grown in the newly developed poly(LLA-co-CL) scaffolds induced expression of osteogenic markers and enhanced bone formation in vivo.

Kaempferol induces chondrogenesis in ATDC5 cells through activation of ERK/BMP-2 signaling pathway.

Science.gov (United States)

Nepal, Manoj; Li, Liang; Cho, Hyoung Kwon; Park, Jong Kun; Soh, Yunjo

2013-12-01

Endochondral bone formation occurs when mesenchymal cells condense to differentiate into chondrocytes, the primary cell types of cartilage. The aim of the present study was to identify novel factors regulating chondrogenesis. We investigated whether kaempferol induces chondrogenic differentiation in clonal mouse chondrogenic ATDC5 cells. Kaempferol treatment stimulated the accumulation of cartilage nodules in a dose-dependent manner. Kaempferol-treated ATDC5 cells stained more intensely with alcian blue staining than control cells, suggesting greater synthesis of matrix proteoglycans in the kaempferol-treated cells. Similarly, kaempferol induced greater activation of alkaline phosphatase activity than control cells, and it enhanced the expression of chondrogenic marker genes, such as collagen type I, collagen type X, OCN, Runx2, and Sox9. Kaempferol induced an acute activation of extracellular signal-regulated kinase (ERK) but not c-jun N-terminal kinase or p38 MAP kinase. PD98059, an inhibitor of MAPK/ERK, decreased in stained cells treated with kaempferol. Furthermore, kaempferol greatly expressed the protein and mRNA levels of BMP-2, suggesting chondrogenesis was stimulated via a BMP-2 pathway. Taken together, our results suggest that kaempferol has chondromodulating effects via an ERK/BMP-2 signaling pathway and could potentially be used as a therapeutic agent for bone growth disorders. Copyright © 2013 Elsevier Ltd. All rights reserved.

Rapamycin inhibits BMP-7-induced osteogenic and lipogenic marker expressions in fetal rat calvarial cells.

Science.gov (United States)

Yeh, Lee-Chuan C; Ma, Xiuye; Ford, Jeffery J; Adamo, Martin L; Lee, John C

2013-08-01

Bone morphogenetic proteins (BMPs) promote osteoblast differentiation and bone formation in vitro and in vivo. BMPs canonically signal through Smad transcription factors, but BMPs may activate signaling pathways traditionally stimulated by growth factor tyrosine kinase receptors. Of these, the mTOR pathway has received considerable attention because BMPs activate P70S6K, a downstream effector of mTOR, suggesting that BMP-induced osteogenesis is mediated by mTOR activation. However, contradictory effects of the mTOR inhibitor rapamycin (RAPA) on bone formation have been reported. Since bone formation is thought to be inversely related to lipid accumulation and mTOR is also important for lipid synthesis, we postulated that BMP-7 may stimulate lipogenic enzyme expression in a RAPA-sensitive mechanism. To test this hypothesis, we determined the effects of RAPA on BMP-7-stimulated expression of osteogenic and lipogenic markers in cultured fetal rat calvarial cells. Our study showed that BMP-7 promoted the expression of osteogenic and lipogenic markers. The effect of BMP-7 on osteogenic markers was greater in magnitude than on lipogenic markers and was temporally more sustained. RAPA inhibited basal and BMP-7-stimulated osteogenic and lipogenic marker expression and bone nodule mineralization. The acetyl CoA carboxylase inhibitor TOFA stimulated the expression of osteoblast differentiation markers, whereas palmitate suppressed their expression. We speculate that the BMP-7-stimulated adipogenesis is part of the normal anabolic response to BMPs, but that inappropriate activation of the lipid biosynthetic pathway by mTOR could have deleterious effects on bone formation and could explain paradoxical effects of RAPA to promote bone formation. Copyright © 2013 Wiley Periodicals, Inc.

BMP pathway regulation of and by macrophages.

Directory of Open Access Journals (Sweden)

Megha Talati

Full Text Available Pulmonary arterial hypertension (PAH is a disease of progressively increasing pulmonary vascular resistance, associated with mutations of the type 2 receptor for the BMP pathway, BMPR2. The canonical signaling pathway for BMPR2 is through the SMAD family of transcription factors. BMPR2 is expressed in every cell type, but the impact of BMPR2 mutations affecting SMAD signaling, such as Bmpr2delx4+, had only previously been investigated in smooth muscle and endothelium. In the present study, we created a mouse with universal doxycycline-inducible expression of Bmpr2delx4+ in order to determine if broader expression had an impact relevant to the development of PAH. We found that the most obvious phenotype was a dramatic, but patchy, increase in pulmonary inflammation. We crossed these double transgenic mice onto an NF-κB reporter strain, and by luciferase assays on live mice, individual organs and isolated macrophages, we narrowed down the origin of the inflammatory phenotype to constitutive activation of tissue macrophages. Study of bone marrow-derived macrophages from mutant and wild-type mice suggested a baseline difference in differentiation state in Bmpr2 mutants. When activated with LPS, both mutant and wild-type macrophages secrete BMP pathway inhibitors sufficient to suppress BMP pathway activity in smooth muscle cells (SMC treated with conditioned media. Functionally, co-culture with macrophages results in a BMP signaling-dependent increase in scratch closure in cultured SMC. We conclude that SMAD signaling through BMP is responsible, in part, for preventing macrophage activation in both live animals and in cells in culture, and that activated macrophages secrete BMP inhibitors in sufficient quantity to cause paracrine effect on vascular smooth muscle.

A BMP7 Variant Inhibits Tumor Angiogenesis In Vitro and In Vivo through Direct Modulation of Endothelial Cell Biology.

Directory of Open Access Journals (Sweden)

Courtney M Tate

Full Text Available Bone morphogenetic proteins (BMPs, members of the TGF-β superfamily, have numerous biological activities including control of growth, differentiation, and vascular development. Using an in vitro co-culture endothelial cord formation assay, we investigated the role of a BMP7 variant (BMP7v in VEGF, bFGF, and tumor-driven angiogenesis. BMP7v treatment led to disruption of neo-endothelial cord formation and regression of existing VEGF and bFGF cords in vitro. Using a series of tumor cell models capable of driving angiogenesis in vitro, BMP7v treatment completely blocked cord formation. Pre-treatment of endothelial cells with BMP7v significantly reduced their cord forming ability, indicating a direct effect on endothelial cell function. BMP7v activated the canonical SMAD signaling pathway in endothelial cells but targeted gene knockdown using shRNA directed against SMAD4 suggests this pathway is not required to mediate the anti-angiogenic effect. In contrast to SMAD activation, BMP7v selectively decreased ERK and AKT activation, significantly decreased endothelial cell migration and down-regulated expression of critical RTKs involved in VEGF and FGF angiogenic signaling, VEGFR2 and FGFR1 respectively. Importantly, in an in vivo angiogenic plug assay that serves as a measurement of angiogenesis, BMP7v significantly decreased hemoglobin content indicating inhibition of neoangiogenesis. In addition, BMP7v significantly decreased angiogenesis in glioblastoma stem-like cell (GSLC Matrigel plugs and significantly impaired in vivo growth of a GSLC xenograft with a concomitant reduction in microvessel density. These data support BMP7v as a potent anti-angiogenic molecule that is effective in the context of tumor angiogenesis.

A BMP7 Variant Inhibits Tumor Angiogenesis In Vitro and In Vivo through Direct Modulation of Endothelial Cell Biology.

Science.gov (United States)

Tate, Courtney M; Mc Entire, Jacquelyn; Pallini, Roberto; Vakana, Eliza; Wyss, Lisa; Blosser, Wayne; Ricci-Vitiani, Lucia; D'Alessandris, Quintino Giorgio; Morgante, Liliana; Giannetti, Stefano; Larocca, Luigi Maria; Todaro, Matilde; Benfante, Antonina; Colorito, Maria Luisa; Stassi, Giorgio; De Maria, Ruggero; Rowlinson, Scott; Stancato, Louis

2015-01-01

Bone morphogenetic proteins (BMPs), members of the TGF-β superfamily, have numerous biological activities including control of growth, differentiation, and vascular development. Using an in vitro co-culture endothelial cord formation assay, we investigated the role of a BMP7 variant (BMP7v) in VEGF, bFGF, and tumor-driven angiogenesis. BMP7v treatment led to disruption of neo-endothelial cord formation and regression of existing VEGF and bFGF cords in vitro. Using a series of tumor cell models capable of driving angiogenesis in vitro, BMP7v treatment completely blocked cord formation. Pre-treatment of endothelial cells with BMP7v significantly reduced their cord forming ability, indicating a direct effect on endothelial cell function. BMP7v activated the canonical SMAD signaling pathway in endothelial cells but targeted gene knockdown using shRNA directed against SMAD4 suggests this pathway is not required to mediate the anti-angiogenic effect. In contrast to SMAD activation, BMP7v selectively decreased ERK and AKT activation, significantly decreased endothelial cell migration and down-regulated expression of critical RTKs involved in VEGF and FGF angiogenic signaling, VEGFR2 and FGFR1 respectively. Importantly, in an in vivo angiogenic plug assay that serves as a measurement of angiogenesis, BMP7v significantly decreased hemoglobin content indicating inhibition of neoangiogenesis. In addition, BMP7v significantly decreased angiogenesis in glioblastoma stem-like cell (GSLC) Matrigel plugs and significantly impaired in vivo growth of a GSLC xenograft with a concomitant reduction in microvessel density. These data support BMP7v as a potent anti-angiogenic molecule that is effective in the context of tumor angiogenesis.

Abrogation of epithelial BMP2 and BMP4 causes Amelogenesis Imperfecta by reducing MMP20 and KLK4 expression.

Science.gov (United States)

Xie, Xiaohua; Liu, Chao; Zhang, Hua; Jani, Priyam H; Lu, Yongbo; Wang, Xiaofang; Zhang, Bin; Qin, Chunlin

2016-05-05

Amelogenesis Imperfecta (AI) can be caused by the deficiencies of enamel matrix proteins, molecules responsible for the transportation and secretion of enamel matrix components, and proteases processing enamel matrix proteins. In the present study, we discovered the double deletion of bone morphogenetic protein 2 (Bmp2) and bone morphogenetic protein 4 (Bmp4) in the dental epithelium by K14-cre resulted in hypoplastic enamel and reduced density in X-ray radiography as well as shortened enamel rods under scanning electron microscopy. Such enamel phenotype was consistent with the diagnosis of hypoplastic amelogenesis imperfecta. Histological and molecular analyses revealed that the removal of matrix proteins in the mutant enamel was drastically delayed, which was coincided with the greatly reduced expression of matrix metalloproteinase 20 (MMP20) and kallikrein 4 (KLK4). Although the expression of multiple enamel matrix proteins was down-regulated in the mutant ameloblasts, the cleavage of ameloblastin was drastically impaired. Therefore, we attributed the AI primarily to the reduction of MMP20 and KLK4. Further investigation found that BMP/Smad4 signaling pathway was down-regulated in the K14-cre;Bmp2(f/f);Bmp4(f/f)ameloblasts, suggesting that the reduced MMP20 and KLK4 expression may be due to the attenuated epithelial BMP/Smad4 signaling.

BMPRIA mediated signaling is essential for temporomandibular joint development in mice.

Directory of Open Access Journals (Sweden)

Shuping Gu

Full Text Available The central importance of BMP signaling in the development and homeostasis of synovial joint of appendicular skeleton has been well documented, but its role in the development of temporomandibular joint (TMJ, also classified as a synovial joint, remains completely unknown. In this study, we investigated the function of BMPRIA mediated signaling in TMJ development in mice by transgenic loss-of- and gain-of-function approaches. We found that BMPRIA is expressed in the cranial neural crest (CNC-derived developing condyle and glenoid fossa, major components of TMJ, as well as the interzone mesenchymal cells. Wnt1-Cre mediated tissue specific inactivation of BmprIa in CNC lineage led to defective TMJ development, including failure of articular disc separation from a hypoplastic condyle, persistence of interzone cells, and failed formation of a functional fibrocartilage layer on the articular surface of the glenoid fossa and condyle, which could be at least partially attributed to the down-regulation of Ihh in the developing condyle and inhibition of apoptosis in the interzone. On the other hand, augmented BMPRIA signaling by Wnt1-Cre driven expression of a constitutively active form of BmprIa (caBmprIa inhibited osteogenesis of the glenoid fossa and converted the condylar primordium from secondary cartilage to primary cartilage associated with ectopic activation of Smad-dependent pathway but inhibition of JNK pathway, leading to TMJ agenesis. Our results present unambiguous evidence for an essential role of finely tuned BMPRIA mediated signaling in TMJ development.

Influence of WFIKKN1 on BMP1-mediated activation of latent myostatin.

Science.gov (United States)

Szláma, György; Vásárhelyi, Viktor; Trexler, Mária; Patthy, László

2016-12-01

The NTR domain of WFIKKN1 protein has been shown to have significant affinity for the prodomain regions of promyostatin and latent myostatin but the biological significance of these interactions remained unclear. In view of its role as a myostatin antagonist, we tested the assumption that WFIKKN1 inhibits the release of myostatin from promyostatin and/or latent myostatin. WFIKKN1 was found to have no effect on processing of promyostatin by furin, the rate of cleavage of latent myostatin by BMP1, however, was significantly enhanced in the presence of WFIKKN1 and this enhancer activity was superstimulated by heparin. Unexpectedly, WFIKKN1 was also cleaved by BMP1 and our studies have shown that the KKN1 fragment generated by BMP1-cleavage of WFIKKN1 contributes most significantly to the observed enhancer activity. Analysis of a pro-TGF-β -based homology model of homodimeric latent myostatin revealed that the BMP1-cleavage sites are buried and not readily accessible to BMP1. In view of this observation, the most plausible explanation for the BMP1-enhancer activity of the KKN1 fragment is that it shifts a conformational equilibrium of latent myostatin from the closed circular structure of the homodimer to a more open form, making the cleavage sites more accessible to BMP1. On the other hand, the observation that the enhancer activity of KKN1 is superstimulated in the presence of heparin is explained by the fact KKN1, latent myostatin, and BMP1 have affinity for heparin and these interactions with heparin increase the local concentrations of the reactants thereby facilitating the action of BMP1. Furin: EC 3.4.21.75; BMP1, bone morphogentic protein 1 or procollagen C-endopeptidase: EC 3.4.24.19. © 2016 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.

Endocardial to myocardial notch-wnt-bmp axis regulates early heart valve development.

Directory of Open Access Journals (Sweden)

Yidong Wang

Full Text Available Endocardial to mesenchymal transformation (EMT is a fundamental cellular process required for heart valve formation. Notch, Wnt and Bmp pathways are known to regulate this process. To further address how these pathways coordinate in the process, we specifically disrupted Notch1 or Jagged1 in the endocardium of mouse embryonic hearts and showed that Jagged1-Notch1 signaling in the endocardium is essential for EMT and early valvular cushion formation. qPCR and RNA in situ hybridization assays reveal that endocardial Jagged1-Notch1 signaling regulates Wnt4 expression in the atrioventricular canal (AVC endocardium and Bmp2 in the AVC myocardium. Whole embryo cultures treated with Wnt4 or Wnt inhibitory factor 1 (Wif1 show that Bmp2 expression in the AVC myocardium is dependent on Wnt activity; Wnt4 also reinstates Bmp2 expression in the AVC myocardium of endocardial Notch1 null embryos. Furthermore, while both Wnt4 and Bmp2 rescue the defective EMT resulting from Notch inhibition, Wnt4 requires Bmp for its action. These results demonstrate that Jagged1-Notch1 signaling in endocardial cells induces the expression of Wnt4, which subsequently acts as a paracrine factor to upregulate Bmp2 expression in the adjacent AVC myocardium to signal EMT.

Delta-like 1/fetal antigen 1(DLK1/FA1) inhibits BMP2 induced osteoblast differentiation through modulation of NFκB signaling pathway

DEFF Research Database (Denmark)

Qiu, Weimin; Abdallah, Basem; Kassem, Moustapha

DLK1/FA1 (delta-like 1/fetal antigen-1) is a negative regulator of bone mass that acts to inhibit osteoblast differentiation and stimulate osteoclast differentiation. However, the molecular mechanisms underlying these effects are not known. Thus, we studied the effect of DLK1/FA1 on different...... osteogenic factors-induced osteoblast differentiation. We identified DLK1/FA1 as an inhibitor of BMP2-induced osteogenesis in mouse myoblast C2C12 cells. Stable overexpression of DLK1/FA1 in C2C12 cells or the addition of its soluble form protein FA1 significantly inhibited BMP2-induced osteogenesis...... as assessed by reduced Alp activity and osteogenic gene expression including Alp, Col1a1, Runx2 and Bglap. In addition, DLK1/FA1 inhibited BMP signaling as demonstrated by reduced gene expression of BMP-responsive genes: Junb and Id1, reduced BMP2 induced luciferase activity in C2C12 BMP luciferase reporter...

A SHH-FOXF1-BMP4 signaling axis regulating growth and differentiation of epithelial and mesenchymal tissues in ureter development.

Science.gov (United States)

Bohnenpoll, Tobias; Wittern, Anna B; Mamo, Tamrat M; Weiss, Anna-Carina; Rudat, Carsten; Kleppa, Marc-Jens; Schuster-Gossler, Karin; Wojahn, Irina; Lüdtke, Timo H-W; Trowe, Mark-Oliver; Kispert, Andreas

2017-08-01

The differentiated cell types of the epithelial and mesenchymal tissue compartments of the mature ureter of the mouse arise in a precise temporal and spatial sequence from uncommitted precursor cells of the distal ureteric bud epithelium and its surrounding mesenchyme. Previous genetic efforts identified a member of the Hedgehog (HH) family of secreted proteins, Sonic hedgehog (SHH) as a crucial epithelial signal for growth and differentiation of the ureteric mesenchyme. Here, we used conditional loss- and gain-of-function experiments of the unique HH signal transducer Smoothened (SMO) to further characterize the cellular functions and unravel the effector genes of HH signaling in ureter development. We showed that HH signaling is not only required for proliferation and SMC differentiation of cells of the inner mesenchymal region but also for survival of cells of the outer mesenchymal region, and for epithelial proliferation and differentiation. We identified the Forkhead transcription factor gene Foxf1 as a target of HH signaling in the ureteric mesenchyme. Expression of a repressor version of FOXF1 in this tissue completely recapitulated the mesenchymal and epithelial proliferation and differentiation defects associated with loss of HH signaling while re-expression of a wildtype version of FOXF1 in the inner mesenchymal layer restored these cellular programs when HH signaling was inhibited. We further showed that expression of Bmp4 in the ureteric mesenchyme depends on HH signaling and Foxf1, and that exogenous BMP4 rescued cell proliferation and epithelial differentiation in ureters with abrogated HH signaling or FOXF1 function. We conclude that SHH uses a FOXF1-BMP4 module to coordinate the cellular programs for ureter elongation and differentiation, and suggest that deregulation of this signaling axis occurs in human congenital anomalies of the kidney and urinary tract (CAKUT).

A SHH-FOXF1-BMP4 signaling axis regulating growth and differentiation of epithelial and mesenchymal tissues in ureter development.

Directory of Open Access Journals (Sweden)

Tobias Bohnenpoll

2017-08-01

Full Text Available The differentiated cell types of the epithelial and mesenchymal tissue compartments of the mature ureter of the mouse arise in a precise temporal and spatial sequence from uncommitted precursor cells of the distal ureteric bud epithelium and its surrounding mesenchyme. Previous genetic efforts identified a member of the Hedgehog (HH family of secreted proteins, Sonic hedgehog (SHH as a crucial epithelial signal for growth and differentiation of the ureteric mesenchyme. Here, we used conditional loss- and gain-of-function experiments of the unique HH signal transducer Smoothened (SMO to further characterize the cellular functions and unravel the effector genes of HH signaling in ureter development. We showed that HH signaling is not only required for proliferation and SMC differentiation of cells of the inner mesenchymal region but also for survival of cells of the outer mesenchymal region, and for epithelial proliferation and differentiation. We identified the Forkhead transcription factor gene Foxf1 as a target of HH signaling in the ureteric mesenchyme. Expression of a repressor version of FOXF1 in this tissue completely recapitulated the mesenchymal and epithelial proliferation and differentiation defects associated with loss of HH signaling while re-expression of a wildtype version of FOXF1 in the inner mesenchymal layer restored these cellular programs when HH signaling was inhibited. We further showed that expression of Bmp4 in the ureteric mesenchyme depends on HH signaling and Foxf1, and that exogenous BMP4 rescued cell proliferation and epithelial differentiation in ureters with abrogated HH signaling or FOXF1 function. We conclude that SHH uses a FOXF1-BMP4 module to coordinate the cellular programs for ureter elongation and differentiation, and suggest that deregulation of this signaling axis occurs in human congenital anomalies of the kidney and urinary tract (CAKUT.

BMP9 inhibits the bone metastasis of breast cancer cells by downregulating CCN2 (connective tissue growth factor, CTGF) expression.

Science.gov (United States)

Ren, Wei; Sun, Xiaoxiao; Wang, Ke; Feng, Honglei; Liu, Yuehong; Fei, Chang; Wan, Shaoheng; Wang, Wei; Luo, Jinyong; Shi, Qiong; Tang, Min; Zuo, Guowei; Weng, Yaguang; He, Tongchuan; Zhang, Yan

2014-03-01

Bone morphogenetic proteins (BMPs), which belong to the transforming growth factor-β superfamily, regulate a wide range of cellular responses including cell proliferation, differentiation, adhesion, migration, and apoptosis. BMP9, the latest BMP to be discovered, is reportedly expressed in a variety of human carcinoma cell lines, but the role of BMP9 in breast cancer has not been fully clarified. In a previous study, BMP9 was found to inhibit the growth, migration, and invasiveness of MDA-MB-231 breast cancer cells. In the current study, the effect of BMP9 on the bone metastasis of breast cancer cells was investigated. After absent or low expression of BMP9 was detected in the MDA-MB-231 breast cancer cells and breast non-tumor adjacent tissues using Western blot and immunohistochemistry, In our previous study, BMP9 could inhibit the proliferation and invasiveness of breast cancer cells MDA-MB-231 in vitro and in vivo. This paper shows that BMP9 inhibit the bone metastasis of breast cancer cells by activating the BMP/Smad signaling pathway and downregulating connective tissue growth factor (CTGF); however, when CTGF expression was maintained, the inhibitory effect of BMP9 on the MDA-MB-231 cells was abolished. Together, these observations indicate that BMP9 is an important mediator of breast cancer bone metastasis and a potential therapeutic target for treating this deadly disease.

Genetic variation in bone morphogenetic protein (BMP) and colon and rectal cancer

Science.gov (United States)

Slattery, Martha L.; Lundgreen, Abbie; Herrick, Jennifer S.; Kadlubar, Susan; Caan, Bette J.; Potter, John D.; Wolff, Roger K.

2011-01-01

Bone morphogenetic proteins (BMP) are part of the TGF-β-signaling pathway; genetic variation in these genes may be involved in colorectal cancer. In this study we evaluated the association between genetic variation in BMP1 (11 tagSNPs), BMP2 (5 tagSNPs), BMP4 (3 tagSNPs), BMPR1A (9 tagSNPs), BMPR1B (21 tagSNPs), BMPR2 (11 tagSNPs), and GDF10 (7 tagSNPs) with risk of colon and rectal cancer and tumor molecular phenotype. We used data from population-based case-control studies (colon cancer n=1574 cases, 1970 controls; rectal cancer n=791 cases, 999 controls). We observed that genetic variation in BMPR1A, BMPR1B, BMPR2, BMP2, and BMP4 was associated with risk of developing colon cancer, with 20 to 30% increased risk for most high-risk genotypes. A summary of high-risk genotypes showed over a twofold increase in colon cancer risk at the upper risk category (OR 2.49 95% CI 1.95, 3.18). BMPR2, BMPR1B, BMP2, and GDF10 were associated with rectal cancer. BMPR2 rs2228545 was associated with an almost twofold increased risk of rectal cancer. The risk associated with the highest category of the summary score for rectal cancer was 2.97 (95% CI 1.87, 4.72). Genes in the BMP-signaling pathway were consistently associated with CIMP+ status in combination with both KRAS-mutated and MSI tumors. BMP genes interacted statistically significantly with other genes in the TGF-β-signaling pathway, including TGFβ1, TGFβR1, Smad 3, Smad 4, and Smad 7. Our data support a role for genetic variation in BMP-related genes in the etiology of colon and rectal cancer. One possible mechanism is via the TGF-β-signaling pathway. PMID:21387313

Metastatic function of BMP-2 in gastric cancer cells: The role of PI3K/AKT, MAPK, the NF-{kappa}B pathway, and MMP-9 expression

Energy Technology Data Exchange (ETDEWEB)

Kang, Myoung Hee [Graduate School of Medicine, Korea University College of Medicine, Korea University, Seoul 136-705 (Korea, Republic of); Oh, Sang Cheul [Division of Oncology/Hematology, Department of Internal Medicine, Korea University College of Medicine, Korea University, Seoul 136-705 (Korea, Republic of); Lee, Hyun Joo [Department of Pathology, Korea University College of Medicine, Korea University, Seoul 136-705 (Korea, Republic of); Kang, Han Na; Kim, Jung Lim [Graduate School of Medicine, Korea University College of Medicine, Korea University, Seoul 136-705 (Korea, Republic of); Kim, Jun Suk [Division of Oncology/Hematology, Department of Internal Medicine, Korea University College of Medicine, Korea University, Seoul 136-705 (Korea, Republic of); Yoo, Young A., E-mail: ydanbi@korea.ac.kr [Brain Korea 21 Program for Biomedical Science, Korea University College of Medicine, Korea University, Seoul 136-705 (Korea, Republic of)

2011-07-15

Bone morphogenetic proteins (BMPs) have been implicated in tumorigenesis and metastatic progression in various types of cancer cells, but the role and cellular mechanism in the invasive phenotype of gastric cancer cells is not known. Herein, we determined the roles of phosphoinositide 3-kinase (PI3K)/AKT, extracellular signal-regulated protein kinase (ERK), nuclear factor (NF)-{kappa}B, and matrix metalloproteinase (MMP) expression in BMP-2-mediated metastatic function in gastric cancer. We found that stimulation of BMP-2 in gastric cancer cells enhanced the phosphorylation of AKT and ERK. Accompanying activation of AKT and ERK kinase, BMP-2 also enhanced phosphorylation/degradation of I{kappa}B{alpha} and the nuclear translocation/activation of NF-{kappa}B. Interestingly, blockade of PI3K/AKT and ERK signaling using LY294002 and PD98059, respectively, significantly inhibited BMP-2-induced motility and invasiveness in association with the activation of NF-{kappa}B. Furthermore, BMP-2-induced MMP-9 expression and enzymatic activity was also significantly blocked by treatment with PI3K/AKT, ERK, or NF-{kappa}B inhibitors. Immunohistochemistry staining of 178 gastric tumor biopsies indicated that expression of BMP-2 and MMP-9 had a significant positive correlation with lymph node metastasis and a poor prognosis. These results indicate that the BMP-2 signaling pathway enhances tumor metastasis in gastric cancer by sequential activation of the PI3K/AKT or MAPK pathway followed by the induction of NF-{kappa}B and MMP-9 activity, indicating that BMP-2 has the potential to be a therapeutic molecular target to decrease metastasis.

Supramolecular Nanofibers Enhance Growth Factor Signaling by Increasing Lipid Raft Mobility

Energy Technology Data Exchange (ETDEWEB)

Newcomb, Christina J.; Sur, Shantanu; Lee, Sungsoo S.; Yu, Jeong Min; Zhou, Yan; Snead, Malcolm L.; Stupp, Samuel I. (NWU); (USC)

2016-04-12

The nanostructures of self-assembling biomaterials have been previously designed to tune the release of growth factors in order to optimize biological repair and regeneration. We report here on the discovery that weakly cohesive peptide nanostructures in terms of intermolecular hydrogen bonding, when combined with low concentrations of osteogenic growth factor, enhance both BMP-2 and Wnt mediated signaling in myoblasts and bone marrow stromal cells, respectively. Conversely, analogous nanostructures with enhanced levels of internal hydrogen bonding and cohesion lead to an overall reduction in BMP-2 signaling. We propose that the mechanism for enhanced growth factor signaling by the nanostructures is related to their ability to increase diffusion within membrane lipid rafts. The phenomenon reported here could lead to new nanomedicine strategies to mediate growth factor signaling for translational targets.

TNF-α Upregulates Expression of BMP-2 and BMP-3 Genes in the Rat Dental Follicle – Implications for Tooth Eruption

Science.gov (United States)

Yao, Shaomian; Prpic, Veronica; Pan, Fenghui; Wise, Gary E.

2011-01-01

The dental follicle appears to regulate both the alveolar bone resorption and bone formation needed for tooth eruption. Tumor necrosis factor-alpha ( TNF-α) gene expression is maximally upregulated at postnatal day 9 in the rat dental follicle of the 1st mandibular molar, a time that correlates with rapid bone growth at the base of the tooth crypt, as well as a minor burst of osteoclastogenesis. TNF-α expression is correlated with the expression of bone morphogenetic protein-2 (BMP-2), a molecule expressed in the dental follicle that can promote bone formation. Because BMP-2 signaling may be augmented by bone morphogenetic protein-3 (BMP-3), it was the objective of this study to determine 1) if the dental follicle expresses BMP-3 and 2) if TNF-α stimulates the dental follicle cells to express BMP-2 and BMP-3. Dental follicles were collected from different postnatal ages of rat pups. Dental follicle cells were incubated with TNF-α to study its dosage and time-course effects on gene expression of BMP-2 and BMP-3, as determined by real-time RT-PCR. Next, immunostaining was conducted to confirm if the protein was synthesized and ELISA of the conditioned medium was conducted to determine if BMP-2 was secreted. We found that BMP-3 expression is correlated with the expression of TNF-α in the dental follicle and TNF-α significantly increased BMP-2 and BMP-3 expression in vitro. Immunostaining and ELISA showed that BMP-2 and BMP-3 were synthesized and secreted. This study suggests that TNF-α can upregulate the expression of bone formation genes that may be needed for tooth eruption. PMID:20067418

3-OST-7 regulates BMP-dependent cardiac contraction.

Directory of Open Access Journals (Sweden)

Shiela C Samson

2013-12-01

Full Text Available The 3-O-sulfotransferase (3-OST family catalyzes rare modifications of glycosaminoglycan chains on heparan sulfate proteoglycans, yet their biological functions are largely unknown. Knockdown of 3-OST-7 in zebrafish uncouples cardiac ventricular contraction from normal calcium cycling and electrophysiology by reducing tropomyosin4 (tpm4 expression. Normal 3-OST-7 activity prevents the expansion of BMP signaling into ventricular myocytes, and ectopic activation of BMP mimics the ventricular noncontraction phenotype seen in 3-OST-7 depleted embryos. In 3-OST-7 morphants, ventricular contraction can be rescued by overexpression of tropomyosin tpm4 but not by troponin tnnt2, indicating that tpm4 serves as a lynchpin for ventricular sarcomere organization downstream of 3-OST-7. Contraction can be rescued by expression of 3-OST-7 in endocardium, or by genetic loss of bmp4. Strikingly, BMP misregulation seen in 3-OST-7 morphants also occurs in multiple cardiac noncontraction models, including potassium voltage-gated channel gene, kcnh2, affected in Romano-Ward syndrome and long-QT syndrome, and cardiac troponin T gene, tnnt2, affected in human cardiomyopathies. Together these results reveal 3-OST-7 as a key component of a novel pathway that constrains BMP signaling from ventricular myocytes, coordinates sarcomere assembly, and promotes cardiac contractile function.

Construction of doxycycline-mediated BMP-2 transgene combining with APA microcapsules for bone repair.

Science.gov (United States)

Qian, Dongyang; Bai, Bo; Yan, Guangbin; Zhang, Shujiang; Liu, Qi; Chen, Yi; Tan, Xiaobo; Zeng, Yanjun

2016-01-01

The repairing of large segmental bone defects is difficult for clinical orthopedists at present. Gene therapy is regarded as a promising method for bone defects repair. The present study aimed to construct an effective and controllable Tet-On expression system for transferring hBMP-2 gene into bone marrow mesenchymal progenitor cells (BMSCs). Meanwhile, with combination of alginate-poly-L-lysine-alginate (APA) microencapsulation technology, we attempted to reduce the influence of immunologic rejection and examine the effect of the Tet-On expression system on osteogenesis of BMSCs. The adenovirus encoding hBMP-2 (ADV-hBMP2) was constructed using the means of molecular cloning. The ADV-hBMP2 and Adeno-X Tet-On virus was respectively transfected to the HEK293 for amplification and afterward BMSCs were co-infected with the virus of ADV-hBMP2 and the Adeno-X Tet-On. The expression of hBMP-2 was measured with induction by doxycycline (DOX) at different concentration by means of RT-PCR and ELISA. Combining Tet-On expression system and APA microcapsules with the use of the pulsed high-voltage electrostatic microcapsule instrument, we examined the expression level of hBMP-2 in APA microcapsules by ELISA as well as the osteogenesis by alizarin red S staining. An effective Tet-On expression system for transferring hBMP-2 gene into BMSCs was constructed successfully. Also, the expression of hBMP-2 could be regulated by concentration of DOX. The data exhibited that BMSCs in APA microcapsules maintained the capability of proliferation and differentiation. The combination of Tet-On expression system and APA microcapsules could promote the osteogenesis of BMSCs. According to the results, microencapsulated Tet-On expression system showed the effective characteristics of secreting hBMP-2 and enhancing osteogenesis, which would provide a promising way for bone repair.

Expressional and functional analyses of transcription factors activated by BMP-4s signaling in early xenopus embryo; BMP-4 shigunaru dentatsu kiko to sono hyoteki kakunai tensha inshi ni kansuru kenkyu

Energy Technology Data Exchange (ETDEWEB)

Maeno, Mitsugu [Niigata University, Niigata (Japan). Faculty of Science

1998-12-16

The expression and physiological function of two transcription factors, GATA-2 and Xmsx-1, in amphibian embryos has been analyzed. The expression of these mRNAs in embryonic cells were firmly regulated by the BMP-4 signaling, that plays a central role in the formation of ventral tissues. The microinjection studies of GATA-2 RNA into embryonic cells suggested that this factor functions in two adjacent germ layers, mesoderm and ectoderm, to participate in blood cell formation in ventral area of embryo. Embryos injected with Xmsx-1 RNA, but not with GATA-2, in dorsal blastomeres exhibited a ventralized phenotype, with microcephaly and swollen abdomen. Thus, Xmsx-1 is a ventralizing agent. However, on the basis of molecular marker analyses, Xmsx-1 did not promote erythropoietic differentiation, but promoted muscle tissue formation. It has been concluded that Xmsx-1 si a target transcription factor of the BMP-4 signaling, but possesses a distinct activity on dorso-ventral patterning of mesodermal tissues. (author)

Traf2 interacts with Smad4 and regulates BMP signaling pathway in MC3T3-E1 osteoblasts

International Nuclear Information System (INIS)

Shimada, Koichi; Ikeda, Kyoko; Ito, Koichi

2009-01-01

Bone morphogenetic proteins (BMPs) play important roles in osteoblast differentiation and maturation. In mammals, the BMP-induced receptor-regulated Smads form complexes with Smad4. These complexes translocate and accumulate in the nucleus, where they regulate the transcription of various target genes. However, the function of Smad4 remains unclear. We performed a yeast two-hybrid screen using Smad4 as bait and a cDNA library derived from bone marrow, to indentify the proteins interacting with Smad4. cDNA clones for Tumor necrosis factor (TNF) receptor-associated factor 2 (Traf2) were identified, and the interaction between the endogenous proteins was confirmed in the mouse osteoblast cell line MC3T3-E1. To investigate the function of Traf2, we silenced it with siRNA. The level of BMP-2 protein in the medium, the expression levels of the Bmp2 gene and BMP-induced transcription factor genes, including Runx2, Dlx5, Msx2, and Sp7, and the phosphorylated-Smad1 protein level were increased in cells transfected with Traf2 siRNA. The nuclear accumulation of Smad1 increased with TNF-α stimulation for 30 min at Traf2 silencing. These results suggest that the TNF-α-stimulated nuclear accumulation of Smad1 may be dependent on Traf2. Thus, the interaction between Traf2 and Smad4 may play a role in the cross-talk between TNF-α and BMP signaling pathways.

Breast cancer cells obtain an osteomimetic feature via epithelial-mesenchymal transition that have undergone BMP2/RUNX2 signaling pathway induction.

Science.gov (United States)

Tan, Cong-Cong; Li, Gui-Xi; Tan, Li-Duan; Du, Xin; Li, Xiao-Qing; He, Rui; Wang, Qing-Shan; Feng, Yu-Mei

2016-11-29

Bone is one of the most common organs of breast cancer metastasis. Cancer cells that mimic osteoblasts by expressing bone matrix proteins and factors have a higher likelihood of metastasizing to bone. However, the molecular mechanisms of osteomimicry formation of cancer cells remain undefined. Herein, we identified a set of bone-related genes (BRGs) that are ectopically co-expressed in primary breast cancer tissues and determined that osteomimetic feature is obtained due to the osteoblast-like transformation of epithelial breast cancer cells that have undergone epithelial-mesenchymal transition (EMT) followed by bone morphogenetic protein-2 (BMP2) stimulation. Furthermore, we demonstrated that breast cancer cells that transformed into osteoblast-like cells with high expression of BRGs showed enhanced chemotaxis, adhesion, proliferation and multidrug resistance in an osteoblast-mimic bone microenvironment in vitro. During these processes, runt-related transcription factor 2 (RUNX2) functioned as a master mediator by suppressing or activating the transcription of BRGs that underlie the dynamic antagonism between the TGF-β/SMAD and BMP/SMAD signaling pathways in breast cancer cells. Our findings suggest a novel mechanism of osteomimicry formation that arises in primary breast tumors, which may explain the propensity of breast cancer to metastasize to the skeleton and contribute to potential strategies for predicting and targeting breast cancer bone metastasis and multidrug resistance.

Leiomyoma-derived transforming growth factor-β impairs bone morphogenetic protein-2-mediated endometrial receptivity.

Science.gov (United States)

Doherty, Leo F; Taylor, Hugh S

2015-03-01

To determine whether transforming growth factor (TGF)-β3 is a paracrine signal secreted by leiomyoma that inhibits bone morphogenetic protein (BMP)-mediated endometrial receptivity and decidualization. Experimental. Laboratory. Women with symptomatic leiomyomas. Endometrial stromal cells (ESCs) and leiomyoma cells were isolated from surgical specimens. Leiomyoma-conditioned media (LCM) was applied to cultured ESC. The TGF-β was blocked by two approaches: TGF-β pan-specific antibody or transfection with a mutant TGF-β receptor type II. Cells were then treated with recombinant human BMP-2 to assess BMP responsiveness. Expression of BMP receptor types 1A, 1B, 2, as well as endometrial receptivity mediators HOXA10 and leukemia inhibitory factor (LIF). Enzyme-linked immunosorbent assay showed elevated TGF-β levels in LCM. LCM treatment of ESC reduced expression of BMP receptor types 1B and 2 to approximately 60% of pretreatment levels. Preincubation of LCM with TGF-β neutralizing antibody or mutant TGF receptor, but not respective controls, prevented repression of BMP receptors. HOXA10 and LIF expression was repressed in recombinant human BMP-2 treated, LCM exposed ESC. Pretreatment of LCM with TGF-β antibody or transfection with mutant TGF receptor prevented HOXA10 and LIF repression. Leiomyoma-derived TGF-β was necessary and sufficient to alter endometrial BMP-2 responsiveness. Blockade of TGF-β prevents repression of BMP-2 receptors and restores BMP-2-stimulated expression of HOXA10 and LIF. Blockade of TGF signaling is a potential strategy to improve infertility and pregnancy loss associated with uterine leiomyoma. Copyright © 2015 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Gremlin-2 is a BMP antagonist that is regulated by the circadian clock

DEFF Research Database (Denmark)

Yeung, Ching-Yan Chloé; Gossan, Nicole; Lu, Yinhui

2014-01-01

knowledge of tendon gene regulation is essential for a complete understanding of FCT biology. Here we show autonomous circadian rhythms in mouse tendon and primary human tenocytes, controlled by an intrinsic molecular circadian clock. Time-series microarrays identified the first circadian transcriptome...... of murine tendon, revealing that 4.6% of the transcripts (745 genes) are expressed in a circadian manner. One of these genes was Grem2, which oscillated in antiphase to BMP signaling. Moreover, recombinant human Gremlin-2 blocked BMP2-induced phosphorylation of Smad1/5 and osteogenic differentiation...... of human tenocytes in vitro. We observed dampened Grem2 expression, deregulated BMP signaling, and spontaneously calcifying tendons in young CLOCKΔ19 arrhythmic mice and aged wild-type mice. Thus, disruption of circadian control, through mutations or aging, of Grem2/BMP signaling becomes a new focus...

Effects of MiR-375-BMPR2 as a Key Factor Downstream of BMP15/GDF9 on the Smad1/5/8 and Smad2/3 Signaling Pathways

Directory of Open Access Journals (Sweden)

Chang Liu

2018-03-01

Full Text Available Background/Aims: Bone morphogenetic protein 15 (BMP15 and growth differentiation factor 9 (GDF9, which are secreted by oocytes, are important regulators of follicular growth and development and ovarian function. These two factors can regulate the proliferation and apoptosis of cumulus cells via modulation of the Smad signaling pathway. Studies have shown that BMP15 and GDF9 can affect the level of miR-375, whereas the target gene of miR-375 is BMPR2, the type II receptor of BMP15 and GDF9. However, whether or how the BMP15/ GDF9-miR-375-BMPR2 pathway affects the proliferation and apoptosis of bovine cumulus cells through regulation of the Smad signaling pathway remains unclear. Methods: In this study, cumulus cells were first obtained from cumulus-oocyte complexes (COCs. Appropriate concentrations of BMP15 and GDF9 were added during the in vitro culture process. Cell Counting Kit-8 (CCK-8 analyses and flow cytometry were used to determine the effects of BMP15/GDF9 on bovine cumulus cells proliferation and apoptosis. Subsequently, miR-375 mimics, miR-375 inhibitor and BMPR2 siRNA were synthesized and used for transfection experiments. Western Blot analysis was used to detect changes before and after transfection in the expression levels of the BMP15/GDF9 type I receptors ALK4, ALK5 and ALK6; the phosphorylation levels of Smad2/3 and Smad1/5/8, which are key signaling pathway proteins downstream of BMP15/GDF9; the expression levels of PTX3, HAS2 and PTGS2, which are key genes involved in cumulus cells proliferation; and Bcl2/Bax, which are genes involved in apoptosis. Results: The addition of 100 ng/mL BMP15 or 200 ng/mL GDF9 or the combined addition of 50 ng/mL BMP15 and 100 ng/mL GDF9 effectively inhibited bovine cumulus cell apoptosis and promoted cell proliferation. BMP15/GDF9 negatively regulated miR-375 expression and positively regulated BMPR2 expression. High levels of miR-375 and inhibition of BMPR2 resulted in increased expression of ALK

Notch signaling mediates the age-associated decrease in adhesion of germline stem cells to the niche.

Directory of Open Access Journals (Sweden)

Chen-Yuan Tseng

2014-12-01

Full Text Available Stem cells have an innate ability to occupy their stem cell niche, which in turn, is optimized to house stem cells. Organ aging is associated with reduced stem cell occupancy in the niche, but the mechanisms involved are poorly understood. Here, we report that Notch signaling is increased with age in Drosophila female germline stem cells (GSCs, and this results in their removal from the niche. Clonal analysis revealed that GSCs with low levels of Notch signaling exhibit increased adhesiveness to the niche, thereby out-competing their neighbors with higher levels of Notch; adhesiveness is altered through regulation of E-cadherin expression. Experimental enhancement of Notch signaling in GSCs hastens their age-dependent loss from the niche, and such loss is at least partially mediated by Sex lethal. However, disruption of Notch signaling in GSCs does not delay GSC loss during aging, and nor does it affect BMP signaling, which promotes self-renewal of GSCs. Finally, we show that in contrast to GSCs, Notch activation in the niche (which maintains niche integrity, and thus mediates GSC retention is reduced with age, indicating that Notch signaling regulates GSC niche occupancy both intrinsically and extrinsically. Our findings expose a novel role of Notch signaling in controlling GSC-niche adhesion in response to aging, and are also of relevance to metastatic cancer cells, in which Notch signaling suppresses cell adhesion.

BMP15 suppresses progesterone production by down-regulating StAR via ALK3 in human granulosa cells.

Science.gov (United States)

Chang, Hsun-Ming; Cheng, Jung-Chien; Klausen, Christian; Leung, Peter C K

2013-12-01

In addition to somatic cell-derived growth factors, oocyte-derived growth differentiation factor (GDF)9 and bone morphogenetic protein (BMP)15 play essential roles in female fertility. However, few studies have investigated their effects on human ovarian steroidogenesis, and fewer still have examined their differential effects or underlying molecular determinants. In the present study, we used immortalized human granulosa cells (SVOG) and human granulosa cell tumor cells (KGN) to compare the effects of GDF9 and BMP15 on steroidogenic enzyme expression and investigate potential mechanisms of action. In SVOG cells, neither GDF9 nor BMP15 affects the mRNA levels of P450 side-chain cleavage enzyme or 3β-hydroxysteroid dehydrogenase. However, treatment with BMP15, but not GDF9, significantly decreases steroidogenic acute regulatory protein (StAR) mRNA and protein levels as well as progesterone production. These suppressive effects, along with the induction of Sma and Mad-related protein (SMAD)1/5/8 phosphorylation, are attenuated by cotreatment with 2 different BMP type I receptor inhibitors (dorsomorphin and DMH-1). Furthermore, depletion of activin receptor-like kinase (ALK)3 using small interfering RNA reverses the effects of BMP15 on SMAD1/5/8 phosphorylation and StAR expression. Similarly, knockdown of ALK3 abolishes BMP15-induced SMAD1/5/8 phosphorylation in KGN cells. These results provide evidence that oocyte-derived BMP15 down-regulates StAR expression and decreases progesterone production in human granulosa cells, likely via ALK3-mediated SMAD1/5/8 signaling. Our findings suggest that oocyte may play a critical role in the regulation of progesterone to prevent premature luteinization during the late stage of follicle development.

Interactions between Bmp-4 and Msx-1 act to restrict gene expression to odontogenic mesenchyme.

Science.gov (United States)

Tucker, A S; Al Khamis, A; Sharpe, P T

1998-08-01

Tooth development is regulated by a reciprocal series of epithelial-mesenchymal interactions. Bmp4 has been identified as a candidate signalling molecule in these interactions, initially as an epithelial signal and then later at the bud stage as a mesenchymal signal (Vainio et al. [1993] Cell 75:45-58). A target gene for Bmp4 signalling is the homeobox gene Msx-1, identified by the ability of recombinant Bmp4 protein to induce expression in mesenchyme. There is, however, no evidence that Bmp4 is the endogenous inducer of Msx-1 expression. Msx-1 and Bmp-4 show dynamic, interactive patterns of expression in oral epithelium and ectomesenchyme during the early stages of tooth development. In this study, we compare the temporal and spatial expression of these two genes to determine whether the changing expression patterns of these genes are consistent with interactions between the two molecules. We show that changes in Bmp-4 expression precede changes in Msx-1 expression. At embryonic day (E)10.5-E11.0, expression patterns are consistent with BMP4 from the epithelium, inducing or maintaining Msx-1 in underlying mesenchyme. At E11.5, Bmp-4 expression shifts from epithelium to mesenchyme and is rapidly followed by localised up-regulation of Msx-1 expression at the sites of Bmp-4 expression. Using cultured explants of developing mandibles, we confirm that exogenous BMP4 is capable of replacing the endogenous source in epithelium and inducing Msx-1 gene expression in mesenchyme. By using noggin, a BMP inhibitor, we show that endogenous Msx-1 expression can be inhibited at E10.5 and E11.5, providing the first evidence that endogenous Bmp-4 from the epithelium is responsible for regulating the early spatial expression of Msx-1. We also show that the mesenchymal shift in Bmp-4 is responsible for up-regulating Msx-1 specifically at the sites of future tooth formation. Thus, we establish that a reciprocal series of interactions act to restrict expression of both genes to future

The zinc transporter SLC39A13/ZIP13 is required for connective tissue development; its involvement in BMP/TGF-beta signaling pathways.

Directory of Open Access Journals (Sweden)

Toshiyuki Fukada

Full Text Available BACKGROUND: Zinc (Zn is an essential trace element and it is abundant in connective tissues, however biological roles of Zn and its transporters in those tissues and cells remain unknown. METHODOLOGY/PRINCIPAL FINDINGS: Here we report that mice deficient in Zn transporter Slc39a13/Zip13 show changes in bone, teeth and connective tissue reminiscent of the clinical spectrum of human Ehlers-Danlos syndrome (EDS. The Slc39a13 knockout (Slc39a13-KO mice show defects in the maturation of osteoblasts, chondrocytes, odontoblasts, and fibroblasts. In the corresponding tissues and cells, impairment in bone morphogenic protein (BMP and TGF-beta signaling were observed. Homozygosity for a SLC39A13 loss of function mutation was detected in sibs affected by a unique variant of EDS that recapitulates the phenotype observed in Slc39a13-KO mice. CONCLUSIONS/SIGNIFICANCE: Hence, our results reveal a crucial role of SLC39A13/ZIP13 in connective tissue development at least in part due to its involvement in the BMP/TGF-beta signaling pathways. The Slc39a13-KO mouse represents a novel animal model linking zinc metabolism, BMP/TGF-beta signaling and connective tissue dysfunction.

Overlapping and differential localization of Bmp-2, Bmp-4, Msx-2 and apoptosis in the endocardial cushion and adjacent tissues of the developing mouse heart.

Science.gov (United States)

Abdelwahid, E; Rice, D; Pelliniemi, L J; Jokinen, E

2001-07-01

The bone morphogenetic proteins BMP-2 and BMP-4 and the homeobox gene MSX-2 are required for normal development of many embryonic tissues. To elucidate their possible roles during the remodeling of the tubular heart into a fully septated four-chambered heart, we have localized the mRNA of Bmp-2, Bmp-4, Msx-2 and apoptotic cells in the developing mouse heart from embryonic day (E)11 to E17. mRNA was localized by in situ hybridization, and apoptotic cells by TUNEL (TDT-mediated dUTP-biotin nick end-labeling) as well as by transmission electron microscopy. By analyzing adjacent serial sections, we demonstrated that the expression of Msx-2 and Bmp-2 strikingly overlapped in the atrioventricular canal myocardium, in the atrioventricular junctional myocardium, and in the maturing myocardium of the atrioventricular valves. Bmp-4 was expressed in the outflow tract myocardium and in the endocardial cushion of the outflow tract ridges from E12 to E14. Msx-2 appeared in the mesenchyme of the atrioventricular endocardial cushion from E11 to E14, while Bmp-2 and Bmp-4 were detected between E11 and E14. Apoptotic cells were also detected in the mesenchyme of the endocardial cushion between E12 and E14. Our results suggest that BMP-2 and MSX-2 are tightly linked to the formation of the atrioventricular junction and valves and that BMP-4 is involved in the development of the outflow tract myocardium and of the endocardial cushion. In addition, BMP-2, BMP-4 and MSX-2 and apoptosis seem to be associated with differentiation of the endocardial cushion.

Essential role of Bmp signaling and its positive feedback loop in the early cell fate evolution of chordates

Czech Academy of Sciences Publication Activity Database

Kozmiková, Iryna; Candiani, S.; Fabian, Peter; Gurská, Daniela; Kozmik, Zbyněk

2013-01-01

Roč. 382, č. 2 (2013), s. 538-554 ISSN 0012-1606 R&D Projects: GA ČR GCP305/10/J064; GA MŠk EE2.3.30.0027 Institutional support: RVO:68378050 Keywords : Bmp signaling * axial patterning * cell fate * chordates * evolution Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.637, year: 2013

Multiple common susceptibility variants near BMP pathway loci GREM1, BMP4, and BMP2 explain part of the missing heritability of colorectal cancer.

Directory of Open Access Journals (Sweden)

Ian P M Tomlinson

2011-06-01

Full Text Available Genome-wide association studies (GWAS have identified 14 tagging single nucleotide polymorphisms (tagSNPs that are associated with the risk of colorectal cancer (CRC, and several of these tagSNPs are near bone morphogenetic protein (BMP pathway loci. The penalty of multiple testing implicit in GWAS increases the attraction of complementary approaches for disease gene discovery, including candidate gene- or pathway-based analyses. The strongest candidate loci for additional predisposition SNPs are arguably those already known both to have functional relevance and to be involved in disease risk. To investigate this proposition, we searched for novel CRC susceptibility variants close to the BMP pathway genes GREM1 (15q13.3, BMP4 (14q22.2, and BMP2 (20p12.3 using sample sets totalling 24,910 CRC cases and 26,275 controls. We identified new, independent CRC predisposition SNPs close to BMP4 (rs1957636, P = 3.93×10(-10 and BMP2 (rs4813802, P = 4.65×10(-11. Near GREM1, we found using fine-mapping that the previously-identified association between tagSNP rs4779584 and CRC actually resulted from two independent signals represented by rs16969681 (P = 5.33×10(-8 and rs11632715 (P = 2.30×10(-10. As low-penetrance predisposition variants become harder to identify-owing to small effect sizes and/or low risk allele frequencies-approaches based on informed candidate gene selection may become increasingly attractive. Our data emphasise that genetic fine-mapping studies can deconvolute associations that have arisen owing to independent correlation of a tagSNP with more than one functional SNP, thus explaining some of the apparently missing heritability of common diseases.

A BMP responsive transcriptional region in the chicken type X collagen gene.

Science.gov (United States)

Volk, S W; Luvalle, P; Leask, T; Leboy, P S

1998-10-01

Bone morphogenetic proteins (BMPs) were originally identified by their ability to induce ectopic bone formation and have been shown to promote both chondrogenesis and chondrocyte hypertrophy. BMPs have recently been found to activate a membrane serine/threonine kinase signaling mechanism in a variety of cell types, but the downstream effectors of BMP signaling in chondrocyte differentiation remain unidentified. We have previously reported that BMP-2 markedly stimulates type X collagen expression in prehypertrophic chick sternal chondrocytes, and that type X collagen mRNA levels in chondrocytes cultured under serum-free (SF) conditions are elevated 3- to 5-fold within 24 h. To better define the molecular mechanisms of induction of chondrocyte hypertrophy by BMPs, we examined the effect of BMPs on type X collagen production by 15-day chick embryo sternal chondrocytes cultured under SF conditions in the presence or absence of 30 ng/ml BMP-2, BMP-4, or BMP-7. Two populations of chondrocytes were used: one representing resting cartilage isolated from the caudal third of the sterna and the second representing prehypertrophic cartilage from the cephalic third of the sterna. BMP-2, BMP-4, and BMP-7 all effectively promoted chondrocyte maturation of cephalic sternal chondrocytes as measured by high levels of alkaline phosphatase, diminished levels of type II collagen, and induction of the hypertrophic chondrocyte-specific marker, type X collagen. To test whether BMP control of type X collagen expression occurs at the transcriptional level, we utilized plasmid constructs containing the chicken collagen X promoter and 5' flanking regions fused to a reporter gene. Constructs were transiently transfected into sternal chondrocytes cultured under SF conditions in the presence or absence of 30 ng/ml BMP-2, BMP-4, or BMP-7. A 533 bp region located 2.4-2.9 kb upstream from the type X collagen transcriptional start site was both necessary and sufficient for strong BMP responsiveness

BMP2 and BMP7 play antagonistic roles in feather induction

Science.gov (United States)

Michon, Frédéric; Forest, Loïc; Collomb, Elodie; Demongeot, Jacques; Dhouailly, Danielle

2008-01-01

Summary During embryonic development, feathers first appear as primordia consisting of an epidermal placode associated with a dermal condensation. In most previous studies, the BMPs have been proposed to function as inhibitors of the formation of cutaneous appendages. We showed that the function of BMPs is quite nuanced: BMP-2 and BMP-7, which are expressed in both skin components, act antagonistically and yet are both involved in the dermal condensations formation. BMP-7, the first to be expressed, is implicated in chemotaxis which leads to cell recruitment to the condensation, whereas BMP-2, which is expressed later, leads to an arrest of cell migration, likely via its modulation of EIIIA Fibronectin domain and α4-Integrin expression. We also propose a mathematical model, a reaction-diffusion system, based on cell proliferation, chemotaxis and the timing of BMP-2 and BMP-7 expression, which simulates the endogenous situation and reproduces the negative effects of excess BMP-2 or BMP-7 on feather patterning. PMID:18635609

DMH1, a small molecule inhibitor of BMP type i receptors, suppresses growth and invasion of lung cancer.

Directory of Open Access Journals (Sweden)

Jijun Hao

Full Text Available The bone morphogenetic protein (BMP signaling cascade is aberrantly activated in human non-small cell lung cancer (NSCLC but not in normal lung epithelial cells, suggesting that blocking BMP signaling may be an effective therapeutic approach for lung cancer. Previous studies demonstrated that some BMP antagonists, which bind to extracellular BMP ligands and prevent their association with BMP receptors, dramatically reduced lung tumor growth. However, clinical application of protein-based BMP antagonists is limited by short half-lives, poor intra-tumor delivery as well as resistance caused by potential gain-of-function mutations in the downstream of the BMP pathway. Small molecule BMP inhibitors which target the intracellular BMP cascades would be ideal for anticancer drug development. In a zebrafish embryo-based structure and activity study, we previously identified a group of highly selective small molecule inhibitors specifically antagonizing the intracellular kinase domain of BMP type I receptors. In the present study, we demonstrated that DMH1, one of such inhibitors, potently reduced lung cell proliferation, promoted cell death, and decreased cell migration and invasion in NSCLC cells by blocking BMP signaling, as indicated by suppression of Smad 1/5/8 phosphorylation and gene expression of Id1, Id2 and Id3. Additionally, DMH1 treatment significantly reduced the tumor growth in human lung cancer xenograft model. In conclusion, our study indicates that small molecule inhibitors of BMP type I receptors may offer a promising novel strategy for lung cancer treatment.

Interactions between BMP-7 and USAG-1 (uterine sensitization-associated gene-1 regulate supernumerary organ formations.

Directory of Open Access Journals (Sweden)

Honoka Kiso

Full Text Available Bone morphogenetic proteins (BMPs are highly conserved signaling molecules that are part of the transforming growth factor (TGF-beta superfamily, and function in the patterning and morphogenesis of many organs including development of the dentition. The functions of the BMPs are controlled by certain classes of molecules that are recognized as BMP antagonists that inhibit BMP binding to their cognate receptors. In this study we tested the hypothesis that USAG-1 (uterine sensitization-associated gene-1 suppresses deciduous incisors by inhibition of BMP-7 function. We learned that USAG-1 and BMP-7 were expressed within odontogenic epithelium as well as mesenchyme during the late bud and early cap stages of tooth development. USAG-1 is a BMP antagonist, and also modulates Wnt signaling. USAG-1 abrogation rescued apoptotic elimination of odontogenic mesenchymal cells. BMP signaling in the rudimentary maxillary incisor, assessed by expressions of Msx1 and Dlx2 and the phosphorylation of Smad protein, was significantly enhanced. Using explant culture and subsequent subrenal capsule transplantation of E15 USAG-1 mutant maxillary incisor tooth primordia supplemented with BMP-7 demonstrated in USAG-1+/- as well as USAG-1-/- rescue and supernumerary tooth development. Based upon these results, we conclude that USAG-1 functions as an antagonist of BMP-7 in this model system. These results further suggest that the phenotypes of USAG-1 and BMP-7 mutant mice reported provide opportunities for regenerative medicine and dentistry.

Induction of osteoblast differentiation by selective activation of kinase-mediated actions of the estrogen receptor.

Science.gov (United States)

Kousteni, Stavroula; Almeida, Maria; Han, Li; Bellido, Teresita; Jilka, Robert L; Manolagas, Stavros C

2007-02-01

Estrogens control gene transcription by cis or trans interactions of the estrogen receptor (ER) with target DNA or via the activation of cytoplasmic kinases. We report that selective activation of kinase-mediated actions of the ER with 4-estren-3alpha,17beta-diol (estren) or an estradiol-dendrimer conjugate, each a synthetic compound that stimulates kinase-mediated ER actions 1,000 to 10,000 times more potently than direct DNA interactions, induced osteoblastic differentiation in established cell lines of uncommitted osteoblast precursors and primary cultures of osteoblast progenitors by stimulating Wnt and BMP-2 signaling in a kinase-dependent manner. In sharp contrast, 17beta-estradiol (E(2)) suppressed BMP-2-induced osteoblast progenitor commitment and differentiation. Consistent with the in vitro findings, estren, but not E(2), stimulated Wnt/beta-catenin-mediated transcription in T-cell factor-lacZ transgenic mice. Moreover, E(2) stimulated BMP signaling in mice in which ERalpha lacks DNA binding activity and classical estrogen response element-mediated transcription (ERalpha(NERKI/-)) but not in wild-type controls. This evidence reveals for the first time the existence of a large signalosome in which inputs from the ER, kinases, bone morphogenetic proteins, and Wnt signaling converge to induce differentiation of osteoblast precursors. ER can either induce it or repress it, depending on whether the activating ligand (and presumably the resulting conformation of the receptor protein) precludes or accommodates ERE-mediated transcription.

Increased Retinal Expression of the Pro-Angiogenic Receptor GPR91 via BMP6 in a Mouse Model of Juvenile Hemochromatosis.

Science.gov (United States)

Arjunan, Pachiappan; Gnanaprakasam, Jaya P; Ananth, Sudha; Romej, Michelle A; Rajalakshmi, Veeranan-Karmegam; Prasad, Puttur D; Martin, Pamela M; Gurusamy, Mariappan; Thangaraju, Muthusamy; Bhutia, Yangzom D; Ganapathy, Vadivel

2016-04-01

Hemochromatosis, an iron-overload disease, occurs as adult and juvenile types. Mutations in hemojuvelin (HJV), an iron-regulatory protein and a bone morphogenetic protein (BMP) coreceptor, underlie most of the juvenile type. Hjv(-/-) mice accumulate excess iron in retina and exhibit aberrant vascularization and angiomas. A succinate receptor, GPR91, is pro-angiogenic in retina. We hypothesized that Hjv(-/-) retinas have increased BMP signaling and increased GPR91 expression as the basis of angiomas. Expression of GPR91 was examined by qPCR, immunofluorescence, and Western blot in wild-type and Hjv(-/-) mouse retinas and pRPE cells. Influence of excess iron and BMP6 on GPR91 expression was investigated in ARPE-19 cells, and wild-type and Hjv(-/-) pRPE cells. Succinate was used to activate GPR91 and determine the effects of GPR91 signaling on VEGF expression. Signaling of BMP6 was studied by the expression of Smad1/5/8 and pSmad4, and the BMP-target gene Id1. The interaction of pSmad4 with GPR91 promoter was studied by ChIP. Expression of GPR91 was higher in Hjv(-/-) retinas and RPE than in wild-type counterparts. Unexpectedly, BMP signaling was increased, not decreased, in Hjv(-/-) retinas and RPE. Bone morphogenetic protein 6 induced GPR91 in RPE, suggesting that increased BMP signaling in Hjv(-/-) retinas was likely responsible for GPR91 upregulation. Exposure of RPE to excess iron and succinate as well as BMP6 and succinate increased VEGF expression. Bone morphogenetic protein 6 promoted the interaction of pSmad4 with GPR91 promoter in RPE. G-protein-coupled receptor 91 is a BMP6 target and Hjv deletion enhances BMP signaling in retina, thus underscoring a role for excess iron and hemochromatosis in abnormal retinal vascularization.

BMP and Hedgehog Regulate Distinct AGM Hematopoietic Stem Cells Ex Vivo

Directory of Open Access Journals (Sweden)

Mihaela Crisan

2016-03-01

Full Text Available Hematopoietic stem cells (HSC, the self-renewing cells of the adult blood differentiation hierarchy, are generated during embryonic stages. The first HSCs are produced in the aorta-gonad-mesonephros (AGM region of the embryo through endothelial to a hematopoietic transition. BMP4 and Hedgehog affect their production and expansion, but it is unknown whether they act to affect the same HSCs. In this study using the BRE GFP reporter mouse strain that identifies BMP/Smad-activated cells, we find that the AGM harbors two types of adult-repopulating HSCs upon explant culture: One type is BMP-activated and the other is a non-BMP-activated HSC type that is indirectly controlled by Hedgehog signaling through the VEGF pathway. Transcriptomic analyses demonstrate that the two HSC types express distinct but overlapping genetic programs. These results revealing the bifurcation in HSC types at early embryonic stages in the AGM explant model suggest that their development is dependent upon the signaling molecules in the microenvironment.

BMP and TGFbeta pathways in human central chondrosarcoma: enhanced endoglin and Smad 1 signaling in high grade tumors

International Nuclear Information System (INIS)

Boeuf, Stephane; Bovée, Judith VMG; Lehner, Burkhard; Akker, Brendy van den; Ruler, Maayke van; Cleton-Jansen, Anne-Marie; Richter, Wiltrud

2012-01-01

As major regulators of normal chondrogenesis, the bone morphogenic protein (BMP) and transforming growth factor β (TGFB) signaling pathways may be involved in the development and progression of central chondrosarcoma. In order to uncover their possible implication, the aim of this study was to perform a systematic quantitative study of the expression of BMPs, TGFBs and their receptors and to assess activity of the corresponding pathways in central chondrosarcoma. Gene expression analysis was performed by quantitative RT-PCR in 26 central chondrosarcoma and 6 healthy articular cartilage samples. Expression of endoglin and nuclear localization of phosphorylated Smad1/5/8 and Smad2 was assessed by immunohistochemical analysis. The expression of TGFB3 and of the activin receptor-like kinase ALK2 was found to be significantly higher in grade III compared to grade I chondrosarcoma. Nuclear phosphorylated Smad1/5/8 and Smad2 were found in all tumors analyzed and the activity of both signaling pathways was confirmed by functional reporter assays in 2 chondrosarcoma cell lines. Immunohistochemical analysis furthermore revealed that phosphorylated Smad1/5/8 and endoglin expression were significantly higher in high-grade compared to low-grade chondrosarcoma and correlated to each other. The BMP and TGFβ signaling pathways were found to be active in central chondrosarcoma cells. The correlation of Smad1/5/8 activity to endoglin expression suggests that, as described in other cell types, endoglin could enhance Smad1/5/8 signaling in high-grade chondrosarcoma cells. Endoglin expression coupled to Smad1/5/8 activation could thus represent a functionally important signaling axis for the progression of chondrosarcoma and a regulator of the undifferentiated phenotype of high-grade tumor cells

Bmp2 deletion causes an amelogenesis imperfecta phenotype via regulating enamel gene expression.

Science.gov (United States)

Guo, Feng; Feng, Junsheng; Wang, Feng; Li, Wentong; Gao, Qingping; Chen, Zhuo; Shoff, Lisa; Donly, Kevin J; Gluhak-Heinrich, Jelica; Chun, Yong Hee Patricia; Harris, Stephen E; MacDougall, Mary; Chen, Shuo

2015-08-01

Although Bmp2 is essential for tooth formation, the role of Bmp2 during enamel formation remains unknown in vivo. In this study, the role of Bmp2 in regulation of enamel formation was investigated by the Bmp2 conditional knock out (Bmp2 cKO) mice. Teeth of Bmp2 cKO mice displayed severe and profound phenotypes with asymmetric and misshaped incisors as well as abrasion of incisors and molars. Scanning electron microscopy analysis showed that the enamel layer was hypoplastic and enamel lacked a typical prismatic pattern. Teeth from null mice were much more brittle as tested by shear and compressive moduli. Expression of enamel matrix protein genes, amelogenin, enamelin, and enamel-processing proteases, Mmp-20 and Klk4 was reduced in the Bmp2 cKO teeth as reflected in a reduced enamel formation. Exogenous Bmp2 up-regulated those gene expressions in mouse enamel organ epithelial cells. This result for the first time indicates Bmp2 signaling is essential for proper enamel development and mineralization in vivo. © 2015 Wiley Periodicals, Inc.

Developmental roles of the BMP1/TLD metalloproteinases.

Science.gov (United States)

Ge, Gaoxiang; Greenspan, Daniel S

2006-03-01

-beta-like morphogens BMP2 and 4 and their invertebrate ortholog decapentaplegic, from latent complexes with the vertebrate extracellular antagonist chordin and its invertebrate ortholog short gastrulation (SOG), respectively. The result is formation of the BMP signaling gradients that form the dorsal-ventral axis in embryogenesis. Thus, BMP1/TLD-like proteinases appear to be key to regulating and orchestrating formation of the ECM and signaling by various TGF-beta-like proteins in morphogenetic and homeostatic events. Copyright 2006 Wiley-Liss, Inc.

BMP and TGFbeta pathways in human central chondrosarcoma: enhanced endoglin and Smad 1 signaling in high grade tumors

Science.gov (United States)

2012-01-01

Background As major regulators of normal chondrogenesis, the bone morphogenic protein (BMP) and transforming growth factor β (TGFB) signaling pathways may be involved in the development and progression of central chondrosarcoma. In order to uncover their possible implication, the aim of this study was to perform a systematic quantitative study of the expression of BMPs, TGFBs and their receptors and to assess activity of the corresponding pathways in central chondrosarcoma. Methods Gene expression analysis was performed by quantitative RT-PCR in 26 central chondrosarcoma and 6 healthy articular cartilage samples. Expression of endoglin and nuclear localization of phosphorylated Smad1/5/8 and Smad2 was assessed by immunohistochemical analysis. Results The expression of TGFB3 and of the activin receptor-like kinase ALK2 was found to be significantly higher in grade III compared to grade I chondrosarcoma. Nuclear phosphorylated Smad1/5/8 and Smad2 were found in all tumors analyzed and the activity of both signaling pathways was confirmed by functional reporter assays in 2 chondrosarcoma cell lines. Immunohistochemical analysis furthermore revealed that phosphorylated Smad1/5/8 and endoglin expression were significantly higher in high-grade compared to low-grade chondrosarcoma and correlated to each other. Conclusions The BMP and TGFβ signaling pathways were found to be active in central chondrosarcoma cells. The correlation of Smad1/5/8 activity to endoglin expression suggests that, as described in other cell types, endoglin could enhance Smad1/5/8 signaling in high-grade chondrosarcoma cells. Endoglin expression coupled to Smad1/5/8 activation could thus represent a functionally important signaling axis for the progression of chondrosarcoma and a regulator of the undifferentiated phenotype of high-grade tumor cells. PMID:23088614

Hydroxyapatite paste Ostim, without elevation of full-thickness flaps, improves alveolar healing stimulating BMP- and VEGF-mediated signal pathways: an experimental study in humans.

Science.gov (United States)

Canuto, R A; Pol, R; Martinasso, G; Muzio, G; Gallesio, G; Mozzati, M

2013-08-01

Tooth extraction is considered as the starting point of jaw atrophy via osteoclast activity stimulation. The maintenance of dental alveolar bone depends on surgery procedure and use of materials to maintain prior space favoring bone regeneration. Among substitutes used in dentistry to fill bone defects, Ostim-Pastes (Ostim) is a nanocrystalline paste tested for treatment of severe clinical conditions. This research first investigated the effect of Ostim on alveolar healing, comparing in the same healthy subjects, an Ostim-filled socket with a not-filled one. Moreover, it also proposed a new surgical protocol for the post-extractive socket treatment using the graft materials without elevation of full-thickness flaps. Fourteen patients were enrolled to bilateral maxillary or mandibular extraction that was performed without elevation of full-thickness flaps. In each patient, one socket was filled using Ostim, and the other one was allowed to undergo natural healing. No suture was carried out. Clinical and biologic parameters were screened at 1, 7, and 14 days. Obtained results evidenced that nanocrystalline hydroxyapatite supports bone regeneration, increasing the synthesis of pro-osteogenic factors as bone morphogenetics protein (BMP)-4, BMP-7, alkaline phosphatase, and osteocalcin. Moreover, filling post-extractive socket with nanocrystalline hydroxyapatite paste leads to a complete epithelialization already at 7 days after extraction, despite the fact that the teeth were extracted without elevation of full-thickness flaps . The improved epithelialization is mediated by increased vascular endothelial growth factor (VEGF) expression. No significant change was observed in inflammatory parameters, with exception of an early and transient IL-1β induction, that could trigger and improve alveolar healing. Clinical and biomolecular observations of this explorative study evidenced that nanocrystalline hydroxyapatite improves alveolar socket healing, increasing angiogenesis

MiR-22 Suppresses BMP7 in the Development of Cirrhosis

Directory of Open Access Journals (Sweden)

Dong Ji

2015-06-01

Full Text Available Background/Aims: New strategies for the prevention and treatment of cirrhosis are urgently needed for improving therapeutic outcome. A role of microRNAs (miRNAs in the pathogenesis of cirrhosis has been recently acknowledged, whereas the exact involved miRNAs as well as the associated molecular signaling pathways have not been determined. Specifically, the studies on the relationship between miR-22 and bone morphogenic protein 7 (BMP7 in the development of cirrhosis are lacking. Methods: We examined the correlation of the levels of miR-22 and bone morphogenic protein 7 (BMP7 in the liver biopsies from patients with cirrhosis. We examined overexpression or suppression of miR-22 on BMP7 in hepatocytes. We examined the binding of miR-22 to the 3'-UTR of BMP7 mRNA. Finally, in a carbon tetrachloride (CCl4-induced cirrhosis model in mice, we gave mice adeno-associated viruses carrying antisense of miR-22, and examined its effects on BMP7 levels and the hallmarks of cirrhosis. Results: The levels of miR-22 and BMP7 in the liver biopsies from patients were strongly and inversely correlated. MiR-22 inhibited BMP7 expression in hepatocytes, through directly binding the 3'-UTR of BMP7 mRNA. Expression of antisense miR-22 significantly attenuated the levels of liver fibrosis, portal hypertension and sodium retention caused by CCl4, possibly through upregulation of BMP7. Conclusions: MiR-22 promotes the development of cirrhosis through BMP7 suppression.

Bmp 2 and bmp 7 induce odonto- and osteogenesis of human tooth germ stem cells.

Science.gov (United States)

Taşlı, P Neslihan; Aydın, Safa; Yalvaç, Mehmet Emir; Sahin, Fikrettin

2014-03-01

Bone morphogenetic proteins (BMPs) initiate, promote, and maintain odontogenesis and osteogenesis. In this study, we studied the effect of bone morphogenic protein 2 (BMP 2) and bone morphogenic protein 7 (BMP 7) as differentiation inducers in tooth and bone regeneration. We compared the effect of BMP 2 and BMP 7 on odontogenic and osteogenic differentiation of human tooth germ stem cells (hTGSCs). Third molar-derived hTGSCs were characterized with mesenchymal stem cell surface markers by flow cytometry. BMP 2 and BMP 7 were transfected into hTGSCs and the cells were seeded onto six-well plates. One day after the transfection, hTGSCs were treated with odontogenic and osteogenic mediums for 14 days. For confirmation of odontogenic and osteogenic differentiation, mRNA levels of BMP2, BMP 7, collagen type 1 (COL1A), osteocalsin (OCN), and dentin sialophosphoprotein (DSPP) genes were measured by quantitative real-time PCR. In addition to this, immunocytochemistry was performed by odontogenic and osteogenic antibodies and mineralization obtained by von Kossa staining. Our results showed that the BMP 2 and BMP 7 both promoted odontogenic and osteogenic differentiation of hTGSCs. Data indicated that BMP 2 treatment and BMP 7 treatment induce odontogenic differentiation without affecting each other, whereas they induce osteogenic differentiation by triggering expression of each other. These findings provide a feasible tool for tooth and bone tissue engineering.

BMP9 and BMP10 are necessary for proper closure of the ductus arteriosus

Science.gov (United States)

Levet, Sandrine; Ouarné, Marie; Ciais, Delphine; Coutton, Charles; Subileau, Mariela; Mallet, Christine; Ricard, Nicolas; Bidart, Marie; Debillon, Thierry; Faravelli, Francesca; Rooryck, Caroline; Feige, Jean-Jacques; Tillet, Emmanuelle; Bailly, Sabine

2015-01-01

The transition to pulmonary respiration after birth requires rapid alterations in the structure of the mammalian cardiovascular system. One dramatic change that occurs is the closure of the ductus arteriosus (DA), an arterial connection in the fetus that directs blood flow away from the pulmonary circulation. Two members of the TGFβ family, bone morphogenetic protein 9 (BMP9) and BMP10, have been recently involved in postnatal angiogenesis, both being necessary for remodeling of newly formed microvascular beds. The aim of the present work was to study whether BMP9 and BMP10 could be involved in closure of the DA. We found that Bmp9 knockout in mice led to an imperfect closure of the DA. Further, addition of a neutralizing anti-BMP10 antibody at postnatal day 1 (P1) and P3 in these pups exacerbated the remodeling defect and led to a reopening of the DA at P4. Transmission electron microscopy images and immunofluorescence stainings suggested that this effect could be due to a defect in intimal cell differentiation from endothelial to mesenchymal cells, associated with a lack of extracellular matrix deposition within the center of the DA. This result was supported by the identification of the regulation by BMP9 and BMP10 of several genes known to be involved in this process. The involvement of these BMPs was further supported by human genomic data because we could define a critical region in chromosome 2 encoding eight genes including BMP10 that correlated with the presence of a patent DA. Together, these data establish roles for BMP9 and BMP10 in DA closure. PMID:26056270

Asymmetric BMP4 signalling improves the realism of kidney organoids.

Science.gov (United States)

Mills, Christopher G; Lawrence, Melanie L; Munro, David A D; Elhendawi, Mona; Mullins, John J; Davies, Jamie A

2017-11-01

We present a strategy for increasing the anatomical realism of organoids by applying asymmetric cues to mimic spatial information that is present in natural embryonic development, and demonstrate it using mouse kidney organoids. Existing methods for making kidney organoids in mice yield developing nephrons arranged around a symmetrical collecting duct tree that has no ureter. We use transplant experiments to demonstrate plasticity in the fate choice between collecting duct and ureter, and show that an environment rich in BMP4 promotes differentiation of early collecting ducts into uroplakin-positive, unbranched, ureter-like epithelial tubules. Further, we show that application of BMP4-releasing beads in one place in an organoid can break the symmetry of the system, causing a nearby collecting duct to develop into a uroplakin-positive, broad, unbranched, ureter-like 'trunk' from one end of which true collecting duct branches radiate and induce nephron development in an arrangement similar to natural kidneys. The idea of using local symmetry-breaking cues to improve the realism of organoids may have applications to organoid systems other than the kidney.

Reconstitution of TGFBR2-Mediated Signaling Causes Upregulation of GDF-15 in HCT116 Colorectal Cancer Cells.

Directory of Open Access Journals (Sweden)

Jennifer Lee

Full Text Available Although inactivating frameshift mutations in the Transforming growth factor beta receptor type 2 (TGFBR2 gene are considered as drivers of microsatellite unstable (MSI colorectal tumorigenesis, consequential alterations of the downstream target proteome are not resolved completely. Applying a click-it chemistry protein labeling approach combined with mass spectrometry in a MSI colorectal cancer model cell line, we identified 21 de novo synthesized proteins differentially expressed upon reconstituted TGFBR2 expression. One candidate gene, the TGF-ß family member Growth differentiation factor-15 (GDF-15, exhibited TGFBR2-dependent transcriptional upregulation causing increased intracellular and extracellular protein levels. As a new TGFBR2 target gene it may provide a link between the TGF-ß branch and the BMP/GDF branch of SMAD-mediated signaling.

Electrostatics and N-glycan-mediated membrane tethering of SCUBE1 is critical for promoting bone morphogenetic protein signalling.

Science.gov (United States)

Liao, Wei-Ju; Tsao, Ku-Chi; Yang, Ruey-Bing

2016-03-01

SCUBE1 (S1), a secreted and membrane-bound glycoprotein, has a modular protein structure composed of an N-terminal signal peptide sequence followed by nine epidermal growth factor (EGF)-like repeats, a spacer region and three cysteine-rich (CR) motifs with multiple potential N-linked glycosylation sites, and one CUB domain at the C-terminus. Soluble S1 is a biomarker of platelet activation but an active participant of thrombosis via its adhesive EGF-like repeats, whereas its membrane-associated form acts as a bone morphogenetic protein (BMP) co-receptor in promoting BMP signal activity. However, the mechanism responsible for the membrane tethering and the biological importance of N-glycosylation of S1 remain largely unknown. In the present study, molecular mapping analysis identified a polycationic segment (amino acids 501-550) in the spacer region required for its membrane tethering via electrostatic interactions possibly with the anionic heparan sulfate proteoglycans. Furthermore, deglycosylation by peptide N-glycosidase F treatment revealed that N-glycans within the CR motif are essential for membrane recruitment through lectin-mediated surface retention. Injection of mRNA encoding zebrafish wild-type but not N-glycan-deficient scube1 restores the expression of haematopoietic and erythroid markers (scl and gata1) in scube1-knockdown embryos. We describe novel mechanisms in targeting S1 to the plasma membrane and demonstrate that N-glycans are required for S1 functions during primitive haematopoiesis in zebrafish. © 2016 Authors; published by Portland Press Limited.

Distinct and overlapping gene regulatory networks in BMP- and HDAC-controlled cell fate determination in the embryonic forebrain

Directory of Open Access Journals (Sweden)

Scholl Catharina

2012-07-01

Full Text Available Abstract Background Both bone morphogenetic proteins (BMPs and histone deacetylases (HDACs have previously been established to play a role in the development of the three major cell types of the central nervous system: neurons, astrocytes, and oligodendrocytes. We have previously established a connection between these two protein families, showing that HDACs suppress BMP-promoted astrogliogenesis in the embryonic striatum. Since HDACs act in the nucleus to effect changes in transcription, an unbiased analysis of their transcriptional targets could shed light on their downstream effects on BMP-signaling. Results Using neurospheres from the embryonic striatum as an in vitro system to analyze this phenomenon, we have performed microarray expression profiling on BMP2- and TSA-treated cultures, followed by validation of the findings with quantitative RT-PCR and protein analysis. In BMP-treated cultures we first observed an upregulation of genes involved in cell-cell communication and developmental processes such as members of BMP and canonical Wnt signaling pathways. In contrast, in TSA-treated cultures we first observed an upregulation of genes involved in chromatin modification and transcription. Interestingly, we could not record direct changes in the protein levels of canonical members of BMP2 signaling, but we did observe an upregulation of both the transcription factor STAT3 and its active isoform phospho-STAT3 at the protein level. Conclusions STAT3 and SMAD1/5/8 interact synergistically to promote astrogliogenesis, and thus we show for the first time that HDACs act to suppress BMP-promoted astrogliogenesis by suppression of the crucial partner STAT3.

Persistent expression of BMP-4 in embryonic chick adrenal cortical cells and its role in chromaffin cell development

Directory of Open Access Journals (Sweden)

Halbach Oliver

2008-10-01

Full Text Available Abstract Background Adrenal chromaffin cells and sympathetic neurons both originate from the neural crest, yet signals that trigger chromaffin development remain elusive. Bone morphogenetic proteins (BMPs emanating from the dorsal aorta are important signals for the induction of a sympathoadrenal catecholaminergic cell fate. Results We report here that BMP-4 is also expressed by adrenal cortical cells throughout chick embryonic development, suggesting a putative role in chromaffin cell development. Moreover, bone morphogenetic protein receptor IA is expressed by both cortical and chromaffin cells. Inhibiting BMP-4 with noggin prevents the increase in the number of tyrosine hydroxylase positive cells in adrenal explants without affecting cell proliferation. Hence, adrenal BMP-4 is likely to induce tyrosine hydroxylase in sympathoadrenal progenitors. To investigate whether persistent BMP-4 exposure is able to induce chromaffin traits in sympathetic ganglia, we locally grafted BMP-4 overexpressing cells next to sympathetic ganglia. Embryonic day 8 chick sympathetic ganglia, in addition to principal neurons, contain about 25% chromaffin-like cells. Ectopic BMP-4 did not increase this proportion, yet numbers and sizes of 'chromaffin' granules were significantly increased. Conclusion BMP-4 may serve to promote specific chromaffin traits, but is not sufficient to convert sympathetic neurons into a chromaffin phenotype.

Microparticle-Mediated Delivery of BMP4 for Generation of Meiosis-Competent Germ Cells from Embryonic Stem Cells.

Science.gov (United States)

Esfandiari, Fereshteh; Ashtiani, Mohammad Kazemi; Sharifi-Tabar, Mehdi; Saber, Maryam; Daemi, Hamed; Ghanian, Mohammad Hossein; Shahverdi, Abdolhossein; Baharvand, Hossein

2017-03-01

Producing meiosis-competent germ cells (GCs) from embryonic stem cells (ESCs) is essential for developing advanced therapies for infertility. Here, a novel approach is presented for generation of GCs from ESCs. In this regard, microparticles (MPs) have been developed from alginate sulfate loaded with bone morphogenetic protein 4 (BMP4). The results here show that BMP4 release from alginate sulfate MPs is significantly retarded by the sulfated groups compared to neat alginate. Then, BMP4-laden MPs are incorporated within the aggregates during differentiation of GCs from ESCs. It is observed that BMP4-laden MPs increase GC differentiation from ESCs at least twofold compared to the conventional soluble delivery method. Interestingly, following meiosis induction, Dazl, an intrinsic factor that enables GCs to enter meiosis, and two essential meiosis genes (Stra8 and Smc1b) are upregulated significantly in MP-induced aggregates compared to aggregates, which are formed by the conventional method. Together, these data show that controlled delivery of BMP4 during ESC differentiation into GC establish meiosis-competent GCs which can serve as an attractive GC source for reproductive medicine. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Bone morphogenetic protein 2 signaling negatively modulates lymphatic development in vertebrate embryos

DEFF Research Database (Denmark)

Dunworth, William P; Cardona-Costa, Jose; Bozkulak, Esra Cagavi

2014-01-01

: Our aim was to delineate the role of bone morphogenetic protein (BMP) 2 signaling in lymphatic development. METHODS AND RESULTS: BMP2 signaling negatively regulates the formation of LECs. Developing LECs lack any detectable BMP signaling activity in both zebrafish and mouse embryos, and excess BMP2...... signaling in zebrafish embryos and mouse embryonic stem cell-derived embryoid bodies substantially decrease the emergence of LECs. Mechanistically, BMP2 signaling induces expression of miR-31 and miR-181a in a SMAD-dependent mechanism, which in turn results in attenuated expression of prospero homeobox...

Ginger Stimulates Hematopoiesis via Bmp Pathway in Zebrafish

Science.gov (United States)

Ferri-Lagneau, Karine F.; Moshal, Karni S.; Grimes, Matthew; Zahora, Braden; Lv, Lishuang; Sang, Shengmin; Leung, TinChung

2012-01-01

Background Anemia is a hematologic disorder with decreased number of erythrocytes. Erythropoiesis, the process by which red blood cells differentiate, are conserved in humans, mice and zebrafish. The only known agents available to treat pathological anemia are erythropoietin and its biologic derivatives. However, erythropoietin therapy elicits unwanted side-effects, high cost and intravenous or subcutaneous injection, warranting the development of a more cost effective and non-peptide alternative. Ginger (Zingiber officinale) has been widely used in traditional medicine; however, to date there is no scientific research documenting the potential of ginger to stimulate hematopoiesis. Methodology/Principal Findings Here, we utilized gata1:dsRed transgenic zebrafish embryos to investigate the effect of ginger extract on hematopoiesis in vivo and we identified its bioactive component, 10-gingerol. We confirmed that ginger and 10-gingerol promote the expression of gata1 in erythroid cells and increase the expression of hematopoietic progenitor markers cmyb and scl. We also demonstrated that ginger and 10-gingerol can promote the hematopoietic recovery from acute hemolytic anemia in zebrafish, by quantifying the number of circulating erythroid cells in the dorsal aorta using video microscopy. We found that ginger and 10-gingerol treatment during gastrulation results in an increase of bmp2b and bmp7a expression, and their downstream effectors, gata2 and eve1. At later stages ginger and 10-gingerol can induce bmp2b/7a, cmyb, scl and lmo2 expression in the caudal hematopoietic tissue area. We further confirmed that Bmp/Smad pathway mediates this hematopoiesis promoting effect of ginger by using the Bmp-activated Bmp type I receptor kinase inhibitors dorsomorphin, LND193189 and DMH1. Conclusions/Significance Our study provides a strong foundation to further evaluate the molecular mechanism of ginger and its bioactive components during hematopoiesis and to investigate their

C. elegans DAF-16/FOXO interacts with TGF-ß/BMP signaling to induce germline tumor formation via mTORC1 activation.

Directory of Open Access Journals (Sweden)

Wenjing Qi

2017-05-01

Full Text Available Activation of the FOXO transcription factor DAF-16 by reduced insulin/IGF signaling (IIS is considered to be beneficial in C. elegans due to its ability to extend lifespan and to enhance stress resistance. In the germline, cell-autonomous DAF-16 activity prevents stem cell proliferation, thus acting tumor-suppressive. In contrast, hypodermal DAF-16 causes a tumorous germline phenotype characterized by hyperproliferation of the germline stem cells and rupture of the adjacent basement membrane. Here we show that cross-talk between DAF-16 and the transforming growth factor ß (TGFß/bone morphogenic protein (BMP signaling pathway causes germline hyperplasia and results in disruption of the basement membrane. In addition to activating MADM/NRBP/hpo-11 gene alone, DAF-16 also directly interacts with both R-SMAD proteins SMA-2 and SMA-3 in the nucleus to regulate the expression of mTORC1 pathway. Knocking-down of BMP genes or each of the four target genes in the hypodermis was sufficient to inhibit germline proliferation, indicating a cell-non-autonomously controlled regulation of stem cell proliferation by somatic tissues. We propose the existence of two antagonistic DAF-16/FOXO functions, a cell-proliferative somatic and an anti-proliferative germline activity. Whereas germline hyperplasia under reduced IIS is inhibited by DAF-16 cell-autonomously, activation of somatic DAF-16 in the presence of active IIS promotes germline proliferation and eventually induces tumor-like germline growth. In summary, our results suggest a novel pathway crosstalk of DAF-16 and TGF-ß/BMP that can modulate mTORC1 at the transcriptional level to cause stem-cell hyperproliferation. Such cell-type specific differences may help explaining why human FOXO activity is considered to be tumor-suppressive in most contexts, but may become oncogenic, e.g. in chronic and acute myeloid leukemia.

C. elegans DAF-16/FOXO interacts with TGF-ß/BMP signaling to induce germline tumor formation via mTORC1 activation.

Science.gov (United States)

Qi, Wenjing; Yan, Yijian; Pfeifer, Dietmar; Donner V Gromoff, Erika; Wang, Yimin; Maier, Wolfgang; Baumeister, Ralf

2017-05-01

Activation of the FOXO transcription factor DAF-16 by reduced insulin/IGF signaling (IIS) is considered to be beneficial in C. elegans due to its ability to extend lifespan and to enhance stress resistance. In the germline, cell-autonomous DAF-16 activity prevents stem cell proliferation, thus acting tumor-suppressive. In contrast, hypodermal DAF-16 causes a tumorous germline phenotype characterized by hyperproliferation of the germline stem cells and rupture of the adjacent basement membrane. Here we show that cross-talk between DAF-16 and the transforming growth factor ß (TGFß)/bone morphogenic protein (BMP) signaling pathway causes germline hyperplasia and results in disruption of the basement membrane. In addition to activating MADM/NRBP/hpo-11 gene alone, DAF-16 also directly interacts with both R-SMAD proteins SMA-2 and SMA-3 in the nucleus to regulate the expression of mTORC1 pathway. Knocking-down of BMP genes or each of the four target genes in the hypodermis was sufficient to inhibit germline proliferation, indicating a cell-non-autonomously controlled regulation of stem cell proliferation by somatic tissues. We propose the existence of two antagonistic DAF-16/FOXO functions, a cell-proliferative somatic and an anti-proliferative germline activity. Whereas germline hyperplasia under reduced IIS is inhibited by DAF-16 cell-autonomously, activation of somatic DAF-16 in the presence of active IIS promotes germline proliferation and eventually induces tumor-like germline growth. In summary, our results suggest a novel pathway crosstalk of DAF-16 and TGF-ß/BMP that can modulate mTORC1 at the transcriptional level to cause stem-cell hyperproliferation. Such cell-type specific differences may help explaining why human FOXO activity is considered to be tumor-suppressive in most contexts, but may become oncogenic, e.g. in chronic and acute myeloid leukemia.

Cross talk between insulin and bone morphogenetic protein signaling systems in brown adipogenesis

DEFF Research Database (Denmark)

Zhang, Hongbin; Schulz, Tim J; Espinoza, Daniel O

2010-01-01

Both insulin and bone morphogenetic protein (BMP) signaling systems are important for adipocyte differentiation. Analysis of gene expression in BMP7-treated fibroblasts revealed a coordinated change in insulin signaling components by BMP7. To further investigate the cross talk between insulin...... BMP7's suppressive effect on pref-1 transcription. Together, these data suggest cross talk between the insulin and BMP signaling systems by which BMP7 can rescue brown adipogenesis in cells with insulin resistance....

BMP-7 enhances cell migration and αvβ3 integrin expression via a c-Src-dependent pathway in human chondrosarcoma cells.

Directory of Open Access Journals (Sweden)

Jui-Chieh Chen

Full Text Available Bone morphogenic protein (BMP-7 is a member of the transforming growth factor (TGF-beta superfamily, which is originally identified based on its ability to induce cartilage and bone formation. In recent years, BMP-7 is also defined as a potent promoter of cell motility, invasion, and metastasis. However, there is little knowledge of the role of BMP-7 and its cellular function in chondrosarcoma cells. In the present study, we investigated the biological impact of BMP-7 on cell motility using transwell assay. In addition, the intracellular signaling pathways were also investigated by pharmacological and genetic approaches. Our results demonstrated that treatment with exogenous BMP-7 markedly increased cell migration by activating c-Src/PI3K/Akt/IKK/NF-κB signaling pathway, resulting in the transactivation of αvβ3 integrin expression. Indeed, abrogation of signaling activation, by chemical inhibition or expression of a kinase dead form of the protein attenuated BMP-7-induced expression of integrin αvβ3 and cell migration. These findings may provide a useful tool for diagnostic/prognostic purposes and even therapeutically in late-stage chondrosarcoma as an anti-metastatic agent.

MRI of transforaminal lumbar interbody fusion: imaging appearance with and without the use of human recombinant bone morphogenetic protein-2 (rhBMP-2)

Energy Technology Data Exchange (ETDEWEB)

Fox, Michael G.; Goldberg, Judd M.; Gaskin, Cree M.; Barr, Michelle S.; Alford, Bennett [University of Virginia, Department of Radiology and Medical Imaging, Charlottesville, VA (United States); Patrie, James T. [University of Virginia, Department of Public Health Sciences, Charlottesville, VA (United States); Shen, Francis H. [University of Virginia, Department of Orthopedic Surgery, Charlottesville, VA (United States)

2014-09-15

To describe the vertebral endplate and intervertebral disc space MRI appearance following TLIF, with and without the use of rhBMP-2, and to determine if the appearance is concerning for discitis/osteomyelitis. After institutional review board approval, 116 TLIF assessments performed on 75 patients with rhBMP-2 were retrospectively and independently reviewed by five radiologists and compared to 73 TLIF assessments performed on 45 patients without rhBMP-2. MRIs were evaluated for endplate signal, disc space enhancement, disc space fluid, and abnormal paraspinal soft tissue. Endplate edema-like signal was reported when T1-weighted hypointensity, T2-weighted hyperintensity, and endplate enhancement were present. Subjective concern for discitis/osteomyelitis on MRI was graded on a five-point scale. Generalized estimating equation binomial regression model analysis was performed with findings correlated with rhBMP-2 use, TLIF level, graft type, and days between TLIF and MRI. The rhBMP-2 group demonstrated endplate edema-like signal (OR 5.66; 95 % CI [1.58, 20.24], p = 0.008) and disc space enhancement (OR 2.40; 95 % CI [1.20, 4.80], p = 0.013) more often after adjusting for the TLIF level, graft type, and the number of days following TLIF. Both groups had a similar temporal distribution for endplate edema-like signal but disc space enhancement peaked earlier in the rhBMP-2 group. Disc space fluid was only present in the rhBMP-2 group. Neither group demonstrated abnormal paraspinal soft tissue and discitis/osteomyelitis was not considered likely in any patient. Endplate edema-like signal and disc space enhancement were significantly more frequent and disc space enhancement developed more rapidly following TLIF when rhBMP-2 was utilized. The concern for discitis/osteomyelitis was similar and minimal in both groups. (orig.)

Activation of the PI3K/Akt pathway mediates bone morphogenetic protein 2-induced invasion of pancreatic cancer cells Panc-1.

Science.gov (United States)

Chen, Xiong; Liao, Jie; Lu, YeBin; Duan, XiaoHui; Sun, WeiJia

2011-06-01

Bone morphogenetic proteins (BMPs) signaling has an emerging role in pancreatic cancer. However, because of the multiple effects of different BMPs, no final conclusions have been made as to the role of BMPs in pancreatic cancer. In our studies, we have focused on bone morphogenetic protein 2(BMP-2) because it induces an epithelial to mesenchymal transition (EMT) and accelerates invasion in the human pancreatic cancer cell line Panc-1. It has been reported that the phosphatidylinositol 3-kinase (PI3K)/Akt pathway mediates invasion of gastric and colon cancer cells, which is unrevealed in pancreatic cancer cells. The objective of our study was to investigate whether BMP-2 mediated invasion might pass through the PI3K/Akt pathway. Our results show that expression of phosphorylation of Akt was increased by treatment with BMP-2, but not Noggin, a BMP-2 antagonist. Then pretreatment of Panc-1 cells with LY294002, an inhibitor of the PI3K/AKT pathway, significantly inhibited BMP-2-induced EMT and invasiveness. The data suggest that BMP-2 accelerates invasion of panc-1 cells via the PI3K/AKT pathway in panc-1 cells, which gives clues to searching new therapy targets in advanced pancreatic cancer.

Bmp suppression in mangrove killifish embryos causes a split in the body axis.

Directory of Open Access Journals (Sweden)

Sulayman Mourabit

Full Text Available Bone morphogenetic proteins (Bmp are major players in the formation of the vertebrate body plan due to their crucial role in patterning of the dorsal-ventral (DV axis. Despite the highly conserved nature of Bmp signalling in vertebrates, the consequences of changing this pathway can be species-specific. Here, we report that Bmp plays an important role in epiboly, yolk syncytial layer (YSL movements, and anterior-posterior (AP axis formation in embryos of the self-fertilizing mangrove killifish, Kryptolebias marmoratus. Stage and dose specific exposures of embryos to the Bmp inhibitor dorsomorphin (DM produced three distinctive morphologies, with the most extreme condition creating the splitbody phenotype, characterised by an extremely short AP axis where the neural tube, somites, and notochord were bilaterally split. In addition, parts of caudal neural tissues were separated from the main body and formed cell islands in the posterior region of the embryo. This splitbody phenotype, which has not been reported in other animals, shows that modification of Bmp may lead to significantly different consequences during development in other vertebrate species.

Enhanced osteogenesis of adipose derived stem cells with Noggin suppression and delivery of BMP-2.

Directory of Open Access Journals (Sweden)

Jiabing Fan

Full Text Available Bone morphogenetic proteins (BMPs are believed to be the most potent osteoinductive factors. However, BMPs are highly pleiotropic molecules and their supra-physiological high dose requirement leads to adverse side effects and inefficient bone formation. Thus, there is a need to develop alternative osteoinductive growth factor strategies that can effectively complement BMP activity. In this study, we intrinsically stimulated BMP signaling in adipose derived stem cells (ASCs by downregulating noggin, a potent BMP antagonist, using an RNAi strategy. ASCs transduced with noggin shRNA significantly enhanced osteogenic differentiation of cells. The potency of endogenous BMPs was subsequently enhanced by stimulating ASCs with exogenous BMPs at a significantly reduced dose. The level of mineralization in noggin shRNA treated ASCs when treated with BMP-2 was comparable to that of control shRNA treated cell treated with 10-fold more BMP-2. The complementary strategy of noggin suppression + BMP-2 to enhance osteogenesis was further confirmed in 3D in vitro environments using scaffolds consisting of chitosan (CH, chondroitin sulfate (CS, and apatite layer on their surfaces designed to slowly release BMP-2. This finding supports the novel therapeutic potential of this complementary strategy in bone regeneration.

Conserved cis-regulatory regions in a large genomic landscape control SHH and BMP-regulated Gremlin1 expression in mouse limb buds

Directory of Open Access Journals (Sweden)

Zuniga Aimée

2012-08-01

Full Text Available Abstract Background Mouse limb bud is a prime model to study the regulatory interactions that control vertebrate organogenesis. Major aspects of limb bud development are controlled by feedback loops that define a self-regulatory signalling system. The SHH/GREM1/AER-FGF feedback loop forms the core of this signalling system that operates between the posterior mesenchymal organiser and the ectodermal signalling centre. The BMP antagonist Gremlin1 (GREM1 is a critical node in this system, whose dynamic expression is controlled by BMP, SHH, and FGF signalling and key to normal progression of limb bud development. Previous analysis identified a distant cis-regulatory landscape within the neighbouring Formin1 (Fmn1 locus that is required for Grem1 expression, reminiscent of the genomic landscapes controlling HoxD and Shh expression in limb buds. Results Three highly conserved regions (HMCO1-3 were identified within the previously defined critical genomic region and tested for their ability to regulate Grem1 expression in mouse limb buds. Using a combination of BAC and conventional transgenic approaches, a 9 kb region located ~70 kb downstream of the Grem1 transcription unit was identified. This region, termed Grem1 Regulatory Sequence 1 (GRS1, is able to recapitulate major aspects of Grem1 expression, as it drives expression of a LacZ reporter into the posterior and, to a lesser extent, in the distal-anterior mesenchyme. Crossing the GRS1 transgene into embryos with alterations in the SHH and BMP pathways established that GRS1 depends on SHH and is modulated by BMP signalling, i.e. integrates inputs from these pathways. Chromatin immunoprecipitation revealed interaction of endogenous GLI3 proteins with the core cis-regulatory elements in the GRS1 region. As GLI3 is a mediator of SHH signal transduction, these results indicated that SHH directly controls Grem1 expression through the GRS1 region. Finally, all cis-regulatory regions within the Grem1

Release and Cytokine Production of BmpB from BmpB-Loaded pH-Sensitive and Mucoadhesive Thiolated Eudragit Microspheres.

Science.gov (United States)

Singh, Bijay; Jiang, Tao; Kim, You-Kyoung; Kang, Sang-Kee; Choi, Yun-Jaie; Cho, Chong-Su

2015-01-01

Swine dysentery is a contagious mucohaemorrhagic colitis of pigs that is caused by anaerobic intestinal spirochaete Brachyspira hyodysenteriae. Recently, an outer membrane lipoprotein of B. hyodysenteriae (BmpB) has been identified, and the mice or pigs immunized with a recombinant BmpB generated antibodies recognizing the native BmpB of B. hyodysenteriae. In this study, we cloned, expressed and purified BmpB protein from E. coli and used it as a vaccine candidate for oral delivery. The BmpB was encapsulated into the pH-sensitive and thiolated Eudragit microspheres (TEMS). The sizes of the microspheres ranged from 5-20 μ. About 22-34% of BmpB were released from the BmpB-loaded TEMS within 24 h at stomach pH 2.0 whereas the release of BmpB from the BmpB-loaded TEMS was 35% in the first one hour and reached 81% within 24 h at intestinal pH 7.2. These data revealed that the BmpB could be protected in the harsh gastric condition. Mucoadhesive experiment in vitro showed that TEMS have high binding affinity with the mucin glycoproteins of porcine intestine. Finally, in vitro production of cytokines from immune cells treated with the BmpB-loaded TEMS suggested that the TEMS would be a promising approach for oral delivery of BmpB as vaccine candidate.

Zygotic LvBMP5-8 is required for skeletal patterning and for left-right but not dorsal-ventral specification in the sea urchin embryo.

Science.gov (United States)

Piacentino, Michael L; Chung, Oliver; Ramachandran, Janani; Zuch, Daniel T; Yu, Jia; Conaway, Evan A; Reyna, Arlene E; Bradham, Cynthia A

2016-04-01

Skeletal patterning in the sea urchin embryo requires coordinated signaling between the pattern-dictating ectoderm and the skeletogenic primary mesenchyme cells (PMCs); recent studies have begun to uncover the molecular basis for this process. Using an unbiased RNA-Seq-based screen, we have previously identified the TGF-ß superfamily ligand, LvBMP5-8, as a skeletal patterning gene in Lytechinus variegatus embryos. This result is surprising, since both BMP5-8 and BMP2/4 ligands have been implicated in sea urchin dorsal-ventral (DV) and left-right (LR) axis specification. Here, we demonstrate that zygotic LvBMP5-8 is required for normal skeletal patterning on the left side, as well as for normal PMC positioning during gastrulation. Zygotic LvBMP5-8 is required for expression of the left-side marker soxE, suggesting that LvBMP5-8 is required for left-side specification. Interestingly, we also find that LvBMP5-8 knockdown suppresses serotonergic neurogenesis on the left side. While LvBMP5-8 overexpression is sufficient to dorsalize embryos, we find that zygotic LvBMP5-8 is not required for normal DV specification or development. In addition, ectopic LvBMP5-8 does not dorsalize LvBMP2/4 morphant embryos, indicating that, in the absence of BMP2/4, BMP5-8 is insufficient to specify dorsal. Taken together, our data demonstrate that zygotic LvBMP5-8 signaling is essential for left-side specification, and for normal left-side skeletal and neural patterning, but not for DV specification. Thus, while both BMP2/4 and BMP5-8 regulate LR axis specification, BMP2/4 but not zygotic BMP5-8 regulates DV axis specification in sea urchin embryos. Copyright © 2016 Elsevier Inc. All rights reserved.

Immobilization of Murine Anti-BMP-2 Monoclonal Antibody on Various Biomaterials for Bone Tissue Engineering

Directory of Open Access Journals (Sweden)

Sahar Ansari

2014-01-01

Full Text Available Biomaterials are widely used as scaffolds for tissue engineering. We have developed a strategy for bone tissue engineering that entails application of immobilized anti-BMP-2 monoclonal antibodies (mAbs to capture endogenous BMPs in vivo and promote antibody-mediated osseous regeneration (AMOR. The purpose of the current study was to compare the efficacy of immobilization of a specific murine anti-BMP-2 mAb on three different types of biomaterials and to evaluate their suitability as scaffolds for AMOR. Anti-BMP-2 mAb or isotype control mAb was immobilized on titanium (Ti microbeads, alginate hydrogel, and ACS. The treated biomaterials were surgically implanted in rat critical-sized calvarial defects. After 8 weeks, de novo bone formation was assessed using micro-CT and histomorphometric analyses. Results showed de novo bone regeneration with all three scaffolds with immobilized anti-BMP-2 mAb, but not isotype control mAb. Ti microbeads showed the highest volume of bone regeneration, followed by ACS. Alginate showed the lowest volume of bone. Localization of BMP-2, -4, and -7 antigens was detected on all 3 scaffolds with immobilized anti-BMP-2 mAb implanted in calvarial defects. Altogether, these data suggested a potential mechanism for bone regeneration through entrapment of endogenous BMP-2, -4, and -7 proteins leading to bone formation using different types of scaffolds via AMOR.

Expression of human bone morphogenetic protein (BMP-2 and BMP-4 genes in transgenic bovine fibroblasts Expressão dos genes bone morphogenetic protein (BMP-2 e BMP-4 em fibroblastos bovinos transgênicos

Directory of Open Access Journals (Sweden)

C. Oleskovicz

2004-08-01

Full Text Available cDNAs dos genes bone morphogenetic protein-2 (BMP-2 e bone morphogenetic protein-4 (BMP-4 foram sintetizados a partir de RNA total extraído de tecidos ósseos de pacientes que apresentavam trauma facial (fraturas do maxilar entre o 7º e o 10º dia pós-trauma e clonados num vetor para expressão em células mamíferas, sob controle do promotor de citomegalovírus (CMV. Os vetores contendo os genes BMP-2 e o BMP-4 foram utilizados para a transfecção de fibroblastos bovinos. mRNAs foram indiretamente detectados por RT-PCR nas células transfectadas. As proteínas BMP-2 e BMP-4 foram detectadas mediante análises de Western blot. Os resultados demonstram a possibilidade de produção desses fatores de crescimento celular em fibroblastos bovinos. Essas células poderão ser utilizadas como fontes doadoras de material genético para a técnica de transferência nuclear na geração de animais transgênicos.

Dorsoventral patterning by the Chordin-BMP pathway: a unified model from a pattern-formation perspective for Drosophila, vertebrates, sea urchins and Nematostella.

Science.gov (United States)

Meinhardt, Hans

2015-09-01

Conserved from Cnidarians to vertebrates, the dorsoventral (DV) axis is patterned by the Chordin-BMP pathway. However, the functions of the pathway's components are very different in different phyla. By modeling it is shown that many observations can be integrated by the assumption that BMP, acting as an inhibitory component in more ancestral systems, became a necessary and activating component for the generation of a secondary and antipodal-located signaling center. The different realizations seen in vertebrates, Drosophila, sea urchins and Nematostella allow reconstruction of a chain of modifications during evolution. BMP-signaling is proposed to be based on a pattern-forming reaction of the activator-depleted substrate type in which BMP-signaling acts via pSmad as the local self-enhancing component and the depletion of the highly mobile BMP-Chordin complex as the long-ranging antagonistic component. Due to the rapid removal of the BMP/Chordin complex during BMP-signaling, an oriented transport and "shuttling" results, although only ordinary diffusion is involved. The system can be self-organizing, allowing organizer formation even from near homogeneous initial situations. Organizers may regenerate after removal. Although connected with some losses of self-regulation, for large embryos as in amphibians, the employment of maternal determinants is an efficient strategy to make sure that only a single organizer of each type is generated. The generation of dorsoventral positional information along a long-extended anteroposterior (AP) axis cannot be achieved directly by a single patch-like organizer. Nature found different solutions for this task. Corresponding models provide a rationale for the well-known reversal in the dorsoventral patterning between vertebrates and insects. Copyright © 2015 Elsevier Inc. All rights reserved.

Serum Proteases Potentiate BMP-Induced Cell Cycle Re-entry of Dedifferentiating Muscle Cells during Newt Limb Regeneration.

Science.gov (United States)

Wagner, Ines; Wang, Heng; Weissert, Philipp M; Straube, Werner L; Shevchenko, Anna; Gentzel, Marc; Brito, Goncalo; Tazaki, Akira; Oliveira, Catarina; Sugiura, Takuji; Shevchenko, Andrej; Simon, András; Drechsel, David N; Tanaka, Elly M

2017-03-27

Limb amputation in the newt induces myofibers to dedifferentiate and re-enter the cell cycle to generate proliferative myogenic precursors in the regeneration blastema. Here we show that bone morphogenetic proteins (BMPs) and mature BMPs that have been further cleaved by serum proteases induce cell cycle entry by dedifferentiating newt muscle cells. Protease-activated BMP4/7 heterodimers that are present in serum strongly induced myotube cell cycle re-entry with protease cleavage yielding a 30-fold potency increase of BMP4/7 compared with canonical BMP4/7. Inhibition of BMP signaling via muscle-specific dominant-negative receptor expression reduced cell cycle entry in vitro and in vivo. In vivo inhibition of serine protease activity depressed cell cycle re-entry, which in turn was rescued by cleaved-mimic BMP. This work identifies a mechanism of BMP activation that generates blastema cells from differentiated muscle. Copyright © 2017 Elsevier Inc. All rights reserved.

Human trabecular meshwork cells express BMP antagonist mRNAs and proteins.

Science.gov (United States)

Tovar-Vidales, Tara; Fitzgerald, Ashley M; Clark, Abbot F

2016-06-01

Glaucoma patients have elevated aqueous humor and trabecular meshwork (TM) levels of transforming growth factor-beta2 (TGF-β2). TGF-β2 has been associated with increased extracellular matrix (ECM) deposition (i.e. fibronectin), which is attributed to the increased resistance of aqueous humor outflow through the TM. We have previously demonstrated that bone morphogenetic protein (BMP) 4 selectively counteracts the profibrotic effect of TGF-β2 with respect to ECM synthesis in the TM, and this action is reversed by the BMP antagonist gremlin. Thus, the BMP and TGF-β signaling pathways antagonize each other's antifibrotic and profibrotic roles. The purpose of this study was to determine whether cultured human TM cells: (a) express other BMP antagonists including noggin, chordin, BMPER, BAMBI, Smurf1 and 2, and (b) whether expression of these proteins is regulated by exogenous TGF-β2 treatment. Primary human trabecular meshwork (TM) cells were grown to confluency and treated with TGF-β2 (5 ng/ml) for 24 or 48 h in serum-free medium. Untreated cell served as controls. qPCR and Western immunoblots (WB) determined that human TM cells expressed mRNAs and proteins for the BMP antagonist proteins: noggin, chordin, BMPER, BAMBI, and Smurf1/2. Exogenous TGF-β2 decreased chordin, BMPER, BAMBI, and Smurf1 mRNA and protein expression. In contrast, TGF-β2 increased secreted noggin and Smurf2 mRNA and protein levels. BMP antagonist members are expressed in the human TM. These molecules may be involved in the normal function of the TM as well as TM pathogenesis. Altered expression of BMP antagonist members may lead to functional changes in the human TM. Copyright © 2016 Elsevier Ltd. All rights reserved.

Regulatory role of melatonin and BMP-4 in prolactin production by rat pituitary lactotrope GH3 cells.

Science.gov (United States)

Ogura-Ochi, Kanako; Fujisawa, Satoshi; Iwata, Nahoko; Komatsubara, Motoshi; Nishiyama, Yuki; Tsukamoto-Yamauchi, Naoko; Inagaki, Kenichi; Wada, Jun; Otsuka, Fumio

2017-08-01

The effects of melatonin on prolactin production and its regulatory mechanism remain uncertain. We investigated the regulatory role of melatonin in prolactin production using rat pituitary lactotrope GH3 cells by focusing on the bone morphogenetic protein (BMP) system. Melatonin receptor activation, induced by melatonin and its receptor agonist ramelteon, significantly suppressed basal and forskolin-induced prolactin secretion and prolactin mRNA expression in GH3 cells. The melatonin MT2 receptor was predominantly expressed in GH3 cells, and the inhibitory effects of melatonin on prolactin production were reversed by treatment with the receptor antagonist luzindole, suggesting functional involvement of MT2 action in the suppression of prolactin release. Melatonin receptor activation also suppressed BMP-4-induced prolactin expression by inhibiting phosphorylation of Smad and transcription of the BMP-target gene Id-1, while BMP-4 treatment upregulated MT2 expression. Melatonin receptor activation suppressed basal, BMP-4-induced and forskolin-induced cAMP synthesis; however, BtcAMP-induced prolactin mRNA expression was not affected by melatonin or ramelteon, suggesting that MT2 activation leads to inhibition of prolactin production through the suppression of Smad signaling and cAMP synthesis. Experiments using intracellular signal inhibitors revealed that the ERK pathway is, at least in part, involved in prolactin induction by GH3 cells. Thus, a new regulatory role of melatonin involving BMP-4 in prolactin secretion was uncovered in lactotrope GH3 cells. Copyright © 2017 Elsevier Inc. All rights reserved.

Progesterone receptor activates Msx2 expression by downregulating TNAP/Akp2 and activating the Bmp pathway in EpH4 mouse mammary epithelial cells.

Directory of Open Access Journals (Sweden)

Jodie M Fleming

Full Text Available Previously we demonstrated that EpH4 mouse mammary epithelial cells induced the homeobox transcription factor Msx2 either when transfected with the progesterone receptor (PR or when treated with Bmp2/4. Msx2 upregulation was unaffected by Wnt inhibitors s-FRP or Dkk1, but was inhibited by the Bmp antagonist Noggin. We therefore hypothesized that PR signaling to Msx2 acts through the Bmp receptor pathway. Herein, we confirm that transcripts for Alk2/ActR1A, a non-canonical BmpR Type I, are upregulated in mammary epithelial cells overexpressing PR (EpH4-PR. Increased phosphorylation of Smads 1,5, 8, known substrates for Alk2 and other BmpR Type I proteins, was observed as was their translocation to the nucleus in EpH4-PR cells. Analysis also showed that Tissue Non-Specific Alkaline Phosphatase (TNAP/Akp2 was also found to be downregulated in EpH4-PR cells. When an Akp2 promoter-reporter construct containing a ½PRE site was transfected into EpH4-PR cells, its expression was downregulated. Moreover, siRNA mediated knockdown of Akp2 increased both Alk2 and Msx2 expression. Collectively these data suggest that PR inhibition of Akp2 results in increased Alk2 activity, increased phosphorylation of Smads 1,5,8, and ultimately upregulation of Msx2. These studies imply that re-activation of the Akp2 gene could be helpful in downregulating aberrant Msx2 expression in PR+ breast cancers.

Strontium doping promotes bioactivity of rhBMP-2 upon calcium phosphate cement via elevated recognition and expression of BMPR-IA.

Science.gov (United States)

Huang, Baolin; Tian, Yu; Zhang, Wenjing; Ma, Yifan; Yuan, Yuan; Liu, Changsheng

2017-11-01

Preserving and improving osteogenic activity of bone morphogenetic protein-2 (BMP-2) upon implants remains one of the key limitations in bone regeneration. With calcium phosphate cement (CPC) as model, we have developed a series of strontium (Sr)-doped CPC (SCPC) to address this issue. The effects of fixed Sr on the bioactivity of recombinant human BMP-2 (rhBMP-2) as well as the underlying mechanism were investigated. The results suggested that the rhBMP-2-induced osteogenic activity was significantly promoted upon SCPCs, especially with a low amount of fixed Sr (SrCO 3 content IA (BMPR-IA) to rhBMP-2 and an increased expression of BMPR-IA in C2C12 model cells. As a result, the activations of BMP-induced signaling pathways were different in C2C12 cells incubated upon CPC/rhBMP-2 and SCPCs/rhBMP-2. These findings explicitly decipher the mechanism of SCPCs promoting osteogenic bioactivity of rhBMP-2 and signify the promising application of the SCPCs/rhBMP-2 matrix in bone regeneration implants. Copyright © 2017 Elsevier B.V. All rights reserved.

Abnormal Uterine Bleeding Is Associated With Increased BMP7 Expression in Human Endometrium.

Science.gov (United States)

Richards, Elliott G; El-Nashar, Sherif A; Schoolmeester, John K; Keeney, Gary L; Mariani, Andrea; Hopkins, Matthew R; Dowdy, Sean C; Daftary, Gaurang S; Famuyide, Abimbola O

2017-05-01

Abnormal uterine bleeding (AUB), a common health concern of women, is a heterogeneous clinical entity that is traditionally categorized into organic and nonorganic causes. Despite varied pharmacologic treatments, few offer sustained efficacy, as most are empiric, unfocused, and do not directly address underlying dysregulated molecular mechanisms. Characterization of such molecular derangements affords the opportunity to develop and use novel, more successful treatments for AUB. Given its implication in other organ systems, we hypothesized that bone morphogenetic protein (BMP) expression is altered in patients with AUB and hence comprehensively investigated dysregulation of BMP signaling pathways by systematically screening 489 samples from 365 patients for differences in the expression of BMP2, 4, 6, and 7 ligands, BMPR1A and B receptors, and downstream SMAD4, 6, and 7 proteins. Expression analysis was correlated clinically with data abstracted from medical records, including bleeding history, age at procedure, ethnicity, body mass index, hormone treatment, and histological diagnosis of fibroids, polyps, adenomyosis, hyperplasia, and cancer. Expression of BMP7 ligand was significantly increased in patients with AUB (H-score: 18.0 vs 26.7; P bleeding (menorrhagia) as their specific AUB pattern demonstrated significantly higher BMP7 expression. Significantly, no differences in the expression of any other BMP ligands, receptors, or SMAD proteins were observed in this large patient cohort. However, expression of BMPR1A, BMPR1B, and SMAD4 was significantly decreased in cancer compared to benign samples. Our study demonstrates that BMP7 is a promising target for future investigation and pharmacologic treatment of AUB.

Antisense targeting of TGF-β1 augments BMP-induced upregulation of osteopontin, type I collagen and Cbfa1 in human Saos-2 cells

International Nuclear Information System (INIS)

Shen, Zhong-Jian; Kook Kim, Sang; Youn Jun, Do; Park, Wan; Ho Kim, Young; Malter, James S.; Jo Moon, Byung

2007-01-01

Despite commonalities in signal transduction in osteoblasts from different species, the role of TGF-β1 on bone formation remains elusive. In particular, the role of autocrine TGF-β1 on human osteoblasts is largely unknown. Here we show the effect of TGF-β1 knock-down on the proliferation and differentiation of osteoblasts induced by BMP2. Treatment with antisense TGF-β1 moderately increased the rate of cell proliferation, which was completely reversed by the exogenous addition of TGF-β1. Notably, TGF-β1 blockade significantly enhanced BMP2-induced upregulation of mRNAs encoding osteopontin, type I collagen and Cbfa1, which was suppressed by exogenous TGF-β1. Moreover, TGF-β1 knock-down increased BMP2-induced phosphorylation of Smad1/5 as well as their nuclear import, which paralleled a reduction of inhibitory Smad6. These data suggest autocrine TGF-β1 antagonizes BMP signaling through modulation of inducible Smad6 and the activity of BMP specific Smad1/5

Functional cardiomyocytes derived from Isl1 cardiac progenitors via Bmp4 stimulation.

Directory of Open Access Journals (Sweden)

Esra Cagavi

Full Text Available As heart failure due to myocardial infarction remains a leading cause of morbidity worldwide, cell-based cardiac regenerative therapy using cardiac progenitor cells (CPCs could provide a potential treatment for the repair of injured myocardium. As adult CPCs may have limitations regarding tissue accessibility and proliferative ability, CPCs derived from embryonic stem cells (ESCs could serve as an unlimited source of cells with high proliferative ability. As one of the CPCs that can be derived from embryonic stem cells, Isl1 expressing cardiac progenitor cells (Isl1-CPCs may serve as a valuable source of cells for cardiac repair due to their high cardiac differentiation potential and authentic cardiac origin. In order to generate an unlimited number of Isl1-CPCs, we used a previously established an ESC line that allows for isolation of Isl1-CPCs by green fluorescent protein (GFP expression that is directed by the mef2c gene, specifically expressed in the Isl1 domain of the anterior heart field. To improve the efficiency of cardiac differentiation of Isl1-CPCs, we studied the role of Bmp4 in cardiogenesis of Isl1-CPCs. We show an inductive role of Bmp directly on cardiac progenitors and its enhancement on early cardiac differentiation of CPCs. Upon induction of Bmp4 to Isl1-CPCs during differentiation, the cTnT+ cardiomyocyte population was enhanced 2.8±0.4 fold for Bmp4 treated CPC cultures compared to that detected for vehicle treated cultures. Both Bmp4 treated and untreated cardiomyocytes exhibit proper electrophysiological and calcium signaling properties. In addition, we observed a significant increase in Tbx5 and Tbx20 expression in differentiation cultures treated with Bmp4 compared to the untreated control, suggesting a link between Bmp4 and Tbx genes which may contribute to the enhanced cardiac differentiation in Bmp4 treated cultures. Collectively these findings suggest a cardiomyogenic role for Bmp4 directly on a pure population of

S100A4 and BMP-2 Co-Dependently Induce Vascular Smooth Muscle Cell Migration via pERK and Chloride Intracellular Channel 4 (CLIC4)

Science.gov (United States)

Spiekerkoetter, Edda; Guignabert, Christophe; de Jesus Perez, Vinicio; Alastalo, Tero-Pekka; Powers, Janine M; Wang, Lingli; Lawrie, Allan; Ambartsumian, Noona; Schmidt, Ann-Marie; Berryman, Mark; Ashley, Richard H; Rabinovitch, Marlene

2009-01-01

Rationale S100A4/Mts1 is implicated in motility of human pulmonary artery smooth muscle cells (hPASMC), through an interaction with the receptor for advanced glycation end products (RAGE). Objective We hypothesized that S100A4/Mts1-mediated hPASMC motility might be enhanced by loss of function of bone morphogenetic protein (BMP) receptor (R) II, observed in pulmonary arterial hypertension (PAH). Methods and Results Both S100A4/Mts1 (500ng/ml) and BMP-2 (10ng/ml) induce migration of hPASMCS in a novel co-dependent manner, in that the response to either ligand is lost with anti-RAGE or BMPRII siRNA. Phosphorylation of ERK is induced by both ligands and is required for motility by inducing MMP2 activity, but phosphoERK1/2 is blocked by anti-RAGE and not by BMPRII siRNA. In contrast, BMPRII siRNA, but not anti-RAGE, reduces expression of intracellular chloride channel 4 (CLIC4), a scaffolding molecule necessary for motility in response to S100A4/Mts1 or BMP-2. Reduced CLIC4 expression does not interfere with S100A4/Mts1 internalization or its interaction with myosin heavy chain IIA (MHCIIA), but does alter alignment of MHCIIA and actin filaments creating the appearance of vacuoles. This abnormality is associated with reduced peripheral distribution and/or delayed activation of RhoA and Rac1, small GTPases required for retraction and extension of lamellipodiae in motile cells. Conclusions Our studies demonstrate how a single ligand (BMP-2 or S100A4/Mts1) can recruit multiple cell surface receptors to relay signals that coordinate events culminating in a functional response, i.e., cell motility. We speculate that this carefully controlled process limits signals from multiple ligands, but could be subverted in disease. PMID:19713532

CEP128 Localizes to the Subdistal Appendages of the Mother Centriole and Regulates TGF-β/BMP Signaling at the Primary Cilium

DEFF Research Database (Denmark)

Mönnich, Maren; Borgeskov, Louise; Breslin, Loretta

2018-01-01

The centrosome is the main microtubule-organizing center in animal cells and comprises a mother and daughter centriole surrounded by pericentriolar material. During formation of primary cilia, the mother centriole transforms into a basal body that templates the ciliary axoneme. Ciliogenesis depends...... on mother centriole-specific distal appendages, whereas the role of subdistal appendages in ciliary function is unclear. Here, we identify CEP128 as a centriole subdistal appendage protein required for regulating ciliary signaling. Loss of CEP128 did not grossly affect centrosomal or ciliary structure...... but caused impaired transforming growth factor-β/bone morphogenetic protein (TGF-β/BMP) signaling in zebrafish and at the primary cilium in cultured mammalian cells. This phenotype is likely the result of defective vesicle trafficking at the cilium as ciliary localization of RAB11 was impaired upon loss...

Functionalization of PCL-3D Electrospun Nanofibrous Scaffolds for Improved BMP2-Induced Bone Formation.

Science.gov (United States)

Miszuk, Jacob M; Xu, Tao; Yao, Qingqing; Fang, Fang; Childs, Josh D; Hong, Zhongkui; Tao, Jianning; Fong, Hao; Sun, Hongli

2018-03-01

Bone morphogenic protein 2 (BMP2) is a key growth factor for bone regeneration, possessing FDA approval for orthopedic applications. BMP2 is often required in supratherapeutic doses clinically, yielding adverse side effects and substantial treatment costs. Considering the crucial role of materials for BMPs delivery and cell osteogenic differentiation, we devote to engineering an innovative bone-matrix mimicking niche to improve low dose of BMP2-induced bone formation. Our previous work describes a novel technique, named thermally induced nanofiber self-agglomeration (TISA), for generating 3D electrospun nanofibrous (NF) polycaprolactone (PCL) scaffolds. TISA process could readily blend PCL with PLA, leading to increased osteogenic capabilities in vitro , however, these bio-inert synthetic polymers produced limited BMP2-induced bone formation in vivo. We therefore hypothesize that functionalization of NF 3D PCL scaffolds with bone-like hydroxyapatite (HA) and BMP2 signaling activator phenamil will provide a favorable osteogenic niche for bone formation at low doses of BMP2. Compared to PCL-3D scaffolds, PCL/HA-3D scaffolds demonstrated synergistically enhanced osteogenic differentiation capabilities of C2C12 cells with phenamil. Importantly, in vivo studies showed this synergism was able to generate significantly increased new bone in an ectopic mouse model, suggesting PCL/HA-3D scaffolds act as a favorable synthetic extracellular matrix for bone regeneration.

Increased bone morphogenetic protein signaling contributes to age-related declines in neurogenesis and cognition.

Science.gov (United States)

Meyers, Emily A; Gobeske, Kevin T; Bond, Allison M; Jarrett, Jennifer C; Peng, Chian-Yu; Kessler, John A

2016-02-01

Aging is associated with decreased neurogenesis in the hippocampus and diminished hippocampus-dependent cognitive functions. Expression of bone morphogenetic protein 4 (BMP4) increases with age by more than 10-fold in the mouse dentate gyrus while levels of the BMP inhibitor, noggin, decrease. This results in a profound 30-fold increase in phosphorylated-SMAD1/5/8, the effector of canonical BMP signaling. Just as observed in mice, a profound increase in expression of BMP4 is observed in the dentate gyrus of humans with no known cognitive abnormalities. Inhibition of BMP signaling either by overexpression of noggin or transgenic manipulation not only increases neurogenesis in aging mice, but remarkably, is associated with a rescue of cognitive deficits to levels comparable to young mice. Additive benefits are observed when combining inhibition of BMP signaling and environmental enrichment. These findings indicate that increased BMP signaling contributes significantly to impairments in neurogenesis and to cognitive decline associated with aging, and identify this pathway as a potential druggable target for reversing age-related changes in cognition. Copyright © 2016 Elsevier Inc. All rights reserved.

Pentagone internalises glypicans to fine-tune multiple signalling pathways

Science.gov (United States)

Norman, Mark; Vuilleumier, Robin; Springhorn, Alexander; Gawlik, Jennifer; Pyrowolakis, George

2016-01-01

Tight regulation of signalling activity is crucial for proper tissue patterning and growth. Here we investigate the function of Pentagone (Pent), a secreted protein that acts in a regulatory feedback during establishment and maintenance of BMP/Dpp morphogen signalling during Drosophila wing development. We show that Pent internalises the Dpp co-receptors, the glypicans Dally and Dally-like protein (Dlp), and propose that this internalisation is important in the establishment of a long range Dpp gradient. Pent-induced endocytosis and degradation of glypicans requires dynamin- and Rab5, but not clathrin or active BMP signalling. Thus, Pent modifies the ability of cells to trap and transduce BMP by fine-tuning the levels of the BMP reception system at the plasma membrane. In addition, and in accordance with the role of glypicans in multiple signalling pathways, we establish a requirement of Pent for Wg signalling. Our data propose a novel mechanism by which morphogen signalling is regulated. DOI: http://dx.doi.org/10.7554/eLife.13301.001 PMID:27269283

Functional expression of BMP7 receptors in oral epithelial cells. Interleukin-17F production in response to BMP7.

Science.gov (United States)

Nishio, Kensuke; Ozawa, Yasumasa; Ito, Hisanori; Kifune, Takashi; Narita, Tatsuya; Iinuma, Toshimitsu; Gionhaku, Nobuhito; Asano, Masatake

2017-10-01

Bone morphogenetic proteins (BMPs) are members of the transforming growth factor-β (TGF-β) superfamily. Recently, BMP7 has been demonstrated to be produced by salivary glands and contribute to embryonic branching in mice. The BMP7 in saliva is thought to be delivered to the oral cavity and is expected to contact with stratified squamous epithelial cells which line the surface of oral mucosa. In this study, we attempted to investigate the effects of BMP7 on oral epithelial cells. The expression of BMP receptors was examined by reverse transcriptase-polymerase chain reaction (RT-PCR). OSCCs were stimulated with human recombinant BMP7 (hrBMP7) and the phosphorylation status of Smad1/5/8 was examined by western blotting. For microarray analysis, Ca9-22 cells were stimulated with 100 ng/mL of hrBMP7 and total RNA was extracted and subjected to real-time PCR. The 5'-untranslated region (5'-UTR) of IL-17 F gene was cloned to pGL4-basic vector and used for luciferase assay. Ca9-22 cells were pre-incubated with DM3189, a specific inhibitor of Smad1/5/8, for inhibition assay. All isoforms of type I and type II BMP receptors were expressed in both Ca9-22 and HSC3 cells and BMP7 stimulation resulted in the phosphorylation of Smad1/5/8 in both cell lines. The microarray analysis revealed the induction of interleukin-17 F (IL-17 F), netrin G2 (NTNG2) and hyaluronan synthase 1 (HAS1). Luciferase assay using the 5'-UTR of the IL-17 F gene revealed transcriptional regulation. Induced IL-17 F production was further confirmed at the protein level by ELISA. Smad1/5/8 inhibitor pretreatment decreased IL-17 F expression levels in the cells.

Cyclic mechanical stretch enhances BMP9-induced osteogenic differentiation of mesenchymal stem cells.

Science.gov (United States)

Song, Yang; Tang, Yinhong; Song, Jinlin; Lei, Mingxing; Liang, Panpan; Fu, Tiwei; Su, Xudong; Zhou, Pengfei; Yang, Li; Huang, Enyi

2018-04-01

The purpose of this study was to investigate whether mechanical stretch can enhance the bone morphogenetic protein 9 (BMP9)-induced osteogenic differentiation in MSCs. Recombinant adenoviruses were used to overexpress the BMP9 in C3H10T1/2 MSCs. Cells were seeded onto six-well BioFlex collagen I-coated plates and subjected to cyclic mechanical stretch [6% elongation at 60 cycles/minute (1 Hz)] in a Flexercell FX-4000 strain unit for up to 12 hours. Immunostaining and confocal microscope were used to detect cytoskeleton organization. Cell cycle progression was checked by flow cytometry. Alkaline phosphatase activity was measured with a Chemiluminescence Assay Kit and was quantified with a histochemical staining assay. Matrix mineralization was examined by Alizarin Red S Staining. Mechanical stretch induces cytoskeleton reorganization and inhibits cell proliferation by preventing cells entry into S phase of the cell cycle. Although mechanical stretch alone does not induce the osteogenic differentiation of C3H10T1/2 MSCs, co-stimulation with mechanical stretch and BMP9 enhances alkaline phosphatase activity. The expression of key lineage-specific regulators (e.g., osteocalcin (OCN), SRY-related HMG-box 9, and runt-related transcription factor 2) is also increased after the co-stimulation, compared to the mechanical stretch stimulation along. Furthermore, mechanical stretch augments the BMP9-mediated bone matrix mineralization of C3H10T1/2 MSCs. Our results suggest that mechanical stretch enhances BMP9-induced osteoblastic lineage specification in C3H10T1/2 MSCs.

Perlecan domain 1 recombinant proteoglycan augments BMP-2 activity and osteogenesis

Directory of Open Access Journals (Sweden)

DeCarlo Arthur A

2012-09-01

Full Text Available Abstract Background Many growth factors, such as bone morphogenetic protein (BMP-2, have been shown to interact with polymers of sulfated disacharrides known as heparan sulfate (HS glycosaminoglycans (GAGs, which are found on matrix and cell-surface proteoglycans throughout the body. HS GAGs, and some more highly sulfated forms of chondroitin sulfate (CS, regulate cell function by serving as co-factors, or co-receptors, in GF interactions with their receptors, and HS or CS GAGs have been shown to be necessary for inducing signaling and GF activity, even in the osteogenic lineage. Unlike recombinant proteins, however, HS and CS GAGs are quite heterogenous due, in large part, to post-translational addition, then removal, of sulfate groups to various positions along the GAG polymer. We have, therefore, investigated whether it would be feasible to deliver a DNA pro-drug to generate a soluble HS/CS proteoglycan in situ that would augment the activity of growth-factors, including BMP-2, in vivo. Results Utilizing a purified recombinant human perlecan domain 1 (rhPln.D1 expressed from HEK 293 cells with HS and CS GAGs, tight binding and dose-enhancement of rhBMP-2 activity was demonstrated in vitro. In vitro, the expressed rhPln.D1 was characterized by modification with sulfated HS and CS GAGs. Dose-enhancement of rhBMP-2 by a pln.D1 expression plasmid delivered together as a lyophilized single-phase on a particulate tricalcium phosphate scaffold for 6 or more weeks generated up to 9 fold more bone volume de novo on the maxillary ridge in a rat model than in control sites without the pln.D1 plasmid. Using a significantly lower BMP-2 dose, this combination provided more than 5 times as much maxillary ridge augmentation and greater density than rhBMP-2 delivered on a collagen sponge (InFuse™. Conclusions A recombinant HS/CS PG interacted strongly and functionally with BMP-2 in binding and cell-based assays, and, in vivo, the pln.247 expression plasmid

Chondrogenesis of Embryonic Stem Cell-Derived Mesenchymal Stem Cells Induced by TGFβ1 and BMP7 Through Increased TGFβ Receptor Expression and Endogenous TGFβ1 Production.

Science.gov (United States)

Lee, Patrick T; Li, Wan-Ju

2017-01-01

For decades stem cells have proven to be invaluable to the study of tissue development. More recently, mesenchymal stem cells (MSCs) derived from embryonic stem cells (ESCs) (ESC-MSCs) have emerged as a cell source with great potential for the future of biomedical research due to their enhanced proliferative capability compared to adult tissue-derived MSCs and effectiveness of musculoskeletal lineage-specific cell differentiation compared to ESCs. We have previously compared the properties and differentiation potential of ESC-MSCs to bone marrow-derived MSCs. In this study, we evaluated the potential of TGFβ1 and BMP7 to induce chondrogenic differentiation of ESC-MSCs compared to that of TGFβ1 alone and further investigated the cellular phenotype and intracellular signaling in response to these induction conditions. Our results showed that the expression of cartilage-associated markers in ESC-MSCs induced by the TGFβ1 and BMP7 combination was increased compared to induction with TGFβ1 alone. The TGFβ1 and BMP7 combination upregulated the expression of TGFβ receptor and the production of endogenous TGFβs compared to TGFβ1 induction. The growth factor combination also increasingly activated both of the TGF and BMP signaling pathways, and inhibition of the signaling pathways led to reduced chondrogenesis of ESC-MSCs. Our findings suggest that by adding BMP7 to TGFβ1-supplemented induction medium, ESC-MSC chondrogenesis is upregulated through increased production of endogenous TGFβ and activities of TGFβ and BMP signaling. J. Cell. Biochem. 118: 172-181, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

The Hedgehog Signal Induced Modulation of Bone Morphogenetic Protein Signaling: An Essential Signaling Relay for Urinary Tract Morphogenesis

Science.gov (United States)

Nakagata, Naomi; Miyagawa, Shinichi; Suzuki, Kentaro; Kitazawa, Sohei; Yamada, Gen

2012-01-01

Background Congenital diseases of the urinary tract are frequently observed in infants. Such diseases present a number of developmental anomalies such as hydroureter and hydronephrosis. Although some genetically-modified mouse models of growth factor signaling genes reproduce urinary phenotypes, the pathogenic mechanisms remain obscure. Previous studies suggest that a portion of the cells in the external genitalia and bladder are derived from peri-cloacal mesenchymal cells that receive Hedgehog (Hh) signaling in the early developmental stages. We hypothesized that defects in such progenitor cells, which give rise to urinary tract tissues, may be a cause of such diseases. Methodology/Principal Findings To elucidate the pathogenic mechanisms of upper urinary tract malformations, we analyzed a series of Sonic hedgehog (Shh) deficient mice. Shh−/− displayed hydroureter and hydronephrosis phenotypes and reduced expression of several developmental markers. In addition, we suggested that Shh modulation at an early embryonic stage is responsible for such phenotypes by analyzing the Shh conditional mutants. Tissue contribution assays of Hh-responsive cells revealed that peri-cloacal mesenchymal cells, which received Hh signal secreted from cloacal epithelium, could contribute to the ureteral mesenchyme. Gain- and loss-of-functional mutants for Hh signaling revealed a correlation between Hh signaling and Bone morphogenetic protein (Bmp) signaling. Finally, a conditional ablation of Bmp receptor type IA (BmprIA) gene was examined in Hh-responsive cell lineages. This system thus made it possible to analyze the primary functions of the growth factor signaling relay. The defective Hh-to-Bmp signaling relay resulted in severe urinary tract phenotypes with a decrease in the number of Hh-responsive cells. Conclusions/Significance This study identified the essential embryonic stages for the pathogenesis of urinary tract phenotypes. These results suggested that Hh

[Review for treatment effect and signaling pathway regulation of kidney-tonifying traditional Chinese medicine on osteoporosis].

Science.gov (United States)

Xiao, Ya-Ping; Zeng, Jie; Jiao, Lin-Na; Xu, Xiao-Yu

2018-01-01

The treatment effect and signaling pathway regulation effects of kidney-tonifying traditional Chinese medicine on osteoporosis have been widely studied, but there is no systematic summary currently. This review comprehensively collected and analyzed the traditional Chinese medicines on the treatment and signaling pathway regulation of osteoporosis in recent ten years, such as Epimedii Folium, Drynariae Rhizoma, Cnidii Fructus, Eucommiae Cortex, Psoraleae Fructus and Dipsaci Radix. Based on the existing findings, the following conclusions were obtained: ①kidney-tonifying traditional Chinese medicine treated osteoporosis mainly through BMP-Smads, Wnt/ β -catenin, MAPK, PI3K/AKT signaling pathway to promote osteoblast bone formation and through OPG/RANKL/ RANK, estrogen, CTSK signaling pathway to inhibit osteoclasts of bone resorption. Epimedii Folium, Drynariae Rhizoma, Cnidii Fructus and Psoraleae Fructus up-regulated the expression of key proteins and genes of BMP-Smads and Wnt/ β -catenin signaling pathways to promote bone formation. Epimedii Folium, Drynariae Rhizoma, Cnidii Fructus, Eucommiae Cortex, Psoraleae Fructus and Dipsaci Radix inhibited the bone resorption by mediating the OPG/RANKL/RANK signaling pathway. ②Kidney-tonifying traditional Chinese medicine prevented and treated osteoporosis through a variety of ways: icariin in Epimedii Folium, naringin in Drynariae Rhizoma, osthole in Cnidii Fructus and psoralen in Psoraleae Fructus can regulate BMP-Smads, Wnt/ β -catenin signaling pathway to promote bone formation, but also activate OPG/RANKL/RANK, CTSK and other signaling pathways to inhibit bone resorption. ③The crosstalk of the signaling pathways and the animal experiments of the traditional Chinese medicine on the prevention and treatment of osteoporosis as well as their multi-target mechanism and comprehensive regulation need further clarification. Copyright© by the Chinese Pharmaceutical Association.

Myocardial Tbx20 regulates early atrioventricular canal formation and endocardial epithelial-mesenchymal transition via Bmp2.

Science.gov (United States)

Cai, Xiaoqiang; Nomura-Kitabayashi, Aya; Cai, Weibin; Yan, Jianyun; Christoffels, Vincent M; Cai, Chen-Leng

2011-12-15

During early embryogenesis, the formation of the cardiac atrioventricular canal (AVC) facilitates the transition of the heart from a linear tube into a chambered organ. However, the genetic pathways underlying this developmental process are poorly understood. The T-box transcription factor Tbx20 is expressed predominantly in the AVC of early heart tube. It was shown that Tbx20 activates Nmyc1 and suppresses Tbx2 expression to promote proliferation and specification of the atrial and ventricular chambers, yet it is not known if Tbx20 is involved in early AVC development. Here, we report that mice lacking Tbx20 in the AVC myocardium fail to form the AVC constriction, and the endocardial epithelial-mesenchymal transition (EMT) is severely perturbed. Tbx20 maintains expression of a variety of genes, including Bmp2, Tbx3 and Hand1 in the AVC myocardium. Intriguingly, we found Bmp2 downstream genes involved in the EMT initiation are also downregulated. In addition, re-expression of Bmp2 in the AVC myocardium substantially rescues the EMT defects resulting from the lack of Tbx20, suggesting Bmp2 is one of the key downstream targets of Tbx20 in AVC development. Our data support a complex signaling network with Tbx20 suppressing Tbx2 in the AVC myocardium but also indirectly promoting Tbx2 expression through Bmp2. The spatiotemporal expression of Tbx2 in the AVC appears to be balanced between these two opposing signals. Overall, our study provides genetic evidence that Tbx20 has essential roles in regulating AVC development that coordinate early cardiac chamber formation. Copyright © 2011 Elsevier Inc. All rights reserved.

Bone Morphogenetic Protein (BMP-4 and BMP-7 regulate differentially Transforming Growth Factor (TGF-β1 in normal human lung fibroblasts (NHLF

Directory of Open Access Journals (Sweden)

Lloyd Clare M

2010-06-01

Full Text Available Abstract Background Airway remodelling is thought to be under the control of a complex group of molecules belonging to the Transforming Growth Factor (TGF-superfamily. The Bone Morphogenetic Proteins (BMPs belong to this family and have been shown to regulate fibrosis in kidney and liver diseases. However, the role of BMPs in lung remodelling remains unclear. BMPs may regulate tissue remodelling in asthma by controlling TGF-β-induced profibrotic functions in lung fibroblasts. Methods Cell cultures were exposed to TGF-β1 alone or in the presence of BMP-4 or BMP-7; control cultures were exposed to medium only. Cell proliferation was assessed by quantification of the incorporation of [3H]-thymidine. The expression of the mRNA encoding collagen type I and IV, tenascin C and fibronectin in normal human lung fibroblasts (NHLF was determined by real-time quantitative PCR and the main results were confirmed by ELISA. Cell differentiation was determined by the analysis of the expression of α-smooth muscle actin (α-SMA by western blot and immunohistochemistry. The effect on matrix metalloproteinase (MMP activity was assessed by zymography. Results We have demonstrated TGF-β1 induced upregulation of mRNAs encoding the extracellular matrix proteins, tenascin C, fibronectin and collagen type I and IV when compared to unstimulated NHLF, and confirmed these results at the protein level. BMP-4, but not BMP-7, reduced TGF-β1-induced extracellular matrix protein production. TGF-β1 induced an increase in the activity of the pro-form of MMP-2 which was inhibited by BMP-7 but not BMP-4. Both BMP-4 and BMP-7 downregulated TGF-β1-induced MMP-13 release compared to untreated and TGF-β1-treated cells. TGF-β1 also induced a myofibroblast-like transformation which was partially inhibited by BMP-7 but not BMP-4. Conclusions Our study suggests that some regulatory properties of BMP-7 may be tissue or cell type specific and unveil a potential regulatory role for

A distinct regulatory region of the Bmp5 locus activates gene expression following adult bone fracture or soft tissue injury.

Science.gov (United States)

Guenther, Catherine A; Wang, Zhen; Li, Emma; Tran, Misha C; Logan, Catriona Y; Nusse, Roel; Pantalena-Filho, Luiz; Yang, George P; Kingsley, David M

2015-08-01

Bone morphogenetic proteins (BMPs) are key signaling molecules required for normal development of bones and other tissues. Previous studies have shown that null mutations in the mouse Bmp5 gene alter the size, shape and number of multiple bone and cartilage structures during development. Bmp5 mutations also delay healing of rib fractures in adult mutants, suggesting that the same signals used to pattern embryonic bone and cartilage are also reused during skeletal regeneration and repair. Despite intense interest in BMPs as agents for stimulating bone formation in clinical applications, little is known about the regulatory elements that control developmental or injury-induced BMP expression. To compare the DNA sequences that activate gene expression during embryonic bone formation and following acute injuries in adult animals, we assayed regions surrounding the Bmp5 gene for their ability to stimulate lacZ reporter gene expression in transgenic mice. Multiple genomic fragments, distributed across the Bmp5 locus, collectively coordinate expression in discrete anatomic domains during normal development, including in embryonic ribs. In contrast, a distinct regulatory region activated expression following rib fracture in adult animals. The same injury control region triggered gene expression in mesenchymal cells following tibia fracture, in migrating keratinocytes following dorsal skin wounding, and in regenerating epithelial cells following lung injury. The Bmp5 gene thus contains an "injury response" control region that is distinct from embryonic enhancers, and that is activated by multiple types of injury in adult animals. Copyright © 2015 Elsevier Inc. All rights reserved.

Crimpy enables discrimination of pre and postsynaptic pools of a BMP at the Drosophila NMJ

Science.gov (United States)

James, Rebecca E.; Hoover, Kendall M.; Bulgari, Dinara; McLaughlin, Colleen N.; Wilson, Christopher G.; Wharton, Kristi A.; Levitan, Edwin S.; Broihier, Heather T.

2014-01-01

Summary Distinct pools of the BMP Glass bottom boat (Gbb) control structure and function of the Drosophila neuromuscular junction. Specifically, motoneuron-derived Gbb regulates baseline neurotransmitter release, while muscle-derived Gbb regulates NMJ growth. Yet how cells differentiate between these ligand pools is not known. Here we present evidence that the neuronal Gbb-binding protein Crimpy (Cmpy) permits discrimination of pre and postsynaptic ligand by serving sequential functions in Gbb signaling. Cmpy first delivers Gbb to dense core vesicles (DCVs) for activity-dependent release from presynaptic terminals. In the absence of Cmpy, Gbb is no longer associated with DCVs and is not released by activity. Electrophysiological analyses demonstrate that Cmpy promotes Gbb's pro-neurotransmission function. Surprisingly, the Cmpy ectodomain is itself released upon DCV exocytosis, arguing that Cmpy serves a second function in BMP signaling. In addition to trafficking Gbb to DCVs, we propose that Gbb/Cmpy co-release from presynaptic terminals defines a neuronal pro-transmission signal. PMID:25453556

Controlled Retention of BMP-2-Derived Peptide on Nanofibers Based on Mussel-Inspired Adhesion for Bone Formation.

Science.gov (United States)

Lee, Jinkyu; Perikamana, Sajeesh Kumar Madhurakkat; Ahmad, Taufiq; Lee, Min Suk; Yang, Hee Seok; Kim, Do-Gyoon; Kim, Kyobum; Kwon, Bosun; Shin, Heungsoo

2017-04-01

Although bone morphogenetic protein-2 (BMP-2) has been frequently used to stimulate bone formation, it has several side effects to be addressed, including the difficulty in optimization of clinically relevant doses and unwanted induction of cancerous signaling processes. In this study, an osteogenic peptide (OP) derived from BMP-2 was investigated as a substitute for BMP-2. In vitro studies showed that OP was able to enhance the osteogenic differentiation and mineralization of human mesenchymal stem cells (hMSCs). The peptides were then conjugated onto biocompatible poly-ι-lactide electrospun nanofibers through polydopamine chemistry. Surface chemical analysis proved that more than 80% of the peptides were stably retained on the nanofiber surface after 8 h of polydopamine coating during at least 28 days, and the amount of peptides that was retained increased depending on the polydopamine coating time. For instance, about 65% of the peptides were retained on nanofibers after 4 h of polydopamine coating. Also, a relatively small dose of peptides could effectively induce bone formation in in vivo critical-sized defects on the calvarial bones of mice. More than 50.4% ± 16.9% of newly formed bone was filled within the defect after treatment with only 10.5 ± 0.6 μg of peptides. Moreover, these groups had similar elastic moduli and contact hardnesses with host bone. Taken together, our results suggest that polydopamine-mediated OP immobilized on nanofibers can modulate the retention of relatively short lengths of peptides, which might make this an effective therapeutic remedy to guide bone regeneration using a relatively small amount of peptides.

Uncovering the Role of BMP Signaling in Melanocyte Development and Melanoma Tumorigenesis

Science.gov (United States)

2016-09-01

specifiers’, genes that are initially expressed broadly in the neural crest and help to maintain neural crest identity (Fig. 2G ) (6). As development...melanoma cells, GDF6 and the BMP pathway negatively regulated SOX9 expression (Fig. 3G ; Fig. S13A-C). Epistasis analyses showed that SOX9 knockdown rescued...criteria, if the tumor volume reached >1,000 mm3; if tumor size or location affected the mobility or general health of animal, the animal was euthanized

Differential phosphorylation of Smad1 integrates BMP and neurotrophin pathways through Erk/Dusp in axon development.

Science.gov (United States)

Finelli, Mattéa J; Murphy, Kevin J; Chen, Lei; Zou, Hongyan

2013-05-30

Sensory axon development requires concerted actions of growth factors for the precise control of axonal outgrowth and target innervation. How developing sensory neurons integrate different cues is poorly understood. We demonstrate here that Smad1 activation is required for neurotrophin-mediated sensory axon growth in vitro and in vivo. Through differential phosphorylation, Smad1 exerts transcriptional selectivity to regulate the expression and activity of Erk1 and Erk2-two key neurotrophin effectors. Specifically, bone morphogenetic proteins (BMPs) signal through carboxy-terminal phosphorylation of Smad1 (pSmad1C) to induce Erk1/2 transcription for enhanced neurotrophin responsiveness. Meanwhile, neurotrophin signaling results in linker phosphorylation of Smad1 (pSmad1L), which in turn upregulates an Erk-specific dual-specificity phosphatase, Dusp6, leading to reduced pErk1/2 and constituting a negative-feedback loop for the prevention of axon overgrowth. Together, the BMP and neurotrophin pathways form a tightly regulated signaling network with a balanced ratio of Erk1/2 and pErk1/2 to direct the precise connections between sensory neurons and peripheral targets. Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.

2,2'-Bis(monoacylglycero) PO4 (BMP), but Not 3,1'-BMP, increases membrane curvature stress to enhance α-tocopherol transfer protein binding to membranes.

Science.gov (United States)

Baptist, Matilda; Panagabko, Candace; Nickels, Jonathan D; Katsaras, John; Atkinson, Jeffrey

2015-03-01

Previous work revealed that α-tocopherol transfer protein (α-TTP) co-localizes with bis(monoacylglycero)phosphate (BMP) in late endosomes. BMP is a lipid unique to late endosomes and is believed to induce membrane curvature and support the multivesicular nature of this organelle. We examined the effect of BMP on α-TTP binding to membranes using dual polarization interferometry and vesicle-binding assay. α-TTP binding to membranes is increased by the curvature-inducing lipid BMP. α-TTP binds to membranes with greater affinity when they contain the 2,2'-BMP versus 3,1'-BMP isomers.

Autologous implantation of BMP2-expressing dermal fibroblasts to improve bone mineral density and architecture in rabbit long bones.

Science.gov (United States)

Ishihara, Akikazu; Weisbrode, Steve E; Bertone, Alicia L

2015-10-01

Cell-mediated gene therapy may treat bone fragility disorders. Dermal fibroblasts (DFb) may be an alternative cell source to stem cells for orthopedic gene therapy because of their rapid cell yield and excellent plasticity with bone morphogenetic protein-2 (BMP2) gene transduction. Autologous DFb or BMP2-expressing autologous DFb were administered in twelve rabbits by two delivery routes; a transcortical intra-medullar infusion into tibiae and delayed intra-osseous injection into femoral drill defects. Both delivery methods of DFb-BMP2 resulted in a successful cell engraftment, increased bone volume, bone mineral density, improved trabecular bone microarchitecture, greater bone defect filling, external callus formation, and trabecular surface area, compared to non-transduced DFb or no cells. Cell engraftment within trabecular bone and bone marrow tissue was most efficiently achieved by intra-osseous injection of DFb-BMP2. Our results suggested that BMP2-expressing autologous DFb have enhanced efficiency of engraftment in target bones resulting in a measurable biologic response by the bone of improved bone mineral density and bone microarchitecture. These results support that autologous implantation of DFb-BMP2 warrants further study on animal models of bone fragility disorders, such as osteogenesis imperfecta and osteoporosis to potentially enhance bone quality, particularly along with other gene modification of these diseases. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

Generation of animal form by the Chordin/Tolloid/BMP gradient: 100 years after D'Arcy Thompson.

Science.gov (United States)

De Robertis, Edward M; Moriyama, Yuki; Colozza, Gabriele

2017-09-01

The classic book "On Growth and Form" by naturalist D'Arcy Thompson was published 100 years ago. To celebrate this landmark, we present experiments in the Xenopus embryo that provide a framework for understanding how simple, quantitative transformations of a morphogen gradient might have affected evolution and morphological diversity of organisms. D'Arcy Thompson proposed that different morphologies might be generated by modifying physical parameters in an underlying system of Cartesian coordinates that pre-existed in Nature and arose during evolutionary history. Chordin is a BMP antagonist secreted by the Spemann organizer located on the dorsal side of the gastrula. Chordin generates a morphogen gradient as first proposed by mathematician Alan Turing. The rate-limiting step of this dorsal-ventral (D-V) morphogen is the degradation of Chordin by the Tolloid metalloproteinase in the ventral side. Chordin is expressed at gastrula on the dorsal side where BMP signaling is low, while at the opposite side peak levels of BMP signaling are reached. In fishes, amphibians, reptiles and birds, high BMP signaling in the ventral region induces transcription of a secreted inhibitor of Tolloid called Sizzled. By depleting Sizzled exclusively in the ventral half of the embryo we were able to expand the ventro-posterior region in an otherwise normal embryo. Conversely, ventral depletion of Tolloid, which stabilizes Chordin, decreased ventral and tail structures, phenocopying the tolloid zebrafish mutation. We explain how historical constraints recorded in the language of DNA become subject to the universal laws of physics when an ancestral reaction-diffusion morphogen gradient dictates form. © 2017 Japanese Society of Developmental Biologists.

BMP7 and SHH regulate Pax2 in mouse retinal astrocytes by relieving TLX repression.

Science.gov (United States)

Sehgal, Rachna; Sheibani, Nader; Rhodes, Simon J; Belecky Adams, Teri L

2009-08-15

Pax2 is essential for development of the neural tube, urogenital system, optic vesicle, optic cup and optic tract. In the eye, Pax2 deficiency is associated with coloboma, a loss of astrocytes in the optic nerve and retina, and abnormal axonal pathfinding of the ganglion cell axons at the optic chiasm. Thus, appropriate expression of Pax2 is essential for astrocyte determination and differentiation. Although BMP7 and SHH have been shown to regulate Pax2 expression, the molecular mechanism by which this regulation occurs is not well understood. In this study, we determined that BMP7 and SHH activate Pax2 expression in mouse retinal astrocyte precursors in vitro. SHH appeared to play a dual role in Pax2 regulation; 1) SHH may regulate BMP7 expression, and 2) the SHH pathway cooperates with the BMP pathway to regulate Pax2 expression. BMP and SHH pathway members can interact separately or together with TLX, a repressor protein in the tailless transcription factor family. Here we show that the interaction of both pathways with TLX relieves the repression of Pax2 expression in mouse retinal astrocytes. Together these data reveal a new mechanism for the cooperative actions of signaling pathways in astrocyte determination and differentiation and suggest interactions of regulatory pathways that are applicable to other developmental programs.

Medium-Term Function of a 3D Printed TCP/HA Structure as a New Osteoconductive Scaffold for Vertical Bone Augmentation: A Simulation by BMP-2 Activation

Directory of Open Access Journals (Sweden)

Mira Moussa

2015-04-01

Full Text Available Introduction: A 3D-printed construct made of orthogonally layered strands of tricalcium phosphate (TCP and hydroxyapatite has recently become available. The material provides excellent osteoconductivity. We simulated a medium-term experiment in a sheep calvarial model by priming the blocks with BMP-2. Vertical bone growth/maturation and material resorption were evaluated. Materials and methods: Titanium hemispherical caps were filled with either bare- or BMP-2 primed constructs and placed onto the calvaria of adult sheep (n = 8. Histomorphometry was performed after 8 and 16 weeks. Results: After 8 weeks, relative to bare constructs, BMP-2 stimulation led to a two-fold increase in bone volume (Bare: 22% ± 2.1%; BMP-2 primed: 50% ± 3% and a 3-fold decrease in substitute volume (Bare: 47% ± 5%; BMP-2 primed: 18% ± 2%. These rates were still observed at 16 weeks. The new bone grew and matured to a haversian-like structure while the substitute material resorbed via cell- and chemical-mediation. Conclusion: By priming the 3D construct with BMP-2, bone metabolism was physiologically accelerated, that is, enhancing vertical bone growth and maturation as well as material bioresorption. The scaffolding function of the block was maintained, leaving time for the bone to grow and mature to a haversian-like structure. In parallel, the material resorbed via cell-mediated and chemical processes. These promising results must be confirmed in clinical tests.

Crimpy enables discrimination of presynaptic and postsynaptic pools of a BMP at the Drosophila neuromuscular junction.

Science.gov (United States)

James, Rebecca E; Hoover, Kendall M; Bulgari, Dinara; McLaughlin, Colleen N; Wilson, Christopher G; Wharton, Kristi A; Levitan, Edwin S; Broihier, Heather T

2014-12-08

Distinct pools of the bone morphogenetic protein (BMP) Glass bottom boat (Gbb) control structure and function of the Drosophila neuromuscular junction. Specifically, motoneuron-derived Gbb regulates baseline neurotransmitter release, whereas muscle-derived Gbb regulates neuromuscular junction growth. Yet how cells differentiate between these ligand pools is not known. Here we present evidence that the neuronal Gbb-binding protein Crimpy (Cmpy) permits discrimination of pre- and postsynaptic ligand by serving sequential functions in Gbb signaling. Cmpy first delivers Gbb to dense core vesicles (DCVs) for activity-dependent release from presynaptic terminals. In the absence of Cmpy, Gbb is no longer associated with DCVs and is not released by activity. Electrophysiological analyses demonstrate that Cmpy promotes Gbb's proneurotransmission function. Surprisingly, the Cmpy ectodomain is itself released upon DCV exocytosis, arguing that Cmpy serves a second function in BMP signaling. In addition to trafficking Gbb to DCVs, we propose that Gbb/Cmpy corelease from presynaptic terminals defines a neuronal protransmission signal. Copyright © 2014 Elsevier Inc. All rights reserved.

Intracellular co-delivery of Sr ion and phenamil drug through mesoporous bioglass nanocarriers synergizes BMP signaling and tissue mineralization.

Science.gov (United States)

Lee, Jung-Hwan; Mandakhbayar, Nandin; El-Fiqi, Ahmed; Kim, Hae-Won

2017-09-15

Inducing differentiation and maturation of resident multipotent stem cells (MSCs) is an important strategy to regenerate hard tissues in mal-calcification conditions. Here we explore a co-delivery approach of therapeutic molecules comprised of ion and drug through a mesoporous bioglass nanoparticle (MBN) for this purpose. Recently, MBN has offered unique potential as a nanocarrier for hard tissues, in terms of high mesoporosity, bone bioactivity (and possibly degradability), tunable delivery of biomolecules, and ionic modification. Herein Sr ion is structurally doped to MBN while drug Phenamil is externally loaded as a small molecule activator of BMP signaling, for the stimulation of osteo/odontogenesis and mineralization of human MSCs derived from dental pulp. The Sr-doped MBN (85Si:10Ca:5Sr) sol-gel processed presents a high mesoporosity with a pore size of ∼6nm. In particular, Sr ion is released slowly at a daily rate of ∼3ppm per mg nanoparticles for up to 7days, a level therapeutically effective for cellular stimulation. The Sr-MBN is internalized to most MSCs via an ATP dependent macropinocytosis within hours, increasing the intracellular levels of Sr, Ca and Si ions. Phenamil is loaded maximally ∼30% into Sr-MBN and then released slowly for up to 7days. The co-delivered molecules (Sr ion and Phenamil drug) have profound effects on the differentiation and maturation of cells, i.e., significantly enhancing expression of osteo/odontogenic genes, alkaline phosphatase activity, and mineralization of cells. Of note, the stimulation is a result of a synergism of Sr and Phenamil, through a Trb3-dependent BMP signaling pathway. This biological synergism is further evidenced in vivo in a mal-calcification condition involving an extracted tooth implantation in dorsal subcutaneous tissues of rats. Six weeks post operation evidences the osseous-dentinal hard tissue formation, which is significantly stimulated by the Sr/Phenamil delivery, based on histomorphometric

Paracrine and autocrine signals promoting full chondrogenic differentiation of a mesoblastic cell line.

Science.gov (United States)

Locker, Morgane; Kellermann, Odile; Boucquey, Marie; Khun, Huot; Huerre, Michel; Poliard, Anne

2004-01-01

triiodothyronine triggers the final differentiation into hypertrophic chondrocytes within a further 15 days. The chondrogenic process is supported by intrinsic expression of Sox9 and BMP family genes. Similarly to the in vivo situation, activation of Sox9 expression and the BMP signaling pathway occurred on mesoblastic condensation. After induction, BMP-activated Smad nuclear translocation persisted throughout the process until the onset of hypertrophy. After dexamethasone addition, Sox9 expression was upregulated. Dexamethasone withdrawal reversed the increase in Sox9 expression and stopped differentiation. Thus, Sox9 seems to be a downstream mediator of dexamethasone action.

Promotion of bone morphogenetic protein signaling by tetraspanins and glycosphingolipids.

Directory of Open Access Journals (Sweden)

Zhiyu Liu

2015-05-01

Full Text Available Bone morphogenetic proteins (BMPs belong to the transforming growth factor β (TGFβ superfamily of secreted molecules. BMPs play essential roles in multiple developmental and homeostatic processes in metazoans. Malfunction of the BMP pathway can cause a variety of diseases in humans, including cancer, skeletal disorders and cardiovascular diseases. Identification of factors that ensure proper spatiotemporal control of BMP signaling is critical for understanding how this pathway is regulated. We have used a unique and sensitive genetic screen to identify the plasma membrane-localized tetraspanin TSP-21 as a key new factor in the C. elegans BMP-like "Sma/Mab" signaling pathway that controls body size and postembryonic M lineage development. We showed that TSP-21 acts in the signal-receiving cells and genetically functions at the ligand-receptor level. We further showed that TSP-21 can associate with itself and with two additional tetraspanins, TSP-12 and TSP-14, which also promote Sma/Mab signaling. TSP-12 and TSP-14 can also associate with SMA-6, the type I receptor of the Sma/Mab pathway. Finally, we found that glycosphingolipids, major components of the tetraspanin-enriched microdomains, are required for Sma/Mab signaling. Our findings suggest that the tetraspanin-enriched membrane microdomains are important for proper BMP signaling. As tetraspanins have emerged as diagnostic and prognostic markers for tumor progression, and TSP-21, TSP-12 and TSP-14 are all conserved in humans, we speculate that abnormal BMP signaling due to altered expression or function of certain tetraspanins may be a contributing factor to cancer development.

FGF and BMP derived from dorsal root ganglia regulate blastema induction in limb regeneration in Ambystoma mexicanum.

Science.gov (United States)

Satoh, Akira; Makanae, Aki; Nishimoto, Yurie; Mitogawa, Kazumasa

2016-09-01

Urodele amphibians have a remarkable organ regeneration ability that is regulated by neural inputs. The identification of these neural inputs has been a challenge. Recently, Fibroblast growth factor (Fgf) and Bone morphogenic protein (Bmp) were shown to substitute for nerve functions in limb and tail regeneration in urodele amphibians. However, direct evidence of Fgf and Bmp being secreted from nerve endings and regulating regeneration has not yet been shown. Thus, it remained uncertain whether they were the nerve factors responsible for successful limb regeneration. To gather experimental evidence, the technical difficulties involved in the usage of axolotls had to be overcome. We achieved this by modifying the electroporation method. When Fgf8-AcGFP or Bmp7-AcGFP was electroporated into the axolotl dorsal root ganglia (DRG), GFP signals were detectable in the regenerating limb region. This suggested that Fgf8 and Bmp7 synthesized in neural cells in the DRG were delivered to the limbs through the long axons. Further knockdown experiments with double-stranded RNA interference resulted in impaired limb regeneration ability. These results strongly suggest that Fgf and Bmp are the major neural inputs that control the organ regeneration ability. Copyright © 2016 Elsevier Inc. All rights reserved.

Role of Bone Morphogenetic Protein 7 (BMP7 in the Modulation of Corneal Stromal and Epithelial Cell Functions

Directory of Open Access Journals (Sweden)

Bhavani S. Kowtharapu

2018-05-01

Full Text Available In the cornea, healing of the wounded avascular surface is an intricate process comprising the involvement of epithelial, stromal and neuronal cell interactions. These interactions result to the release of various growth factors that play prominent roles during corneal wound healing response. Bone morphogenetic proteins (BMPs are unique multi-functional potent growth factors of the transforming growth factor-beta (TGF-β superfamily. Treatment of corneal epithelial cells with substance P and nerve growth factor resulted to an increase in the expression of BMP7 mRNA. Since BMP7 is known to modulate the process of corneal wound healing, in this present study, we investigated the influence of exogenous rhBMP7 on human corneal epithelial cell and stromal cell (SFs function. To obtain a high-fidelity expression profiling of activated biomarkers and pathways, transcriptome-wide gene-level expression profiling of epithelial cells in the presence of BMP7 was performed. Gene ontology analysis shows BMP7 stimulation activated TGF-β signaling and cell cycle pathways, whereas biological processes related to cell cycle, microtubule and intermediate filament cytoskeleton organization were significantly impacted in corneal epithelial cells. Scratch wound healing assay showed increased motility and migration of BMP7 treated epithelial cells. BMP7 stimulation studies show activation of MAPK cascade proteins in epithelial cells and SFs. Similarly, a difference in the expression of claudin, Zink finger E-box-binding homeobox 1 was observed along with phosphorylation levels of cofilin in epithelial cells. Stimulation of SFs with BMP7 activated them with increased expression of α-smooth muscle actin. In addition, an elevated phosphorylation of epidermal growth factor receptor following BMP7 stimulation was also observed both in corneal epithelial cells and SFs. Based on our transcriptome analysis data on epithelial cells and the results obtained in SFs, we

Mesenchymal stem cells with rhBMP-2 inhibits the growth of canine osteosarcoma cells.

Science.gov (United States)

Rici, Rose Eli Grassi; Alcântara, Dayane; Fratini, Paula; Wenceslau, Cristiane Valverde; Ambrósio, Carlos Eduardo; Miglino, Maria Angelica; Maria, Durvanei Augusto

2012-02-22

The bone morphogenetic proteins (BMPs) belong to a unique group of proteins that includes the growth factor TGF-β. BMPs play important roles in cell differentiation, cell proliferation, and inhibition of cell growth. They also participate in the maturation of several cell types, depending on the microenvironment and interactions with other regulatory factors. Depending on their concentration gradient, the BMPs can attract various types of cells and act as chemotactic, mitogenic, or differentiation agents. BMPs can interfere with cell proliferation and the formation of cartilage and bone. In addition, BMPs can induce the differentiation of mesenchymal progenitor cells into various cell types, including chondroblasts and osteoblasts. The aim of this study was to analyze the effects of treatment with rhBMP-2 on the proliferation of canine mesenchymal stem cells (cMSCs) and the tumor suppression properties of rhBMP-2 in canine osteocarcoma (OST) cells. Osteosarcoma cell lines were isolated from biopsies and excisions of animals with osteosarcoma and were characterized by the Laboratory of Biochemistry and Biophysics, Butantan Institute. The mesenchymal stem cells were derived from the bone marrow of canine fetuses (cMSCs) and belong to the University of São Paulo, College of Veterinary Medicine (FMVZ-USP) stem cell bank. After expansion, the cells were cultured in a 12-well Transwell system; cells were treated with bone marrow mesenchymal stem cells associated with rhBMP2. Expression of the intracytoplasmic and nuclear markers such as Caspase-3, Bax, Bad, Bcl-2, Ki-67, p53, Oct3/4, Nanog, Stro-1 were performed by flow citometry. We evaluated the regenerative potential of in vitro treatment with rhBMP-2 and found that both osteogenic induction and tumor regression occur in stem cells from canine bone marrow. rhBMP-2 inhibits the proliferation capacity of OST cells by mechanisms of apoptosis and tumor suppression mediated by p53. We propose that rhBMP-2 has great

Homeobox protein MSX-1 inhibits expression of bone morphogenetic protein 2, bone morphogenetic protein 4, and lymphoid enhancer-binding factor 1 via Wnt/β-catenin signaling to prevent differentiation of dental mesenchymal cells during the late bell stage.

Science.gov (United States)

Feng, Xiao-Yu; Wu, Xiao-Shan; Wang, Jin-Song; Zhang, Chun-Mei; Wang, Song-Lin

2018-02-01

Homeobox protein MSX-1 (hereafter referred to as MSX-1) is essential for early tooth-germ development. Tooth-germ development is arrested at bud stage in Msx1 knockout mice, which prompted us to study the functions of MSX-1 beyond this stage. Here, we investigated the roles of MSX-1 during late bell stage. Mesenchymal cells of the mandibular first molar were isolated from mice at embryonic day (E)17.5 and cultured in vitro. We determined the expression levels of β-catenin, bone morphogenetic protein 2 (Bmp2), Bmp4, and lymphoid enhancer-binding factor 1 (Lef1) after knockdown or overexpression of Msx1. Our findings suggest that knockdown of Msx1 promoted expression of Bmp2, Bmp4, and Lef1, resulting in elevated differentiation of odontoblasts, which was rescued by blocking the expression of these genes. In contrast, overexpression of Msx1 decreased the expression of Bmp2, Bmp4, and Lef1, leading to a reduction in odontoblast differentiation. The regulation of Bmp2, Bmp4, and Lef1 by Msx1 was mediated by the Wnt/β-catenin signaling pathway. Additionally, knockdown of Msx1 impaired cell proliferation and slowed S-phase progression, while overexpression of Msx1 also impaired cell proliferation and prolonged G1-phase progression. We therefore conclude that MSX-1 maintains cell proliferation by regulating transition of cells from G1-phase to S-phase and prevents odontoblast differentiation by inhibiting expression of Bmp2, Bmp4, and Lef1 at the late bell stage via the Wnt/β-catenin signaling pathway. © 2017 Eur J Oral Sci.

Signaling hierarchy regulating human endothelial cell development.

Science.gov (United States)

Kelly, Melissa A; Hirschi, Karen K

2009-05-01

Our present knowledge of the regulation of mammalian endothelial cell differentiation has been largely derived from studies of mouse embryonic development. However, unique mechanisms and hierarchy of signals that govern human endothelial cell development are unknown and, thus, explored in these studies. Using human embryonic stem cells as a model system, we were able to reproducibly and robustly generate differentiated endothelial cells via coculture on OP9 marrow stromal cells. We found that, in contrast to studies in the mouse, bFGF and VEGF had no specific effects on the initiation of human vasculogenesis. However, exogenous Ihh promoted endothelial cell differentiation, as evidenced by increased production of cells with cobblestone morphology that coexpress multiple endothelial-specific genes and proteins, form lumens, and exhibit DiI-AcLDL uptake. Inhibition of BMP signaling using Noggin or BMP4, specifically, using neutralizing antibodies suppressed endothelial cell formation; whereas, addition of rhBMP4 to cells treated with the hedgehog inhibitor cyclopamine rescued endothelial cell development. Our studies revealed that Ihh promoted human endothelial cell differentiation from pluripotent hES cells via BMP signaling, providing novel insights applicable to modulating human endothelial cell formation and vascular regeneration for human clinical therapies.

Mesenchymal Stem Cells Mitigate Cirrhosis through BMP7

Directory of Open Access Journals (Sweden)

Bing Li

2015-01-01

Full Text Available Background/Aims: Transplantation of mesenchymal stem cells (MSCs has therapeutic effects on various diseases, while its effect on developing cirrhosis as well as the underlying mechanism remained largely unknown. Methods: Twenty C57BL/6 mice were randomly separated into 2 groups of ten each. One group received transplantation of MSCs, while the other group received saline as control. The mice then received intraperitoneal injection of carbon tetrachloride (CCl4 twice per week for 8 weeks to develop cirrhosis. After another 4 weeks, the levels of cirrhosis in these mice were evaluated by liver fibrosis area, portal pressure, sodium balance and excretion. Transcripts of transforming growth factor β 1 (TGFβ1 and bone morphogenic protein 7 (BMP7 in the mouse livers were quantified by RT-qPCR. BMP7-depleted MSCs were prepared and applied in this model, and compared to MSCs. Results: Liver fibrosis, portal hypertension and sodium retention that were developed by CCl4, were all significantly alleviated by MSCs transplantation, which decreased TGFβ1 levels and increased BMP7 levels in the injured liver. MSCs were found to express extremely high levels of BMP7. Knockdown of BMP7 in MSCs completely abolished the protective effect of MSCs against CCl4-induced cirrhosis. Conclusions: MSCs mitigate cirrhosis through their production of BMP7 against the fibrogenic effect of TGFβ1 in the injured liver.

BMP9 ameliorates amyloidosis and the cholinergic defect in a mouse model of Alzheimer's disease.

Science.gov (United States)

Burke, Rebecca M; Norman, Timothy A; Haydar, Tarik F; Slack, Barbara E; Leeman, Susan E; Blusztajn, Jan Krzysztof; Mellott, Tiffany J

2013-11-26

Bone morphogenetic protein 9 (BMP9) promotes the acquisition of the cholinergic phenotype in basal forebrain cholinergic neurons (BFCN) during development and protects these neurons from cholinergic dedifferentiation following axotomy when administered in vivo. A decline in BFCN function occurs in patients with Alzheimer's disease (AD) and contributes to the AD-associated memory deficits. We infused BMP9 intracerebroventricularly for 7 d in transgenic AD model mice expressing green fluorescent protein specifically in cholinergic neurons (APP.PS1/CHGFP) and in wild-type littermate controls (WT/CHGFP). We used 5-mo-old mice, an age when the AD transgenics display early amyloid deposition and few cholinergic defects, and 10-mo-old mice, by which time these mice exhibit established disease. BMP9 infusion reduced the number of Aβ42-positive amyloid plaques in the hippocampus and cerebral cortex of 5- and 10-mo-old APP.PS1/CHGFP mice and reversed the reductions in choline acetyltransferase protein levels in the hippocampus of 10-mo-old APP.PS1/CHGFP mice. The treatment increased cholinergic fiber density in the hippocampus of both WT/CHGFP and APP.PS1/CHGFP mice at both ages. BMP9 infusion also increased hippocampal levels of neurotrophin 3, insulin-like growth factor 1, and nerve growth factor and of the nerve growth factor receptors, tyrosine kinase receptor A and p75/NGFR, irrespective of the genotype of the mice. These data show that BMP9 administration is effective in reducing the Aβ42 amyloid plaque burden, reversing cholinergic neuron abnormalities, and generating a neurotrophic milieu for BFCN in a mouse model of AD and provide evidence that the BMP9-signaling pathway may constitute a therapeutic target for AD.

Immortalization and characterization of mouse floxed Bmp2/4 osteoblasts

International Nuclear Information System (INIS)

Wu, Li-An; Yuan, Guohua; Yang, Guobin; Ortiz-Gonzalez, Iris; Yang, Wuchen; Cui, Yong; MacDougall, Mary; Donly, Kevin J.; Harris, Stephen; Chen, Shuo

2009-01-01

Generation of a floxed Bmp2/4 osteoblast cell line is a valuable tool for studying the modulatory effects of Bmp2 and Bmp4 on osteoblast differentiation as well as relevant molecular events. In this study, primary floxed Bmp2/4 mouse osteoblasts were cultured and transfected with simian virus 40 large T-antigen. Transfection was verified by polymerase chain reaction (PCR) and immunohistochemistry. To examine the characteristics of the transfected cells, morphology, proliferation and mineralization were analyzed, expression of cell-specific genes including Runx2, ATF4, Dlx3, Osx, dentin matrix protein 1, bone sialoprotein, osteopontin, osteocalcin, osteonectin and collagen type I was detected. These results show that transfected floxed Bmp2/4 osteoblasts bypassed senescence with a higher proliferation rate, but retain the genotypic and phenotypic characteristics similar to the primary cells. Thus, we for the first time demonstrate the establishment of an immortalized mouse floxed Bmp2/4 osteoblast cell line.

Immortalization and characterization of mouse floxed Bmp2/4 osteoblasts

Energy Technology Data Exchange (ETDEWEB)

Wu, Li-An [Department of Pediatric Dentistry, The University of Texas Health Science Center at San Antonio, TX (United States); Department of Pediatric Dentistry, School of Stomatology, The Fourth Military Medical University, Xi-an (China); Yuan, Guohua; Yang, Guobin [Department of Pediatric Dentistry, The University of Texas Health Science Center at San Antonio, TX (United States); Key Laboratory of Oral Biomedical Engineering Ministry of Education, Wuhan (China); Ortiz-Gonzalez, Iris [Department of Pediatric Dentistry, The University of Texas Health Science Center at San Antonio, TX (United States); Yang, Wuchen; Cui, Yong [Department of Periodontics, Dental School, The University of Texas Health Science Center at San Antonio, TX (United States); MacDougall, Mary [Department of Oral/Maxillofacial Surgery, University of Alabama, Birmingham, AL (United States); Donly, Kevin J. [Department of Pediatric Dentistry, The University of Texas Health Science Center at San Antonio, TX (United States); Harris, Stephen [Department of Periodontics, Dental School, The University of Texas Health Science Center at San Antonio, TX (United States); Chen, Shuo, E-mail: chens0@uthscsa.edu [Department of Pediatric Dentistry, The University of Texas Health Science Center at San Antonio, TX (United States)

2009-08-14

Generation of a floxed Bmp2/4 osteoblast cell line is a valuable tool for studying the modulatory effects of Bmp2 and Bmp4 on osteoblast differentiation as well as relevant molecular events. In this study, primary floxed Bmp2/4 mouse osteoblasts were cultured and transfected with simian virus 40 large T-antigen. Transfection was verified by polymerase chain reaction (PCR) and immunohistochemistry. To examine the characteristics of the transfected cells, morphology, proliferation and mineralization were analyzed, expression of cell-specific genes including Runx2, ATF4, Dlx3, Osx, dentin matrix protein 1, bone sialoprotein, osteopontin, osteocalcin, osteonectin and collagen type I was detected. These results show that transfected floxed Bmp2/4 osteoblasts bypassed senescence with a higher proliferation rate, but retain the genotypic and phenotypic characteristics similar to the primary cells. Thus, we for the first time demonstrate the establishment of an immortalized mouse floxed Bmp2/4 osteoblast cell line.

Growth differentiation factor 3 is induced by bone morphogenetic protein 6 (BMP-6) and BMP-7 and increases luteinizing hormone receptor messenger RNA expression in human granulosa cells.

Science.gov (United States)

Shi, Jia; Yoshino, Osamu; Osuga, Yutaka; Akiyama, Ikumi; Harada, Miyuki; Koga, Kaori; Fujimoto, Akihisa; Yano, Tetsu; Taketani, Yuji

2012-04-01

To examine the relevance of growth differentiation factor 3 (GDF-3) and bone morphogenetic protein (BMP) cytokines in human ovary. Molecular studies. Research laboratory. Eight women undergoing salpingo-oophorectomy and 30 women undergoing ovarian stimulation for in vitro fertilization. Localizing GDF-3 protein in human ovaries; granulosa cells (GC) cultured with GDF-3, BMP-6, or BMP-7 followed by RNA extraction. The localization of GDF-3 protein in normal human ovaries via immunohistochemical analysis, GDF-3 messenger RNA (mRNA) expression evaluation via quantitative real-time reverse transcription and polymerase chain reaction (RT-PCR), and evaluation of the effect of GDF-3 on leuteinizing hormone (LH) receptor mRNA expression via quantitative real-time RT-PCR. In the ovary, BMP cytokines, of the transforming growth factor beta (TGF-β) superfamily, are known as a luteinization inhibitor by suppressing LH receptor expression in GC. Growth differentiation factor 3, a TGF-β superfamily cytokine, is recognized as an inhibitor of BMP cytokines in other cells. Immunohistochemical analysis showed that GDF-3 was strongly detected in the GC of antral follicles. An in vitro assay revealed that BMP-6 or BMP-7 induced GDF-3 mRNA in GC. Also, GDF-3 increased LH receptor mRNA expression and inhibited the effect of BMP-7, which suppressed the LH receptor mRNA expression in GC. GDF-3, induced with BMP-6 and BMP-7, might play a role in folliculogenesis by inhibiting the effect of BMP cytokines. Copyright © 2012 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Mesenchymal stem cells with rhBMP-2 inhibits the growth of canine osteosarcoma cells

Directory of Open Access Journals (Sweden)

Grassi Rici Rose

2012-02-01

Full Text Available Abstract Background The bone morphogenetic proteins (BMPs belong to a unique group of proteins that includes the growth factor TGF-β. BMPs play important roles in cell differentiation, cell proliferation, and inhibition of cell growth. They also participate in the maturation of several cell types, depending on the microenvironment and interactions with other regulatory factors. Depending on their concentration gradient, the BMPs can attract various types of cells and act as chemotactic, mitogenic, or differentiation agents. BMPs can interfere with cell proliferation and the formation of cartilage and bone. In addition, BMPs can induce the differentiation of mesenchymal progenitor cells into various cell types, including chondroblasts and osteoblasts. The aim of this study was to analyze the effects of treatment with rhBMP-2 on the proliferation of canine mesenchymal stem cells (cMSCs and the tumor suppression properties of rhBMP-2 in canine osteocarcoma (OST cells. Osteosarcoma cell lines were isolated from biopsies and excisions of animals with osteosarcoma and were characterized by the Laboratory of Biochemistry and Biophysics, Butantan Institute. The mesenchymal stem cells were derived from the bone marrow of canine fetuses (cMSCs and belong to the University of São Paulo, College of Veterinary Medicine (FMVZ-USP stem cell bank. After expansion, the cells were cultured in a 12-well Transwell system; cells were treated with bone marrow mesenchymal stem cells associated with rhBMP2. Expression of the intracytoplasmic and nuclear markers such as Caspase-3, Bax, Bad, Bcl-2, Ki-67, p53, Oct3/4, Nanog, Stro-1 were performed by flow citometry. Results We evaluated the regenerative potential of in vitro treatment with rhBMP-2 and found that both osteogenic induction and tumor regression occur in stem cells from canine bone marrow. rhBMP-2 inhibits the proliferation capacity of OST cells by mechanisms of apoptosis and tumor suppression mediated by p

Control of germline stem cell self-renewal and differentiation in the Drosophila ovary: concerted actions of niche signals and intrinsic factors.

Science.gov (United States)

Xie, Ting

2013-01-01

In the Drosophila ovary, germline stem cells (GSCs) physically interact with their niche composed of terminal filament cells, cap cells, and possibly GSC-contacting escort cells (ECs). A GSC divides to generate a self-renewing stem cell that remains in the niche and a differentiating daughter that moves away from the niche. The GSC niche provides a bone morphogenetic protein (BMP) signal that maintains GSC self-renewal by preventing stem cell differentiation via repression of the differentiation-promoting gene bag of marbles (bam). In addition, it expresses E-cadherin, which mediates cell adhesion for anchoring GSCs in the niche, enabling continuous self-renewal. GSCs themselves also express different classes of intrinsic factors, including signal transducers, transcription factors, chromatin remodeling factors, translation regulators, and miRNAs, which control self-renewal by strengthening interactions with the niche and repressing various differentiation pathways. Differentiated GSC daughters, known as cystoblasts (CBs), also express distinct classes of intrinsic factors to inhibit self-renewal and promote germ cell differentiation. Surprisingly, GSC progeny are also dependent on their surrounding ECs for proper differentiation at least partly by preventing BMP from diffusing to the differentiated germ cell zone and by repressing ectopic BMP expression. Therefore, both GSC self-renewal and CB differentiation are controlled by collaborative actions of extrinsic signals and intrinsic factors. Copyright © 2012 Wiley Periodicals, Inc.

Deficiency of retinaldehyde dehydrogenase 1 induces BMP2 and increases bone mass in vivo.

Directory of Open Access Journals (Sweden)

Shriram Nallamshetty

Full Text Available The effects of retinoids, the structural derivatives of vitamin A (retinol, on post-natal peak bone density acquisition and skeletal remodeling are complex and compartment specific. Emerging data indicates that retinoids, such as all trans retinoic acid (ATRA and its precursor all trans retinaldehyde (Rald, exhibit distinct and divergent transcriptional effects in metabolism. Despite these observations, the role of enzymes that control retinoid metabolism in bone remains undefined. In this study, we examined the skeletal phenotype of mice deficient in retinaldehyde dehydrogenase 1 (Aldh1a1, the enzyme responsible for converting Rald to ATRA in adult animals. Bone densitometry and micro-computed tomography (µCT demonstrated that Aldh1a1-deficient (Aldh1a1(-/- female mice had higher trabecular and cortical bone mass compared to age and sex-matched control C57Bl/6 wild type (WT mice at multiple time points. Histomorphometry confirmed increased cortical bone thickness and demonstrated significantly higher bone marrow adiposity in Aldh1a1(-/- mice. In serum assays, Aldh1a1(-/- mice also had higher serum IGF-1 levels. In vitro, primary Aldh1a1(-/- mesenchymal stem cells (MSCs expressed significantly higher levels of bone morphogenetic protein 2 (BMP2 and demonstrated enhanced osteoblastogenesis and adipogenesis versus WT MSCs. BMP2 was also expressed at higher levels in the femurs and tibias of Aldh1a1(-/- mice with accompanying induction of BMP2-regulated responses, including expression of Runx2 and alkaline phosphatase, and Smad phosphorylation. In vitro, Rald, which accumulates in Aldh1a1(-/- mice, potently induced BMP2 in WT MSCs in a retinoic acid receptor (RAR-dependent manner, suggesting that Rald is involved in the BMP2 increases seen in Aldh1a1 deficiency in vivo. Collectively, these data implicate Aldh1a1 as a novel determinant of cortical bone density and marrow adiposity in the skeleton in vivo through modulation of BMP signaling.

Deficiency of retinaldehyde dehydrogenase 1 induces BMP2 and increases bone mass in vivo.

Science.gov (United States)

Nallamshetty, Shriram; Wang, Hong; Rhee, Eun-Jung; Kiefer, Florian W; Brown, Jonathan D; Lotinun, Sutada; Le, Phuong; Baron, Roland; Rosen, Clifford J; Plutzky, Jorge

2013-01-01

The effects of retinoids, the structural derivatives of vitamin A (retinol), on post-natal peak bone density acquisition and skeletal remodeling are complex and compartment specific. Emerging data indicates that retinoids, such as all trans retinoic acid (ATRA) and its precursor all trans retinaldehyde (Rald), exhibit distinct and divergent transcriptional effects in metabolism. Despite these observations, the role of enzymes that control retinoid metabolism in bone remains undefined. In this study, we examined the skeletal phenotype of mice deficient in retinaldehyde dehydrogenase 1 (Aldh1a1), the enzyme responsible for converting Rald to ATRA in adult animals. Bone densitometry and micro-computed tomography (µCT) demonstrated that Aldh1a1-deficient (Aldh1a1(-/-) ) female mice had higher trabecular and cortical bone mass compared to age and sex-matched control C57Bl/6 wild type (WT) mice at multiple time points. Histomorphometry confirmed increased cortical bone thickness and demonstrated significantly higher bone marrow adiposity in Aldh1a1(-/-) mice. In serum assays, Aldh1a1(-/-) mice also had higher serum IGF-1 levels. In vitro, primary Aldh1a1(-/-) mesenchymal stem cells (MSCs) expressed significantly higher levels of bone morphogenetic protein 2 (BMP2) and demonstrated enhanced osteoblastogenesis and adipogenesis versus WT MSCs. BMP2 was also expressed at higher levels in the femurs and tibias of Aldh1a1(-/-) mice with accompanying induction of BMP2-regulated responses, including expression of Runx2 and alkaline phosphatase, and Smad phosphorylation. In vitro, Rald, which accumulates in Aldh1a1(-/-) mice, potently induced BMP2 in WT MSCs in a retinoic acid receptor (RAR)-dependent manner, suggesting that Rald is involved in the BMP2 increases seen in Aldh1a1 deficiency in vivo. Collectively, these data implicate Aldh1a1 as a novel determinant of cortical bone density and marrow adiposity in the skeleton in vivo through modulation of BMP signaling.

Cell saver filtering of extravasated rhBMP-2 after degenerative scoliosis reconstruction

Directory of Open Access Journals (Sweden)

Gabriel Liu, MBBCh, MSc, FRCS, FAMS (Orth

2015-06-01

Full Text Available RhBMP-2 is a bone fusion enhancer commonly used in scoliosis reconstruction surgery. It is delivered via an absorbable collagen sponge but has been known to migrate away from its delivery site. RhBMP-2 extravasation in surgical drainage has been noted during first two days post-surgery. Cell savers are widely used in scoliosis reconstruction to limit transfusion requirements and are commonly deployed in cases where rhBMP-2 is used for fusion augmentation. It is not known whether rhBMP-2 is present in salvaged blood or filtered away during cell saver recycling. Through this case series of four patients who underwent scoliosis reconstruction, we assess cell saver efficacy in filtering rhBMP-2 molecules by quantifying the amount of rhBMP-2 present in salvaged blood obtained after postoperative drainage recycling by OrthoPAT® cell saver and comparing it to rhBMP-2 leakage in postoperative drainage without cell saver recycling. We report an almost 10-fold reduction of rhBMP-2 concentration in salvaged blood obtained after cell saver recycling of postoperative drainage, suggesting cell saver effectiveness in filtering rhBMP-2 molecules.

Negative regulation of RIG-I-mediated antiviral signaling by TRK-fused gene (TFG) protein

International Nuclear Information System (INIS)

Lee, Na-Rae; Shin, Han-Bo; Kim, Hye-In; Choi, Myung-Soo; Inn, Kyung-Soo

2013-01-01

Highlights: •TRK-fused gene product (TFG) interacts with TRIM25 upon viral infection. •TFG negatively regulates RIG-I mediated antiviral signaling. •TFG depletion leads to enhanced viral replication. •TFG act downstream of MAVS. -- Abstract: RIG-I (retinoic acid inducible gene I)-mediated antiviral signaling serves as the first line of defense against viral infection. Upon detection of viral RNA, RIG-I undergoes TRIM25 (tripartite motif protein 25)-mediated K63-linked ubiquitination, leading to type I interferon (IFN) production. In this study, we demonstrate that TRK-fused gene (TFG) protein, previously identified as a TRIM25-interacting protein, binds TRIM25 upon virus infection and negatively regulates RIG-I-mediated type-I IFN signaling. RIG-I-mediated IFN production and nuclear factor (NF)-κB signaling pathways were upregulated by the suppression of TFG expression. Furthermore, vesicular stomatitis virus (VSV) replication was significantly inhibited by small inhibitory hairpin RNA (shRNA)-mediated knockdown of TFG, supporting the suppressive role of TFG in RIG-I-mediated antiviral signaling. Interestingly, suppression of TFG expression increased not only RIG-I-mediated signaling but also MAVS (mitochondrial antiviral signaling protein)-induced signaling, suggesting that TFG plays a pivotal role in negative regulation of RNA-sensing, RIG-I-like receptor (RLR) family signaling pathways

Functional studies of signaling pathways in peri-implantation development of the mouse embryo by RNAi

Directory of Open Access Journals (Sweden)

Bell Graham

2005-12-01

Full Text Available Abstract Background Studies of gene function in the mouse have relied mainly on gene targeting via homologous recombination. However, this approach is difficult to apply in specific windows of time, and to simultaneously knock-down multiple genes. Here we report an efficient method for dsRNA-mediated gene silencing in late cleavage-stage mouse embryos that permits examination of phenotypes at post-implantation stages. Results We show that introduction of Bmp4 dsRNA into intact blastocysts by electroporation recapitulates the genetic Bmp4 null phenotype at gastrulation. It also reveals a novel role for Bmp4 in the regulation the anterior visceral endoderm specific gene expression and its positioning. We also show that RNAi can be used to simultaneously target several genes. When applied to the three murine isoforms of Dishevelled, it leads to earlier defects than previously observed in double knock-outs. These include severe delays in post-implantation development and defects in the anterior midline and neural folds at headfold stages. Conclusion Our results indicate that the BMP4 signalling pathway contributes to the development of the anterior visceral endoderm, and reveal an early functional redundancy between the products of the murine Dishevelled genes. The proposed approach constitutes a powerful tool to screen the functions of genes that govern the development of the mouse embryo.

BMP9 inhibits proliferation and metastasis of HER2-positive SK-BR-3 breast cancer cells through ERK1/2 and PI3K/AKT pathways.

Science.gov (United States)

Ren, Wei; Liu, Yuehong; Wan, Shaoheng; Fei, Chang; Wang, Wei; Chen, Yingying; Zhang, Zhihui; Wang, Ting; Wang, Jinshu; Zhou, Lan; Weng, Yaguang; He, Tongchuan; Zhang, Yan

2014-01-01

Bone morphogenetic protein 9 (BMP9), a member of TGF-β superfamily, is reported to inhibit the growth and migration of prostate cancer, osteosarcoma and triple-negative MDA-MB-231 breast cancer cells. However, little is known about the effect of on the biological behaviors of HER2-positive SK-BR-3 breast cancer cells and the underlying mechanisms. This study aimed to investigate the effects of BMP9 on the proliferation and metastasis of SK-BR-3 cells with BMP9 over-expression or BMP9 down-regulated expression. Results indicated that exogenously expressed BMP9 inhibited the proliferation and metastasis of SK-BR-3 cells while decreased endogenous BMP9 expression in SK-BR-3 cells promoted the proliferation and migration of breast cancer cells in vitro and in vivo. In SK-BR-3 cells with BMP9 over-expression, the phosphorylation of HER2, ERK1/2 and AKT was markedly suppressed and the HER2 expression decreased at both mRNA and protein levels, while opposite results were observed in SK-BR-3 cells with BMP9 knock down. When the phosphorylation of ERK1/2 and PI3K/AKT was inhibited by PD98059 and LY294002, respectively, the decreased proliferation and invasion induced by BMP9 knock down were eliminated. These findings suggest that BMP9 can inhibit the proliferation and metastasis of SK-BR-3 cells via inactivating ERK1/2 and PI3K/AKT signaling pathways. Thus, BMP9 may serve as a useful agent in the treatment of HER-2 positive breast cancer.

DMPD: Negative regulation of cytoplasmic RNA-mediated antiviral signaling. [Dynamic Macrophage Pathway CSML Database

Lifescience Database Archive (English)

Full Text Available 18703349 Negative regulation of cytoplasmic RNA-mediated antiviral signaling. Komur...Show Negative regulation of cytoplasmic RNA-mediated antiviral signaling. PubmedID 18703349 Title Negative r...egulation of cytoplasmic RNA-mediated antiviral signaling. Authors Komuro A, Bamm

ROS and ROS-Mediated Cellular Signaling

Directory of Open Access Journals (Sweden)

Jixiang Zhang

2016-01-01

Full Text Available It has long been recognized that an increase of reactive oxygen species (ROS can modify the cell-signaling proteins and have functional consequences, which successively mediate pathological processes such as atherosclerosis, diabetes, unchecked growth, neurodegeneration, inflammation, and aging. While numerous articles have demonstrated the impacts of ROS on various signaling pathways and clarify the mechanism of action of cell-signaling proteins, their influence on the level of intracellular ROS, and their complex interactions among multiple ROS associated signaling pathways, the systemic summary is necessary. In this review paper, we particularly focus on the pattern of the generation and homeostasis of intracellular ROS, the mechanisms and targets of ROS impacting on cell-signaling proteins (NF-κB, MAPKs, Keap1-Nrf2-ARE, and PI3K-Akt, ion channels and transporters (Ca2+ and mPTP, and modifying protein kinase and Ubiquitination/Proteasome System.

Transcription regulation of the vegf gene by the BMP/Smad pathway in the angioblast of zebrafish embryos

International Nuclear Information System (INIS)

He Chen; Chen Xiaozhuo

2005-01-01

Vascular endothelial growth factor (VEGF) is a mitogen that is critically involved in vasculogenesis, angiogenesis, and hematopoiesis. However, what and how transcription factors participate in the regulation of vegf gene expression are not fully understood. Here we report the cloning and sequencing of the zebrafish vegf promoter which revealed that the promoter contains a number of bone morphogenetic protein (BMP)-activated Smad binding elements (SBE), implicating Smad1 and Smad5 in the regulation of BMP-induced expression of vegf. Electrophoretic mobility shift assays of adding recombinant Smad proteins to the SBE-containing DNA oligonucleotides that represent portions of zebrafish vegf promoter resulted in mobility shift of the oligonucleotides. These changes demonstrate potential interactions between Smad1/5 and the vegf promoter. Reporter activity assays using the wild-type or SBE-deleted vegf promoters to drive the luciferase reporter gene expression revealed that Smad1 stimulated while Smad5 repressed the vegf promoter activity in zebrafish embryos. These data indicate that the BMP/Smad signaling pathway is involved in the regulation of zebrafish vegf transcription. In addition, we demonstrate that transgenic expression of human BMP4 in zebrafish embryos induced an expansion of the posterior intermediate cell mass (ICM, also commonly called blood island), a population of cells containing endothelial and hematopoietic precursors. In the expanded ICM, vegf and VEGF receptor 2 (flk-1) were ectopically co-expressed, suggesting that an autocrine/paracrine regulation of vegf expression may exist and contribute to the BMP-induced hemangiogenic cell proliferation

BMP4 and LGL1 are Down Regulated in an Ovine Model of Congenital Diaphragmatic Hernia

Directory of Open Access Journals (Sweden)

Heather eEmmerton-Coughlin

2014-11-01

Full Text Available Background/Purpose: The molecular pathophysiology of lung hypoplasia in congenital diaphragmatic hernia (CDH remains poorly understood. The Wnt signaling pathway and downstream targets, such as bone morphogenetic proteins (BMP 4 and other factors such as late gestation lung protein 1 (LGL1, are essential to normal lung development. Nitrofen-induced hypoplastic CDH rodent lungs demonstrate down regulation of the Wnt pathway including BMP4 and reduced LGL1 expression. The aim of the current study was to examine the molecular pathophysiology associated with a surgically induced CDH in an ovine model. Methods: Left thoracotomy was performed at 80 days in 14 fetal sheep; CDH was created in 7 experimental animals. Lungs were harvested at 136 days (term=145d. Lung weight and mean terminal bronchiole density (MTBD were measured to determine the degree of pulmonary hypoplasia. Quantitative real time PCR was undertaken to analyze Wnt2, Wnt7b, BMP4 and LGL1 mRNA expression. Results: Total lung weight was decreased while MTBD was increased in the CDH group (p<0.05, confirming pulmonary hypoplasia. BMP4 and LGL1 mRNA was significantly reduced in CDH lungs (p<0.05. Wnt2 mRNA was decreased, although not significantly (p<0.06. Conclusions: For the first time, down regulation of BMP4 and Lgl1 are reported in an ovine CDH model. In contrast to other animal models, these changes are persistent to near term. These findings suggest that mechanical compression from herniated viscera may play a more important role in causing pulmonary hypoplasia in CDH, rather than a primary defect in lung organogenesis.

TGF-β prevents phosphate-induced osteogenesis through inhibition of BMP and Wnt/β-catenin pathways.

Directory of Open Access Journals (Sweden)

Fátima Guerrero

Full Text Available BACKGROUND: Transforming growth factor-β (TGF-β is a key cytokine during differentiation of mesenchymal stem cells (MSC into vascular smooth muscle cells (VSMC. High phosphate induces a phenotypic transformation of vascular smooth muscle cells (VSMC into osteogenic-like cells. This study was aimed to evaluate signaling pathways involved during VSMC differentiation of MSC in presence or not of high phosphate. RESULTS: Our results showed that TGF-β induced nuclear translocation of Smad3 as well as the expression of vascular smooth muscle markers, such as smooth muscle alpha actin, SM22α, myocardin, and smooth muscle-myosin heavy chain. The addition of high phosphate to MSC promoted nuclear translocation of Smad1/5/8 and the activation of canonical Wnt/β-catenin in addition to an increase in BMP-2 expression, calcium deposition and alkaline phosphatase activity. The administration of TGF-β to MSC treated with high phosphate abolished all these effects by inhibiting canonical Wnt, BMP and TGF-β pathways. A similar outcome was observed in high phosphate-treated cells after the inhibition of canonical Wnt signaling with Dkk-1. Conversely, addition of both Wnt/β-catenin activators CHIR98014 and lithium chloride enhanced the effect of high phosphate on BMP-2, calcium deposition and alkaline phosphatase activity. CONCLUSIONS: Full VSMC differentiation induced by TGF-β may not be achieved when extracellular phosphate levels are high. Moreover, TGF-β prevents high phosphate-induced osteogenesis by decreasing the nuclear translocation of Smad 1/5/8 and avoiding the activation of Wnt/β-catenin pathway.

Bone Morphogenetic Protein (BMP-7 expression is decreased in human hypertensive nephrosclerosis

Directory of Open Access Journals (Sweden)

Cohen Clemens D

2010-11-01

Full Text Available Abstract Background Bone Morphogenetic Protein (BMP-7 is protective in different animal models of acute and chronic kidney disease. Its role in human kidneys, and in particular hypertensive nephrosclerosis, has thus far not been described. Methods BMP-7 mRNA was quantified using real-time PCR and localised by immunostaining in tissue samples from normal and nephrosclerotic human kidneys. The impact of angiotensin (AT-II and the AT-II receptor antagonist telmisartan on BMP-7 mRNA levels and phosphorylated Smad 1/5/8 (pSmad 1/5/8 expression was quantified in proximal tubular cells (HK-2. Functional characteristics of BMP-7 were evaluated by testing its influence on TGF-β induced epithelial-to-mesenchymal transition (EMT, expression of TGF-β receptor type I (TGF-βRI and phosphorylated Smad 2 (pSmad 2 as well as on TNF-α induced apoptosis of proximal tubular cells. Results BMP-7 was predominantly found in the epithelia of the distal tubule and the collecting duct and was less abundant in proximal tubular cells. In sclerotic kidneys, BMP-7 was significantly decreased as demonstrated by real-time PCR and immunostaining. AT-II stimulation in HK-2 cells led to a significant decrease of BMP-7 and pSmad 1/5/8, which was partially ameliorated upon co-incubation with telmisartan. Only high concentrations of BMP-7 (100 ng/ml were able to reverse TNF-α-induced apoptosis and TGF-β-induced EMT in human proximal tubule cells possibly due to a decreased expression of TGF-βRI. In addition, BMP-7 was able to reverse TGF-β-induced phosphorylation of Smad 2. Conclusions The findings suggest a protective role for BMP-7 by counteracting the TGF-β and TNF-α-induced negative effects. The reduced expression of BMP-7 in patients with hypertensive nephrosclerosis may imply loss of protection and regenerative potential necessary to counter the disease.

Differentiation of Odontoblast-Like Cells From Mouse Induced Pluripotent Stem Cells by Pax9 and Bmp4 Transfection.

Science.gov (United States)

Seki, Daisuke; Takeshita, Nobuo; Oyanagi, Toshihito; Sasaki, Shutaro; Takano, Ikuko; Hasegawa, Masakazu; Takano-Yamamoto, Teruko

2015-09-01

The field of tooth regeneration has progressed in recent years, and human tooth regeneration could become viable in the future. Because induced pluripotent stem (iPS) cells can differentiate into odontogenic cells given appropriate conditions, iPS cells are a potential cell source for tooth regeneration. However, a definitive method to induce iPS cell-derived odontogenic cells has not been established. We describe a novel method of odontoblast differentiation from iPS cells using gene transfection. We generated mouse iPS cell-derived neural crest-like cells (iNCLCs), which exhibited neural crest markers. Next, we differentiated iNCLCs into odontoblast-like cells by transfection of Pax9 and Bmp4 expression plasmids. Exogenous Pax9 upregulated expression of Msx1 and dentin matrix protein 1 (Dmp1) in iNCLCs but not bone morphogenetic protein 4 (Bmp4) or dentin sialophosphoprotein (Dspp). Exogenous Bmp4 upregulated expression of Msx1, Dmp1, and Dspp in iNCLCs, but not Pax9. Moreover, cotransfection of Pax9 and Bmp4 plasmids in iNCLCs revealed a higher expression of Pax9 than when Pax9 plasmid was used alone. In contrast, exogenous Pax9 downregulated Bmp4 overexpression. Cotransfection of Pax9 and Bmp4 synergistically upregulated Dmp1 expression; however, Pax9 overexpression downregulated exogenous Bmp4-induced Dspp expression. Together, these findings suggest that an interaction between exogenous Pax9- and Bmp4-induced signaling modulated Dmp1 and Dspp expression. In conclusion, transfection of Pax9 and Bmp4 expression plasmids in iNCLCs induced gene expression associated with odontoblast differentiation, suggesting that iNCLCs differentiated into odontoblast-like cells. The iPS cell-derived odontoblast-like cells could be a useful cell source for tooth regeneration. It has been reported that induced pluripotent stem (iPS) cells differentiate into odontogenic cells by administration of recombinant growth factors and coculture with odontogenic cells. Therefore, they can

Cigarette smoke regulates VEGFR2-mediated survival signaling in rat lungs

Directory of Open Access Journals (Sweden)

Stevenson Christopher S

2010-02-01

Full Text Available Abstract Background Vascular endothelial growth factor (VEGF and VEGF receptor 2 (VEGFR2-mediated survival signaling is critical to endothelial cell survival, maintenance of the vasculature and alveolar structure and regeneration of lung tissue. Reduced VEGF and VEGFR2 expression in emphysematous lungs has been linked to increased endothelial cell death and vascular regression. Previously, we have shown that CS down-regulated the VEGFR2 and its downstream signaling in mouse lungs. However, the VEGFR2-mediated survival signaling in response to oxidants/cigarette smoke (CS is not known. We hypothesized that CS exposure leads to disruption of VEGFR2-mediated endothelial survival signaling in rat lungs. Methods Adult male Sprague-Dawley rats were exposed CS for 3 days, 8 weeks and 6 months to investigate the effect of CS on VEGFR2-mediated survival signaling by measuring the Akt/PI3-kinase/eNOS downstream signaling in rat lungs. Results and Discussion We show that CS disrupts VEGFR2/PI3-kinase association leading to decreased Akt and eNOS phosphorylation. This may further alter the phosphorylation of the pro-apoptotic protein Bad and increase the Bad/Bcl-xl association. However, this was not associated with a significant lung cell death as evidenced by active caspase-3 levels. These data suggest that although CS altered the VEGFR2-mediated survival signaling in the rat lungs, but it was not sufficient to cause lung cell death. Conclusion The rat lungs exposed to CS in acute, sub-chronic and chronic levels may be representative of smokers where survival signaling is altered but was not associated with lung cell death whereas emphysema is known to be associated with lung cell apoptosis.

DMPD: IRAK1: a critical signaling mediator of innate immunity. [Dynamic Macrophage Pathway CSML Database

Lifescience Database Archive (English)

Full Text Available 17890055 IRAK1: a critical signaling mediator of innate immunity. Gottipati S, Rao ...IRAK1: a critical signaling mediator of innate immunity. PubmedID 17890055 Title IRAK1: a critical signaling mediator

Bone regeneration by implantation of adipose-derived stromal cells expressing BMP-2

International Nuclear Information System (INIS)

Li Huiwu; Dai Kerong; Tang Tingting; Zhang Xiaoling; Yan Mengning; Lou Jueren

2007-01-01

In this study, we reported that the adipose-derived stromal cells (ADSCs) genetically modified by bone morphogenetic protein 2 (BMP-2) healed critical-sized canine ulnar bone defects. First, the osteogenic and adipogenic differentiation potential of the ADSCs derived from canine adipose tissue were demonstrated. And then the cells were modified by the BMP-2 gene and the expression and bone-induction ability of BMP-2 were identified. Finally, the cells modified by BMP-2 gene were applied to a β-tricalcium phosphate (TCP) carrier and implanted into ulnar bone defects in the canine model. After 16 weeks, radiographic, histological, and histomorphometry analysis showed that ADSCs modified by BMP-2 gene produced a significant increase of newly formed bone area and healed or partly healed all of the bone defects. We conclude that ADSCs modified by the BMP-2 gene can enhance the repair of critical-sized bone defects in large animals

Agmatine modulates melanogenesis via MITF signaling pathway.

Science.gov (United States)

Kwon, Eun-Jeong; Kim, Moon-Moo

2017-01-01

Agmatine contained in soybean is also found in Manaca, an anti-aging plant, inhabited in Amazon and induces vasodilation by the promotion of NO synthesis in blood vessel. However, the research of agmatine on melanin synthesis related to hair greying is lacking. The aim of this study was to investigate the melanogenic effect of agmatine via regulation of MITF signaling pathway in B16F1 cells. It was determined whether agmatine regulates melanin synthesis at cellular level in addition to the effect of agmatine on mushroom tyrosinase in vitro in the presence of different concentrations of agmatine. Furthermore, the effect of agmatine on the protein expressions of tyrosinase, TRP-1, TRP-2, BMP-4, BMP-6, C-KIT, p-p38, MITF and C-FOS were examined by western blot analysis. In addition, immunofluorescence staining was carried out to visualize the location of MITF expression in cell. Agmatine at 256μM or more increased melanin synthesis as well as tyrosinase activity. Moreover, whereas agmatine increased the expression levels of TRP-1, BMP-6, p-p38 and MITF, it reduced the expression level of BMP-4. It was also found that agmatine enhanced the expression level of MITF in nucleus. These results suggest that agmatine could induce melanin synthesis though the regulation of MITF transcription factor via BMP-6/p38 signaling pathway. Copyright © 2016 Elsevier B.V. All rights reserved.

Cathepsin H indirectly regulates morphogenetic protein-4 (BMP-4) in various human cell lines

Science.gov (United States)

Rojnik, Matija; Jevnikar, Zala; Mirkovic, Bojana; Janes, Damjan; Zidar, Nace; Kikelj, Danijel; Kos, Janko

2011-01-01

Background Cathepsin H is a cysteine protease considered to play a major role in tumor progression, however, its precise function in tumorigenesis is unclear. Cathepsin H was recently proposed to be involved in processing of bone morphogenetic protein 4 (BMP-4) in mice. In order to clarify whether cathepsin H also regulates BMP-4 in humans, its impact on BMP-4 expression, processing and degradation was investigated in prostate cancer (PC-3), osteosarcoma (HOS) and pro-monocytic (U937) human cell lines. Materials and methods BMP-4 expression was founded to be regulated by cathepsin H using PCR array technology and confirmed by real time PCR. Immunoassays including Western blot and confocal microscopy were used to evaluate the influence of cathepsin H on BMP-4 processing. Results In contrast to HOS, the expression of BMP-4 mRNA in U937 and PC3 cells was significantly decreased by cathepsin H. The different regulation of BMP-4 synthesis could be associated with the absence of the mature 28 kDa cathepsin H form in HOS cells, where only the intermediate 30 kDa form was observed. No co-localization of BMP-4 and cathepsin H was observed in human cell lines and the multistep processing of BMP-4 was not altered in the presence of specific cathepsin H inhibitor. Isolated cathepsin H does not cleave mature recombinant BMP-4, neither with its amino- nor its endopeptidase activity. Conclusions Our results exclude direct proteolytic processing of BMP-4 by cathepsin H, however, they provide support for its involvement in the regulation of BMP-4 expression. PMID:22933963

Twisted Gastrulation as a BMP Modulator during Mammary Gland Development and Tumorigenesis

Science.gov (United States)

2014-05-01

tissues ( Bonilla -Claudio et al., 2012). In WT TEBs (Fig. 8A–D) and mature ducts (Fig. 8I–L) almost all luminal cells expressed GATA-3. In contrast, Twsg1...PubMed: 12952901] Bonilla -Claudio M, Wang J, Bai Y, Klysik E, Selever J, Martin JF. Bmp signaling regulates a dosedependent transcriptional...deficient mice. Arch Oral Biol. 2006; 51:433–8. [PubMed: 16289463] Metallo CM, Ji L, de Pablo JJ, Palecek SP. Retinoic Acid and Bone Morphogenetic

Comparison of the osteogenesis and fusion rates between activin A/BMP-2 chimera (AB204) and rhBMP-2 in a beagle's posterolateral lumbar spine model.

Science.gov (United States)

Zheng, Guang Bin; Yoon, Byung-Hak; Lee, Jae Hyup

2017-10-01

Activin A/BMP-2 chimera (AB204) could promote bone healing more effectively than recombinant bone morphogenetic protein 2 (rhBMP-2) with much lower dose in a rodent model, but there is no report about the effectiveness of AB204 in a large animal model. The purpose of this study was to compare the osteogenesis and fusion rate between AB204 and rhBMP-2 using biphasic calcium phosphate (BCP) as a carrier in a beagle's posterolateral lumbar fusion model. This is a randomized control animal study. Seventeen male beagle dogs were included. Bilateral posterolateral fusion was performed at the L1-L2 and L4-L5 levels. Biphasic calcium phosphate (2 cc), rhBMP-2 (50 µg)+BCP (2 cc), or AB204 (50 µg)+BCP (2 cc) were implanted into the intertransverse space randomly. X-ray was performed at 4 and 8 weeks. After 8 weeks, the animals were sacrificed, and new bone formation and fusion rate were evaluated by manual palpation, computed tomography (CT), and undecalcified histology. The AB204 group showed significantly higher fusion rate (90%) than the rhBMP-2 group (15%) or the Osteon group (6.3%) by manual palpation. On x-ray and CT assessment, fusion rate and the volume of newly formed bone were also significantly higher in AB204 group than other groups. In contrast, more osteolysis was found in rhBMP-2 group (40%) than in AB204 group (10%) on CT study. In histologic results, new bone formation was sufficient between transverse processes in AB204 group, and obvious trabeculation and bone remodeling were observed. But in rhBMP-2 group, new bone formation was less than AB204 group and osteolysis was observed between the intertransverse spaces. A low dose of AB204 with BCP as a carrier significantly promotes the fusion rate in a large animal model when compared with the rhBMP-2. These findings demonstrate that AB204 could be an alternative to rhBMP-2 to improve fusion rate. Copyright © 2017 Elsevier Inc. All rights reserved.

Distinct steps of neural induction revealed by Asterix, Obelix and TrkC, genes induced by different signals from the organizer.

Directory of Open Access Journals (Sweden)

Sonia Pinho

2011-04-01

Full Text Available The amniote organizer (Hensen's node can induce a complete nervous system when grafted into a peripheral region of a host embryo. Although BMP inhibition has been implicated in neural induction, non-neural cells cannot respond to BMP antagonists unless previously exposed to a node graft for at least 5 hours before BMP inhibitors. To define signals and responses during the first 5 hours of node signals, a differential screen was conducted. Here we describe three early response genes: two of them, Asterix and Obelix, encode previously undescribed proteins of unknown function but Obelix appears to be a nuclear RNA-binding protein. The third is TrkC, a neurotrophin receptor. All three genes are induced by a node graft within 4-5 hours but they differ in the extent to which they are inducible by FGF: FGF is both necessary and sufficient to induce Asterix, sufficient but not necessary to induce Obelix and neither sufficient nor necessary for induction of TrkC. These genes are also not induced by retinoic acid, Noggin, Chordin, Dkk1, Cerberus, HGF/SF, Somatostatin or ionomycin-mediated Calcium entry. Comparison of the expression and regulation of these genes with other early neural markers reveals three distinct "epochs", or temporal waves, of gene expression accompanying neural induction by a grafted organizer, which are mirrored by specific stages of normal neural plate development. The results are consistent with neural induction being a cascade of responses elicited by different signals, culminating in the formation of a patterned nervous system.

Requirement of Xmsx-1 in the BMP-triggered ventralization of Xenopus embryos.

Science.gov (United States)

Yamamoto, T S; Takagi, C; Ueno, N

2000-03-01

Signaling triggered by polypeptide growth factors leads to the activation of their target genes. Several homeobox genes are known to be induced in response to polypeptide growth factors in early Xenopus development. In particular, Xmsx-1, an amphibian homologue of vertebrate Msx-1, is well characterized as a target gene of bone morphogenetic protein (BMP). Here, using a dominant-negative form of Xmsx-1 (VP-Xmsx-1), which is a fusion protein made with the virus-derived VP16 activation domain, we have examined whether Xmsx-1 activity is required in the endogenous ventralizing pathway. VP-Xmsx-1 induced a secondary body axis, complete with muscle and neural tissues, when overexpressed in ventral blastomeres, suggesting that Xmsx-1 activity is necessary for both mesoderm and ectoderm to be ventralized. We have also examined the epistatic relationship between Xmsx-1 and another ventralizing homeobox protein, Xvent-1, and show that Xmsx-1 is likely to be acting upstream of Xvent-1. We propose that Xmsx-1 is required in the BMP-stimulated ventralization pathway that involves the downstream activation of Xvent-1.

Convergence of bone morphogenetic protein and laminin-1 signaling pathways promotes proliferation and colony formation by fetal mouse pancreatic cells

International Nuclear Information System (INIS)

Jiang Fangxu; Harrison, Leonard C.

2005-01-01

We previously reported that bone morphogenetic proteins (BMPs), members of the transforming growth factor superfamily, together with the basement membrane glycoprotein laminin-1 (Ln-1), promote proliferation of fetal pancreatic cells and formation of colonies containing peripheral insulin-positive cells. Here, we further investigate the cross-talk between BMP and Ln-1 signals. By RT-PCR, receptors for BMP (BMPR) (excepting BMPR-1B) and Ln-1 were expressed in the fetal pancreas between E13.5 and E17.5. Specific blocking antibodies to BMP-4 and -6 and selective BMP antagonists partially inhibited colony formation by fetal pancreas cells. Colony formation induced by BMP-6 and Ln-1 was completely abolished in a dose-dependent manner by blocking Ln-1 binding to its α 6 integrin and α-dystroglycan receptors or by blocking the Ln-1 signaling molecules, phosphatidyl-inositol-3-kinase (P13K) and MAP kinase kinase-1. These results demonstrate a convergence of BMP and Ln-1 signaling through P13K and MAP kinase pathways to induce proliferation and colony formation in E15.5 fetal mouse pancreatic cells

Identification of potential pathway mediation targets in Toll-like receptor signaling.

Directory of Open Access Journals (Sweden)

Fan Li

2009-02-01

Full Text Available Recent advances in reconstruction and analytical methods for signaling networks have spurred the development of large-scale models that incorporate fully functional and biologically relevant features. An extended reconstruction of the human Toll-like receptor signaling network is presented herein. This reconstruction contains an extensive complement of kinases, phosphatases, and other associated proteins that mediate the signaling cascade along with a delineation of their associated chemical reactions. A computational framework based on the methods of large-scale convex analysis was developed and applied to this network to characterize input-output relationships. The input-output relationships enabled significant modularization of the network into ten pathways. The analysis identified potential candidates for inhibitory mediation of TLR signaling with respect to their specificity and potency. Subsequently, we were able to identify eight novel inhibition targets through constraint-based modeling methods. The results of this study are expected to yield meaningful avenues for further research in the task of mediating the Toll-like receptor signaling network and its effects.

Alveolar ridge and maxillary sinus augmentation using rhBMP-2: a systematic review.

Science.gov (United States)

Freitas, Rubens Moreno de; Spin-Neto, Rubens; Marcantonio Junior, Elcio; Pereira, Luís Antônio Violin Dias; Wikesjö, Ulf M E; Susin, Cristiano

2015-01-01

The aim of this systematic review was to evaluate clinical and safety data for recombinant human bone morphogenetic protein-2 (rhBMP-2) in an absorbable collagen sponge (ACS) carrier when used for alveolar ridge/maxillary sinus augmentation in humans. Clinical studies/case series published 1980 through June 2012 using rhBMP-2/ACS were searched. Studies meeting the following criteria were considered eligible for inclusion: >10 subjects at baseline and maxillary sinus or alveolar ridge augmentation not concomitant with implant placement. Seven of 69 publications were eligible for review. rhBMP-2/ACS yielded clinically meaningful bone formation for maxillary sinus augmentation that would allow placement of regular dental implants without consistent differences between rhBMP-2 concentrations. Nevertheless, the statistical analysis showed that sinus augmentation following autogenous bone graft was significantly greater (mean bone height: 1.6 mm, 95% CI: 0.5-2.7 mm) than for rhBMP-2/ACS (rhBMP-2 at 1.5 mg/mL). In extraction sockets, rhBMP-2/ACS maintained alveolar ridge height while enhancing alveolar ridge width. Safety reports did not represent concerns for the proposed indications. rhBMP-2/ACS appears a promising alternative to autogenous bone grafts for alveolar ridge/maxillary sinus augmentation; dose and carrier optimization may expand its efficacy, use, and clinical application. © 2013 Wiley Periodicals, Inc.

Bone morphogenetic protein 9 as a key regulator of liver progenitor cells in DDC-induced cholestatic liver injury.

Science.gov (United States)

Addante, Annalisa; Roncero, Cesáreo; Almalé, Laura; Lazcanoiturburu, Nerea; García-Álvaro, María; Fernández, Margarita; Sanz, Julián; Hammad, Seddik; Nwosu, Zeribe C; Lee, Se-Jin; Fabregat, Isabel; Dooley, Steven; Ten Dijke, Peter; Herrera, Blanca; Sánchez, Aránzazu

2018-05-11

Bone morphogenetic protein 9 (BMP9) interferes with liver regeneration upon acute injury, while promoting fibrosis upon carbon tetrachloride-induced chronic injury. We have now addressed the role of BMP9 in 3,5 diethoxicarbonyl-1,4 dihydrocollidine (DDC)-induced cholestatic liver injury, a model of liver regeneration mediated by hepatic progenitor cell (known as oval cell), exemplified as ductular reaction and oval cell expansion. WT and BMP9KO mice were submitted to DDC diet. Livers were examined for liver injury, fibrosis, inflammation and oval cell expansion by serum biochemistry, histology, RT-qPCR and western blot. BMP9 signalling and effects in oval cells were studied in vitro using western blot and transcriptional assays, plus functional assays of DNA synthesis, cell viability and apoptosis. Crosslinking assays and short hairpin RNA approaches were used to identify the receptors mediating BMP9 effects. Deletion of BMP9 reduces liver damage and fibrosis, but enhances inflammation upon DDC feeding. Molecularly, absence of BMP9 results in overactivation of PI3K/AKT, ERK-MAPKs and c-Met signalling pathways, which together with an enhanced ductular reaction and oval cell expansion evidence an improved regenerative response and decreased damage in response to DDC feeding. Importantly, BMP9 directly targets oval cells, it activates SMAD1,5,8, decreases cell growth and promotes apoptosis, effects that are mediated by Activin Receptor-Like Kinase 2 (ALK2) type I receptor. We identify BMP9 as a negative regulator of oval cell expansion in cholestatic injury, its deletion enhancing liver regeneration. Likewise, our work further supports BMP9 as an attractive therapeutic target for chronic liver diseases. © 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Importance of a diffusion-dominant small volume to activate cell-secreted soluble factor signaling in embryonic stem cell culture in microbioreactors: a mathematical model based study.

Science.gov (United States)

Chowdhury, Mohammad Mahfuz; Fujii, Teruo; Sakai, Yasuyuki

2013-07-01

In our previous studies, we observed that cell-secreted BMP4 had a prominent influence on mouse embryonic stem cell (mESC) behaviors in a membrane-based two-chambered microbioreactor (MB), but not in a macro-scale culture (6-well plate/6WP). In this study, we investigated how the physical aspects of these cultures regulated BMP4 signaling by developing mathematical models of the cultures. The models estimated signaling activity in the cultures by considering size of the undifferentiated mESC colonies and their growth, diffusion of BMP4, and BMP4 trafficking process in the colonies. The models successfully depicted measured profile of BMP4 concentration in the culture medium which was two times higher in the MB than that in the 6WP during 5-day culture. The models estimated that, owing to the small volume and the membrane, cells were exposed to a higher BMP4 concentration in the top chamber of the MB than that in the 6WP culture. The higher concentration of BMP4 induced a higher concentration of BMP4-bound receptor in the colony in the MB than in the 6WP, thereby leading to the higher activation of BMP4 signaling in the MB. The models also predicted that the size of the MB, but not that of the 6WP, was suitable for maximizing BMP4 accumulation and upregulating its signaling. This study will be helpful in analyzing culture systems, designing microfluidic devices for controlling ESC or other cell behavior. Copyright © 2013 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Wnt/β-Catenin Signaling Defines Organizing Centers that Orchestrate Growth and Differentiation of the Regenerating Zebrafish Caudal Fin

Directory of Open Access Journals (Sweden)

Daniel Wehner

2014-02-01

Full Text Available Zebrafish regenerate their fins via the formation of a population of progenitor cells, the blastema. Wnt/β-catenin signaling is essential for blastemal cell proliferation and patterning of the overlying epidermis. Yet, we find that β-catenin signaling is neither active in the epidermis nor the majority of the proliferative blastemal cells. Rather, tissue-specific pathway interference indicates that Wnt signaling in the nonproliferative distal blastema is required for cell proliferation in the proximal blastema, and signaling in cells lining the osteoblasts directs osteoblast differentiation. Thus, Wnt signaling regulates epidermal patterning, blastemal cell proliferation, and osteoblast maturation indirectly via secondary signals. Gene expression profiling, chromatin immunoprecipitation, and functional rescue experiments suggest that Wnt/β-catenin signaling acts through Fgf and Bmp signaling to control epidermal patterning, whereas retinoic acid and Hedgehog signals mediate its effects on blastemal cell proliferation. We propose that Wnt signaling orchestrates fin regeneration by defining organizing centers that instruct cellular behaviors of adjacent tissues.

BMP4 density gradient in disk-shaped confinement

Science.gov (United States)

Bozorgui, Behnaz; Teimouri, Hamid; Kolomeisky, Anatoly B.

We present a quantitative model that explains the scaling of BMP4 gradients during gastrulation and the recent experimental observation that geometric confinement of human embryonic stem cells is sufficient to recapitulate much of germ layer patterning. Based on a assumption that BMP4 diffusion rate is much smaller than the diffusion rate of it's inhibitor molecules, our results confirm that the length-scale which defines germ layer territories does not depend on system size.

Cytoskeletal Reorganization Drives Mesenchymal Condensation and Regulates Downstream Molecular Signaling.

Directory of Open Access Journals (Sweden)

Poulomi Ray

Full Text Available Skeletal condensation occurs when specified mesenchyme cells self-organize over several days to form a distinctive cartilage template. Here, we determine how and when specified mesenchyme cells integrate mechanical and molecular information from their environment, forming cartilage condensations in the pharyngeal arches of chick embryos. By disrupting cytoskeletal reorganization, we demonstrate that dynamic cell shape changes drive condensation and modulate the response of the condensing cells to Fibroblast Growth Factor (FGF, Bone Morphogenetic Protein (BMP and Transforming Growth Factor beta (TGF-β signaling pathways. Rho Kinase (ROCK-driven actomyosin contractions and Myosin II-generated differential cell cortex tension regulate these cell shape changes. Disruption of the condensation process inhibits the differentiation of the mesenchyme cells into chondrocytes, demonstrating that condensation regulates the fate of the mesenchyme cells. We also find that dorsal and ventral condensations undergo distinct cell shape changes. BMP signaling is instructive for dorsal condensation-specific cell shape changes. Moreover, condensations exhibit ventral characteristics in the absence of BMP signaling, suggesting that in the pharyngeal arches ventral morphology is the ground pattern. Overall, this study characterizes the interplay between cytoskeletal dynamics and molecular signaling in a self-organizing system during tissue morphogenesis.

Curcumin induces osteoblast differentiation through mild-endoplasmic reticulum stress-mediated such as BMP2 on osteoblast cells.

Science.gov (United States)

Son, Hyo-Eun; Kim, Eun-Jung; Jang, Won-Gu

2018-01-15

Curcumin (diferuloylmethane or [1E,6E]-1,7-bis[4-hydroxy-3-methoxyphenyl]-1,6heptadiene-3,5-dione) is a phenolic natural product derived from the rhizomes of the turmeric plant, Curcuma longa. It is reported to have various biological actions such as anti-oxidative, anti-inflammatory, and anti-cancer effects. However, the molecular mechanism of osteoblast differentiation by curcumin has not yet been reported. The cytotoxicity of curcumin was identified using the 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Expression of osteogenic markers and endoplasmic reticulum (ER) stress markers in C3H1-T1/2 cells were measured using reverse-transcriptase polymerase chain reaction (RT-PCR) and Western blotting. Alkaline phosphatase (ALP) staining was performed to assess ALP activity in C3H10T1/2 cells. Transcriptional activity was detected using a luciferase reporter assay. Curcumin increased the expression of genes such as distal-less homeobox 5 (Dlx5), runt-related transcription factor 2 (Runx2), ALP, and osteocalcin (OC), which subsequently induced osteoblast differentiation in C3H10T1/2 cells. In addition, ALP activity and mineralization was found to be increased by curcumin treatment. Curcumin also induced mild ER stress similar to bone morphogenetic protein 2 (BMP2) function in osteoblast cells. Next, we confirmed that curcumin increased mild ER stress and osteoblast differentiation similar to BMP2 in C3H10T1/2 mesenchymal stem cells. Transient transfection studies also showed that curcumin increased ATF6-Luc activity, while decreasing the activities of CREBH-Luc and SMILE-Luc. In addition, similar to BMP2, curcumin induced the phosphorylation of Smad 1/5/9. Overall, these results demonstrate that curcumin-induced mild ER stress increases osteoblast differentiation via ATF6 expression in C3H10T1/2 cells. Copyright © 2017. Published by Elsevier Inc.

Thyroid hormone-induced hypertrophy in mesenchymal stem cell chondrogenesis is mediated by bone morphogenetic protein-4.

Science.gov (United States)

Karl, Alexandra; Olbrich, Norman; Pfeifer, Christian; Berner, Arne; Zellner, Johannes; Kujat, Richard; Angele, Peter; Nerlich, Michael; Mueller, Michael B

2014-01-01

Chondrogenic differentiating mesenchymal stem cells (MSCs) express markers of hypertrophic growth plate chondrocytes. As hypertrophic cartilage undergoes ossification, this is a concern for the application of MSCs in articular cartilage tissue engineering. To identify mechanisms that elicit this phenomenon, we used an in vitro hypertrophy model of chondrifying MSCs for differential gene expression analysis and functional experiments with the focus on bone morphogenetic protein (BMP) signaling. Hypertrophy was induced in chondrogenic MSC pellet cultures by transforming growth factor β (TGFβ) and dexamethasone withdrawal and addition of triiodothyronine. Differential gene expression analysis of BMPs and their receptors was performed. Based on these results, the in vitro hypertrophy model was used to investigate the effect of recombinant BMP4 and the BMP inhibitor Noggin. The enhancement of hypertrophy could be shown clearly by an increased cell size, alkaline phosphatase activity, and collagen type X deposition. Upon induction of hypertrophy, BMP4 and the BMP receptor 1B were upregulated. Addition of BMP4 further enhanced hypertrophy in the absence, but not in the presence of TGFβ and dexamethasone. Thyroid hormone induced hypertrophy by upregulation of BMP4 and this induced enhancement of hypertrophy could be blocked by the BMP antagonist Noggin. BMP signaling is an important modulator of the late differentiation stages in MSC chondrogenesis and the thyroid hormone induces this pathway. As cartilage tissue engineering constructs will be exposed to this factor in vivo, this study provides important insight into the biology of MSC-based cartilage. Furthermore, the possibility to engineer hypertrophic cartilage may be helpful for critical bone defect repair.

Expression and functional study of extracellular BMP antagonists during the morphogenesis of the digits and their associated connective tissues.

Directory of Open Access Journals (Sweden)

Carlos I Lorda-Diez

Full Text Available The purpose of this study is to gain insight into the role of BMP signaling in the diversification of the embryonic limb mesodermal progenitors destined to form cartilage, joints, and tendons. Given the importance of extracellular BMP modulators in in vivo systems, we performed a systematic search of those expressed in the developing autopod during the formation of the digits. Here, we monitored the expression of extracellular BMP modulators including: Noggin, Chordin, Chordin-like 1, Chordin-like 2, Twisted gastrulation, Dan, BMPER, Sost, Sostdc1, Follistatin, Follistatin-like 1, Follistatin-like 5 and Tolloid. These factors show differential expression domains in cartilage, joints and tendons. Furthermore, they are induced in specific temporal patterns during the formation of an ectopic extra digit, preceding the appearance of changes that are identifiable by conventional histology. The analysis of gene regulation, cell proliferation and cell death that are induced by these factors in high density cultures of digit progenitors provides evidence of functional specialization in the control of mesodermal differentiation but not in cell proliferation or apoptosis. We further show that the expression of these factors is differentially controlled by the distinct signaling pathways acting in the developing limb at the stages covered by this study. In addition, our results provide evidence suggesting that TWISTED GASTRULATION cooperates with CHORDINS, BMPER, and NOGGIN in the establishment of tendons or cartilage in a fashion that is dependent on the presence or absence of TOLLOID.

Dexamethasone, BMP-2, and 1,25-dihydroxyvitamin D enhance a more differentiated osteoblast phenotype

DEFF Research Database (Denmark)

Jørgensen, Niklas Rye; Henriksen, Z; Sørensen, O H

2004-01-01

. Osteoblast phenotypes were induced by either dexamethasone (Dex) or bone morphogenetic protein-2 (BMP-2). Bone marrow was obtained from biopsies at the posterior iliac spine. Cells were isolated by gradient centrifugation and grown to confluence. Cells were treated with 1 nM 1,25-dihydroxyvitamin D (vitamin...... activity was increased by Dex, but not by BMP-2 treatment. P1NP production was decreased after Dex treatment, while BMP-2 had no effect on P1NP levels. Osteocalcin production was low in cultures not stimulated with vitamin D. Dex or BMP-2 treatment alone did not affect the basic osteocalcin levels......, but in combination with vitamin D, BMP-2 increased the osteocalcin production, while Dex treatment completely suppressed osteocalcin production. Further, PTH-induced cAMP production was greatly enhanced by Dex treatment, whereas BMP-2 did not affect cAMP production. Finally, in vitro mineralization was greatly...

Gremlin-1 induces BMP-independent tumor cell proliferation, migration, and invasion.

Directory of Open Access Journals (Sweden)

Minsoo Kim

Full Text Available Gremlin-1, a bone morphogenetic protein (BMP antagonist, is overexpressed in various cancerous tissues but its role in carcinogenesis has not been established. Here, we report that gremlin-1 binds various cancer cell lines and this interaction is inhibited by our newly developed gremlin-1 antibody, GRE1. Gremlin-1 binding to cancer cells was unaffected by the presence of BMP-2, BMP-4, and BMP-7. In addition, the binding was independent of vascular endothelial growth factor receptor-2 (VEGFR2 expression on the cell surface. Addition of gremlin-1 to A549 cells induced a fibroblast-like morphology and decreased E-cadherin expression. In a scratch wound healing assay, A549 cells incubated with gremlin-1 or transfected with gremlin-1 showed increased migration, which was inhibited in the presence of the GRE1 antibody. Gremlin-1 transfected A549 cells also exhibited increased invasiveness as well as an increased growth rate. These effects were also inhibited by the addition of the GRE1 antibody. In conclusion, this study demonstrates that gremlin-1 directly interacts with cancer cells in a BMP- and VEGFR2-independent manner and can induce cell migration, invasion, and proliferation.

Image Size Variation Influence on Corrupted and Non-viewable BMP Image

Science.gov (United States)

Azmi, Tengku Norsuhaila T.; Azma Abdullah, Nurul; Rahman, Nurul Hidayah Ab; Hamid, Isredza Rahmi A.; Chai Wen, Chuah

2017-08-01

Image is one of the evidence component seek in digital forensics. Joint Photographic Experts Group (JPEG) format is most popular used in the Internet because JPEG files are very lossy and easy to compress that can speed up Internet transmitting processes. However, corrupted JPEG images are hard to recover due to the complexities of determining corruption point. Nowadays Bitmap (BMP) images are preferred in image processing compared to another formats because BMP image contain all the image information in a simple format. Therefore, in order to investigate the corruption point in JPEG, the file is required to be converted into BMP format. Nevertheless, there are many things that can influence the corrupting of BMP image such as the changes of image size that make the file non-viewable. In this paper, the experiment indicates that the size of BMP file influences the changes in the image itself through three conditions, deleting, replacing and insertion. From the experiment, we learnt by correcting the file size, it can able to produce a viewable file though partially. Then, it can be investigated further to identify the corruption point.

DMPD: Signalling pathways mediating type I interferon gene expression. [Dynamic Macrophage Pathway CSML Database

Lifescience Database Archive (English)

Full Text Available 17904888 Signalling pathways mediating type I interferon gene expression. Edwards M...hways mediating type I interferon gene expression. PubmedID 17904888 Title Signalling pathways...R, Slater L, Johnston SL. Microbes Infect. 2007 Sep;9(11):1245-51. Epub 2007 Jul 1. (.png) (.svg) (.html) (.csml) Show Signalling pat

Meeting report - TGF-β superfamily: signaling in development and disease.

Science.gov (United States)

Zhang, Ying E; Newfeld, Stuart J

2013-11-01

The latest advances on the transforming growth factor β (TGF-β) and bone morphogenetic protein (BMP) signaling pathways were reported at the July 2013 FASEB Summer Research Conference 'The TGF-β Superfamily: Development and Disease'. The meeting was held in Steamboat Springs, Colorado, USA at 6700 feet above sea level in the Rocky Mountains. This was the seventh biannual meeting in the series. In attendance were investigators from a broad range of disciplines with a common interest in the mechanics of TGF-β and BMP signaling pathways, their normal developmental and homeostatic functions, and the diseases associated with pathway misregulation.

Plant cell surface receptor-mediated signaling - a common theme amid diversity.

Science.gov (United States)

He, Yunxia; Zhou, Jinggeng; Shan, Libo; Meng, Xiangzong

2018-01-29

Sessile plants employ a diverse array of plasma membrane-bound receptors to perceive endogenous and exogenous signals for regulation of plant growth, development and immunity. These cell surface receptors include receptor-like kinases (RLKs) and receptor-like proteins (RLPs) that harbor different extracellular domains for perception of distinct ligands. Several RLK and RLP signaling pathways converge at the somatic embryogenesis receptor kinases (SERKs), which function as shared co-receptors. A repertoire of receptor-like cytoplasmic kinases (RLCKs) associate with the receptor complexes to relay intracellular signaling. Downstream of the receptor complexes, mitogen-activated protein kinase (MAPK) cascades are among the key signaling modules at which the signals converge, and these cascades regulate diverse cellular and physiological responses through phosphorylation of different downstream substrates. In this Review, we summarize the emerging common theme that underlies cell surface receptor-mediated signaling pathways in Arabidopsis thaliana : the dynamic association of RLKs and RLPs with specific co-receptors and RLCKs for signal transduction. We further discuss how signaling specificities are maintained through modules at which signals converge, with a focus on SERK-mediated receptor signaling. © 2018. Published by The Company of Biologists Ltd.

Risk variants in BMP4 promoters for nonsyndromic cleft lip/palate in a Chilean population

Directory of Open Access Journals (Sweden)

Suazo José

2011-12-01

Full Text Available Abstract Background Bone morphogenetic protein 4 gene (BMP4 plays a key role during maxillofacial development, since orofacial clefts are observed in animals when this gene is conditionally inactivated. We recently reported the existence of association between nonsyndromic cleft lip/palate (NSCLP and BMP4 polymorphisms by detecting transmission deviations for haplotypes that include a region containing a BMP4 promoter in case-parent trios. The aim of the present study was to search for possible causal mutations within BMP4 promoters (BMP4.1 and BMP4.2. Methods We analyzed the sequence of BMP4.1 and BMP4.2 in 167 Chilean NSCLP cases and 336 controls. Results We detected three novel variants in BMP4.1 (c.-5514G > A, c.-5365C > T and c.-5049C > T which could be considered as cleft risk factors due to their absence in controls. Additionally, rs2855530 G allele (BMP4.2 carriers showed an increased risk for NSCLP restricted to males (OR = 1.52; 95% C.I. = 1.07-2.15; p = 0.019. For this same SNP the dominant genotype model showed a higher frequency of G/G+G/C and a lower frequency of C/C in cases than controls in the total sample (p = 0.03 and in the male sample (p = 0.003. Bioinformatic prediction analysis showed that all the risk variants detected in this study could create new transcription factor binding motifs. Conclusions The sex-dependent association between rs2855530 and NSCLP could indirectly be related to the differential gene expression observed between sexes in animal models. We concluded that risk variants detected herein could potentially alter BMP4 promoter activity in NSCLP. Further functional and developmental studies are necessary to support this hypothesis.

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

DEFF Research Database (Denmark)

Hansson, Mattias; Petersen, Dorthe Rønn; 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...... is found at the lowest activin concentration. The expression of Gsc and other anterior markers induced by activin is prevented by treatment with BMP4, which induces T expression and subsequent mesodermal development. We show that canonical Wnt signaling is required only during late stages of activin....... 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...

Id4 functions downstream of Bmp signaling to restrict TCF function in endocardial cells during atrioventricular valve development.

Science.gov (United States)

Ahuja, Suchit; Dogra, Deepika; Stainier, Didier Y R; Reischauer, Sven

2016-04-01

The atrioventricular canal (AVC) connects the atrial and ventricular chambers of the heart and its formation is critical for the development of the cardiac valves, chamber septation and formation of the cardiac conduction system. Consequently, problems in AVC formation can lead to congenital defects ranging from cardiac arrhythmia to incomplete cardiac septation. While our knowledge about early heart tube formation is relatively comprehensive, much remains to be investigated about the genes that regulate AVC formation. Here we identify a new role for the basic helix-loop-helix factor Id4 in zebrafish AVC valve development and function. id4 is first expressed in the AVC endocardium and later becomes more highly expressed in the atrial chamber. TALEN induced inactivation of id4 causes retrograde blood flow at the AV canal under heat induced stress conditions, indicating defects in AV valve function. At the molecular level, we found that id4 inactivation causes misexpression of several genes important for AVC and AV valve formation including bmp4 and spp1. We further show that id4 appears to control the number of endocardial cells that contribute to the AV valves by regulating Wnt signaling in the developing AVC endocardium. Copyright © 2016 Elsevier Inc. All rights reserved.

Low-dose rhBMP2/7 heterodimer to reconstruct peri-implant bone defects: a micro-CT evaluation.

Science.gov (United States)

Wang, Jingxiao; Zheng, Yuanna; Zhao, Juan; Liu, Tie; Gao, Lixia; Gu, Zhiyuan; Wu, Gang

2012-01-01

To delineate the dynamic micro-architectures of bone induced by low-dose bone morphogenetic protein (BMP)-2/7 heterodimer in peri-implant bone defects compared to BMP2 and BMP7 homodimer. Peri-implant bone defects (8 mm in diameter, 4 mm in depth) were created surrounding SLA-treated titanium implants (3.1 mm in diameter, 10 mm in length) in minipig's calvaria. We administrated collagen sponges with adsorbed low-dose (30 ng/mm(3) ) BMP2/7 to treat the defects using BMP2, BMP7 or no BMP as controls.2, 3 and 6 weeks after implantation, we adopted micro-computer tomography to evaluate the micro-architectures of new bone using the following parameters: relative bone volume (BV/TV), trabecular number (Tb.N), trabecular thickness (Tb.Th), trabecular separation (Tb.Sp), connectivity density, and structure mode index (SMI). Bone implant contact (BIC) was also revealed histologically. Consistent with 2 and 3 weeks, after 6 weeks post-operation, BMP2/7 resulted in significantly higher BV/TV (63.033 ± 2.055%) and significantly lower SMI (-4.405 ± 0.500) than BMP2 (BV/TV: 43.133 ± 2.001%; SMI: -0.086 ± 0.041) and BMP7 (BV/TV: 41.467 ± 1.850%; SMI: -0.044 ± 0.016) respectively. Significant differences were also found in Tb.N, Tb.Th and Tb.Sp at all time points. At 2 weeks, BMP2/7 resulted in significantly higher BIC than the controls. Low-dose BMP2/7 heterodimer facilitated more rapid bone regeneration in better quality in peri-implant bone defects than BMP2 and BMP7 homodimers. © 2011 John Wiley & Sons A/S.

BMP antagonism by Noggin is required in presumptive notochord cells for mammalian foregut morphogenesis.

Science.gov (United States)

Fausett, Sarah R; Brunet, Lisa J; Klingensmith, John

2014-07-01

Esophageal atresia with tracheoesophageal fistula (EA/TEF) is a serious human birth defect, in which the esophagus ends before reaching the stomach, and is aberrantly connected with the trachea. Several mouse models of EA/TEF have recently demonstrated that proper dorsal/ventral (D/V) patterning of the primitive anterior foregut endoderm is essential for correct compartmentalization of the trachea and esophagus. Here we elucidate the pathogenic mechanisms underlying the EA/TEF that occurs in mice lacking the BMP antagonist Noggin, which display correct dorsal/ventral patterning. To clarify the mechanism of this malformation, we use spatiotemporal manipulation of Noggin and BMP receptor 1A conditional alleles during foregut development. Surprisingly, we find that the expression of Noggin in the compartmentalizing endoderm is not required to generate distinct tracheal and esophageal tubes. Instead, we show that Noggin and BMP signaling attenuation are required in the early notochord to correctly resolve notochord cells from the dorsal foregut endoderm, which in turn, appears to be a prerequisite for foregut compartmentalization. Collectively, our findings support an emerging model for a mechanism underlying EA/TEF in which impaired notochord resolution from the early endoderm causes the foregut to be hypo-cellular just prior to the critical period of compartmentalization. Our further characterizations suggest that Noggin may regulate a cell rearrangement process that involves reciprocal E-cadherin and Zeb1 expression in the resolving notochord cells. Copyright © 2014. Published by Elsevier Inc.

Designer TGFβ superfamily ligands with diversified functionality.

Directory of Open Access Journals (Sweden)

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.

Comparison of newly developed anti-bone morphogenetic protein 4 llama-derived antibodies with commercially available BMP4 inhibitors.

Science.gov (United States)

Calpe, Silvia; Correia, Ana C P; Sancho-Serra, Maria Del Carmen; Krishnadath, Kausilia K

2016-01-01

Due to improved understanding of the role of bone morphogenetic protein 4 (BMP4) in an increasing number of diseases, the development of selective inhibitors of BMP4 is an attractive therapeutic option. The currently available BMP4 inhibitors are not suitable as therapeutics because of their low specificity and low effectiveness. Here, we compared newly generated anti-BMP4 llama-derived antibodies (VHHs) with 3 different types of commercially available BMP4 inhibitors, natural antagonists, small molecule BMPR inhibitors and conventional anti-BMP4 monoclonal antibodies. We found that the anti-BMP4 VHHs were as effective as the natural antagonist or small molecule inhibitors, but had higher specificity. We also showed that commercial anti-BMP4 antibodies were inferior in terms of both specificity and effectiveness. These findings might result from the fact that the VHHs C4C4 and C8C8 target a small region within the BMPR1 epitope of BMP4, whereas the commercial antibodies target other areas of the BMP4 molecule. Our results show that the newly developed anti-BMP4 VHHs are promising antibodies with better specificity and effectivity for inhibition of BMP4, making them an attractive tool for research and for therapeutic applications.

Vitamin C-linker-conjugated tripeptide AHK stimulates BMP-2-induced osteogenic differentiation of mouse myoblast C2C12 cells.

Science.gov (United States)

Jung, Jung-Il; Park, Kyeong-Yong; Lee, Yura; Park, Mira; Kim, Jiyeon

2018-03-15

Vitamin C-linker-conjugated Ala-His-Lys tripeptide (Vit C-AHK) is a derivative of Vitamin C-conjugated tripeptides, which were originally developed as a component of a product for collagen synthesis enhancement or human dermal fibroblast growth. Here, we investigated the effect of Vit C-AHK on bone morphogenetic protein (BMP)-2-induced osteoblast differentiation in a cell culture model. Vit C-AHK enhanced proliferation of C2C12 cells and induction of BMP-2-induced alkaline phosphatase, a typical marker of osteoblast differentiation. Vit C-AHK also stimulated the phosphorylation and translocation of Smad1/5/8 to the nucleus and phosphorylation of mitogen-activated protein kinases (MAPKs) including ERK1/2 and p38. In addition, Vit C-AHK enhanced the BMP-2-induced mRNA expression of osteoblast differentiation-related genes such as ALP, BMP-2, Osteocalcin, and Runx2. Our results suggest that Vit C-AHK exerts an enhancing effect on osteoblast proliferation and differentiation through activation of Smad1/5/8 and MAPK ERK1/2 and p38 signaling and without significant cytotoxicity. These results provide important data for the development of peptide-based bone-regenerative agents and treatment of bone-related disorders. Copyright © 2018 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.

Site-Directed Immobilization of BMP-2: Two Approaches for the Production of Innovative Osteoinductive Scaffolds.

Science.gov (United States)

Tabisz, Barbara; Schmitz, Werner; Schmitz, Michael; Luehmann, Tessa; Heusler, Eva; Rybak, Jens-Christoph; Meinel, Lorenz; Fiebig, Juliane E; Mueller, Thomas D; Nickel, Joachim

2017-03-13

The regenerative potential of bone is strongly impaired in pathological conditions, such as nonunion fractures. To support bone regeneration various scaffolds have been developed in the past, which have been functionalized with osteogenic growth factors such as bone morphogenetic proteins (BMPs). However, most of them required supra-physiological levels of these proteins leading to burst releases, thereby causing severe side effects. Site-specific, covalent coupling of BMP2 to implant materials might be an optimal strategy in order to overcome these problems. Therefore, we created a BMP-2 variant (BMP2-K3Plk) containing a noncanonical amino acid (propargyl-l-lysine) substitution introduced by genetic code expansion that allows for site-specific and covalent immobilization onto polymeric scaffold materials. To directly compare different coupling strategies, we also produced a BMP2 variant containing an additional cysteine residue (BMP2-A2C) allowing covalent coupling by thioether formation. The BMP2-K3Plk mutant was coupled to functionalized beads by a copper-catalyzed azide-alkyne cycloaddition (CuAAC) either directly or via a short biotin-PEG linker both with high specificity. After exposing the BMP-coated beads to C2C12 cells, ALP expression appeared locally restricted in close proximity to these beads, showing that both coupled BMP2 variants trigger cell differentiation. The advantage of our approach over non-site-directed immobilization techniques is the ability to produce fully defined osteogenic surfaces, allowing for lower BMP2 loads and concomitant higher bioactivities, for example, due to controlled orientation toward BMP2 receptors. Such products might provide superior bone healing capabilities with potential safety advantages as of homogeneous product outcome.

Combination of Mineral Trioxide Aggregate and Platelet-rich Fibrin Promotes the Odontoblastic Differentiation and Mineralization of Human Dental Pulp Cells via BMP/Smad Signaling Pathway.

Science.gov (United States)

Woo, Su-Mi; Kim, Won-Jae; Lim, Hae-Soon; Choi, Nam-Ki; Kim, Sun-Hun; Kim, Seon-Mi; Jung, Ji-Yeon

2016-01-01

Recent reports have shown that the combined use of platelet-rich fibrin (PRF), an autologous fibrin matrix, and mineral trioxide aggregate (MTA) as root filling material is beneficial for the endodontic management of an open apex. However, the potential of the combination of MTA and PRF as an odontogenic inducer in human dental pulp cells (HDPCs) in vitro has not yet been studied. The purpose of this study was to evaluate the effect of the combination of MTA and PRF on odontoblastic maturation in HDPCs. HDPCs extracted from third molars were directly cultured with MTA and PRF extract (PRFe). Odontoblastic differentiation of HDPCs was evaluated by measuring the alkaline phosphatase (ALP) activity, and the expression of odontogenesis-related genes was detected using reverse-transcription polymerase chain reaction or Western blot. Mineralization formation was assessed by alizarin red staining. HDPCs treated with MTA and PRFe significantly up-regulated the expression of dentin sialoprotein and dentin matrix protein-1 and enhanced ALP activity and mineralization compared with those with MTA or PRFe treatment alone. In addition, the combination of MTA and PRFe induced the activation of bone morphogenic proteins (BMP)/Smad, whereas LDN193189, the bone morphogenic protein inhibitor, attenuated dentin sialophosphoprotein and dentin matrix protein-1 expression, ALP activity, and mineralization enhanced by MTA and PRFe treatment. This study shows that the combination of MTA and PRF has a synergistic effect on the stimulation of odontoblastic differentiation of HDPCs via the modulation of the BMP/Smad signaling pathway. Copyright © 2016 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

Bone morphogenetic protein-2 (BMP-2 and transforming growth factor-β1 (TGF-β1 alter connexin 43 phosphorylation in MC3T3-E1 Cells

Directory of Open Access Journals (Sweden)

Rudkin George H

2001-07-01

Full Text Available Abstract Background Bone morphogenetic proteins (BMPs and transforming growth factor-βs (TGF-βs are important regulators of bone repair and regeneration. BMP-2 and TGF-β1 have been shown to inhibit gap junctional intercellular communication (GJIC in MC3T3-E1 cells. Connexin 43 (Cx43 has been shown to mediate GJIC in osteoblasts and it is the predominant gap junctional protein expressed in these murine osteoblast-like cells. We examined the expression, phosphorylation, and subcellular localization of Cx43 after treatment with BMP-2 or TGF-β1 to investigate a possible mechanism for the inhibition of GJIC. Results Northern blot analysis revealed no detectable change in the expression of Cx43 mRNA. Western blot analysis demonstrated no significant change in the expression of total Cx43 protein. However, significantly higher ratios of unphosphorylated vs. phosphorylated forms of Cx43 were detected after BMP-2 or TGF-β1 treatment. Immunofluorescence and cell protein fractionation revealed no detectable change in the localization of Cx43 between the cytosol and plasma membrane. Conclusions BMP-2 and TGF-β1 do not alter expression of Cx43 at the mRNA or protein level. BMP-2 and TGF-β1 may inhibit GJIC by decreasing the phosphorylated form of Cx43 in MC3T3-E1 cells.

BMP9-Induced Osteogenetic Differentiation and Bone Formation of Muscle-Derived Stem Cells

Directory of Open Access Journals (Sweden)

Li Xiang

2012-01-01

Full Text Available Efficient osteogenetic differentiation and bone formation from muscle-derived stem cells (MDSCs should have potential clinical applications in treating nonunion fracture healing or bone defects. Here, we investigate osteogenetic differentiation ability of MDSCs induced by bone morphogenetic protein 9 (BMP9 in vitro and bone formation ability in rabbit radius defects repairing model. Rabbit's MDSCs were extracted by type I collagenase and trypsin methods, and BMP9 was introduced into MDSCs by infection with recombinant adenovirus. Effects of BMP9-induced osteogenetic differentiation of MDSCs were identified with alkaline phosphatase (ALP activity and expression of later marker. In stem-cell implantation assay, MDSCs have also shown valuable potential bone formation ability induced by BMP9 in rabbit radius defects repairing test. Taken together, our findings suggest that MDSCs are potentiated osteogenetic stem cells which can be induced by BMP9 to treat large segmental bone defects, nonunion fracture, and/or osteoporotic fracture.

Insulin-like growth factor-1 (IGF-1) enhances the osteogenic activity of bone morphogenetic protein-6 (BMP-6) in vitro and in vivo, and together have a stronger osteogenic effect than when IGF-1 is combined with BMP-2.

Science.gov (United States)

Rico-Llanos, Gustavo A; Becerra, Jose; Visser, Rick

2017-07-01

Bone morphogenetic protein-2 (BMP-2) is widely used in orthopedic surgery and bone tissue engineering because of its strong osteogenic activity. However, BMP-2 treatments have several drawbacks and many groups are actively exploring alternatives. Since BMP-6 has been demonstrated to be more osteoinductive, its use, either alone or together with other growth factors, might be an interesting option. In this work, we have compared the effect of BMP-2, BMP-6, or insulin-like growth factor-1 (IGF-1), either alone or in combination. Murine preosteoblasts were treated with 15 nM IGF-1 and/or 6 nM BMP-2 or -6 and the expression of osteogenic marker genes, proliferation, and alkaline phosphatase (ALP) activity in vitro were analyzed. The results showed that IGF-1 greatly enhanced the BMP-induced osteogenic differentiation of these cells in general and that the ALP activity in the cultures was higher when the combination was made with BMP-6 than with BMP-2. Furthermore, we tested the osteogenic potential of these treatments in vivo by loading 25 pmoles of IGF-1 and/or 10 pmoles of BMP-2 or -6 onto absorbable collagen sponges and implanting them into an ectopic bone formation model in rats. This study revealed that only BMP-6 was able to induce bone formation at the used dose and that the addition of IGF-1 contributed to an increase of the mineralization in the implants. Hence, the combination of BMP-6 with IGF-1 might be a better alternative than BMP-2 for orthopedic surgery or bone tissue engineering approaches. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 1867-1875, 2017. © 2017 Wiley Periodicals, Inc.

The mediator complex in genomic and non-genomic signaling in cancer.

Science.gov (United States)

Weber, Hannah; Garabedian, Michael J

2018-05-01

Mediator is a conserved, multi-subunit macromolecular machine divided structurally into head, middle, and tail modules, along with a transiently associating kinase module. Mediator functions as an integrator of transcriptional regulatory activity by interacting with DNA-bound transcription factors and with RNA polymerase II (Pol II) to both activate and repress gene expression. Mediator has been shown to affect multiple steps in transcription, including chromatin looping between enhancers and promoters, pre-initiation complex formation, transcriptional elongation, and mRNA splicing. Individual Mediator subunits participate in regulation of gene expression by the estrogen and androgen receptors and are altered in a number of endocrine cancers, including breast and prostate cancer. In addition to its role in genomic signaling, MED12 has been implicated in non-genomic signaling by interacting with and activating TGF-beta receptor 2 in the cytoplasm. Recent structural studies have revealed extensive inter-domain interactions and complex architecture of the Mediator-Pol II complex, suggesting that Mediator is capable of reorganizing its conformation and composition to fit cellular needs. We propose that alterations in Mediator subunit expression that occur in various cancers could impact the organization and function of Mediator, resulting in changes in gene expression that promote malignancy. A better understanding of the role of Mediator in cancer could reveal new approaches to the diagnosis and treatment of Mediator-dependent endocrine cancers, especially in settings of therapy resistance. Copyright © 2017 Elsevier Inc. All rights reserved.

Regulation of Msx genes by a Bmp gradient is essential for neural crest specification.

Science.gov (United States)

Tribulo, Celeste; Aybar, Manuel J; Nguyen, Vu H; Mullins, Mary C; Mayor, Roberto

2003-12-01

There is evidence in Xenopus and zebrafish embryos that the neural crest/neural folds are specified at the border of the neural plate by a precise threshold concentration of a Bmp gradient. In order to understand the molecular mechanism by which a gradient of Bmp is able to specify the neural crest, we analyzed how the expression of Bmp targets, the Msx genes, is regulated and the role that Msx genes has in neural crest specification. As Msx genes are directly downstream of Bmp, we analyzed Msx gene expression after experimental modification in the level of Bmp activity by grafting a bead soaked with noggin into Xenopus embryos, by expressing in the ectoderm a dominant-negative Bmp4 or Bmp receptor in Xenopus and zebrafish embryos, and also through Bmp pathway component mutants in the zebrafish. All the results show that a reduction in the level of Bmp activity leads to an increase in the expression of Msx genes in the neural plate border. Interestingly, by reaching different levels of Bmp activity in animal cap ectoderm, we show that a specific concentration of Bmp induces msx1 expression to a level similar to that required to induce neural crest. Our results indicate that an intermediate level of Bmp activity specifies the expression of Msx genes in the neural fold region. In addition, we have analyzed the role that msx1 plays on neural crest specification. As msx1 has a role in dorsoventral pattering, we have carried out conditional gain- and loss-of-function experiments using different msx1 constructs fused to a glucocorticoid receptor element to avoid an early effect of this factor. We show that msx1 expression is able to induce all other early neural crest markers tested (snail, slug, foxd3) at the time of neural crest specification. Furthermore, the expression of a dominant negative of Msx genes leads to the inhibition of all the neural crest markers analyzed. It has been previously shown that snail is one of the earliest genes acting in the neural crest

Allergen-Induced Dermatitis Causes Alterations in Cutaneous Retinoid-Mediated Signaling in Mice

Science.gov (United States)

Gericke, Janine; Ittensohn, Jan; Mihály, Johanna; Dubrac, Sandrine; Rühl, Ralph

2013-01-01

Nuclear receptor-mediated signaling via RARs and PPARδ is involved in the regulation of skin homeostasis. Moreover, activation of both RAR and PPARδ was shown to alter skin inflammation. Endogenous all-trans retinoic acid (ATRA) can activate both receptors depending on specific transport proteins: Fabp5 initiates PPARδ signaling whereas Crabp2 promotes RAR signaling. Repetitive topical applications of ovalbumin (OVA) in combination with intraperitoneal injections of OVA or only intraperitoneal OVA applications were used to induce allergic dermatitis. In our mouse model, expression of IL-4, and Hbegf increased whereas expression of involucrin, Abca12 and Spink5 decreased in inflamed skin, demonstrating altered immune response and epidermal barrier homeostasis. Comprehensive gene expression analysis showed alterations of the cutaneous retinoid metabolism and retinoid-mediated signaling in allergic skin immune response. Notably, ATRA synthesis was increased as indicated by the elevated expression of retinaldehyde dehydrogenases and increased levels of ATRA. Consequently, the expression pattern of genes downstream to RAR was altered. Furthermore, the increased ratio of Fabp5 vs. Crabp2 may indicate an up-regulation of the PPARδ pathway in allergen-induced dermatitis in addition to the altered RAR signaling. Thus, our findings suggest that ATRA levels, RAR-mediated signaling and signaling involved in PPARδ pathways are mainly increased in allergen-induced dermatitis and may contribute to the development and/or maintenance of allergic skin diseases. PMID:23977003

Phenotypic variability in patients with osteogenesis imperfecta caused by BMP1 mutations.

Science.gov (United States)

Pollitt, Rebecca C; Saraff, Vrinda; Dalton, Ann; Webb, Emma A; Shaw, Nick J; Sobey, Glenda J; Mughal, M Zulf; Hobson, Emma; Ali, Farhan; Bishop, Nicholas J; Arundel, Paul; Högler, Wolfgang; Balasubramanian, Meena

2016-12-01

Osteogenesis Imperfecta (OI) is an inherited bone fragility disorder most commonly associated with autosomal dominant mutations in the type I collagen genes. Autosomal recessive mutations in a number of genes have also been described, including the BMP1 gene that encodes the mammalian Tolloid (mTLD) and its shorter isoform bone morphogenic protein-1 (BMP1). To date, less than 20 individuals with OI have been identified with BMP1 mutations, with skeletal phenotypes ranging from mild to severe and progressively deforming. In the majority of patients, bone fragility was associated with increased bone mineral density (BMD); however, the full range of phenotypes associated with BMP1 remains unclear. Here, we describe three children with mutations in BMP1 associated with a highly variable phenotype: a sibship homozygous for the c.2188delC mutation that affects only the shorter BMP1 isoform and a further patient who is compound heterozygous for a c.1293C>G nonsense mutation and a c.1148G>A missense mutation in the CUB1 domain. These individuals had recurrent fractures from early childhood, are hypermobile and have no evidence of dentinogenesis imperfecta. The homozygous siblings with OI had normal areal BMD by dual energy X-ray absorptiometry whereas the third patient presented with a high bone mass phenotype. Intravenous bisphosphonate therapy was started in all patients, but discontinued in two patients and reduced in another due to concerns about increasing bone stiffness leading to chalk-stick fractures. Given the association of BMP1-related OI with very high bone material density, concerns remain whether anti-resorptive therapy is indicated in this ultra-rare form of OI.© 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Key mediators of intracellular amino acids signaling to mTORC1 activation.

Science.gov (United States)

Duan, Yehui; Li, Fengna; Tan, Kunrong; Liu, Hongnan; Li, Yinghui; Liu, Yingying; Kong, Xiangfeng; Tang, Yulong; Wu, Guoyao; Yin, Yulong

2015-05-01

Mammalian target of rapamycin complex 1 (mTORC1) is activated by amino acids to promote cell growth via protein synthesis. Specifically, Ras-related guanosine triphosphatases (Rag GTPases) are activated by amino acids, and then translocate mTORC1 to the surface of late endosomes and lysosomes. Ras homolog enriched in brain (Rheb) resides on this surface and directly activates mTORC1. Apart from the presence of intracellular amino acids, Rag GTPases and Rheb, other mediators involved in intracellular amino acid signaling to mTORC1 activation include human vacuolar sorting protein-34 (hVps34) and mitogen-activating protein kinase kinase kinase kinase-3 (MAP4K3). Those molecular links between mTORC1 and its mediators form a complicate signaling network that controls cellular growth, proliferation, and metabolism. Moreover, it is speculated that amino acid signaling to mTORC1 may start from the lysosomal lumen. In this review, we discussed the function of these mediators in mTORC1 pathway and how these mediators are regulated by amino acids in details.

Mesenchymal Wnt/β-catenin signaling limits tooth number.

Science.gov (United States)

Järvinen, Elina; Shimomura-Kuroki, Junko; Balic, Anamaria; Jussila, Maria; Thesleff, Irma

2018-02-21

Tooth agenesis is one of the predominant developmental anomalies in humans, usually affecting the permanent dentition generated by sequential tooth formation and, in most cases, caused by mutations perturbing epithelial Wnt/β-catenin signaling. In addition, loss-of-function mutations in the Wnt feedback inhibitor AXIN2 lead to human tooth agenesis. We have investigated the functions of Wnt/β-catenin signaling during sequential formation of molar teeth using mouse models. Continuous initiation of new teeth, which is observed after genetic activation of Wnt/β-catenin signaling in the oral epithelium, was accompanied by enhanced expression of Wnt antagonists and a downregulation of Wnt/β-catenin signaling in the dental mesenchyme. Genetic and pharmacological activation of mesenchymal Wnt/β-catenin signaling negatively regulated sequential tooth formation, an effect partly mediated by Bmp4. Runx2 , a gene whose loss-of-function mutations result in sequential formation of supernumerary teeth in the human cleidocranial dysplasia syndrome, suppressed the expression of Wnt inhibitors Axin2 and Drapc1 in dental mesenchyme. Our data indicate that increased mesenchymal Wnt signaling inhibits the sequential formation of teeth, and suggest that Axin2 / Runx2 antagonistic interactions modulate the level of mesenchymal Wnt/β-catenin signaling, underlying the contrasting dental phenotypes caused by human AXIN2 and RUNX2 mutations. © 2018. Published by The Company of Biologists Ltd.

Alk2/ACVR1 and Alk3/BMPR1A Provide Essential Function for Bone Morphogenetic Protein-Induced Retinal Angiogenesis.

Science.gov (United States)

Lee, Heon-Woo; Chong, Diana C; Ola, Roxana; Dunworth, William P; Meadows, Stryder; Ka, Jun; Kaartinen, Vesa M; Qyang, Yibing; Cleaver, Ondine; Bautch, Victoria L; Eichmann, Anne; Jin, Suk-Won

2017-04-01

Increasing evidence suggests that bone morphogenetic protein (BMP) signaling regulates angiogenesis. Here, we aimed to define the function of BMP receptors in regulating early postnatal angiogenesis by analysis of inducible, endothelial-specific deletion of the BMP receptor components Bmpr2 (BMP type 2 receptor), Alk1 (activin receptor-like kinase 1), Alk2 , and Alk3 in mouse retinal vessels. Expression analysis of several BMP ligands showed that proangiogenic BMP ligands are highly expressed in postnatal retinas. Consistently, BMP receptors are also strongly expressed in retina with a distinct pattern. To assess the function of BMP signaling in retinal angiogenesis, we first generated mice carrying an endothelial-specific inducible deletion of Bmpr2 . Postnatal deletion of Bmpr2 in endothelial cells substantially decreased the number of angiogenic sprouts at the vascular front and branch points behind the front, leading to attenuated radial expansion. To identify critical BMPR1s (BMP type 1 receptors) associated with BMPR2 in retinal angiogenesis, we generated endothelial-specific inducible deletion of 3 BMPR1s abundantly expressed in endothelial cells and analyzed the respective phenotypes. Among these, endothelial-specific deletion of either Alk2 / acvr1 or Alk3 / Bmpr1a caused a delay in radial expansion, reminiscent of vascular defects associated with postnatal endothelial-specific deletion of BMPR2, suggesting that ALK2/ACVR1 and ALK3/BMPR1A are likely to be the critical BMPR1s necessary for proangiogenic BMP signaling in retinal vessels. Our data identify BMP signaling mediated by coordination of ALK2/ACVR1, ALK3/BMPR1A, and BMPR2 as an essential proangiogenic cue for retinal vessels. © 2017 The Authors.

BMP15 Prevents Cumulus Cell Apoptosis Through CCL2 and FBN1 in Porcine Ovaries

Directory of Open Access Journals (Sweden)

Bo Zhai

2013-07-01

Full Text Available Background: Bone morphogenetic protein-15 (BMP15 is a maternal gene necessary for mammalian reproduction. BMP15 expression increased in oocytes accompanied by follicle growth and development. The function and regulation mechanism of BMP15 in porcine cumulus cell apoptosis process is still unclear now. Methods: In this study, flow cytometry (FCM was used to analyze the effects of BMP15 with different concentrations to cumulus cell apoptosis. High-throughput sequencing technology was carried out to screen regulatory genes linked closely with BMP15. In order to confirm the function of (MCP-1/CCL2 and FBN1 in cumulus cell apoptosis, RNA interference (RNAi method was used to inhibit the expression of (MCP-1/CCL2 and FBN1. Apoptosis and proliferation of cumulus cell treated with siRNA transfection technology were measured by FCM, 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide, quantitative real time-PCR (RT-qPCR and western blotting. Results: The results showed that the apoptosis levels of cumulus cell treated by BMP15 decreased significantly in a dose-dependent manner. The expression of related genes protein 1 (MCP-1/CCL2 and fibrillin1 (FBN1 were both regulated by BMP15. After transfection, the proliferation of porcine cumulus cells increased significantly and apoptosis of cumulus cells was prevented while FBN1 was silenced after BMP15 treatment. The proliferation of cumulus cells decreased significantly and apoptosis rate of cumulus cells increased significantly while CCL2 was silenced. Conclusion: The results obtained in this study firstly demonstrated that CCL2 and FBN1 are important regulatory factors of BMP15 in preventing cumulus cell apoptosis in porcine ovaries.

Lunatic fringe-mediated Notch signaling regulates adult hippocampal neural stem cell maintenance.

Science.gov (United States)

Semerci, Fatih; Choi, William Tin-Shing; Bajic, Aleksandar; Thakkar, Aarohi; Encinas, Juan Manuel; Depreux, Frederic; Segil, Neil; Groves, Andrew K; Maletic-Savatic, Mirjana

2017-07-12

Hippocampal neural stem cells (NSCs) integrate inputs from multiple sources to balance quiescence and activation. Notch signaling plays a key role during this process. Here, we report that Lunatic fringe ( Lfng), a key modifier of the Notch receptor, is selectively expressed in NSCs. Further, Lfng in NSCs and Notch ligands Delta1 and Jagged1, expressed by their progeny, together influence NSC recruitment, cell cycle duration, and terminal fate. We propose a new model in which Lfng-mediated Notch signaling enables direct communication between a NSC and its descendants, so that progeny can send feedback signals to the 'mother' cell to modify its cell cycle status. Lfng-mediated Notch signaling appears to be a key factor governing NSC quiescence, division, and fate.

Bmp2 in osteoblasts of periosteum and trabecular bone links bone formation to vascularization and mesenchymal stem cells

Science.gov (United States)

Yang, Wuchen; Guo, Dayong; Harris, Marie A.; Cui, Yong; Gluhak-Heinrich, Jelica; Wu, Junjie; Chen, Xiao-Dong; Skinner, Charles; Nyman, Jeffry S.; Edwards, James R.; Mundy, Gregory R.; Lichtler, Alex; Kream, Barbara E.; Rowe, David W.; Kalajzic, Ivo; David, Val; Quarles, Darryl L.; Villareal, Demetri; Scott, Greg; Ray, Manas; Liu, S.; Martin, James F.; Mishina, Yuji; Harris, Stephen E.

2013-01-01

Summary We generated a new Bmp2 conditional-knockout allele without a neo cassette that removes the Bmp2 gene from osteoblasts (Bmp2-cKOob) using the 3.6Col1a1-Cre transgenic model. Bones of Bmp2-cKOob mice are thinner, with increased brittleness. Osteoblast activity is reduced as reflected in a reduced bone formation rate and failure to differentiate to a mature mineralizing stage. Bmp2 in osteoblasts also indirectly controls angiogenesis in the periosteum and bone marrow. VegfA production is reduced in Bmp2-cKOob osteoblasts. Deletion of Bmp2 in osteoblasts also leads to defective mesenchymal stem cells (MSCs), which correlates with the reduced microvascular bed in the periosteum and trabecular bones. Expression of several MSC marker genes (α-SMA, CD146 and Angiopoietin-1) in vivo, in vitro CFU assays and deletion of Bmp2 in vitro in α-SMA+ MSCs support our conclusions. Critical roles of Bmp2 in osteoblasts and MSCs are a vital link between bone formation, vascularization and mesenchymal stem cells. PMID:23843612

Root signals that mediate mutualistic interactions in the rhizosphere.

Science.gov (United States)

Rasmann, Sergio; Turlings, Ted Cj

2016-08-01

A recent boom in research on belowground ecology is rapidly revealing a multitude of fascinating interactions, in particular in the rhizosphere. Many of these interactions are mediated by photo-assimilates that are excreted by plant roots. Root exudates are not mere waste products, but serve numerous functions to control abiotic and biotic processes. These functions range from changing the chemical and physical properties of the soil, inhibiting the growth of competing plants, combatting herbivores, and regulating the microbial community. Particularly intriguing are root-released compounds that have evolved to serve mutualistic interactions with soil-dwelling organisms. These mutually beneficial plant-mediated signals are not only of fundamental ecological interest, but also exceedingly important from an agronomical perspective. Here, we attempt to provide an overview of the plant-produced compounds that have so far been implicated in mutualistic interactions. We propose that these mutualistic signals may have evolved from chemical defenses and we point out that they can be (mis)used by specialized pathogens and herbivores. We speculate that many more signals and interactions remain to be uncovered and that a good understanding of the mechanisms and ecological implications can be the basis for exploitation and manipulation of the signals for crop improvement and protection. Copyright © 2016 Elsevier Ltd. All rights reserved.

Sustained release of BMP-2 in bioprinted alginate for osteogenicity in mice and rats.

Directory of Open Access Journals (Sweden)

Michelle T Poldervaart

Full Text Available The design of bioactive three-dimensional (3D scaffolds is a major focus in bone tissue engineering. Incorporation of growth factors into bioprinted scaffolds offers many new possibilities regarding both biological and architectural properties of the scaffolds. This study investigates whether the sustained release of bone morphogenetic protein 2 (BMP-2 influences osteogenicity of tissue engineered bioprinted constructs. BMP-2 loaded on gelatin microparticles (GMPs was used as a sustained release system, which was dispersed in hydrogel-based constructs and compared to direct inclusion of BMP-2 in alginate or control GMPs. The constructs were supplemented with goat multipotent stromal cells (gMSCs and biphasic calcium phosphate to study osteogenic differentiation and bone formation respectively. BMP-2 release kinetics and bioactivity showed continuous release for three weeks coinciding with osteogenicity. Osteogenic differentiation and bone formation of bioprinted GMP containing constructs were investigated after subcutaneous implantation in mice or rats. BMP-2 significantly increased bone formation, which was not influenced by the release timing. We showed that 3D printing of controlled release particles is feasible and that the released BMP-2 directs osteogenic differentiation in vitro and in vivo.

Differential expression of a BMP4 reporter allele in anterior fungiform versus posterior circumvallate taste buds of mice

Directory of Open Access Journals (Sweden)

Barlow Linda A

2010-10-01

Full Text Available Abstract Background Bone Morphogenetic Protein 4 (BMP4 is a diffusible factor which regulates embryonic taste organ development. However, the role of BMP4 in taste buds of adult mice is unknown. We utilized transgenic mice with LacZ under the control of the BMP4 promoter to reveal the expression of BMP4 in the tongues of adult mice. Further we evaluate the pattern of BMP4 expression with that of markers of specific taste bud cell types and cell proliferation to define and compare the cell populations expressing BMP4 in anterior (fungiform papillae and posterior (circumvallate papilla tongue. Results BMP4 is expressed in adult fungiform and circumvallate papillae, i.e., lingual structures composed of non-taste epithelium and taste buds. Unexpectedly, we find both differences and similarities with respect to expression of BMP4-driven ß-galactosidase. In circumvallate papillae, many fusiform cells within taste buds are BMP4-ß-gal positive. Further, a low percentage of BMP4-expressing cells within circumvallate taste buds is immunopositive for markers of each of the three differentiated taste cell types (I, II and III. BMP4-positive intragemmal cells also expressed a putative marker of immature taste cells, Sox2, and consistent with this finding, intragemmal cells expressed BMP4-ß-gal within 24 hours after their final mitosis, as determined by BrdU birthdating. By contrast, in fungiform papillae, BMP4-ß-gal positive cells are never encountered within taste buds. However, in both circumvallate and fungiform papillae, BMP4-ß-gal expressing cells are located in the perigemmal region, comprising basal and edge epithelial cells adjacent to taste buds proper. This region houses the proliferative cell population that gives rise to adult taste cells. However, perigemmal BMP4-ß-gal cells appear mitotically silent in both fungiform and circumvallate taste papillae, as we do not find evidence of their active proliferation using cell cycle immunomarkers

Differential expression of a BMP4 reporter allele in anterior fungiform versus posterior circumvallate taste buds of mice.

Science.gov (United States)

Nguyen, Ha M; Barlow, Linda A

2010-10-13

Bone Morphogenetic Protein 4 (BMP4) is a diffusible factor which regulates embryonic taste organ development. However, the role of BMP4 in taste buds of adult mice is unknown. We utilized transgenic mice with LacZ under the control of the BMP4 promoter to reveal the expression of BMP4 in the tongues of adult mice. Further we evaluate the pattern of BMP4 expression with that of markers of specific taste bud cell types and cell proliferation to define and compare the cell populations expressing BMP4 in anterior (fungiform papillae) and posterior (circumvallate papilla) tongue. BMP4 is expressed in adult fungiform and circumvallate papillae, i.e., lingual structures composed of non-taste epithelium and taste buds. Unexpectedly, we find both differences and similarities with respect to expression of BMP4-driven ß-galactosidase. In circumvallate papillae, many fusiform cells within taste buds are BMP4-ß-gal positive. Further, a low percentage of BMP4-expressing cells within circumvallate taste buds is immunopositive for markers of each of the three differentiated taste cell types (I, II and III). BMP4-positive intragemmal cells also expressed a putative marker of immature taste cells, Sox2, and consistent with this finding, intragemmal cells expressed BMP4-ß-gal within 24 hours after their final mitosis, as determined by BrdU birthdating. By contrast, in fungiform papillae, BMP4-ß-gal positive cells are never encountered within taste buds. However, in both circumvallate and fungiform papillae, BMP4-ß-gal expressing cells are located in the perigemmal region, comprising basal and edge epithelial cells adjacent to taste buds proper. This region houses the proliferative cell population that gives rise to adult taste cells. However, perigemmal BMP4-ß-gal cells appear mitotically silent in both fungiform and circumvallate taste papillae, as we do not find evidence of their active proliferation using cell cycle immunomarkers and BrdU birthdating. Our data suggest that

Manipulation of Fgf and Bmp signaling in teleost fishes suggests potential pathways for the evolutionary origin of multicuspid teeth.

Science.gov (United States)

Jackman, William R; Davies, Shelby H; Lyons, David B; Stauder, Caitlin K; Denton-Schneider, Benjamin R; Jowdry, Andrea; Aigler, Sharon R; Vogel, Scott A; Stock, David W

2013-01-01

Teeth with two or more cusps have arisen independently from an ancestral unicuspid condition in a variety of vertebrate lineages, including sharks, teleost fishes, amphibians, lizards, and mammals. One potential explanation for the repeated origins of multicuspid teeth is the existence of multiple adaptive pathways leading to them, as suggested by their different uses in these lineages. Another is that the addition of cusps required only minor changes in genetic pathways regulating tooth development. Here we provide support for the latter hypothesis by demonstrating that manipulation of the levels of Fibroblast growth factor (Fgf) or Bone morphogenetic protein (Bmp) signaling produces bicuspid teeth in the zebrafish (Danio rerio), a species lacking multicuspid teeth in its ancestry. The generality of these results for teleosts is suggested by the conversion of unicuspid pharyngeal teeth into bicuspid teeth by similar manipulations of the Mexican Tetra (Astyanax mexicanus). That these manipulations also produced supernumerary teeth in both species supports previous suggestions of similarities in the molecular control of tooth and cusp number. We conclude that despite their apparent complexity, the evolutionary origin of multicuspid teeth is positively constrained, likely requiring only slight modifications of a pre-existing mechanism for patterning the number and spacing of individual teeth. © 2013 Wiley Periodicals, Inc.

Nitric oxide-mediated bystander signal transduction induced by heavy-ion microbeam irradiation

Science.gov (United States)

Tomita, Masanori; Matsumoto, Hideki; Funayama, Tomoo; Yokota, Yuichiro; Otsuka, Kensuke; Maeda, Munetoshi; Kobayashi, Yasuhiko

2015-07-01

In general, a radiation-induced bystander response is known to be a cellular response induced in non-irradiated cells after receiving bystander signaling factors released from directly irradiated cells within a cell population. Bystander responses induced by high-linear energy transfer (LET) heavy ions at low fluence are an important health problem for astronauts in space. Bystander responses are mediated via physical cell-cell contact, such as gap-junction intercellular communication (GJIC) and/or diffusive factors released into the medium in cell culture conditions. Nitric oxide (NO) is a well-known major initiator/mediator of intercellular signaling within culture medium during bystander responses. In this study, we investigated the NO-mediated bystander signal transduction induced by high-LET argon (Ar)-ion microbeam irradiation of normal human fibroblasts. Foci formation by DNA double-strand break repair proteins was induced in non-irradiated cells, which were co-cultured with those irradiated by high-LET Ar-ion microbeams in the same culture plate. Foci formation was suppressed significantly by pretreatment with an NO scavenger. Furthermore, NO-mediated reproductive cell death was also induced in bystander cells. Phosphorylation of NF-κB and Akt were induced during NO-mediated bystander signaling in the irradiated and bystander cells. However, the activation of these proteins depended on the incubation time after irradiation. The accumulation of cyclooxygenase-2 (COX-2), a downstream target of NO and NF-κB, was observed in the bystander cells 6 h after irradiation but not in the directly irradiated cells. Our findings suggest that Akt- and NF-κB-dependent signaling pathways involving COX-2 play important roles in NO-mediated high-LET heavy-ion-induced bystander responses. In addition, COX-2 may be used as a molecular marker of high-LET heavy-ion-induced bystander cells to distinguish them from directly irradiated cells, although this may depend on the time

Nonsense-Mediated RNA Decay Influences Human Embryonic Stem Cell Fate

Directory of Open Access Journals (Sweden)

Chih-Hong Lou

2016-06-01

Full Text Available Nonsense-mediated RNA decay (NMD is a highly conserved pathway that selectively degrades specific subsets of RNA transcripts. Here, we provide evidence that NMD regulates early human developmental cell fate. We found that NMD factors tend to be expressed at higher levels in human pluripotent cells than in differentiated cells, raising the possibility that NMD must be downregulated to permit differentiation. Loss- and gain-of-function experiments in human embryonic stem cells (hESCs demonstrated that, indeed, NMD downregulation is essential for efficient generation of definitive endoderm. RNA-seq analysis identified NMD target transcripts induced when NMD is suppressed in hESCs, including many encoding signaling components. This led us to test the role of TGF-β and BMP signaling, which we found NMD acts through to influence definitive endoderm versus mesoderm fate. Our results suggest that selective RNA decay is critical for specifying the developmental fate of specific human embryonic cell lineages.

Binding Interactions of Keratin-Based Hair Fiber Extract to Gold, Keratin, and BMP-2.

Directory of Open Access Journals (Sweden)

Roche C de Guzman

Full Text Available Hair-derived keratin biomaterials composed mostly of reduced keratin proteins (kerateines have demonstrated their utility as carriers of biologics and drugs for tissue engineering. Electrostatic forces between negatively-charged keratins and biologic macromolecules allow for effective drug retention; attraction to positively-charged growth factors like bone morphogenetic protein 2 (BMP-2 has been used as a strategy for osteoinduction. In this study, the intermolecular surface and bulk interaction properties of kerateines were investigated. Thiol-rich kerateines were chemisorbed onto gold substrates to form an irreversible 2-nm rigid layer for surface plasmon resonance analysis. Kerateine-to-kerateine cohesion was observed in pH-neutral water with an equilibrium dissociation constant (KD of 1.8 × 10(-4 M, indicating that non-coulombic attractive forces (i.e. hydrophobic and van der Waals were at work. The association of BMP-2 to kerateine was found to be greater (KD = 1.1 × 10(-7 M, within the range of specific binding. Addition of salts (phosphate-buffered saline; PBS shortened the Debye length or the electrostatic field influence which weakened the kerateine-BMP-2 binding (KD = 3.2 × 10(-5 M. BMP-2 in bulk kerateine gels provided a limited release in PBS (~ 10% dissociation in 4 weeks, suggesting that electrostatic intermolecular attraction was significant to retain BMP-2 within the keratin matrix. Complete dissociation between kerateine and BMP-2 occurred when the PBS pH was lowered (to 4.5, below the keratin isoelectric point of 5.3. This phenomenon can be attributed to the protonation of keratin at a lower pH, leading to positive-positive repulsion. Therefore, the dynamics of kerateine-BMP-2 binding is highly dependent on pH and salt concentration, as well as on BMP-2 solubility at different pH and molarity. The study findings may contribute to our understanding of the release kinetics of drugs from keratin biomaterials and allow for the

Vitamin D receptor signaling is required for heart development in zebrafish embryo

Energy Technology Data Exchange (ETDEWEB)

Kwon, Hye-Joo, E-mail: hjkwon@pnu.edu.sa [Biology Department, Texas A& M University, College Station, TX77843-3258 (United States); Biology Department, Princess Nourah University, Riyadh 11671 (Saudi Arabia)

2016-02-12

Vitamin D has been found to be associated with cardiovascular diseases. However, the role of vitamin D in heart development during embryonic period is largely unknown. Vitamin D induces its genomic effects through its nuclear receptor, the vitamin D receptor (VDR). The present study investigated the role of VDR on heart development by antisense-mediated knockdown approaches in zebrafish model system. In zebrafish embryos, two distinct VDR genes (vdra and vdrb) have been identified. Knockdown of vdra has little effect on heart development, whereas disrupting vdrb gene causes various cardiac phenotypes, characterized by pericardial edema, slower heart rate and laterality defects. Depletion of both vdra and vdrb (vdra/b) produce additive, but not synergistic effects. To determine whether atrioventricular (AV) cardiomyocytes are properly organized in these embryos, the expression of bmp4, which marks the developing AV boundary at 48 h post-fertilization, was examined. Notably, vdra/b-deficient embryos display ectopic expression of bmp4 towards the ventricle or throughout atrial and ventricular chambers. Taken together, these results suggest that VDR signaling plays an essential role in heart development. - Highlights: • VDR signaling is involved in embryonic heart development. • Knockdown of vdrb, but not vdra, causes decreased heart rate in zebrafish embryo. • Loss of vdr results in cardiac laterality defects. • Loss of vdra/b alters atrioventricular boundary formation. • Loss of vdra/b causes abnormal cardiac looping.

Vitamin D receptor signaling is required for heart development in zebrafish embryo

International Nuclear Information System (INIS)

Kwon, Hye-Joo

2016-01-01

Vitamin D has been found to be associated with cardiovascular diseases. However, the role of vitamin D in heart development during embryonic period is largely unknown. Vitamin D induces its genomic effects through its nuclear receptor, the vitamin D receptor (VDR). The present study investigated the role of VDR on heart development by antisense-mediated knockdown approaches in zebrafish model system. In zebrafish embryos, two distinct VDR genes (vdra and vdrb) have been identified. Knockdown of vdra has little effect on heart development, whereas disrupting vdrb gene causes various cardiac phenotypes, characterized by pericardial edema, slower heart rate and laterality defects. Depletion of both vdra and vdrb (vdra/b) produce additive, but not synergistic effects. To determine whether atrioventricular (AV) cardiomyocytes are properly organized in these embryos, the expression of bmp4, which marks the developing AV boundary at 48 h post-fertilization, was examined. Notably, vdra/b-deficient embryos display ectopic expression of bmp4 towards the ventricle or throughout atrial and ventricular chambers. Taken together, these results suggest that VDR signaling plays an essential role in heart development. - Highlights: • VDR signaling is involved in embryonic heart development. • Knockdown of vdrb, but not vdra, causes decreased heart rate in zebrafish embryo. • Loss of vdr results in cardiac laterality defects. • Loss of vdra/b alters atrioventricular boundary formation. • Loss of vdra/b causes abnormal cardiac looping.

Synthetic scaffold coating with adeno-associated virus encoding BMP2 to promote endogenous bone repair.

Science.gov (United States)

Dupont, Kenneth M; Boerckel, Joel D; Stevens, Hazel Y; Diab, Tamim; Kolambkar, Yash M; Takahata, Masahiko; Schwarz, Edward M; Guldberg, Robert E

2012-03-01

Biomaterial scaffolds functionalized to stimulate endogenous repair mechanisms via the incorporation of osteogenic cues offer a potential alternative to bone grafting for the treatment of large bone defects. We first quantified the ability of a self-complementary adeno-associated viral vector encoding bone morphogenetic protein 2 (scAAV2.5-BMP2) to enhance human stem cell osteogenic differentiation in vitro. In two-dimensional culture, scAAV2.5-BMP2-transduced human mesenchymal stem cells (hMSCs) displayed significant increases in BMP2 production and alkaline phosphatase activity compared with controls. hMSCs and human amniotic-fluid-derived stem cells (hAFS cells) seeded on scAAV2.5-BMP2-coated three-dimensional porous polymer Poly(ε-caprolactone) (PCL) scaffolds also displayed significant increases in BMP2 production compared with controls during 12 weeks of culture, although only hMSC-seeded scaffolds displayed significantly increased mineral formation. PCL scaffolds coated with scAAV2.5-BMP2 were implanted into critically sized immunocompromised rat femoral defects, both with or without pre-seeding of hMSCs, representing ex vivo and in vivo gene therapy treatments, respectively. After 12 weeks, defects treated with acellular scAAV2.5-BMP2-coated scaffolds displayed increased bony bridging and had significantly higher bone ingrowth and mechanical properties compared with controls, whereas defects treated with scAAV2.5-BMP2 scaffolds pre-seeded with hMSCs failed to display significant differences relative to controls. When pooled, defect treatment with scAAV2.5-BMP2-coated scaffolds, both with or without inclusion of pre-seeded hMSCs, led to significant increases in defect mineral formation at all time points and increased mechanical properties compared with controls. This study thus presents a novel acellular bone-graft-free endogenous repair therapy for orthotopic tissue-engineered bone regeneration.

Inositol trisphosphate receptor mediated spatiotemporal calcium signalling.

Science.gov (United States)

Miyazaki, S

1995-04-01

Spatiotemporal Ca2+ signalling in the cytoplasm is currently understood as an excitation phenomenon by analogy with electrical excitation in the plasma membrane. In many cell types, Ca2+ waves and Ca2+ oscillations are mediated by inositol 1,4,5-trisphosphate (IP3) receptor/Ca2+ channels in the endoplasmic reticulum membrane, with positive feedback between cytosolic Ca2+ and IP3-induced Ca2+ release creating a regenerative process. Remarkable advances have been made in the past year in the analysis of subcellular Ca2+ microdomains using confocal microscopy and of Ca2+ influx pathways that are functionally coupled to IP3-induced Ca2+ release. Ca2+ signals can be conveyed into the nucleus and mitochondria. Ca2+ entry from outside the cell allows repetitive Ca2+ release by providing Ca2+ to refill the endoplasmic reticulum stores, thus giving rise to frequency-encoded Ca2+ signals.

DMPD: Modulation of Toll-interleukin 1 receptor mediated signaling. [Dynamic Macrophage Pathway CSML Database

Lifescience Database Archive (English)

Full Text Available 15662540 Modulation of Toll-interleukin 1 receptor mediated signaling. Li X, Qin J.... J Mol Med. 2005 Apr;83(4):258-66. Epub 2005 Jan 21. (.png) (.svg) (.html) (.csml) Show Modulation of Toll-i...nterleukin 1 receptor mediated signaling. PubmedID 15662540 Title Modulation of Toll-interleukin 1 receptor

[Preparation of vanilline cross-linked rhBMP-2/chitosan microspheres and its effect on mesenchymal stem cells].

Science.gov (United States)

Wu, Gui; Wang, Hai; Qiu, Guixing; Yu, Xin; Su, Xinlin; Ma, Pei; Yin, Bo; Wu, Zhihong

2015-06-02

To prepare rhBMP-2/chitosan microspheres (rhBMP-2 CMs) with vanilline as a cross-linking reagent and study the biocompatibility and drug release characteristic of microspheres in vitro. Emulsion cross-linking method was utilized to prepare rhBMP-2 CMs, Scanning electron microscope (SEM) was used to observe the microstructure of microspheres.Leaching solution of microspheres and blank culture medium were designated as experimental and control groups respectively. Both groups were cultured with human mesenchymal stem cells (hMSCs) to determine its cytotoxicity and its effect on the proliferation of hMSCs. Dynamic immersion method was used to examine the in vitro release characteristic of rhBMP-2. And the alkaline phosphatase (ALP) activity of hMSCs was determined to reveal the bioactivity of released rhBMP-2. The rhBMP-2 CMs were spherical under SEM.After treating with leaching solution for 24 and 48 h, there was no inter-group statistical difference in optical density (OD) values at both timepoints (24 h:0.72 ± 0.07 vs 0.73 ± 0.05, P > 0.05; 48 h:1.19 ± 0.11 vs 1.27 ± 0.06, P > 0.05). After culturing with leaching solution for 1, 3 and 7 days, the number of cells increased with time for both groups. And the OD values were not statistically different at each timepoint. Five milligram rhBMP-2 CMs soaked for 19 days with a gradual release of rhBMP-2. The concentration of rhBMP-2 was 216.1 ± 20.0 ng/ml at Day 19. At Days 3 and 7, the ALP activities of hMSCs were (0.50 ± 0.07) and (0.68 ± 0.06) µmol pNPP·min⁻¹·mg⁻¹ protein respectively and both were higher than that of blank culture medium group (0.14 ± 0.01) (P < 0.05). With an excellent biocompatibility, rhBMP-2 CMs may be an ideal carrier for control-released rhBMP-2 and encapsulated rhBMP-2 remains bioactive.

Alk2/ACVR1 and Alk3/BMPR1A Provide Essential Function for Bone Morphogenetic Protein Induced Retinal Angiogenesis

Science.gov (United States)

Lee, Heon-Woo; Chong, Diana C.; Ola, Roxana; Dunworth, William P.; Meadows, Stryder; Ka, Jun; Kaartinen, Vesa M.; Qyang, Yibing; Cleaver, Ondine; Bautch, Victoria L.; Eichmann, Anne; Jin, Suk-Won

2017-01-01

Objective Increasing evidence suggests that Bone Morphogenetic Protein (BMP) signaling regulates angiogenesis. Here, we aimed to define the function of BMP receptors in regulating early post-natal angiogenesis by analysis of inducible, endothelial specific deletion of the BMP receptor components Bmpr2, Alk1, Alk2 and Alk3 in mouse retinal vessels. Approach and Results Expression analysis of several BMP ligands showed that pro-angiogenic BMP ligands are highly expressed in postnatal retinas. Consistently, BMP receptors are also strongly expressed in retina with a distinct pattern. To assess the function of BMP signaling in retinal angiogenesis, we first generated mice carrying an endothelial-specific inducible deletion of BMP Type 2 receptor (Bmpr2). Postnatal deletion of Bmpr2 in endothelial cells substantially decreased the number of angiogenic sprouts at the vascular front and branchpoints behind the front, leading to attenuated radial expansion. To identify critical BMPR1s associated with BMPR2 in retinal angiogenesis, we generated endothelial-specific inducible deletion of three BMPR1s abundantly expressed in endothelial cells and analyzed the respective phenotypes. Among these, endothelial specific deletion of either Alk2/acvr1 or Alk3/Bmpr1a caused a delay in radial expansion, reminiscent of vascular defects associated with postnatal endothelial specific deletion of BMPR2, suggesting that ALK2/ACVR1 and ALK3/BMPR1A are likely to be the critical BMPR1s necessary for pro-angiogenic BMP signaling in retinal vessels. Conclusions Our data identify BMP signaling mediated by coordination of ALK2/ACVR1, ALK3/BMPR1A, and BMPR2 as an essential pro-angiogenic cue for retinal vessels. PMID:28232325

The Ihh signal is essential for regulating proliferation and hypertrophy of cultured chicken chondrocytes.

Science.gov (United States)

Ma, R S; Zhou, Z L; Luo, J W; Zhang, H; Hou, J F

2013-10-01

The Indian hedgehog (Ihh) signal plays a vital role in regulating proliferation and hypertrophy of chondrocytes. To investigate its function in postnatal chicken (Gallus gallus) chondrocytes, cyclopamine was used to inhibit Ihh signaling. The MTT and ALP assays revealed the downgrade-proliferation and upgrade-differentiation of chondrocytes. To further elucidate the mechanism, the mRNA expression levels of Ihh, parathyroid hormone related protein (PTHrP), Gli-2, Bcl-2, Bone Morphogenetic Protein 6 (BMP-6), type X collagen (Col X) and type II collagen (Col II) were detected by quantitative real-time RT-PCR analysis, and the protein expressions of Ihh, Col X, and Col II were determined using Western blot analysis. After the Ihh signal was blocked, chondrocytes demonstrated high expression levels of PTHrP and Col X and low levels of Gli-2, BMP-6, Bcl-2 and Col II although Ihh expression was increased. Based on these results, the Ihh signal is essential for balancing chicken chondrocyte proliferation and hypertrophy, and the regulatory function of PTHrP acts in an Ihh-dependent manner. Furthermore, BMP-6 and Bcl-2 played roles in maintaining the development of chondrocytes and may be downstream regulatory factors of Ihh signaling. © 2013.

Iron regulation of hepcidin despite attenuated Smad1,5,8 signaling in mice without transferrin receptor 2 or Hfe

Science.gov (United States)

Corradini, Elena; Rozier, Molly; Meynard, Delphine; Odhiambo, Adam; Lin, Herbert Y.; Feng, Qi; Migas, Mary C.; Britton, Robert S.; Babitt, Jodie L.; Fleming, Robert E.

2011-01-01

Background & Aims HFE and transferrin receptor 2 (TFR2) are each necessary for the normal relationship between body iron status and liver hepcidin expression. In murine Hfe and Tfr2 knockout models of hereditary hemochromatosis (HH), signal transduction to hepcidin via the bone morphogenetic protein 6 (Bmp6)/Smad1,5,8 pathway is attenuated. We examined the effect of dietary iron on regulation of hepcidin expression via the Bmp6/Smad1,5,8 pathway using mice with targeted disruption of Tfr2, Hfe, or both genes. Methods Hepatic iron concentrations and mRNA expression of Bmp6 and hepcidin were compared with wild-type mice in each of the HH models on standard or iron-loading diets. Liver phospho-Smad (P-Smad)1,5,8 and Id1 mRNA levels were measured as markers of Bmp/Smad signaling. Results While Bmp6 expression was increased, liver hepcidin and Id1 expression were decreased in each of the HH models compared with wild-type mice. Each of the HH models also demonstrated attenuated P-Smad1,5,8 levels relative to liver iron status. Mice with combined Hfe/Tfr2 disruption were most affected. Dietary iron loading increased hepcidin and Id1 expression in each of the HH models. Compared with wild-type mice, HH mice demonstrated attenuated (Hfe knockout) or no increases in P-Smad1,5,8 levels in response to dietary iron loading. Conclusions These observations demonstrate that Tfr2 and Hfe are each required for normal signaling of iron status to hepcidin via Bmp6/Smad1,5,8 pathway. Mice with combined loss of Hfe and Tfr2 up-regulate hepcidin in response to dietary iron loading without increases in liver BMP6 mRNA or steady-state P-Smad1,5,8 levels. PMID:21745449

COUP-TFII mediates progesterone regulation of uterine implantation by controlling ER activity.

Directory of Open Access Journals (Sweden)

Isao Kurihara

2007-06-01

Full Text Available Progesterone and estrogen are critical regulators of uterine receptivity. To facilitate uterine remodeling for embryo attachment, estrogen activity in the uterine epithelia is attenuated by progesterone; however, the molecular mechanism by which this occurs is poorly defined. COUP-TFII (chicken ovalbumin upstream promoter transcription factor II; also known as NR2F2, a member of the nuclear receptor superfamily, is highly expressed in the uterine stroma and its expression is regulated by the progesterone-Indian hedgehog-Patched signaling axis that emanates from the epithelium. To further assess COUP-TFII uterine function, a conditional COUP-TFII knockout mouse was generated. This mutant mouse is infertile due to implantation failure, in which both embryo attachment and uterine decidualization are impaired. Using this animal model, we have identified a novel genetic pathway in which BMP2 lies downstream of COUP-TFII. Epithelial progesterone-induced Indian hedgehog regulates stromal COUP-TFII, which in turn controls BMP2 to allow decidualization to manifest in vivo. Interestingly, enhanced epithelial estrogen activity, which impedes maturation of the receptive uterus, was clearly observed in the absence of stromal-derived COUP-TFII. This finding is consistent with the notion that progesterone exerts its control of implantation through uterine epithelial-stromal cross-talk and reveals that stromal-derived COUP-TFII is an essential mediator of this complex cross-communication pathway. This finding also provides a new signaling paradigm for steroid hormone regulation in female reproductive biology, with attendant implications for furthering our understanding of the molecular mechanisms that underlie dysregulation of hormonal signaling in such human reproductive disorders as endometriosis and endometrial cancer.

Effect of a Novel Nonviral Gene Delivery of BMP-2 on Bone Healing

Directory of Open Access Journals (Sweden)

P. Schwabe

2012-01-01

Full Text Available Background. Gene therapeutic drug delivery approaches have been introduced to improve the efficiency of growth factors at the site of interest. This study investigated the efficacy and safety of a new nonviral copolymer-protected gene vector (COPROG for the stimulation of bone healing. Methods. In vitro, rat osteoblasts were transfected with COPROG + luciferase plasmid or COPROG + hBMP-2 plasmid. In vivo, rat tibial fractures were intramedullary stabilized with uncoated versus COPROG+hBMP-2-plasmid-coated titanium K-wires. The tibiae were prepared for biomechanical and histological analyses at days 28 and 42 and for transfection/safety study at days 2, 4, 7, 28, and 42. Results. In vitro results showed luciferase expression until day 21, and hBMP-2-protein was measured from day 2 – day 10. In vivo, the local application of hBMP-2-plasmid showed a significantly higher maximum load after 42 days compared to that in the control. The histomorphometric analysis revealed a significantly less mineralized periosteal callus area in the BMP-2 group compared to the control at day 28. The rt-PCR showed no systemic biodistribution of luciferase RNA. Conclusion. A positive effect on fracture healing by nonviral BMP-2 plasmid application from COPROG-coated implants could be shown in this study; however, the effect of the vector may be improved with higher plasmid concentrations. Transfection showed no biodistribution to distant organs and was considered to be safe.

Single-cell analysis reveals a link between CD3- and CD59-mediated signaling pathways in Jurkat T cells

International Nuclear Information System (INIS)

Lipp, A. M.

2012-01-01

Elevation of intracellular free calcium concentration ([Ca2+]i) is a key signal during T cell activation and is commonly used as a read-out parameter for stimulation of T cell signaling. Upon T cell stimulation a variety of calcium signals is produced by individual cells of the T cell population and the type of calcium signal strongly influences cell fate decisions. The heterogeneous nature of T cells is masked in ensemble measurements, which highlights the need for single-cell measurements. In this study we used single-cell calcium measurements in Jurkat cells to investigate signaling pathways, which are triggered by different proteins, namely CD3 and CD59. By application of an automated cluster algorithm the presented assay provides unbiased analysis of a large data set of individual calcium time traces generated by the whole cell population. By using this method we could demonstrate that the Jurkat population generates heterogeneous calcium signals in a stimulus-dependent manner. Furthermore, our data revealed the existence of a link between CD3- and CD59-mediated signaling pathways. Single-cell calcium measurements in Jurkat cells expressing different levels of the T cell receptor (TCR) complex indicated that CD59-mediated calcium signaling is critically dependent on TCR surface expression levels. In addition, triggering CD59-mediated calcium signaling resulted in down-regulation of TCR surface expression levels, which is known to happen upon direct TCR triggering too. Moreover, by using siRNA-mediated protein knock-downs and protein knock-out Jurkat mutants we could show that CD3- and CD59-mediated calcium signaling require identical key proteins. We therefore explored by which mechanism CD59-mediated signaling couples into TCR-mediated signaling. Fluorescence recovery after photobleaching (FRAP) experiments and live-cell protein-protein interaction assays provided no evidence of a direct physical interaction between CD3- and CD59-mediated signaling pathways

Uncertainty in BMP evaluation and optimization for watershed management

Science.gov (United States)

Chaubey, I.; Cibin, R.; Sudheer, K.; Her, Y.

2012-12-01

Use of computer simulation models have increased substantially to make watershed management decisions and to develop strategies for water quality improvements. These models are often used to evaluate potential benefits of various best management practices (BMPs) for reducing losses of pollutants from sources areas into receiving waterbodies. Similarly, use of simulation models in optimizing selection and placement of best management practices under single (maximization of crop production or minimization of pollutant transport) and multiple objective functions has increased recently. One of the limitations of the currently available assessment and optimization approaches is that the BMP strategies are considered deterministic. Uncertainties in input data (e.g. precipitation, streamflow, sediment, nutrient and pesticide losses measured, land use) and model parameters may result in considerable uncertainty in watershed response under various BMP options. We have developed and evaluated options to include uncertainty in BMP evaluation and optimization for watershed management. We have also applied these methods to evaluate uncertainty in ecosystem services from mixed land use watersheds. In this presentation, we will discuss methods to to quantify uncertainties in BMP assessment and optimization solutions due to uncertainties in model inputs and parameters. We have used a watershed model (Soil and Water Assessment Tool or SWAT) to simulate the hydrology and water quality in mixed land use watershed located in Midwest USA. The SWAT model was also used to represent various BMPs in the watershed needed to improve water quality. SWAT model parameters, land use change parameters, and climate change parameters were considered uncertain. It was observed that model parameters, land use and climate changes resulted in considerable uncertainties in BMP performance in reducing P, N, and sediment loads. In addition, climate change scenarios also affected uncertainties in SWAT

The Role of Cgrp-Receptor Component Protein (Rcp in Cgrp-Mediated Signal Transduction

Directory of Open Access Journals (Sweden)

M. A. Prado

2001-01-01

Full Text Available The calcitonin gene-related peptide (CGRP-receptor component protein (RCP is a 17-kDa intracellular peripheral membrane protein required for signal transduction at CGRP receptors. To determine the role of RCP in CGRP-mediated signal transduction, RCP was depleted from NIH3T3 cells using antisense strategy. Loss of RCP protein correlated with loss of cAMP production by CGRP in the antisense cells. In contrast, loss of RCP had no effect on CGRP-mediated binding; therefore RCP is not acting as a chaperone for the CGRP receptor. Instead, RCP is a novel signal transduction molecule that couples the CGRP receptor to the cellular signal transduction machinery. RCP thus represents a prototype for a new class of signal transduction proteins that are required for regulation of G protein-coupled receptors.

Dual specificity of activin type II receptor ActRIIb in dorso-ventral patterning during zebrafish embryogenesis.

Science.gov (United States)

Nagaso, H; Suzuki, A; Tada, M; Ueno, N

1999-04-01

Members of the transforming growth factor-beta (TGF-beta) superfamily are thought to regulate specification of a variety of tissue types in early embryogenesis. These effects are mediated through a cell surface receptor complex, consisting of two classes of ser/thr kinase receptor, type I and type II. In the present study, cDNA encoding zebrafish activin type II receptors, ActRIIa and ActRIIb was cloned and characterized. Overexpression of ActRIIb in zebrafish embryos caused dorsalization of embryos, as observed in activin-overexpressing embryos. However, in blastula stage embryos, ActRIIb induced formation of both dorsal and ventro-lateral mesoderm. It has been suggested that these inducing signals from ActRIIb are mediated through each specific type I receptor, TARAM-A and BMPRIA, depending on activin and bone morphogenetic protein (BMP), respectively. In addition, it was shown that a kinase-deleted form of ActRIIb (dnActRIIb) suppressed both activin- and BMP-like signaling pathways. These results suggest that ActRIIb at least has dual roles in both activin and BMP signaling pathways during zebrafish embryogenesis.

Expression of BMP-2 in Vascular Endothelial Cells of Recipient May Predict Delayed Graft Function After Renal Transplantation

Directory of Open Access Journals (Sweden)

Nikolina Basic-Jukic

2016-11-01

Full Text Available Background/Aims: Delayed graft function (DGF is associated with adverse outcomes after renal transplantation. Bone morphogenetic protein-2 (BMP-2 is involved in both endothelial function and immunological events. We compared expression of BMP-2 in epigastric artery of renal transplant recipients with immediate graft function (IGF and DGF. Methods: 79 patients were included in this prospective study. Patients were divided in IGF group (64 patients and DGF group (15 patients. BMP-2 expression in intima media (BMP2m and endothelium (BMP2e of epigastric artery was assessed by immunohistochemistry. Results: Lower intensity of BMP2e staining was recorded in DGF compared to IGF. In DGF patients, 93% had no expression of BMP2e and 7% had 1st grade expression, compared to 45% and 41% in IGF group, respectively (P=0.001 (Pst grade expression. Patients who had BMP2e staining positive had lower odds for DGF (OR 0.059 [0.007, 0.477] and this remained significant even after adjustment for donor and recipient variables, cold ischemia time, and immunological matching (OR 0.038 [0.003, 0.492]. Conclusions: Our results demonstrate that BMP-2 expression in endothelial cells of epigastric arteries may predict development of DGF.

Calcium-mediated signaling and calmodulin-dependent kinase regulate hepatocyte-inducible nitric oxide synthase expression.

Science.gov (United States)

Zhang, Baochun; Crankshaw, Will; Nesemeier, Ryan; Patel, Jay; Nweze, Ikenna; Lakshmanan, Jaganathan; Harbrecht, Brian G

2015-02-01

Induced nitric oxide synthase (iNOS) is induced in hepatocytes by shock and inflammatory stimuli. Excessive NO from iNOS mediates shock-induced hepatic injury and death, so understanding the regulation of iNOS will help elucidate the pathophysiology of septic shock. In vitro, cytokines induce iNOS expression through activation of signaling pathways including mitogen-activated protein kinases and nuclear factor κB. Cytokines also induce calcium (Ca(2+)) mobilization and activate calcium-mediated intracellular signaling pathways, typically through activation of calmodulin-dependent kinases (CaMK). Calcium regulates NO production in macrophages but the role of calcium and calcium-mediated signaling in hepatocyte iNOS expression has not been defined. Primary rat hepatocytes were isolated, cultured, and induced to produce NO with proinflammatory cytokines. Calcium mobilization and Ca(2+)-mediated signaling were altered with ionophore, Ca(2+) channel blockers, and inhibitors of CaMK. The Ca(2+) ionophore A23187 suppressed cytokine-stimulated NO production, whereas Ethylene glycol tetraacetic acid and nifedipine increased NO production, iNOS messenger RNA, and iNOS protein expression. Inhibition of CaMK with KN93 and CBD increased NO production but the calcineurin inhibitor FK 506 decreased iNOS expression. These data demonstrate that calcium-mediated signaling regulates hepatocyte iNOS expression and does so through a mechanism independent of calcineurin. Changes in intracellular calcium levels may regulate iNOS expression during hepatic inflammation induced by proinflammatory cytokines. Copyright © 2015 Elsevier Inc. All rights reserved.

Polymorphism in exon2 of BMP15 gene in Iranian sangsari sheep

Directory of Open Access Journals (Sweden)

zana pirkhezranian

2015-04-01

Full Text Available Fertility rate is an economically important trait in sheep, which is influenced by genetic and environment. So far, three genes have been identified that affects this trait, one of them would be the BMP family, the most famous one is BMP15. Different mutations in the BMP15 gene, increases reproductive performance and growth rate in sheep. The aim of this study was to investigate the genetic and phylogenetic of BMP15 gene sequence in Iranian Sangsari sheep. For this purpose, the blood samples from 20 animal of Damghan station were collected. After DNA extracting, a segment of 222 bp of exon 2 of BMP15 gene was amplified using polymerase chain reaction. Then, all of the PCR products were sequenced. The results showed existence of four haplotypes and three significant mutations of the gene that which one of them was seen for first. In order to determine the genetic distance of Sansari sheep with other animals especially sheep breeds about 103 sequences were taken from Genebank, Then, phylogenetic trees were drawn. Genetic distances and nucleotide differences were calculated. The results showed that goat, cattle and buffalo have minimum genetic distance and monkey, human and mouse have maximum distance with Sangsari sheep and native Hindi and Kashmiri sheep have not any differences with Iranian Sangsari sheep.

BMP2-loaded hollow hydroxyapatite microspheres exhibit enhanced osteoinduction and osteogenicity in large bone defects.

Science.gov (United States)

Xiong, Long; Zeng, Jianhua; Yao, Aihua; Tu, Qiquan; Li, Jingtang; Yan, Liang; Tang, Zhiming

2015-01-01

The regeneration of large bone defects is an osteoinductive, osteoconductive, and osteogenic process that often requires a bone graft for support. Limitations associated with naturally autogenic or allogenic bone grafts have demonstrated the need for synthetic substitutes. The present study investigates the feasibility of using novel hollow hydroxyapatite microspheres as an osteoconductive matrix and a carrier for controlled local delivery of bone morphogenetic protein 2 (BMP2), a potent osteogenic inducer of bone regeneration. Hollow hydroxyapatite microspheres (100±25 μm) with a core (60±18 μm) and a mesoporous shell (180±42 m(2)/g surface area) were prepared by a glass conversion technique and loaded with recombinant human BMP2 (1 μg/mg). There was a gentle burst release of BMP2 from microspheres into the surrounding phosphate-buffered saline in vitro within the initial 48 hours, and continued at a low rate for over 40 days. In comparison with hollow hydroxyapatite microspheres without BMP2 or soluble BMP2 without a carrier, BMP2-loaded hollow hydroxyapatite microspheres had a significantly enhanced capacity to reconstitute radial bone defects in rabbit, as shown by increased serum alkaline phosphatase; quick and complete new bone formation within 12 weeks; and great biomechanical flexural strength. These results indicate that BMP2-loaded hollow hydroxyapatite microspheres could be a potential new option for bone graft substitutes in bone regeneration.

Trends Analysis of rhBMP Utilization in Single-Level Posterior Lumbar Interbody Fusion in the United States.

Science.gov (United States)

Lao, Lifeng; Cohen, Jeremiah R; Buser, Zorica; Brodke, Darrel S; Youssef, Jim A; Park, Jong-Beom; Yoon, S Tim; Wang, Jeffrey C; Meisel, Hans-Joerg

2017-10-01

Retrospective study. Recombinant human bone morphogenetic protein-2 (rhBMP-2) has been widely used in spinal fusion surgery, but there is little information on rhBMP-2 utilization in single-level posterior lumbar interbody fusion (PLIF). The purpose of our study was to evaluate the trends and demographics of rhBMP-2 utilization in single-level PLIF. Patients who underwent single-level PLIF from 2005 to 2011 were identified by searching ICD-9 diagnosis and procedure codes in the PearlDiver Patient Records Database, a national database of orthopedic insurance records. The year of procedure, age, gender, and region of the United States were recorded for each patient. Results were reported for each variable as the incidence of procedures identified per 100 000 patients searched in the database. A total of 2735 patients had single-level PLIF. The average rate of single-level PLIF with rhBMP-2 maintained at a relatively stable level (28% to 31%) from 2005 to 2009, but decreased in 2010 (9.9%) and 2011 (11.8%). The overall incidence of single-level PLIF without rhBMP-2 (0.68 cases per 100 000 patients) was statistically higher ( P level PLIF with rhBMP-2 (0.21 cases per 100 000 patients). The average rate of single-level PLIF with rhBMP-2 utilization was the highest in West (30.1%), followed by Midwest (26.9%), South (20.5%), and Northeast (17.8%). The highest incidence of single-level PLIF with rhBMP-2 was observed in the age group level PLIF. There was a 3-fold increase in the rate of PLIF without rhBMP-2 compared to PLIF with rhBMP-2, with both procedures being mainly done in patients less than 65 years of age.

Role of CD137 signaling in dengue virus-mediated apoptosis

International Nuclear Information System (INIS)

Nagila, Amar; Netsawang, Janjuree; Srisawat, Chatchawan; Noisakran, Sansanee; Morchang, Atthapan; Yasamut, Umpa; Puttikhunt, Chunya; Kasinrerk, Watchara

2011-01-01

Highlights: → For the first time the role of CD137 in dengue virus (DENV) infection. → Induction of DENV-mediated apoptosis by CD137 signaling. → Sensitization to CD137-mediated apoptosis by dengue virus capsid protein (DENV C). → Nuclear localization of DENV C is required for CD137-mediated apoptosis. -- Abstract: Hepatic dysfunction is a well recognized feature of dengue virus (DENV) infection. However, molecular mechanisms of hepatic injury are still poorly understood. A complex interaction between DENV and the host immune response contributes to DENV-mediated tissue injury. DENV capsid protein (DENV C) physically interacts with the human death domain-associated protein Daxx. A double substitution mutation in DENV C (R85A/K86A) abrogates Daxx interaction, nuclear localization and apoptosis. Therefore we compared the expression of cell death genes between HepG2 cells expressing DENV C and DENV C (R85A/K86A) using a real-time PCR array. Expression of CD137, which is a member of the tumor necrosis factor receptor family, increased significantly in HepG2 cells expressing DENV C compared to HepG2 cells expressing DENV C (R85A/K86A). In addition, CD137-mediated apoptotic activity in HepG2 cells expressing DENV C was significantly increased by anti-CD137 antibody compared to that of HepG2 cells expressing DENV C (R85A/K86A). In DENV-infected HepG2 cells, CD137 mRNA and CD137 positive cells significantly increased and CD137-mediated apoptotic activity was increased by anti-CD137 antibody. This work is the first to demonstrate the contribution of CD137 signaling to DENV-mediated apoptosis.

Role of CD137 signaling in dengue virus-mediated apoptosis

Energy Technology Data Exchange (ETDEWEB)

Nagila, Amar [Medical Molecular Biology Unit, Office for Research and Development, Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok (Thailand); Department of Biochemistry, Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok (Thailand); Netsawang, Janjuree [Faculty of Medical Technology, Rangsit University, Bangkok (Thailand); Srisawat, Chatchawan [Department of Biochemistry, Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok (Thailand); Noisakran, Sansanee [Dengue Hemorrhagic Fever Research Unit, Office for Research and Development, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok (Thailand); Medical Biotechnology Unit, National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Bangkok (Thailand); Morchang, Atthapan; Yasamut, Umpa [Medical Molecular Biology Unit, Office for Research and Development, Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok (Thailand); Department of Immunology, Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok (Thailand); Puttikhunt, Chunya [Dengue Hemorrhagic Fever Research Unit, Office for Research and Development, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok (Thailand); Medical Biotechnology Unit, National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Bangkok (Thailand); Kasinrerk, Watchara [Division of Clinical Immunology, Department of Medical Technology, Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai (Thailand); Biomedical Technology Research Center, National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency at Chiang Mai University, Chiang Mai (Thailand); and others

2011-07-08

Highlights: {yields} For the first time the role of CD137 in dengue virus (DENV) infection. {yields} Induction of DENV-mediated apoptosis by CD137 signaling. {yields} Sensitization to CD137-mediated apoptosis by dengue virus capsid protein (DENV C). {yields} Nuclear localization of DENV C is required for CD137-mediated apoptosis. -- Abstract: Hepatic dysfunction is a well recognized feature of dengue virus (DENV) infection. However, molecular mechanisms of hepatic injury are still poorly understood. A complex interaction between DENV and the host immune response contributes to DENV-mediated tissue injury. DENV capsid protein (DENV C) physically interacts with the human death domain-associated protein Daxx. A double substitution mutation in DENV C (R85A/K86A) abrogates Daxx interaction, nuclear localization and apoptosis. Therefore we compared the expression of cell death genes between HepG2 cells expressing DENV C and DENV C (R85A/K86A) using a real-time PCR array. Expression of CD137, which is a member of the tumor necrosis factor receptor family, increased significantly in HepG2 cells expressing DENV C compared to HepG2 cells expressing DENV C (R85A/K86A). In addition, CD137-mediated apoptotic activity in HepG2 cells expressing DENV C was significantly increased by anti-CD137 antibody compared to that of HepG2 cells expressing DENV C (R85A/K86A). In DENV-infected HepG2 cells, CD137 mRNA and CD137 positive cells significantly increased and CD137-mediated apoptotic activity was increased by anti-CD137 antibody. This work is the first to demonstrate the contribution of CD137 signaling to DENV-mediated apoptosis.

Expression of BMP-2 in Vascular Endothelial Cells of Recipient May Predict Delayed Graft Function After Renal Transplantation.

Science.gov (United States)

Basic-Jukic, Nikolina; Gulin, Marijana; Hudolin, Tvrtko; Kastelan, Zeljko; Katalinic, Lea; Coric, Marijana; Veda, Marija Varnai; Ivkovic, Vanja; Kes, Petar; Jelakovic, Bojan

2016-01-01

Delayed graft function (DGF) is associated with adverse outcomes after renal transplantation. Bone morphogenetic protein-2 (BMP-2) is involved in both endothelial function and immunological events. We compared expression of BMP-2 in epigastric artery of renal transplant recipients with immediate graft function (IGF) and DGF. 79 patients were included in this prospective study. Patients were divided in IGF group (64 patients) and DGF group (15 patients). BMP-2 expression in intima media (BMP2m) and endothelium (BMP2e) of epigastric artery was assessed by immunohistochemistry. Lower intensity of BMP2e staining was recorded in DGF compared to IGF. In DGF patients, 93% had no expression of BMP2e and 7% had 1st grade expression, compared to 45% and 41% in IGF group, respectively (P=0.001) (P<0.001 for no expression and P = 0.015 for 1st grade expression). Patients who had BMP2e staining positive had lower odds for DGF (OR 0.059 [0.007, 0.477]) and this remained significant even after adjustment for donor and recipient variables, cold ischemia time, and immunological matching (OR 0.038 [0.003, 0.492]). Our results demonstrate that BMP-2 expression in endothelial cells of epigastric arteries may predict development of DGF. © 2016 The Author(s) Published by S. Karger AG, Basel.

ALK1 signaling inhibits angiogenesis by cooperating with the Notch pathway.

Science.gov (United States)

Larrivée, Bruno; Prahst, Claudia; Gordon, Emma; del Toro, Raquel; Mathivet, Thomas; Duarte, Antonio; Simons, Michael; Eichmann, Anne

2012-03-13

Activin receptor-like kinase 1 (ALK1) is an endothelial-specific member of the TGF-β/BMP receptor family that is inactivated in patients with hereditary hemorrhagic telangiectasia (HHT). How ALK1 signaling regulates angiogenesis remains incompletely understood. Here we show that ALK1 inhibits angiogenesis by cooperating with the Notch pathway. Blocking Alk1 signaling during postnatal development in mice leads to retinal hypervascularization and the appearance of arteriovenous malformations (AVMs). Combined blockade of Alk1 and Notch signaling further exacerbates hypervascularization, whereas activation of Alk1 by its high-affinity ligand BMP9 rescues hypersprouting induced by Notch inhibition. Mechanistically, ALK1-dependent SMAD signaling synergizes with activated Notch in stalk cells to induce expression of the Notch targets HEY1 and HEY2, thereby repressing VEGF signaling, tip cell formation, and endothelial sprouting. Taken together, these results uncover a direct link between ALK1 and Notch signaling during vascular morphogenesis that may be relevant to the pathogenesis of HHT vascular lesions. Copyright © 2012 Elsevier Inc. All rights reserved.

Best Management Practices (BMP) Monitoring Manual Field Guide: Implementation and Effectiveness for Protection of Water Resources

Science.gov (United States)

David Welsch; Roger Ryder; Tim Post

2006-01-01

The specific purpose of the BMP protocol is to create an economical, standardized, and repeatable BMP monitoring process that is completely automated, from data gathering through report generation, in order to provide measured data, ease of use, and compatibility with State BMP programs.The protocol was developed to meet the following needs:? Document the use and...

Defective fluid shear stress mechanotransduction mediates hereditary hemorrhagic telangiectasia

Science.gov (United States)

Baeyens, Nicolas; Larrivée, Bruno; Ola, Roxana; Hayward-Piatkowskyi, Brielle; Dubrac, Alexandre; Huang, Billy; Ross, Tyler D.; Coon, Brian G.; Min, Elizabeth; Tsarfati, Maya; Tong, Haibin; Eichmann, Anne

2016-01-01

Morphogenesis of the vascular system is strongly modulated by mechanical forces from blood flow. Hereditary hemorrhagic telangiectasia (HHT) is an inherited autosomal-dominant disease in which arteriovenous malformations and telangiectasias accumulate with age. Most cases are linked to heterozygous mutations in Alk1 or Endoglin, receptors for bone morphogenetic proteins (BMPs) 9 and 10. Evidence suggests that a second hit results in clonal expansion of endothelial cells to form lesions with poor mural cell coverage that spontaneously rupture and bleed. We now report that fluid shear stress potentiates BMPs to activate Alk1 signaling, which correlates with enhanced association of Alk1 and endoglin. Alk1 is required for BMP9 and flow responses, whereas endoglin is only required for enhancement by flow. This pathway mediates both inhibition of endothelial proliferation and recruitment of mural cells; thus, its loss blocks flow-induced vascular stabilization. Identification of Alk1 signaling as a convergence point for flow and soluble ligands provides a molecular mechanism for development of HHT lesions. PMID:27646277

Bone Morphogenic Protein-2 (rhBMP2)-Loaded Silk Fibroin Scaffolds to Enhance the Osteoinductivity in Bone Tissue Engineering

Science.gov (United States)

Du, Guang-Yu; He, Sheng-Wei; Sun, Chuan-Xiu; Mi, Li-Dong

2017-10-01

There is an increasing demand for formulations of silk fibroin (SF) scaffolds in biomedical applications. SF was crosslinked via glutaraldehyde with osteoinductive recombinant human bone morphogenic protein-2 (rhBMP2) of different ratios viz. (i) 3% SF with no rhBMP2 (SF), (ii) 3% SF with equal amount of rhBMP2 (SF+BMP2), and (iii) 12% SF with 3% of rhBMP2 (4SF+BMP2), and these solutions were used in electrospinning-based fabrication of nanoscaffolds for evaluating increased osteoinductive potential of SF scaffolds with rhBMP2. Stress-strain relationship suggested there is no loss in mechanical strength of fibers with addition of rhBMP2, and mechanical strength of scaffold was improved with increase in concentration of SF. rhBMP2 association increased the water retention capacity of scaffold as evident from swelling studies. Viability of hMSCs was found to be higher in conjugated scaffolds, and scaffolds do not exhibit any cytotoxicity towards guest cells. Cells were found to have higher alkaline phosphatase activity in conjugated scaffolds under in vitro and in vivo conditions which establishes the increased osteoinductivity of the novel construct. The scaffolds were found to be effective for in vivo bone formation as well.

BMP-6 inhibits growth of mature human B cells; induction of Smad phosphorylation and upregulation of Id1

Directory of Open Access Journals (Sweden)

Kersten Christian

2005-05-01

Full Text Available Abstract Background Bone morphogenetic proteins (BMPs belong to the TGF-β superfamily and are secreted proteins with pleiotropic roles in many different cell types. A potential role of BMP-6 in the immune system has been implied by various studies of malignant and rheumatoid diseases. In the present study, we explored the role of BMP-6 in normal human peripheral blood B cells. Results The B cells were found to express BMP type I and type II receptors and BMP-6 rapidly induced phosphorylation of Smad1/5/8. Furthermore, Smad-phosphorylation was followed by upregulation of Id1 mRNA and Id1 protein, whereas Id2 and Id3 expression was not affected. Furthermore, we found that BMP-6 had an antiproliferative effect both in naïve (CD19+CD27- and memory B cells (CD19+CD27+ stimulated with anti-IgM alone or the combined action of anti-IgM and CD40L. Additionally, BMP-6 induced cell death in activated memory B cells. Importantly, the antiproliferative effect of BMP-6 in B-cells was completely neutralized by the natural antagonist, noggin. Furthermore, B cells were demonstrated to upregulate BMP-6 mRNA upon stimulation with anti-IgM. Conclusion In mature human B cells, BMP-6 inhibited cell growth, and rapidly induced phosphorylation of Smad1/5/8 followed by an upregulation of Id1.

Breast Cancer Research Program (BCRP) - Predoctoral Traineeship - Elucidating the Role of the Type III Transforming Growth Factor-beta Receptor in Bone Morphogenetic Signaling in Breast Cancer

Science.gov (United States)

2008-03-01

phosphorylation in the pancreatic cancer cell model, Panc -1 (data not shown). This data emphasize that TβRIII’s role in BMP signaling is likely to be cell... Panc -1 cells were adenovirally infected with TβRIII, followed by BMP-4-induced EMT. The cells were then plated in Matrigel invasion chambers. A...BMP-2, while loss of TβRIII in Panc -1 (pancreatic cancer cells) increased cell sensitivity to BMP-2 and had no effect on MDA-MB-231 (breast cancer

Probabilistic Determination of Green Infrastructure Pollutant Removal Rates from the International Stormwater BMP Database

Science.gov (United States)

Gilliom, R.; Hogue, T. S.; McCray, J. E.

2017-12-01

There is a need for improved parameterization of stormwater best management practices (BMP) performance estimates to improve modeling of urban hydrology, planning and design of green infrastructure projects, and water quality crediting for stormwater management. Percent removal is commonly used to estimate BMP pollutant removal efficiency, but there is general agreement that this approach has significant uncertainties and is easily affected by site-specific factors. Additionally, some fraction of monitored BMPs have negative percent removal, so it is important to understand the probability that a BMP will provide the desired water quality function versus exacerbating water quality problems. The widely used k-C* equation has shown to provide a more adaptable and accurate method to model BMP contaminant attenuation, and previous work has begun to evaluate the strengths and weaknesses of the k-C* method. However, no systematic method exists for obtaining first-order removal rate constants needed to use the k-C* equation for stormwater BMPs; thus there is minimal application of the method. The current research analyzes existing water quality data in the International Stormwater BMP Database to provide screening-level parameterization of the k-C* equation for selected BMP types and analysis of factors that skew the distribution of efficiency estimates from the database. Results illustrate that while certain BMPs are more likely to provide desired contaminant removal than others, site- and design-specific factors strongly influence performance. For example, bioretention systems show both the highest and lowest removal rates of dissolved copper, total phosphorous, and total nitrogen. Exploration and discussion of this and other findings will inform the application of the probabilistic pollutant removal rate constants. Though data limitations exist, this research will facilitate improved accuracy of BMP modeling and ultimately aid decision-making for stormwater quality

Forestry BMP Implementation Costs for Virginia

Science.gov (United States)

R.M. Shaffer; H.L. Haney; E.G. Worrell; W.M. Aust

1998-01-01

Forestry Best Management Practices (BMPs) are operational techniques used to protect water quality during timber harvesting operations. The implementation cost of BMPs is important to loggers, forest landowners, and the forest industry. This study provides an estimate of BMP implementation cost on a per harvested acre basis for the coastal plain, Piedmont, and...

Haploinsufficient Bmp4 ocular phenotypes include anterior segment dysgenesis with elevated intraocular pressure

Directory of Open Access Journals (Sweden)

Nusinowitz Steven

2001-11-01

Full Text Available Abstract Background Glaucoma is a blinding disease usually associated with high intraocular pressure (IOP. In some families, abnormal anterior segment development contributes to glaucoma. The genes causing anterior segment dysgenesis and glaucoma in most of these families are not identified and the affected developmental processes are poorly understood. Bone morphogenetic proteins (BMPs participate in various developmental processes. We tested the importance of Bmp4 gene dosage for ocular development and developmental glaucoma. Results Bmp4+/- mice have anterior segment abnormalities including malformed, absent or blocked trabecular meshwork and Schlemm's canal drainage structures. Mice with severe drainage structure abnormalities, over 80% or more of their angle's extent, have elevated IOP. The penetrance and severity of abnormalities is strongly influenced by genetic background, being most severe on the C57BL/6J background and absent on some other backgrounds. On the C57BL/6J background there is also persistence of the hyaloid vasculature, diminished numbers of inner retinal cells, and absence of the optic nerve. Conclusions We demonstrate that heterozygous deficiency of BMP4 results in anterior segment dysgenesis and elevated IOP. The abnormalities are similar to those in human patients with developmental glaucoma. Thus, BMP4 is a strong candidate to contribute to Axenfeld-Rieger anomaly and other developmental conditions associated with human glaucoma. BMP4 also participates in posterior segment development and wild-type levels are usually critical for optic nerve development on the C57BL/6J background. Bmp4+/- mice are useful for studying various components of ocular development, and may allow identification of strain specific modifiers affecting a variety of ocular phenotypes.

Low-dose rhBMP2/7 heterodimer to reconstruct peri-implant bone defects: a micro-CT evaluation

NARCIS (Netherlands)

Wang, J.; Zheng, Y.; Zhao, J.; Liu, T.; Gao, L.; Gu, Z.; Wu, G.

2012-01-01

Objectives To delineate the dynamic micro-architectures of bone induced by low-dose bone morphogenetic protein (BMP)-2/7 heterodimer in peri-implant bone defects compared to BMP2 and BMP7 homodimer. Material and Methods Peri-implant bone defects (8 mm in diameter, 4 mm in depth) were created

Factors controlling headspace pressure in a manual manometric BMP method can be used to produce a methane output comparable to AMPTS.

Science.gov (United States)

Himanshu, H; Voelklein, M A; Murphy, J D; Grant, J; O'Kiely, P

2017-08-01

The manual manometric biochemical methane potential (mBMP) test uses the increase in pressure to calculate the gas produced. This gas production may be affected by the headspace volume in the incubation bottle and by the overhead pressure measurement and release (OHPMR) frequency. The biogas and methane yields of cellulose, barley, silage and slurry were compared with three incubation bottle headspace volumes (50, 90 and 180ml; constant 70ml total medium) and four OHPMR frequencies (daily, each third day, weekly and solely at the end of experiment). The methane yields of barley, silage and slurry were compared with those from an automated volumetric method (AMPTS). Headspace volume and OHPMR frequency effects on biogas yield were mediated mainly through headspace pressure, with the latter having a negative effect on the biogas yield measured and relatively little effect on methane yield. Two mBMP treatments produced methane yields equivalent to AMPTS. Copyright © 2017 Elsevier Ltd. All rights reserved.

Notch Signaling Mediates Skeletal Muscle Atrophy in Cancer Cachexia Caused by Osteosarcoma

Directory of Open Access Journals (Sweden)

Xiaodong Mu

2016-01-01

Full Text Available Skeletal muscle atrophy in cancer cachexia is mediated by the interaction between muscle stem cells and various tumor factors. Although Notch signaling has been known as a key regulator of both cancer development and muscle stem cell activity, the potential involvement of Notch signaling in cancer cachexia and concomitant muscle atrophy has yet to be elucidated. The murine K7M2 osteosarcoma cell line was used to generate an orthotopic model of sarcoma-associated cachexia, and the role of Notch signaling was evaluated. Skeletal muscle atrophy was observed in the sarcoma-bearing mice, and Notch signaling was highly active in both tumor tissues and the atrophic skeletal muscles. Systemic inhibition of Notch signaling reduced muscle atrophy. In vitro coculture of osteosarcoma cells with muscle-derived stem cells (MDSCs isolated from normal mice resulted in decreased myogenic potential of MDSCs, while the application of Notch inhibitor was able to rescue this repressed myogenic potential. We further observed that Notch-activating factors reside in the exosomes of osteosarcoma cells, which activate Notch signaling in MDSCs and subsequently repress myogenesis. Our results revealed that signaling between tumor and muscle via the Notch pathway may play an important role in mediating the skeletal muscle atrophy seen in cancer cachexia.

Coordinated Proliferation and Differentiation of Human-Induced Pluripotent Stem Cell-Derived Cardiac Progenitor Cells Depend on Bone Morphogenetic Protein Signaling Regulation by GREMLIN 2.

Science.gov (United States)

Bylund, Jeffery B; Trinh, Linh T; Awgulewitsch, Cassandra P; Paik, David T; Jetter, Christopher; Jha, Rajneesh; Zhang, Jianhua; Nolan, Kristof; Xu, Chunhui; Thompson, Thomas B; Kamp, Timothy J; Hatzopoulos, Antonis K

2017-05-01

Heart development depends on coordinated proliferation and differentiation of cardiac progenitor cells (CPCs), but how the two processes are synchronized is not well understood. Here, we show that the secreted Bone Morphogenetic Protein (BMP) antagonist GREMLIN 2 (GREM2) is induced in CPCs shortly after cardiac mesoderm specification during differentiation of human pluripotent stem cells. GREM2 expression follows cardiac lineage differentiation independently of the differentiation method used, or the origin of the pluripotent stem cells, suggesting that GREM2 is linked to cardiogenesis. Addition of GREM2 protein strongly increases cardiomyocyte output compared to established procardiogenic differentiation methods. Our data show that inhibition of canonical BMP signaling by GREM2 is necessary to promote proliferation of CPCs. However, canonical BMP signaling inhibition alone is not sufficient to induce cardiac differentiation, which depends on subsequent JNK pathway activation specifically by GREM2. These findings may have broader implications in the design of approaches to orchestrate growth and differentiation of pluripotent stem cell-derived lineages that depend on precise regulation of BMP signaling.

Coordinated Proliferation and Differentiation of Human-Induced Pluripotent Stem Cell-Derived Cardiac Progenitor Cells Depend on Bone Morphogenetic Protein Signaling Regulation by GREMLIN 2

Science.gov (United States)

Bylund, Jeffery B.; Trinh, Linh T.; Awgulewitsch, Cassandra P.; Paik, David T.; Jetter, Christopher; Jha, Rajneesh; Zhang, Jianhua; Nolan, Kristof; Xu, Chunhui; Thompson, Thomas B.; Kamp, Timothy J.

2017-01-01

Heart development depends on coordinated proliferation and differentiation of cardiac progenitor cells (CPCs), but how the two processes are synchronized is not well understood. Here, we show that the secreted Bone Morphogenetic Protein (BMP) antagonist GREMLIN 2 (GREM2) is induced in CPCs shortly after cardiac mesoderm specification during differentiation of human pluripotent stem cells. GREM2 expression follows cardiac lineage differentiation independently of the differentiation method used, or the origin of the pluripotent stem cells, suggesting that GREM2 is linked to cardiogenesis. Addition of GREM2 protein strongly increases cardiomyocyte output compared to established procardiogenic differentiation methods. Our data show that inhibition of canonical BMP signaling by GREM2 is necessary to promote proliferation of CPCs. However, canonical BMP signaling inhibition alone is not sufficient to induce cardiac differentiation, which depends on subsequent JNK pathway activation specifically by GREM2. These findings may have broader implications in the design of approaches to orchestrate growth and differentiation of pluripotent stem cell-derived lineages that depend on precise regulation of BMP signaling. PMID:28125926

Bone morphogenetic protein-9 suppresses growth of myeloma cells by signaling through ALK2 but is inhibited by endoglin

International Nuclear Information System (INIS)

Olsen, O E; Wader, K F; Misund, K; Våtsveen, T K; Rø, T B; Mylin, A K; Turesson, I; Størdal, B F; Moen, S H; Standal, T; Waage, A; Sundan, A; Holien, T

2014-01-01

Multiple myeloma is a malignancy of plasma cells predominantly located in the bone marrow. A number of bone morphogenetic proteins (BMPs) induce apoptosis in myeloma cells in vitro, and with this study we add BMP-9 to the list. BMP-9 has been found in human serum at concentrations that inhibit cancer cell growth in vitro. We here show that the level of BMP-9 in serum was elevated in myeloma patients (median 176 pg/ml, range 8–809) compared with healthy controls (median 110 pg/ml, range 8–359). BMP-9 was also present in the bone marrow and was able to induce apoptosis in 4 out of 11 primary myeloma cell samples by signaling through ALK2. BMP-9-induced apoptosis in myeloma cells was associated with c-MYC downregulation. The effects of BMP-9 were counteracted by membrane-bound (CD105) or soluble endoglin present in the bone marrow microenvironment, suggesting a mechanism for how myeloma cells can evade the tumor suppressing activity of BMP-9 in multiple myeloma

Regulation of α5 and αV Integrin Expression by GDF-5 and BMP-7 in Chondrocyte Differentiation and Osteoarthritis.

Directory of Open Access Journals (Sweden)

David Garciadiego-Cázares

Full Text Available The Integrin β1 family is the major receptors of the Extracellular matrix (ECM, and the synthesis and degradation balance of ECM is seriously disrupted during Osteoarthritis (OA. In this scenario, integrins modify their pattern expression and regulate chondrocyte differentiation in the articular cartilage. Members of the Transforming growth factor beta (Tgf-β Superfamily, such as Growth differentiation factor 5 (Gdf-5 and Bone morphogenetic protein 7 (Bmp-7, play a key role in joint formation and could regulate the integrin expression during chondrocyte differentiation and osteoarthritis progression in an experimental OA rat model. Decrease of α5 integrin expression in articular cartilage was related with chondrocyte dedifferentiation during OA progression, while increase of α1, α2, and α3 integrin expression was related with fibrous areas in articular cartilage during OA. Hypertrophic chondrocytes expressed αV integrin and was increased in the articular cartilage of rats with OA. Integrin expression during chondrocyte differentiation was also analyzed in a micromass culture system of mouse embryo mesenchymal cells, micromass cultures was treated with Gdf-5 or Bmp-7 for 4 and 6 days, respectively. Gdf-5 induced the expression of the α5 sub-unit, while Bmp-7 induced the expression of the αV sub-unit. This suggests a switch in signaling for prehypertrophic chondrocyte differentiation towards hypertrophy, where Gdf-5 could maintain the articular chondrocyte phenotype and Bmp-7 would induce hypertrophy. Decrease of Ihh expression during late stages of OA in rat model suggest that the ossification in OA rat knees and endochondral ossification could be activated by Bmp-7 and αV integrin in absence of Ihh. Thus, chondrocyte phenotype in articular cartilage is similar to prehypetrophic chondrocyte in growth plate, and is preserved due to the presence of Indian hedgehog (Ihh, Gdf-5 and α5 integrin to maintain articular cartilage and prevent

Characteristics and stimulation potential with BMP-2 and BMP-7 of tenocyte-like cells isolated from the rotator cuff of female donors.

Directory of Open Access Journals (Sweden)

Franka Klatte-Schulz

Full Text Available Tendon bone healing of the rotator cuff is often associated with non-healing or recurrent defects, which seems to be influenced by the patient's age and sex. The present study aims to examine cellular biological characteristics of tenocyte-like cells that may contribute to this impaired rotator cuff healing. Moreover, a therapeutic approach using growth factors could possibly stimulate tendon bone healing. Therefore, our second aim was to identify patient groups who would particularly benefit from growth factor stimulation. Tenocyte-like cells isolated from supraspinatus tendons of female donors younger and older than 65 years of age were characterized with respect to different cellular biological parameters, such as cell density, cell count, marker expression, collagen-I protein synthesis, and stem cell potential. Furthermore, cells of the donor groups were stimulated with BMP-2 and BMP-7 (200 and 1000 ng/ml in 3D-culture and analyzed for cell count, marker expression and collagen-I protein synthesis. Female donors older than 65 years of age showed significantly decreased cell count and collagen-I protein synthesis compared to cells from donors younger than 65 years. Cellular biological parameters including cell count, collagen-I and -III expression, and collagen-I protein synthesis of cells from both donor groups were stimulated with BMP-2 and BMP-7. The cells from donors older than 65 years revealed a decreased stimulation potential for cell count compared to the younger group. Cells from female donors older than 65 years of age showed inferior cellular biological characteristics. This may be one reason for a weaker healing potential observed in older female patients and should be taken into consideration for tendon bone healing of the rotator cuff.

Rac1 promotes chondrogenesis by regulating STAT3 signaling pathway.

Science.gov (United States)

Kim, Hyoin; Sonn, Jong Kyung

2016-09-01

The small GTPase protein Rac1 is involved in a wide range of biological processes including cell differentiation. Previously, Rac1 was shown to promote chondrogenesis in micromass cultures of limb mesenchyme. However, the pathways mediating Rac1's role in chondrogenesis are not fully understood. This study aimed to explore the molecular mechanisms by which Rac1 regulates chondrogenic differentiation. Phosphorylation of signal transducer and activator of transcription 3 (STAT3) was increased as chondrogenesis proceeded in micromass cultures of chick wing bud mesenchyme. Inhibition of Rac1 with NSC23766, janus kinase 2 (JAK2) with AG490, or STAT3 with stattic inhibited chondrogenesis and reduced phosphorylation of STAT3. Conversely, overexpression of constitutively active Rac1 (Rac L61) increased phosphorylation of STAT3. Rac L61 expression resulted in increased expression of interleukin 6 (IL-6), and treatment with IL-6 increased phosphorylation of STAT3. NSC23766, AG490, and stattic prohibited cell aggregation, whereas expression of Rac L61 increased cell aggregation, which was reduced by stattic treatment. Our studies indicate that Rac1 induces STAT3 activation through expression and action of IL-6. Overexpression of Rac L61 increased expression of bone morphogenic protein 4 (BMP4). BMP4 promoted chondrogenesis, which was inhibited by K02288, an activin receptor-like kinase-2 inhibitor, and increased phosphorylation of p38 MAP kinase. Overexpression of Rac L61 also increased phosphorylation of p38 MAPK, which was reduced by K02288. These results suggest that Rac1 activates STAT3 by expression of IL-6, which in turn increases expression and activity of BMP4, leading to the promotion of chondrogenesis. © 2016 International Federation for Cell Biology.

Poly(lactic-co-glycolide) polymer constructs cross-linked with human BMP-6 and VEGF protein significantly enhance rat mandible defect repair.

Science.gov (United States)

Das, Anusuya; Fishero, Brian A; Christophel, J Jared; Li, Ching-Ju; Kohli, Nikita; Lin, Yong; Dighe, Abhijit S; Cui, Quanjun

2016-04-01

We have previously shown that the combined delivery of mesenchymal stem cells (MSCs), vascular endothelial growth factor (VEGF) and bone morphogenetic protein 6 (BMP-6) induces significantly more bone formation than that induced by the delivery of any single factor or a combination of any two factors. We now determine whether the exogenous addition of VEGF and BMP-6 is sufficient for bone healing when MSCs are not provided. Poly(lactic-co-glycolic acid) (PLAGA) microsphere-based three-dimensional scaffolds (P) were fabricated by thermal sintering of PLAGA microspheres. The scaffolds were chemically cross-linked with 200 ng recombinant human VEGF (P(VEGF)) or BMP-6 (P(BMP-6)) or both (P(VEGF+BMP-6)) by the EDC-NHS-MES method. Release of the proteins from the scaffolds was detected for 21 days in vitro which confirmed their comparable potential to supply the proteins in vivo. The scaffolds were delivered to a critical-sized mandibular defect created in 32 Sprague Dawley rats. Significant bone regeneration was observed only in rats with P(VEGF+BMP-6) scaffolds at weeks 2, 8 and 12 as revealed by micro-computer tomography. Vascular ingrowth was higher in the P(VEGF+BMP-6) group as seen by microfil imaging than in other groups. Trichrome staining revealed that a soft callus formed in P(VEGF), P(BMP-6) and P(VEGF+BMP-6) but not in P. MSCs isolated from rat femurs displayed expression of the bone-specific marker osteocalcin when cultured with P(VEGF), P(BMP-6), or P(VEGF+BMP-6) but not with P. Robust mineralization and increased alkaline phosphatase gene expression were seen in rat MSCs when cultured on P(VEGF+BMP-6) but not on P, P(VEGF), or P(BMP-6). Thus, unlike the delivery of VEGF or BMP-6 alone, the combined delivery of VEGF and BMP-6 to the bone defect significantly enhanced bone repair through the enhancement of angiogenesis and the differentiation of endogenously recruited MSCs into the bone repair site.

BMP2 induces PANC-1 cell invasion by MMP-2 overexpression through ROS and ERK.

Science.gov (United States)

Liu, Jun; Ben, Qi-Wen; Yao, Wei-Yan; Zhang, Jian-Jun; Chen, Da-Fan; He, Xiang-Yi; Li, Lei; Yuan, Yao-Zong

2012-06-01

The emerging roles of bone morphogenetic proteins (BMPs) in the initiation and progression of multiple cancers have drawn great attention in cancer research. We hypothesized that BMP2 promotes cancer metastasis by modulating MMP-2 secretion and activity through intracellular ROS regulation and ERK activation in human pancreatic cancer. Our data show that stimulation of PANC-1 cells with BMP2 induced MMP-2 secretion and activation, associated with decreased E-cadherin expression, resulting in epithelial-to-mesenchymal transformation (EMT) and cell invasion. Blockade of ROS by the ROS scavenger, 2-MPG, abolished cell invasion, inhibited the EMT process and decreased MMP-2 expression, suggesting ROS accumulation caused an increase in MMP-2 expression in BMP2-stimulated PANC-1 cell invasion. Furthermore, treatment of PANC-1 cells with 2-MPG or ERK inhibitor PD98059 reduced the phosphorylation of ERK, resulting in attenuation of BMP2-induced cell invasion and MMP-2 activation. Taken together, these results suggest that BMP2 induces the cell invasion of PANC-1 cells by enhancing MMP-2 secretion and acting through ROS accumulation and ERK activation.

Effect of growth factors (BMP-4/7 & bFGF on proliferation & osteogenic differentiation of bone marrow stromal cells

Directory of Open Access Journals (Sweden)

Shaohui Yuan

2013-01-01

Full Text Available Background & objectives: BMP (bone morphogenetic protein-4/7 and bFGF (basic fibroblast growth factor significantly promote the osteogenic activity and the proliferation of rabbit BMSCs (bone marrow stromal cells, respectively. However, their synergistic effects on the proliferation and the differentiation of BMSCs remain unclear. In the present study, the effects of bFGF and BMP-4/7 were investigated on the proliferation and the differentiation of rat BMSCs in vitro. Methods: BMSCs were isolated from New Zealand white rabbits and cultured to the third passage. The samples were divided into five groups according to the material implanted: (A 80 ng/ml BMP-4/7; (B 80 ng/ml bFGF; (C 30 ng/ml BMP-4/7 and 30 ng/ml bFGF; (D 50 ng/ml BMP-4/7 and 50 ng/ml bFGF; and (E 80 ng/ml BMP-4/7 and 80 ng/ml bFGF. Cell proliferation was analyzed using methyl thiazolyl tetrazolium (MTT assay. Alkaline phosphatase activity and osteocalcin (OC dynamics were also measured. Results: BMP-4/7 alone significantly (P<0.05 promoted the proliferation of BMSCs. At the same time, it also promoted or inhibited the osteogenic differentiation of BMSCs. The synergistic effects of BMP-4/7 and bFGF significantly promoted both the proliferation and the osteogenic differentiation of BMSCs. The treatment of the synergistic effects was dose and time dependent. Interpretation & conclusions: A rational combination of BMP-4/7 and bFGF can promote the proliferation and the osteogenic differentiation of BMSCs. In addition, the synergistic functions are effective.

PI3K/Akt is involved in brown adipogenesis mediated by growth differentiation factor-5 in association with activation of the Smad pathway

Energy Technology Data Exchange (ETDEWEB)

Hinoi, Eiichi; Iezaki, Takashi; Fujita, Hiroyuki; Watanabe, Takumi; Odaka, Yoshiaki; Ozaki, Kakeru; Yoneda, Yukio, E-mail: yyoneda@p.kanazawa-u.ac.jp

2014-07-18

Highlights: • Akt is preferentially phosphorylated in BAT and sWAT of aP2-GDF5 mice. • PI3K/Akt signaling is involved in GDF5-induced brown adipogenesis. • PI3K/Akt signaling regulates GDF5-induced Smad5 phosphorylation. - Abstract: We have previously demonstrated promotion by growth differentiation factor-5 (GDF5) of brown adipogenesis for systemic energy expenditure through a mechanism relevant to activating the bone morphological protein (BMP) receptor/mothers against decapentaplegic homolog (Smad)/peroxisome proliferator-activated receptor gamma co-activator 1α (PGC-1α) pathway. Here, we show the involvement of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway in brown adipogenesis mediated by GDF5. Overexpression of GDF5 in cells expressing adipocyte protein-2 markedly accelerated the phosphorylation of Smad1/5/8 and Akt in white and brown adipose tissues. In brown adipose tissue from heterozygous GDF5{sup Rgsc451} mutant mice expressing a dominant-negative (DN) GDF5 under obesogenic conditions, the basal phosphorylation of Smad1/5/8 and Akt was significantly attenuated. Exposure to GDF5 not only promoted the phosphorylation of both Smad1/5/8 and Akt in cultured brown pre-adipocytes, but also up-regulated Pgc1a and uncoupling protein-1 expression in a manner sensitive to the PI3K/Akt inhibitor Ly294002 as well as retroviral infection with DN-Akt. GDF5 drastically promoted BMP-responsive luciferase reporter activity in a Ly294002-sensitive fashion. Both Ly294002 and DN-Akt markedly inhibited phosphorylation of Smad5 in the nuclei of brown pre-adipocytes. These results suggest that PI3K/Akt signals play a role in the GDF5-mediated brown adipogenesis through a mechanism related to activation of the Smad pathway.

Lead induces chondrogenesis and alters transforming growth factor-beta and bone morphogenetic protein signaling in mesenchymal cell populations.

Science.gov (United States)

Zuscik, Michael J; Ma, Lin; Buckley, Taylor; Puzas, J Edward; Drissi, Hicham; Schwarz, Edward M; O'Keefe, Regis J

2007-09-01

It has been established that skeletal growth is stunted in lead-exposed children. Because chondrogenesis is a seminal step during skeletal development, elucidating the impact of Pb on this process is the first step toward understanding the mechanism of Pb toxicity in the skeleton. The aim of this study was to test the hypothesis that Pb alters chondrogenic commitment of mesenchymal cells and to assess the effects of Pb on various signaling pathways. We assessed the influence of Pb on chondrogenesis in murine limb bud mesenchymal cells (MSCs) using nodule formation assays and gene analyses. The effects of Pb on transforming growth factor-beta (TGF-beta) and bone morphogenetic protein (BMP) signaling was studied using luciferase-based reporters and Western analyses, and luciferase-based assays were used to study cyclic adenosine monophosphate response element binding protein (CREB), beta-catenin, AP-1, and nuclear factor-kappa B (NF-kappaB) signaling. We also used an ectopic bone formation assay to determine how Pb affects chondrogenesis in vivo. Pb-exposed MSCs showed enhanced basal and TGF-beta/BMP induction of chondrogenesis, evidenced by enhanced nodule formation and up-regulation of Sox-9, type 2 collagen, and aggrecan, all key markers of chondrogenesis. We observed enhanced chondrogenesis during ectopic bone formation in mice preexposed to Pb via drinking water. In MSCs, Pb enhanced TGF-beta but inhibited BMP-2 signaling, as measured by luciferase reporter assays and Western analyses of Smad phosphorylation. Although Pb had no effect on basal CREB or Wnt/beta-catenin pathway activity, it induced NFkappaB signaling and inhibited AP-1 signaling. The in vitro and in vivo induction of chondrogenesis by Pb likely involves modulation and integration of multiple signaling pathways including TGF-beta, BMP, AP-1, and NFkappaB.

Antibody-Mediated Osseous Regeneration for Bone Tissue Engineering in Canine Segmental Defects

Directory of Open Access Journals (Sweden)

A. Khojasteh

2018-01-01

Full Text Available Among many applications of therapeutic monoclonal antibodies (mAbs, a unique approach for regenerative medicine has entailed antibody-mediated osseous regeneration (AMOR. In an effort to identify a clinically relevant model of craniofacial defect, the present study investigated the efficacy of mAb specific for bone morphogenetic protein- (BMP- 2 to repair canine segmental mandibular continuity defect model. Accordingly, a 15 mm unilateral segmental defect was created in mandible and fixated with a titanium plate. Anorganic bovine bone mineral with 10% collagen (ABBM-C was functionalized with 25 μg/mL of either chimeric anti-BMP-2 mAb or isotype-matched mAb (negative control. Recombinant human (rh BMP-2 served as positive control. Morphometric analyses were performed on computed tomography (CT and histologic images. Bone densities within healed defect sites at 12 weeks after surgery were 1360.81 ± 10.52 Hounsfield Unit (HU, 1044.27 ± 141.16 HU, and 839.45 ± 179.41 HU, in sites with implanted anti-BMP-2 mAb, rhBMP-2, and isotype mAb groups, respectively. Osteoid bone formation in anti-BMP-2 mAb (42.99% ± 8.67 and rhBMP-2 (48.97% ± 2.96 groups was not significantly different but was higher (p<0.05 than in sites with isotype control mAb (26.8% ± 5.35. In view of the long-term objective of translational application of AMOR in humans, the results of the present study demonstrated the feasibility of AMOR in a large clinically relevant animal model.

A single peroxisomal targeting signal mediates matrix protein import in diatoms.

Directory of Open Access Journals (Sweden)

Nicola H Gonzalez

Full Text Available Peroxisomes are single membrane bound compartments. They are thought to be present in almost all eukaryotic cells, although the bulk of our knowledge about peroxisomes has been generated from only a handful of model organisms. Peroxisomal matrix proteins are synthesized cytosolically and posttranslationally imported into the peroxisomal matrix. The import is generally thought to be mediated by two different targeting signals. These are respectively recognized by the two import receptor proteins Pex5 and Pex7, which facilitate transport across the peroxisomal membrane. Here, we show the first in vivo localization studies of peroxisomes in a representative organism of the ecologically relevant group of diatoms using fluorescence and transmission electron microscopy. By expression of various homologous and heterologous fusion proteins we demonstrate that targeting of Phaeodactylum tricornutum peroxisomal matrix proteins is mediated only by PTS1 targeting signals, also for proteins that are in other systems imported via a PTS2 mode of action. Additional in silico analyses suggest this surprising finding may also apply to further diatoms. Our data suggest that loss of the PTS2 peroxisomal import signal is not reserved to Caenorhabditis elegans as a single exception, but has also occurred in evolutionary divergent organisms. Obviously, targeting switching from PTS2 to PTS1 across different major eukaryotic groups might have occurred for different reasons. Thus, our findings question the widespread assumption that import of peroxisomal matrix proteins is generally mediated by two different targeting signals. Our results implicate that there apparently must have been an event causing the loss of one targeting signal even in the group of diatoms. Different possibilities are discussed that indicate multiple reasons for the detected targeting switching from PTS2 to PTS1.

BMP7 transfection induces in-vitro osteogenic differentiation of dental pulp mesenchymal stem cells

Directory of Open Access Journals (Sweden)

Ka Po John Yau

2013-01-01

Full Text Available Objective: To assess whether in-vitro osteogenic differentiation of human dental pulp mesenchymal stem cells can be induced by transient transfection with the gene encoding human bone morphogenic protein 7 (BMP7. Materials and Methods: A mesenchymal stem cell population was isolated from the dental pulp of two extracted permanent premolars, expanded and characterized. The human BMP7 gene, as a recombinant pcDNA3.1/V5-His-TOPO-BMP7 plasmid, was transfected into the cells. Three negative controls were used: No plasmid, empty vector, and an unrelated vector encoding green fluorescent protein. After the interval of 24 and 48 h, mRNA levels of alkaline phosphatase and osteocalcin as markers of in-vitro osteogenic differentiation were measured by real-time polymerase chain reaction and standardized against β-actin mRNA levels. Results: The level of alkaline phosphatase mRNA was significantly higher for the BMP7 group than for all three negative controls 48 h after transfection (706.9 vs. 11.24 for untransfected cells, 78.05 for empty vector, and 73.10 for green fluorescent protein vector. The level of osteocalcin mRNA was significantly higher for the BMP7 group than for all three negative controls 24 h after transfection (1.0, however, decreased after another 24 h. Conclusions: In-vitro osteoblastic differentiation of human dental pulp mesenchymal stem cells, as indicated by expression of alkaline phosphatase and osteocalcin, can be induced by transient transfection with the BMP7 gene.

Biochemicalmethane potential (BMP) of solid organic substrates

DEFF Research Database (Denmark)

Raposo, F.; Fernández-Cegrí, V.; de la Rubia, M.A.

2011-01-01

BACKGROUND: This paper describes results obtained for different participating research groups in an interlaboratory study related to biochemical methane potential (BMP). In this research work, all experimental conditions influencing the test such as inoculum, substrate characteristics and experim...... significantly according to the experimental approaches....

Paracrine-mediated osteoclastogenesis by the osteosarcoma MG63 cell line: is RANKL/RANK signalling really important?

Science.gov (United States)

Costa-Rodrigues, J; Teixeira, C A; Fernandes, M H

2011-08-01

Although in the past little attention has been paid to the influence of osteosarcoma cells in osteoclast function, recent studies suggest a close relationship between osteosarcoma aggressiveness and osteoclastic activity. The present study addresses the paracrine effects of MG63 cells, a human osteosarcoma-derived cell line, on the differentiation of peripheral blood osteoclast precursor cells (PBMC). PBMC were cultured for 21 days in the presence of conditioned media from MG63 cell cultures (CM) collected at 48 h (CM_MG1), 7 days (CM_MG2) and 14 days (CM_MG3). MG63 cell cultures displayed the expression of ALP and BMP-2 and, also, the osteoclastogenic genes M-CSF and RANKL, although with a low expression of RANKL. PBMC cultures supplemented with CM presented an evident osteoclastogenic behavior, which was dependent on the culture period of the MG63 cells. The inductive effect appeared to be more relevant for the differentiation and activation genes, c-myc and c-src, and lower for genes associated with osteoclast function. In addition, PBMC cultures displayed increased functional parameters, including calcium phosphate resorbing activity. Assessment of the PBMC cultures in the presence of U0126, PDTC, and indomethacin suggested that in addition to MEK and NFkB pathways, other signaling mechanisms, probably not involving RANKL/RANK interaction, might be activated in the presence of conditioned medium from MG63. In conclusion, MG63 cell line appears to induce a significant paracrine-mediated osteoclastogenic response. Understanding the mechanisms underlying the interaction of osteosarcoma cells and osteoclasts may contribute to the development of new potential approaches in the treatment of such bone metabolic diseases.

Molecular Signaling Pathways Mediating Osteoclastogenesis Induced by Prostate Cancer Cells

International Nuclear Information System (INIS)

Rafiei, Shahrzad; Komarova, Svetlana V

2013-01-01

Advanced prostate cancer commonly metastasizes to bone leading to osteoblastic and osteolytic lesions. Although an osteolytic component governed by activation of bone resorbing osteoclasts is prominent in prostate cancer metastasis, the molecular mechanisms of prostate cancer-induced osteoclastogenesis are not well-understood. We studied the effect of soluble mediators released from human prostate carcinoma cells on osteoclast formation from mouse bone marrow and RAW 264.7 monocytes. Soluble factors released from human prostate carcinoma cells significantly increased viability of naïve bone marrow monocytes, as well as osteoclastogenesis from precursors primed with receptor activator of nuclear factor κ-B ligand (RANKL). The prostate cancer-induced osteoclastogenesis was not mediated by RANKL as it was not inhibited by osteoprotegerin (OPG). However inhibition of TGFβ receptor I (TβRI), or macrophage-colony stimulating factor (MCSF) resulted in attenuation of prostate cancer-induced osteoclastogenesis. We characterized the signaling pathways induced in osteoclast precursors by soluble mediators released from human prostate carcinoma cells. Prostate cancer factors increased basal calcium levels and calcium fluctuations, induced nuclear localization of nuclear factor of activated t-cells (NFAT)c1, and activated prolonged phosphorylation of ERK1/2 in RANKL-primed osteoclast precursors. Inhibition of calcium signaling, NFATc1 activation, and ERK1/2 phosphorylation significantly reduced the ability of prostate cancer mediators to stimulate osteoclastogenesis. This study reveals the molecular mechanisms underlying the direct osteoclastogenic effect of prostate cancer derived factors, which may be beneficial in developing novel osteoclast-targeting therapeutic approaches

Expression characteristics of BMP2, BMPR-IA and Noggin in different stages of hair follicle in yak skin.

Science.gov (United States)

Song, Liang-Li; Cui, Yan; Yu, Si-Jiu; Liu, Peng-Gang; Liu, Jun; Yang, Xue; He, Jun-Feng; Zhang, Qian

2018-05-01

Bone morphogenetic protein 2 (BMP2), BMP receptor-IA (BMPR-IA), and the BMP2 antagonist Noggin are important proteins involved in regulating the hair follicle (HF) cycle in skin. In order to explore the expression profiles of BMP2, BMPR-IA, and Noggin in the HF cycle of yak skin, we collected adult yak skin in the telogen, proanagen, and midanagen phases of HFs and evaluated gene and protein expression by real-time quantitative polymerase chain reaction (qRT-PCR), western blotting, and immunohistochemistry. qRT-PCR and western blotting results showed that BMP2 and BMPR-IA expression levels were highest in the telogen of HFs and higher than that of Noggin in the same phase. The expression of Noggin was significantly higher in proanagen and midanagen phases of HFs than in the telogen phase, with the highest expression observed in the proanagen phase. Moreover, the expression of Noggin in the proanagen phase was significantly higher than those of BMP2 and BMPR-IA during the same phase. Immunohistochemistry results showed that BMP2, BMPR-IA, and Noggin were expressed in the skin epidermis, sweat glands, sebaceous glands, HF outer root sheath, and hair matrix. In summary, the characteristic expression profiles of BMP2, BMPR-IA, and Noggin suggested that BMP2 and BMPR-IA had inhibitory effects on the growth of HFs in yaks, whereas Noggin promoted the growth of yak HFs, mainly by affecting skin epithelial cell activity. These results provide a basis for further studies of HF development and cycle transition in yak skin. Copyright © 2017. Published by Elsevier Inc.

Disruption of Smad4 in neural crest cells leads to mid-gestation death with pharyngeal arch, craniofacial and cardiac defects

Science.gov (United States)

Nie, Xuguang; Deng, Chu-xia; Wang, Qin; Jiao, Kai

2008-01-01

TGFβ/BMP signaling pathways are essential for normal development of neural crest cells (NCCs). Smad4 encodes the only common Smad protein in mammals, which is a critical nuclear mediator of TGFβ/BMP signaling. In this work, we sought to investigate the roles of Smad4 for development of NCCs. To overcome the early embryonic lethality of Smad4 null mice, we specifically disrupted Smad4 in NCCs using a Cre/loxP system. The mutant mice died at mid-gestation with defects in facial primordia, pharyngeal arches, outflow tract and cardiac ventricles. Further examination revealed that mutant embryos displayed severe molecular defects starting from E9.5. Expression of multiple genes, including Msx1, 2, Ap-2α, Pax3, and Sox9, which play critical roles for NCC development, was downregulated by NCC disruption of Smad4. Moreover, increased cell death was observed in pharyngeal arches from E10.5. However, the cell proliferation rate in these areas was not substantially altered. Taken together, these findings provide compelling genetic evidence that Smad4-mediated activities of TGFβ/BMP signals are essential for appropriate NCC development. PMID:18334251

Bone morphogenetic protein signaling and olig1/2 interact to regulate the differentiation and maturation of adult oligodendrocyte precursor cells.

Science.gov (United States)

Cheng, Xiaoxin; Wang, Yaping; He, Qian; Qiu, Mengsheng; Whittemore, Scott R; Cao, Qilin

2007-12-01

Promotion of remyelination is an important therapeutic strategy for the treatment of the demyelinating neurological disorders. Adult oligodendrocyte precursor cells (OPCs), which normally reside quiescently in the adult central nervous system (CNS), become activated and proliferative after demyelinating lesions. However, the extent of endogenous remyelination is limited because of the failure of adult OPCs to mature into myelinating oligodendrocytes (OLs) in the demyelinated CNS. Understanding the molecular mechanisms that regulate the differentiation of adult OPCs could lead to new therapeutic strategies to treat these disorders. In this study, we established a stable culture of adult spinal cord OPCs and developed a reliable in vitro protocol to induce their sequential differentiation. Adult OPCs expressed bone morphogenetic protein (BMP) type Ia, Ib, and II receptor subunits, which are required for BMP signal transduction. BMP2 and 4 promoted dose-dependent astrocyte differentiation of adult OPCs with concurrent suppression of OL differentiation. Treatment of OPCs with BMP2 and 4 increased ID4 expression and decreased the expression of olig1 and olig2. Overexpression of olig1 or olig2 blocked the astrocyte differentiation of adult OPCs induced by BMP2 and 4. Furthermore, overexpression of both olig1 and olig2, but not olig1 or olig2 alone, rescued OL differentiation from inhibition by BMP2 and 4. Our results demonstrated that downregulation of olig1 and olig2 is an important mechanism by which BMP2 and 4 inhibit OL differentiation of adult OPCs. These data suggest that blocking BMP signaling combined with olig1/2 overexpression could be a useful therapeutic strategy to enhance endogenous remyelination and facilitate functional recovery in CNS demyelinated disorders. Disclosure of potential conflicts of interest is found at the end of this article.

Genome-wide identification of microRNA targets in human ES cells reveals a role for miR-302 in modulating BMP response

Science.gov (United States)

Lipchina, Inna; Elkabetz, Yechiel; Hafner, Markus; Sheridan, Robert; Mihailovic, Aleksandra; Tuschl, Thomas; Sander, Chris; Studer, Lorenz; Betel, Doron

2011-01-01

MicroRNAs are important regulators in many cellular processes, including stem cell self-renewal. Recent studies demonstrated their function as pluripotency factors with the capacity for somatic cell reprogramming. However, their role in human embryonic stem (ES) cells (hESCs) remains poorly understood, partially due to the lack of genome-wide strategies to identify their targets. Here, we performed comprehensive microRNA profiling in hESCs and in purified neural and mesenchymal derivatives. Using a combination of AGO cross-linking and microRNA perturbation experiments, together with computational prediction, we identified the targets of the miR-302/367 cluster, the most abundant microRNAs in hESCs. Functional studies identified novel roles of miR-302/367 in maintaining pluripotency and regulating hESC differentiation. We show that in addition to its role in TGF-β signaling, miR-302/367 promotes bone morphogenetic protein (BMP) signaling by targeting BMP inhibitors TOB2, DAZAP2, and SLAIN1. This study broadens our understanding of microRNA function in hESCs and is a valuable resource for future studies in this area. PMID:22012620

Cyst-Like Osteolytic Formations in Recombinant Human Bone Morphogenetic Protein-2 (rhBMP-2) Augmented Sheep Spinal Fusion.

Science.gov (United States)

Pan, Hsin Chuan; Lee, Soonchul; Ting, Kang; Shen, Jia; Wang, Chenchao; Nguyen, Alan; Berthiaume, Emily A; Zara, Janette N; Turner, A Simon; Seim, Howard B; Kwak, Jin Hee; Zhang, Xinli; Soo, Chia

2017-07-01

Multiple case reports using recombinant human bone morphogenetic protein-2 (rhBMP-2) have reported complications. However, the local adverse effects of rhBMP-2 application are not well documented. In this report we show that, in addition to promoting lumbar spinal fusion through potent osteogenic effects, rhBMP-2 augmentation promotes local cyst-like osteolytic formations in sheep trabecular bones that have undergone anterior lumbar interbody fusion. Three months after operation, conventional computed tomography showed that the trabecular bones of the rhBMP-2 application groups could fuse, whereas no fusion was observed in the control group. Micro-computed tomography analysis revealed that the core implant area's bone volume fraction and bone mineral density increased proportionately with rhBMP-2 dose. Multiple cyst-like bone voids were observed in peri-implant areas when using rhBMP-2 applications, and these sites showed significant bone mineral density decreases in relation to the unaffected regions. Biomechanically, these areas decreased in strength by 32% in comparison with noncystic areas. Histologically, rhBMP-2-affected void sites had an increased amount of fatty marrow, thinner trabecular bones, and significantly more adiponectin- and cathepsin K-positive cells. Despite promoting successful fusion, rhBMP-2 use in clinical applications may result in local adverse structural alterations and compromised biomechanical changes to the bone. Copyright © 2017 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

Bone marrow concentrate promotes bone regeneration with a suboptimal-dose of rhBMP-2.

Science.gov (United States)

Egashira, Kazuhiro; Sumita, Yoshinori; Zhong, Weijian; I, Takashi; Ohba, Seigo; Nagai, Kazuhiro; Asahina, Izumi

2018-01-01

Bone marrow concentrate (BMC), which is enriched in mononuclear cells (MNCs) and platelets, has recently attracted the attention of clinicians as a new optional means for bone engineering. We previously reported that the osteoinductive effect of bone morphogenetic protein-2 (BMP-2) could be enhanced synergistically by co-transplantation of peripheral blood (PB)-derived platelet-rich plasma (PRP). This study aims to investigate whether BMC can effectively promote bone formation induced by low-dose BMP-2, thereby reducing the undesirable side-effects of BMP-2, compared to PRP. Human BMC was obtained from bone marrow aspirates using an automated blood separator. The BMC was then seeded onto β-TCP granules pre-adsorbed with a suboptimal-dose (minimum concentration to induce bone formation at 2 weeks in mice) of recombinant human (rh) BMP-2. These specimens were transplanted subcutaneously to the dorsal skin of immunodeficient-mice and the induction of ectopic bone formation was assessed 2 and 4 weeks post-transplantation. Transplantations of five other groups [PB, PRP, platelet-poor plasma (PPP), bone marrow aspirate (BM), and BM-PPP] were employed as experimental controls. Then, to clarify the effects on vertical bone augmentation, specimens from the six groups were transplanted for on-lay placement on the craniums of mice. The results indicated that BMC, which contained an approximately 2.5-fold increase in the number of MNCs compared to PRP, could accelerate ectopic bone formation until 2 weeks post-transplantation. On the cranium, the BMC group promoted bone augmentation with a suboptimal-dose of rhBMP-2 compared to other groups. Particularly in the BMC specimens harvested at 4 weeks, we observed newly formed bone surrounding the TCP granules at sites far from the calvarial bone. In conclusion, the addition of BMC could reduce the amount of rhBMP-2 by one-half via its synergistic effect on early-phase osteoinduction. We propose here that BMC transplantation

Volatile-Mediated within-Plant Signaling in Hybrid Aspen: Required for Systemic Responses.

Science.gov (United States)

Li, Tao; Blande, James D

2017-04-01

Plant volatiles play crucial roles in signaling between plants and their associated community members, but their role in within-plant signaling remains largely unexplored, particularly under field conditions. Using a system comprising the hybrid aspen (Populus tremula x tremuloides) and the specialized herbivorous leaf beetle (Phratora laticollis) and, combining field, greenhouse and laboratory experiments, we examined whether local damage triggered systemic responses in undamaged branches that lack vascular connection to the damaged branches, and to what extent this was caused by airborne volatile signals versus internal signals. An experiment tracing dye through the vasculature of saplings revealed no downward movement of the dye from upper to lower branches, suggesting a lack of vascular connectivity among branches. However, we found under both field and laboratory conditions that herbivore feeding on upper branches elicited volatile emissions by undamaged lower branches. Greenhouse experiments manipulating air contact between damaged and undamaged branches showed that systemic induction of volatiles was almost eliminated when air contact was interrupted. Our findings clearly demonstrate that herbivore-induced volatiles overcome vascular constraints and mediate within-plant signaling. Further, we found that volatile signaling led to induction of different classes of volatiles under field and environment controlled conditions, with a weaker response observed in the field. This difference not only reflects the dose- and time-dependent nature of volatile signaling, but also points out that future studies should focus more on field observations to better understand the ecological role of volatile-mediated within-plant signaling.

Pannexin 1 channels mediate 'find-me' signal release and membrane permeability during apoptosis.

Science.gov (United States)

Chekeni, Faraaz B; Elliott, Michael R; Sandilos, Joanna K; Walk, Scott F; Kinchen, Jason M; Lazarowski, Eduardo R; Armstrong, Allison J; Penuela, Silvia; Laird, Dale W; Salvesen, Guy S; Isakson, Brant E; Bayliss, Douglas A; Ravichandran, Kodi S

2010-10-14

Apoptotic cells release 'find-me' signals at the earliest stages of death to recruit phagocytes. The nucleotides ATP and UTP represent one class of find-me signals, but their mechanism of release is not known. Here, we identify the plasma membrane channel pannexin 1 (PANX1) as a mediator of find-me signal/nucleotide release from apoptotic cells. Pharmacological inhibition and siRNA-mediated knockdown of PANX1 led to decreased nucleotide release and monocyte recruitment by apoptotic cells. Conversely, PANX1 overexpression enhanced nucleotide release from apoptotic cells and phagocyte recruitment. Patch-clamp recordings showed that PANX1 was basally inactive, and that induction of PANX1 currents occurred only during apoptosis. Mechanistically, PANX1 itself was a target of effector caspases (caspases 3 and 7), and a specific caspase-cleavage site within PANX1 was essential for PANX1 function during apoptosis. Expression of truncated PANX1 (at the putative caspase cleavage site) resulted in a constitutively open channel. PANX1 was also important for the 'selective' plasma membrane permeability of early apoptotic cells to specific dyes. Collectively, these data identify PANX1 as a plasma membrane channel mediating the regulated release of find-me signals and selective plasma membrane permeability during apoptosis, and a new mechanism of PANX1 activation by caspases.

A cascade of morphogenic signaling initiated by the meninges controls corpus callosum formation.

Science.gov (United States)

Choe, Youngshik; Siegenthaler, Julie A; Pleasure, Samuel J

2012-02-23

The corpus callosum is the most prominent commissural connection between the cortical hemispheres, and numerous neurodevelopmental disorders are associated with callosal agenesis. By using mice either with meningeal overgrowth or selective loss of meninges, we have identified a cascade of morphogenic signals initiated by the meninges that regulates corpus callosum development. The meninges produce BMP7, an inhibitor of callosal axon outgrowth. This activity is overcome by the induction of expression of Wnt3 by the callosal pathfinding neurons, which antagonize the inhibitory effects of BMP7. Wnt3 expression in the cingulate callosal pathfinding axons is developmentally regulated by another BMP family member, GDF5, which is produced by the adjacent Cajal-Retzius neurons and turns on before outgrowth of the callosal axons. The effects of GDF5 are in turn under the control of a soluble GDF5 inhibitor, Dan, made by the meninges. Thus, the meninges and medial neocortex use a cascade of signals to regulate corpus callosum development. Copyright © 2012 Elsevier Inc. All rights reserved.

PDL1 Signals through Conserved Sequence Motifs to Overcome Interferon-Mediated Cytotoxicity

Directory of Open Access Journals (Sweden)

Maria Gato-Cañas

2017-08-01

Full Text Available PDL1 blockade produces remarkable clinical responses, thought to occur by T cell reactivation through prevention of PDL1-PD1 T cell inhibitory interactions. Here, we find that PDL1 cell-intrinsic signaling protects cancer cells from interferon (IFN cytotoxicity and accelerates tumor progression. PDL1 inhibited IFN signal transduction through a conserved class of sequence motifs that mediate crosstalk with IFN signaling. Abrogation of PDL1 expression or antibody-mediated PDL1 blockade strongly sensitized cancer cells to IFN cytotoxicity through a STAT3/caspase-7-dependent pathway. Moreover, somatic mutations found in human carcinomas within these PDL1 sequence motifs disrupted motif regulation, resulting in PDL1 molecules with enhanced protective activities from type I and type II IFN cytotoxicity. Overall, our results reveal a mode of action of PDL1 in cancer cells as a first line of defense against IFN cytotoxicity.

Repair of Cranial Bone Defects Using rhBMP2 and Submicron Particle of Biphasic Calcium Phosphate Ceramics with Through-Hole

Directory of Open Access Journals (Sweden)

Byung-Chul Jeong

2015-01-01

Full Text Available Recently a submicron particle of biphasic calcium phosphate ceramic (BCP with through-hole (donut-shaped BCP (d-BCP was developed for improving the osteoconductivity. This study was performed to examine the usefulness of d-BCP for the delivery of osteoinductive rhBMP2 and the effectiveness on cranial bone regeneration. The d-BCP was soaked in rhBMP2 solution and then freeze-dried. Scanning electron microscope (SEM, energy dispersive spectroscopy (EDS, and Raman spectroscopy analyses confirmed that rhBMP2 was well delivered onto the d-BCP surface and the through-hole. The bioactivity of the rhBMP2/d-BCP composite was validated in MC3T3-E1 cells as an in vitro model and in critical-sized cranial defects in C57BL/6 mice. When freeze-dried d-BCPs with rhBMP2 were placed in transwell inserts and suspended above MC3T3-E1, alkaline phosphatase activity and osteoblast-specific gene expression were increased compared to non-rhBMP2-containing d-BCPs. For evaluating in vivo effectiveness, freeze-dried d-BCPs with or without rhBMP2 were implanted into critical-sized cranial defects. Microcomputed tomography and histologic analysis showed that rhBMP2-containing d-BCPs significantly enhanced cranial bone regeneration compared to non-rhBMP2-containing control. These results suggest that a combination of d-BCP and rhBMP2 can accelerate bone regeneration, and this could be used to develop therapeutic strategies in hard tissue healing.

Repair of Cranial Bone Defects Using rhBMP2 and Submicron Particle of Biphasic Calcium Phosphate Ceramics with Through-Hole.

Science.gov (United States)

Jeong, Byung-Chul; Choi, Hyuck; Hur, Sung-Woong; Kim, Jung-Woo; Oh, Sin-Hye; Kim, Hyun-Seung; Song, Soo-Chang; Lee, Keun-Bae; Park, Kwang-Bum; Koh, Jeong-Tae

2015-01-01

Recently a submicron particle of biphasic calcium phosphate ceramic (BCP) with through-hole (donut-shaped BCP (d-BCP)) was developed for improving the osteoconductivity. This study was performed to examine the usefulness of d-BCP for the delivery of osteoinductive rhBMP2 and the effectiveness on cranial bone regeneration. The d-BCP was soaked in rhBMP2 solution and then freeze-dried. Scanning electron microscope (SEM), energy dispersive spectroscopy (EDS), and Raman spectroscopy analyses confirmed that rhBMP2 was well delivered onto the d-BCP surface and the through-hole. The bioactivity of the rhBMP2/d-BCP composite was validated in MC3T3-E1 cells as an in vitro model and in critical-sized cranial defects in C57BL/6 mice. When freeze-dried d-BCPs with rhBMP2 were placed in transwell inserts and suspended above MC3T3-E1, alkaline phosphatase activity and osteoblast-specific gene expression were increased compared to non-rhBMP2-containing d-BCPs. For evaluating in vivo effectiveness, freeze-dried d-BCPs with or without rhBMP2 were implanted into critical-sized cranial defects. Microcomputed tomography and histologic analysis showed that rhBMP2-containing d-BCPs significantly enhanced cranial bone regeneration compared to non-rhBMP2-containing control. These results suggest that a combination of d-BCP and rhBMP2 can accelerate bone regeneration, and this could be used to develop therapeutic strategies in hard tissue healing.

Effect of heat treatment at 600 degree C for 10 hours on human BMP

International Nuclear Information System (INIS)

Izawa, H.; Hachiya, Y.; Muramatsu, K.; Narita, Y.; Kawai, T.

1999-01-01

Viral infection are an extremely serious in allogeneic bone transplantations. While it is essential to kill viruses such as HIV in allogeneic bone graft, the osteoinductive activity must be preserved. Heat treatment of allogeneic bone graft at 60 degree C for 10 hours is effective in killing viruses such as HIV, but it is unclear to what extent the activity of human bone morphogenetic protein (hBMP) is preserved. In this experiment crude hBMP was extracted from both heated and non-heated human bones which were decalcified by the Urist method. Gelatin capsules containing 5mg of crude hBMP were transplanted into the thigh muscles of 5 week old mice. Human bone samples heated in a water bath at 60 degree C for 10 hours and non-heated samples were each transplanted into 5 mice. At 20 days after transplantation, the heterotopic bone formation was compared by evaluation of X-ray and histologicic analysis. X-rays showed heterotopic bone formation in both heated and non-heated samples. Further, histologic analysis showed that peripheral osteoid tissue had developed into laminar bone formation and interlaminar bone marrow was observed. Heterotopic bone formation was induced by crude hBMP from heated bones in a similar way to crude hBMP from non-heated bones observed in X-ray. There was no significant difference in histologic analysis. The crude hBMP, extracted from bones which were heat-treated at 60 degree C for 10 hours induced heterotopic bone formation similar to that in non-heated bone observed by X-ray and histologic analysis at 20 days after transplantation. This demostrates that the heat-treated bone preserved osteoinduction

2-N, 6-O-sulfated chitosan-assisted BMP-2 immobilization of PCL scaffolds for enhanced osteoinduction

Energy Technology Data Exchange (ETDEWEB)

Cao, Lingyan [Key Laboratory for Ultrafine Materials of Ministry of Education, East China University of Science and Technology, Shanghai 200237 (China); Engineering Research Center for Biomedical Materials of Ministry of Education, East China University of Science and Technology, Shanghai 200237 (China); CSIRO Manufacturing, Bayview Avenue, Clayton, Victoria 3168 (Australia); Department of Prosthodontics, College of Stomatology, Ninth People' s Hospital, School of Medicine, Shanghai Jiao Tong University, 639 Zhizaoju Road, Shanghai 200011 (China); Yu, Yuanman [Key Laboratory for Ultrafine Materials of Ministry of Education, East China University of Science and Technology, Shanghai 200237 (China); Engineering Research Center for Biomedical Materials of Ministry of Education, East China University of Science and Technology, Shanghai 200237 (China); Wang, Jing, E-mail: biomatwj@163.com [Key Laboratory for Ultrafine Materials of Ministry of Education, East China University of Science and Technology, Shanghai 200237 (China); Engineering Research Center for Biomedical Materials of Ministry of Education, East China University of Science and Technology, Shanghai 200237 (China); Werkmeister, Jerome A [CSIRO Manufacturing, Bayview Avenue, Clayton, Victoria 3168 (Australia); McLean, Keith M, E-mail: Keith.McLean@csiro.au [CSIRO Manufacturing, Bayview Avenue, Clayton, Victoria 3168 (Australia); Liu, Changsheng, E-mail: liucs@ecust.edu.cn [Key Laboratory for Ultrafine Materials of Ministry of Education, East China University of Science and Technology, Shanghai 200237 (China); Engineering Research Center for Biomedical Materials of Ministry of Education, East China University of Science and Technology, Shanghai 200237 (China)

2017-05-01

The aim of this study was to develop a 2-N, 6-O-sulfated chitosan (26SCS) modified electrospun fibrous PCL scaffold for bone morphogenetic protein-2 (BMP-2) delivery to improve osteoinduction. The PCL scaffold was modified by an aminolysis reaction using ethylenediamine (ED) and 26SCS was immobilized via electrostatic interactions (PCL-N-S). Scaffolds were characterized by scanning electron microscopy (SEM), atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS) and contact angle measurements. In vitro BMP-2 adsorption and release kinetics indicated that modified PCL-N-S scaffolds showed higher levels of binding of BMP-2 (about 30–100 times), moderative burst release (about one third), and prolonged releasing time compared to the unmodified PCL scaffold. The bioactivity of released BMP-2 determined by alkaline phosphatase (ALP) activity assay was maintained and improved 8– 12 times with increasing concentration of immobilized 26SCS on the scaffolds. In vitro studies demonstrated that bone marrow mesenchymal stem cells (BMSCs) attached more readily to the PCL-N-S scaffolds with increased spreading. In conclusion, 26SCS modified PCL scaffolds can be a potent system for the sustained and bioactive delivery of BMP-2. - Graphical abstract: Limited self-regenerating capacity of human body makes the reconstruction of critical size bone defect a significant challenge. Although bone morphogenetic protein-2 (BMP-2) is an important differentiation factor inducing bone regeneration, it's short half-life in vivo and potent side effect at high dosage still show lots of concerns in the clinical use. Herein, modification of electrospun PCL scaffolds was presented through immobilizing of sulfated chitosan (26SCS). The modified scaffolds effectively improve the binding capacity of BMP-2 and exhibited an enhanced bioactivity and sustained release in vitro. Thus, the use of 26SCS modified PCL scaffolds combined with BMP-2 could be a useful scaffold for tissue

Cardiogenic induction of pluripotent stem cells streamlined through a conserved SDF-1/VEGF/BMP2 integrated network.

Directory of Open Access Journals (Sweden)

Anca Chiriac

Full Text Available BACKGROUND: Pluripotent stem cells produce tissue-specific lineages through programmed acquisition of sequential gene expression patterns that function as a blueprint for organ formation. As embryonic stem cells respond concomitantly to diverse signaling pathways during differentiation, extraction of a pro-cardiogenic network would offer a roadmap to streamline cardiac progenitor output. METHODS AND RESULTS: To resolve gene ontology priorities within precursor transcriptomes, cardiogenic subpopulations were here generated according to either growth factor guidance or stage-specific biomarker sorting. Innate expression profiles were independently delineated through unbiased systems biology mapping, and cross-referenced to filter transcriptional noise unmasking a conserved progenitor motif (55 up- and 233 down-regulated genes. The streamlined pool of 288 genes organized into a core biological network that prioritized the "Cardiovascular Development" function. Recursive in silico deconvolution of the cardiogenic neighborhood and associated canonical signaling pathways identified a combination of integrated axes, CXCR4/SDF-1, Flk-1/VEGF and BMP2r/BMP2, predicted to synchronize cardiac specification. In vitro targeting of the resolved triad in embryoid bodies accelerated expression of Nkx2.5, Mef2C and cardiac-MHC, enhanced beating activity, and augmented cardiogenic yield. CONCLUSIONS: Transcriptome-wide dissection of a conserved progenitor profile thus revealed functional highways that coordinate cardiogenic maturation from a pluripotent ground state. Validating the bioinformatics algorithm established a strategy to rationally modulate cell fate, and optimize stem cell-derived cardiogenesis.

Electrospun PLLA nanofiber scaffolds and their use in combination with BMP-2 for reconstruction of bone defects.

Directory of Open Access Journals (Sweden)

Markus D Schofer

Full Text Available Adequate migration and differentiation of mesenchymal stem cells is essential for regeneration of large bone defects. To achieve this, modern graft materials are becoming increasingly important. Among them, electrospun nanofiber scaffolds are a promising approach, because of their high physical porosity and potential to mimic the extracellular matrix (ECM.The objective of the present study was to examine the impact of electrospun PLLA nanofiber scaffolds on bone formation in vivo, using a critical size rat calvarial defect model. In addition we analyzed whether direct incorporation of bone morphogenetic protein 2 (BMP-2 into nanofibers could enhance the osteoinductivity of the scaffolds. Two critical size calvarial defects (5 mm were created in the parietal bones of adult male Sprague-Dawley rats. Defects were either (1 left unfilled, or treated with (2 bovine spongiosa, (3 PLLA scaffolds alone or (4 PLLA/BMP-2 scaffolds. Cranial CT-scans were taken at fixed intervals in vivo. Specimens obtained after euthanasia were processed for histology, histomorphometry and immunostaining (Osteocalcin, BMP-2 and Smad5.PLLA scaffolds were well colonized with cells after implantation, but only showed marginal ossification. PLLA/BMP-2 scaffolds showed much better bone regeneration and several ossification foci were observed throughout the defect. PLLA/BMP-2 scaffolds also stimulated significantly faster bone regeneration during the first eight weeks compared to bovine spongiosa. However, no significant differences between these two scaffolds could be observed after twelve weeks. Expression of osteogenic marker proteins in PLLA/BMP-2 scaffolds continuously increased throughout the observation period. After twelve weeks osteocalcin, BMP-2 and Smad5 were all significantly higher in the PLLA/BMP-2 group than in all other groups.Electrospun PLLA nanofibers facilitate colonization of bone defects, while their use in combination with BMP-2 also increases bone

Congenital amegakaryocytic thrombocytopenia iPS cells exhibit defective MPL-mediated signaling.

Science.gov (United States)

Hirata, Shinji; Takayama, Naoya; Jono-Ohnishi, Ryoko; Endo, Hiroshi; Nakamura, Sou; Dohda, Takeaki; Nishi, Masanori; Hamazaki, Yuhei; Ishii, Ei-ichi; Kaneko, Shin; Otsu, Makoto; Nakauchi, Hiromitsu; Kunishima, Shinji; Eto, Koji

2013-09-01

Congenital amegakaryocytic thrombocytopenia (CAMT) is caused by the loss of thrombopoietin receptor-mediated (MPL-mediated) signaling, which causes severe pancytopenia leading to bone marrow failure with onset of thrombocytopenia and anemia prior to leukopenia. Because Mpl(-/-) mice do not exhibit the human disease phenotype, we used an in vitro disease tracing system with induced pluripotent stem cells (iPSCs) derived from a CAMT patient (CAMT iPSCs) and normal iPSCs to investigate the role of MPL signaling in hematopoiesis. We found that MPL signaling is essential for maintenance of the CD34+ multipotent hematopoietic progenitor (MPP) population and development of the CD41+GPA+ megakaryocyte-erythrocyte progenitor (MEP) population, and its role in the fate decision leading differentiation toward megakaryopoiesis or erythropoiesis differs considerably between normal and CAMT cells. Surprisingly, complimentary transduction of MPL into normal or CAMT iPSCs using a retroviral vector showed that MPL overexpression promoted erythropoiesis in normal CD34+ hematopoietic progenitor cells (HPCs), but impaired erythropoiesis and increased aberrant megakaryocyte production in CAMT iPSC-derived CD34+ HPCs, reflecting a difference in the expression of the transcription factor FLI1. These results demonstrate that impaired transcriptional regulation of the MPL signaling that normally governs megakaryopoiesis and erythropoiesis underlies CAMT.

Drosophila motor neuron retraction during metamorphosis is mediated by inputs from TGF-β/BMP signaling and orphan nuclear receptors.

Directory of Open Access Journals (Sweden)

Ana Boulanger

Full Text Available Larval motor neurons remodel during Drosophila neuro-muscular junction dismantling at metamorphosis. In this study, we describe the motor neuron retraction as opposed to degeneration based on the early disappearance of β-Spectrin and the continuing presence of Tubulin. By blocking cell dynamics with a dominant-negative form of Dynamin, we show that phagocytes have a key role in this process. Importantly, we show the presence of peripheral glial cells close to the neuro-muscular junction that retracts before the motor neuron. We show also that in muscle, expression of EcR-B1 encoding the steroid hormone receptor required for postsynaptic dismantling, is under the control of the ftz-f1/Hr39 orphan nuclear receptor pathway but not the TGF-β signaling pathway. In the motor neuron, activation of EcR-B1 expression by the two parallel pathways (TGF-β signaling and nuclear receptor triggers axon retraction. We propose that a signal from a TGF-β family ligand is produced by the dismantling muscle (postsynapse compartment and received by the motor neuron (presynaptic compartment resulting in motor neuron retraction. The requirement of the two pathways in the motor neuron provides a molecular explanation for the instructive role of the postsynapse degradation on motor neuron retraction. This mechanism insures the temporality of the two processes and prevents motor neuron pruning before postsynaptic degradation.

Drosophila motor neuron retraction during metamorphosis is mediated by inputs from TGF-β/BMP signaling and orphan nuclear receptors.

Science.gov (United States)

Boulanger, Ana; Farge, Morgane; Ramanoudjame, Christophe; Wharton, Kristi; Dura, Jean-Maurice

2012-01-01

Larval motor neurons remodel during Drosophila neuro-muscular junction dismantling at metamorphosis. In this study, we describe the motor neuron retraction as opposed to degeneration based on the early disappearance of β-Spectrin and the continuing presence of Tubulin. By blocking cell dynamics with a dominant-negative form of Dynamin, we show that phagocytes have a key role in this process. Importantly, we show the presence of peripheral glial cells close to the neuro-muscular junction that retracts before the motor neuron. We show also that in muscle, expression of EcR-B1 encoding the steroid hormone receptor required for postsynaptic dismantling, is under the control of the ftz-f1/Hr39 orphan nuclear receptor pathway but not the TGF-β signaling pathway. In the motor neuron, activation of EcR-B1 expression by the two parallel pathways (TGF-β signaling and nuclear receptor) triggers axon retraction. We propose that a signal from a TGF-β family ligand is produced by the dismantling muscle (postsynapse compartment) and received by the motor neuron (presynaptic compartment) resulting in motor neuron retraction. The requirement of the two pathways in the motor neuron provides a molecular explanation for the instructive role of the postsynapse degradation on motor neuron retraction. This mechanism insures the temporality of the two processes and prevents motor neuron pruning before postsynaptic degradation.

Concurrent inhibition of kit- and FcepsilonRI-mediated signaling: coordinated suppression of mast cell activation

DEFF Research Database (Denmark)

Jensen, Bettina M; Beaven, Michael A; Iwaki, Shoko

2008-01-01

Although primarily required for the growth, differentiation, and survival of mast cells, Kit ligand (stem cell factor) is also required for optimal antigen-mediated mast cell activation. Therefore, concurrent inhibition of Kit- and FcepsilonRI-mediated signaling would be an attractive approach...... characterized Kit inhibitor imatinib mesylate (imatinib). In contrast to imatinib, however, hypothemycin also effectively inhibited FcepsilonRI-mediated degranulation and cytokine production in addition to the potentiation of these responses via Kit. The effect of hypothemycin on Kit-mediated responses could...... be explained by its inhibition of Kit kinase activity, whereas the inhibitory effects on FcepsilonRI-dependent signaling were at the level of Btk activation. Because hypothemycin also significantly reduced the mouse passive cutaneous anaphylaxis response in vivo, these data provide proof of principle...

Proteomic analysis of the signaling pathway mediated by the heterotrimeric Gα protein Pga1 of Penicillium chrysogenum.

Science.gov (United States)

Carrasco-Navarro, Ulises; Vera-Estrella, Rosario; Barkla, Bronwyn J; Zúñiga-León, Eduardo; Reyes-Vivas, Horacio; Fernández, Francisco J; Fierro, Francisco

2016-10-06

The heterotrimeric Gα protein Pga1-mediated signaling pathway regulates the entire developmental program in Penicillium chrysogenum, from spore germination to the formation of conidia. In addition it participates in the regulation of penicillin biosynthesis. We aimed to advance the understanding of this key signaling pathway using a proteomics approach, a powerful tool to identify effectors participating in signal transduction pathways. Penicillium chrysogenum mutants with different levels of activity of the Pga1-mediated signaling pathway were used to perform comparative proteomic analyses by 2D-DIGE and LC-MS/MS. Thirty proteins were identified which showed differences in abundance dependent on Pga1 activity level. By modifying the intracellular levels of cAMP we could establish cAMP-dependent and cAMP-independent pathways in Pga1-mediated signaling. Pga1 was shown to regulate abundance of enzymes in primary metabolic pathways involved in ATP, NADPH and cysteine biosynthesis, compounds that are needed for high levels of penicillin production. An in vivo phosphorylated protein containing a pleckstrin homology domain was identified; this protein is a candidate for signal transduction activity. Proteins with possible roles in purine metabolism, protein folding, stress response and morphogenesis were also identified whose abundance was regulated by Pga1 signaling. Thirty proteins whose abundance was regulated by the Pga1-mediated signaling pathway were identified. These proteins are involved in primary metabolism, stress response, development and signal transduction. A model describing the pathways through which Pga1 signaling regulates different cellular processes is proposed.

Comparative expression analyses of bone morphogenetic protein 4 (BMP4) expressions in muscles of tilapia and common carp indicate that BMP4 plays a role in the intermuscular bone distribution in a dose-dependent manner.

Science.gov (United States)

Su, Shengyan; Dong, Zaijie

2018-01-01

Intermuscular bones in fish negatively influence both meet processing and attractiveness to consumers. Tilapia (Oreochromis niloticus) and common carp (Cyprinus carpio) are both major farmed fish species globally, but whereas the former does not possess intermuscular bones, the latter does. Therefore, these two species might present a good model to study the genetic control of distribution of intermuscular bones in fish. Bone morphogenetic protein 4 (BMP4) gene is associated with tissue ossification and bone regeneration in mammals, but in fish its role in ossification remains understudied. To study the relationship between BMP4 and bone distribution in fish, we determined the expression of BMP4 in muscle tissues of common carp and tilapia on transcriptional and translational levels. As the gene has been merely predicted in silico from the genome of common carp, we have cloned and characterized it. The gene (GenBank: HQ446455) contains one intron and two exons, which encode a 400-amino acid protein with high homology to other known BMP4 protein sequences. Phylogenetic analysis showed that common carp clustered within the Cypriniformes clade (zebrafish was the closest ortholog) and tilapia within the Percomorpha clade. Using microCT scanning, we confirmed that intermuscular bones could be observed only in common carp (none in tilapia), but only in dorsal and caudal muscles (none in the ventral muscle). Expression levels of BMP4 in the muscles of common carp were in agreement with this observation both on transcriptional (qPCR) and translational (immunohistochemistry) level: higher in dorsal and caudal muscles, and lower in the ventral muscle. In tilapia, expression of BMP4 gene was also detectable in all three muscles, but expression levels in all three muscles were comparable to the one observed in the ventral muscle of carp, i.e., very low. Therefore, among the six studied muscles, the expression of BMP4 was high only in the two that possess intermuscular bones

NF-kappaB specifically activates BMP-2 gene expression in growth plate chondrocytes in vivo and in a chondrocyte cell line in vitro.

Science.gov (United States)

Feng, Jian Q; Xing, Lianping; Zhang, Jiang-Hong; Zhao, Ming; Horn, Diane; Chan, Jeannie; Boyce, Brendan F; Harris, Stephen E; Mundy, Gregory R; Chen, Di

2003-08-01

Bone morphogenetic protein-2 (BMP-2) regulates growth plate chondrogenesis during development and postnatal bone growth, but the control mechanisms of BMP-2 expression in growth plate chondrocytes are unknown. Here we have used both in vitro and in vivo approaches to demonstrate that transcription factor, NF-kappaB, regulates BMP-2 gene expression in chondrocytes. Two putative NF-kappaB response elements were found in the -2712/+165 region of the BMP-2 gene. Cotransfection of mutant I-kappaBalpha expression plasmids with BMP-2 promoter-luciferase reporters into TMC-23 chondrocyte cell line suppressed BMP-2 transcription. Mutations in NF-kappaB response elements in the BMP-2 gene lead to decreases in BMP-2 promoter activity. Electrophoretic mobility shift assay using nuclear extracts from TMC-23 chondrocytic cells revealed that the NF-kappaB subunits p50 and p65 bound to the NF-kappaB response elements of the BMP-2 gene. Thus, NF-kappaB may positively regulate BMP-2 gene transcription. Consistent with these findings, expression of BMP-2 mRNA was significantly reduced in growth plate chondrocytes in NF-kappaB p50/p52 dKO mice, which associated with decreased numbers of 5-bromo-2'-deoxyuridine (BrdUrd)-positive cells in the proliferating zone of growth plate in these mice. Therefore, in postnatal growth plate chondrocytes, expression of BMP-2 is regulated by NF-kappaB, which may play an important role in chondrogenesis.