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Sample records for nh2-terminal kinase mediates

  1. n-Butyrate inhibits Jun NH(2)-terminal kinase activation and cytokine transcription in mast cells

    International Nuclear Information System (INIS)

    Diakos, Christos; Prieschl, Eva E.; Saeemann, Marcus D.; Boehmig, Georg A.; Csonga, Robert; Sobanov, Yury; Baumruker, Thomas; Zlabinger, Gerhard J.

    2006-01-01

    Mast cells are well known to contribute to type I allergic conditions but only recently have been brought in association with chronic relapsing/remitting autoimmune diseases such as celiac disease and ulcerative colitis. Since the bacterial metabolite n-butyrate is considered to counteract intestinal inflammation we investigated the effects of this short chain fatty acid on mast cell activation. Using RNAse protection assays and reporter gene technology we show that n-butyrate downregulates TNF-α transcription. This correlates with an impaired activation of the Jun NH(2)-terminal kinase (JNK) but not other MAP kinases such as ERK and p38 that are largely unaffected by n-butyrate. As a consequence, we observed a decreased nuclear activity of AP-1 and NF-AT transcription factors. These results indicate that n-butyrate inhibits critical inflammatory mediators in mast cells by relatively selectively targeting the JNK signalling

  2. n-Butyrate inhibits Jun NH(2)-terminal kinase activation and cytokine transcription in mast cells.

    Science.gov (United States)

    Diakos, Christos; Prieschl, Eva E; Säemann, Marcus D; Böhmig, Georg A; Csonga, Robert; Sobanov, Yury; Baumruker, Thomas; Zlabinger, Gerhard J

    2006-10-20

    Mast cells are well known to contribute to type I allergic conditions but only recently have been brought in association with chronic relapsing/remitting autoimmune diseases such as celiac disease and ulcerative colitis. Since the bacterial metabolite n-butyrate is considered to counteract intestinal inflammation we investigated the effects of this short chain fatty acid on mast cell activation. Using RNAse protection assays and reporter gene technology we show that n-butyrate downregulates TNF-alpha transcription. This correlates with an impaired activation of the Jun NH(2)-terminal kinase (JNK) but not other MAP kinases such as ERK and p38 that are largely unaffected by n-butyrate. As a consequence, we observed a decreased nuclear activity of AP-1 and NF-AT transcription factors. These results indicate that n-butyrate inhibits critical inflammatory mediators in mast cells by relatively selectively targeting the JNK signalling.

  3. SH3-binding protein 5 mediates the neuroprotective effect of the secreted bioactive peptide humanin by inhibiting c-Jun NH2-terminal kinase.

    Science.gov (United States)

    Takeshita, Yuji; Hashimoto, Yuichi; Nawa, Mikiro; Uchino, Hiroyuki; Matsuoka, Masaaki

    2013-08-23

    Humanin is a secreted bioactive peptide that suppresses cell toxicity caused by a variety of insults. The neuroprotective effect of Humanin against Alzheimer disease (AD)-related death is mediated by the binding of Humanin to its heterotrimeric Humanin receptor composed of ciliary neurotrophic receptor α, WSX-1, and gp130, as well as the activation of intracellular signaling pathways including a JAK2 and STAT3 signaling axis. Despite the elucidation of the signaling pathways by which Humanin mediates its neuroprotection, the transcriptional targets of Humanin that behaves as effectors of Humanin remains undefined. In the present study, Humanin increased the mRNA and protein expression of SH3 domain-binding protein 5 (SH3BP5), which has been known to be a JNK interactor, in neuronal cells. Similar to Humanin treatment, overexpression of SH3BP5 inhibited AD-related neuronal death, while siRNA-mediated knockdown of endogenous SH3BP5 expression attenuated the neuroprotective effect of Humanin. These results indicate that SH3BP5 is a downstream effector of Humanin. Furthermore, biochemical analysis has revealed that SH3BP5 binds to JNK and directly inhibits JNK through its two putative mitogen-activated protein kinase interaction motifs (KIMs).

  4. SH3-binding Protein 5 Mediates the Neuroprotective Effect of the Secreted Bioactive Peptide Humanin by Inhibiting c-Jun NH2-terminal Kinase*

    Science.gov (United States)

    Takeshita, Yuji; Hashimoto, Yuichi; Nawa, Mikiro; Uchino, Hiroyuki; Matsuoka, Masaaki

    2013-01-01

    Humanin is a secreted bioactive peptide that suppresses cell toxicity caused by a variety of insults. The neuroprotective effect of Humanin against Alzheimer disease (AD)-related death is mediated by the binding of Humanin to its heterotrimeric Humanin receptor composed of ciliary neurotrophic receptor α, WSX-1, and gp130, as well as the activation of intracellular signaling pathways including a JAK2 and STAT3 signaling axis. Despite the elucidation of the signaling pathways by which Humanin mediates its neuroprotection, the transcriptional targets of Humanin that behaves as effectors of Humanin remains undefined. In the present study, Humanin increased the mRNA and protein expression of SH3 domain-binding protein 5 (SH3BP5), which has been known to be a JNK interactor, in neuronal cells. Similar to Humanin treatment, overexpression of SH3BP5 inhibited AD-related neuronal death, while siRNA-mediated knockdown of endogenous SH3BP5 expression attenuated the neuroprotective effect of Humanin. These results indicate that SH3BP5 is a downstream effector of Humanin. Furthermore, biochemical analysis has revealed that SH3BP5 binds to JNK and directly inhibits JNK through its two putative mitogen-activated protein kinase interaction motifs (KIMs). PMID:23861391

  5. Specific effects of c-Jun NH2-terminal kinase-interacting protein 1 in neuronal axons

    Directory of Open Access Journals (Sweden)

    Shu Tang

    2016-01-01

    Full Text Available c-Jun NH2-terminal kinase (JNK-interacting protein 3 plays an important role in brain-derived neurotrophic factor/tropomyosin-related kinase B (TrkB anterograde axonal transport. It remains unclear whether JNK-interacting protein 1 mediates similar effects, or whether JNK-interacting protein 1 affects the regulation of TrkB anterograde axonal transport. In this study, we isolated rat embryonic hippocampus and cultured hippocampal neurons in vitro. Coimmunoprecipitation results demonstrated that JNK-interacting protein 1 formed TrkB complexes in vitro and in vivo. Immunocytochemistry results showed that when JNK-interacting protein 1 was highly expressed, the distribution of TrkB gradually increased in axon terminals. However, the distribution of TrkB reduced in axon terminals after knocking out JNK-interacting protein 1. In addition, there were differences in distribution of TrkB after JNK-interacting protein 1 was knocked out compared with not. However, knockout of JNK-interacting protein 1 did not affect the distribution of TrkB in dendrites. These findings confirm that JNK-interacting protein 1 can interact with TrkB in neuronal cells, and can regulate the transport of TrkB in axons, but not in dendrites.

  6. cJun NH2-terminal kinase 1 (JNK1): roles in metabolic regulation of insulin resistance

    OpenAIRE

    Sabio, Guadalupe; Davis, Roger J.

    2010-01-01

    The cJun NH2-terminal kinase isoform JNK1 is implicated in the mechanism of obesity-induced insulin resistance. Feeding a high fat diet causes activation of the JNK1 signaling pathway, insulin resistance, and obesity in mice. Germ-line ablation of Jnk1 prevents both diet-induced obesity and insulin resistance. Genetic analysis indicates that the effects of JNK1 on insulin resistance can be separated from effects of JNK1 on obesity. Emerging research indicates that JNK1 plays multiple roles in...

  7. Serpin squamous cell carcinoma antigen inhibits UV-induced apoptosis via suppression of c-JUN NH2-terminal kinase

    OpenAIRE

    Katagiri, Chika; Nakanishi, Jotaro; Kadoya, Kuniko; Hibino, Toshihiko

    2006-01-01

    Protection from ultraviolet (UV) irradiation is a fundamental issue for living organisms. Although melanin's critical role in the protection of basal keratinocytes is well understood, other factors remain essentially unknown. We demonstrate that up-regulation of squamous cell carcinoma antigen-1 (SCCA1) suppresses c-Jun NH2-terminal kinase-1 (JNK1) and thus blocks UV-induced keratinocyte apoptosis. We found that serpin SCCA1 is markedly elevated in the top layers of sun-exposed or UV-irradiat...

  8. Serpin squamous cell carcinoma antigen inhibits UV-induced apoptosis via suppression of c-JUN NH2-terminal kinase.

    Science.gov (United States)

    Katagiri, Chika; Nakanishi, Jotaro; Kadoya, Kuniko; Hibino, Toshihiko

    2006-03-27

    Protection from ultraviolet (UV) irradiation is a fundamental issue for living organisms. Although melanin's critical role in the protection of basal keratinocytes is well understood, other factors remain essentially unknown. We demonstrate that up-regulation of squamous cell carcinoma antigen-1 (SCCA1) suppresses c-Jun NH2-terminal kinase-1 (JNK1) and thus blocks UV-induced keratinocyte apoptosis. We found that serpin SCCA1 is markedly elevated in the top layers of sun-exposed or UV-irradiated epidermis. UV-induced apoptosis was significantly decreased when SCCA was overexpressed in 3T3/J2 cells. It was significantly increased when SCCA was down-regulated with small interfering RNA in HaCaT keratinocytes. A search for SCCA-interacting molecules showed specific binding with phosphorylated JNK. Interestingly, SCCA1 specifically suppressed the kinase activity of JNK1. Upon exposure of keratinocytes to UV, SCCA1 was bound to JNK1 and transferred to the nucleus. Involucrin promoter-driven SCCA1 transgenic mice showed remarkable resistance against UV irradiation. These findings reveal an unexpected serpin function and define a novel UV protection mechanism in human skin.

  9. Triptolide, a diterpenoid triepoxide, induces antitumor proliferation via activation of c-Jun NH2-terminal kinase 1 by decreasing phosphatidylinositol 3-kinase activity in human tumor cells

    International Nuclear Information System (INIS)

    Miyata, Yoshiki; Sato, Takashi; Ito, Akira

    2005-01-01

    Triptolide, a diterpenoid triepoxide extracted from the Chinese herb Tripterygium wilfordii Hook f., exerts antitumorigenic actions against several tumor cells, but the intracellular target signal molecule(s) for this antitumorigenesis activity of triptolide remains to be identified. In the present study, we demonstrated that triptolide, in a dose-dependent manner, inhibited the proliferation of human fibrosarcoma HT-1080, human squamous carcinoma SAS, and human uterine cervical carcinoma SKG-II cells. In addition, triptolide was found to decrease phosphatidylinositol 3-kinase (PI3K) activity. A PI3K inhibitor, LY-294002, mimicked the triptolide-induced antiproliferative activity in HT-1080, SAS, and SKG-II cells. There was no change in the activity of Akt or protein kinase C (PKC), both of which are downstream effectors in the PI3K pathway. Furthermore, the phosphorylation of Ras, Raf, and mitogen-activated protein/extracellular signal-regulated kinase 1/2 was not modified in HT-1080 cells treated with triptolide. However, the phosphorylation of c-Jun NH 2 -terminal kinase 1 (JNK1) was found to increase in both triptolide- and LY-294002-treated cells. Furthermore, the triptolide-induced inhibition of HT-1080 cell proliferation was not observed by JNK1 siRNA-treatment. These results provide novel evidence that PI3K is a crucial target molecule in the antitumorigenic action of triptolide. They further suggest a possible triptolide-induced inhibitory signal for tumor cell proliferation that is initiated by the decrease in PI3K activity, which in turn leads to the augmentation of JNK1 phosphorylation via the Akt and/or PKC-independent pathway(s). Moreover, it is likely that the activation of JNK1 is required for the triptolide-induced inhibition of tumor proliferation

  10. Protein kinase B/Akt activates c-Jun NH(2)-terminal kinase by increasing NO production in response to shear stress

    Science.gov (United States)

    Go, Y. M.; Boo, Y. C.; Park, H.; Maland, M. C.; Patel, R.; Pritchard, K. A. Jr; Fujio, Y.; Walsh, K.; Darley-Usmar, V.; Jo, H.

    2001-01-01

    Laminar shear stress activates c-Jun NH(2)-terminal kinase (JNK) by the mechanisms involving both nitric oxide (NO) and phosphatidylinositide 3-kinase (PI3K). Because protein kinase B (Akt), a downstream effector of PI3K, has been shown to phosphorylate and activate endothelial NO synthase, we hypothesized that Akt regulates shear-dependent activation of JNK by stimulating NO production. Here, we examined the role of Akt in shear-dependent NO production and JNK activation by expressing a dominant negative Akt mutant (Akt(AA)) and a constitutively active mutant (Akt(Myr)) in bovine aortic endothelial cells (BAEC). As expected, pretreatment of BAEC with the PI3K inhibitor (wortmannin) prevented shear-dependent stimulation of Akt and NO production. Transient expression of Akt(AA) in BAEC by using a recombinant adenoviral construct inhibited the shear-dependent stimulation of NO production and JNK activation. However, transient expression of Akt(Myr) by using a recombinant adenoviral construct did not induce JNK activation. This is consistent with our previous finding that NO is required, but not sufficient on its own, to activate JNK in response to shear stress. These results and our previous findings strongly suggest that shear stress triggers activation of PI3K, Akt, and endothelial NO synthase, leading to production of NO, which (along with O(2-), which is also produced by shear) activates Ras-JNK pathway. The regulation of Akt, NO, and JNK by shear stress is likely to play a critical role in its antiatherogenic effects.

  11. Excitatory transmission onto AgRP neurons is regulated by cJun NH2-terminal kinase 3 in response to metabolic stress.

    Science.gov (United States)

    Vernia, Santiago; Morel, Caroline; Madara, Joseph C; Cavanagh-Kyros, Julie; Barrett, Tamera; Chase, Kathryn; Kennedy, Norman J; Jung, Dae Young; Kim, Jason K; Aronin, Neil; Flavell, Richard A; Lowell, Bradford B; Davis, Roger J

    2016-02-24

    The cJun NH2-terminal kinase (JNK) signaling pathway is implicated in the response to metabolic stress. Indeed, it is established that the ubiquitously expressed JNK1 and JNK2 isoforms regulate energy expenditure and insulin resistance. However, the role of the neuron-specific isoform JNK3 is unclear. Here we demonstrate that JNK3 deficiency causes hyperphagia selectively in high fat diet (HFD)-fed mice. JNK3 deficiency in neurons that express the leptin receptor LEPRb was sufficient to cause HFD-dependent hyperphagia. Studies of sub-groups of leptin-responsive neurons demonstrated that JNK3 deficiency in AgRP neurons, but not POMC neurons, was sufficient to cause the hyperphagic response. These effects of JNK3 deficiency were associated with enhanced excitatory signaling by AgRP neurons in HFD-fed mice. JNK3 therefore provides a mechanism that contributes to homeostatic regulation of energy balance in response to metabolic stress.

  12. Eupalmerin acetate, a novel anticancer agent from Caribbean gorgonian octocorals, induces apoptosis in malignant glioma cells via the c-Jun NH2-terminal kinase pathway.

    Science.gov (United States)

    Iwamaru, Arifumi; Iwado, Eiji; Kondo, Seiji; Newman, Robert A; Vera, Burnilda; Rodríguez, Abimael D; Kondo, Yasuko

    2007-01-01

    The marine ecosystem is a vast but largely untapped resource for potential naturally based medicines. We tested 15 compounds derived from organisms found in the Caribbean Sea (14 gorgonian octocoral-derived compounds and one sponge-derived compound) for their anticancer effects on human malignant glioma U87-MG and U373-MG cells. Eupalmerin acetate (EPA) was chosen as the lead compound based on its longer-term stability and greater cytotoxicity than those of the other compounds we tested in these cell types. EPA induced G(2)-M cell cycle arrest and apoptosis via the mitochondrial pathway; it translocated Bax from the cytoplasm to the mitochondria and dissipated the mitochondrial transmembrane potential in both cell types. EPA was found to increase phosphorylated c-Jun NH(2)-terminal kinase (JNK) by >50% in both U87-MG and U373-MG cells. A specific JNK inhibitor, SP600125, inhibited EPA-induced apoptosis, confirming the involvement of the JNK pathway in EPA-induced apoptotic cell death. Furthermore, 7 days of daily intratumoral injections of EPA significantly suppressed the growth of s.c. malignant glioma xenografts (P < 0.01, on day 19). These results indicate that EPA is therapeutically effective against malignant glioma cells in vitro and in vivo and that it, or a similar marine-based compound, may hold promise as a clinical anticancer agent.

  13. Cloning and Functional Characterization of c-Jun NH2-Terminal Kinase from the Mediterranean Species of the Whitefly Bemisia tabaci Complex

    Directory of Open Access Journals (Sweden)

    Xiao-Wei Wang

    2013-06-01

    Full Text Available c-Jun NH2-terminal kinase (JNK signaling is a highly conserved pathway that controls gene transcription in response to a wide variety of biological and environmental stresses. In this study, a JNK from the invasive Mediterranean (MED species of the whitefly Bemisia tabaci complex was cloned and characterized. The full-length JNK cDNA of MED consists of 1565 bp, with an 1176 bp open reading frame encoding 392 amino acids. Comparison of JNK amino acid sequences among different species showed that the sequences of JNKs are highly conserved. To reveal its biological function, the gene expression and functional activation of JNK were analyzed during various stress conditions. Quantitative RT-PCR analysis showed that the relative expression level of JNK remained hardly unchanged when the insects were transferred from cotton (a suitable host plant to tobacco (an unsuitable host plant, infected with bacteria and treated with high and low temperatures. However, the mRNA level of JNK significantly increased when treated with fungal pathogens. Furthermore, we found that the amount of phosphorylated JNK increased significantly after fungal infection, while there is no obvious change for phosphorylated p38 and ERK. Our results indicate that the whitefly JNK plays an important role in whitefly’s immune responses to fungal infection.

  14. Bex2 regulates cell proliferation and apoptosis in malignant glioma cells via the c-Jun NH2-terminal kinase pathway

    International Nuclear Information System (INIS)

    Zhou, Xiuping; Meng, Qingming; Xu, Xuebin; Zhi, Tongle; Shi, Qiong; Wang, Yong; Yu, Rutong

    2012-01-01

    Highlights: ► The expression levels of Bex2 markedly increased in glioma tissues. ► Bex2 over-expression promoted cell proliferation, while its down-regulation inhibited cell growth. ► Bex2 down-regulation promoted cell apoptosis via JNK/c-Jun signaling pathway. -- Abstract: The function of Bex2, a member of the Brain Expressed X-linked gene family, in glioma is controversial and its mechanism is largely unknown. We report here that Bex2 regulates cell proliferation and apoptosis in malignant glioma cells via the c-Jun NH2-terminal kinase (JNK) pathway. The expression level of Bex2 is markedly increased in glioma tissues. We observed that Bex2 over-expression promotes cell proliferation, while down-regulation of Bex2 inhibits cell growth. Furthermore, Bex2 down-regulation promotes cell apoptosis and activates the JNK pathway; these effects were abolished by administration of the JNK specific inhibitor, (SP600125). Thus, Bex2 may be an important player during the development of glioma.

  15. Sodium hydrogen exchange 1 (NHE-1) regulates connexin 43 expression in cardiomyocytes via reverse mode sodium calcium exchange and c-Jun NH2-terminal kinase-dependent pathways.

    Science.gov (United States)

    Stanbouly, Seta; Kirshenbaum, Lorrie A; Jones, Douglas L; Karmazyn, Morris

    2008-10-01

    Connexin 43, the major connexin isoform in gap junctions of cardiac ventricular myocytes, undergoes changes in distribution and expression in cardiac diseases. The Na(+)-H(+) exchanger (NHE-1), a key mediator of hypertrophy and heart failure, has been shown to be localized in the cardiomyocyte gap junctional regions; however, whether NHE-1 regulates gap junction proteins in the hypertrophied cardiomyocyte is not known. To address this question, neonatal rat ventricular myocytes were treated with phenylephrine (PE) for 24 h to induce hypertrophy. Increased Cx43 expression observed with PE treatment (132.4 +/- 6.3% compared to control; P isopropyl-3-(methylsulfonyl)benzoyl-guanidine methanesulfonate]. PE-induced hypertrophy was associated with mitogen-activated protein kinase c-Jun NH(2)-terminal kinase (JNK) 1/2 activation, whereas inhibition of JNK1/2 with either SP600125 [anthra(1,9-cd)pyrazol-6(2H)-one 1,9-pyrazoloanthrone] or small interfering RNA significantly increased PE-induced up-regulation of Cx43 protein levels. Inhibition of reverse mode Na(+)-Ca(2+) exchange (NCX) with KB-R7943 [2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl]isothiourea mesylate] partially reversed JNK1/2 activation (195.2 +/- 21.4 versus 143.7 +/- 14.4% with KB-R7943; P < 0.05) and augmented up-regulation of Cx43 protein (121.1 +/- 8.3 versus 215.9 +/- 25.6% with KB-R7943; P < 0.05) in the presence of PE. Our results demonstrate that NHE-1 negatively regulates Cx43 protein expression in PE-induced cardiomyocyte hypertrophy via a JNK1/2-dependent pathway, which is probably activated by reverse mode NCX activity.

  16. Dual inhibitory roles of geldanamycin on the c-Jun NH2-terminal kinase 3 signal pathway through suppressing the expression of mixed-lineage kinase 3 and attenuating the activation of apoptosis signal-regulating kinase 1 via facilitating the activation of Akt in ischemic brain injury.

    Science.gov (United States)

    Wen, X-R; Li, C; Zong, Y-Y; Yu, C-Z; Xu, J; Han, D; Zhang, G-Y

    2008-10-15

    It is well documented that heat-shock protein (hsp90) plays an essential role in maintaining stability and activity of its clients. Recent studies have shown that geldanamycin (GA), an inhibitor of hsp90, could decrease the protein of mixed-lineage kinase (MLK) 3 and activate Akt; our previous research documented that MLK3 and Akt and subsequent c-Jun N-terminal kinase (JNK) were involved in neuronal cell death in ischemic brain injury. Here, we investigated whether GA could decrease the protein of MLK3 and activate Akt in rat four-vessel occlusion ischemic model. Our results showed that global cerebral ischemia followed by reperfusion could enhance the association of hsp90 with MLK3, the association of hsp90 with Src, and JNK3 activation. As a result, GA decreased the protein of MLK3 and down-regulated JNK activation. On the other hand, Src kinase was activated and phosphorylated Cbl, which then recruited the p85 subunit of phosphatidylinositol 3-kinase (PI-3K), resulting in PI-3K activation, and as a consequence increased Akt activation, which inhibited ASK1 activation and down-regulated JNK3 activation. In summary, our results indicated that GA showed a dual inhibitory role on JNK3 activation and exerted strong neuroprotection in vivo and in vitro, which provides a new possible approach for stroke therapy.

  17. The human receptor for urokinase plasminogen activator. NH2-terminal amino acid sequence and glycosylation variants

    DEFF Research Database (Denmark)

    Behrendt, N; Rønne, E; Ploug, M

    1990-01-01

    , but no N-acetyl-D-galactosamine. Glycosylation is responsible for substantial heterogeneity in the receptor on phorbol ester-stimulated U937 cells, and also for molecular weight variations among various cell lines. The amino acid composition and the NH2-terminal amino acid sequence are reported...... amino-terminal fragment; 2) the identical electrophoretic mobilities observed for cross-linked conjugates, formed between either the purified protein or the u-PA receptor on intact U937 cells and the above ligands; 3) the identity of the apparent molecular weight of the purified protein...

  18. NH2-terminal amino acid distribution and amino acid composition of Streptococcus faecalis R soluble and ribosomal proteins.

    Science.gov (United States)

    Samuel, C E; Murray, C L; Rabinowitz, J C

    1973-10-01

    The NH(2)-terminal amino acid distribution of Streptococcus faecalis R soluble and ribosomal proteins isolated from cells at different stages of growth on either folate-sufficient or folate-deficient medium was determined by the dinitrophenyl method. The NH(2)-terminal residues do not follow the random distribution observed for the total amino acid composition of S. faecalis soluble and ribosomal proteins. Methionine and alanine occur most frequently; serine, threonine, aspartic and glutamic acids, and glycine are also present at the NH(2)-terminal position of S. faecalis R proteins. The absence of folic acid yields cells that are incapable of formylating methionyl-transfer ribonucelic acid tRNA(f) (Met), but does not affect either the qualitative or quantitative NH(2)-terminal distribution of total soluble or total ribosomal proteins compared to cells grown with folate. A small quantitative difference was observed in the frequency of distribution of certain amino acids at the NH(2)-termini between log and stationary phase soluble proteins. The amino acid residues found at the NH(2)-terminal position of S. faecalis proteins are qualitatively similar to those reported for several other organisms.

  19. An anti-NH2-terminal antibody localizes NBCn1 to heart endothelia and skeletal and vascular smooth muscle cells

    DEFF Research Database (Denmark)

    Damkier, Helle Hasager; Nielsen, Søren; Prætorius, Jeppe

    2006-01-01

    The electroneutral sodium bicarbonate cotransporter NBCn1 or NBC3 was originally cloned from rat aorta and from human skeletal muscle. NBCn1 (or NBC3) has been localized to the basolateral membrane of various epithelia, but thus far it has been impossible to detect the protein in these tissues...... and vasculature in skeletal muscle. Analysis of variable NBCn1 splicing by RT-PCR revealed that an NH2-terminal sequence, the cassette III, seems absent from cardiovascular NBCn1 and that both cassettes I and III are variable in most epithelia, whereas cassette II is absent from epithelial NBCn1. Thus...

  20. NH2-terminal monoiodination of hexadecapeptide gastrin: a simple procedure for preparation of 125I-gastrin for radioimmunoassays and receptor studies

    International Nuclear Information System (INIS)

    Rehfeld, J.F.

    1980-01-01

    A simple procedure has been developed for monoiodination of gastrin to a high specific radioactivity. The COOH-terminal hexadecapeptide fragment of gastrin-17 was coupled at the NH 2 -terminal glycine residue to iodinated 3-(4-hydroxyphenyl) propionic acid N-hydroxysuccinimide ester. The labelled hexadecepeptide was then efficiently separated from unlabelled hexapeptide and other reagents by chromatography on Sephadex G-50 superfine columns. The mean specific radioactivity was 560 μCi/nmol (n = 5). The tracer was stable and useful for three to four months. The present NH 2 -terminal monoiodination procedure is a valuable supplement to our previous technique for monoiodination of gastrin at tyrosine-12, in that the NH 2 -terminal labelled gastrin is well bound also to antisera and receptors specific for the tyrosine containing sequence of gastrin. In addition the present prcedure requires only simple gel filtration instead of ion-exchange chromatography in order to achieve adequate separation of labelled from unlabelled gastrin. (Auth.)

  1. Procalcitonin NH2-terminal cleavage peptide has no mitogenic effect on normal human osteoblast-like cells

    International Nuclear Information System (INIS)

    Hassager, C.; Bonde, S.K.; Anderson, M.A.; Rink, H.; Spelsberg, T.C.; Riggs, B.L.

    1991-01-01

    The NH2-terminal cleavage peptide of procalcitonin (N-proCT) recently was reported to be a bone cell mitogen. The authors have investigated the effect of N-proCT on the proliferation of normal human cells that have the phenotype of mature osteoblasts (hOB cells). N-proCT treatment for 24, 48, or 96 h in concentrations from 1 nM to 1 microM did not significantly increase [3H]thymidine uptake (means ranged from -19% to 38% of control, no significant differences) in hOB cells (6-10 cell strains per experiment) plated at four different densities. However, the hOB cells responded significantly to treatment with transforming growth factor β (3 ng/ml), bovine insulin (300 micrograms/ml), or 30% fetal calf serum, which were included in all experiments as positive controls. The [3H]thymidine uptake data were confirmed in a direct cell count experiment tested at 96 h. Thus they data do not support the hypothesis that N-proCT is a potent mitogen for normal human osteoblasts

  2. Treponema pallidum subsp. pallidum TP0136 protein is heterogeneous among isolates and binds cellular and plasma fibronectin via its NH2-terminal end.

    Science.gov (United States)

    Ke, Wujian; Molini, Barbara J; Lukehart, Sheila A; Giacani, Lorenzo

    2015-03-01

    Adherence-mediated colonization plays an important role in pathogenesis of microbial infections, particularly those caused by extracellular pathogens responsible for systemic diseases, such as Treponema pallidum subsp. pallidum (T. pallidum), the agent of syphilis. Among T. pallidum adhesins, TP0136 is known to bind fibronectin (Fn), an important constituent of the host extracellular matrix. To deepen our understanding of the TP0136-Fn interaction dynamics, we used two naturally-occurring sequence variants of the TP0136 protein to investigate which region of the protein is responsible for Fn binding, and whether TP0136 would adhere to human cellular Fn in addition to plasma Fn and super Fn as previously reported. Fn binding assays were performed with recombinant proteins representing the two full-length TP0136 variants and their discrete regions. As a complementary approach, we tested inhibition of T. pallidum binding to Fn by recombinant full-length TP0136 proteins and fragments, as well as by anti-TP0136 immune sera. Our results show that TP0136 adheres more efficiently to cellular Fn than to plasma Fn, that the TP0136 NH2-terminal conserved region of the protein is primarily responsible for binding to plasma Fn but that binding sites for cellular Fn are also present in the protein's central and COOH-terminal regions. Additionally, message quantification studies show that tp0136 is highly transcribed during experimental infection, and that its message level increases in parallel to the host immune pressure on the pathogen, which suggests a possible role for this protein in T. pallidum persistence. In a time where syphilis incidence is high, our data will help in the quest to identify suitable targets for development of a much needed vaccine against this important disease.

  3. Inducible STAT3 NH2 terminal mono-ubiquitination promotes BRD4 complex formation to regulate apoptosis.

    Science.gov (United States)

    Ray, Sutapa; Zhao, Yingxin; Jamaluddin, Mohammad; Edeh, Chukwudi B; Lee, Chang; Brasier, Allan R

    2014-07-01

    Signal Transducers and Activator of Transcription-3 (STAT3) are latent transcription factors that are regulated by post-translational modifications (PTMs) in response to cellular activation by the IL-6 superfamily of cytokines to regulate cell cycle progression and/or apoptosis. Here we observe that STAT3 is inducibly mono-ubiquitinated and investigate its consequences. Using domain mapping and highly specific selected reaction monitoring-mass spectrometric assays, we identify lysine (K) 97 in its NH2-terminal domain as the major mono-ubiquitin conjugation site. We constructed a mono-ubiquitinated mimic consisting of a deubiquitinase-resistant monomeric ubiquitin fused to the NH2 terminus of STAT3 (ubiquitinated-STAT3 FP). In complex assays of ectopically expressed ubi-STAT3-FP, we observed enhanced complex formation with bromodomain-containing protein 4 (BRD4), a component of the activated positive transcriptional elongation factor (P-TEFb) complex. Chromatin immunoprecipitation experiments in STAT3(+/-) and STAT3(-/-) MEFs showed BRD4 recruitment to STAT3-dependent suppressor of cytokine signaling-3 gene (SOCS3). The effect of a selective small molecule inhibitor of BRD4, JQ1, to inhibit SOCS3 expression demonstrated the functional role of BRD4 for STAT3-dependent transcription. Additionally, ectopic ubiquitinated-STAT3 FP expression upregulated BCL2, BCL2L1, APEX1, SOD2, CCND1 and MYC expression indicating the role of ubiquitinated STAT3 in anti-apoptosis and cellular proliferation. Finally we observed that ubiquitinated-STAT3 FP suppressed TNFα-induced apoptotic cell death, indicating the functional importance of mono-ubiquitinated STAT3 in antiapoptotic gene expression. We conclude that STAT3 mono-ubiquitination is a key trigger in BRD4-dependent antiapoptotic and pro-proliferative gene expression programs. Thus, inhibiting the STAT3 mono-ubiquitination-BRD4 pathway may be a novel therapeutic target for the treatment of STAT3-dependent proliferative diseases

  4. Peptide affinity analysis of proteins that bind to an unstructured NH2-terminal region of the osmoprotective transcription factor NFAT5

    Science.gov (United States)

    DuMond, Jenna F.; Ramkissoon, Kevin; Zhang, Xue; Izumi, Yuichiro; Wang, Xujing; Eguchi, Koji; Gao, Shouguo; Mukoyama, Masashi; Ferraris, Joan D.

    2016-01-01

    NFAT5 is an osmoregulated transcription factor that particularly increases expression of genes involved in protection against hypertonicity. Transcription factors often contain unstructured regions that bind co-regulatory proteins that are crucial for their function. The NH2-terminal region of NFAT5 contains regions predicted to be intrinsically disordered. We used peptide aptamer-based affinity chromatography coupled with mass spectrometry to identify protein preys pulled down by one or more overlapping 20 amino acid peptide baits within a predicted NH2-terminal unstructured region of NFAT5. We identify a total of 467 unique protein preys that associate with at least one NH2-terminal peptide bait from NFAT5 in either cytoplasmic or nuclear extracts from HEK293 cells treated with elevated, normal, or reduced NaCl concentrations. Different sets of proteins are pulled down from nuclear vs. cytoplasmic extracts. We used GeneCards to ascertain known functions of the protein preys. The protein preys include many that were previously known, but also many novel ones. Consideration of the novel ones suggests many aspects of NFAT5 regulation, interaction and function that were not previously appreciated, for example, hypertonicity inhibits NFAT5 by sumoylating it and the NFAT5 protein preys include components of the CHTOP complex that desumoylate proteins, an action that should contribute to activation of NFAT5. PMID:26757802

  5. Cell-induced potentiation of the plasminogen activation system is abolished by a monoclonal antibody that recognizes the NH2-terminal domain of the urokinase receptor

    DEFF Research Database (Denmark)

    Rønne, E; Behrendt, N; Ellis, V

    1991-01-01

    -u-PA and plasminogen with U937 cells. This antibody, which is also the only one to completely inhibit the binding of DFP-inactivated [125I]-u-PA to U937 cells, is directed against the u-PA binding NH2-terminal domain of u-PAR, a well-defined fragment formed by limited chymotrypsin digestion of purified u......-PAR, demonstrating the functional independence of the u-PA binding domain as well as the critical role of u-PAR in the assembly of the cell-surface plasminogen activation system....

  6. Increased phosphorylation of skeletal muscle glycogen synthase at NH2-terminal sites during physiological hyperinsulinemia in type 2 diabetes

    DEFF Research Database (Denmark)

    Højlund, Kurt; Staehr, Peter; Hansen, Bo Falck

    2003-01-01

    -hyperinsulinemic clamps. Analysis using phospho-specific antibodies revealed that insulin decreases phosphorylation of sites 3a + 3b in human muscle, and this was accompanied by activation of Akt and inhibition of glycogen synthase kinase-3alpha. In type 2 diabetic subjects these effects of insulin were fully intact...

  7. Dysregulation of glycogen synthase COOH- and NH2-terminal phosphorylation by insulin in obesity and type 2 diabetes mellitus

    DEFF Research Database (Denmark)

    Højlund, Kurt; Birk, Jesper Bratz; Klein, Ditte Kjærsgaard

    2009-01-01

    Context: Insulin-stimulated glucose disposal is impaired in obesity and type 2 diabetes mellitus (T2DM) and is tightly linked to impaired skeletal muscle glucose uptake and storage. Impaired activation of glycogen synthase (GS) by insulin is a well-established defect in both obesity and T2DM......, but the underlying mechanisms remain unclear. Design and Participants: Insulin action was investigated in a matched cohort of lean healthy, obese nondiabetic, and obese type 2 diabetic subjects by the euglycemic-hyperinsulinemic clamp technique combined with muscle biopsies. Activity, site-specific phosphorylation....... The exaggerated insulin resistance in T2DM compared with obese subjects was not reflected by differences in site 3 phosphorylation but was accompanied by a significantly higher site 1b phosphorylation during insulin stimulation. Hyperphosphorylation of another Ca(2+)/calmodulin-dependent kinase-II target...

  8. Isolation and characterization of the normal crossreacting antigen: homology of its NH2-terminal amino acid sequence with that of carcinoembryonic antigen.

    Science.gov (United States)

    Engvall, E; Shively, J E; Wrann, M

    1978-01-01

    The normal antigen, NCA, which crossreacts with the carcinoembryonic antigen, CEA, was purified from normal spleen tissue by an immunochemical purification method using insolubilized antibodies to either CEA or NCA. The highly purified NCA obtained was extensively characterized by immunological tests. The molecular weight of NCA determined by chromatography on Sephadex G-200 was approximately 100,000. The total amount of carbohydrate in NCA was 30%, compared to 60% in CEA. NCA and CEA also differed in sugar composition. The amino acid composition of NCA was nearly identical to that of CEA, except for the apparent presence of methionine in NCA but not in CEA. The sequence of the first 26 NH2-terminal amino acids in NCA was identical to that of CEA except at position 21, where alanine was found in NCA instead of valine in CEA. Images PMID:77016

  9. Increased phosphorylation of skeletal muscle glycogen synthase at NH2-terminal sites during physiological hyperinsulinemia in type 2 diabetes

    DEFF Research Database (Denmark)

    Højlund, Kurt; Staehr, Peter; Hansen, Bo Falck

    2003-01-01

    In type 2 diabetes, insulin activation of muscle glycogen synthase (GS) is impaired. This defect plays a major role for the development of insulin resistance and hyperglycemia. In animal muscle, insulin activates GS by reducing phosphorylation at both NH(2)- and COOH-terminal sites, but the mecha...... in the phosphorylation of sites 2 + 2a. This phosphorylation abnormality likely caused the impaired GS activation and glucose storage, thereby contributing to skeletal muscle insulin resistance, and may therefore play a pathophysiological role in type 2 diabetes.......In type 2 diabetes, insulin activation of muscle glycogen synthase (GS) is impaired. This defect plays a major role for the development of insulin resistance and hyperglycemia. In animal muscle, insulin activates GS by reducing phosphorylation at both NH(2)- and COOH-terminal sites....... Despite that, insulin-mediated glucose disposal and storage were reduced and activation of GS was virtually absent in type 2 diabetic subjects. Insulin did not decrease phosphorylation of sites 2 + 2a in healthy human muscle, whereas in diabetic muscle insulin infusion in fact caused a marked increase...

  10. Suppression of STAT3 NH2-terminal domain chemosensitizes medulloblastoma cells by activation of protein inhibitor of activated STAT3 via de-repression by microRNA-21.

    Science.gov (United States)

    Ray, Sutapa; Coulter, Don W; Gray, Shawn D; Sughroue, Jason A; Roychoudhury, Shrabasti; McIntyre, Erin M; Chaturvedi, Nagendra K; Bhakat, Kishor K; Joshi, Shantaram S; McGuire, Timothy R; Sharp, John G

    2018-04-01

    Medulloblastoma (MB) is a malignant pediatric brain tumor with poor prognosis. Signal transducers and activators of transcription-3 (STAT3) is constitutively activated in MB where it functions as an oncoprotein, mediating cancer progression and metastasis. Here, we have delineated the functional role of activated STAT3 in MB, by using a cell permeable STAT3-NH 2 terminal domain inhibitor (S3-NTDi) that specifically perturbs the structure/function of STAT3. We have implemented several biochemical experiments using human MB tumor microarray (TMA) and pediatric MB cell lines, derived from high-risk SHH-TP53-mutated and MYC-amplified Non-WNT/SHH tumors. Treatment of MB cells with S3-NTDi leads to growth inhibition, cell cycle arrest, and apoptosis. S3-NTDi downregulated expression of STAT3 target genes, delayed migration of MB cells, attenuated epithelial-mesenchymal transition (EMT) marker expressions and reduced cancer stem-cell associated protein expressions in MB-spheres. To elucidate mechanisms, we showed that S3-NTDi induce expression of pro-apoptotic gene, C/EBP-homologous protein (CHOP), and decrease association of STAT3 to the proximal promoter of CCND1 and BCL2. Of note, S3-NTDi downregulated microRNA-21, which in turn, de-repressed Protein Inhibitor of Activated STAT3 (PIAS3), a negative regulator of STAT3 signaling pathway. Furthermore, combination therapy with S3-NTDi and cisplatin significantly decreased highly aggressive MYC-amplified MB cell growth and induced apoptosis by downregulating STAT3 regulated proliferation and anti-apoptotic gene expression. Together, our results revealed an important role of STAT3 in regulating MB pathogenesis. Disruption of this pathway with S3-NTDi, therefore, may serves as a promising candidate for targeted MB therapy by enhancing chemosensitivity of MB cells and potentially improving outcomes in high-risk patients. © 2017 Wiley Periodicals, Inc.

  11. Proline residues in the HIV-1 NH2-terminal capsid domain: structure determinants for proper core assembly and subsequent steps of early replication.

    Science.gov (United States)

    Fitzon, T; Leschonsky, B; Bieler, K; Paulus, C; Schröder, J; Wolf, H; Wagner, R

    2000-03-15

    that proline residues in the NH(2)-terminal capsid domain represent critical structure determinants for proper formation of functional virion cores and subsequent stages of early replication. Copyright 2000 Academic Press.

  12. Andrographolide sensitizes cancer cells to TRAIL-induced apoptosis via p53-mediated death receptor 4 up-regulation.

    Science.gov (United States)

    Zhou, Jing; Lu, Guo-Dong; Ong, Chye-Sun; Ong, Choon-Nam; Shen, Han-Ming

    2008-07-01

    Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is an important member of the tumor necrosis factor subfamily with great potential in cancer therapy. Andrographolide (Andro), a diterpenoid lactone isolated from a traditional herbal medicine Andrographis paniculata, is known to possess potent anti-inflammatory and anticancer activities. Here, we showed that pretreatment with Andro significantly enhances TRAIL-induced apoptosis in various human cancer cell lines, including those TRAIL-resistant cells. Such sensitization is achieved through transcriptional up-regulation of death receptor 4 (DR4), a death receptor of TRAIL. In search of the molecular mechanisms responsible for DR4 up-regulation, we found that the tumor suppressor p53 plays an essential role in DR4 transcriptional activation. Andro is capable of activating p53 via increased p53 phosphorylation and protein stabilization, a process mediated by enhanced reactive oxygen species production and subsequent c-Jun NH(2)-terminal kinase activation. Pretreatment with an antioxidant (N-acetylcysteine) or a c-Jun NH(2)-terminal kinase inhibitor (SP600125) effectively prevented Andro-induced p53 activation and DR4 up-regulation and eventually blocked the Andro-induced sensitization on TRAIL-induced apoptosis. Taken together, these results present a novel anticancer effect of Andro and support its potential application in cancer therapy to overcome TRAIL resistance.

  13. Emerging roles of protein kinases in microglia-mediated neuroinflammation.

    Science.gov (United States)

    Lee, Sun-Hwa; Suk, Kyoungho

    2017-12-15

    Neuroinflammation is mediated by resident central nervous system glia, neurons, peripherally derived immune cells, blood-brain barrier, and inflammatory mediators secreted from these cells. Neuroinflammation has been implicated in stroke and neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, multiple sclerosis, and amyotrophic lateral sclerosis. Protein kinases have been one of the most exploited therapeutic targets in the current pharmacological research, especially in studies on cancer and inflammation. To date, 32 small-molecule protein kinase inhibitors have been approved by the United States Food and Drug Administration for the treatment of cancer and inflammation. However, there is no drug effectively targeting neuroinflammation and/or neurodegenerative diseases. Recent studies have advanced several protein kinases as important drug targets in neuroinflammation and/or neurodegenerative diseases. Here, we review emerging protein kinases potentially involved in neuroinflammation and subsequent neurodegenerative diseases. Copyright © 2017 Elsevier Inc. All rights reserved.

  14. Mitogen-activated protein kinases mediate Mycobacterium ...

    Indian Academy of Sciences (India)

    CD44, an adhesion molecule, has been reported to be a binding site for Mycobacterium tuberculosis (M. tuberculosis) in macrophages and it also mediates mycobacterial phagocytosis, macrophage recruitment and protective immunity against pulmonary tuberculosis in vivo. However, the signalling pathways that are ...

  15. Mitogen-activated protein kinases mediate Mycobacterium

    Indian Academy of Sciences (India)

    CD44, an adhesion molecule, has been reported to be a binding site for Mycobacterium tuberculosis (M. tuberculosis) in macrophages and it also mediates mycobacterial phagocytosis, macrophage recruitment and protective immunity against pulmonary tuberculosis in vivo. However, the signalling pathways that are ...

  16. Nuclear localization of Lyn tyrosine kinase mediated by inhibition of its kinase activity

    International Nuclear Information System (INIS)

    Ikeda, Kikuko; Nakayama, Yuji; Togashi, Yuuki; Obata, Yuuki; Kuga, Takahisa; Kasahara, Kousuke; Fukumoto, Yasunori; Yamaguchi, Naoto

    2008-01-01

    Src-family kinases, cytoplasmic enzymes that participate in various signaling events, are found at not only the plasma membrane but also subcellular compartments, such as the nucleus, the Golgi apparatus and late endosomes/lysosomes. Lyn, a member of the Src-family kinases, is known to play a role in DNA damage response and cell cycle control in the nucleus. However, it is still unclear how the localization of Lyn to the nucleus is regulated. Here, we investigated the mechanism of the distribution of Lyn between the cytoplasm and the nucleus in epitheloid HeLa cells and hematopoietic THP-1 cells. Lyn was definitely detected in purified nuclei by immunofluorescence and immunoblotting analyses. Nuclear accumulation of Lyn was enhanced upon treatment of cells with leptomycin B (LMB), an inhibitor of Crm1-mediated nuclear export. Moreover, Lyn mutants lacking the sites for lipid modification were highly accumulated in the nucleus upon LMB treatment. Intriguingly, inhibition of the kinase activity of Lyn by SU6656, Csk overexpression, or point mutation in the ATP-binding site induced an increase in nuclear Lyn levels. These results suggest that Lyn being imported into and rapidly exported from the nucleus preferentially accumulates in the nucleus by inhibition of the kinase activity and lipid modification

  17. Mouse model of testosterone-induced muscle fiber hypertrophy: involvement of p38 mitogen-activated protein kinase-mediated Notch signaling.

    Science.gov (United States)

    Brown, Danielle; Hikim, Amiya P Sinha; Kovacheva, Ekaterina L; Sinha-Hikim, Indrani

    2009-04-01

    As a prerequisite for studies using mutant mice, we established a mouse model for investigating the molecular mechanisms by which testosterone (T) promotes muscle growth. Groups of six adult male mice (C57BL/6) received one of the following treatments: 1) vehicle (sterile distilled water; normal control) and 2) GnRH antagonist with empty (sham control) or 2 cm T- filled implant. Mice were killed 2, 6, and 8 weeks after treatment. T treatment for 8 weeks resulted in a significant (Phypertrophy was accompanied by up-regulation of the Notch ligand Delta 1 and activation of Notch signaling, as evidenced by increase in activated forms of Notch 1 and Notch 2. Consistent with this, we also observed an increase in the number of proliferating cell nuclear antigen (PCNA)-positive nuclei in muscles of T-treated mice, indicating that activation of Notch signaling enhanced cell proliferation. T supplementation not only triggered p38 mitogen-activated protein kinase (MAPK) activation but also concurrently inhibited c-Jun NH(2)-terminal kinase (JNK) activation within 2 weeks of treatment. Concomitant administration of SB203580, a p38 MAPK inhibitor, effectively blocked T-induced activation of Notch signaling and significantly (Phypertrophy through activation of Notch signaling and the inactivation of JNK together with the activation of p38 MAPK may be critical for T-induced activation of Notch signaling and, as a consequence, muscle fiber hypertrophy.

  18. Mefloquine neurotoxicity is mediated by non-receptor tyrosine kinase.

    Science.gov (United States)

    Milatovic, Dejan; Jenkins, Jerry W; Hood, Jonathan E; Yu, Yingchun; Rongzhu, Lu; Aschner, Michael

    2011-10-01

    Among several available antimalarial drugs, mefloquine has proven to be effective against drug-resistant Plasmodium falciparum and remains the drug of choice for both therapy and chemoprophylaxis. However, mefloquine is known to cause adverse neurological and/or psychiatric symptoms, which offset its therapeutic advantage. The exact mechanisms leading to the adverse neurological effects of mefloquine are poorly defined. Alterations in neurotransmitter release and calcium homeostasis, the inhibition of cholinesterases and the interaction with adenosine A(2A) receptors have been hypothesized to play prominent roles in mediating the deleterious effects of this drug. Our recent data have established that mefloquine can also trigger oxidative damage and subsequent neurodegeneration in rat cortical primary neurons. Furthermore, we have utilized a system biology-centered approach and have constructed a pathway model of cellular responses to mefloquine, identifying non-receptor tyrosine kinase 2 (Pyk2) as a critical target in mediating mefloquine neurotoxicity. In this study, we sought to establish an experimental validation of Pyk2 using gene-silencing techniques (siRNA). We have examined whether the downregulation of Pyk2 in primary rat cortical neurons alters mefloquine neurotoxicity by evaluating cell viability, apoptosis and oxidative stress. Results from our study have confirmed that mefloquine neurotoxicity is associated with apoptotic response and oxidative injury, and we have demonstrated that mefloquine affects primary rat cortical neurons, at least in part, via Pyk2. The implication of these findings may prove beneficial in suppressing the neurological side effects of mefloquine and developing effective therapeutic modalities to offset its adverse effects. Copyright © 2011 Elsevier Inc. All rights reserved.

  19. Mammalian STE20-like kinase 2, not kinase 1, mediates photoreceptor cell death during retinal detachment

    Science.gov (United States)

    Matsumoto, H; Murakami, Y; Kataoka, K; Lin, H; Connor, K M; Miller, J W; Zhou, D; Avruch, J; Vavvas, D G

    2014-01-01

    Photoreceptor cell death is the definitive cause of vision loss in retinal detachment (RD). Mammalian STE20-like kinase (MST) is a master regulator of both cell death and proliferation and a critical factor in development and tumorigenesis. However, to date the role of MST in neurodegeneration has not been fully explored. Utilizing MST1−/− and MST2−/− mice we identified MST2, but not MST1, as a regulator of photoreceptor cell death in a mouse model of RD. MST2−/− mice demonstrated significantly decreased photoreceptor cell death and outer nuclear layer (ONL) thinning after RD. Additionally, caspase-3 activation was attenuated in MST2−/− mice compared to control mice after RD. The transcription of p53 upregulated modulator of apoptosis (PUMA) and Fas was also reduced in MST2−/− mice post-RD. Retinas of MST2−/− mice displayed suppressed nuclear relocalization of phosphorylated YAP after RD. Consistent with the reduction of photoreceptor cell death, MST2−/− mice showed decreased levels of proinflammatory cytokines such as monocyte chemoattractant protein 1 and interleukin 6 as well as attenuated inflammatory CD11b cell infiltration during the early phase of RD. These results identify MST2, not MST1, as a critical regulator of caspase-mediated photoreceptor cell death in the detached retina and indicate its potential as a future neuroprotection target. PMID:24874741

  20. Critical roles of cellular glutathione homeostasis and jnk activation in andrographolide-mediated apoptotic cell death in human hepatoma cells.

    Science.gov (United States)

    Ji, Lili; Shen, Kaikai; Jiang, Ping; Morahan, Grant; Wang, Zhengtao

    2011-08-01

    Andrographolide (ANDRO), isolated from the traditional herbal medicine Andrographis paniculata, is reported to have the potential therapeutic effects for hepatocellular carcinoma (HCC) in our previous reports. Here, we investigated the mechanism of ANDRO-mediated apoptotic cell death, focusing on the involvement of cellular reduced glutathione (GSH) homeostasis and c-Jun NH(2) -Terminal kinase (JNK). Buthionine sulfoximine (BSO), an inhibitor of cellular GSH biosynthesis, significantly augmented ANDRO-induced cytotoxicity in hepatoma Hep3B and HepG2 cells. BSO depleted cellular GSH, and augmented ANDRO-induced apoptosis, inhibition of colony formation and JNK activation in Hep3B cells. All these effects could be reversed by GSH monoethyl ester (GSH.EE), whose deacetylation replenishes cellular GSH. BSO also augmented ANDRO-induced activation of apoptosis signal-regulating kinase 1 (ASK1), mitogen-activated protein kinase kinase-4 (MKK4) and c-Jun, which are all up-stream or down-stream signals of JNK. Further results showed that JNK inhibitor SP600125 and 420116 both reversed ANDRO-induced cytotoxicity, and SP600125 also decreased ANDRO-increased intracellular GSH and GCL activity. Finally, we showed that in nude mice bearing xenografted Hep3B tumors, BSO improved the inhibition of tumor growth by ANDRO. Taken together, our results suggest that there is a crosstalk between JNK activation and cellular GSH homeostasis, and ANDRO targets this to induce cytotoxicity in hepatoma cells. Copyright © 2011 Wiley-Liss, Inc.

  1. Dissociation of Akt1 from its negative regulator JIP1 is mediated through the ASK1-MEK-JNK signal transduction pathway during metabolic oxidative stress: a negative feedback loop.

    Science.gov (United States)

    Song, Jae J; Lee, Yong J

    2005-07-04

    We have previously observed that metabolic oxidative stress-induced death domain-associated protein (Daxx) trafficking is mediated by the ASK1-SEK1-JNK1-HIPK1 signal transduction pathway. The relocalized Daxx from the nucleus to the cytoplasm during glucose deprivation participates in a positive regulatory feedback loop by binding to apoptosis signal-regulating kinase (ASK) 1. In this study, we report that Akt1 is involved in a negative regulatory feedback loop during glucose deprivation. Akt1 interacts with c-Jun NH(2)-terminal kinase (JNK)-interacting protein (JIP) 1, and Akt1 catalytic activity is inhibited. The JNK2-mediated phosphorylation of JIP1 results in the dissociation of Akt1 from JIP1 and subsequently restores Akt1 enzyme activity. Concomitantly, Akt1 interacts with stress-activated protein kinase/extracellular signal-regulated kinase (SEK) 1 (also known as MKK4) and inhibits SEK1 activity. Knockdown of SEK1 leads to the inhibition of JNK activation, JIP1-JNK2 binding, and the dissociation of Akt1 from JIP1 during glucose deprivation. Knockdown of JIP1 also leads to the inhibition of JNK activation, whereas the knockdown of Akt1 promotes JNK activation during glucose deprivation. Altogether, our data demonstrate that Akt1 participates in a negative regulatory feedback loop by interacting with the JIP1 scaffold protein.

  2. Tauroursodeoxycholate Protects Rat Hepatocytes from Bile Acid-Induced Apoptosis via β1-Integrin- and Protein Kinase A-Dependent Mechanisms

    Directory of Open Access Journals (Sweden)

    Annika Sommerfeld

    2015-05-01

    Full Text Available Background/Aims: Ursodeoxycholic acid, which in vivo is rapidly converted into its taurine conjugate, is frequently used for the treatment of cholestatic liver disease. Apart from its choleretic effects, tauroursodeoxycholate (TUDC can protect hepatocytes from bile acid-induced apoptosis, but the mechanisms underlying its anti-apoptotic effects are poorly understood. Methods: These mechanisms were investigated in perfused rat liver and isolated rat hepatocytes. Results: It was found that TUDC inhibited the glycochenodeoxycholate (GCDC-induced activation of the CD95 death receptor at the level of association between CD95 and the epidermal growth factor receptor. This was due to a rapid TUDC-induced β1-integrin-dependent cyclic AMP (cAMP signal with induction of the dual specificity mitogen-activated protein (MAP kinase phosphatase 1 (MKP-1, which prevented GCDC-induced phosphorylation of mitogen-activated protein kinase kinase 4 (MKK4 and c-jun-NH2-terminal kinase (JNK activation. Furthermore, TUDC induced a protein kinase A (PKA-mediated serine/threonine phosphorylation of the CD95, which was recently identified as an internalization signal for CD95. Furthermore, TUDC inhibited GCDC-induced CD95 targeting to the plasma membrane in a β1-integrin-and PKA-dependent manner. In line with this, the β1-integrin siRNA knockdown in sodium taurocholate cotransporting polypeptide (Ntcp-transfected HepG2 cells abolished the protective effect of TUDC against GCDC-induced apoptosis. Conclusion: TUDC exerts its anti-apoptotic effect via a β1-integrin-mediated formation of cAMP, which prevents CD95 activation by hydrophobic bile acids at the levels of JNK activation and CD95 serine/threonine phosphorylation.

  3. Leptin Enhances Synthesis of Proinflammatory Mediators in Human Osteoarthritic Cartilage—Mediator Role of NO in Leptin-Induced PGE2, IL-6, and IL-8 Production

    Directory of Open Access Journals (Sweden)

    Katriina Vuolteenaho

    2009-01-01

    Full Text Available Obesity is an important risk factor for osteoarthritis (OA in weight-bearing joints, but also in hand joints, pointing to an obesity-related metabolic factor that influences on the pathogenesis of OA. Leptin is an adipokine regulating energy balance, and it has recently been related also to arthritis and inflammation as a proinflammatory factor. In the present paper, the effects of leptin on human OA cartilage were studied. Leptin alone or in combination with IL-1 enhanced the expression of iNOS and COX-2, and production of NO, PGE2, IL-6, and IL-8. The results suggest that the effects of leptin are mediated through activation of transcription factor nuclear factor κB (NF-κB and mitogen-activated protein kinase (MAPK pathway c-Jun NH2-terminal kinase (JNK. Interestingly, inhibition of leptin-induced NO production with a selective iNOS inhibitor 1400 W inhibited also the production of IL-6, IL-8, and PGE2, and this was reversed by exogenously added NO-donor SNAP, suggesting that the effects of leptin on IL-6, IL-8, and PGE2 production are dependent on NO. These findings support the idea of leptin as a factor enhancing the production of proinflammatory factors in OA cartilage and as an agent contributing to the obesity-associated increased risk for osteoarthritis.

  4. Tec kinase mediating IL-8 transcription in monocytes stimulated with LPS.

    Science.gov (United States)

    Wang, Guang-Qing; Yang, Xiao-Yan; Jia, Yi-Tao; Xia, Zhao-Fan

    2009-08-01

    The purpose of present study was to explore the possibility of Tec kinase as a mediator for IL-8 transcription in monocytes stimulated with LPS. Plasmids of mouse Tec kinase IV or Tec kinase IV with inactivating point mutations generated with QuikChange site-directed mutagenesis were co-transfected with IL-8 promoter driven luciferase construct into RAW264.7 cells, then luciferase activity was measured with a luminometer. The results shown Tec kinase could significantly enhance IL-8 transcription. Furthermore, point inactivating mutation in SH2, PH or PTK domain almost completely abolish the effects of Tec kinase on the transcription of IL-8. In the transfection experiment, PD98059, a MEK1 inhibitor, decreased the transcription of IL-8 in a dose dependent pattern. When siRNA for Tec kinase was transfected into THP-1 cells, it could efficiently block the production of IL-8 from THP-1 cells (p Tec kinase may mediate the transcription of IL-8 in monocyte stimulated with LPS.

  5. The cyclin-dependent kinase 8 module sterically blocks Mediator interactions with RNA polymerase II

    DEFF Research Database (Denmark)

    Elmlund, Hans; Baraznenok, Vera; Lindahl, Martin

    2006-01-01

    CDK8 (cyclin-dependent kinase 8), along with CycC, Med12, and Med13, form a repressive module (the Cdk8 module) that prevents RNA polymerase II (pol II) interactions with Mediator. Here, we report that the ability of the Cdk8 module to prevent pol II interactions is independent of the Cdk8......-dependent kinase activity. We use electron microscopy and single-particle reconstruction to demonstrate that the Cdk8 module forms a distinct structural entity that binds to the head and middle region of Mediator, thereby sterically blocking interactions with pol II....

  6. A Protein Scaffold Coordinates SRC-Mediated JNK Activation in Response to Metabolic Stress

    Directory of Open Access Journals (Sweden)

    Shashi Kant

    2017-09-01

    Full Text Available Obesity is a major risk factor for the development of metabolic syndrome and type 2 diabetes. How obesity contributes to metabolic syndrome is unclear. Free fatty acid (FFA activation of a non-receptor tyrosine kinase (SRC-dependent cJun NH2-terminal kinase (JNK signaling pathway is implicated in this process. However, the mechanism that mediates SRC-dependent JNK activation is unclear. Here, we identify a role for the scaffold protein JIP1 in SRC-dependent JNK activation. SRC phosphorylation of JIP1 creates phosphotyrosine interaction motifs that bind the SH2 domains of SRC and the guanine nucleotide exchange factor VAV. These interactions are required for SRC-induced activation of VAV and the subsequent engagement of a JIP1-tethered JNK signaling module. The JIP1 scaffold protein, therefore, plays a dual role in FFA signaling by coordinating upstream SRC functions together with downstream effector signaling by the JNK pathway.

  7. A Protein Scaffold Coordinates SRC-Mediated JNK Activation in Response to Metabolic Stress.

    Science.gov (United States)

    Kant, Shashi; Standen, Claire L; Morel, Caroline; Jung, Dae Young; Kim, Jason K; Swat, Wojciech; Flavell, Richard A; Davis, Roger J

    2017-09-19

    Obesity is a major risk factor for the development of metabolic syndrome and type 2 diabetes. How obesity contributes to metabolic syndrome is unclear. Free fatty acid (FFA) activation of a non-receptor tyrosine kinase (SRC)-dependent cJun NH 2 -terminal kinase (JNK) signaling pathway is implicated in this process. However, the mechanism that mediates SRC-dependent JNK activation is unclear. Here, we identify a role for the scaffold protein JIP1 in SRC-dependent JNK activation. SRC phosphorylation of JIP1 creates phosphotyrosine interaction motifs that bind the SH2 domains of SRC and the guanine nucleotide exchange factor VAV. These interactions are required for SRC-induced activation of VAV and the subsequent engagement of a JIP1-tethered JNK signaling module. The JIP1 scaffold protein, therefore, plays a dual role in FFA signaling by coordinating upstream SRC functions together with downstream effector signaling by the JNK pathway. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  8. DMPD: Protein kinase C epsilon: a new target to control inflammation andimmune-mediated disorders. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 14643884 Protein kinase C epsilon: a new target to control inflammation andimmune-m...g) (.html) (.csml) Show Protein kinase C epsilon: a new target to control inflammation andimmune-mediated di...sorders. PubmedID 14643884 Title Protein kinase C epsilon: a new target to control inflammation

  9. Dynamic Allostery Mediated by a Conserved Tryptophan in the Tec Family Kinases.

    Directory of Open Access Journals (Sweden)

    Nikita Chopra

    2016-03-01

    Full Text Available Bruton's tyrosine kinase (Btk is a Tec family non-receptor tyrosine kinase that plays a critical role in immune signaling and is associated with the immunological disorder X-linked agammaglobulinemia (XLA. Our previous findings showed that the Tec kinases are allosterically activated by the adjacent N-terminal linker. A single tryptophan residue in the N-terminal 17-residue linker mediates allosteric activation, and its mutation to alanine leads to the complete loss of activity. Guided by hydrogen/deuterium exchange mass spectrometry results, we have employed Molecular Dynamics simulations, Principal Component Analysis, Community Analysis and measures of node centrality to understand the details of how a single tryptophan mediates allostery in Btk. A specific tryptophan side chain rotamer promotes the functional dynamic allostery by inducing coordinated motions that spread across the kinase domain. Either a shift in the rotamer population, or a loss of the tryptophan side chain by mutation, drastically changes the coordinated motions and dynamically isolates catalytically important regions of the kinase domain. This work also identifies a new set of residues in the Btk kinase domain with high node centrality values indicating their importance in transmission of dynamics essential for kinase activation. Structurally, these node residues appear in both lobes of the kinase domain. In the N-lobe, high centrality residues wrap around the ATP binding pocket connecting previously described Catalytic-spine residues. In the C-lobe, two high centrality node residues connect the base of the R- and C-spines on the αF-helix. We suggest that the bridging residues that connect the catalytic and regulatory architecture within the kinase domain may be a crucial element in transmitting information about regulatory spine assembly to the catalytic machinery of the catalytic spine and active site.

  10. Dynamic Allostery Mediated by a Conserved Tryptophan in the Tec Family Kinases.

    Science.gov (United States)

    Chopra, Nikita; Wales, Thomas E; Joseph, Raji E; Boyken, Scott E; Engen, John R; Jernigan, Robert L; Andreotti, Amy H

    2016-03-01

    Bruton's tyrosine kinase (Btk) is a Tec family non-receptor tyrosine kinase that plays a critical role in immune signaling and is associated with the immunological disorder X-linked agammaglobulinemia (XLA). Our previous findings showed that the Tec kinases are allosterically activated by the adjacent N-terminal linker. A single tryptophan residue in the N-terminal 17-residue linker mediates allosteric activation, and its mutation to alanine leads to the complete loss of activity. Guided by hydrogen/deuterium exchange mass spectrometry results, we have employed Molecular Dynamics simulations, Principal Component Analysis, Community Analysis and measures of node centrality to understand the details of how a single tryptophan mediates allostery in Btk. A specific tryptophan side chain rotamer promotes the functional dynamic allostery by inducing coordinated motions that spread across the kinase domain. Either a shift in the rotamer population, or a loss of the tryptophan side chain by mutation, drastically changes the coordinated motions and dynamically isolates catalytically important regions of the kinase domain. This work also identifies a new set of residues in the Btk kinase domain with high node centrality values indicating their importance in transmission of dynamics essential for kinase activation. Structurally, these node residues appear in both lobes of the kinase domain. In the N-lobe, high centrality residues wrap around the ATP binding pocket connecting previously described Catalytic-spine residues. In the C-lobe, two high centrality node residues connect the base of the R- and C-spines on the αF-helix. We suggest that the bridging residues that connect the catalytic and regulatory architecture within the kinase domain may be a crucial element in transmitting information about regulatory spine assembly to the catalytic machinery of the catalytic spine and active site.

  11. Arabidopsis mitogen-activated protein kinase kinases MKK1 and MKK2 have overlapping functions in defense signaling mediated by MEKK1, MPK4, and MKS1

    DEFF Research Database (Denmark)

    Qiu, Jin-Long; Zhou, Lu; Yun, Byung-Wook

    2008-01-01

    by biochemical and molecular analyses implicating the kinases in jasmonate- and salicylate-dependent defense responses, mediated in part via the MPK4 substrate MKS1. In addition, transcriptome analyses delineate overlapping and specific effects of the kinases on global gene expression patterns demonstrating both...

  12. Indirubin Inhibits LPS-Induced Inflammation via TLR4 Abrogation Mediated by the NF-kB and MAPK Signaling Pathways.

    Science.gov (United States)

    Lai, Jin-Lun; Liu, Yu-Hui; Liu, Chang; Qi, Ming-Pu; Liu, Rui-Ning; Zhu, Xi-Fang; Zhou, Qiu-Ge; Chen, Ying-Yu; Guo, Ai-Zhen; Hu, Chang-Min

    2017-02-01

    Indirubin plays an important role in the treatment of many chronic diseases and exhibits strong anti-inflammatory activity. However, the molecular mode of action during mastitis prophylaxis remains poorly understood. In this study, a lipopolysaccharide (LPS)-induced mastitis mouse model showed that indirubin attenuated histopathological changes in the mammary gland, local tissue necrosis, and neutrophil infiltration. Moreover, indirubin significantly downregulated the production of interleukin (IL)-1β, IL-6, and tumor necrosis factor-α (TNF-α). We explored the mechanism whereby indirubin exerts protective effects against LPS-induced inflammation of mouse mammary epithelial cells (MMECs). The addition of different concentrations of indirubin before exposure of cells to LPS for 1 h significantly attenuated inflammation and reduced the concentrations of the three inflammatory cytokines in a dose-dependent manner. Indirubin downregulated LPS-induced cyclooxygenase-2 (COX-2) and Toll-like receptor 4 (TLR4) expression, inhibited phosphorylation of the LPS-induced nuclear transcription factor-kappa B (NF-kB) P65 protein and its inhibitor IkBα of the NF-kB signaling pathway. Furthermore, indirubin suppressed phosphorylation of P38, extracellular signal-regulated kinase (ERK), and c-Jun NH 2 -terminal kinase (JNK) of the mitogen-activated protein kinase (MAPK) signal pathways. Thus, indirubin effectively suppressed LPS-induced inflammation via TLR4 abrogation mediated by the NF-kB and MAPK signaling pathways and may be useful for mastitis prophylaxis.

  13. Fibroblast Growth Factor 21 Mediates Glycemic Regulation by Hepatic JNK

    Directory of Open Access Journals (Sweden)

    Santiago Vernia

    2016-03-01

    Full Text Available The cJun NH2-terminal kinase (JNK-signaling pathway is implicated in metabolic syndrome, including dysregulated blood glucose concentration and insulin resistance. Fibroblast growth factor 21 (FGF21 is a target of the hepatic JNK-signaling pathway and may contribute to the regulation of glycemia. To test the role of FGF21, we established mice with selective ablation of the Fgf21 gene in hepatocytes. FGF21 deficiency in the liver caused marked loss of FGF21 protein circulating in the blood. Moreover, the protective effects of hepatic JNK deficiency to suppress metabolic syndrome in high-fat diet-fed mice were not observed in mice with hepatocyte-specific FGF21 deficiency, including reduced blood glucose concentration and reduced intolerance to glucose and insulin. Furthermore, we show that JNK contributes to the regulation of hepatic FGF21 expression during fasting/feeding cycles. These data demonstrate that the hepatokine FGF21 is a key mediator of JNK-regulated metabolic syndrome.

  14. Tec family kinases: regulation of FcεRI-mediated mast-cell activation.

    Science.gov (United States)

    Ellmeier, Wilfried; Abramova, Anastasia; Schebesta, Alexandra

    2011-06-01

    Mast cells express the high-affinity receptor for IgE (FcεRI) and are key players in type I hypersensitivity reactions. They are critically involved in the development of allergic rhinitis, allergic asthma and systemic anaphylaxis, however, they also regulate normal physiological processes that link innate and adaptive immune responses. Thus, their activation has to be tightly controlled. One group of signaling molecules that are activated upon FcεRI stimulation is formed by Tec family kinases, and three members of this kinase family (Btk, Itk and Tec) are expressed in mast cells. Many studies have revealed important functions of Tec kinases in signaling pathways downstream of the antigen receptors in lymphocytes. This review summarizes the current knowledge about the function of Tec family kinases in FcεRI-mediated signaling pathways in mast cell. © 2011 The Authors Journal compilation © 2011 FEBS.

  15. Phosphorylation of Src by phosphoinositide 3-kinase regulates beta-adrenergic receptor-mediated EGFR transactivation.

    Science.gov (United States)

    Watson, Lewis J; Alexander, Kevin M; Mohan, Maradumane L; Bowman, Amber L; Mangmool, Supachoke; Xiao, Kunhong; Naga Prasad, Sathyamangla V; Rockman, Howard A

    2016-10-01

    β2-Adrenergic receptors (β2AR) transactivate epidermal growth factor receptors (EGFR) through formation of a β2AR-EGFR complex that requires activation of Src to mediate signaling. Here, we show that both lipid and protein kinase activities of the bifunctional phosphoinositide 3-kinase (PI3K) enzyme are required for β2AR-stimulated EGFR transactivation. Mechanistically, the generation of phosphatidylinositol (3,4,5)-tris-phosphate (PIP3) by the lipid kinase function stabilizes β2AR-EGFR complexes while the protein kinase activity of PI3K regulates Src activation by direct phosphorylation. The protein kinase activity of PI3K phosphorylates serine residue 70 on Src to enhance its activity and induce EGFR transactivation following βAR stimulation. This newly identified function for PI3K, whereby Src is a substrate for the protein kinase activity of PI3K, is of importance since Src plays a key role in pathological and physiological signaling. Copyright © 2016 Elsevier Inc. All rights reserved.

  16. The role of phosphatidylinositol 3-kinase in neural cell adhesion molecule-mediated neuronal differentiation and survival

    DEFF Research Database (Denmark)

    Ditlevsen, Dorte K; Køhler, Lene B; Pedersen, Martin V

    2003-01-01

    The neural cell adhesion molecule, NCAM, is known to stimulate neurite outgrowth from primary neurones and PC12 cells presumably through signalling pathways involving the fibroblast growth factor receptor (FGFR), protein kinase A (PKA), protein kinase C (PKC), the Ras-mitogen activated protein...... that phosphatidylinositol 3-kinase (PI3K) is required for NCAM-mediated neurite outgrowth from PC12-E2 cells and from cerebellar and dopaminergic neurones in primary culture, and that the thr/ser kinase Akt/protein kinase B (PKB) is phosphorylated downstream of PI3K after stimulation with C3. Moreover, we present data...

  17. Protein Kinase G facilitates EGFR-mediated cell death in MDA-MB-468 cells

    Energy Technology Data Exchange (ETDEWEB)

    Jackson, Nicole M.; Ceresa, Brian P., E-mail: brian.ceresa@louisville.edu

    2016-08-15

    The Epidermal Growth Factor Receptor (EGFR) is a transmembrane receptor tyrosine kinase with critical implications in cell proliferation, migration, wound healing and the regulation of apoptosis. However, the EGFR has been shown to be hyper-expressed in a number of human malignancies. The MDA-MB-468 metastatic breast cell line is one example of this. This particular cell line hyper-expresses the EGFR and undergoes EGFR-mediated apoptosis in response to EGF ligand. The goal of this study was to identify the kinases that could be potential intermediates for the EGFR-mediated induction of apoptosis intracellularly. After identifying Cyclic GMP-dependent Protein Kinase G (PKG) as a plausible intermediate, we wanted to determine the temporal relationship of these two proteins in the induction of apoptosis. We observed a dose-dependent decrease in MDA-MB-468 cell viability, which was co-incident with increased PKG activity as measured by VASPSer239 phosphorylation. In addition, we observed a dose dependent decrease in cell viability, as well as an increase in apoptosis, in response to two different PKG agonists, 8-Bromo-cGMP and 8-pCPT-cGMP. MDA-MB-468 cells with reduced PKG activity had attenuated EGFR-mediated apoptosis. These findings indicate that PKG does not induce cell death via transphosphorylation of the EGFR. Instead, PKG activity occurs following EGFR activation. Together, these data indicate PKG as an intermediary in EGFR-mediated cell death, likely via apoptotic pathway.

  18. Protein Kinase G facilitates EGFR-mediated cell death in MDA-MB-468 cells

    International Nuclear Information System (INIS)

    Jackson, Nicole M.; Ceresa, Brian P.

    2016-01-01

    The Epidermal Growth Factor Receptor (EGFR) is a transmembrane receptor tyrosine kinase with critical implications in cell proliferation, migration, wound healing and the regulation of apoptosis. However, the EGFR has been shown to be hyper-expressed in a number of human malignancies. The MDA-MB-468 metastatic breast cell line is one example of this. This particular cell line hyper-expresses the EGFR and undergoes EGFR-mediated apoptosis in response to EGF ligand. The goal of this study was to identify the kinases that could be potential intermediates for the EGFR-mediated induction of apoptosis intracellularly. After identifying Cyclic GMP-dependent Protein Kinase G (PKG) as a plausible intermediate, we wanted to determine the temporal relationship of these two proteins in the induction of apoptosis. We observed a dose-dependent decrease in MDA-MB-468 cell viability, which was co-incident with increased PKG activity as measured by VASPSer239 phosphorylation. In addition, we observed a dose dependent decrease in cell viability, as well as an increase in apoptosis, in response to two different PKG agonists, 8-Bromo-cGMP and 8-pCPT-cGMP. MDA-MB-468 cells with reduced PKG activity had attenuated EGFR-mediated apoptosis. These findings indicate that PKG does not induce cell death via transphosphorylation of the EGFR. Instead, PKG activity occurs following EGFR activation. Together, these data indicate PKG as an intermediary in EGFR-mediated cell death, likely via apoptotic pathway.

  19. Hypochlorous acid via peroxynitrite activates protein kinase Cθ and insulin resistance in adipocytes.

    Science.gov (United States)

    Zhou, Jun; Wang, Qilong; Ding, Ye; Zou, Ming-Hui

    2015-02-01

    We recently reported that genetic deletion of myeloperoxidase (MPO) alleviates obesity-related insulin resistance in mice in vivo. How MPO impairs insulin sensitivity in adipocytes is poorly characterized. As hypochlorous acid (HOCl) is a principal oxidant product generated by MPO, we evaluated the effects of HOCl on insulin signaling in adipocytes differentiated from 3T3-L1 cells. Exposure of 3T3-L1 adipocytes to exogenous HOCl (200 μmol/l) attenuated insulin-stimulated 2-deoxyglucose uptake, GLUT4 translocation, and insulin signals, including tyrosine phosphorylation of insulin receptor substrate 1 (IRS1) and phosphorylation of Akt. Furthermore, treatment with HOCl induced phosphorylation of IRS1 at serine 307, inhibitor κB kinase (IKK), c-Jun NH2-terminal kinase (JNK), and phosphorylation of PKCθ (PKCθ). In addition, genetic and pharmacological inhibition of IKK and JNK abolished serine phosphorylation of IRS1 and impairment of insulin signaling by HOCl. Furthermore, knockdown of PKCθ using siRNA transfection suppressed phosphorylation of IKK and JNK and consequently attenuated the HOCl-impaired insulin signaling pathway. Moreover, activation of PKCθ by peroxynitrite was accompanied by increased phosphorylation of IKK, JNK, and IRS1-serine 307. In contrast, ONOO(-) inhibitors abolished HOCl-induced phosphorylation of PKCθ, IKK, JNK, and IRS1-serine 307, as well as insulin resistance. Finally, high-fat diet (HFD)-induced insulin resistance was associated with enhanced phosphorylation of PKCθ, IKK, JNK, and IRS1 at serine 307 in white adipose tissues from WT mice, all of which were not found in Mpo knockout mice fed HFDs. We conclude that HOCl impairs insulin signaling pathway by increasing ONOO(-) mediated phosphorylation of PKCθ, resulting in phosphorylation of IKK/JNK and consequent serine phosphorylation of IRS1 in adipocytes. © 2015 The authors.

  20. Regulation of Ubiquitination-Mediated Protein Degradation by Survival Kinases in Cancer

    International Nuclear Information System (INIS)

    Yamaguchi, Hirohito; Hsu, Jennifer L.; Hung, Mien-Chie

    2012-01-01

    The ubiquitin–proteasome system is essential for multiple physiological processes via selective degradation of target proteins and has been shown to plays a critical role in human cancer. Activation of oncogenic factors and inhibition of tumor suppressors have been shown to be essential for cancer development, and protein ubiquitination has been linked to the regulation of oncogenic factors and tumor suppressors. Three kinases, AKT, extracellular signal-regulated kinase, and IκB kinase, we refer to as oncokinases, are activated in multiple human cancers. We and others have identified several key downstream targets that are commonly regulated by these oncokinases, some of which are regulated directly or indirectly via ubiquitin-mediated proteasome degradation, including FOXO3, β-catenin, myeloid cell leukemia-1, and Snail. In this review, we summarize these findings from our and other groups and discuss potential future studies and applications in the clinic.

  1. The apoptotic mechanism of action of the sphingosine kinase 1 selective inhibitor SKI-178 in human acute myeloid leukemia cell lines.

    Science.gov (United States)

    Dick, Taryn E; Hengst, Jeremy A; Fox, Todd E; Colledge, Ashley L; Kale, Vijay P; Sung, Shen-Shu; Sharma, Arun; Amin, Shantu; Loughran, Thomas P; Kester, Mark; Wang, Hong-Gang; Yun, Jong K

    2015-03-01

    We previously developed SKI-178 (N'-[(1E)-1-(3,4-dimethoxyphenyl)ethylidene]-3-(4-methoxxyphenyl)-1H-pyrazole-5-carbohydrazide) as a novel sphingosine kinase-1 (SphK1) selective inhibitor and, herein, sought to determine the mechanism-of-action of SKI-178-induced cell death. Using human acute myeloid leukemia (AML) cell lines as a model, we present evidence that SKI-178 induces prolonged mitosis followed by apoptotic cell death through the intrinsic apoptotic cascade. Further examination of the mechanism of action of SKI-178 implicated c-Jun NH2-terminal kinase (JNK) and cyclin-dependent protein kinase 1 (CDK1) as critical factors required for SKI-178-induced apoptosis. In cell cycle synchronized human AML cell lines, we demonstrate that entry into mitosis is required for apoptotic induction by SKI-178 and that CDK1, not JNK, is required for SKI-178-induced apoptosis. We further demonstrate that the sustained activation of CDK1 during prolonged mitosis, mediated by SKI-178, leads to the simultaneous phosphorylation of the prosurvival Bcl-2 family members, Bcl-2 and Bcl-xl, as well as the phosphorylation and subsequent degradation of Mcl-1. Moreover, multidrug resistance mediated by multidrug-resistant protein1 and/or prosurvival Bcl-2 family member overexpression did not affect the sensitivity of AML cells to SKI-178. Taken together, these findings highlight the therapeutic potential of SKI-178 targeting SphK1 as a novel therapeutic agent for the treatment of AML, including multidrug-resistant/recurrent AML subtypes. Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.

  2. Acute ethanol exposure-induced autophagy-mediated cardiac injury via activation of the ROS-JNK-Bcl-2 pathway.

    Science.gov (United States)

    Zhu, Zhongxin; Huang, Yewei; Lv, Lingchun; Tao, Youli; Shao, Minglong; Zhao, Congcong; Xue, Mei; Sun, Jia; Niu, Chao; Wang, Yang; Kim, Sunam; Cong, Weitao; Mao, Wei; Jin, Litai

    2018-02-01

    Binge drinking is associated with increased cardiac autophagy, and often triggers heart injury. Given the essential role of autophagy in various cardiac diseases, this study was designed to investigate the role of autophagy in ethanol-induced cardiac injury and the underlying mechanism. Our study showed that ethanol exposure enhanced the levels of LC3-II and LC3-II positive puncta and promoted cardiomyocyte apoptosis in vivo and in vitro. In addition, we found that ethanol induced autophagy and cardiac injury largely via the sequential triggering of reactive oxygen species (ROS) accumulation, activation of c-Jun NH2-terminal kinase (JNK), phosphorylation of Bcl-2, and dissociation of the Beclin 1/Bcl-2 complex. By contrast, inhibition of ethanol-induced autophagic flux with pharmacologic agents in the hearts of mice and cultured cells significantly alleviated ethanol-induced cardiomyocyte apoptosis and heart injury. Elimination of ROS with the antioxidant N-acetyl cysteine (NAC) or inhibition of JNK with the JNK inhibitor SP600125 reduced ethanol-induced autophagy and subsequent autophagy-mediated apoptosis. Moreover, metallothionein (MT), which can scavenge reactive oxygen and nitrogen species, also attenuated ethanol-induced autophagy and cell apoptosis in MT-TG mice. In conclusion, our findings suggest that acute ethanol exposure induced autophagy-mediated heart toxicity and injury mainly through the ROS-JNK-Bcl-2 signaling pathway. © 2017 Wiley Periodicals, Inc.

  3. Induction of apoptosis in renal cell carcinoma by reactive oxygen species: involvement of extracellular signal-regulated kinase 1/2, p38delta/gamma, cyclooxygenase-2 down-regulation, and translocation of apoptosis-inducing factor.

    LENUS (Irish Health Repository)

    Ambrose, Monica

    2012-02-03

    Renal cell carcinoma (RCC) is the most common malignancy of the kidney. Unfortunately, RCCs are highly refractory to conventional chemotherapy, radiation therapy, and even immunotherapy. Thus, novel therapeutic targets need to be sought for the successful treatment of RCCs. We now report that 6-anilino-5,8-quinolinequinone (LY83583), an inhibitor of cyclic GMP production, induced growth arrest and apoptosis of the RCC cell line 786-0. It did not prove deleterious to normal renal epithelial cells, an important aspect of chemotherapy. To address the cellular mechanism(s), we used both genetic and pharmacological approaches. LY83583 induced a time- and dose-dependent increase in RCC apoptosis through dephosphorylation of mitogen-activated protein kinase kinase 1\\/2 and its downstream extracellular signal-regulated kinases (ERK) 1 and -2. In addition, we observed a decrease in Elk-1 phosphorylation and cyclooxygenase-2 (COX-2) down-regulation. We were surprised that we failed to observe an increase in either c-Jun NH(2)-terminal kinase or p38alpha and -beta mitogen-activated protein kinase activation. In contradiction, reintroduction of p38delta by stable transfection or overexpression of p38gamma dominant negative abrogated the apoptotic effect. Cell death was associated with a decrease and increase in Bcl-x(L) and Bax expression, respectively, as well as release of cytochrome c and translocation of apoptosis-inducing factor. These events were associated with an increase in reactive oxygen species formation. The antioxidant N-acetyl l-cysteine, however, opposed LY83583-mediated mitochondrial dysfunction, ERK1\\/2 inactivation, COX-2 down-regulation, and apoptosis. In conclusion, our results suggest that LY83583 may represent a novel therapeutic agent for the treatment of RCC, which remains highly refractory to antineoplastic agents. Our data provide a molecular basis for the anticancer activity of LY83583.

  4. Functional Roles of p38 Mitogen-Activated Protein Kinase in Macrophage-Mediated Inflammatory Responses

    Directory of Open Access Journals (Sweden)

    Yanyan Yang

    2014-01-01

    Full Text Available Inflammation is a natural host defensive process that is largely regulated by macrophages during the innate immune response. Mitogen-activated protein kinases (MAPKs are proline-directed serine and threonine protein kinases that regulate many physiological and pathophysiological cell responses. p38 MAPKs are key MAPKs involved in the production of inflammatory mediators, including tumor necrosis factor-α (TNF-α and cyclooxygenase-2 (COX-2. p38 MAPK signaling plays an essential role in regulating cellular processes, especially inflammation. In this paper, we summarize the characteristics of p38 signaling in macrophage-mediated inflammation. In addition, we discuss the potential of using inhibitors targeting p38 expression in macrophages to treat inflammatory diseases.

  5. Protein kinase C is activated in glomeruli from streptozotocin diabetic rats. Possible mediation by glucose

    International Nuclear Information System (INIS)

    Craven, P.A.; DeRubertis, F.R.

    1989-01-01

    Glomerular inositol content and the turnover of polyphosphoinositides was reduced by 58% in 1-2 wk streptozotocin diabetic rats. Addition of inositol to the incubation medium increased polyphosphoinositide turnover in glomeruli from diabetic rats to control values. Despite the reduction in inositol content and polyphosphoinositide turnover, protein kinase C was activated in glomeruli from diabetic rats, as assessed by an increase in the percentage of enzyme activity associated with the particulate cell fraction. Total protein kinase C activity was not different between glomeruli from control and diabetic rats. Treatment of diabetic rats with insulin to achieve near euglycemia prevented the increase in particulate protein kinase C. Moreover, incubation of glomeruli from control rats with glucose (100-1,000 mg/dl) resulted in a progressive increase in labeled diacylglycerol production and in the percentage of protein kinase C activity which was associated with the particulate fraction. These results support a role for hyperglycemia per se in the enhanced state of activation of protein kinase C seen in glomeruli from diabetic rats. Glucose did not appear to increase diacylglycerol by stimulating inositol phospholipid hydrolysis in glomeruli. Other pathways for diacylglycerol production, including de novo synthesis and phospholipase C mediated hydrolysis of phosphatidylcholine or phosphatidyl-inositol-glycan are not excluded

  6. Specificity is complex and time consuming: mutual exclusivity in tyrosine kinase-mediated signaling.

    Science.gov (United States)

    O'Rourke, Lisa; Ladbury, John E

    2003-06-01

    Most fundamental cellular processes are transduced through tyrosine kinase (TK)-mediated pathways. For transduction without corruption, the protein-protein interactions involved have to be mutually exclusive. Many of these proteins bind via homologous domains whose binding characteristics suggest that their innate specificity is not sufficiently high to account for the integrity of signal transduction. Stimulation of TK-mediated signals is often accompanied by recruitment of a precise, multimolecular protein complex that is itself capable of imposing specificity. Furthermore, this complex provides protection against phosphatase activity, controlling the longevity of the active signaling complex, and thus influencing outcomes in subsequent downstream events.

  7. TGF-βRI kinase activity mediates Emdogain-stimulated in vitro osteoclastogenesis.

    Science.gov (United States)

    Gruber, Reinhard; Roos, Gilles; Caballé-Serrano, Jordi; Miron, Rick; Bosshardt, Dieter D; Sculean, Anton

    2014-07-01

    Emdogain, containing an extract of fetal porcine enamel matrix proteins, is a potent stimulator of in vitro osteoclastogenesis. The underlying molecular mechanisms are, however, unclear. Here, we have addressed the role of transforming growth factor-beta receptor type 1 (TGF-βRI) kinase activity on osteoclastogenesis in murine bone marrow cultures. Inhibition of TGF-βRI kinase activity with SB431542 abolished the effect of Emdogain on osteoclastogenesis induced by receptor activator of nuclear factor kappa-B ligand or tumor necrosis factor-alpha. SB431542 also suppressed the Emdogain-mediated increase of OSCAR, a co-stimulatory protein, and dendritic cell-specific transmembrane protein and Atp6v0d2, the latter two being involved in cell fusion. Similar to transforming growth factor-beta1 (TGF-β), Emdogain could not compensate for the inhibition of IL-4 and IFNγ on osteoclast formation. When using the murine macrophage cell line RAW246.7, SB431542 and the smad-3 inhibitor SIS3 blocked Emdogain-stimulated expression of the transcription factor NFATc1. Taken together, the data suggest that TGF-βRI kinase activity is necessary to mediate in vitro effects of Emdogain on osteoclastogenesis. Based on these in vitro data, we can speculate that at least part of the clinical effects of Emdogain on osteoclastogenesis is mediated via TGF-β signaling.

  8. Lithium potentiates GSK-3β activity by inhibiting phosphoinositide 3-kinase-mediated Akt phosphorylation

    International Nuclear Information System (INIS)

    Tian, Nie; Kanno, Takeshi; Jin, Yu; Nishizaki, Tomoyuki

    2014-01-01

    Highlights: • Lithium suppresses Akt activity by reducing PI3K-mediated Akt phosphorylation. • Lithium enhances GSK-3β activity by reducing Akt-mediated GSK-3β phosphorylation. • Lithium suppresses GSK-3β activity through its direct inhibition. - Abstract: Accumulating evidence has pointed to the direct inhibitory action of lithium, an anti-depressant, on GSK-3β. The present study investigated further insight into lithium signaling pathways. In the cell-free assay Li 2 CO 3 significantly inhibited phosphoinositide 3-kinase (PI3K)-mediated phosphorylation of Akt1 at Ser473, but Li 2 CO 3 did not affect PI3K-mediated PI(3,4,5)P 3 production and 3-phosphoinositide-dependent protein kinase 1 (PDK1)-mediated phosphorylation of Akt1 at Thr308. This indicates that lithium could enhance GSK-3β activity by suppressing Akt-mediated Ser9 phosphorylation of GSK-3β in association with inhibition of PI3K-mediated Akt activation. There was no direct effect of Li 2 CO 3 on Akt1-induced phosphorylation of GSK-3β at Ser9, but otherwise Li 2 CO 3 significantly reduced GSK-3β-mediated phosphorylation of β-catenin at Ser33/37 and Thr41. This indicates that lithium directly inhibits GSK-3β in an Akt-independent manner. In rat hippocampal slices Li 2 CO 3 significantly inhibited phosphorylation of Akt1/2 at Ser473/474, GSK-3β at Ser9, and β-catenin at Ser33/37 and Thr41. Taken together, these results indicate that lithium exerts its potentiating and inhibiting bidirectional actions on GSK-3β activity

  9. TOP 1 and 2, polysaccharides from Taraxacum officinale, inhibit NFκB-mediated inflammation and accelerate Nrf2-induced antioxidative potential through the modulation of PI3K-Akt signaling pathway in RAW 264.7 cells.

    Science.gov (United States)

    Park, Chung Mu; Cho, Chung Won; Song, Young Sun

    2014-04-01

    Anti-inflammatory and anti-oxidative activities of polysaccharides from Taraxacum officinale (TOP 1 and 2) were analyzed in RAW 264.7 cells. First, lipopolysaccharide (LPS) was applied to identify anti-inflammatory activity of TOPs, which reduced expression of inducible nitric oxide synthase (iNOS) and tumor necrosis factor (TNF)-α. TOPs treatment inhibited phosphorylation of inflammatory transcription factor, nuclear factor (NF)κB, and its upstream signaling molecule, PI3K/Akt. Second, cytoprotective potential of TOPs against oxidative stress was investigated via heme oxygenase (HO)-1 induction. HO-1, one of phase II enzymes shows antioxidative activity, was potently induced by TOPs treatment, which was in accordance with the nuclear translocation of nuclear factor-erythroid 2 p45-related factor 2 (Nrf2). In addition, TOPs treatment phosphorylated PI3K/Akt with slight activation of c-Jun NH2-terminal kinase (JNK). TOPs-mediated HO-1 induction protected macrophage cells from oxidative stress-induced cell death, which was confirmed by SnPP and CoPP (HO-1 inhibitor and inducer, respectively). Consequently, TOPs potently inhibited NFκB-mediated inflammation and accelerated Nrf2-mediated antioxidative potential through the modulation of PI3K/Akt pathway, which would contribute to their promising strategy for novel anti-inflammatory and anti-oxidative agents. Copyright © 2014. Published by Elsevier Ltd.

  10. Heme-induced Trypanosoma cruzi proliferation is mediated by CaM kinase II

    Energy Technology Data Exchange (ETDEWEB)

    Souza, C.F. [Laboratorio de Imunomodulacao e Protozoologia, Instituto Oswaldo Cruz, Fiocruz (Brazil); Carneiro, A.B.; Silveira, A.B. [Laboratorio de Sinalizacao Celular, Instituto de Bioquimica Medica, UFRJ (Brazil); Laranja, G.A.T. [Laboratorio de Interacao Tripanosomatideos e Vetores, Departamento de Bioquimica, IBRAG, UERJ, 20551-030 Rio de Janeiro (Brazil); Silva-Neto, M.A.C. [Laboratorio de Sinalizacao Celular, Instituto de Bioquimica Medica, UFRJ (Brazil); INCT, Entomologia Molecular (Brazil); Costa, S.C. Goncalves da [Laboratorio de Imunomodulacao e Protozoologia, Instituto Oswaldo Cruz, Fiocruz (Brazil); Paes, M.C., E-mail: mcpaes@uerj.br [Laboratorio de Interacao Tripanosomatideos e Vetores, Departamento de Bioquimica, IBRAG, UERJ, 20551-030 Rio de Janeiro (Brazil); INCT, Entomologia Molecular (Brazil)

    2009-12-18

    Trypanosoma cruzi, the etiologic agent of Chagas disease, is transmitted through triatomine vectors during their blood-meal on vertebrate hosts. These hematophagous insects usually ingest approximately 10 mM of heme bound to hemoglobin in a single meal. Blood forms of the parasite are transformed into epimastigotes in the crop which initiates a few hours after parasite ingestion. In a previous work, we investigated the role of heme in parasite cell proliferation and showed that the addition of heme significantly increased parasite proliferation in a dose-dependent manner . To investigate whether the heme effect is mediated by protein kinase signalling pathways, parasite proliferation was evaluated in the presence of several protein kinase (PK) inhibitors. We found that only KN-93, a classical inhibitor of calcium-calmodulin-dependent kinases (CaMKs), blocked heme-induced cell proliferation. KN-92, an inactive analogue of KN-93, was not able to block this effect. A T. cruzi CaMKII homologue is most likely the main enzyme involved in this process since parasite proliferation was also blocked when Myr-AIP, an inhibitory peptide for mammalian CaMKII, was included in the cell proliferation assay. Moreover, CaMK activity increased in parasite cells with the addition of heme as shown by immunological and biochemical assays. In conclusion, the present results are the first strong indications that CaMKII is involved in the heme-induced cell signalling pathway that mediates parasite proliferation.

  11. Protein kinase D1 signaling in angiogenic gene expression and VEGF-mediated angiogenesis

    Directory of Open Access Journals (Sweden)

    Bin eRen MD, Phd, FAHA

    2016-05-01

    Full Text Available Protein kinase D 1 (PKD-1 is a signaling kinase important in fundamental cell functions including migration, proliferation and differentiation. PKD-1 is also a key regulator of gene expression and angiogenesis that is essential for cardiovascular development and tumor progression. Further understanding molecular aspects of PKD-1 signaling in the regulation of angiogenesis may have translational implications in obesity, cardiovascular disease and cancer. The author will summarize and provide the insights into molecular mechanisms by which PKD-1 regulates transcriptional expression of angiogenic genes, focusing on the transcriptional regulation of CD36 by PKD-1-FoxO1 signaling axis along with the potential implications of this axis in arterial differentiation and morphogenesis. He will also discuss a new concept of dynamic balance between proangiogenic and antiangiogenic signaling in determining angiogenic switch, and stress how PKD-1 signaling regulates VEGF signaling-mediated angiogenesis.

  12. Torilin Inhibits Inflammation by Limiting TAK1-Mediated MAP Kinase and NF-κB Activation

    Directory of Open Access Journals (Sweden)

    Mehari Endale

    2017-01-01

    Full Text Available Torilin, a sesquiterpene isolated from the fruits of Torilis japonica, has shown antimicrobial, anticancer, and anti-inflammatory properties. However, data on the mechanism of torilin action against inflammation is limited. This study aimed at determining the anti-inflammatory property of torilin in LPS-induced inflammation using in vitro model of inflammation. We examined torilin’s effect on expression levels of inflammatory mediators and cytokines in LPS-stimulated RAW 264.7 macrophages. The involvement of NF-kB and AP-1, MAP kinases, and adaptor proteins were assessed. Torilin strongly inhibited LPS-induced NO release, iNOS, PGE2, COX-2, NF-α, IL-1β, IL-6, and GM-CSF gene and protein expressions. In addition, MAPKs were also suppressed by torilin pretreatment. Involvement of ERK1/2, P38MAPK, and JNK1/2 was further confirmed by PD98059, SB203580, and SP600125 mediated suppression of iNOS and COX-2 proteins. Furthermore, torilin attenuated NF-kB and AP-1 translocation, DNA binding, and reporter gene transcription. Interestingly, torilin inhibited TAK1 kinase activation with the subsequent suppression of MAPK-mediated JNK, p38, ERK1/2, and AP-1 (ATF-2 and c-jun activation and IKK-mediated I-κBα degradation, p65/p50 activation, and translocation. Together, the results revealed the suppression of NF-κB and AP-1 regulated inflammatory mediator and cytokine expressions, suggesting the test compound’s potential as a candidate anti-inflammatory agent.

  13. Non-opsonic phagocytosis of Legionella pneumophila by macrophages is mediated by phosphatidylinositol 3-kinase.

    Directory of Open Access Journals (Sweden)

    Souvenir D Tachado

    Full Text Available BACKGROUND: Legionella pneumophila, is an intracellular pathogen that causes Legionnaires' disease in humans, a potentially lethal pneumonia. L. pneumophila has the ability to enter and replicate in the host and is essential for pathogenesis. METHODOLOGY/PRINCIPAL FINDINGS: Phagocytosis was measured by cell invasion assays. Construction of PI3K mutant by PCR cloning and expression of dominant negative mutant was detected by Western blot. PI3K activity was measured by 32P labeling and detection of phospholipids products by thin layer chromatography. Infection of macrophages with virulent L. pneumophila stimulated the formation of phosphatidylinositol 3-phosphate (PIP3, a phosphorylated lipid product of PI3K whereas two structurally distinct phosphatidylinositol 3 kinase (PI3K inhibitors, wortmannin and LY294002, reduced L. pneumophila entry into macrophages in a dose-dependent fashion. Furthermore, PI3K activation led to Akt stimulation, a serine/threonine kinase, which was also inhibited by wortmannin and LY294002. In contrast, PI3K and protein kinase B (PKB/Akt activities were lower in macrophages infected with an avirulent bacterial strain. Only virulent L. pneumophila increased lipid kinase activity present in immunoprecipitates of the p85alpha subunit of class I PI3K and tyrosine phosphorylated proteins. In addition, macrophages expressing a specific dominant negative mutant of PI3K reduced L. pneumophila entry into these cells. CONCLUSION/SIGNIFICANCE: Entry of L. pneumophila is mediated by PI3K/Akt signaling pathway. These results suggest an important role for PI3K and Akt in the L. pneumophila infection process. They point to possible novel strategies for undermining L. pneumophila host uptake and reducing pathogenesis of Legionnaires' disease.

  14. 5-ALA mediated photodynamic therapy induces autophagic cell death via AMP-activated protein kinase

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    Lin Yu-Hsin

    2010-04-01

    Full Text Available Abstract Photodynamic therapy (PDT has been developed as an anticancer treatment, which is based on the tumor-specific accumulation of a photosensitizer that induces cell death after irradiation of light with a specific wavelength. Depending on the subcellular localization of the photosensitizer, PDT could trigger various signal transduction cascades and induce cell death such as apoptosis, autophagy, and necrosis. In this study, we report that both AMP-activated protein kinase (AMPK and mitogen-activated protein kinase (MAPK signaling cascades are activated following 5-aminolevulinic acid (ALA-mediated PDT in both PC12 and CL1-0 cells. Although the activities of caspase-9 and -3 are elevated, the caspase inhibitor zVAD-fmk did not protect cells against ALA-PDT-induced cell death. Instead, autophagic cell death was found in PC12 and CL1-0 cells treated with ALA-PDT. Most importantly, we report here for the first time that it is the activation of AMPK, but not MAPKs that plays a crucial role in mediating autophagic cell death induced by ALA-PDT. This novel observation indicates that the AMPK pathway play an important role in ALA-PDT-induced autophagy.

  15. ROS and CDPK-like kinase-mediated activation of MAP kinase in rice roots exposed to lead.

    Science.gov (United States)

    Huang, Tsai-Lien; Huang, Hao-Jen

    2008-04-01

    Lead (Pb2+) is a cytotoxic metal ion in plants, the mechanism of which is not yet established. The aim of this study is to investigate the signalling pathways that are activated by elevated concentrations of Pb2+ in rice roots. Root growth was stunted and cell death was accelerated when exposed to different dosages of Pb2+ during extended time periods. Using ROS-sensitive dye and Ca2+ indicator, we demonstrated that Pb2+ induced ROS production and Ca2+ accumulation, respectively. In addition, Pb2+ elicited a remarkable increase in myelin basic protein (MBP) kinase activities. By immunoblot and immunoprecipitation analysis, 40- and 42-kDa MBP kinases that were activated by Pb2+ were identified to be mitogen-activated protein (MAP) kinases. Pre-treatment of rice roots with an antioxidant and a NADPH oxidase inhibitor, glutathione (GSH) and diphenylene iodonium (DPI), effectively reduced Pb2+-induced cell death and MAP kinase activation. Moreover, calcium-dependent protein kinase (CDPK) antagonist, W7, attenuated Pb2+-induced cell death and MAP kinase activation. These results suggested that the ROS and CDPK may function in the Pb2+-triggered cell death and MAP kinase signalling pathway in rice roots.

  16. Src tyrosine kinases contribute to serotonin-mediated contraction by regulating calcium-dependent pathways in rat skeletal muscle arteries.

    Science.gov (United States)

    Zavaritskaya, Olga; Lubomirov, Lubomir T; Altay, Serdar; Schubert, Rudolf

    2017-06-01

    The Src tyrosine kinase family contributes to the signalling mechanism mediating serotonin (5-hydroxytryptamine (5-HT))-induced vasoconstriction. These kinases were reported to influence the calcium sensitivity of the contractile apparatus. Whether Src kinases affect also the intracellular calcium concentration during constriction of intact arteries is unknown. Thus, we tested the hypothesis that constriction of arteries is associated with a Src kinase-dependent alteration of the intracellular calcium concentration. Contractility of gracilis arteries of Wistar rats was studied using isometric and isobaric myography. The intracellular calcium concentration was measured simultaneously with tension by FURA-2 fluorimetry. Inhibition of Src kinases with 10 μM PP2, 30 μM dasatinib and 100 μM AZM 475271 resulted in a strong attenuation of 5-HT-induced contractions. Vessel incubation with 10 μM PP3, an inactive analogue of PP2, had no effect. Removal of the endothelium did not alter vessel contractile responses to 5-HT nor the action of the Src-kinase inhibitor PP2. The PP2-mediated inhibition of 5-HT-induced contraction was associated with a reduced response of [Ca 2+ ] i to 5-HT. In particular, inhibition of Src kinases attenuates 5-HT-induced calcium influx as well as calcium release from intracellular stores. In contrast, the calcium sensitivity of the contractile apparatus and the filling state of the sarcoplasmic reticulum were not influenced by Src kinases during 5-HT-induced contractions. We conclude that Src kinase activation is a powerful mechanism to produce vasoconstriction of small skeletal muscle arteries of rats. This effect is endothelium-independent. The data further suggest that the action of c-Src kinases is associated with a change in the intracellular calcium concentration that involves Ca 2+ entry and Ca 2+ release pathways.

  17. Differential Requirements for Src-Family Kinases in SYK or ZAP70-Mediated SLP-76 Phosphorylation in Lymphocytes.

    Science.gov (United States)

    Fasbender, Frank; Claus, Maren; Wingert, Sabine; Sandusky, Mina; Watzl, Carsten

    2017-01-01

    In a synthetic biology approach using Schneider (S2) cells, we show that SLP-76 is directly phosphorylated at tyrosines Y113 and Y128 by SYK in the presence of ITAM-containing adapters such as CD3ζ, DAP12, or FcεRγ. This phosphorylation was dependent on at least one functional ITAM and a functional SH2 domain within SYK. Inhibition of Src-kinases by inhibitors PP1 and PP2 did not reduce SLP-76 phosphorylation in S2 cells, suggesting an ITAM and SYK dependent, but Src-kinase independent signaling pathway. This direct ITAM/SYK/SLP-76 signaling pathway therefore differs from previously described ITAM signaling. However, the SYK-family kinase ZAP70 required the additional co-expression of the Src-family kinases Fyn or Lck to efficiently phosphorylate SLP-76 in S2 cells. This difference in Src-family kinase dependency of SYK versus ZAP70-mediated ITAM-based signaling was further demonstrated in human lymphocytes. ITAM signaling in ZAP70-expressing T cells was dependent on the activity of Src-family kinases. In contrast, Src-family kinases were partially dispensable for ITAM signaling in SYK-expressing B cells or in natural killer cells, which express SYK and ZAP70. This demonstrates that SYK can signal using a Src-kinase independent ITAM-based signaling pathway, which may be involved in calibrating the threshold for lymphocyte activation.

  18. Phosphorylation-mediated control of histone chaperone ASF1 levels by Tousled-like kinases.

    Directory of Open Access Journals (Sweden)

    Maxim Pilyugin

    Full Text Available Histone chaperones are at the hub of a diverse interaction networks integrating a plethora of chromatin modifying activities. Histone H3/H4 chaperone ASF1 is a target for cell-cycle regulated Tousled-like kinases (TLKs and both proteins cooperate during chromatin replication. However, the precise role of post-translational modification of ASF1 remained unclear. Here, we identify the TLK phosphorylation sites for both Drosophila and human ASF1 proteins. Loss of TLK-mediated phosphorylation triggers hASF1a and dASF1 degradation by proteasome-dependent and independent mechanisms respectively. Consistent with this notion, introduction of phosphorylation-mimicking mutants inhibits hASF1a and dASF1 degradation. Human hASF1b is also targeted for proteasome-dependent degradation, but its stability is not affected by phosphorylation indicating that other mechanisms are likely to be involved in control of hASF1b levels. Together, these results suggest that ASF1 cellular levels are tightly controlled by distinct pathways and provide a molecular mechanism for post-translational regulation of dASF1 and hASF1a by TLK kinases.

  19. Apoptosis signal-regulating kinase 1 mediates denbinobin-induced apoptosis in human lung adenocarcinoma cells

    Directory of Open Access Journals (Sweden)

    Pan Shiow-Lin

    2009-05-01

    Full Text Available Abstract In the present study, we explore the role of apoptosis signal-regulating kinase 1 (ASK1 in denbinobin-induced apoptosis in human lung adenocarcinoma (A549 cells. Denbinobin-induced cell apoptosis was attenuated by an ASK1 dominant-negative mutant (ASK1DN, two antioxidants (N-acetyl-L-cysteine (NAC and glutathione (GSH, a c-Jun N-terminal kinase (JNK inhibitor (SP600125, and an activator protein-1 (AP-1 inhibitor (curcumin. Treatment of A549 cells with denbinobin caused increases in ASK1 activity and reactive oxygen species (ROS production, and these effects were inhibited by NAC and GSH. Stimulation of A549 cells with denbinobin caused JNK activation; this effect was markedly inhibited by NAC, GSH, and ASK1DN. Denbinobin induced c-Jun phosphorylation, the formation of an AP-1-specific DNA-protein complex, and Bim expression. Bim knockdown using a bim short interfering RNA strategy also reduced denbinobin-induced A549 cell apoptosis. The denbinobin-mediated increases in c-Jun phosphorylation and Bim expression were inhibited by NAC, GSH, SP600125, ASK1DN, JNK1DN, and JNK2DN. These results suggest that denbinobin might activate ASK1 through ROS production to cause JNK/AP-1 activation, which in turn induces Bim expression, and ultimately results in A549 cell apoptosis.

  20. Regulation of ubiquitination-mediated protein degradation by survival kinases in cancer

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    Hirohito eYamaguchi

    2012-02-01

    Full Text Available The ubiquitin-proteasome system is essential for multiple physiological processes via selective degradation of target proteins and has been shown to plays a critical role in human cancer. Activation of oncogenic factors and inhibition of tumor suppressors have been shown to be essential for cancer development, and protein ubiquitination has been linked to the regulation of oncogenic factors and tumor suppressors. Three kinases, Akt, ERK, and IKK, we refer to as oncokinases, are activated in multiple human cancers. We and others have identified several key downstream targets that are commonly regulated by these oncokinases, some of which are regulated directly or indirectly via ubiquitin-mediated proteasome degradation, including FOXO3, β-catenin, Mcl-1, and Snail. In this review, we summarize these findings from our and other groups and discuss potential future studies and applications in the clinic.

  1. Light-mediated Reversible Modulation of the Mitogen-activated Protein Kinase Pathway during Cell Differentiation and Xenopus Embryonic Development.

    Science.gov (United States)

    Krishnamurthy, Vishnu V; Turgeon, Aurora J; Khamo, John S; Mondal, Payel; Sharum, Savanna R; Mei, Wenyan; Yang, Jing; Zhang, Kai

    2017-06-15

    Kinase activity is crucial for a plethora of cellular functions, including cell proliferation, differentiation, migration, and apoptosis. During early embryonic development, kinase activity is highly dynamic and widespread across the embryo. Pharmacological and genetic approaches are commonly used to probe kinase activities. Unfortunately, it is challenging to achieve superior spatial and temporal resolution using these strategies. Furthermore, it is not feasible to control the kinase activity in a reversible fashion in live cells and multicellular organisms. Such a limitation remains a bottleneck for achieving a quantitative understanding of kinase activity during development and differentiation. This work presents an optogenetic strategy that takes advantage of a bicistronic system containing photoactivatable proteins Arabidopsis thaliana cryptochrome 2 (CRY2) and the N-terminal domain of cryptochrome-interacting basic-helix-loop-helix (CIBN). Reversible activation of the mitogen-activated protein kinase (MAPK) signaling pathway is achieved through light-mediated protein translocation in live cells. This approach can be applied to mammalian cell cultures and live vertebrate embryos. This bicistronic system can be generalized to control the activity of other kinases with similar activation mechanisms and can be applied to other model systems.

  2. Protective features of resveratrol on human spermatozoa cryopreservation may be mediated through 5' AMP-activated protein kinase activation.

    Science.gov (United States)

    Shabani Nashtaei, M; Amidi, F; Sedighi Gilani, M A; Aleyasin, A; Bakhshalizadeh, Sh; Naji, M; Nekoonam, S

    2017-03-01

    motility parameters. These findings, although preliminary, suggest that resveratrol-induced improvement of cryopreserved sperm functions may be mediated through activation of AMP-activated protein kinase, indicating the importance of AMP-activated protein kinase activity for human spermatozoa functions. Further investigations are required to elucidate the mechanism by which resveratrol ameliorates oxidative stress-mediated damages in an AMP-activated protein kinase-dependent mechanism. © 2016 American Society of Andrology and European Academy of Andrology.

  3. Myristoylation of Src kinase mediates Src-induced and high-fat diet-accelerated prostate tumor progression in mice.

    Science.gov (United States)

    Kim, Sungjin; Yang, Xiangkun; Li, Qianjin; Wu, Meng; Costyn, Leah; Beharry, Zanna; Bartlett, Michael G; Cai, Houjian

    2017-11-10

    Exogenous fatty acids provide substrates for energy production and biogenesis of the cytoplasmic membrane, but they also enhance cellular signaling during cancer cell proliferation. However, it remains controversial whether dietary fatty acids are correlated with tumor progression. In this study, we demonstrate that increased Src kinase activity is associated with high-fat diet-accelerated progression of prostate tumors and that Src kinases mediate this pathological process. Moreover, in the in vivo prostate regeneration assay, host SCID mice carrying Src(Y529F)-transduced regeneration tissues were fed a low-fat diet or a high-fat diet and treated with vehicle or dasatinib. The high-fat diet not only accelerated Src-induced prostate tumorigenesis in mice but also compromised the inhibitory effect of the anticancer drug dasatinib on Src kinase oncogenic potential in vivo We further show that myristoylation of Src kinase is essential to facilitate Src-induced and high-fat diet-accelerated tumor progression. Mechanistically, metabolism of exogenous myristic acid increased the biosynthesis of myristoyl CoA and myristoylated Src and promoted Src kinase-mediated oncogenic signaling in human cells. Of the fatty acids tested, only exogenous myristic acid contributed to increased intracellular myristoyl CoA levels. Our results suggest that targeting Src kinase myristoylation, which is required for Src kinase association at the cellular membrane, blocks dietary fat-accelerated tumorigenesis in vivo Our findings uncover the molecular basis of how the metabolism of myristic acid stimulates high-fat diet-mediated prostate tumor progression. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  4. Rho-kinase regulates extracellular matrix-mediated osteogenic differentiation of periodontal ligament cells.

    Science.gov (United States)

    Ugawa, Yuki; Yamamoto, Tadashi; Kawamura, Mari; Yamashiro, Keisuke; Shimoe, Masayuki; Tomikawa, Kazuya; Hongo, Shoichi; Maeda, Hiroshi; Takashiba, Shogo

    2017-06-01

    The periodontal ligament (PDL) cells contain heterogeneous mesenchymal cell populations, which have the ability to differentiate into cells that produce adjacent mineralized tissues and abundant extracellular matrix (ECM). ECM is essential not only for the homeostasis of the periodontal tissue, but also for controlling the differentiation of the PDL cells. The process of differentiation involves mechanotransduction, which links the ECM to the cytoskeleton. The present study investigated the roles of Rho-associated coiled-coil containing protein kinase (ROCK) signaling, a crucial regulator of the cytoskeleton, during ECM-mediated osteogenic differentiation of PDL cells in vitro. The PDL cells were isolated from human periodontal ligaments of extracted teeth and cultured in osteogenic medium with or without Y-27632, a pharmacological inhibitor of ROCK. ECM-coated plates were used for ECM-mediated differentiation. The osteogenic phenotype was evaluated at different time points by real-time RT-PCR for the gene encoding alkaline phosphatase (ALP) and an ALP activity assay. The effects of ROCK on cytoskeletal changes and ECM synthesis were examined by immunofluorescence analysis. Y-27632 significantly inhibited ALP at the mRNA and protein activity levels in the late stage of differentiation; concomitantly, the actin filament content and the extracellular levels of collagen-I and fibronectin were markedly decreased by Y-27632. Exogenous collagen-I and fibronectin temporally increased ALP activity, with fibronectin showing a more pronounced effect. Importantly, ECM-mediated differentiation was almost completely inhibited by Y-27632. These findings indicated that ECM-mediated differentiation is dependent on ROCK signaling, and ROCK signaling contributes to the establishment of the ECM microenvironment for PDL cell differentiation. © 2017 International Federation for Cell Biology.

  5. The prostate-derived sterile 20-like kinase (PSK) regulates microtubule organization and stability.

    Science.gov (United States)

    Mitsopoulos, Costas; Zihni, Ceniz; Garg, Ritu; Ridley, Anne J; Morris, Jonathan D H

    2003-05-16

    Sterile 20 (STE20) protein kinases, which include germinal center kinases and p21-activated protein kinases, are known to activate mitogen-activated protein kinase pathways (c-Jun NH(2)-terminal kinase, p38, or extracellular signal-regulated kinase), leading to changes in gene transcription. Some STE20s can also regulate the cytoskeleton, and we have shown that the germinal center kinase-like kinase prostate-derived STE20-like kinase (PSK) affects actin cytoskeletal organization. Here, we demonstrate that PSK colocalizes with microtubules; and that this localization is disrupted by the microtubule depolymerizing agent nocodazole. The association of PSK with microtubules results in the production of stabilized perinuclear microtubule cables that are nocodazole-resistant and contain increased levels of acetylated alpha-tubulin. Kinase-defective PSK (K57A) or the C terminus of PSK (amino acids 745-1235) lacking the kinase domain are sufficient for microtubule binding and stabilization, demonstrating that the catalytic activity of the protein is not required. The localization of PSK to microtubules occurs via its C terminus, and PSK binds and phosphorylates alpha- and beta-tubulin in vitro. The N terminus of PSK (1-940) is unable to bind or stabilize microtubules, demonstrating that PSK must associate with microtubules for their reorganization to occur. These results demonstrate that PSK interacts with microtubules and affects their organization and stability independently of PSK kinase activity.

  6. Sucrose nonfermenting AMPK-related kinase (SNARK) mediates contraction-stimulated glucose transport in mouse skeletal muscle

    DEFF Research Database (Denmark)

    Koh, Ho-Jin; Toyoda, Taro; Fujii, Nobuharu

    2010-01-01

    The signaling mechanisms that mediate the important effects of contraction to increase glucose transport in skeletal muscle are not well understood, but are known to occur through an insulin-independent mechanism. Muscle-specific knockout of LKB1, an upstream kinase for AMPK and AMPK-related prot...

  7. Bruton's tyrosine kinase and phospholipase C gamma 2 mediate chemokine-controlled B cell migration and homing

    NARCIS (Netherlands)

    de Gorter, David J. J.; Beuling, Esther A.; Kersseboom, Rogier; Middendorp, Sabine; van Gils, Janine M.; Hendriks, Rudolf W.; Pals, Steven T.; Spaargaren, Marcel

    2007-01-01

    Control of integrin-mediated adhesion and migration by chemokines plays a critical role in B cell development, differentiation, and function; however, the underlying signaling mechanisms are poorly defined. Here we show that the chemokine SDF-1 induced activation of Bruton's tyrosine kinase (Btk)

  8. β3-Adrenoceptor-mediated relaxation of rat and human urinary bladder : roles of BKCa channels and Rho kinase

    NARCIS (Netherlands)

    Cernecka, Hana; Kersten, Kim; Maarsingh, Harm; Elzinga, Carolina R.; de Jong, Igle Jan; Korstanje, Cees; Michel, Martin C.; Schmidt, Martina

    Previous studies suggest that the large-conductance Ca2+-activated K+ (BKCa) channel and Rho-kinase play major roles in the control of urinary bladder tone. Here, we investigated their involvement in beta-adrenoceptor (AR)-mediated relaxation of rat and human bladder. Concentration-response curves

  9. A novel spleen tyrosine kinase inhibitor blocks c-Jun N-terminal kinase-mediated gene expression in synoviocytes

    NARCIS (Netherlands)

    Cha, Hoon-Suk; Boyle, David L.; Inoue, Tomoyuki; Schoot, Reineke; Tak, Paul P.; Pine, Polly; Firestein, Gary S.

    2006-01-01

    Spleen tyrosine kinase (Syk) is a key regulator of cell signaling induced by cytokines or Fc receptor engagement. However, the role of Syk in rheumatoid arthritis (RA) is not known yet. We investigated the pathways activated by Syk in tumor necrosis factor-alpha (TNFalpha)-stimulated fibroblast-like

  10. Energetics of Src homology domain interactions in receptor tyrosine kinase-mediated signaling.

    Science.gov (United States)

    Ladbury, John E; Arold, Stefan T

    2011-01-01

    Intracellular signaling from receptor tyrosine kinases (RTK) on extracellular stimulation is fundamental to all cellular processes. The protein-protein interactions which form the basis of this signaling are mediated through a limited number of polypeptide domains. For signal transduction without corruption, based on a model where signaling pathways are considered as linear bimolecular relays, these interactions have to be highly specific. This is particularly the case when one considers that any cell may have copies of similar binding domains found in numerous proteins. In this work, an overview of the thermodynamics of binding of two of the most common of these domains (SH2 and SH3 domains) is given. This, coupled with insight from high-resolution structural detail, provides a comprehensive survey of how recognition of cognate binding sites for these domains occurs. Based on the data presented, we conclude that specificity offered by these interactions of SH2 and SH3 domains is limited and not sufficient to enforce mutual exclusivity in RTK-mediated signaling. This may explain the current lack of success in pharmaceutical intervention to inhibit the interactions of these domains when they are responsible for aberrant signaling and the resulting disease states such as cancer. Copyright © 2011 Elsevier Inc. All rights reserved.

  11. A Raf-like protein kinase BHP mediates blue light-dependent stomatal opening.

    Science.gov (United States)

    Hayashi, Maki; Inoue, Shin-Ichiro; Ueno, Yoshihisa; Kinoshita, Toshinori

    2017-03-30

    Stomata in the plant epidermis open in response to blue light and affect photosynthesis and plant growth by regulating CO 2 uptake and transpiration. In stomatal guard cells under blue light, plasma membrane H + -ATPase is phosphorylated and activated via blue light-receptor phototropins and a signaling mediator BLUS1, and H + -ATPase activation drives stomatal opening. However, details of the signaling between phototropins and H + -ATPase remain largely unknown. In this study, through a screening of specific inhibitors for the blue light-dependent H + -ATPase phosphorylation in guard cells, we identified a Raf-like protein kinase, BLUE LIGHT-DEPENDENT H + -ATPASE PHOSPHORYLATION (BHP). Guard cells in the bhp mutant showed impairments of stomatal opening and H + -ATPase phosphorylation in response to blue light. BHP is abundantly expressed in the cytosol of guard cells and interacts with BLUS1 both in vitro and in vivo. Based on these results, BHP is a novel signaling mediator in blue light-dependent stomatal opening, likely downstream of BLUS1.

  12. Myosin light chain kinase mediates intestinal barrier disruption following burn injury.

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    Chuanli Chen

    Full Text Available BACKGROUND: Severe burn injury results in the loss of intestinal barrier function, however, the underlying mechanism remains unclear. Myosin light chain (MLC phosphorylation mediated by MLC kinase (MLCK is critical to the pathophysiological regulation of intestinal barrier function. We hypothesized that the MLCK-dependent MLC phosphorylation mediates the regulation of intestinal barrier function following burn injury, and that MLCK inhibition attenuates the burn-induced intestinal barrier disfunction. METHODOLOGY/PRINCIPAL FINDINGS: Male balb/c mice were assigned randomly to either sham burn (control or 30% total body surface area (TBSA full thickness burn without or with intraperitoneal injection of ML-9 (2 mg/kg, an MLCK inhibitor. In vivo intestinal permeability to fluorescein isothiocyanate (FITC-dextran was measured. Intestinal mucosa injury was assessed histologically. Tight junction proteins ZO-1, occludin and claudin-1 was analyzed by immunofluorescent assay. Expression of MLCK and phosphorylated MLC in ileal mucosa was assessed by Western blot. Intestinal permeability was increased significantly after burn injury, which was accompanied by mucosa injury, tight junction protein alterations, and increase of both MLCK and MLC phosphorylation. Treatment with ML-9 attenuated the burn-caused increase of intestinal permeability, mucosa injury, tight junction protein alterations, and decreased MLC phosphorylation, but not MLCK expression. CONCLUSIONS/SIGNIFICANCE: The MLCK-dependent MLC phosphorylation mediates intestinal epithelial barrier dysfunction after severe burn injury. It is suggested that MLCK-dependent MLC phosphorylation may be a critical target for the therapeutic treatment of intestinal epithelial barrier disruption after severe burn injury.

  13. The double-stranded RNA-activated protein kinase mediates viral-induced encephalitis

    International Nuclear Information System (INIS)

    Scheuner, Donalyn; Gromeier, Matthias; Davies, Monique V.; Dorner, Andrew J.; Song Benbo; Patel, Rupali V.; Wimmer, Eckard J.; McLendon, Roger E.; Kaufman, Randal J.

    2003-01-01

    The double-stranded (ds) RNA-activated protein kinase (PKR) plays an important role in control of viral infections and cell growth. We have studied the role of PKR in viral infection in mice that are defective in the PKR signaling pathway. Transgenic mice were derived that constitutively express a trans-dominant-negative kinase-defective mutant PKR under control of the β-actin promoter. The trans-dominant-negative PKR mutant expressing transgenic mice do not have a detectable phenotype, similar to observations with PKR knock-out mice. The requirement for PKR in viral pathogenesis was studied by intracerebral infection of mice with a mouse-adapted poliovirus. Histopathological analysis revealed diffuse encephalomyelitis with severe inflammatory lesions throughout the central nervous system (CNS) in infected wild-type mice. In contrast, histopathological evaluation of virus-injected trans-dominant-negative PKR transgenic mice as well as PKR knock-out mice yielded no signs of tissue damage associated with inflammatory host responses. However, the virus did replicate in both models of PKR-deficient mice at a level equal to that observed in wild-type infected mice. Although the results indicate a clear difference in susceptibility to poliovirus-induced encephalitis, this difference manifests clinically as a slight delay in fatal neuropathy in trans-dominant-negative PKR transgenic and PKR knock-out animals. Our observations support the finding that viral-induced PKR activation may play a significant role in pathogenesis by mediating the host response to viral CNS infection. They support PKR to be an effective target to control tissue damage due to deleterious host responses to viral infection

  14. Activation of JNK and c-Jun is involved in glucose oxidase-mediated cell death of human lymphoma cells.

    Science.gov (United States)

    Son, Young-Ok; Jang, Yong-Suk; Shi, Xianglin; Lee, Jeong-Chae

    2009-12-31

    Mitogen-activated protein kinases (MAPK) affect the activation of activator protein-1 (AP-1), which plays an important role in regulating a range of cellular processes. However, the roles of these signaling factors on hydrogen peroxide (H(2)O(2))-induced cell death are unclear. This study examined the effects of H(2)O(2) on the activation of MAPK and AP-1 by exposing the cells to H(2)O(2) generated by either glucose oxidase or a bolus addition. Exposing BJAB or Jurkat cells to H(2)O(2) affected the activities of MAPK differently according to the method of H(2)O(2) exposure. H(2)O(2) increased the AP-1-DNA binding activity in these cells, where continuously generated H(2)O(2) led to an increase in mainly the c-Fos, FosB and c-Jun proteins. The c-Jun-NH(2)-terminal kinase (JNK)-mediated activation of c-Jun was shown to be related to the H(2)O(2)-induced cell death. However, the suppression of H(2)O(2)-induced oxidative stress by either JNK inhibitor or c-Jun specific antisense transfection was temporary in the cells exposed to glucose oxidase but not to a bolus H(2)O(2). This was associated with the disruption of death signaling according to the severe and prolonged depletion of reduced glutathione. Overall, these results suggest that H(2)O(2) may decide differently the mode of cell death by affecting the intracellular redox state of thiol-containing antioxidants, and this depends more closely on the duration exposed to H(2)O(2) than the concentration of this agent.

  15. Induction of matrix metalloproteinase-2 by tenascin-X deficiency is mediated through the c-Jun N-terminal kinase and protein tyrosine kinase phosphorylation pathway

    International Nuclear Information System (INIS)

    Matsumoto, Ken-ichi; Minamitani, Takeharu; Orba, Yasuko; Sato, Mami; Sawa, Hirofumi; Ariga, Hiroyoshi

    2004-01-01

    The results of our previous study showed that tumor invasion and metastasis are promoted in extracellular matrix (ECM) tenascin-X-deficient (TNX-/-) mice via increased expression of matrix metalloproteinases (MMPs). However, little is known about the relationship between TNX deficiency and activation of MMP genes. In this study, we investigated the molecular mechanism by which TNX deficiency activates the MMP-2 gene. We examined the intracellular signaling pathways that regulate gene expression of the proteinase in isolated fibroblasts. Results of gelatin zymography showed that MMP-2 was induced to a greater extent in TNX-/- fibroblasts embedded in type I collagen than in wild-type fibroblasts. RT-PCR analysis revealed that the increased level of MMP-2 expression was caused at the transcription level. Conversely, stable overexpression of TNX in a fibroblast cell line reduced MMP-2 expression and suppressed MMP-2 promoter activity. In addition, treatment of TNX-/- fibroblasts with SP600125, a c-Jun N-terminal kinase (JNK) inhibitor, and genistein, a tyrosine kinase inhibitor, suppressed the increased level of proMMP-2 and increased MMP-2 promoter activity in TNX-/- fibroblasts. Furthermore, increased activation of JNK and tyrosine phosphorylation of certain proteins were observed in TNX-/- fibroblasts. These findings suggest that induction of MMP-2 by TNX deficiency is mediated, at least in part, through the JNK and protein tyrosine kinase phosphorylation pathway

  16. Versican G1 domain enhances adenoviral-mediated transgene expression and can be modulated by inhibitors of the Janus kinase (JAK)/STAT and Src family kinase pathways.

    Science.gov (United States)

    Akinfenwa, Patricia Y; Bond, Wesley S; Ildefonso, Cristhian J; Hurwitz, Mary Y; Hurwitz, Richard L

    2017-09-01

    To examine the biochemical influences that may contribute to the success of gene therapy for ocular disorders, the role of versican, a vitreous component, in adenoviral-mediated transgene expression was examined. Versican is a large chondroitin sulfate-containing, hyaluronic acid-binding proteoglycan present in the extracellular matrix and in ocular vitreous body. Y79 retinoblastoma cells and CD44-negative SK-N-DZ neuroblastoma cells transduced with adenoviral vectors in the presence of versican respond with an activation of transgene expression. Proteolysis of versican generates a hyaluronan-binding G1 domain. The addition of recombinant versican G1 to SK-N-DZ cells results in a similar activation of transgene expression, and treatment with dasatinib, an inhibitor of Src family kinases, also mimics the effects of versican. Enhancement is accompanied by an increase in signal transducer and activator of transcription 5 (STAT5) phosphorylation and is abrogated by treatment with C188-9, a STAT3/5 inhibitor, or with ruxolitinib, a Janus kinase 1/2 (JAK1/2) inhibitor. These data implicate versican G1 in enhancing adenoviral vector transgene expression in a hyaluronic acid-CD44 independent manner that is down-regulated by inhibitors of the JAK/STAT pathway and enhanced by inhibitors of the Src kinase pathway. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  17. Fbxl12 triggers G1 arrest by mediating degradation of calmodulin kinase I.

    Science.gov (United States)

    Mallampalli, Rama K; Kaercher, Leah; Snavely, Courtney; Pulijala, Roopa; Chen, Bill B; Coon, Tiffany; Zhao, Jing; Agassandian, Marianna

    2013-10-01

    Cell cycle progression through its regulatory control by changes in intracellular Ca(2+) levels at the G1/S transition mediates cellular proliferation and viability. Ca(2+)/CaM-dependent kinase 1 (CaMKI) appears critical in regulating the assembly of the cyclin D1/cdk4 complex essential for G1 progression, but how this occurs is unknown. Cyclin D1/cdk4 assembly in the early G1 phase is also regulated via binding to p27. Here, we show that a ubiquitin E3 ligase component, F-box protein Fbxl12, mediates CaMKI degradation via a proteasome-directed pathway leading to disruption of cyclin D1/cdk4 complex assembly and resultant G1 arrest in lung epithelia. We also demonstrate that i) CaMKI phosphorylates p27 at Thr(157) and Thr(198) in human cells and at Thr(170) and Thr(197) in mouse cells to modulate its subcellular localization; ii) Fbxl12-induced CaMKI degradation attenuates p27 phosphorylation at these sites in early G1 and iii) activation of CaMKI during G1 transition followed by p27 phosphorylation appears to be upstream to other p27 phosphorylation events, an effect abrogated by Fbxl12 overexpression. Lastly, known inducers of G1 arrest significantly increase Fbxl12 levels in cells. Thus, Fbxl12 may be a previously uncharacterized, functional growth inhibitor regulating cell cycle progression that might be used for mechanism-based therapy. Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.

  18. Inhibition of c-Jun N-terminal kinase sensitizes tumor cells to flavonoid-induced apoptosis through down-regulation of JunD

    International Nuclear Information System (INIS)

    Kook, Sung-Ho; Son, Young-Ok; Jang, Yong-Suk; Lee, Kyung-Yeol; Lee, Seung-Ah; Kim, Beom-Soo; Lee, Hyun-Jeong; Lee, Jeong-Chae

    2008-01-01

    Reduction of susceptibility to apoptosis signals is a crucial step in carcinogenesis. Therefore, sensitization of tumor cells to apoptosis is a promising therapeutic strategy. c-Jun NH 2 -terminal kinase (JNK) has been implicated in stress-induced apoptosis. However, many studies also emphasize the role of JNK on cell survival, although its mechanisms are not completely understood. Previously, we found that inhibition of JNK activity promotes flavonoid-mediated apoptosis of human osteosarcoma cells. We thus determined whether inhibition of JNK sensitizes tumor cells to a bioflavonoid-induced apoptosis, and whether this effect of JNK is a general effect. As the results, quercetin and genistein as well as a flavonoid fraction induced apoptosis of tumor cells, which was further accelerated by specific JNK inhibitor, SP600125 or by small interfering RNA specific to JNK1/2. This effect was specific to types of cells because it was further apparent in tumorigenic cell lines. Inhibition of JNK by SP600125 also reduced flavonoid-stimulated nuclear induction of JunD which was known to have protective role in apoptosis, whereas JNK inhibition alone had little effect on apoptosis. The flavonoid-induced apoptosis of tumor cells was significantly enhanced by transfecting them with antisense JunD oligonucleotides. These results suggest that inhibition of JNK facilitates flavonoid-induced apoptosis through down-regulation of JunD, which is further sensitive to tumor cells. Therefore, combination with a specific JNK inhibitor further enhances the anti-cancer and chemopreventive potential of bio-flavonoids

  19. High Glucose-Mediated Tyrosine Nitration of PI3-Kinase: A Molecular Switch of Survival and Apoptosis in Endothelial Cells

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    Sally L. Elshaer

    2018-03-01

    Full Text Available Diabetes and hyperglycemia are associated with increased retinal oxidative and nitrative stress and vascular cell death. Paradoxically, high glucose stimulates expression of survival and angiogenic growth factors. Therefore, we examined the hypothesis that high glucose-mediated tyrosine nitration causes inhibition of the survival protein PI3-kinase, and in particular, its regulatory p85 subunit in retinal endothelial cell (EC cultures. Retinal EC were cultured in high glucose (HG, 25 mM for 3 days or peroxynitrite (PN, 100 µM overnight in the presence or absence of a peroxynitrite decomposition catalyst (FeTPPs, 2.5 µM, or the selective nitration inhibitor epicatechin (100 µM. Apoptosis of ECs was assessed using TUNEL assay and caspase-3 activity. Immunoprecipitation and Western blot were used to assess protein expression and tyrosine nitration of p85 subunit and its interaction with the p110 subunit. HG or PN accelerated apoptosis of retinal ECs compared to normal glucose (NG, 5 mM controls. HG- or PN-treated cells also showed significant increases in tyrosine nitration on the p85 subunit of PI3-kinase that inhibited its association with the catalytic p110 subunit and impaired PI3-kinase/Akt kinase activity. Decomposing peroxynitrite or blocking tyrosine nitration of p85 restored the activity of PI3-kinase, and prevented apoptosis and activation of p38 MAPK. Inhibiting p38 MAPK or overexpression of the constitutively activated Myr-Akt construct prevented HG- or peroxynitrite-mediated apoptosis. In conclusion, HG impairs pro-survival signals and causes accelerated EC apoptosis, at least in part via tyrosine nitration and inhibition of PI3-kinase. Inhibitors of nitration can be used in adjuvant therapy to delay diabetic retinopathy and microvascular complication.

  20. Protein Kinases Possibly Mediate Hypergravity-Induced Changes in F-Actin Expression by Endothelial Cells

    Science.gov (United States)

    Love, Felisha D.; Melhado, Caroline D.; Bosah, Francis N.; Harris-Hooker, Sandra A.; Sanford, Gary L.

    1998-01-01

    Basic cellular functions such as electrolyte concentration, cell growth rate, glucose utilization, bone formation, response to growth stimulation, and exocytosis are modified in microgravity. These studies indicate that microgravity affects a number of physiological systems and included in this are cell signaling mechanisms. Rijken and coworkers performed growth factor studies that showed PKC signaling and actin microfilament organization appears to be sensitive to microgravity, suggesting that the inhibition of signal transduction by microgravity may be related to alterations in actin microfilament organization. However, similar studies have not been done for vascular cells. Vascular endothelial cells play critical roles in providing nutrients to organ and tissues and in wound repair. The major deterrent to ground-based microgravity studies is that it is impossible to achieved true microgravity for longer than a few minutes on earth. Hence, it has not been possible to conduct prolonged microgravity studies except for two models that simulate certain aspects of microgravity. However, hypergravity is quite easily achieved. Several researchers have shown that hypergravity will increase the proliferation of several different cell lines while decreasing cell motility and slowing liver regeneration following partial hepatectomy, These studies indicate the hypergravity also alters the behavior of most cells. Several investigators have shown that hypergravity affects the activation of several protein kinases (PKs) in cells. In this study, we investigated whether hypergravity alters the expression of f-actin by bovine aortic endothelial cells (BAECs) and the role of PK's (calmodulin 11 dependent, PKA and PKC) as mediators of these effects.

  1. Histamine induces activation of protein kinase D that mediates tissue factor expression and activity in human aortic smooth muscle cells

    Science.gov (United States)

    Hao, Feng; Wu, Daniel Dongwei; Xu, Xuemin

    2012-01-01

    Histamine, an inflammatory mediator, has been shown to influence the pathogenesis of vascular wall cells. However, the molecular basis of its influence is not well understood. Our data reveal that histamine markedly induces protein kinase D (PKD) activation in human aortic smooth muscle cells. PKD belongs to a family of serine/threonine protein kinases, and its function in vascular disease is largely unknown. Our data show that histamine-induced PKD phosphorylation is dependent on the activation of histamine receptor 1 and protein kinase C (PKC). To determine the role of PKD in the histamine pathway, we employed a small-interfering RNA approach to downregulate PKD expression and found that PKD1 and PKD2 are key mediators for expression of tissue factor (TF), which is the key initiator of blood coagulation and is important for thrombosis. Our results show that PKD2 predominantly mediates histamine-induced TF expression via the p38 mitogen-activated protein kinase (MAPK) pathway, whereas PKD1 mediates histamine-induced TF expression through a p38 MAPK-independent pathway. We demonstrate that histamine induces TF expression via the PKC-dependent PKD activation. Our data provide the first evidence that PKD is a new component in histamine signaling in live cells and that PKD has a novel function in the histamine signaling pathway leading to gene expression, as evidenced by TF expression. Importantly, our data reveal a regulatory link from histamine to PKD and TF, providing new insights into the mechanisms of coagulation and the development of atherothrombosis. PMID:23001835

  2. Dihydrotestosterone regulating apolipoprotein M expression mediates via protein kinase C in HepG2 cells

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    Yi-zhou Ye

    2012-12-01

    Full Text Available Abstract Background Administration of androgens decreases plasma concentrations of high-density lipid cholesterol (HDL-C. However, the mechanisms by which androgens mediate lipid metabolism remain unknown. This present study used HepG2 cell cultures and ovariectomized C57BL/6 J mice to determine whether apolipoprotein M (ApoM, a constituent of HDL, was affected by dihydrotestosterone (DHT. Methods HepG2 cells were cultured in the presence of either DHT, agonist of protein kinase C (PKC, phorbol-12-myristate-13-acetate (PMA, blocker of androgen receptor flutamide together with different concentrations of DHT, or DHT together with staurosporine at different concentrations for 24 hrs. Ovariectomized C57BL/6 J mice were treated with DHT or vehicle for 7d or 14d and the levels of plasma ApoM and livers ApoM mRNA were measured. The mRNA levels of ApoM, ApoAI were determined by real-time RT-PCR. ApoM and ApoAI were determined by western blotting analysis. Results Addition of DHT to cell culture medium selectively down-regulated ApoM mRNA expression and ApoM secretion in a dose-dependent manner. At 10 nM DHT, the ApoM mRNA levels were about 20% lower than in untreated cells and about 40% lower at 1000 nM DHT than in the control cells. The secretion of ApoM into the medium was reduced to a similar extent. The inhibitory effect of DHT on ApoM secretion was not blocked by the classical androgen receptor blocker flutamide but by an antagonist of PKC, Staurosporine. Agonist of PKC, PMA, also reduced ApoM. At 0.5 μM PMA, the ApoM mRNA levels and the secretion of ApoM into the medium were about 30% lower than in the control cells. The mRNA expression levels and secretion of another HDL-associated apolipoprotein AI (ApoAI were not affected by DHT. The levels of plasma ApoM and liver ApoM mRNA of DHT-treated C57BL/6 J mice were lower than those of vehicle-treated mice. Conclusions DHT directly and selectively down-regulated the level of ApoM mRNA and the

  3. Transient receptor potential canonical 3 (TRPC3) mediates thrombin-induced astrocyte activation and upregulates its own expression in cortical astrocytes.

    Science.gov (United States)

    Shirakawa, Hisashi; Sakimoto, Shinya; Nakao, Kenji; Sugishita, Aiko; Konno, Masakazu; Iida, Shota; Kusano, Ayaka; Hashimoto, Emina; Nakagawa, Takayuki; Kaneko, Shuji

    2010-09-29

    Reactive astrogliosis, defined by abnormal morphology and excessive cell proliferation, is a characteristic response of astrocytes to CNS injuries, including intracerebral hemorrhage. Thrombin, a major blood-derived serine protease, leaks into the brain parenchyma upon blood-brain barrier disruption and can induce brain injury and astrogliosis. Transient receptor potential canonical (TRPC) channels, Ca(2+)-permeable, nonselective cation channels, are expressed in astrocytes and involved in Ca(2+) influx after receptor stimulation; however, their pathophysiological functions in reactive astrocytes remain unknown. We investigated the pathophysiological roles of TRPC in thrombin-activated cortical astrocytes. Application of thrombin (1 U/ml, 20 h) upregulated TRPC3 protein, which was associated with increased Ca(2+) influx after thapsigargin treatment. Pharmacological manipulations revealed that the TRPC3 upregulation was mediated by protease-activated receptor 1 (PAR-1), extracellular signal-regulated protein kinase, c-Jun NH(2)-terminal kinase, and nuclear factor-κB signaling and required de novo protein synthesis. The Ca(2+) signaling blockers BAPTA-AM, cyclopiazonic acid, and 2-aminoethoxydiphenyl borate and a selective TRPC3 inhibitor, pyrazole-3, attenuated TRPC3 upregulation, suggesting that Ca(2+) signaling through TRPC3 contributes to its increased expression. Thrombin-induced morphological changes at 3 h upregulated S100B, a marker of reactive astrocytes, at 20 h and increased astrocytic proliferation by 72 h, all of which were inhibited by Ca(2+)-signaling blockers and specific knockdown of TRPC3 using small interfering RNA. Intracortical injection of SFLLR-NH(2), a PAR-1 agonist peptide, induced proliferation of astrocytes, most of which were TRPC3 immunopositive. These results suggest that thrombin dynamically upregulates TRPC3 and that TRPC3 contributes to the pathological activation of astrocytes in part through a feedforward upregulation of its own

  4. Induction of viral, 7-methyl-guanosine cap-independent translation and oncolysis by mitogen-activated protein kinase-interacting kinase-mediated effects on the serine/arginine-rich protein kinase.

    Science.gov (United States)

    Brown, Michael C; Bryant, Jeffrey D; Dobrikova, Elena Y; Shveygert, Mayya; Bradrick, Shelton S; Chandramohan, Vidyalakshmi; Bigner, Darell D; Gromeier, Matthias

    2014-11-01

    Protein synthesis, the most energy-consuming process in cells, responds to changing physiologic priorities, e.g., upon mitogen- or stress-induced adaptations signaled through the mitogen-activated protein kinases (MAPKs). The prevailing status of protein synthesis machinery is a viral pathogenesis factor, particularly for plus-strand RNA viruses, where immediate translation of incoming viral RNAs shapes host-virus interactions. In this study, we unraveled signaling pathways centered on the ERK1/2 and p38α MAPK-interacting kinases MNK1/2 and their role in controlling 7-methyl-guanosine (m(7)G) "cap"-independent translation at enterovirus type 1 internal ribosomal entry sites (IRESs). Activation of Raf-MEK-ERK1/2 signals induced viral IRES-mediated translation in a manner dependent on MNK1/2. This effect was not due to MNK's known functions as eukaryotic initiation factor (eIF) 4G binding partner or eIF4E(S209) kinase. Rather, MNK catalytic activity enabled viral IRES-mediated translation/host cell cytotoxicity through negative regulation of the Ser/Arg (SR)-rich protein kinase (SRPK). Our investigations suggest that SRPK activity is a major determinant of type 1 IRES competency, host cell cytotoxicity, and viral proliferation in infected cells. We are targeting unfettered enterovirus IRES activity in cancer with PVSRIPO, the type 1 live-attenuated poliovirus (PV) (Sabin) vaccine containing a human rhinovirus type 2 (HRV2) IRES. A phase I clinical trial of PVSRIPO with intratumoral inoculation in patients with recurrent glioblastoma (GBM) is showing early promise. Viral translation proficiency in infected GBM cells is a core requirement for the antineoplastic efficacy of PVSRIPO. Therefore, it is critically important to understand the mechanisms controlling viral cap-independent translation in infected host cells. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  5. Spatial cycles mediated by UNC119 solubilisation maintain Src family kinases plasma membrane localisation.

    Science.gov (United States)

    Konitsiotis, Antonios D; Roßmannek, Lisaweta; Stanoev, Angel; Schmick, Malte; Bastiaens, Philippe I H

    2017-07-24

    The peripheral membrane proto-oncogene Src family protein tyrosine kinases relay growth factor signals to the cytoplasm of mammalian cells. We unravel the spatial cycles of solubilisation, trapping on perinuclear membrane compartments and vesicular transport that counter entropic equilibration to endomembranes for maintaining the enrichment and activity of Src family protein tyrosine kinases at the plasma membrane. The solubilising factor UNC119 sequesters myristoylated Src family protein tyrosine kinases from the cytoplasm, enhancing their diffusion to effectively release Src family protein tyrosine kinases on the recycling endosome by localised Arl2/3 activity. Src is then trapped on the recycling endosome via electrostatic interactions, whereas Fyn is quickly released to be kinetically trapped on the Golgi by palmitoyl acyl-transferase activity. Vesicular trafficking from these compartments restores enrichment of the Src family protein tyrosine kinases to the plasma membrane. Interference with these spatial cycles by UNC119 knockdown disrupts Src family protein tyrosine kinase localisation and signalling activity, indicating that UNC119 could be a drug target to affect oncogenic Src family protein tyrosine kinase signalling.The peripheral membrane proto-oncogene Src family protein tyrosine kinases (SFKs) transmit growth factor signals to the cytoplasm. Here the authors show that the solubilising factor UNC119 sequesters myristoylated SFKs to maintain its enrichment at the plasma membrane to enable signal transduction.

  6. Comparison of agrobacterium mediated wheat and barley transformation with nucleoside diphosphate kinase 2 (NDPK2) gene

    International Nuclear Information System (INIS)

    Waheed, U.; Shah, M.M.; Smedley, M.; Harwood, W.

    2016-01-01

    An efficient and reliable transformation system is imperative for improvement of important crop species like barley and wheat. Wheat transformation is complex due to larger genome size and polyploidy while barley has a limitation of genotypic dependency. The objective of current study was to compare the relative transformation efficiency of wheat and barley using specific expression vector pBRACT 214-NDPK2 constructed through gateway cloning carrying Nucleoside Diphosphate Kinase 2 (NDPK2) gene. The vector was used to compare the transformation response in both crops using immature embryos through Agrobacterium mediated transformation. Both wheat and barley showed different responses towards callus induction and regeneration. Immature embryos of 1.5 to 2 mm in diameter was found optimum for wheat callus induction while 1 to 1.5 mm for barley. Both embryogenic and non-embryogenic calli were found in wheat with significantly greater tendency for embryogenecity in barley. The overall regeneration response was found different for all transformed wheat and barley cultivars. Wheat cultivars showed good response initially that drastically slowed down in later stages with the exception of Fielder that reached to the green shoots with good roots. The barley transformed lines showed good regeneration response as compared to wheat. PCR analysis of putative transformants using genomic DNA showed a maximum of 27% transformation efficiency in barely. No true transformation response was obtained in all cultivars of wheat used in this study. The protocol developed for wheat and barley transformation will greatly be helpful in crop improvement programme through genetic engineering especially in diploid relatives of cereals. (author)

  7. Sustained protein kinase D activation mediates respiratory syncytial virus-induced airway barrier disruption.

    Science.gov (United States)

    Rezaee, Fariba; DeSando, Samantha A; Ivanov, Andrei I; Chapman, Timothy J; Knowlden, Sara A; Beck, Lisa A; Georas, Steve N

    2013-10-01

    Understanding the regulation of airway epithelial barrier function is a new frontier in asthma and respiratory viral infections. Despite recent progress, little is known about how respiratory syncytial virus (RSV) acts at mucosal sites, and very little is known about its ability to influence airway epithelial barrier function. Here, we studied the effect of RSV infection on the airway epithelial barrier using model epithelia. 16HBE14o- bronchial epithelial cells were grown on Transwell inserts and infected with RSV strain A2. We analyzed (i) epithelial apical junction complex (AJC) function, measuring transepithelial electrical resistance (TEER) and permeability to fluorescein isothiocyanate (FITC)-conjugated dextran, and (ii) AJC structure using immunofluorescent staining. Cells were pretreated or not with protein kinase D (PKD) inhibitors. UV-irradiated RSV served as a negative control. RSV infection led to a significant reduction in TEER and increase in permeability. Additionally it caused disruption of the AJC and remodeling of the apical actin cytoskeleton. Pretreatment with two structurally unrelated PKD inhibitors markedly attenuated RSV-induced effects. RSV induced phosphorylation of the actin binding protein cortactin in a PKD-dependent manner. UV-inactivated RSV had no effect on AJC function or structure. Our results suggest that RSV-induced airway epithelial barrier disruption involves PKD-dependent actin cytoskeletal remodeling, possibly dependent on cortactin activation. Defining the mechanisms by which RSV disrupts epithelial structure and function should enhance our understanding of the association between respiratory viral infections, airway inflammation, and allergen sensitization. Impaired barrier function may open a potential new therapeutic target for RSV-mediated lung diseases.

  8. Laminin α2-mediated focal adhesion kinase activation triggers Alport glomerular pathogenesis.

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    Duane Delimont

    Full Text Available It has been known for some time that laminins containing α1 and α2 chains, which are normally restricted to the mesangial matrix, accumulate in the glomerular basement membranes (GBM of Alport mice, dogs, and humans. We show that laminins containing the α2 chain, but not those containing the α1 chain activates focal adhesion kinase (FAK on glomerular podocytes in vitro and in vivo. CD151-null mice, which have weakened podocyte adhesion to the GBM rendering these mice more susceptible to biomechanical strain in the glomerulus, also show progressive accumulation of α2 laminins in the GBM, and podocyte FAK activation. Analysis of glomerular mRNA from both models demonstrates significant induction of MMP-9, MMP-10, MMP-12, MMPs linked to GBM destruction in Alport disease models, as well as the pro-inflammatory cytokine IL-6. SiRNA knockdown of FAK in cultured podocytes significantly reduced expression of MMP-9, MMP-10 and IL-6, but not MMP-12. Treatment of Alport mice with TAE226, a small molecule inhibitor of FAK activation, ameliorated fibrosis and glomerulosclerosis, significantly reduced proteinuria and blood urea nitrogen levels, and partially restored GBM ultrastructure. Glomerular expression of MMP-9, MMP-10 and MMP-12 mRNAs was significantly reduced in TAE226 treated animals. Collectively, this work identifies laminin α2-mediated FAK activation in podocytes as an important early event in Alport glomerular pathogenesis and suggests that FAK inhibitors, if safe formulations can be developed, might be employed as a novel therapeutic approach for treating Alport renal disease in its early stages.

  9. Rho Kinase ROCK2 Mediates Acid-Induced NADPH Oxidase NOX5-S Expression in Human Esophageal Adenocarcinoma Cells.

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    Jie Hong

    Full Text Available Mechanisms of the progression from Barrett's esophagus (BE to esophageal adenocarcinoma (EA are not fully understood. We have shown that NOX5-S may be involved in this progression. However, how acid upregulates NOX5-S is not well known. We found that acid-induced increase in NOX5-S expression was significantly decreased by the Rho kinase (ROCK inhibitor Y27632 in BE mucosal biopsies and FLO-1 EA cells. In addition, acid treatment significantly increased the Rho kinase activity in FLO-1 cells. The acid-induced increase in NOX5-S expression and H2O2 production was significantly decreased by knockdown of Rho kinase ROCK2, but not by knockdown of ROCK1. Conversely, the overexpression of the constitutively active ROCK2, but not the constitutively active ROCK1, significantly enhanced the NOX5-S expression and H2O2 production. Moreover, the acid-induced increase in Rho kinase activity and in NOX5-S mRNA expression was blocked by the removal of calcium in both FLO-1 and OE33 cells. The calcium ionophore A23187 significantly increased the Rho kinase activity and NOX5-S mRNA expression. We conclude that acid-induced increase in NOX5-S expression and H2O2 production may depend on the activation of ROCK2, but not ROCK1, in EA cells. The acid-induced activation of Rho kinase may be mediated by the intracellular calcium increase. It is possible that persistent acid reflux present in BE patients may increase the intracellular calcium, activate ROCK2 and thereby upregulate NOX5-S. High levels of reactive oxygen species derived from NOX5-S may cause DNA damage and thereby contribute to the progression from BE to EA.

  10. Receptor-like kinases as surface regulators for RAC/ROP-mediated pollen tube growth and interaction with the pistil

    Science.gov (United States)

    Zou, Yanjiao; Aggarwal, Mini; Zheng, Wen-Guang; Wu, Hen-Ming; Cheung, Alice Y.

    2011-01-01

    Background RAC/ROPs are RHO-type GTPases and are known to play diverse signalling roles in plants. Cytoplasmic RAC/ROPs are recruited to the cell membrane and activated in response to extracellular signals perceived and mediated by cell surface-located signalling assemblies, transducing the signals to regulate cellular processes. More than any other cell types in plants, pollen tubes depend on continuous interactions with an extracellular environment produced by their surrounding tissues as they grow within the female organ pistil to deliver sperm to the female gametophyte for fertilization. Scope We review studies on pollen tube growth that provide compelling evidence indicating that RAC/ROPs are crucial for regulating the cellular processes that underlie the polarized cell growth process. Efforts to identify cell surface regulators that mediate extracellular signals also point to RAC/ROPs being the molecular switches targeted by growth-regulating female factors for modulation to mediate pollination and fertilization. We discuss a large volume of work spanning more than two decades on a family of pollen-specific receptor kinases and some recent studies on members of the FERONIA family of receptor-like kinases (RLKs). Significance The research described shows the crucial roles that two RLK families play in transducing signals from growth regulatory factors to the RAC/ROP switch at the pollen tube apex to mediate and target pollen tube growth to the female gametophyte and signal its disintegration to achieve fertilization once inside the female chamber. PMID:22476487

  11. Aspirin Augments IgE-Mediated Histamine Release from Human Peripheral Basophils via Syk Kinase Activation

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    Hiroaki Matsuo

    2013-01-01

    Conclusions: Aspirin enhanced histamine release from basophils via increased Syk kinase activation, and that the augmentation of histamine release by NSAIDs or FAs may be one possible cause of worsening symptoms in patients with chronic urticaria and FDEIA.

  12. Wall-associated kinase-like polypeptide mediates nutritional status perception and response

    Science.gov (United States)

    Yang, Zhenbiao; Karr, Stephen

    2014-02-11

    The disclosure relates to methods for modulating plant growth and organogenesis using dominant-negative receptor-like kinases. The disclosure further provides a method for increasing plant yield relative to corresponding wild type plants comprising modulating the expression in a plant of a nucleic acid encoding a Wall-Associated Kinase-like 14 polypeptide or a homolog thereof, and selecting for plants having increased yield or growth on a nutrient deficient substrate.

  13. Ca(2+) calmodulin kinase and calcineurin mediate IGF-1-induced skeletal muscle dihydropyridine receptor alpha(1S) transcription.

    Science.gov (United States)

    Zheng, Z; Wang, Z M; Delbono, O

    2004-01-15

    The skeletal muscle L-type Ca(2+) channel or dihydropyridine(DHP)-sensitive receptor is a key molecule involved in membrane voltage-sensing, sarcoplasmic reticulum Ca(2+) release, and muscle contraction. Previous work from our laboratory has shown that the insulin-like growth factor-1 (IGF-1) increases skeletal muscle L-type Ca(2+) channel or dihydropyridine-sensitive receptor DHPRalpha(1S) transcriptional activity by acting on the cyclic AMP response element binding protein (CREB) element of the promoter region; however, the cellular signaling mediating this process is not known. In this study, we investigated the signaling pathway whereby IGF-1 enhances the expression of DHPRalpha(1S) in C2C12 myotubes, using a molecular, pharmacological and electrophysiological approach. We found that inhibition of the Ca(2+)/Calmodulin (CaM)-dependent protein kinase or calcineurin, influenced IGF-1-induced increase in DHPRalpha(1S) expression, as detected by recording the luminescence of the DHPRalpha(1S) promoter-luciferase fusion construct and by immunoblot analysis of the DHPR alpha1 subunit. IGF-1 significantly increased CaM kinase and calcineurin activity and the cellular levels of phosphorylated CREB in a time-dependent manner. The role of CaM kinase and calcineurin in DHPRalpha(1S) expression was confirmed by functional recording of the effects of the inhibition of the kinase and phosphatase on IGF-1-mediated enhancement of charge movement. These results support the conclusion that IGF-1 controls CREB phosphorylation by activating a phosphorylation and dephosphorylation cascade, which ultimately modulates the DHPRalpha(1S) gene transcription.

  14. A novel role of protein tyrosine kinase2 in mediating chloride secretion in human airway epithelial cells.

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    Lihua Liang

    Full Text Available Ca(2+ activated Cl(- channels (CaCC are up-regulated in cystic fibrosis (CF airway surface epithelia. The presence and functional properties of CaCC make it a possible therapeutic target to compensate for the deficiency of Cl(- secretion in CF epithelia. CaCC is activated by an increase in cytosolic Ca(2+, which not only activates epithelial CaCCs, but also inhibits epithelial Na(+ hyperabsorption, which may also be beneficial in CF. Our previous study has shown that spiperone, a known antipsychotic drug, activates CaCCs and stimulates Cl(- secretion in polarized human non-CF and CF airway epithelial cell monolayers in vitro, and in Cystic Fibrosis Transmembrane Conductance Regulator (CFTR knockout mice in vivo. Spiperone activates CaCC not by acting in its well-known role as an antagonist of either 5-HT2 or D2 receptors, but through a protein tyrosine kinase-coupled phospholipase C-dependent pathway. Moreover, spiperone independently activates CFTR through a novel mechanism. Herein, we performed a mass spectrometry analysis and identified the signaling molecule that mediates the spiperone effect in activating chloride secretion through CaCC and CFTR. Proline-rich tyrosine kinase 2 (PYK2 is a non-receptor protein tyrosine kinase, which belongs to the focal adhesion kinase family. The inhibition of PYK2 notably reduced the ability of spiperone to increase intracellular Ca(2+ and Cl(- secretion. In conclusion, we have identified the tyrosine kinase, PYK2, as the modulator, which plays a crucial role in the activation of CaCC and CFTR by spiperone. The identification of this novel role of PYK2 reveals a new signaling pathway in human airway epithelial cells.

  15. The expression of COX-2 in VEGF-treated endothelial cells is mediated through protein tyrosine kinase

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    Pravit Akarasereenont

    2002-01-01

    Full Text Available Cyclooxygenase (COX, existing as the COX-1 and COX-2 isoforms, converts arachidonic acid to prostaglandin H2, which is then further metabolized to various prostaglandins. Vascular endothelial growth factor (VEGF has been shown to play important roles in inflammation and is upregulated by the prostaglandin E series through COX-2 in several cell types. Here, we have investigated the effects of VEGF on the COX isoform expressed in human umbilical vein endothelial cells (HUVEC. The signalling mechanism of the COX isoform expressed in endothelial cells activated with VEGF will be also investigated using the tyrosine kinase inhibitor, genistein, and protein kinase C inhibitor, staurosporine. The activity of COX2 was assessed by measuring the production of 6-keto-prostaglandin F1α in the presence of exogenous arachidonic acids (10 μM, 10 min by enzyme immunoassay. The expression of COX isoform protein was detected by immunoblot using specific antibodies. Untreated HUVEC contained no COX-2 protein. In HUVEC treated with VEGF (0.01-50 ng/ml, COX-2 protein, but not COX-1, and COX activity were increased in a dose-dependent manner. Interestingly, the increased COX-2 protein and activity in response to VEGF (10 ng/ml was inhibited by the tyrosine kinase inhibitor, genistein (0.05-5 μg/ml, but not by the protein kinase C inhibitor, staurosporine (0.1-10 ng/ml. Thus, the induction of COX-2 by VEGF in endothelial cells was mediated through protein tyrosine kinase, and the uses of specific COX-2 inhibitors in these conditions, in which VEGF was involved, might have a role.

  16. Differential effects of vasopressin and phenylephrine on protein kinase C-mediated protein phosphorylations in isolated hepatocytes

    International Nuclear Information System (INIS)

    Cooper, R.H.; Johanson, R.A.; Wiliamson, J.R.

    1986-01-01

    Receptor-mediated breakdown of inositol lipids produces two intracellular signals, diacylglycerol, which activates protein kinase C, and inositol trisphosphate, which causes release of intracellular vesicular Ca 2+ . This study examined the effects of Ca 2+ -ionophores, vasopressin, phenylephrine, and phorbol ester (PMA) on hepatocyte protein phosphorylations. [ 32 P] Phosphoproteins from hepatocytes prelabeled with 32 P were resolved by 2-dimensional SDS-PAGE and corresponding autoradiographs were quantitated by densitometric analysis. The phosphorylation of five proteins, a plasma membrane bound 16 kDa protein with pI 6.4, a cytosolic 16 kDa protein with pI 5.8, and proteins with Mr's of 36 kDa, 52 kDa, and 68 kDa, could be attributed to phosphorylation by protein kinase C since the phosphorylation was stimulated by PMA. When the vasopressin concentration was varied, low vasopressin stimulated the phosphorylation of only the membrane bound 16 kDa protein of the above set of proteins, while higher vasopressin concentrations were required to stimulate the phosphorylation of all five proteins. Phenylephrine, even at supramaximal concentrations, stimulated the phosphorylation of only the membrane bound 16 kDa protein. These results suggest that phenylephrine is a less potent activator of protein kinase C than vasopressin by virtue of limited or localized diacylglycerol production

  17. The regulatory effect of nucleoside diphosphate kinase on G-protein and G-protein mediated phospholipase C.

    Science.gov (United States)

    Zhang, D; Chang, K

    1995-03-01

    The effect of nucleoside diphosphate kinase (NDPK) on the activity of guanine nucleotide regulatory protein (G-protein) mediated phospholipase C (PLC) and on the [35S] GTPT tau S binding of G-protein was investigated in this work in order to demonstrate the mechanism behind the regulation of G-protein and its effector PLC by NDPK. The stimulation of PLC in turkey erythrocyte membrane by both GTP and GTP tau S indicated that the PLC stimulation was mediated by G-protein. NDPK alone stimulated PLC activity, as well as the stimulation in the presence of GTP and GDP, in a dose-dependent manner. However, NDPK inhibited GTP tau S-stimulated PLC. Furthermore, NDPK inhibited [35S]GTP tau S binding of purified Gi-protein in a non-competitive manner. A hypothesis implying an important role of direct interaction of G-protein and NDPK in the regulation of their functions is suggested and discussed.

  18. T cell responses affected by aminopeptidase N (CD13)-mediated trimming of major histocompatibility complex class II-bound peptides

    DEFF Research Database (Denmark)

    Larsen, S L; Pedersen, L O; Buus, S

    1996-01-01

    the exopeptidase Aminopeptidase N (APN, CD13) as one of the enzymes involved in the observed cell-surface antigen processing. The NH2-terminal end of the longer peptide could, even while bound to major histocompatibility complex (MHC) class II molecules, be digested by APN with dramatic consequences for T cell......Endocytosed protein antigens are believed to be fragmented in what appears to be a balance between proteolysis and MHC-mediated epitope protection, and the resulting peptide-MHC complexes are transported to the surface of the antigen-presenting cells (APC) and presented to T cells. The events...

  19. The tight junction protein ZO-2 and Janus kinase 1 mediate intercellular communications in vascular smooth muscle cells

    Energy Technology Data Exchange (ETDEWEB)

    Tkachuk, Natalia; Tkachuk, Sergey; Patecki, Margret [Department of Nephrology, Hannover Medical School, Hannover D-30625 (Germany); Kusch, Angelika [Department of Nephrology and Intensive Care Medicine, Charite Campus Virchow-Klinikum, Berlin D-13353 (Germany); Korenbaum, Elena; Haller, Hermann [Department of Nephrology, Hannover Medical School, Hannover D-30625 (Germany); Dumler, Inna, E-mail: dumler.inna@mh-hannover.de [Department of Nephrology, Hannover Medical School, Hannover D-30625 (Germany)

    2011-07-08

    Highlights: {yields} The tight junction protein ZO-2 associates with Jak1 in vascular smooth muscle cells via ZO-2 N-terminal fragment. {yields} Jak1 mediates ZO-2 tyrosine phosphorylation and ZO-2 localization to the sites of homotypic intercellular contacts. {yields} The urokinase receptor uPAR regulates ZO-2/Jak1 functional association. {yields} The ZO-2/Jak1/uPAR signaling complex is required for vascular smooth muscle cells functional network formation. -- Abstract: Recent evidence points to a multifunctional role of ZO-2, the tight junction protein of the MAGUK (membrane-associated guanylate kinase-like) family. Though ZO-2 has been found in cell types lacking tight junction structures, such as vascular smooth muscle cells (VSMC), little is known about ZO-2 function in these cells. We provide evidence that ZO-2 mediates specific homotypic cell-to-cell contacts between VSMC. Using mass spectrometry we found that ZO-2 is associated with the non-receptor tyrosine kinase Jak1. By generating specific ZO-2 constructs we further found that the N-terminal fragment of ZO-2 molecule is responsible for this interaction. Adenovirus-based expression of Jak1 inactive mutant demonstrated that Jak1 mediates ZO-2 tyrosine phosphorylation. By means of RNA silencing, expression of Jak1 mutant form and fluorescently labeled ZO-2 fusion protein we further specified that active Jak1, but not Jak1 inactive mutant, mediates ZO-2 localization to the sites of intercellular contacts. We identified the urokinase receptor uPAR as a pre-requisite for these cellular events. Functional requirement of the revealed signaling complex for VSMC network formation was confirmed in experiments using Matrigel and in contraction assay. Our findings imply involvement of the ZO-2 tight junction independent signaling complex containing Jak1 and uPAR in VSMC intercellular communications. This mechanism may contribute to vascular remodeling in occlusive cardiovascular diseases and in arteriogenesis.

  20. Dendritic cells sensitize TCRs through self-MHC-mediated Src family kinase activation

    Czech Academy of Sciences Publication Activity Database

    Meraner, P.; Hořejší, Václav; Wolpl, A.; Fischer, G.F.; Stingl, G.; Maurer, D.

    2007-01-01

    Roč. 178, č. 4 (2007), s. 2262-2271 ISSN 0022-1767 Institutional research plan: CEZ:AV0Z50520514 Keywords : TCR * dendritic cells * Src kinases Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 6.068, year: 2007

  1. P-glycoprotein Mediates Ceritinib Resistance in Anaplastic Lymphoma Kinase-rearranged Non-small Cell Lung Cancer

    Directory of Open Access Journals (Sweden)

    Ryohei Katayama

    2016-01-01

    Full Text Available The anaplastic lymphoma kinase (ALK fusion oncogene is observed in 3%–5% of non-small cell lung cancer (NSCLC. Crizotinib and ceritinib, a next-generation ALK tyrosine kinase inhibitor (TKI active against crizotinib-refractory patients, are clinically available for the treatment of ALK-rearranged NSCLC patients, and multiple next-generation ALK-TKIs are currently under clinical evaluation. These ALK-TKIs exhibit robust clinical activity in ALK-rearranged NSCLC patients; however, the emergence of ALK-TKI resistance restricts the therapeutic effect. To date, various secondary mutations or bypass pathway activation-mediated resistance have been identified, but large parts of the resistance mechanism are yet to be identified. Here, we report the discovery of p-glycoprotein (P-gp/ABCB1 overexpression as a ceritinib resistance mechanism in ALK-rearranged NSCLC patients. P-gp exported ceritinib and its overexpression conferred ceritinib and crizotinib resistance, but not to PF-06463922 or alectinib, which are next-generation ALK inhibitors. Knockdown of ABCB1 or P-gp inhibitors sensitizes the patient-derived cancer cells to ceritinib, in vitro and in vivo. P-gp overexpression was identified in three out of 11 cases with in ALK-rearranged crizotinib or ceritinib resistant NSCLC patients. Our study suggests that alectinib, PF-06463922, or P-gp inhibitor with ceritinib could overcome the ceritinib or crizotinib resistance mediated by P-gp overexpression.

  2. Neutrophil FcγRIIA promotes IgG-mediated glomerular neutrophil capture via Abl/Src kinases.

    Science.gov (United States)

    Nishi, Hiroshi; Furuhashi, Kazuhiro; Cullere, Xavier; Saggu, Gurpanna; Miller, Mark J; Chen, Yunfeng; Rosetti, Florencia; Hamilton, Samantha L; Yang, Lihua; Pittman, Spencer P; Liao, Jiexi; Herter, Jan M; Berry, Jeffrey C; DeAngelo, Daniel J; Zhu, Cheng; Tsokos, George C; Mayadas, Tanya N

    2017-10-02

    The kidney glomerular capillaries are frequent sites of immune complex deposition and subsequent neutrophil accumulation in post-infectious and rapidly progressive glomerulonephritis. However, the mechanisms of neutrophil recruitment remain enigmatic, and there is no targeted therapeutic to avert this proximal event in glomerular inflammation. The uniquely human activating Fc receptor FcγRIIA promotes glomerular neutrophil accumulation and damage in anti-glomerular basement membrane-induced (anti-GBM-induced) glomerulonephritis when expressed on murine neutrophils. Here, we found that neutrophils are directly captured by immobilized IgG antibodies under physiological flow conditions in vitro through FcγRIIA-dependent, Abl/Src tyrosine kinase-mediated F-actin polymerization. Biophysical measurements showed that the lifetime of FcγRIIA-IgG bonds increased under mechanical force in an F-actin-dependent manner, which could enable the capture of neutrophils under physiological flow. Kidney intravital microscopy revealed that circulating neutrophils, which were similar in diameter to glomerular capillaries, abruptly arrested following anti-GBM antibody deposition via neutrophil FcγRIIA and Abl/Src kinases. Accordingly, inhibition of Abl/Src with bosutinib reduced FcγRIIA-mediated glomerular neutrophil accumulation and renal injury in experimental, crescentic anti-GBM nephritis. These data identify a pathway of neutrophil recruitment within glomerular capillaries following IgG deposition that may be targeted by bosutinib to avert glomerular injury.

  3. LKB1 mediates the development of conventional and innate T cells via AMP-dependent kinase autonomous pathways.

    Directory of Open Access Journals (Sweden)

    Marouan Zarrouk

    Full Text Available The present study has examined the role of the serine/threonine kinase LKB1 in the survival and differentiation of CD4/8 double positive thymocytes. LKB1-null DPs can respond to signals from the mature α/β T-cell-antigen receptor and initiate positive selection. However, in the absence of LKB1, thymocytes fail to mature to conventional single positive cells causing severe lymphopenia in the peripheral lymphoid tissues. LKB1 thus appears to be dispensable for positive selection but important for the maturation of positively selected thymocytes. LKB1 also strikingly prevented the development of invariant Vα14 NKT cells and innate TCR αβ gut lymphocytes. Previous studies with gain of function mutants have suggested that the role of LKB1 in T cell development is mediated by its substrate the AMP-activated protein kinase (AMPK. The present study now analyses the impact of AMPK deletion in DP thymocytes and shows that the role of LKB1 during the development of both conventional and innate T cells is mediated by AMPK-independent pathways.

  4. Trophoblast cell fusion and differentiation are mediated by both the protein kinase C and a pathways.

    Directory of Open Access Journals (Sweden)

    Waka Omata

    Full Text Available The syncytiotrophoblast of the human placenta is an epithelial barrier that interacts with maternal blood and is a key for the transfer of nutrients and other solutes to the developing fetus. The syncytiotrophoblast is a true syncytium and fusion of progenitor cytotrophoblasts is the cardinal event leading to the formation of this layer. BeWo cells are often used as a surrogate for cytotrophoblasts, since they can be induced to fuse, and then express certain differentiation markers associated with trophoblast syncytialization. Dysferlin, a syncytiotrophoblast membrane repair protein, is up-regulated in BeWo cells induced to fuse by treatment with forskolin; this fusion is thought to occur through cAMP/protein kinase A-dependent mechanisms. We hypothesized that dysferlin may also be up-regulated in response to fusion through other pathways. Here, we show that BeWo cells can also be induced to fuse by treatment with an activator of protein kinase C, and that this fusion is accompanied by increased expression of dysferlin. Moreover, a dramatic synergistic increase in dysferlin expression is observed when both the protein kinase A and protein kinase C pathways are activated in BeWo cells. This synergy in fusion is also accompanied by dramatic increases in mRNA for the placental fusion proteins syncytin 1, syncytin 2, as well as dysferlin. Dysferlin, however, was shown to be dispensable for stimulus-induced BeWo cell syncytialization, since dysferlin knockdown lines fused to the same extent as control cells. The classical trophoblast differentiation marker human chorionic gonadotropin was also monitored and changes in the expression closely parallel that of dysferlin in all of the experimental conditions employed. Thus different biochemical markers of trophoblast fusion behave in concert supporting the hypothesis that activation of both protein kinase C and A pathways lead to trophoblastic differentiation.

  5. Ribosomal Protein S6 Kinase (RSK-2 as a central effector molecule in RON receptor tyrosine kinase mediated epithelial to mesenchymal transition induced by macrophage-stimulating protein

    Directory of Open Access Journals (Sweden)

    Zhang Rui-Wen

    2011-05-01

    Full Text Available Abstract Background Epithelial to mesenchymal transition (EMT occurs during cancer cell invasion and malignant metastasis. Features of EMT include spindle-like cell morphology, loss of epithelial cellular markers and gain of mesenchymal phenotype. Activation of the RON receptor tyrosine kinase by macrophage-stimulating protein (MSP has been implicated in cellular EMT program; however, the major signaling determinant(s responsible for MSP-induced EMT is unknown. Results The study presented here demonstrates that RSK2, a downstream signaling protein of the Ras-Erk1/2 pathway, is the principal molecule that links MSP-activated RON signaling to complete EMT. Using MDCK cells expressing RON as a model, a spindle-shape based screen was conducted, which identifies RSK2 among various intracellular proteins as a potential signaling molecule responsible for MSP-induced EMT. MSP stimulation dissociated RSK2 with Erk1/2 and promoted RSK2 nuclear translocation. MSP strongly induced RSK2 phosphorylation in a dose-dependent manner. These effects relied on RON and Erk1/2 phosphorylation, which is significantly potentiated by transforming growth factor (TGF-β1, an EMT-inducing cytokine. Specific RSK inhibitor SL0101 completely prevented MSP-induced RSK phosphorylation, which results in inhibition of MSP-induced spindle-like morphology and suppression of cell migration associated with EMT. In HT-29 cancer cells that barely express RSK2, forced RSK2 expression results in EMT-like phenotype upon MSP stimulation. Moreover, specific siRNA-mediated silencing of RSK2 but not RSK1 in L3.6pl pancreatic cancer cells significantly inhibited MSP-induced EMT-like phenotype and cell migration. Conclusions MSP-induced RSK2 activation is a critical determinant linking RON signaling to cellular EMT program. Inhibition of RSK2 activity may provide a therapeutic opportunity for blocking RON-mediated cancer cell migration and subsequent invasion.

  6. Inhibition of PIM1 kinase attenuates inflammation-induced pro-labour mediators in human foetal membranes in vitro.

    Science.gov (United States)

    Lim, Ratana; Barker, Gillian; Lappas, Martha

    2017-06-01

    Does proviral integration site for Moloney murine leukaemic virus (PIM)1 kinase play a role in regulating the inflammatory processes of human labour and delivery? PIM1 kinase plays a critical role in foetal membranes in regulating pro-inflammatory and pro-labour mediators. Infection and inflammation have strong causal links to preterm delivery by stimulating pro-inflammatory cytokines and collagen degrading enzymes, which can lead to rupture of membranes. PIM1 has been shown to have a role in immune regulation and inflammation in non-gestational tissues; however, its role has not been explored in the field of human labour. PIM1 expression was analysed in myometrium and/or foetal membranes obtained at term and preterm (n = 8-9 patients per group). Foetal membranes, freshly isolated amnion cells and primary myometrial cells were used to investigate the effect of PIM1 inhibition on pro-labour mediators (n = 5 patients per treatment group). Foetal membranes, from term and preterm, were obtained from non-labouring and labouring women, and from preterm pre-labour rupture of membranes (PPROM) (n = 9 per group). Amnion was collected from women with and without preterm chorioamnionitis (n = 8 per group). Expression of PIM1 kinase was determined by qRT-PCR and western blotting. To determine the effect of PIM1 kinase inhibition on the expression of pro-inflammatory and pro-labour mediators induced by bacterial products lipopolysaccharide (LPS) (10 μg/ml) and flagellin (1 μg/ml) and pro-inflammatory cytokine tumour necrosis factor (TNF) (10 ng/ml), chemical inhibitors SMI-4a (20 μM) and AZD1208 (50 μM) were used in foetal membrane explants and siRNA against PIM1 was used in primary amnion cells. Statistical significance was set at P membranes after spontaneous term labour compared to no labour at term and in amnion with preterm chorioamnionitis compared to preterm with no chorioamnionitis. There was no change in PIM1 expression with preterm labour or PPROM

  7. Positional signaling mediated by a receptor-like kinase in Arabidopsis.

    Science.gov (United States)

    Kwak, Su-Hwan; Shen, Ronglai; Schiefelbein, John

    2005-02-18

    The position-dependent specification of root epidermal cells in Arabidopsis provides an elegant paradigm for cell patterning during development. Here, we describe a new gene, SCRAMBLED (SCM), required for cells to appropriately interpret their location within the developing root epidermis. SCM encodes a receptor-like kinase protein with a predicted extracellular domain of six leucine-rich repeats and an intracellular serine-threonine kinase domain. SCM regulates the expression of the GLABRA2, CAPRICE, WEREWOLF, and ENHANCER OF GLABRA3 transcription factor genes that define the cell fates. Further, the SCM gene is expressed throughout the developing root. Therefore, SCM likely enables developing epidermal cells to detect positional cues and establish an appropriate cell-type pattern.

  8. Induction of rat hepatic zinc thionein by phorbol ester-mediated protein kinase C pathway

    Energy Technology Data Exchange (ETDEWEB)

    Garrett, S.H.; Funk, A.E.; Brady, F.O.

    1986-05-01

    Metallothionein (MT) exists in rat liver mainly as a zinc protein. The levels of this protein fluctuate in response to a variety of internal and external stimuli. Among these inducers of MT are metals, glucocorticoids, catecholamines, and polypeptide hormones. Metals and glucocorticoids are primary inducers of MT, while the others operate either via adenylate cyclase/cAMP/cAMP-dependent protein kinase, or via phospholipase C/inositol 1,4,5-triphosphate, diacylglycerol/Ca/sup 2 +/-dependent protein kinase, protein kinase C. The authors have examined the role of the protein kinase C pathway in the induction of MT by using a phorbol ester, 12-O-tetradecanoyl-phorbol 13-acetate (TPA), to activate it. In vivo TPA is a good inducer of Zn/sub 7/-MT with an ED/sub 0.5/ of 26.5 nmoles/kg b.w. Maximal levels reached were about 7..mu..g Zn in MT/g liver, an induction increase of 8 to 10-fold. An inactive compound, 4..beta..-phorbol, and the vehicle (DMSO) did not stimulate the synthesis of Zn/sub 7/-MT. This induction by TPA requires de novo protein synthesis, as demonstrated by a cycloheximide/(/sup 35/S)-cysteine experiment. TPA stimulated Zn incorporation by 8.6-fold and (/sup 35/S)-cysteine incorporation by 4.8-fold during an 11h induction. These increases were blocked 100% by treatment with cycloheximide at -1 and +5h. These experiments have been repeated in cultured hepatocytes, using (/sup 35/S)-cysteine incorporation, slab SDS-PAGE, and autoradiography to quantitate MT levels.

  9. Tec-kinase-mediated phosphorylation of fibroblast growth factor 2 is essential for unconventional secretion.

    Science.gov (United States)

    Ebert, Antje D; Laussmann, Mareike; Wegehingel, Sabine; Kaderali, Lars; Erfle, Holger; Reichert, Jürgen; Lechner, Johannes; Beer, Hans-Dietmar; Pepperkok, Rainer; Nickel, Walter

    2010-06-01

    Fibroblast growth factor 2 (FGF2) is a potent mitogen that is exported from cells by an endoplasmic reticulum (ER)/Golgi-independent mechanism. Unconventional secretion of FGF2 occurs by direct translocation across plasma membranes, a process that depends on the phosphoinositide phosphatidylinositol 4,5-biphosphate (PI(4,5)P(2)) at the inner leaflet as well as heparan sulfate proteoglycans at the outer leaflet of plasma membranes; however, additional core and regulatory components of the FGF2 export machinery have remained elusive. Here, using a highly effective RNAi screening approach, we discovered Tec kinase as a novel factor involved in unconventional secretion of FGF2. Tec kinase does not affect FGF2 secretion by an indirect mechanism, but rather forms a heterodimeric complex with FGF2 resulting in phosphorylation of FGF2 at tyrosine 82, a post-translational modification shown to be essential for FGF2 membrane translocation to cell surfaces. Our findings suggest a crucial role for Tec kinase in regulating FGF2 secretion under various physiological conditions and, therefore, provide a new perspective for the development of a novel class of antiangiogenic drugs targeting the formation of the FGF2/Tec complex.

  10. Mycobacterium tuberculosis Ser/Thr protein kinase B mediates an oxygen-dependent replication switch

    Energy Technology Data Exchange (ETDEWEB)

    Ortega, Corrie; Liao, Reiling; Anderson, Lindsey N.; Rustad, Tige; Ollodart, Anja R.; Wright, Aaron T.; Sherman, David R.; Grundner, Christoph

    2014-01-07

    In the majority of cases, Mycobacterium tuberculosis (Mtb) infections are clinically latent, characterized by little or no bacterial replication and drug tolerance. Low oxygen tension is a major host factor inducing bacteriostasis, but the molecular mechanisms driving oxygen-dependent replication are poorly understood. Mtb encodes eleven serine/threonine protein kinases, a family of signaling molecules known to regulate similar replicative adaptations in other bacteria. Here, we tested the role of serine/threonine phosphorylation in the Mtb response to altered oxygen status, using an in vitro model of latency (hypoxia) and reactivation (reaeration). Broad kinase inhibition compromised survival of Mtb in hypoxia. Activity-based protein profiling and genetic mutation identified PknB as the kinase critical for surviving hypoxia. Mtb replication was highly sensitive to changes in PknB levels in aerated culture, and even more so in hypoxia. A mutant overexpressing PknB specifically in hypoxia showed a 10-fold loss in viability in low oxygen conditions. In contrast, chemically reducing PknB activity during hypoxia specifically compromised resumption of growth during reaeration. These data support a model in which PknB activity is reduced to achieve bacteriostasis, and elevated when replication resumes. Together, these data show that phosphosignaling controls replicative transitions associated with latency and reactivation, that PknB is a major regulator of these transitions, and that PknB could provide a highly vulnerable therapeutic target at every step of the Mtb life cycle - active disease, latency, and reactivation.

  11. Polymeric immunoglobulin receptor-mediated invasion of Streptococcus pneumoniae into host cells requires a coordinate signaling of SRC family of protein-tyrosine kinases, ERK, and c-Jun N-terminal kinase.

    Science.gov (United States)

    Agarwal, Vaibhav; Asmat, Tauseef M; Dierdorf, Nina I; Hauck, Christof R; Hammerschmidt, Sven

    2010-11-12

    Streptococcus pneumoniae are commensals of the human nasopharynx with the capacity to invade mucosal respiratory cells. PspC, a pneumococcal surface protein, interacts with the human polymeric immunoglobulin receptor (pIgR) to promote bacterial adherence to and invasion into epithelial cells. Internalization of pneumococci requires the coordinated action of actin cytoskeleton rearrangements and the retrograde machinery of pIgR. Here, we demonstrate the involvement of Src protein-tyrosine kinases (PTKs), focal adhesion kinase (FAK), extracellular signal-regulated kinase (ERK), and c-Jun N-terminal kinase (JNK) but not p38 mitogen-activated protein kinases (MAPK) in pneumococcal invasion via pIgR. Pharmacological inhibitors of PTKs and MAPKs and genetic interference with Src PTK and FAK functions caused a significant reduction of pIgR-mediated pneumococcal invasion but did not influence bacterial adhesion to host cells. Furthermore, pneumococcal ingestion by host cells induces activation of ERK1/2 and JNK. In agreement with activated JNK, its target molecule and DNA-binding protein c-Jun was phosphorylated. We also show that functionally active Src PTK is essential for activation of ERK1/2 upon pneumococcal infections. In conclusion, these data illustrate the importance of a coordinated signaling between Src PTKs, ERK1/2, and JNK during PspC-pIgR-mediated uptake of pneumococci by host epithelial cells.

  12. A Truncated Fragment of Src Protein Kinase Generated by Calpain-mediated Cleavage Is a Mediator of Neuronal Death in Excitotoxicity*

    Science.gov (United States)

    Hossain, M. Iqbal; Roulston, Carli L.; Kamaruddin, M. Aizuddin; Chu, Percy W. Y.; Ng, Dominic C. H.; Dusting, Gregory J.; Bjorge, Jeffrey D.; Williamson, Nicholas A.; Fujita, Donald J.; Cheung, Steve N.; Chan, Tung O.; Hill, Andrew F.; Cheng, Heung-Chin

    2013-01-01

    Excitotoxicity resulting from overstimulation of glutamate receptors is a major cause of neuronal death in cerebral ischemic stroke. The overstimulated ionotropic glutamate receptors exert their neurotoxic effects in part by overactivation of calpains, which induce neuronal death by catalyzing limited proteolysis of specific cellular proteins. Here, we report that in cultured cortical neurons and in vivo in a rat model of focal ischemic stroke, the tyrosine kinase Src is cleaved by calpains at a site in the N-terminal unique domain. This generates a truncated Src fragment of ∼52 kDa, which we localized predominantly to the cytosol. A cell membrane-permeable fusion peptide derived from the unique domain of Src prevents calpain from cleaving Src in neurons and protects against excitotoxic neuronal death. To explore the role of the truncated Src fragment in neuronal death, we expressed a recombinant truncated Src fragment in cultured neurons and examined how it affects neuronal survival. Expression of this fragment, which lacks the myristoylation motif and unique domain, was sufficient to induce neuronal death. Furthermore, inactivation of the prosurvival kinase Akt is a key step in its neurotoxic signaling pathway. Because Src maintains neuronal survival, our results implicate calpain cleavage as a molecular switch converting Src from a promoter of cell survival to a mediator of neuronal death in excitotoxicity. Besides unveiling a new pathological action of Src, our discovery of the neurotoxic action of the truncated Src fragment suggests new therapeutic strategies with the potential to minimize brain damage in ischemic stroke. PMID:23400779

  13. Tec kinases regulate actin assembly and cytokine expression in LPS-stimulated human neutrophils via JNK activation.

    Science.gov (United States)

    Zemans, Rachel L; Arndt, Patrick G

    2009-01-01

    The acute inflammatory response involves neutrophils wherein recognition of bacterial products, such as lipopolysaccharide (LPS), activates intracellular signaling pathways. We have shown that the mitogen-activated protein kinase (MAPK) c-Jun NH(2) terminal kinase (JNK) is activated by LPS in neutrophils and plays a critical role in monocyte chemoattractant protein (MCP)-1 expression and actin assembly. As the Tec family kinases are expressed in neutrophils and regulate activation of the MAPKs in other cell systems, we hypothesized that the Tec kinases are an upstream component of the signaling pathway leading to LPS-induced MAPKs activation in neutrophils. Herein, we show that the Tec kinases are activated in LPS-stimulated human neutrophils and that inhibition of the Tec kinases, with leflunomide metabolite analog (LFM-A13), decreased LPS-induced JNK, but not p38, activity. Furthermore, LPS-induced actin polymerization as well as MCP-1, tumor necrosis factor-alpha, interleukin-6, and interleukin-1beta expression are dependent on Tec kinase activity.

  14. Propofol reduced myocardial contraction of vertebrates partly by mediating the cyclic AMP-dependent protein kinase phosphorylation pathway

    International Nuclear Information System (INIS)

    Sun, Xiaotong; Zhang, Xinyu; Bo, Qiyu; Meng, Tao; Lei, Zhen; Li, Jingxin; Hou, Yonghao; Yu, Xiaoqian; Yu, Jingui

    2016-01-01

    Propofol inhibits myocardial contraction in a dose dependent manner. The present study is designed to examine the effect of propofol on PKA mediated myocardial contraction in the absence of adrenoreceptor agonist. The contraction of isolated rat heart was measured in the presence or absence of PKA inhibitor H89 or propofol, using a pressure transducer. The levels of cAMP and PKA kinase activity were detected by ELISA. The mRNA and total protein or phosphorylation level of PKA and downstream proteins were tested in the presence or absence of PKA inhibitor H89 or propofol, using RT-PCR, QPCR and western blotting. The phosphorylation level of PKA was examined thoroughly using immunofluorescence and PKA activity non-radioactive detection kit. Propofol induced a dose-dependent negative contractile response on the rat heart. The inhibitory effect of high concentration propofol (50 μM) with 45% decease of control could be partly reversed by the PKA inhibitor H89 (10 μM) and the depressant effect of propofol decreased from 45% to 10%. PKA kinase activity was inhibited by propofol in a dose-dependent manner. Propofol also induced a decrease in phosphorylation of PKA, which was also inhibited by H89, but did not alter the production of cAMP and the mRNA levels of PKA. The downstream proteins of PKA, PLN and RyR2 were phosphorylated to a lesser extent with propofol or H89 than control. These results demonstrated that propofol induced a negative myocardial contractile response partly by mediating the PKA phosphorylation pathway.

  15. EGFR tyrosine kinase inhibitory peptide attenuates Helicobacter pylori-mediated hyper-proliferation in AGS enteric epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Himaya, S.W.A. [Marine Bio-Process Research Center, Pukyong National University, Nam-Gu, Busan, 608-737 (Korea, Republic of); Dewapriya, Pradeep [Department of Chemistry, Pukyong National University, Nam-Gu, Busan, 608-737 (Korea, Republic of); Kim, Se-Kwon, E-mail: sknkim@pknu.ac.kr [Marine Bio-Process Research Center, Pukyong National University, Nam-Gu, Busan, 608-737 (Korea, Republic of); Department of Chemistry, Pukyong National University, Nam-Gu, Busan, 608-737 (Korea, Republic of)

    2013-06-15

    Helicobacter pylori infection is one of the most critical causes of stomach cancer. The current study was conducted to explore the protective effects of an isolated active peptide H-P-6 (Pro-Gln-Pro-Lys-Val-Leu-Asp-Ser) from microbial hydrolysates of Chlamydomonas sp. against H. pylori-induced carcinogenesis. The peptide H-P-6 has effectively suppressed H. pylori-induced hyper-proliferation and migration of gastric epithelial cells (AGS). However, the peptide did not inhibit the viability of the bacteria or invasion into AGS cells. Therefore, the effect of the peptide on regulating H. pylori-induced molecular signaling was investigated. The results indicated that H. pylori activates the EGFR tyrosine kinase signaling and nuclear translocation of the β-catenin. The EGFR activation has led to the up-regulation of PI3K/Akt signaling pathway. Moreover, the nuclear translocation levels of β-catenin were significantly increased as a result of Akt mediated down-regulation of GSK3/β protein levels in the cytoplasm. Both of these consequences have resulted in increased expression of cell survival and migration related genes such as c-Myc, cyclin-D, MMP-2 and matrilysin. Interestingly, the isolated peptide potently inhibited H. pylori-mediated EGFR activation and thereby down-regulated the subsequent P13K/Akt signaling leading to β-catenin nuclear translocation. The effect of the peptide was confirmed with the use of EGFR tyrosine kinase inhibitor AG1487 and molecular docking studies. Collectively this study identifies a potent peptide which regulates the H. pylori-induced hyper-proliferation and migration of AGS cells at molecular level. - Highlights: • Chlamydomonas sp. derived peptide H-P-6 inhibits H. pylori-induced pathogenesis. • H-P-6 suppresses H. pylori-induced hyper-proliferation and migration of AGS cells. • The peptide inhibits H. pylori-induced EGFR activation.

  16. The Antiproliferative Activity of Kinase Inhibitors in Chronic Myeloid Leukemia Cells Is Mediated by FOXO Transcription Factors

    Science.gov (United States)

    Pellicano, Francesca; Scott, Mary T; Helgason, G Vignir; Hopcroft, Lisa E M; Allan, Elaine K; Aspinall-O'Dea, Mark; Copland, Mhairi; Pierce, Andrew; Huntly, Brian J P; Whetton, Anthony D; Holyoake, Tessa L

    2014-01-01

    Chronic myeloid leukemia (CML) is initiated and maintained by the tyrosine kinase BCR-ABL which activates a number of signal transduction pathways, including PI3K/AKT signaling and consequently inactivates FOXO transcription factors. ABL-specific tyrosine kinase inhibitors (TKIs) induce minimal apoptosis in CML progenitor cells, yet exert potent antiproliferative effects, through as yet poorly understood mechanisms. Here, we demonstrate that in CD34+ CML cells, FOXO1 and 3a are inactivated and relocalized to the cytoplasm by BCR-ABL activity. TKIs caused a decrease in phosphorylation of FOXOs, leading to their relocalization from cytoplasm (inactive) to nucleus (active), where they modulated the expression of key FOXO target genes, such as Cyclin D1, ATM, CDKN1C, and BCL6 and induced G1 arrest. Activation of FOXO1 and 3a and a decreased expression of their target gene Cyclin D1 were also observed after 6 days of in vivo treatment with dasatinib in a CML transgenic mouse model. The over-expression of FOXO3a in CML cells combined with TKIs to reduce proliferation, with similar results seen for inhibitors of PI3K/AKT/mTOR signaling. While stable expression of an active FOXO3a mutant induced a similar level of quiescence to TKIs alone, shRNA-mediated knockdown of FOXO3a drove CML cells into cell cycle and potentiated TKI-induced apoptosis. These data demonstrate that TKI-induced G1 arrest in CML cells is mediated through inhibition of the PI3K/AKT pathway and reactivation of FOXOs. This enhanced understanding of TKI activity and induced progenitor cell quiescence suggests that new therapeutic strategies for CML should focus on manipulation of this signaling network. Stem Cells 2014;32:2324–2337 PMID:24806995

  17. The kinase LKB1 mediates glucose homeostasis in liver and therapeutic effects of metformin.

    Science.gov (United States)

    Shaw, Reuben J; Lamia, Katja A; Vasquez, Debbie; Koo, Seung-Hoi; Bardeesy, Nabeel; Depinho, Ronald A; Montminy, Marc; Cantley, Lewis C

    2005-12-09

    The Peutz-Jegher syndrome tumor-suppressor gene encodes a protein-threonine kinase, LKB1, which phosphorylates and activates AMPK [adenosine monophosphate (AMP)-activated protein kinase]. The deletion of LKB1 in the liver of adult mice resulted in a nearly complete loss of AMPK activity. Loss of LKB1 function resulted in hyperglycemia with increased gluconeogenic and lipogenic gene expression. In LKB1-deficient livers, TORC2, a transcriptional coactivator of CREB (cAMP response element-binding protein), was dephosphorylated and entered the nucleus, driving the expression of peroxisome proliferator-activated receptor-gamma coactivator 1alpha (PGC-1alpha), which in turn drives gluconeogenesis. Adenoviral small hairpin RNA (shRNA) for TORC2 reduced PGC-1alpha expression and normalized blood glucose levels in mice with deleted liver LKB1, indicating that TORC2 is a critical target of LKB1/AMPK signals in the regulation of gluconeogenesis. Finally, we show that metformin, one of the most widely prescribed type 2 diabetes therapeutics, requires LKB1 in the liver to lower blood glucose levels.

  18. Cyclic AMP (cAMP)-mediated stimulation of adipocyte differentiation requires the synergistic action of Epac- and cAMP-dependent protein kinase-dependent processes

    DEFF Research Database (Denmark)

    Petersen, Rasmus Koefoed; Madsen, Lise; Pedersen, Lone Møller

    2008-01-01

    AMP-dependent stimulation of adipocyte differentiation. Epac, working via Rap, acted synergistically with cAMP-dependent protein kinase (protein kinase A [PKA]) to promote adipogenesis. The major role of PKA was to down-regulate Rho and Rho-kinase activity, rather than to enhance CREB phosphorylation. Suppression of Rho......-kinase impaired proadipogenic insulin/insulin-like growth factor 1 signaling, which was restored by activation of Epac. This interplay between PKA and Epac-mediated processes not only provides novel insight into the initiation and tuning of adipocyte differentiation, but also demonstrates a new mechanism of c......AMP signaling whereby cAMP uses both PKA and Epac to achieve an appropriate cellular response....

  19. The extracellular regulated kinases (ERK) 1/2 mediate cannabinoid-induced inhibition of gap junctional communication in endothelial cells

    Science.gov (United States)

    Brandes, R P; Popp, R; Ott, G; Bredenkötter, D; Wallner, C; Busse, R; Fleming, I

    2002-01-01

    Cannabinoids are potent inhibitors of endothelium-derived hyperpolarizing factor (EDHF)-mediated relaxations. We set out to study the mechanism underlying this effect and the possible role of cannabinoid-induced changes in intercellular gap junction communication.In cultured endothelial cells, Δ9-tetrahydrocannabinol (Δ9-THC) and the cannabinoid receptor agonist HU210, increased the phosphorylation of extracellular regulated kinases 1/2 (ERK1/2) and inhibited gap junctional communication, as determined by Lucifer Yellow dye transfer and electrical capacity measurements.Δ9-THC elicited a pronounced increase in the phosphorylation of connexin 43, which was sensitive to PD98059 and U0126, two inhibitors of ERK1/2 activation. Inhibition of ERK1/2 also prevented the Δ9-THC-induced inhibition of gap junctional communication.Δ9-THC prevented both the bradykinin-induced hyperpolarization and the nitric oxide and prostacyclin-independent relaxation of pre-contracted rings of porcine coronary artery. These effects were prevented by PD98059 as well as U0126.In the absence of Δ9-THC, neither PD98059 nor U0126 affected the NO-mediated relaxation of coronary artery rings but both substances induced a leftward shift in the concentration – relaxation curve to bradykinin when diclofenac and Nωnitro-L-arginine were present. Moreover, PD98059 and U0126 prolonged the bradykinin-induced hyperpolarization of porcine coronary arteries, without affecting the magnitude of the response.These results indicate that the cannabinoid-induced activation of ERK1/2, which leads to the phosphorylation of connexin 43 and inhibition of gap junctional communication, may partially account for the Δ9-THC-induced inhibition of EDHF-mediated relaxation. Moreover, the activation of ERK1/2 by endothelial cell agonists such as bradykinin, appears to exert a negative feedback inhibition on EDHF-mediated responses. PMID:12086980

  20. Malignant transformation of mature T cells after gammaretrovirus mediated transfer of nucleophosmin-anaplastic lymphoma kinase oncogene

    Directory of Open Access Journals (Sweden)

    Ashok Kumar

    2015-01-01

    Full Text Available Background: Gene therapy has been in use to cure hereditary and acquired diseases by incorporating the desired gene into the cells with the help of gammaretroviral vectors. Despite the success of this therapy in X-linked severe combined immunodeficiency syndrome, few patients developed leukemia as a major adverse event due to retroviral insertional mutagenesis within stem cells. In experimental animals also, retroviral-mediated gene transfer technique resulted in the development of leukemia. On the other hand, evidence suggests that mature T cells (TC are relatively resistant to transformation even after retroviral-mediated transfer of potent oncogenes Tcl1, ΔTrkA and LMO2 with no reported side effects yet. Aims: To further address the safety issue for TC use in gene therapy, this study investigated susceptibility of mature polyclonal TC to malignant transformation by the retroviral-mediated transfer of nucleophosmin-anaplastic lymphoma kinase (NPM-ALK oncogene. Materials and Methods: Wild-type mature TC, isolated from C57BL/6 donor mice (genetic background Ly5.1 were transduced with gamma-retroviral vectors encoding the potent TC oncogene NPM-ALK or the control vector enhanced green fluorescent protein eGFP. The cells were then transplanted into RAG-1 deficient recipient mice (genetic background Ly5.2. Results: Two out of five mice from NPM-ALK oncogene group developed leukemia/lymphoma after latency periods (153 and 250 days, respectively. None of the mice from the control group developed any malignancy throughout the observational period. Conclusion: Mature polyclonal TC are relatively susceptible to malignant transformation after gamma-retroviral mediated transfer of NPM-ALK oncogene; hence safety of TC use in gene therapy should be further investigated to avoid the possible side-effect of development of leukemia/lymphoma.

  1. Mitogen-Activated Protein Kinases Mediate Upregulation of Hypothalamic AT1 Receptors in Heart Failure Rats

    Science.gov (United States)

    Wei, Shun-Guang; Yu, Yang; Zhang, Zhi-Hua; Weiss, Robert M.; Felder, Robert B.

    2009-01-01

    In heart failure (HF), angiotensin type-1 receptor (AT1-R) expression is upregulated in brain regions regulating sympathetic drive, blood pressure and body fluid homeostasis. However, the mechanism by which brain AT1-R are upregulated in HF remains unknown. The present study examined the hypothesis that the angiotensin II (ANG II)-triggered mitogen-activated protein kinases (MAPK) p44/42, p38 and c-Jun N-terminal kinase (JNK) contribute to upregulation of the AT1-R in the hypothalamus of rats with HF. AT1-R protein, AT1-R mRNA and AT1-R immunoreactivity increased in the paraventricular nucleus of hypothalamus (PVN) and the subfornical organ (SFO) of rats with ischemia-induced HF, compared with sham-operated controls. Phosphorylated p44/42 MAPK, JNK, and p38 MAPK also increased in PVN and SFO. A 4-week intracerebroventricular (ICV) infusion of the AT1-R antagonist losartan decreased AT1-R protein and phosphorylation of p44/42 MAPK, JNK and p38 MAPK in the HF rats. A 4-week ICV infusion of the p44/42 MAPK inhibitor PD98059 or the JNK inhibitor SP600125 significantly decreased AT1-R protein and AT1-R immunoreactivity in the PVN and SFO, but the p38 MAPK inhibitor SB203580 did not. Treatment with ICV losartan, PD98059 and SP600125 had no effect on AT1-R expression by Western blot in sham-operated rats. In untreated HF rats 4 weeks after coronary ligation, a 3-hour ICV infusion of PD98059, SP600125 or losartan reduced AT1-R mRNA in PVN and SFO. These data indicate that MAPK plays an important role in the upregulation of AT1-R in the rat forebrain in heart failure, and suggest that ANG II upregulates its own receptor by this mechanism. PMID:18768402

  2. Requirement for Ras/Rac1-Mediated p38 and c-Jun N-Terminal Kinase Signaling in Stat3 Transcriptional Activity Induced by the Src Oncoprotein

    Science.gov (United States)

    Turkson, James; Bowman, Tammy; Adnane, Jalila; Zhang, Yi; Djeu, Julie Y.; Sekharam, Madhavi; Frank, David A.; Holzman, Lawrence B.; Wu, Jie; Sebti, Said; Jove, Richard

    1999-01-01

    Signal transducers and activators of transcription (STATs) are transcription factors that mediate normal biologic responses to cytokines and growth factors. However, abnormal activation of certain STAT family members, including Stat3, is increasingly associated with oncogenesis. In fibroblasts expressing the Src oncoprotein, activation of Stat3 induces specific gene expression and is required for cell transformation. Although the Src tyrosine kinase induces constitutive Stat3 phosphorylation on tyrosine, activation of Stat3-mediated gene regulation requires both tyrosine and serine phosphorylation of Stat3. We investigated the signaling pathways underlying the constitutive Stat3 activation in Src oncogenesis. Expression of Ras or Rac1 dominant negative protein blocks Stat3-mediated gene regulation induced by Src in a manner consistent with dependence on p38 and c-Jun N-terminal kinase (JNK). Both of these serine/threonine kinases and Stat3 serine phosphorylation are constitutively induced in Src-transformed fibroblasts. Furthermore, inhibition of p38 and JNK activities suppresses constitutive Stat3 serine phosphorylation and Stat3-mediated gene regulation. In vitro kinase assays with purified full-length Stat3 as the substrate show that both JNK and p38 can phosphorylate Stat3 on serine. Moreover, inhibition of p38 activity and thus of Stat3 serine phosphorylation results in suppression of transformation by v-Src but not v-Ras, consistent with a requirement for Stat3 serine phosphorylation in Src transformation. Our results demonstrate that Ras- and Rac1-mediated p38 and JNK signals are required for Stat3 transcriptional activity induced by the Src oncoprotein. These findings delineate a network of tyrosine and serine/threonine kinase signaling pathways that converge on Stat3 in the context of oncogenesis. PMID:10523640

  3. Pazopanib, a novel multi-kinase inhibitor, shows potent antitumor activity in colon cancer through PUMA-mediated apoptosis.

    Science.gov (United States)

    Zhang, Lingling; Wang, Huanan; Li, Wei; Zhong, Juchang; Yu, Rongcheng; Huang, Xinfeng; Wang, Honghui; Tan, Zhikai; Wang, Jiangang; Zhang, Yingjie

    2017-01-10

    Colon cancer is still the third most common cancer which has a high mortality but low five-year survival rate. Novel tyrosine kinase inhibitors (TKI) such as pazopanib become effective antineoplastic agents that show promising clinical activity in a variety of carcinoma, including colon cancer. However, the precise underlying mechanism against tumor is unclear. Here, we demonstrated that pazopanib promoted colon cancer cell apoptosis through inducing PUMA expression. Pazopanib induced p53-independent PUMA activation by inhibiting PI3K/Akt signaling pathway, thereby activating Foxo3a, which subsequently bound to the promoter of PUMA to activate its transcription. After induction, PUMA activated Bax and triggered the intrinsic mitochondrial apoptosis pathway. Furthermore, administration of pazopanib highly suppressed tumor growth in a xenograft model. PUMA deletion in cells and tumors led to resistance of pazopanib, indicating PUMA-mediated pro-apoptotic and anti-tumor effects in vitro and in vivo. Combing pazopanib with some conventional or novel drugs, produced heightened and synergistic antitumor effects that were associated with potentiated PUMA induction via different pathways. Taken together, these results establish a critical role of PUMA in mediating the anticancer effects of pazopanib in colon cancer cells and provide the rationale for clinical evaluation.

  4. Immunomodulatory role of interleukin-10 in visceral leishmaniasis: defective activation of protein kinase C-mediated signal transduction events.

    Science.gov (United States)

    Bhattacharyya, S; Ghosh, S; Jhonson, P L; Bhattacharya, S K; Majumdar, S

    2001-03-01

    Leishmania donovani, an intracellular protozoan parasite, challenges host defense mechanisms by impairing the signal transduction of macrophages. In this study we investigated whether interleukin-10 (IL-10)-mediated alteration of signaling events in a murine model of visceral leishmaniasis is associated with macrophage deactivation. Primary in vitro cultures of macrophages infected with leishmanial parasites markedly elevated the endogenous release of IL-10. Treatment with either L. donovani or recombinant IL-10 (rIL-10) inhibited both the activity and expression of the Ca2+-dependent protein kinase C (PKC) isoform. However, preincubation with neutralizing anti-IL-10 monoclonal antibody (MAb) restored the PKC activity in the parasitized macrophage. Furthermore, we observed that coincubation of macrophages with rIL-10 and L. donovani increased the intracellular parasite burden, which was abrogated by anti-IL-10 MAb. Consistent with these observations, generation of superoxide (O2-) and nitric oxide and the release of murine tumor necrosis factor-alpha were attenuated in response to L. donovani or rIL-10 treatment. On the other hand, preincubation of the infected macrophages with neutralizing anti-IL-10 MAb significantly blocked the inhibition of nitric oxide and murine tumor necrosis factor-alpha release by the infected macrophages. These findings imply that infection with L. donovani induces endogenous secretion of murine IL-10, which in turn facilitates the intracellular survival of the protozoan and orchestrates several immunomodulatory roles via selective impairment of PKC-mediated signal transduction.

  5. CD36 and Fyn kinase mediate malaria-induced lung endothelial barrier dysfunction in mice infected with Plasmodium berghei.

    Directory of Open Access Journals (Sweden)

    Ifeanyi U Anidi

    Full Text Available Severe malaria can trigger acute lung injury characterized by pulmonary edema resulting from increased endothelial permeability. However, the mechanism through which lung fluid conductance is altered during malaria remains unclear. To define the role that the scavenger receptor CD36 may play in mediating this response, C57BL/6J (WT and CD36-/- mice were infected with P. berghei ANKA and monitored for changes in pulmonary endothelial barrier function employing an isolated perfused lung system. WT lungs demonstrated a >10-fold increase in two measures of paracellular fluid conductance and a decrease in the albumin reflection coefficient (σalb compared to control lungs indicating a loss of barrier function. In contrast, malaria-infected CD36-/- mice had near normal fluid conductance but a similar reduction in σalb. In WT mice, lung sequestered iRBCs demonstrated production of reactive oxygen species (ROS. To determine whether knockout of CD36 could protect against ROS-induced endothelial barrier dysfunction, mouse lung microvascular endothelial monolayers (MLMVEC from WT and CD36-/- mice were exposed to H2O2. Unlike WT monolayers, which showed dose-dependent decreases in transendothelial electrical resistance (TER from H2O2 indicating loss of barrier function, CD36-/- MLMVEC demonstrated dose-dependent increases in TER. The differences between responses in WT and CD36-/- endothelial cells correlated with important differences in the intracellular compartmentalization of the CD36-associated Fyn kinase. Malaria infection increased total lung Fyn levels in CD36-/- lungs compared to WT, but this increase was due to elevated production of the inactive form of Fyn further suggesting a dysregulation of Fyn-mediated signaling. The importance of Fyn in CD36-dependent endothelial signaling was confirmed using in vitro Fyn knockdown as well as Fyn-/- mice, which were also protected from H2O2- and malaria-induced lung endothelial leak, respectively. Our

  6. Phosphatidylinositol 3-kinase is essential for kit ligand-mediated survival, whereas interleukin-3 and flt3 ligand induce expression of antiapoptotic Bcl-2 family genes

    DEFF Research Database (Denmark)

    Karlsson, Richard; Engström, Maria; Jönsson, Maria

    2003-01-01

    Cytokines such as interleukin 3 (IL-3), kit ligand (KL), and flt3 ligand (FL) promote survival of hematopoietic stem cells and myeloid progenitor cells. In many cell types, members of the Bcl-2 gene family are major regulators of survival, but the mediating mechanisms are not fully understood....... Using two myeloid progenitor cell lines, FDCP-mix and FDC-P1, as well as primary mouse bone marrow progenitors, we demonstrate that KL-mediated survival is dependent on the activation of phosphatidylinositol-3 (PI-3) kinase. The inhibitor LY294002 was able to completely abolish survival mediated by KL...

  7. AMP-activated protein kinase (AMPK mediates nutrient regulation of thioredoxin-interacting protein (TXNIP in pancreatic beta-cells.

    Directory of Open Access Journals (Sweden)

    Maayan Shaked

    Full Text Available Thioredoxin-interacting protein (TXNIP regulates critical biological processes including inflammation, stress and apoptosis. TXNIP is upregulated by glucose and is a critical mediator of hyperglycemia-induced beta-cell apoptosis in diabetes. In contrast, the saturated long-chain fatty acid palmitate, although toxic to the beta-cell, inhibits TXNIP expression. The mechanisms involved in the opposing effects of glucose and fatty acids on TXNIP expression are unknown. We found that both palmitate and oleate inhibited TXNIP in a rat beta-cell line and islets. Palmitate inhibition of TXNIP was independent of fatty acid beta-oxidation or esterification. AMP-activated protein kinase (AMPK has an important role in cellular energy sensing and control of metabolic homeostasis; therefore we investigated its involvement in nutrient regulation of TXNIP. As expected, glucose inhibited whereas palmitate stimulated AMPK. Pharmacologic activators of AMPK mimicked fatty acids by inhibiting TXNIP. AMPK knockdown increased TXNIP expression in presence of high glucose with and without palmitate, indicating that nutrient (glucose and fatty acids effects on TXNIP are mediated in part via modulation of AMPK activity. TXNIP is transcriptionally regulated by carbohydrate response element-binding protein (ChREBP. Palmitate inhibited glucose-stimulated ChREBP nuclear entry and recruitment to the Txnip promoter, thereby inhibiting Txnip transcription. We conclude that AMPK is an important regulator of Txnip transcription via modulation of ChREBP activity. The divergent effects of glucose and fatty acids on TXNIP expression result in part from their opposing effects on AMPK activity. In light of the important role of TXNIP in beta-cell apoptosis, its inhibition by fatty acids can be regarded as an adaptive/protective response to glucolipotoxicity. The finding that AMPK mediates nutrient regulation of TXNIP may have important implications for the pathophysiology and treatment

  8. Tyrosine kinase inhibitors as modulators of trastuzumab-mediated antibody-dependent cell-mediated cytotoxicity in breast cancer cell lines.

    Science.gov (United States)

    Collins, Denis M; Gately, Kathy; Hughes, Clare; Edwards, Connla; Davies, Anthony; Madden, Stephen F; O'Byrne, Kenneth J; O'Donovan, Norma; Crown, John

    2017-09-01

    Trastuzumab is an anti-HER2 monoclonal antibody (mAb) therapy capable of antibody-dependent cell-mediated cytotoxicity (ADCC) and used in the treatment of HER2+ breast cancer. Through interactions with FcƴR+ immune cell subsets, trastuzumab functions as a passive immunotherapy. The EGFR/HER2-targeting tyrosine kinase inhibitor (TKI) lapatinib and the next generation TKIs afatinib and neratinib, can alter HER2 levels, potentially modulating the ADCC response to trastuzumab. Using LDH-release assays, we investigated the impact of antigen modulation, assay duration and peripheral blood mononuclear cell (PBMC) activity on trastuzumab-mediated ADCC in breast cancer models of maximal (SKBR3) and minimal (MCF-7) target antigen expression to determine if modulating the ADCC response to trastuzumab using TKIs may be a viable approach for enhancing tumor immune reactivity. HER2 levels were determined in lapatinib, afatinib and neratinib-treated SKBR3 and MCF-7 using high content analysis (HCA). Trastuzumab-mediated ADCC was assessed following treatment with TKIs utilising a colorimetric LDH release-based protocol at 4 and 12h timepoints. PBMC activity was assessed against non-MHC-restricted K562 cells. A flow cytometry-based method (CFSE/7-AAD) was also used to measure trastuzumab-mediated ADCC in medium-treated SKBR3 and MCF-7. HER2 antigen levels were significantly altered by the three TKIs in both cell line models. The TKIs significantly reduced LDH levels directly in SKBR3 cells but not MCF-7. Lapatinib and neratinib augment trastuzumab-related ADCC in SKBR3 but the effect was not consistent with antigen expression levels and was dependent on volunteer PBMC activity (vs. K562). A 12h assay timepoint produced more consistent results. Trastuzumab-mediated ADCC (PBMC:target cell ratio of 10:1) was measured at 7.6±4.7% (T12) by LDH assay and 19±3.2 % (T12) using the flow cytometry-based method in the antigen-low model MCF-7. In the presence of effector cells with high

  9. Expression profiling of MAP kinase-mediated meiotic progression in Caenorhabditis elegans.

    Directory of Open Access Journals (Sweden)

    Stefanie W Leacock

    2006-11-01

    Full Text Available The LET-60 (Ras/LIN-45 (Raf/MPK-1 (MAP kinase signaling pathway plays a key role in the development of multiple tissues in Caenorhabditis elegans. For the most part, the identities of the downstream genes that act as the ultimate effectors of MPK-1 signaling have remained elusive. A unique allele of mpk-1, ga111, displays a reversible, temperature-sensitive, tissue-specific defect in progression through meiotic prophase I. We performed gene expression profiling on mpk-1(ga111 animals to identify candidate downstream effectors of MPK-1 signaling in the germ line. This analysis delineated a cohort of genes whose expression requires MPK-1 signaling in germ cells in the pachytene stage of meiosis I. RNA in situ hybridization analysis shows that these genes are expressed in the germ line in an MPK-1-dependent manner and have a spatial expression pattern consistent with the location of activated MPK-1. We found that one MPK-1 signaling-responsive gene encoding a C2H2 zinc finger protein plays a role in meiotic chromosome segregation downstream of MPK-1. Additionally, discovery of genes responsive to MPK-1 signaling permitted us to order MPK-1 signaling relative to several events occurring in pachytene, including EFL-1/DPL-1 gene regulation and X chromosome reactivation. This study highlights the utility of applying global gene expression methods to investigate genes downstream of commonly used signaling pathways in vivo.

  10. Axl, a receptor tyrosine kinase, mediates flow-induced vascular remodeling.

    Science.gov (United States)

    Korshunov, Vyacheslav A; Mohan, Amy M; Georger, Mary A; Berk, Bradford C

    2006-06-09

    Intima-media thickening (IMT) in response to hemodynamic stress is a physiological process that requires coordinated signaling among endothelial, inflammatory, and vascular smooth muscle cells (VSMC). Axl, a receptor tyrosine kinase, whose ligand is Gas6, is highly induced in VSMC after carotid injury. Because Axl regulates cell migration, phagocytosis and apoptosis, we hypothesized that Axl would play a role in IMT. Vascular remodeling in mice deficient in Axl (Axl(-/-)) and wild-type littermates (Axl(+/+)) was induced by ligation of the left carotid artery (LCA) branches maintaining flow via the left occipital artery. Both genotypes had similar baseline hemodynamic parameters and carotid artery structure. Partial ligation altered blood flow equally in both genotypes: increased by 60% in the right carotid artery (RCA) and decreased by 80% in the LCA. There were no significant differences in RCA remodeling between genotypes. However, in the LCA Axl(-/-) developed significantly smaller intima+media compared with Axl(+/+) (31+/-4 versus 42+/-6x10(-6) microm3, respectively). Quantitative immunohistochemistry of Axl(-/-) LCA showed increased apoptosis compared with Axl(+/+) (5-fold). As expected, p-Akt was decreased in Axl(-/-), whereas there was no difference in Gas6 expression. Cell composition also changed significantly, with increases in CD45+ cells and decreases in VSMC, macrophages, and neutrophils in Axl(-/-) compared with Axl(+/+). These data demonstrate an important role for Axl in flow-dependent remodeling by regulating vascular apoptosis and vascular inflammation.

  11. Distinct Receptor Tyrosine Kinase Subsets Mediate Anti-HER2 Drug Resistance in Breast Cancer*

    Science.gov (United States)

    Alexander, Peter B.; Chen, Rui; Gong, Chang; Yuan, Lifeng; Jasper, Jeff S.; Ding, Yi; Markowitz, Geoffrey J.; Yang, Pengyuan; Xu, Xin; McDonnell, Donald P.; Song, Erwei; Wang, Xiao-Fan

    2017-01-01

    Targeted inhibitors of the human epidermal growth factor receptor 2 (HER2), such as trastuzumab and lapatinib, are among the first examples of molecularly targeted cancer therapy and have proven largely effective for the treatment of HER2-positive breast cancers. However, approximately half of those patients either do not respond to these therapies or develop secondary resistance. Although a few signaling pathways have been implicated, a comprehensive understanding of mechanisms underlying HER2 inhibitor drug resistance is still lacking. To address this critical question, we undertook a concerted approach using patient expression data sets, HER2-positive cell lines, and tumor samples biopsied both before and after trastuzumab treatment. Together, these methods revealed that high expression and activation of a specific subset of receptor tyrosine kinases (RTKs) was strongly associated with poor clinical prognosis and the development of resistance. Mechanistically, these RTKs are capable of maintaining downstream signal transduction to promote tumor growth via the suppression of cellular senescence. Consequently, these findings provide the rationale for the design of therapeutic strategies for overcoming drug resistance in breast cancer via combinational inhibition of the limited number of targets from this specific subset of RTKs. PMID:27903634

  12. Tyrosine kinase Btk regulates E-selectin–mediated integrin activation and neutrophil recruitment by controlling phospholipase C (PLC) γ2 and PI3Kγ pathways

    Science.gov (United States)

    Mueller, Helena; Stadtmann, Anika; Van Aken, Hugo; Hirsch, Emilio; Wang, Demin; Ley, Klaus

    2010-01-01

    Selectins mediate leukocyte rolling, trigger β2-integrin activation, and promote leukocyte recruitment into inflamed tissue. E-selectin binding to P-selectin glycoprotein ligand 1 (PSGL-1) leads to activation of an immunoreceptor tyrosine-based activation motif (ITAM)–dependent pathway, which in turn activates the spleen tyrosine kinase (Syk). However, the signaling pathway linking Syk to integrin activation after E-selectin engagement is unknown. To identify the pathway, we used different gene-deficient mice in autoperfused flow chamber, intravital microscopy, peritonitis, and biochemical studies. We report here that the signaling pathway downstream of Syk divides into a phospholipase C (PLC) γ2– and phosphoinositide 3-kinase (PI3K) γ–dependent pathway. The Tec family kinase Bruton tyrosine kinase (Btk) is required for activating both pathways, generating inositol-3,4,5-trisphosphate (IP3), and inducing E-selectin–mediated slow rolling. Inhibition of this signal-transduction pathway diminished Gαi-independent leukocyte adhesion to and transmigration through endothelial cells in inflamed postcapillary venules of the cremaster. Gαi-independent neutrophil recruitment into the inflamed peritoneal cavity was reduced in Btk−/− and Plcg2−/− mice. Our data demonstrate the functional importance of this newly identified signaling pathway mediated by E-selectin engagement. PMID:20167705

  13. Activation of Focal Adhesion Kinase and Src Mediates Acquired Sorafenib Resistance in A549 Human Lung Adenocarcinoma Xenografts.

    Science.gov (United States)

    Zhou, Qingyu; Guo, Xiaofang; Choksi, Riya

    2017-12-01

    Despite encouraging clinical results with sorafenib monotherapy in patients with KRAS- mutant non-small-cell lung cancer (NSCLC), the overall survival benefit of this drug is limited by the inevitable development of acquired resistance. The exact mechanism underlying acquired sorafenib resistance in KRAS -mutant NSCLC is unclear. In this study, the mechanism of acquired sorafenib resistance was explored using a biologically relevant xenograft model, which was established by using the A549 human lung adenocarcinoma cell line and an in vivo-derived, sorafenib-resistant A549 subline (A549/SRFres). Results from the initial study demonstrated that sorafenib treatment significantly decreased E-cadherin ( P A549/SRFres tumors, whereas expression levels of phospho-protein kinase B (AKT), phospho-focal adhesion kinase (FAK), and phospho-Src were elevated in sorafenib-treated A549 and A549/SRFres tumors. We next examined whether concomitant dasatinib treatment could overcome acquired sorafenib resistance by blocking the FAK/Src escape route that mediates resistance. Despite the observed in vitro synergy between sorafenib and dasatinib, the in vivo antitumor effect of half-dose sorafenib-dasatinib combination therapy was inferior to that of the full-dose sorafenib treatment. Although the sorafenib-dasatinib combination effectively inhibited Src and AKT phosphorylation, it did not block the Y576/577-FAK phosphorylation, nor did it decrease vimentin protein expression; unexpectedly, it increased Y397-FAK phosphorylation and MMP9 protein expression in tumors. These results suggest that acquired sorafenib resistance in KRAS -mutant A549 xenografts involves the compensatory activation of FAK and Src, and Src inhibition alone is insufficient to diminish sorafenib-promoted epithelial-mesenchymal transition process and invasive potentials in tumors. Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.

  14. Angiotensin II Facilitates Matrix Metalloproteinase-9-Mediated Myosin Light Chain Kinase Degradation in Pressure Overload-Induced Cardiac Hypertrophy

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    Shun Wang

    2017-12-01

    Full Text Available Background/Aims: Angiotensin II (Ang II has been shown to promote cardiac remodeling during the process of hypertrophy. Myosin light chain kinase (MLCK, a specific kinase for the phosphorylation of myosin light chain 2 (MLC2, plays an important role in regulating cardiac muscle contraction and hypertrophy. However, whether Ang II could facilitate cardiac hypertrophy by altering the expression of MLCK remains unclear. This study aimed to investigate this effect and the underlying mechanisms. Methods: Cardiac hypertrophy was induced via pressure overload in rats, which were then evaluated via histological and biochemical measurements and echocardiography. Angiotensin-converting enzyme inhibitor (ACEI was used to inhibit Ang II. Neonatal rat cardiomyocytes were stimulated with Ang II to induce hypertrophy and were treated with a matrix metalloproteinase 9 (MMP9 inhibitor. Myocyte hypertrophy was evaluated using immunofluorescence and qRT-PCR. Degradation of recombinant human MLCK by recombinant human MMP9 was tested using a cleavage assay. The expression levels of MLCK, MLC2, phospho-myosin light chain 2 (p-MLC2, myosin phosphatase 2 (MYPT2, and calmodulin (CaM were measured using western blotting. Results: ACEI improved cardiac function and remodeling and increased the levels of MLCK and p-MLC2 as well as reduced the expression of MMP9 in pressure overload-induced cardiac hypertrophy. Moreover, the MMP9 inhibitor alleviated myocyte hypertrophy and upregulated the levels of MLCK and p-MLC2 in Ang II-induced cardiomyocyte hypertrophy. Recombinant human MLCK was concentration- and time-dependently degraded by recombinant human MMP9 in vitro, and this process was prevented by the MMP9 inhibitor. Conclusion: Our results suggest that Ang II is involved in the degradation of MLCK in pressure overload-induced cardiac hypertrophy and that this process was mediated by MMP9.

  15. Atypical protein kinase C mediates activation of NF-E2-related factor 2 in response to oxidative stress.

    Science.gov (United States)

    Numazawa, Satoshi; Ishikawa, Makie; Yoshida, Aya; Tanaka, Sachiko; Yoshida, Takemi

    2003-08-01

    Transcription factor NF-E2-related factor 2 (Nrf2) regulates the induction of antioxidative proteins, including heme oxygenase-1 (HO-1). Nrf2 is sequestered in the cytoplasm by Keap1 under unstimulated conditions but translocates into the nucleus and transactivates the antioxidant responsive element (ARE) upon exposure to oxidative insults. It has recently been demonstrated that in vitro phosphorylation of Nrf2 on Ser40 by protein kinase C (PKC) facilitates the dissociation of Nrf2 from the Keap1 complex (Huang HC, Nguyen T, and Pickett CB. J Biol Chem 277: 42769-42774, 2002). The present study was designed to examine whether PKC is involved in oxidative stress-mediated nuclear translocation of Nrf2 in vivo and, if so, which PKC isoforms are involved. Induction of HO-1 gene expression by phorone, a glutathione depletor, and 4-hydroxy-2,3-nonenal (4-HNE), an end product of lipid peroxidation, was suppressed by a specific PKC inhibitor, Ro-31-8220, at concentrations that inhibit all isoforms in WI-38 cells. The induction of HO-1 was not affected by prolonged exposure of the cells to 12-O-tetradecanoylphorbol-13 acetate (TPA), suggesting that TPA-insensitive atypical PKC (aPKC) isoforms are involved. An immunocomplex kinase assay revealed that phorone and 4-HNE increased aPKCiota activity. In COS-7 cells, 4-HNE induced nuclear translocation of the Nrf2-green fluorescent protein (GFP) fusion protein, but not the Nrf2(S40A)-GFP mutant. In the absence of oxidative insults, the Nrf2(S40E)-GFP mutant was distributed in the nucleus. The Nrf2-GFP accumulation in the nucleus was induced by coexpression of aPKCiota, but not by a kinase inactive mutant aPKCiota(K274W). The activity of an ARE-driven reporter was increased by coexpression of aPKCiota, and this effect was eliminated by Ro-31-8220 in HepG2 cells. The reporter activity induced by 4-HNE was inhibited by coexpression of aPKCiota(K274W). These results suggest that phosphorylation of Nrf2 Ser40 by aPKC(s) is involved in

  16. Muscarinic receptor-mediated activation of p70 S6 kinase 1 (S6K1) in 1321N1 astrocytoma cells: permissive role of phosphoinositide 3-kinase.

    OpenAIRE

    Tang, Xiuwen; Wang, Lijun; Proud, Christopher G; Downes, C Peter

    2003-01-01

    In 1321N1 astrocytoma cells, carbachol stimulation of M3 muscarinic cholinergic receptors, coupled to phospholipase C, evoked a persistent 10-20-fold activation of p70 S6 kinase (S6K1). This response was abolished by chelation of cytosolic Ca2+ and reproduced by the Ca2+ ionophore ionomycin, but was not prevented by down-regulation or inhibition of protein kinase C. Carbachol-stimulated activation and phosphorylation of S6K1 at Thr389 were prevented by rapamycin, an inhibitor of mTOR (mammali...

  17. The MAP kinase JNK2 mediates cigarette smoke-induced arterial thrombosis.

    Science.gov (United States)

    Breitenstein, Alexander; Stämpfli, Simon F; Reiner, Martin F; Shi, Yi; Keller, Stephan; Akhmedov, Alexander; Schaub Clerigué, Ariane; Spescha, Remo D; Beer, Hans-Jürg; Lüscher, Thomas F; Tanner, Felix C; Camici, Giovanni G

    2017-01-05

    Despite public awareness of its deleterious effects, smoking remains a major cause of death. Indeed, it is a risk factor for atherothrombotic complications and in line with this, the introduction of smoking ban in public areas reduced smoking-associated cardiovascular complications. Nonetheless, smoking remains a major concern, and molecular mechanisms by which it causes cardiovascular disease are not known. Peripheral blood monocytes from healthy smokers displayed increased JNK2 and tissue factor (TF) gene expression compared to non-smokers (n=15, pthrombosis was investigated. Wild-type mice exposed to smoke displayed reduced time to thrombotic arterial occlusion (n=8; pthrombosis and ROS production. These results underscore a major role of JNK2 in smoke-mediated thrombus formation and may offer an attractive target to prevent smoke-related thrombosis in those subjects which do not manage quitting.

  18. The pan-ErbB tyrosine kinase inhibitor canertinib induces caspase-mediated cell death in human T-cell leukemia (Jurkat) cells

    Energy Technology Data Exchange (ETDEWEB)

    Trinks, Cecilia, E-mail: Cecilia.trinks@liu.se [Division of Oncology, Department of Clinical and Experimental Medicine, Faculty of Health Sciences, Linkoeping University, Linkoeping (Sweden); Severinsson, Emelie A., E-mail: Emelie.severinsson@liu.se [Division of Oncology, Department of Clinical and Experimental Medicine, Faculty of Health Sciences, Linkoeping University, Linkoeping (Sweden); Holmlund, Birgitta, E-mail: Birgitta.holmlund@lio.se [Department of Oncology, County Council of Ostergoetland, Linkoeping (Sweden); Green, Anna, E-mail: Anna.green@liu.se [Division of Cell Biology, Department of Clinical and Experimental Medicine, Faculty of Health Sciences, Linkoeping University, Linkoeping (Sweden); Green, Henrik, E-mail: Henrik.green@liu.se [Clinical Pharmacology, Division of Drug Research, Department of Medical and Health Sciences, Faculty of Health Sciences, Linkoeping University, Linkoeping (Sweden); Joensson, Jan-Ingvar, E-mail: Jan-ingvar.jonsson@liu.se [Experimental Hematology Unit, Department of Clinical and Experimental Medicine, Faculty of Health Sciences, Linkoeping University, Linkoeping (Sweden); Hallbeck, Anna-Lotta, E-mail: Anna-Lotta.Hallbeck@lio.se [Division of Oncology, Department of Clinical and Experimental Medicine, Faculty of Health Sciences, Linkoeping University, Linkoeping (Sweden); Department of Oncology, County Council of Ostergoetland, Linkoeping (Sweden); Walz, Thomas M., E-mail: Thomas.Walz@lio.se [Division of Oncology, Department of Clinical and Experimental Medicine, Faculty of Health Sciences, Linkoeping University, Linkoeping (Sweden); Department of Oncology, County Council of Ostergoetland, Linkoeping (Sweden)

    2011-07-08

    Highlights: {yields} Canertinib induces caspase-mediated apoptosis in T-cell leukemia cells in vitro. {yields} Canertinib mediates activation of the intrinsic apoptotic pathway. {yields} Canertinib induces apoptosis in an ErbB receptor independent manner. {yields} Lymphocyte specific proteins as well as survival kinases are inhibited. {yields} Canertinib may act as a multi-kinase inhibiting drug in human T-cell malignancies. -- Abstract: Canertinib is a novel ErbB-receptor inhibitor currently in clinical development for the treatment of solid tumors overexpressing ErbB-receptors. We have recently demonstrated that canertinib displays anti-proliferative and pro-apoptotic effects in human myeloid leukemia cells devoid of ErbB-receptors. The mechanism mediating these effects are however unknown. In this study, we show that canertinib is able to act as a multi-kinase inhibitor by inhibition of several intracellular kinases involved in T-cell signaling such as Akt, Erk1/2 and Zap-70, and reduced Lck protein expression in the human T-cell leukemia cell line Jurkat. Treatment with canertinib at a concentration of 2 {mu}M caused accumulation of Jurkat cells in the G{sub 1} cell cycle phase and increased doses induced apoptosis in a time-dependent manner. Apoptotic signs of treated cells were detected by Annexin V staining and cleavage of PARP, caspase-3, -8, -9, -10 and Bid. A subset of the pro-apoptotic signals mediated by canertinib could be significantly reduced by specific caspase inhibitors. Taken together, these results demonstrate the dual ability of canertinib to downregulate important signaling pathways and to activate caspase-mediated intrinsic apoptosis pathway in human T-cell leukemia cells.

  19. Protein Kinase C-dependent Ubiquitination and Clathrin-mediated Endocytosis of the Cationic Amino Acid Transporter CAT-1*

    Science.gov (United States)

    Vina-Vilaseca, Arnau; Bender-Sigel, Julia; Sorkina, Tatiana; Closs, Ellen Ildicho; Sorkin, Alexander

    2011-01-01

    Cationic amino acid transporter 1 (CAT-1) is responsible for the bulk of the uptake of cationic amino acids in most mammalian cells. Activation of protein kinase C (PKC) leads to down-regulation of the cell surface CAT-1. To examine the mechanisms of PKC-induced down-regulation of CAT-1, a functional mutant of CAT-1 (CAT-1-HA-GFP) was generated in which a hemagglutinin antigen (HA) epitope tag was introduced into the second extracellular loop and GFP was attached to the carboxyl terminus. CAT-1-HA-GFP was stably expressed in porcine aorthic endothelial and human epithelial kidney (HEK) 293 cells. Using the HA antibody internalization assay we have demonstrated that PKC-dependent endocytosis was strongly inhibited by siRNA depletion of clathrin heavy chain, indicating that CAT-1-HA-GFP internalization requires clathrin-coated pits. Internalized CAT-1-HA-GFP was accumulated in early, recycling, and late endosomes. PKC activation also resulted in ubiquitination of CAT-1. CAT-1 ubiquitination and endocytosis in phorbol ester-stimulated porcine aorthic endothelial and HEK293 cells were inhibited by siRNA knockdown of NEDD4-2 and NEDD4-1 E3 ubiquitin ligases, respectively. In contrast, ubiquitination and endocytosis of the dopamine transporter was dependent on NEDD4-2 in all cell types tested. Altogether, our data suggest that ubiquitination mediated by NEDD4-2 or NEDD4-1 leading to clathrin-mediated endocytosis is the common mode of regulation of various transporter proteins by PKC. PMID:21212261

  20. Impaired vascular function in sepsis-surviving rats mediated by oxidative stress and Rho-Kinase pathway

    Directory of Open Access Journals (Sweden)

    Priscila de Souza

    2016-12-01

    Full Text Available We investigated long-lasting changes in endothelial and vascular function in adult rat survivors of severe sepsis induced by cecal ligation and puncture (CLP model. For this, male Wistar rats (200–350 g had their cecum punctured once (non-transfixing hole with a 14-gauge needle. Performed in this way, a mortality rate around 30% was achieved in the first 72 h. The survivors, together with age-matched control rats (not subjected to CLP, were maintained in our holding room for 60 days (S60 group and had the descending thoracic aorta processed for functional, histological, biochemical or molecular analyses. Endothelium-intact aortic rings obtained from sepsis-surviving S60 group displayed increased angiotensin II-induced contraction, accompanied by decreased activity of the endogenous superoxide dismutase, augmented reactive oxygen species generation, and increased levels of tyrosine nitration compared with vessels from control group. The superoxide scavengers superoxide dismutase and tempol, and the antioxidant apocynin, were able to avoid this enhanced contractility to angiotensin II in aortic rings from the S60 group. In addition, aortic rings from the S60 group presented reduced sensitivity to Y-27632, a Rho-kinase (ROCK inhibitor. Immunoblot analyses revealed augmented RhoA and ROCK II, and high levels of phosphorylation of myosin phosphatase target subunit 1 in vessels from S60 rats. In conclusion, aortic rings from sepsis-surviving rats display endothelial dysfunction mediated by the increased production of reactive oxygen species, which in turn reduces the bioavailability of nitric oxide and increases the formation of peroxynitrite, and enhances RhoA-ROCK-mediated calcium sensitization, leading to augmented contractile responses to angiotensin II. Notably, this is the first study demonstrating long-term dysfunction in the vasculature of sepsis-surviving rats, which take place or remain beyond the acute septic insult.

  1. Glucagon Receptor Antagonism Improves Glucose Metabolism and Cardiac Function by Promoting AMP-Mediated Protein Kinase in Diabetic Mice

    Directory of Open Access Journals (Sweden)

    Ankit X. Sharma

    2018-02-01

    Full Text Available The antidiabetic potential of glucagon receptor antagonism presents an opportunity for use in an insulin-centric clinical environment. To investigate the metabolic effects of glucagon receptor antagonism in type 2 diabetes, we treated Leprdb/db and Lepob/ob mice with REMD 2.59, a human monoclonal antibody and competitive antagonist of the glucagon receptor. As expected, REMD 2.59 suppresses hepatic glucose production and improves glycemia. Surprisingly, it also enhances insulin action in both liver and skeletal muscle, coinciding with an increase in AMP-activated protein kinase (AMPK-mediated lipid oxidation. Furthermore, weekly REMD 2.59 treatment over a period of months protects against diabetic cardiomyopathy. These functional improvements are not derived simply from correcting the systemic milieu; nondiabetic mice with cardiac-specific overexpression of lipoprotein lipase also show improvements in contractile function after REMD 2.59 treatment. These observations suggest that hyperglucagonemia enables lipotoxic conditions, allowing the development of insulin resistance and cardiac dysfunction during disease progression.

  2. Phosphorylation of p62 by AMP-activated protein kinase mediates autophagic cell death in adult hippocampal neural stem cells.

    Science.gov (United States)

    Ha, Shinwon; Jeong, Seol-Hwa; Yi, Kyungrim; Chung, Kyung Min; Hong, Caroline Jeeyeon; Kim, Seong Who; Kim, Eun-Kyoung; Yu, Seong-Woon

    2017-08-18

    In the adult brain, programmed death of neural stem cells is considered to be critical for tissue homeostasis and cognitive function and is dysregulated in neurodegeneration. Previously, we have reported that adult rat hippocampal neural (HCN) stem cells undergo autophagic cell death (ACD) following insulin withdrawal. Because the apoptotic capability of the HCN cells was intact, our findings suggested activation of unique molecular mechanisms linking insulin withdrawal to ACD rather than apoptosis. Here, we report that phosphorylation of autophagy-associated protein p62 by AMP-activated protein kinase (AMPK) drives ACD and mitophagy in HCN cells. Pharmacological inhibition of AMPK or genetic ablation of the AMPK α2 subunit by clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 genome editing suppressed ACD, whereas AMPK activation promoted ACD in insulin-deprived HCN cells. We found that following insulin withdrawal AMPK phosphorylated p62 at a novel site, Ser-293/Ser-294 (in rat and human p62, respectively). Phosphorylated p62 translocated to mitochondria and induced mitophagy and ACD. Interestingly, p62 phosphorylation at Ser-293 was not required for staurosporine-induced apoptosis in HCN cells. To the best of our knowledge, this is the first report on the direct phosphorylation of p62 by AMPK. Our data suggest that AMPK-mediated p62 phosphorylation is an ACD-specific signaling event and provide novel mechanistic insight into the molecular mechanisms in ACD. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  3. Arabidopsis ZED1-related kinases mediate the temperature-sensitive intersection of immune response and growth homeostasis.

    Science.gov (United States)

    Wang, Zhicai; Cui, Dayong; Liu, Jing; Zhao, Jingbo; Liu, Cheng; Xin, Wei; Li, Yuan; Liu, Na; Ren, Dongtao; Tang, Dingzhong; Hu, Yuxin

    2017-07-01

    Activation of the immune response in plants antagonizes growth and development in the absence of pathogens, and such an autoimmune phenotype is often suppressed by the elevation of ambient temperature. However, molecular regulation of the ambient temperature-sensitive intersection of immune response and growth is largely elusive. A genetic screen identified an Arabidopsis mutant, zed1-D, by its high temperature-dependent growth retardation. A combination of molecular, cytological and genetic approaches was used to investigate the molecular basis behind the temperature-sensitive growth and immune response in zed1-D. A dominant mutation in HOPZ-ETI-DEFICIENT 1 (ZED1) is responsible for a high temperature-dependent autoimmunity and growth retardation in zed1-D. The autoimmune phenotype in zed1-D is dependent on the HOPZ-ACTIVATED RESISTANCE 1 (ZAR1). ZED1 and some ZED1-related kinases (ZRKs) are induced by elevated temperature and function cooperatively to suppress the immune response by modulating the transcription of SUPPRESSOR OF NPR1-1 CONSTITUTIVE 1 (SNC1) in the absence of pathogens. Our data reveal a previously unidentified role of ZRKs in the ambient temperature-sensitive immune response in the absence of pathogens, and thus reveals a possible molecular mechanism underlying the temperature-mediated intersection of immune response and growth in plants. © 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.

  4. Integrin-linked kinase mediates force transduction in cardiomyocytes by modulating SERCA2a/PLN function.

    Science.gov (United States)

    Traister, Alexandra; Li, Mark; Aafaqi, Shabana; Lu, Mingliang; Arab, Sara; Radisic, Milica; Gross, Gil; Guido, Fiorella; Sherret, John; Verma, Subodh; Slorach, Cameron; Mertens, Luc; Hui, Wei; Roy, Anna; Delgado-Olguín, Paul; Hannigan, Gregory; Maynes, Jason T; Coles, John G

    2014-09-11

    Human dilated cardiomyopathy (DCM) manifests as a profound reduction in biventricular cardiac function that typically progresses to death or cardiac transplantation. There is no effective mechanism-based therapy currently available for DCM, in part because the transduction of mechanical load into dynamic changes in cardiac contractility (termed mechanotransduction) remains an incompletely understood process during both normal cardiac function and in disease states. Here we show that the mechanoreceptor protein integrin-linked kinase (ILK) mediates cardiomyocyte force transduction through regulation of the key calcium regulatory protein sarcoplasmic/endoplasmic reticulum Ca(2+)ATPase isoform 2a (SERCA-2a) and phosphorylation of phospholamban (PLN) in the human heart. A non-oncogenic ILK mutation with a synthetic point mutation in the pleckstrin homology-like domain (ILK(R211A)) is shown to enhance global cardiac function through SERCA-2a/PLN. Thus, ILK serves to link mechanoreception to the dynamic modulation of cardiac contractility through a previously undiscovered interaction with the functional SERCA-2a/PLN module that can be exploited to rescue impaired mechanotransduction in DCM.

  5. The phosphatidylinositol-3 kinase pathway is not essential for insulin-like growth factor I receptor-mediated clonogenic radioresistance

    International Nuclear Information System (INIS)

    Yu, Dong; Watanabe, Hiroshi; Shibuya, Hitoshi; Miura, Masahiko

    2002-01-01

    The insulin-like growth factor I receptor (IGF-IR) is known to induce clonogenic radioresistance in cells following ionizing irradiation. To explore the downstream signaling pathways, we focused on the phosphatidylinositol-3 kinase (PI3-K) pathway, which is thought to be the primary cell survival signal originating from the receptor. For this purpose, R- cells deficient in the endogenous IGF-IR were used as a recipient of the human IGF-IR with or without mutations at potential PI3-K activation sites: NPXY 950 and Y 1316 XXM. Mutats with double mutation at Y950/Y1316 exhibited not abrogated, but reduced activation of insulin receptor substance-1 (IRS-1), PI3-K, and Akt upon IGF-I stimulation. However, the mutants had the same clonogenic radioresistance as cells with wild type (WT) receptors. Neither wortmannin nor LY294002, specific inhibitors of PI3-K, affected the radioresistance of cells with WT receptors at concentrations specific for PI3-K. Collectively, these results indicate that the PI3-K pathway is not essential for IGF-IR-mediated clonogenic radioresistance. (author)

  6. P21-activated kinase 7 mediates cisplatin-resistance of esophageal squamous carcinoma cells with Aurora-A overexpression.

    Directory of Open Access Journals (Sweden)

    Shun He

    Full Text Available Aurora-A overexpression is common in various types of cancers and has been shown to be involved in tumorigenesis through different signaling pathways, yet how the deregulation affects cancer therapeutics remains elusive. Here we showed that overexpression of Aurora-A rendered esophageal cancer cells resistance to cisplatin (CDDP by inhibiting apoptosis. By using an apoptosis array, we identified a downstream gene, p21-activated kinase 7 (PAK7. PAK7 was upregulated by Aurora-A overexpression at both mRNA and protein levels. Importantly, the expression levels of Aurora-A and PAK7 were correlated in ESCC primary samples. Chromatin immunoprecipitation (ChIP assay revealed that binding of E2F1 to the promoter of PAK7 was significantly enhanced upon Aurora-A activation, and knockdown of transcription factor E2F1 decreased PAK7 expression, suggesting that Aurora-A regulated PAK7 through E2F1. Furthermore, we demonstrated that PAK7 knockdown led to increased apoptosis, and Aurora-A-induced resistance to CDDP was reversed by downregulation of PAK7, suggesting PAK7 was a downstream player of Aurora-A that mediated chemoresistance of ESCC cells to CDDP. Our data suggest that PAK7 may serve as an attractive candidate for therapeutics in ESCC patients with Aurora-A abnormality.

  7. Macrophage Akt1 Kinase-Mediated Mitophagy Modulates Apoptosis Resistance and Pulmonary Fibrosis.

    Science.gov (United States)

    Larson-Casey, Jennifer L; Deshane, Jessy S; Ryan, Alan J; Thannickal, Victor J; Carter, A Brent

    2016-03-15

    Idiopathic pulmonary fibrosis (IPF) is a devastating lung disorder with increasing incidence. Mitochondrial oxidative stress in alveolar macrophages is directly linked to pulmonary fibrosis. Mitophagy, the selective engulfment of dysfunctional mitochondria by autophagasomes, is important for cellular homeostasis and can be induced by mitochondrial oxidative stress. Here, we show Akt1 induced macrophage mitochondrial reactive oxygen species (ROS) and mitophagy. Mice harboring a conditional deletion of Akt1 in macrophages (Akt1(-/-)Lyz2-cre) and Park2(-/-) mice had impaired mitophagy and reduced active transforming growth factor-β1 (TGF-β1). Although Akt1 increased TGF-β1 expression, mitophagy inhibition in Akt1-overexpressing macrophages abrogated TGF-β1 expression and fibroblast differentiation. Importantly, conditional Akt1(-/-)Lyz2-cre mice and Park2(-/-) mice had increased macrophage apoptosis and were protected from pulmonary fibrosis. Moreover, IPF alveolar macrophages had evidence of increased mitophagy and displayed apoptosis resistance. These observations suggest that Akt1-mediated mitophagy contributes to alveolar macrophage apoptosis resistance and is required for pulmonary fibrosis development. Copyright © 2016 Elsevier Inc. All rights reserved.

  8. PME-1 protects extracellular signal-regulated kinase pathway activity from protein phosphatase 2A-mediated inactivation in human malignant glioma.

    Science.gov (United States)

    Puustinen, Pietri; Junttila, Melissa R; Vanhatupa, Sari; Sablina, Anna A; Hector, Melissa E; Teittinen, Kaisa; Raheem, Olayinka; Ketola, Kirsi; Lin, Shujun; Kast, Juergen; Haapasalo, Hannu; Hahn, William C; Westermarck, Jukka

    2009-04-01

    Extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase pathway activity is regulated by the antagonist function of activating kinases and inactivating protein phosphatases. Sustained ERK pathway activity is commonly observed in human malignancies; however, the mechanisms by which the pathway is protected from phosphatase-mediated inactivation in the tumor tissue remain obscure. Here, we show that methylesterase PME-1-mediated inhibition of the protein phosphatase 2A promotes basal ERK pathway activity and is required for efficient growth factor response. Mechanistically, PME-1 is shown to support ERK pathway signaling upstream of Raf, but downstream of growth factor receptors and protein kinase C. In malignant gliomas, PME-1 expression levels correlate with both ERK activity and cell proliferation in vivo. Moreover, PME-1 expression significantly correlates with disease progression in human astrocytic gliomas (n=222). Together, these observations identify PME-1 expression as one mechanism by which ERK pathway activity is maintained in cancer cells and suggest an important functional role for PME-1 in the disease progression of human astrocytic gliomas.

  9. Src subfamily kinases regulate nuclear export and degradation of transcription factor Nrf2 to switch off Nrf2-mediated antioxidant activation of cytoprotective gene expression.

    Science.gov (United States)

    Niture, Suryakant K; Jain, Abhinav K; Shelton, Phillip M; Jaiswal, Anil K

    2011-08-19

    Nrf2 (NF-E2-related factor 2) is a nuclear transcription factor that in response to chemical and radiation stress regulates coordinated induction of a battery of cytoprotective gene expressions leading to cellular protection. In this study, we investigated the role of Src kinases in the regulation of Nrf2 and downstream signaling. siRNA-mediated inhibition of Fyn, Src, Yes, and Fgr, but not Lyn, in mouse hepatoma Hepa-1 cells, led to nuclear accumulation of Nrf2 and up-regulation of Nrf2 downstream gene expression. Mouse embryonic fibroblasts with combined deficiency of Fyn/Src/Yes/Fgr supported results from siRNA. In addition, steady-state overexpression of Fyn, Src, and Yes phosphorylated Nrf2Tyr568 that triggered nuclear export and degradation of Nrf2 and down-regulation of Nrf2 downstream gene expression. Exposure of cells to antioxidant, oxidant, or UV radiation increased nuclear import of Fyn, Src, and Yes kinases, which phosphorylated Nrf2Tyr568 resulting in nuclear export and degradation of Nrf2. Further analysis revealed that stress-activated GSK3β acted upstream to the Src kinases and phosphorylated the Src kinases, leading to their nuclear localization and Nrf2 phosphorylation. The overexpression of Src kinases in Hepa-1 cells led to decreased Nrf2, increased apoptosis, and decreased cell survival. Mouse embryonic fibroblasts deficient in Src kinases showed nuclear accumulation of Nrf2, induction of Nrf2 and downstream gene expression, reduced apoptosis, and increased cell survival. The studies together demonstrate that Src kinases play a critical role in nuclear export and degradation of Nrf2, thereby providing a negative feedback mechanism to switch off Nrf2 activation and restore normal cellular homeostasis.

  10. Role of the PKCα-c-Src tyrosine kinase pathway in the mediation of p120-catenin degradation in ventilator-induced lung injury.

    Science.gov (United States)

    Zhao, Tao; Zhao, Hongwei; Li, Gang; Zheng, Shengfa; Liu, Mengjie; Gu, Changping; Wang, Yuelan

    2016-11-01

    Ventilator-induced lung injury (VILI) is commonly associated with respiratory barrier dysfunction; however, the mechanisms have not been fully elucidated. This study aimed to determine the order and components of the signalling pathway that mediates the degradation of adherin junction of p120-catenin in VILI. For the in vivo study, C57BL/6 mice were pre-treated with inhibitors for 60 min prior to 4 h of mechanical ventilation. For the in vitro study, mouse lung epithelial 12 (MLE-12) cells were pre-treated with inhibitors for 60 min or small interfering RNA (siRNA) for 48 h prior to cyclic stretch at 20% for 4 h. The protein levels of protein kinase Cα (PKCα), activated c-Src and p120-catenin were determined via western blot analysis. Lung injury was determined via HE staining, immunofluorescence, wet/dry ratio and lung injury scores. High tidal volume mechanical ventilation and 20% cyclic stretch resulted in the degradation of p120-catenin. Inhibitors of PKCα blocked c-Src kinase activation and p120-catenin degradation in VILI. Inhibitors of c-Src kinase or PP2 or siRNA blocked p120-catenin degradation but not PKCα activation. The current findings demonstrates that PKCα and c-Src kinase participate in VILI. PKCα activation phosphorylates c-Src kinase and further decreases p120-catenin in VILI. © 2016 Asian Pacific Society of Respirology.

  11. Phosphorylation of the norepinephrine transporter at threonine 258 and serine 259 is linked to protein kinase C-mediated transporter internalization

    DEFF Research Database (Denmark)

    Jayanthi, Lankupalle D; Annamalai, Balasubramaniam; Samuvel, Devadoss J

    2006-01-01

    ester (beta-PMA)-induced phosphorylation of NET occurs on serine and threonine residues. Beta-PMA treatment inhibited NE transport, reduced plasma membrane hNET levels, and stimulated hNET phosphorylation in human placental trophoblast cells expressing the WT-hNET. Substance P-mediated activation......Recently, we have demonstrated the phosphorylation- and lipid raft-mediated internalization of the native norepinephrine transporter (NET) following protein kinase C (PKC) activation (Jayanthi, L. D., Samuvel, D. J., and Ramamoorthy, S. (2004) J. Biol. Chem. 279, 19315-19326). Here we tested...

  12. Adenosine A2A receptor-dependent proliferation of pulmonary endothelial cells is mediated through calcium mobilization, PI3-kinase and ERK1/2 pathways

    International Nuclear Information System (INIS)

    Ahmad, Aftab; Schaack, Jerome B.; White, Carl W.; Ahmad, Shama

    2013-01-01

    Highlights: •A 2A receptor-induced pulmonary endothelial growth is mediated by PI3K and ERK1/2. •Cytosolic calcium mobilization is also critical for pulmonary endothelial growth. •Effectors of A 2A receptor, like tyrosine kinases and cAMP increase PI3K/Akt signaling. •Activation of A 2A receptor can contribute to vascular remodeling. -- Abstract: Hypoxia and HIF-2α-dependent A 2A receptor expression and activation increase proliferation of human lung microvascular endothelial cells (HLMVECs). This study was undertaken to investigate the signaling mechanisms that mediate the proliferative effects of A 2A receptor. A 2A receptor-mediated proliferation of HLMVECs was inhibited by intracellular calcium chelation, and by specific inhibitors of ERK1/2 and PI3-kinase (PI3K). The adenosine A 2A receptor agonist CGS21680 caused intracellular calcium mobilization in controls and, to a greater extent, in A 2A receptor-overexpressing HLMVECs. Adenoviral-mediated A 2A receptor overexpression as well as receptor activation by CGS21680 caused increased PI3K activity and Akt phosphorylation. Cells overexpressing A 2A receptor also manifested enhanced ERK1/2 phosphorylation upon CGS21680 treatment. A 2A receptor activation also caused enhanced cAMP production. Likewise, treatment with 8Br-cAMP increased PI3K activity. Hence A 2A receptor-mediated cAMP production and PI3K and Akt phosphorylation are potential mediators of the A 2A -mediated proliferative response of HLMVECs. Cytosolic calcium mobilization and ERK1/2 phosphorylation are other critical effectors of HLMVEC proliferation and growth. These studies underscore the importance of adenosine A 2A receptor in activation of survival and proliferative pathways in pulmonary endothelial cells that are mediated through PI3K/Akt and ERK1/2 pathways

  13. Conformational Stability of the NH2-Terminal Propeptide of the Precursor of Pulmonary Surfactant Protein SP-B.

    Directory of Open Access Journals (Sweden)

    Ángeles Bañares-Hidalgo

    Full Text Available Assembly of pulmonary surfactant lipid-protein complexes depends on conformational changes coupled with proteolytic maturation of proSP-B, the precursor of pulmonary surfactant protein B (SP-B, along the surfactant biogenesis pathway in pneumocytes. Conformational destabilization of the N-terminal propeptide of proSP-B (SP-BN triggers exposure of the mature SP-B domain for insertion into surfactant lipids. We have studied the conformational stability during GdmCl- or urea-promoted unfolding of SP-BN with trp fluorescence and circular dichroism spectroscopies. Binding of the intermediate states to bis-ANS suggests their molten globule-like character. ΔG0H2O was ~ 12.7 kJ·mol-1 either with urea or GdmCl. None of the thermal transitions of SP-BN detected by CD correspond to protein unfolding. Differential scanning calorimetry of SP-BN evidenced two endothermic peaks involved in oligomer dissociation as confirmed with 2 M urea. Ionic strength was relevant since at 150 mM NaCl, the process originating the endotherm at the highest temperature was irreversible (Tm2 = 108.5°C with an activation energy of 703.8 kJ·mol-1. At 500 mM NaCl the process became reversible (Tm2 = 114.4°C and data were fitted to the Non-two States model with two subpeaks. No free thiols in the propeptide could be titrated by DTNB with or without 5.7 M GdmCl, indicating disulfide bonds establishment.

  14. The NH2-terminal php domain of the alpha subunit of the Escherichia coli replicase binds the epsilon proofreading subunit.

    Science.gov (United States)

    Wieczorek, Anna; McHenry, Charles S

    2006-05-05

    The alpha subunit of the replicase of all bacteria contains a php domain, initially identified by its similarity to histidinol phosphatase but of otherwise unknown function (Aravind, L., and Koonin, E. V. (1998) Nucleic Acids Res. 26, 3746-3752). Deletion of 60 residues from the NH2 terminus of the alpha php domain destroys epsilon binding. The minimal 255-residue php domain, estimated by sequence alignment with homolog YcdX, is insufficient for epsilon binding. However, a 320-residue segment including sequences that immediately precede the polymerase domain binds epsilon with the same affinity as the 1160-residue full-length alpha subunit. A subset of mutations of a conserved acidic residue (Asp43 in Escherichia coli alpha) present in the php domain of all bacterial replicases resulted in defects in epsilon binding. Using sequence alignments, we show that the prototypical gram+ Pol C, which contains the polymerase and proofreading activities within the same polypeptide chain, has an epsilon-like sequence inserted in a surface loop near the center of the homologous YcdX protein. These findings suggest that the php domain serves as a platform to enable coordination of proofreading and polymerase activities during chromosomal replication.

  15. An anti-NH2-terminal antibody localizes NBCn1 to heart endothelia and skeletal and vascular smooth muscle cells

    DEFF Research Database (Denmark)

    Damkier, Helle Hasager; Nielsen, Søren; Prætorius, Jeppe

    2006-01-01

    The electroneutral sodium bicarbonate cotransporter NBCn1 or NBC3 was originally cloned from rat aorta and from human skeletal muscle. NBCn1 (or NBC3) has been localized to the basolateral membrane of various epithelia, but thus far it has been impossible to detect the protein in these tissues...

  16. Calcium/Calmodulin-Dependent Protein Kinase IV Mediates IFN-γ-Induced Immune Behaviors in Skeletal Muscle Cells

    Directory of Open Access Journals (Sweden)

    RuiCai Gu

    2018-03-01

    Full Text Available Background/Aims: Whether calcium/calmodulin-dependent protein kinase IV (CaMKIV plays a role in regulating immunologic features of muscle cells in inflammatory environment, as it does for immune cells, remains mostly unknown. In this study, we investigated the influence of endogenous CaMKIV on the immunological characteristics of myoblasts and myotubes received IFN-γ stimulation. Methods: C2C12 and murine myogenic precursor cells (MPCs were cultured and differentiated in vitro, in the presence of pro-inflammatory IFN-γ. CaMKIV shRNA lentivirus transfection was performed to knockdown CaMKIV gene in C2C12 cells. pEGFP-N1-CaMKIV plasmid was delivered into knockout cells for recovering intracellular CaMKIV gene level. CREB1 antagonist KG-501 was used to block CREB signal. qPCR, immunoblot analysis, or immunofluorescence was used to detect mRNA and protein levels of CaMKIV, immuno-molecules, or pro-inflammatory cytokines and chemokines. Co-stimulatory molecules expression was assessed by FACS analysis. Results: IFN-γ induces the expression or up-regulation of MHC-I/II and TLR3, and the up-regulation of CaMKIV level in muscle cells. In contrast, CaMKIV knockdown in myoblasts and myotubes leads to expression inhibition of the above immuno-molecules. As well, CaMKIV knockdown selectively inhibits pro-inflammatory cytokines/chemokines, and co-stimulatory molecules expression in IFN-γ treated myoblasts and myotubes. Finally, CaMKIV knockdown abolishes IFN-γ induced CREB pathway molecules accumulation in differentiated myotubes. Conclusions: CaMKIV can be induced to up-regulate in muscle cells under inflammatory condition, and positively mediates intrinsic immune behaviors of muscle cells triggered by IFN-γ.

  17. Bortezomib induces neuropathic pain through protein kinase C-mediated activation of presynaptic NMDA receptors in the spinal cord.

    Science.gov (United States)

    Xie, Jing-Dun; Chen, Shao-Rui; Chen, Hong; Pan, Hui-Lin

    2017-09-01

    Chemotherapeutic drugs, including bortezomib, often cause painful peripheral neuropathy, which is a severe dose-limiting adverse effect experienced by many cancer patients. The glutamate N-methyl-d-aspartate receptors (NMDARs) at the spinal cord level are critically involved in the synaptic plasticity associated with neuropathic pain. In this study, we determined whether treatment with bortezomib, a proteasome inhibitor, affects the NMDAR activity of spinal dorsal horn neurons. Systemic treatment with bortezomib in rats did not significantly affect postsynaptic NMDAR currents elicited by puff application of NMDA directly to dorsal horn neurons. Bortezomib treatment markedly increased the baseline frequency of miniature excitatory postsynaptic currents (EPSCs), which was completely normalized by the NMDAR antagonist 2-amino-5-phosphonopentanoic acid (AP5). AP5 also reduced the amplitude of monosynaptic EPSCs evoked by dorsal root stimulation in bortezomib-treated, but not vehicle-treated, rats. Furthermore, inhibition of protein kinase C (PKC) with chelerythrine fully reversed the increased frequency of miniature EPSCs and the amplitude of evoked EPSCs in bortezomib-treated rats. Intrathecal injection of AP5 and chelerythrine both profoundly attenuated mechanical allodynia and hyperalgesia induced by systemic treatment with bortezomib. In addition, treatment with bortezomib induced striking membrane translocation of PKC-βII, PKC-δ, and PKC-ε in the dorsal root ganglion. Our findings indicate that bortezomib treatment potentiates nociceptive input from primary afferent nerves via PKC-mediated tonic activation of presynaptic NMDARs. Targeting presynaptic NMDARs and PKC at the spinal cord level may be an effective strategy for treating chemotherapy-induced neuropathic pain. Copyright © 2017 Elsevier Ltd. All rights reserved.

  18. Prenatal testosterone exposure induces hypertension in adult females via androgen receptor-dependent protein kinase Cδ-mediated mechanism.

    Science.gov (United States)

    Blesson, Chellakkan S; Chinnathambi, Vijayakumar; Hankins, Gary D; Yallampalli, Chandra; Sathishkumar, Kunju

    2015-03-01

    Prenatal exposure to excess testosterone induces hyperandrogenism in adult females and predisposes them to hypertension. We tested whether androgens induce hypertension through transcriptional regulation and signaling of protein kinase C (PKC) in the mesenteric arteries. Pregnant Sprague-Dawley rats were injected with vehicle or testosterone propionate (0.5 mg/kg per day from gestation days 15 to 19, SC) and their 6-month-old adult female offspring were examined. Plasma testosterone levels (0.84±0.04 versus 0.42±0.09 ng/mL) and blood pressures (111.6±1.3 versus 104.5±2.4 mm Hg) were significantly higher in prenatal testosterone-exposed rats compared with controls. This was accompanied with enhanced expression of PKCδ mRNA (1.5-fold) and protein (1.7-fold) in the mesenteric arteries of prenatal testosterone-exposed rats. In addition, mesenteric artery contractile responses to PKC activator, phorbol-12,13-dibutyrate, was significantly greater in prenatal testosterone-exposed rats. Treatment with androgen receptor antagonist flutamide (10 mg/kg, SC, BID for 10 days) significantly attenuated hypertension, PKCδ expression, and the exaggerated vasoconstriction in prenatal testosterone-exposed rats. In vitro exposure of testosterone to cultured mesenteric artery smooth muscle cells dose dependently upregulated PKCδ expression. Analysis of PKCδ gene revealed a putative androgen responsive element in the promoter upstream to the transcription start site and an enhancer element in intron-1. Chromatin immunoprecipitation assays showed that androgen receptors bind to these elements in response to testosterone stimulation. Furthermore, luciferase reporter assays showed that the enhancer element is highly responsive to androgens and treatment with flutamide reverses reporter activity. Our studies identified a novel androgen-mediated mechanism for the control of PKCδ expression via transcriptional regulation that controls vasoconstriction and blood pressure. © 2014

  19. MicroRNA-451 Negatively Regulates Hepatic Glucose Production and Glucose Homeostasis by Targeting Glycerol Kinase-Mediated Gluconeogenesis.

    Science.gov (United States)

    Zhuo, Shu; Yang, Mengmei; Zhao, Yanan; Chen, Xiaofang; Zhang, Feifei; Li, Na; Yao, Pengle; Zhu, Tengfei; Mei, Hong; Wang, Shanshan; Li, Yu; Chen, Shiting; Le, Yingying

    2016-11-01

    MicroRNAs (miRNAs) are a new class of regulatory molecules implicated in type 2 diabetes, which is characterized by insulin resistance and hepatic glucose overproduction. We show that miRNA-451 (miR-451) is elevated in the liver tissues of dietary and genetic mouse models of diabetes. Through an adenovirus-mediated gain- and loss-of-function study, we found that miR-451 negatively regulates hepatic gluconeogenesis and blood glucose levels in normal mice and identified glycerol kinase (Gyk) as a direct target of miR-451. We demonstrate that miR-451 and Gyk regulate hepatic glucose production, the glycerol gluconeogenesis axis, and the AKT-FOXO1-PEPCK/G6Pase pathway in an opposite manner; Gyk could reverse the effect of miR-451 on hepatic gluconeogenesis and AKT-FOXO1-PEPCK/G6Pase pathway. Moreover, overexpression of miR-451 or knockdown of Gyk in diabetic mice significantly inhibited hepatic gluconeogenesis, alleviated hyperglycemia, and improved glucose tolerance. Further studies showed that miR-451 is upregulated by glucose and insulin in hepatocytes; the elevation of hepatic miR-451 in diabetic mice may contribute to inhibiting Gyk expression. This study provides the first evidence that miR-451 and Gyk regulate the AKT-FOXO1-PEPCK/G6Pase pathway and play critical roles in hepatic gluconeogenesis and glucose homeostasis and identifies miR-451 and Gyk as potential therapeutic targets against hyperglycemia in diabetes. © 2016 by the American Diabetes Association.

  20. Spinal release of tumour necrosis factor activates c-Jun N-terminal kinase and mediates inflammation-induced hypersensitivity.

    Science.gov (United States)

    Bas, D B; Abdelmoaty, S; Sandor, K; Codeluppi, S; Fitzsimmons, B; Steinauer, J; Hua, X Y; Yaksh, T L; Svensson, C I

    2015-02-01

    Mounting evidence points to individual contributions of tumour necrosis factor-alpha (TNF) and the c-Jun N-terminal kinase (JNK) pathway to the induction and maintenance of various pain states. Here we explore the role of spinal TNF and JNK in carrageenan-induced hypersensitivity. As links between TNF and JNK have been demonstrated in vitro, we investigated if TNF regulates spinal JNK activity in vivo. TNF levels in lumbar cerebrospinal fluid (CSF) were measured by enzyme-linked immunosorbent assay, spinal TNF gene expression by real-time polymerase chain reaction and TNF protein expression, JNK and c-Jun phosphorylation by western blotting. The role of spinal TNF and JNK in inflammation-induced mechanical and thermal hypersensitivity was assessed by injecting the TNF inhibitor etanercept and the JNK inhibitors SP600125 and JIP-1 intrathecally (i.t.). TNF-mediated regulation of JNK activity was examined by assessing the effect of i.t. etanercept on inflammation-induced spinal JNK activity. TNF levels were increased in CSF and spinal cord following carrageenan-induced inflammation. While JNK phosphorylation followed the same temporal pattern as TNF, c-jun was only activated at later time points. Intrathecal injection of TNF and JNK inhibitors attenuated carrageenan-induced mechanical and thermal hypersensitivity. TNF stimulation induced JNK phosphorylation in cultured spinal astrocytes and blocking the spinal actions of TNF in vivo by i.t. injection of etanercept reduced inflammation-induced spinal JNK activity. Here we show that spinal JNK activity is dependent on TNF and that both TNF and the JNK signalling pathways modulate pain-like behaviour induced by peripheral inflammation. © 2014 The Authors. European Journal of Pain published by John Wiley & Sons Ltd on behalf of European Pain Federation - EFIC®.

  1. {delta}-Opioid receptor-stimulated Akt signaling in neuroblastoma x glioma (NG108-15) hybrid cells involves receptor tyrosine kinase-mediated PI3K activation

    Energy Technology Data Exchange (ETDEWEB)

    Heiss, Anika; Ammer, Hermann [Institute of Pharmacology, Toxicology and Pharmacy Ludwig-Maximilians-University of Munich Koeniginstrasse 16 80539 Muenchen Federal Republic of Germany (Germany); Eisinger, Daniela A., E-mail: eisinger@pharmtox.vetmed.uni-muenchen.de [Institute of Pharmacology, Toxicology and Pharmacy Ludwig-Maximilians-University of Munich Koeniginstrasse 16 80539 Muenchen Federal Republic of Germany (Germany)

    2009-07-15

    {delta}-Opioid receptor (DOR) agonists possess cytoprotective properties, an effect associated with activation of the 'pro-survival' kinase Akt. Here we delineate the signal transduction pathway by which opioids induce Akt activation in neuroblastoma x glioma (NG108-15) hybrid cells. Exposure of the cells to both [D-Pen{sup 2,5}]enkephalin and etorphine resulted in a time- and dose-dependent increase in Akt activity, as measured by means of an activation-specific antibody recognizing phosphoserine-473. DOR-mediated Akt signaling is blocked by the opioid antagonist naloxone and involves inhibitory G{sub i/o} proteins, because pre-treatment with pertussis toxin, but not over-expression of the G{sub q/11} scavengers EBP50 and GRK2-K220R, prevented this effect. Further studies with Wortmannin and LY294002 revealed that phophoinositol-3-kinase (PI3K) plays a central role in opioid-induced Akt activation. Opioids stimulate Akt activity through transactivation of receptor tyrosine kinases (RTK), because pre-treatment of the cells with inhibitors for neurotrophin receptor tyrosine kinases (AG879) and the insulin-like growth factor receptor IGF-1 (AG1024), but not over-expression of the G{beta}{gamma} scavenger phosducin, abolished this effect. Activated Akt translocates to the nuclear membrane, where it promotes GSK3 phosphorylation and prevents caspase-3 cleavage, two key events mediating inhibition of cell apoptosis and enhancement of cell survival. Taken together, these results demonstrate that in NG108-15 hybrid cells DOR agonists possess cytoprotective properties mediated by activation of the RTK/PI3K/Akt signaling pathway.

  2. Focal adhesion kinase-mediated activation of glycogen synthase kinase 3β regulates IL-33 receptor internalization and IL-33 signaling

    OpenAIRE

    Zhao, Jing; Wei, Jianxin; Bowser, Rachel K; Traister, Russell S; Fan, Ming-Hui; Zhao, Yutong

    2014-01-01

    IL-33, a relatively new member of the IL-1 cytokine family, plays a crucial role in allergic inflammation and acute lung injury. ST2L, the receptor for IL-33, is expressed on immune effector cells and lung epithelia, and plays a critical role in triggering inflammation. We have previously shown that ST2L stability is regulated by the ubiquitin-proteasome system, however its upstream internalization has not been studied. Here, we demonstrate that glycogen synthase kinase 3β (GSK3β) regulates S...

  3. The F-box Protein KIB1 Mediates Brassinosteroid-Induced Inactivation and Degradation of GSK3-like Kinases in Arabidopsis.

    Science.gov (United States)

    Zhu, Jia-Ying; Li, Yuyao; Cao, Dong-Mei; Yang, Hongjuan; Oh, Eunkyoo; Bi, Yang; Zhu, Shengwei; Wang, Zhi-Yong

    2017-06-01

    The glycogen synthase kinase-3 (GSK3) family kinases are central cellular regulators highly conserved in all eukaryotes. In Arabidopsis, the GSK3-like kinase BIN2 phosphorylates a range of proteins to control broad developmental processes, and BIN2 is degraded through unknown mechanism upon receptor kinase-mediated brassinosteroid (BR) signaling. Here we identify KIB1 as an F-box E3 ubiquitin ligase that promotes the degradation of BIN2 while blocking its substrate access. Loss-of-function mutations of KIB1 and its homologs abolished BR-induced BIN2 degradation and caused severe BR-insensitive phenotypes. KIB1 directly interacted with BIN2 in a BR-dependent manner and promoted BIN2 ubiquitination in vitro. Expression of an F-box-truncated KIB1 caused BIN2 accumulation but dephosphorylation of its substrate BZR1 and activation of BR responses because KIB1 blocked BIN2 binding to BZR1. Our study demonstrates that KIB1 plays an essential role in BR signaling by inhibiting BIN2 through dual mechanisms of blocking substrate access and promoting degradation. Copyright © 2017 Elsevier Inc. All rights reserved.

  4. Pressure-induced vascular oxidative stress is mediated through activation of integrin-linked kinase 1/betaPIX/Rac-1 pathway.

    Science.gov (United States)

    Vecchione, Carmine; Carnevale, Daniela; Di Pardo, Alba; Gentile, Maria Teresa; Damato, Antonio; Cocozza, Germana; Antenucci, Giovanna; Mascio, Giada; Bettarini, Umberto; Landolfi, Alessandro; Iorio, Luca; Maffei, Angelo; Lembo, Giuseppe

    2009-11-01

    High blood pressure induces a mechanical stress on vascular walls and evokes oxidative stress and vascular dysfunction. The aim of this study was to characterize the intracellular signaling causing vascular oxidative stress in response to pressure. In carotid arteries subjected to high pressure levels, we observed not only an impaired vasorelaxation, increased superoxide production, and NADPH oxidase activity, but also a concomitant activation of Rac-1, a small G protein. Selective inhibition of Rac-1, with an adenovirus carrying a dominant-negative Rac-1 mutant, significantly reduced NADPH oxidase activity and oxidative stress and, more importantly, rescued vascular function in carotid arteries at high pressure. The analysis of molecular events associated with mechanotransduction demonstrated at high pressure levels an overexpression of integrin-linked kinase 1 and its recruitment to plasma membrane interacting with paxillin. The inhibition of integrin-linked kinase 1 by small interfering RNA impaired Rac-1 activation and rescued oxidative stress-induced vascular dysfunction in response to high pressure. Finally, we showed that betaPIX, a guanine-nucleotide exchange factor, is the intermediate molecule recruited by integrin-linked kinase 1, converging the intracellular signaling toward Rac-1-mediated oxidative vascular dysfunction during pressure overload. Our data demonstrate that biomechanical stress evoked by high blood pressure triggers an integrin-linked kinase 1/betaPIX/Rac-1 signaling, thus generating oxidative vascular dysfunction.

  5. Receptor binding and cell-mediated metabolism of [125I]monoiodoglucagon by isolated canine hepatocytes

    International Nuclear Information System (INIS)

    Hagopian, W.A.; Tager, H.S.

    1984-01-01

    A reverse-phase HPLC method has been developed to purify 125 I-labeled products resulting from the chloramine-T-based iodination of glucagon. In addition the products [( 125 I)iodoTyr 10 13 ]glucagon, [( 125 I)iodoTyr 13 ]glucagon, and [( 125 I)iodoTyr 10 ]glucagon) have been used to study the receptor binding of glucagon and the cell-mediated metabolism of the hormone by isolated canine hepatocytes. It was concluded that (a) not withstanding apparent differences in affinities exhibited by the three peptides, the interactions with the glucagon receptor are functionally equivalent, and (b) the cell-mediated metabolism of receptor-bound glucagon involves the formation of hormone-derived peptides in which the biologically important NH 2 -terminal region of the hormone has been modified by limited proteolytic cleavage

  6. Osteitis fibrosa is mediated by Platelet-Derived Growth Factor-A via a phosphoinositide 3-kinase-dependent signaling pathway in a rat model for chronic hyperparathyroidism.

    Science.gov (United States)

    Lowry, Malcolm B; Lotinun, Sutada; Leontovich, Alexey A; Zhang, Minzhi; Maran, Avudaiappan; Shogren, Kristen L; Palama, Brett K; Marley, Kevin; Iwaniec, Urszula T; Turner, Russell T

    2008-11-01

    Abnormal secretion of PTH by the parathyroid glands contributes to a variety of common skeletal disorders. Prior studies implicate platelet-derived growth factor-A (PDGF-A) as an important mediator of selective PTH actions on bone. The present studies used targeted gene profiling and small-molecule antagonists directed against candidate gene products to elucidate the roles of specific PTH-regulated genes and signaling pathways. A group of 29 genes in rats continuously infused with PTH and cotreated with the PDGF receptor antagonist trapidil were differentially expressed compared with PTH treatment alone. Several of the identified genes were functionally clustered as regulators of fibroblast differentiation and extracellular matrix modeling, including the matrix cross-linking enzyme lysyl oxidase (LOX). Treatment with beta-aminopropionitrile, an irreversible inhibitor of LOX activity, dramatically reduced diffuse mineralization but had no effect on PTH-induced fibrosis. In contrast, the receptor tyrosine kinase inhibitor Gleevec and the phosphoinositide 3-kinase inhibitor wortmannin each reduced bone marrow fibrosis. In summary, the present studies support the hypotheses that PTH-induced bone marrow fibrosis is mediated by PDGF-A via a phosphoinositide 3-kinase-dependent signaling pathway and that increased LOX gene expression plays a key role in abnormal mineralization, a hallmark of chronic hyperparathyroidism.

  7. Protein kinase A-mediated cell proliferation in brown preadipocytes is independent of Erk1/2, PI{sub 3}K and mTOR

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Yanling; Sato, Masaaki; Guo, Yuan; Bengtsson, Tore; Nedergaard, Jan, E-mail: jan@metabol.su.se

    2014-10-15

    The physiological agonist norepinephrine promotes cell proliferation of brown preadipocytes during the process of tissue recruitment. In a primary culture system, cAMP mediates these adrenergic effects. In the present study, we demonstrated that, in contrast to other systems where the mitogenic effect of cAMP requires the synergistic action of (serum) growth factors, especially insulin/IGF, the cAMP effect in brown preadipocytes was independent of serum and insulin. Protein kinase A, rather than Epac, mediated the cAMP mitogenic effect. The Erk 1/2 family of MAPK, the PI{sub 3}K system and the mTOR complexes were all activated by cAMP, but these activations were not necessary for cAMP-induced cell proliferation; a protein kinase C isoform may be involved in mediating cAMP-activated cell proliferation. We conclude that the generally acknowledged cellular mediators for induction of cell proliferation are not involved in this process in the brown preadipocyte system; this conclusion may be of relevance both for examination of mechanisms for induction of brown adipose tissue recruitment but also for understanding the mechanism behind e.g. certain endocrine neoplasias. - Highlights: • cAMP can mimick norepinephrine-induced proliferation of brown preadipocytes. • The cAMP-induced proliferation can occur in the absence of serum, of any other growth factors, and of insulin. • Erk1/2, PI{sub 3}K and mTOR are cAMP activated but not involved in induction of proliferation. • A Protein Kinase C member may be in the signalling cascade. • This pathway analysis may also be of importance for certain endocrine hyper- and neoplasias.

  8. A role for the non-receptor tyrosine kinase ACK1 in TNF-alpha-mediated apoptosis and proliferation in human intestinal epithelial caco-2 cells.

    Science.gov (United States)

    Zhao, Xinmei; Lv, Chaolan; Chen, Shengbo; Zhi, Fachao

    2017-09-16

    The roles of tumor necrosis factor alpha (TNF-alpha) and its mediators in cellular processes related to intestinal diseases remain elusive. In this study, we aimed to determine the biological role of activated Cdc42-associated kinase 1 (ACK1) in TNF-alpha-mediated apoptosis and proliferation in Caco-2 cells. ACK1 expression was knocked down using ACK1-specific siRNAs, and ACK1 activity was disrupted using a small molecule ACK1 inhibitor. The Terminal deoxynucleotidyl transferase biotin-dUTP Nick End Labeling (TUNEL) and the BrdU incorporation assays were used to measure apoptosis and cell proliferation, respectively. ACK1-specific siRNA and the pharmacological ACK1 inhibitor significantly abrogated the TNF-alpha-mediated anti-apoptotic effects and proliferation of Caco-2 cells. Interestingly, TNF-alpha activated ACK1 at tyrosine 284 (Tyr284), and the ErbB family of proteins was implicated in ACK1 activation in Caco-2 cells. ACK1-Tyr284 was required for protein kinase B (AKT) activation, and ACK1 signaling was mediated through recruiting and phosphorylating the down-stream adaptor protein AKT, which likely promoted cell proliferation in response to TNF-alpha. Moreover, ACK1 activated AKT and Src enhanced nuclear factor-кB (NF-кB) activity, suggesting a correlation between NF-кB signaling and TNF-alpha-mediated apoptosis in Caco-2 cells. Our results demonstrate that ACK1 plays an important role in modulating TNF-alpha-induced aberrant cell proliferation and apoptosis, mediated in part by ACK1 activation. ACK1 and its down-stream effectors may hold promise as therapeutic targets in the prevention and treatment of gastrointestinal cancers, in particular, those induced by chronic intestinal inflammation. © 2017 The Authors. Cell Biology International Published by John Wiley & Sons Ltd on behalf of International Federation of Cell Biology.

  9. Receptor protein tyrosine phosphatase alpha activates Src-family kinases and controls integrin-mediated responses in fibroblasts

    DEFF Research Database (Denmark)

    Su, J; Muranjan, M; Sap, J

    1999-01-01

    BACKGROUND: Fyn and c-Src are two of the most widely expressed Src-family kinases. Both are strongly implicated in the control of cytoskeletal organization and in the generation of integrin-dependent signalling responses in fibroblasts. These proteins are representative of a large family...... of tyrosine kinases, the activity of which is tightly controlled by inhibitory phosphorylation of a carboxyterminal tyrosine residue (Tyr527 in chicken c-Src); this phosphorylation induces the kinases to form an inactive conformation. Whereas the identity of such inhibitory Tyr527 kinases has been well...... established, no corresponding phosphatases have been identified that, under physiological conditions, function as positive regulators of c-Src and Fyn in fibroblasts. RESULTS: Receptor protein tyrosine phosphatase alpha (RPTPalpha) was inactivated by homologous recombination. Fibroblasts derived from...

  10. Contraction-mediated glycogenolysis in mouse skeletal muscle lacking creatine kinase: the role of phosphorylase b activation.

    NARCIS (Netherlands)

    Katz, A.; Andersson, D.C.; Yu, J.; Norman, B.; Sandstrom, M.E.; Wieringa, B.; Westerblad, H.

    2003-01-01

    Skeletal muscle that is deficient in creatine kinase (CK-/-) exhibits accelerated glycogenolysis during contraction. Understanding this phenomenon could provide insight into the control of glycogenolysis during contraction. Therefore, glycogen breakdown was investigated in isolated extensor

  11. Extracellular Signal-regulated Kinase 5 (ERK5) Mediates Prolactin-stimulated Adult Neurogenesis in the Subventricular Zone and Olfactory Bulb*

    Science.gov (United States)

    Wang, Wenbin; Pan, Yung-Wei; Wietecha, Tomasz; Zou, Junhui; Abel, Glen M.; Kuo, Chay T.; Xia, Zhengui

    2013-01-01

    Prolactin-stimulated adult neurogenesis in the subventricular zone (SVZ) and olfactory bulb (OB) mediates several reproductive behaviors including mating/pregnancy, dominant male pheromone preference in females, and paternal recognition of offspring. However, downstream signaling mechanisms underlying prolactin-induced adult neurogenesis are completely unknown. We report here for the first time that prolactin activates extracellular signal-regulated kinase 5 (ERK5), a MAP kinase that is specifically expressed in the neurogenic regions of the adult mouse brain. Knockdown of ERK5 by retroviral infection of shRNA attenuates prolactin-stimulated neurogenesis in SVZ-derived adult neural stem/progenitor cells (aNPCs). Inducible erk5 deletion in adult neural stem cells of transgenic mice inhibits neurogenesis in the SVZ and OB following prolactin infusion or mating/pregnancy. These results identify ERK5 as a novel and critical signaling mechanism underlying prolactin-induced adult neurogenesis. PMID:23223235

  12. Acrolein-induced activation of mitogen-activated protein kinase signaling is mediated by alkylation of thioredoxin reductase and thioredoxin 1

    Directory of Open Access Journals (Sweden)

    Matthew J. Randall

    2013-01-01

    Full Text Available Cigarette smoking remains a major health concern worldwide, and many of the adverse effects of cigarette smoke (CS can be attributed to its abundant electrophilic aldehydes, such as acrolein (2-propenal. Previous studies indicate that acrolein readily reacts with thioredoxin reductase 1 (TrxR1, a critical enzyme involved in regulation of thioredoxin (Trx-mediated redox signaling, by alkylation at its selenocysteine (Sec residue. Because alkylation of Sec within TrxR1 has significant implications for its enzymatic function, we explored the potential importance of TrxR1 alkylation in acrolein-induced activation or injury of bronchial epithelial cells. Exposure of human bronchial epithelial HBE1 cells to acrolein (1–30 μM resulted in dose-dependent loss of TrxR thioredoxin reductase activity, which coincided with its alkylation, as determined by biotin hydrazide labeling, and was independent of initial GSH status. To test the involvement of TrxR1 in acrolein responses in HBE1 cells, we suppressed TrxR1 using siRNA silencing or augmented TrxR1 by cell supplementation with sodium selenite. Acrolein exposure of HBE1 cells induced dose-dependent activation of the MAP kinases, extracellular regulated kinase (ERK, c-Jun N-terminal kinase (JNK, and p38, and activation of JNK was markedly enhanced after selenite-mediated induction of TrxR1, and was associated with increased alkylation of TrxR1. Conversely, siRNA silencing of TrxR1 significantly suppressed the ability of acrolein to activate JNK, and also appeared to attenuate acrolein-dependent activation of ERK and p38. Alteration of initial TrxR1 levels by siRNA or selenite supplementation also affected initial Trx1 redox status and acrolein-mediated alkylation of Trx1, but did not significantly affect acrolein-mediated activation of HO-1 or cytotoxicity. Collectively, our findings indicate that alkylation of TrxR1 and/or Trx1 may contribute directly to acrolein-mediated activation of MAP kinases

  13. Acrolein-induced activation of mitogen-activated protein kinase signaling is mediated by alkylation of thioredoxin reductase and thioredoxin 1.

    Science.gov (United States)

    Randall, Matthew J; Spiess, Page C; Hristova, Milena; Hondal, Robert J; van der Vliet, Albert

    2013-01-01

    Cigarette smoking remains a major health concern worldwide, and many of the adverse effects of cigarette smoke (CS) can be attributed to its abundant electrophilic aldehydes, such as acrolein (2-propenal). Previous studies indicate that acrolein readily reacts with thioredoxin reductase 1 (TrxR1), a critical enzyme involved in regulation of thioredoxin (Trx)-mediated redox signaling, by alkylation at its selenocysteine (Sec) residue. Because alkylation of Sec within TrxR1 has significant implications for its enzymatic function, we explored the potential importance of TrxR1 alkylation in acrolein-induced activation or injury of bronchial epithelial cells. Exposure of human bronchial epithelial HBE1 cells to acrolein (1-30 μM) resulted in dose-dependent loss of TrxR thioredoxin reductase activity, which coincided with its alkylation, as determined by biotin hydrazide labeling, and was independent of initial GSH status. To test the involvement of TrxR1 in acrolein responses in HBE1 cells, we suppressed TrxR1 using siRNA silencing or augmented TrxR1 by cell supplementation with sodium selenite. Acrolein exposure of HBE1 cells induced dose-dependent activation of the MAP kinases, extracellular regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38, and activation of JNK was markedly enhanced after selenite-mediated induction of TrxR1, and was associated with increased alkylation of TrxR1. Conversely, siRNA silencing of TrxR1 significantly suppressed the ability of acrolein to activate JNK, and also appeared to attenuate acrolein-dependent activation of ERK and p38. Alteration of initial TrxR1 levels by siRNA or selenite supplementation also affected initial Trx1 redox status and acrolein-mediated alkylation of Trx1, but did not significantly affect acrolein-mediated activation of HO-1 or cytotoxicity. Collectively, our findings indicate that alkylation of TrxR1 and/or Trx1 may contribute directly to acrolein-mediated activation of MAP kinases such as JNK, and

  14. Acrolein-induced activation of mitogen-activated protein kinase signaling is mediated by alkylation of thioredoxin reductase and thioredoxin 1☆☆☆

    Science.gov (United States)

    Randall, Matthew J.; Spiess, Page C.; Hristova, Milena; Hondal, Robert J.; van der Vliet, Albert

    2013-01-01

    Cigarette smoking remains a major health concern worldwide, and many of the adverse effects of cigarette smoke (CS) can be attributed to its abundant electrophilic aldehydes, such as acrolein (2-propenal). Previous studies indicate that acrolein readily reacts with thioredoxin reductase 1 (TrxR1), a critical enzyme involved in regulation of thioredoxin (Trx)-mediated redox signaling, by alkylation at its selenocysteine (Sec) residue. Because alkylation of Sec within TrxR1 has significant implications for its enzymatic function, we explored the potential importance of TrxR1 alkylation in acrolein-induced activation or injury of bronchial epithelial cells. Exposure of human bronchial epithelial HBE1 cells to acrolein (1–30 μM) resulted in dose-dependent loss of TrxR thioredoxin reductase activity, which coincided with its alkylation, as determined by biotin hydrazide labeling, and was independent of initial GSH status. To test the involvement of TrxR1 in acrolein responses in HBE1 cells, we suppressed TrxR1 using siRNA silencing or augmented TrxR1 by cell supplementation with sodium selenite. Acrolein exposure of HBE1 cells induced dose-dependent activation of the MAP kinases, extracellular regulated1 kinase (ERK), c-Jun N-terminal kinase (JNK), and p38, and activation of JNK was markedly enhanced after selenite-mediated induction of TrxR1, and was associated with increased alkylation of TrxR1. Conversely, siRNA silencing of TrxR1 significantly suppressed the ability of acrolein to activate JNK, and also appeared to attenuate acrolein-dependent activation of ERK and p38. Alteration of initial TrxR1 levels by siRNA or selenite supplementation also affected initial Trx1 redox status and acrolein-mediated alkylation of Trx1, but did not significantly affect acrolein-mediated activation of HO-1 or cytotoxicity. Collectively, our findings indicate that alkylation of TrxR1 and/or Trx1 may contribute directly to acrolein-mediated activation of MAP kinases such as JNK

  15. Focal adhesion kinase-mediated activation of glycogen synthase kinase 3β regulates IL-33 receptor internalization and IL-33 signaling.

    Science.gov (United States)

    Zhao, Jing; Wei, Jianxin; Bowser, Rachel K; Traister, Russell S; Fan, Ming-Hui; Zhao, Yutong

    2015-01-15

    IL-33, a relatively new member of the IL-1 cytokine family, plays a crucial role in allergic inflammation and acute lung injury. Long form ST2 (ST2L), the receptor for IL-33, is expressed on immune effector cells and lung epithelia and plays a critical role in triggering inflammation. We have previously shown that ST2L stability is regulated by the ubiquitin-proteasome system; however, its upstream internalization has not been studied. In this study, we demonstrate that glycogen synthase kinase 3β (GSK3β) regulates ST2L internalization and IL-33 signaling. IL-33 treatment induced ST2L internalization, and an effect was attenuated by inhibition or downregulation of GSK3β. GSK3β was found to interact with ST2L on serine residue 446 in response to IL-33 treatment. GSK3β binding site mutant (ST2L(S446A)) and phosphorylation site mutant (ST2L(S442A)) are resistant to IL-33-induced ST2L internalization. We also found that IL-33 activated focal adhesion kinase (FAK). Inhibition of FAK impaired IL-33-induced GSK3β activation and ST2L internalization. Furthermore, inhibition of ST2L internalization enhanced IL-33-induced cytokine release in lung epithelial cells. These results suggest that modulation of the ST2L internalization by FAK/GSK3β might serve as a unique strategy to lessen pulmonary inflammation. Copyright © 2015 by The American Association of Immunologists, Inc.

  16. Regulation of ERK-mediated signal transduction by p38 MAP kinase in human monocytic THP-1 cells.

    Science.gov (United States)

    Numazawa, Satoshi; Watabe, Masahiko; Nishimura, Satoshi; Kurosawa, Masahiro; Izuno, Makoto; Yoshida, Takemi

    2003-05-01

    SB 203580 has been widely used to specifically shut down the p38 MAP kinase-dependent pathway, although it is capable of inducing c-Raf kinase activity in cells. The present study demonstrates that SB 203580 activates members of the ERK cascade, c-Raf, MEK, and ERK, in human monocytic THP-1 cells. The activation of these kinases was sustained for at least 24 h after SB 203580 treatment and was also observed in U937 cells, suggesting that c-Raf efficiently transduces the signal even in the presence of the inhibitor in these cells. However, the expression of ERK cascade-dependent genes, such as c-fos and IL-1beta, was extremely limited. Analysis of the cellular distribution of ERK in SB 203580-treated cells indicated that nuclear translocation of phosphorylated ERK was impaired. Also, nuclear translocation of ERK induced by 12-O-tetradecanoyl-phorbol-13-acetate (TPA) was inhibited by SB 239063, which does not associate with c-Raf and is highly selective for p38 MAP kinase. In addition, the forced expression of the dominant negative mutant of p38 MAP kinase suppressed serum responsive element-dependent transactivation induced by TPA. These results suggest that the steady-state level of p38 MAP kinase activity modulates ERK signaling.

  17. Focal adhesion kinase, a downstream mediator of Raf-1 signaling, suppresses cellular adhesion, migration, and neuroendocrine markers in BON carcinoid cells.

    Science.gov (United States)

    Ning, Li; Chen, Herbert; Kunnimalaiyaan, Muthusamy

    2010-05-01

    We have recently reported that activation of the Raf-1/mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase 1/2 (MEK1/2)/ERK1/2 signaling cascade in gastrointestinal carcinoid cell line (BON) alters cellular morphology and neuroendocrine phenotype. The mechanisms by which Raf-1 mediates these changes in carcinoid cells are unclear. Here, we report that activation of the Raf-1 signaling cascade in BON cells induced the expression of focal adhesion kinase (FAK) protein, suppressed the production of neuroendocrine markers, and resulted in significant decreases in cellular adhesion and migration. Importantly, inactivation of MEK1/2 by 1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio]butadiene or abolition of FAK induction in Raf-1-activated BON cells by targeted siRNA led to reversal of the Raf-1-mediated reduction in neuroendocrine markers and cellular adhesion and migration. Phosphorylation site-specific antibodies detected the phosphorylated FAK(Tyr407), but not FAK(Tyr397), in these Raf-1-activated cells, indicating that FAK(Tyr407) may be associated with changes in the neuroendocrine phenotype. Overexpression of constitutively active FAK plasmids (wild-type FAK or FAK(Tyr397) mutant) into BON cells reduced neuroendocrine markers, whereas the FAK(Tyr407) mutant plasmid did not show any decrease in the levels of neuroendocrine markers, indicating that phosphorylation of FAK at the Tyr(407) residue may be important for these effects. Our results showed for the first time that FAK is an essential downstream effector of the Raf-1/MEK1/2/ERK1/2 signaling cascade and negatively regulated the neuroendocrine and metastatic phenotype in BON cells. (c)2010 AACR.

  18. Src tyrosine kinase mediates platelet-derived growth factor BB-induced and redox-dependent migration in metanephric mesenchymal cells

    Science.gov (United States)

    Gorin, Yves

    2013-01-01

    The adult kidney is derived from the interaction between the metanephric blastema and the ureteric bud. Platelet-derived growth factor (PDGF) receptor β is essential for the development of the mature glomerular tuft, as mice deficient for this receptor lack mesangial cells. This study investigated the role of Src tyrosine kinase in PDGF-mediated reactive oxygen species (ROS) generation and migration of metanephric mesenchymal cells (MMCs). Cultured embryonic MMCs from wild-type and PDGF receptor-deficient embryos were established. Migration was determined via wound-healing assay. Unlike PDGF AA, PDGF BB-induced greater migration in MMCs with respect to control. This was abrogated by neutralizing an antibody to PDGF BB. Phosphatidylinositol 3-kinase (PI3K) inhibitors suppressed PDGF BB-induced migration. Conversely, mitogen-activated protein kinase/extracellular signal-regulated kinase (MEK) inhibitors had no effect. Src inhibitors inhibited PDGF-induced cell migration, PI3K activity, and Akt phosphorylation. Adenoviral dominant negative Src (AD DN Src) abrogated PDGF BB-induced Akt phosphorylation. Hydrogen peroxide stimulated cell migration. PDGF BB-induced wound closure was inhibited by the antioxidants N-acetyl-l-cysteine, tiron, and the flavoprotein inhibitor diphenyleneiodonium. These cells express the NADPH oxidase homolog Nox4. Inhibiting Nox4 with antisense oligonucleotides or small interfering RNA (siRNA) suppressed PDGF-induced wound closure. Inhibition of Src with siRNA reduced PDGF BB-induced ROS generation as assessed by 2′,7′-dichlorodihydrofluorescein diacetate fluorescence. Furthermore, PDGF BB-stimulated ROS generation and migration were similarly suppressed by Ad DN Src. In MMCs, PDGF BB-induced migration is mediated by PI3K and Src in a redox-dependent manner involving Nox4. Src may be upstream to PI3K and Nox4. PMID:24197068

  19. Sustained activation of the AKT/mTOR and MAP kinase pathways mediate resistance to the Src inhibitor, dasatinib, in thyroid cancer.

    Science.gov (United States)

    Mishall, Katie M; Beadnell, Thomas C; Kuenzi, Brent M; Klimczak, Dorothy M; Superti-Furga, Giulio; Rix, Uwe; Schweppe, Rebecca E

    2017-11-28

    New targeted therapies are needed for advanced thyroid cancer. Our lab has shown that Src is a key mediator of tumorigenic processes in thyroid cancer. However, single-agent Src inhibitors have had limited efficacy in solid tumors. In order to more effectively target Src in the clinic, our lab has previously generated four thyroid cancer cell lines that are resistant to dasatinib through gradual dose escalation. We further tested two additional Src inhibitors and shown the dasatinib-resistant (DasRes) cells exhibit cross-resistance to saracatinib, but are sensitive to bosutinib, suggesting that unique off-targets of bosutinib play an important role in mediating sensitivity to bosutinib. To identify the kinases targeted by dasatinib and bosutinib, we utilized an unbiased compound centric chemical proteomics screen. We identified 33 kinases that were enriched in the bosutinib pull down. Using the STRING database to map protein-protein interactions of the unique bosutinib targets, we identified a signaling axis which included mTOR, FAK, and MEK. Inhibition of the mTOR, MEK, and Src/FAK nodes simultaneously was the most effective at reducing cell growth and survival. Overall, these studies have identified key mediators of Src inhibitor resistance, and show that targeting these signaling nodes are necessary for anti-tumor efficacy.

  20. Prostaglandin E2 and the protein kinase A pathway mediate arachidonic acid induction of c-fos in human prostate cancer cells

    Science.gov (United States)

    Chen, Y.; Hughes-Fulford, M.

    2000-01-01

    Arachidonic acid (AA) is the precursor for prostaglandin E2 (PGE2) synthesis and increases growth of prostate cancer cells. To further elucidate the mechanisms involved in AA-induced prostate cell growth, induction of c-fos expression by AA was investigated in a human prostate cancer cell line, PC-3. c-fos mRNA was induced shortly after addition of AA, along with a remarkable increase in PGE2 production. c-fos expression and PGE2 production induced by AA was blocked by a cyclo-oxygenase inhibitor, flurbiprofen, suggesting that PGE2 mediated c-fos induction. Protein kinase A (PKA) inhibitor H-89 abolished induction of c-fos expression by AA, and partially inhibited PGE2 production. Protein kinase C (PKC) inhibitor GF109203X had no significant effect on c-fos expression or PGE2 production. Expression of prostaglandin (EP) receptors, which mediate signal transduction from PGE2 to the cells, was examined by reverse transcription polymerase chain reaction in several human prostate cell lines. EP4 and EP2, which are coupled to the PKA signalling pathway, were expressed in all cells tested. Expression of EP1, which activates the PKC pathway, was not detected. The current study showed that induction of the immediate early gene c-fos by AA is mediated by PGE2, which activates the PKA pathway via the EP2/4 receptor in the PC-3 cells.

  1. Herpes simplex virus internalization into epithelial cells requires Na+/H+ exchangers and p21-activated kinases but neither clathrin- nor caveolin-mediated endocytosis.

    Science.gov (United States)

    Devadas, Deepika; Koithan, Thalea; Diestel, Randi; Prank, Ute; Sodeik, Beate; Döhner, Katinka

    2014-11-01

    Herpes simplex virus 1 (HSV-1) is an alphaherpesvirus that has been reported to infect some epithelial cell types by fusion at the plasma membrane but others by endocytosis. To determine the molecular mechanisms of productive HSV-1 cell entry, we perturbed key endocytosis host factors using specific inhibitors, RNA interference (RNAi), or overexpression of dominant negative proteins and investigated their effects on HSV-1 infection in the permissive epithelial cell lines Vero, HeLa, HEp-2, and PtK2. HSV-1 internalization required neither endosomal acidification nor clathrin- or caveolin-mediated endocytosis. In contrast, HSV-1 gene expression and internalization were significantly reduced after treatment with 5-(N-ethyl-N-isopropyl)amiloride (EIPA). EIPA blocks the activity of Na(+)/H(+) exchangers, which are plasma membrane proteins implicated in all forms of macropinocytosis. HSV-1 internalization furthermore required the function of p21-activated kinases that contribute to macropinosome formation. However, in contrast to some forms of macropinocytosis, HSV-1 did not enlist the activities of protein kinase C (PKC), tyrosine kinases, C-terminal binding protein 1, or dynamin to activate its internalization. These data suggest that HSV-1 depends on Na(+)/H(+) exchangers and p21-activated kinases either for macropinocytosis or for local actin rearrangements required for fusion at the plasma membrane or subsequent passage through the actin cortex underneath the plasma membrane. After initial replication in epithelial cells, herpes simplex viruses (HSVs) establish latent infections in neurons innervating these regions. Upon primary infection and reactivation from latency, HSVs cause many human skin and neurological diseases, particularly in immunocompromised hosts, despite the availability of effective antiviral drugs. Many viruses use macropinocytosis for virus internalization, and many host factors mediating this entry route have been identified, although the

  2. Vav3 modulates B cell receptor responses by regulating phosphoinositide 3-kinase activation.

    Science.gov (United States)

    Inabe, Kazunori; Ishiai, Masamichi; Scharenberg, Andrew M; Freshney, Norman; Downward, Julian; Kurosaki, Tomohiro

    2002-01-21

    To elucidate the mechanism(s) by which Vav3, a new member of the Vav family proteins, participates in B cell antigen receptor (BCR) signaling, we have generated a B cell line deficient in Vav3. Here we report that Vav3 influences phosphoinositide 3-kinase (PI3K) function through Rac1 in that phosphatidylinositol-3,4,5-trisphosphate (PIP3) generation was attenuated by loss of Vav3 or by expression of a dominant negative form of Rac1. The functional interaction between PI3K and Rac1 was also demonstrated by increased PI3K activity in the presence of GTP-bound Rac1. In addition, we show that defects of calcium mobilization and c-Jun NH2-terminal kinase (JNK) activation in Vav3-deficient cells are relieved by deletion of a PIP3 hydrolyzing enzyme, SH2 domain-containing inositol polyphosphate 5'-phosphatase (SHIP). Hence, our results suggest a role for Vav3 in regulating the B cell responses by promoting the sustained production of PIP3 and thereby calcium flux.

  3. Vav3 Modulates B Cell Receptor Responses by Regulating Phosphoinositide 3-Kinase Activation

    Science.gov (United States)

    Inabe, Kazunori; Ishiai, Masamichi; Scharenberg, Andrew M.; Freshney, Norman; Downward, Julian; Kurosaki, Tomohiro

    2002-01-01

    To elucidate the mechanism(s) by which Vav3, a new member of the Vav family proteins, participates in B cell antigen receptor (BCR) signaling, we have generated a B cell line deficient in Vav3. Here we report that Vav3 influences phosphoinositide 3-kinase (PI3K) function through Rac1 in that phosphatidylinositol-3,4,5-trisphosphate (PIP3) generation was attenuated by loss of Vav3 or by expression of a dominant negative form of Rac1. The functional interaction between PI3K and Rac1 was also demonstrated by increased PI3K activity in the presence of GTP-bound Rac1. In addition, we show that defects of calcium mobilization and c-Jun NH2-terminal kinase (JNK) activation in Vav3-deficient cells are relieved by deletion of a PIP3 hydrolyzing enzyme, SH2 domain-containing inositol polyphosphate 5′-phosphatase (SHIP). Hence, our results suggest a role for Vav3 in regulating the B cell responses by promoting the sustained production of PIP3 and thereby calcium flux. PMID:11805146

  4. AMP-activated protein kinase mediates T cell activation-induced expression of FasL and COX-2 via protein kinase C theta-dependent pathway in human Jurkat T leukemia cells.

    Science.gov (United States)

    Lee, Jung Yeon; Choi, A-Young; Oh, Young Taek; Choe, Wonchae; Yeo, Eui-Ju; Ha, Joohun; Kang, Insug

    2012-06-01

    AMP-activated protein kinase (AMPK), an important regulator of energy homeostasis, is known to be activated during T cell activation. T cell activation by T cell receptor (TCR) engagement or its pharmacological mimics, PMA plus ionomycin (PMA/Io), induces immunomodulatory FasL and cyclooxygenase-2 (COX-2) expression. In this study, we examined the role and mechanisms of AMPK in PMA/Io-induced expression of FasL and COX-2 in Jurkat T human leukemic cells. Inhibition of AMPK by a pharmacological agent, compound C, or AMPKα1 siRNA suppressed expression of FasL and COX-2 mRNAs and proteins in PMA/Io-activated Jurkat cells. It also reduced secretion of FasL protein and prostaglandin E2, a main product of COX-2, in Jurkat cells and peripheral blood lymphocytes activated with PMA/Io or monoclonal anti-CD3 plus anti-CD28. Consistently, inhibition of AMPK blocked promoter activities of FasL and COX-2 in activated Jurkat cells. As protein kinase C theta (PKCθ) is a central molecule for TCR signaling, we examined any possible cross-talk between AMPK and PKCθ in activated T cells. Of particular importance, we found that inhibition of AMPK blocked phosphorylation and activation of PKCθ, suggesting that AMPK is an upstream kinase of PKCθ. Moreover, we showed that AMPK was directly associated with PKCθ and phosphorylated Thr538 of PKCθ in PMA/Io-stimulated Jurkat cells. We also showed that inhibition of PKCθ by rottlerin or dominant negative PKCθ reduced AMPK-mediated transcriptional activation of NF-AT and AP-1 in activated Jurkat cells. Taken together, these results suggest that AMPK regulates expression of FasL and COX-2 via the PKCθ and NF-AT and AP-1 pathways in activated Jurkat cells. Copyright © 2012 Elsevier Inc. All rights reserved.

  5. The Botrytis cinerea PAK kinase BcCla4 mediates morphogenesis, growth and cell cycle regulating processes downstream of BcRac.

    Science.gov (United States)

    Minz-Dub, Anna; Sharon, Amir

    2017-05-01

    Rac proteins are involved in a variety of cellular processes. Effector proteins that interact with active Rac convey the GTPase-generated signal to downstream developmental cascades and processes. Here we report on the analysis of the main effector and signal cascade downstream of BcRac, the Rac homolog of the grey mold fungus Botrytis cinerea. Several lines of evidence highlighted the p21-activated kinase Cla4 as an important effector of Rac in fungi. Analysis of Δbccla4 strains revealed that the BcCla4 protein was sufficient to mediate all of the examined BcRac-driven processes, including hyphal growth and morphogenesis, conidia production and pathogenicity. In addition, the Δbccla4 strains had altered nuclei content, a phenomenon that was previously observed in Δbcrac isolates, thus connecting the BcRac/BcCla4 module with cell cycle control. Further analyses revealed that BcRac/BcCla4 control mitotic entry through changes in phosphorylation status of the cyclin dependent kinase BcCdk1. The complete cascade includes the kinase BcWee1, which is downstream of BcCla4 and upstream of BcCdk1. These results provide a mechanistic insight on the connection of cell cycle, morphogenesis and pathogenicity in fungi, and position BcCla4 as the most essential effector and central regulator of all of these processes downstream of BcRac. © 2017 John Wiley & Sons Ltd.

  6. Induction of protection against paraquat-induced oxidative damage by abscisic acid in maize leaves is mediated through mitogen-activated protein kinase.

    Science.gov (United States)

    Ding, Hai-Dong; Zhang, Xiao-Hua; Xu, Shu-Cheng; Sun, Li-Li; Jiang, Ming-Yi; Zhang, A-Ying; Jin, Yin-Gen

    2009-10-01

    Mitogen-activated protein kinase (MAPK) cascade has been shown to be important components in stress signal transduction pathway. In the present study, protection of maize seedlings (Zea mays L.) against paraquat-generated oxidative toxicity by abscisic acid (ABA), its association with MAPK and ZmMPK5, a candidate for MAPK were investigated. Treatment of maize leaves with exogenous ABA led to significant decreases in the content of malondialdehyde, the percentage of ion leakage and the level of protein oxidation (in terms of carbonyl groups) under paraquat (PQ) stress. However, such decreases were blocked by the pretreatment with two MAPK kinase inhibitors PD98059 and U0126. The damage caused by PQ was further aggravated by inhibitors. Two inhibitors also suppressed the total activities of the antioxidant enzymes superoxide dismutase (SOD, EC 1.15.1.1), catalase (CAT, EC 1.11.1.6), ascorbate peroxidase (APX, EC 1.11.1.11), and glutathione reductase (GR, EC 1.6.4.2). Besides, treatment with PQ stimulated the activation of a 46 kDa MAPK, which was identified as ZmMPK5 by in-gel kinase assay with immunoprecipitation. These results reveal that ABA-induced protection against PQ-generated oxidative damage is mediated through MAPK cascade in maize leaves, in which ZmMPK5, a candidate for MAPK, is demonstrated to be involved.

  7. p38 mitogen-activated protein kinase is involved in arginase-II-mediated eNOS-uncoupling in obesity.

    Science.gov (United States)

    Yu, Yi; Rajapakse, Angana G; Montani, Jean-Pierre; Yang, Zhihong; Ming, Xiu-Fen

    2014-07-18

    Endothelial nitric oxide synthase (eNOS)-uncoupling links obesity-associated insulin resistance and type-II diabetes to the increased incidence of cardiovascular disease. Studies have indicated that increased arginase is involved in eNOS-uncoupling through competing with the substrate L-arginine. Given that arginase-II (Arg-II) exerts some of its biological functions through crosstalk with signal transduction pathways, and that p38 mitogen-activated protein kinase (p38mapk) is involved in eNOS-uncoupling, we investigated here whether p38mapk is involved in Arg-II-mediated eNOS-uncoupling in a high fat diet (HFD)-induced obesity mouse model. Obesity was induced in wild type (WT) and Arg-II-deficient (Arg-II(-/-)) mice on C57BL/6 J background by high-fat diet (HFD, 55% fat) for 14 weeks starting from age of 7 weeks. The entire aortas were isolated and subjected to 1) immunoblotting analysis of the protein level of eNOS, Arg-II and p38mapk activation; 2) arginase activity assay; 3) endothelium-dependent and independent vasomotor responses; 4) en face staining of superoxide anion and NO production with Dihydroethidium and 4,5-Diaminofluorescein Diacetate, respectively, to assess eNOS-uncoupling. To evaluate the role of p38mapk, isolated aortas were treated with p38mapk inhibitor SB203580 (10 μmol/L, 1 h) prior to the analysis. In addition, the role of p38mapk in Arg-II-induced eNOS-uncoupling was investigated in cultured human endothelial cells overexpressing Arg-II in the absence or presence of shRNA against p38mapk. HFD enhanced Arg-II expression/activity and p38mapk activity, which was associated with eNOS-uncoupling as revealed by decreased NO and enhanced L-NAME-inhibitable superoxide in aortas of WT obese mice. In accordance, WT obese mice revealed decreased endothelium-dependent relaxations to acetylcholine despite of higher eNOS protein level, whereas Arg-II(-/-) obese mice were protected from HFD-induced eNOS-uncoupling and endothelial dysfunction, which

  8. rse, a novel receptor-type tyrosine kinase with homology to Axl/Ufo, is expressed at high levels in the brain.

    Science.gov (United States)

    Mark, M R; Scadden, D T; Wang, Z; Gu, Q; Goddard, A; Godowski, P J

    1994-04-08

    We have isolated cDNA clones that encode the human and murine forms of a novel receptor-type tyrosine kinase termed Rse. Sequence analysis indicates that human Rse contains 890 amino acids, with an extracellular region composed of two immunoglobulin-like domains followed by two fibronectin type III domains. Murine Rse contains 880 amino acids and shares 90% amino acid identity with its human counterpart. Rse is structurally similar to the receptor-type tyrosine kinase Axl/Ufo, and the two proteins have 35 and 63% sequence identity in their extracellular and intracellular domains, respectively. To study the synthesis and activation of this putative receptor-type tyrosine kinase, we constructed a version of Rse (termed gD-Rse, where gD represents glycoprotein D) that contains an NH2-terminal epitope tag. NIH3T3 cells were engineered to express gD-Rse, which could be detected at the cell surface by fluorescence-activated cell sorting. Moreover, gD-Rse was rapidly phosphorylated on tyrosine residues upon incubation of the cells with an antibody directed against the epitope tag, suggesting that rse encodes an active tyrosine kinase. In the human tissues we examined, the highest level of expression of rse mRNA was observed in the brain; rse mRNA was also detected in the premegakaryocytopoietic cell lines CMK11-5 and Dami. The gene for rse was localized to human chromosome 15.

  9. Signaling Rho-kinase mediates inflammation and apoptosis in T cells and renal tubules in cisplatin nephrotoxicity.

    Science.gov (United States)

    Nozaki, Yuji; Kinoshita, Koji; Hino, Shoichi; Yano, Tomohiro; Niki, Kaoru; Hirooka, Yasuaki; Kishimoto, Kazuya; Funauchi, Masanori; Matsumura, Itaru

    2015-04-15

    Nephrotoxicity is a frequent complication of cisplatin-induced chemotherapy, in which T cells are known to promote acute kidney injury (AKI). Apoptosis and necrosis of tubules and inflammatory events also contribute to the nephrotoxicity. A delineation of the mechanisms that underlie the inappropriate renal and tubular inflammation can thus provide important insights into potential therapies for cisplatin-induced AKI. Rho-kinases are known to act as molecular switches controlling several critical cellular functions, including cell migration, cytokine production, and apoptosis. Here, we show that the Rho-kinase inhibitor fasudil attenuated cisplatin nephrotoxicity, resulting in less histological damage, improved renal function, and the infiltration of fewer leukocytes into the kidney. Renal nuclear factor-κB activation and apoptosis were reduced, and the expressions of proinflammatory renal cytokine and chemokine mRNA were decreased. Urinary and renal kidney injury molecule-1 (Kim-1) expression was also reduced, a finding that is consistent with diminished kidney injury. In the current study, we also showed that fasudil could be protective of the impaired tubules. In vitro, fasudil reduced the apoptosis (annexin-V+PI cells) and cytokine production (tumor necrosis factor+ cells) in T cells and the apoptosis (annexin-V+PI cells) and tubular damage (Kim-1+ cells) in proximal tubular cells by flow cytometric analysis. As Rho-kinase plays an important role in promoting cisplatin nephrotoxicity, inhibiting Rho-kinase may be a therapeutic strategy for preventing cisplatin-induced AKI. Copyright © 2015 the American Physiological Society.

  10. A phosphoserine/threonine-binding pocket in AGC kinases and PDK1 mediates activation by hydrophobic motif phosphorylation

    DEFF Research Database (Denmark)

    Frödin, Morten; Antal, Torben L; Dümmler, Bettina A

    2002-01-01

    docking site that recruits and activates PDK1, which then phosphorylates the activation loop. Here, we discover a pocket in the kinase domain of PDK1 that recognizes the phosphoserine/phosphothreonine in the hydrophobic motif by identifying two oppositely positioned arginine and lysine residues that bind...

  11. A novel hybrid kinase is essential for regulating the σB-mediated stress response of Bacillus cereus.

    NARCIS (Netherlands)

    Been, de M.W.H.J.; Tempelaars, Marcel; Schaik, van W.; Moezelaar, Roy; Siezen, R.J.; Abee, Tjakko

    2009-01-01

    A common bacterial strategy for monitoring environmental challenges is to use two-component systems, which consist of a sensor histidine kinase (HK) and a response regulator (RR). In the food-borne pathogen Bacillus cereus, the alternative sigma factor σB is activated by the RR RsbY. Here we present

  12. A novel hybrid kinase is essential for regulating the sigmaB-mediated stress response of Bacillus cereus

    NARCIS (Netherlands)

    Been, de M.W.H.J.; Tempelaars, M.H.; Schaik, van W.; Moezelaar, R.; Siezen, R.J.; Abee, T.

    2010-01-01

    A common bacterial strategy for monitoring environmental challenges is to use two-component systems, which consist of a sensor histidine kinase (HK) and a response regulator (RR). In the food-borne pathogen Bacillus cereus, the alternative sigma factor sB is activated by the RR RsbY. Here we present

  13. Investigation of the Role of Breast Tumor Kinase (Brk) in ERK5 and p38-Mediated Breast Cancer Cell

    National Research Council Canada - National Science Library

    Lofgren, Kristopher

    2007-01-01

    ...). Little is known about the role of Brk in breast cancer. Data from our lab has shown that shRNA knockdown of endogenous Brk in T47D breast cancer cells blocked ERK5 and p38 mitogen activated protein kinase (MAPK...

  14. Use of double-stranded RNA-mediated interference to determine the substrates of protein tyrosine kinases and phosphatases.

    Science.gov (United States)

    Muda, Marco; Worby, Carolyn A; Simonson-Leff, Nancy; Clemens, James C; Dixon, Jack E

    2002-08-15

    Despite the wealth of information generated by genome-sequencing projects, the identification of in vivo substrates of specific protein kinases and phosphatases is hampered by the large number of candidate enzymes, overlapping enzyme specificity and sequence similarity. In the present study, we demonstrate the power of RNA interference (RNAi) to dissect signal transduction cascades involving specific kinases and phosphatases. RNAi is used to identify the cellular tyrosine kinases upstream of the phosphorylation of Down-Syndrome cell-adhesion molecule (Dscam), a novel cell-surface molecule of the immunoglobulin-fibronectin super family, which has been shown to be important for axonal path-finding in Drosophila. Tyrosine phosphorylation of Dscam recruits the Src homology 2 domain of the adaptor protein Dock to the receptor. Dock, the ortho- logue of mammalian Nck, is also essential for correct axonal path-finding in Drosophila. We further determined that Dock is tyrosine-phosphorylated in vivo and identified DPTP61F as the protein tyrosine phosphatase responsible for maintaining Dock in its non-phosphorylated state. The present study illustrates the versatility of RNAi in the identification of the physiological substrates for protein kinases and phosphatases.

  15. Mammary gland-specific ablation of focal adhesion kinase reduces the incidence of p53-mediated mammary tumour formation.

    NARCIS (Netherlands)

    Miltenburg, van M.H.; Nimwegen, van M.J.; Tijdens, R.B.; Lalai, R.A.; Kuiper, R.; Klarenbeek, S.; Schouten, P.C.; Vries, de A.; Jonkers, J.M.M.; Water, van de B.

    2014-01-01

    BACKGROUND Elevated expression of focal adhesion kinase (FAK) occurs in numerous human cancers including colon-, cervix- and breast cancer. Although several studies have implicated FAK in mammary tumour formation induced by ectopic oncogene expression, evidence supporting a role for FAK in

  16. Sodium butyrate-mediated differentiation of colorectal cancer cells: regulation of PKC-betaII by PI3-kinase

    Czech Academy of Sciences Publication Activity Database

    Turečková, Jolana; Vojtěchová, Martina; Kučerová, Dana; Velek, Jiří; Tuháčková, Zdena

    2005-01-01

    Roč. 15, č. 2 (2005), s. 329-335 ISSN 1107-3756 R&D Projects: GA ČR(CZ) GP301/02/D159; GA AV ČR(CZ) KSK5020115 Keywords : phosphatidylinositol 3-kinase * PKCbetaII * adenocarcinoma Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.090, year: 2005

  17. Analysis of IRS-1-mediated phosphatidylinositol 3-kinase activation in the adipose tissue of polycystic ovary syndrome patients complicated with insulin resistance

    International Nuclear Information System (INIS)

    Chu Yongli; Qiu Hongyu; Sun Yongyu; Li Min; Li Hongfa

    2004-01-01

    Objective: To investigate the insulin receptor substance-1 (IRS-1)-mediated phosphatidylinositol-3 (PI-3) kinase activity in adipose tissue of polycystic ovary syndrome (PCOS) patients, and to explore molecular mechanisms of insulin resistance of PCOS. Methods: Blood and adipose tissue samples from patients with PCOS with insulin resistance (n=19), PCOS without insulin resistance (n=10) and controls (n=15) were collected. Serum luteinizing hormone (LH), follicle stimulating hormone (FSH), testosterone (T) were measured by chemiluminescence assay. Fasting insulin (FIN) was measured by radioimmunoassay. Fasting plasma glucose (FPG) was measured by oxidase assay. Insulin resistance index (IR) was calculated using homeostasis model assessment (HOMA) to analyze the relationship between these markers and insulin resistance. The tyrosine phosphorylation of IRS-1 was measured by immunoprecipitation and enhanced chemiluminescent immunoblotting technique. PI-3 kinase activity was detected by immunoprecipitation, thin-layer chromatography and gamma scintillation counting. The results were analyzed by statistical methods. Results: 1) The levels of serum LH, LH/FSH, T, FIN and HOMA-IR in PCOS without insulin resistance were significantly higher than those of control group (all P<0.05); the levels of serum LH, LH/FSH, T, FIN and HOMA-IR in PCOS with insulin resistance were significantly higher than those of PCOS without insulin resistance (all P<0.05). 2) The tyrosine phosphorylation analysis of IRS-1 showed that IRS-1 tyrosine phosphorylation was significantly decreased in PCOS with insulin resistance compared to that of PCOS without insulin resistance and control groups (P<0.01). 3) PI-3 kinase activity was significantly decreased (P<0.01) and negatively correlated with HOMA-IR. Conclusion: In consequence of the weaker signal caused by the change of upper stream signal molecule IRS-1 tyrosine phosphorylation, PI-3 kinase activity decreased, it affects the insulin signal

  18. Cyclin-dependent kinase 5-mediated phosphorylation of CHIP promotes the tAIF-dependent death pathway in rotenone-treated cortical neurons.

    Science.gov (United States)

    Kim, Chiho; Lee, Juhyung; Ko, Yeon Uk; Oh, Young J

    2018-01-01

    Cyclin-dependent kinase 5 (Cdk5) is a proline-directed serine/threonine kinase. Its dysregulation has been implicated in various neurodegenerative diseases. We previously reported that phosphorylation of the C-terminus of the Hsc70-interacting protein (CHIP) by Cdk5 promotes truncated apoptosis-inducing factor (tAIF)-mediated neuronal death induced by oxidative stress. Here, we determined whether this Cdk5-dependent cell death signaling pathway is present in experimental models of Parkinson's disease. First, we showed that rotenone activates Cdk5 in primary cultures of cortical neurons and causes tAIF-dependent neuronal cell death. This event was attenuated by negative regulation of endogenous Cdk5 activity by the pharmacological Cdk5 inhibitor, roscovitine, or by lentiviral knockdown of Cdk5. Cdk5 phosphorylates CHIP at Ser20 in rotenone-treated neurons. Consequently, overexpression of CHIP S20A , but not CHIP WT , attenuates tAIF-induced cell death in rotenone-treated cortical neurons. Taken together, these results indicate that phosphorylation of CHIP at Ser20 by Cdk5 activation inhibits CHIP-mediated tAIF degradation, thereby contributing to tAIF-induced neuronal cell death following rotenone treatment. Copyright © 2017 Elsevier B.V. All rights reserved.

  19. A role for protein kinase PKR in the mediation of Epstein-Barr virus latent membrane protein-1-induced IL-6 and IL-10 expression.

    Science.gov (United States)

    Lin, San San; Lee, Davy C W; Law, Anna H Y; Fang, Jun Wei; Chua, Daniel T T; Lau, Allan S Y

    2010-05-01

    Expression of Epstein-Barr virus-encoded oncogenic latent membrane protein 1 (LMP1) has been substantially associated with tumorigenic transformation in the virus-infected cells. The pathogenic complexity of LMP1 is partly due to the cytokine dysregulation including IL-6 and IL-10 in perturbing the host immune responses. Here we have identified an important signaling event mediated by a dsRNA-dependent serine/threonine protein kinase, PKR, in regulating LMP1-induced IL-6 and IL-10 expression. We first demonstrated that PKR plays a significant role in mediating LMP1-induced cytokine expression by using a PKR inhibitor 2-aminopurine, and the specific role of PKR involved was confirmed by the use of siRNA oligos targeting PKR and/or a dominant-negative PKR mutant. We next revealed that PKR activity mediates LMP1-enhanced NF-kappaB nuclear translocation resulting in cytokine induction. We further demonstrated at the chromatin level that LMP1 can significantly elevate the phosphorylation of histone H3 on serine 10 (Ser 10), and the process was dependent on PKR activity. Our findings thus suggest that PKR plays an important role in mediating the cytokine gene expression induced by LMP1 through NF-kappaB activation and histone H3 Ser 10 phosphorylation. Copyright (c) 2010 Elsevier Ltd. All rights reserved.

  20. Receptor protein tyrosine phosphatase alpha activates Src-family kinases and controls integrin-mediated responses in fibroblasts

    DEFF Research Database (Denmark)

    Su, J; Muranjan, M; Sap, J

    1999-01-01

    BACKGROUND: Fyn and c-Src are two of the most widely expressed Src-family kinases. Both are strongly implicated in the control of cytoskeletal organization and in the generation of integrin-dependent signalling responses in fibroblasts. These proteins are representative of a large family of tyros......BACKGROUND: Fyn and c-Src are two of the most widely expressed Src-family kinases. Both are strongly implicated in the control of cytoskeletal organization and in the generation of integrin-dependent signalling responses in fibroblasts. These proteins are representative of a large family...... established, no corresponding phosphatases have been identified that, under physiological conditions, function as positive regulators of c-Src and Fyn in fibroblasts. RESULTS: Receptor protein tyrosine phosphatase alpha (RPTPalpha) was inactivated by homologous recombination. Fibroblasts derived from...

  1. A cross-talk between TrkB and Ret tyrosine kinases receptors mediates neuroblastoma cells differentiation.

    Directory of Open Access Journals (Sweden)

    Carla Lucia Esposito

    Full Text Available Understanding the interplay between intracellular signals initiated by multiple receptor tyrosine kinases (RTKs to give the final cell phenotype is a major pharmacological challenge. Retinoic acid (RA-treatment of neuroblastoma (NB cells implicates activation of Ret and TrkB RTKs as critical step to induce cell differentiation. By studying the signaling interplay between TrkB and Ret as paradigmatic example, here we demonstrate the existence of a cross-talk mechanism between the two unrelated receptors that is needed to induce the cell differentiation. Indeed, we show that TrkB receptor promotes Ret phosphorylation by a mechanism that does not require GDNF. This reveals to be a key mechanism, since blocking either TrkB or Ret by small interfering RNA causes a failure in NB biochemical and morphological differentiation. Our results provide the first evidence that a functional transactivation between distinct tyrosine kinases receptors is required for an important physiological process.

  2. Activation of AMP-Activated Protein Kinase α and Extracelluar Signal-Regulated Kinase Mediates CB-PIC-Induced Apoptosis in Hypoxic SW620 Colorectal Cancer Cells

    Directory of Open Access Journals (Sweden)

    Sung-Yun Cho

    2013-01-01

    Full Text Available Here, antitumor mechanism of cinnamaldehyde derivative CB-PIC was elucidated in human SW620 colon cancer cells. CB-PIC significantly exerted cytotoxicity, increased sub-G1 accumulation, and cleaved PARP with apoptotic features, while it enhanced the phosphorylation of AMPK alpha and ACC as well as activated the ERK in hypoxic SW620 cells. Furthermore, CB-PIC suppressed the expression of HIF1 alpha, Akt, and mTOR and activated the AMPK phosphorylation in hypoxic SW620 cells. Conversely, silencing of AMPKα blocked PARP cleavage and ERK activation induced by CB-PIC, while ERK inhibitor PD 98059 attenuated the phosphorylation of AMPKα in hypoxic SW620 cells, implying cross-talk between ERK and AMPKα. Furthermore, cotreatment of CB-PIC and metformin enhanced the inhibition of HIF1α and Akt/mTOR and the activation of AMPKα and pACC in hypoxic SW620 cells. In addition, CB-PIC suppressed the growth of SW620 cells inoculated in BALB/c athymic nude mice, and immunohistochemistry revealed that CB-PIC treatment attenuated the expression of Ki-67, CD34, and CAIX and increased the expression of pAMPKα in CB-PIC-treated group. Interestingly, CP-PIC showed better antitumor activity in SW620 colon cancer cells under hypoxia than under normoxia, since it may be applied to chemoresistance. Overall, our findings suggest that activation of AMPKα and ERK mediates CB-PIC-induced apoptosis in hypoxic SW620 colon cancer cells.

  3. Basal and Activated Calcium Sensitization Mediated by RhoA/Rho Kinase Pathway in Rats with Genetic and Salt Hypertension

    Czech Academy of Sciences Publication Activity Database

    Behuliak, Michal; Bencze, Michal; Vaněčková, Ivana; Kuneš, Jaroslav; Zicha, Josef

    2017-01-01

    Roč. 2017, January (2017), č. článku 8029728. ISSN 2314-6133 R&D Projects: GA ČR(CZ) GP14-16225P; GA MZd(CZ) NV15-25396A Institutional support: RVO:67985823 Keywords : calcium sensitization * RhoA/Rho kinase * fasudil * calcium influx * nifedipine * BAY K8644 Subject RIV: FA - Cardiovascular Diseases incl. Cardiotharic Surgery OBOR OECD: Cardiac and Cardiovascular systems Impact factor: 2.476, year: 2016

  4. Dibenzoylmethane exerts metabolic activity through regulation of AMP-activated protein kinase (AMPK-mediated glucose uptake and adipogenesis pathways.

    Directory of Open Access Journals (Sweden)

    Nami Kim

    Full Text Available Dibenzoylmethane (DBM has been shown to exert a variety of beneficial effects on human health. However, the mechanism of action is poorly understood. In this study, DBM increased phosphorylation of AMP-activated protein kinase (AMPK and stimulated glucose uptake in a skeletal muscle cell line. Both knockdown of AMPK with siRNA and inhibition with AMPK inhibitor blocked DBM-induced glucose uptake. DBM increased the concentration of intracellular calcium and glucose uptake due to DBM was abolished by STO-609 (a calcium/calmodulin-dependent protein kinase inhibitor. DBM stimulated phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK, which was blocked by pretreatment with compound C, an AMPK inhibitor. The expression of glucose transporter type 4 (GLUT4 was increased by DBM. The translocation of GLUT4 to the plasma membrane was also increased by DBM in AMPK dependently. In addition, DBM suppressed weight gain and prevented fat accumulation in the liver and abdomen in mice fed a high-fat diet. In pre-adipocyte cells, DBM decreased the activity of acetyl-CoA carboxylase (ACC, the rate-limiting enzyme of fatty acid synthesis. Expression of the adipogenic gene, fatty acid synthase (FAS, was suppressed by DBM in an AMPK-dependent manner. These results showed that the beneficial metabolic effects of DBM might be due to regulation of glucose uptake via AMPK in skeletal muscle and inhibition of adipogenesis in pre-adipocytes.

  5. Opa+ Neisseria gonorrhoeae exhibits reduced survival in human neutrophils via Src family kinase-mediated bacterial trafficking into mature phagolysosomes.

    Science.gov (United States)

    Johnson, M Brittany; Ball, Louise M; Daily, Kylene P; Martin, Jennifer N; Columbus, Linda; Criss, Alison K

    2015-05-01

    During gonorrhoeal infection, there is a heterogeneous population of Neisseria gonorrhoeae (Gc) varied in their expression of opacity-associated (Opa) proteins. While Opa proteins are important for bacterial attachment and invasion of epithelial cells, Opa+ Gc has a survival defect after exposure to neutrophils. Here, we use constitutively Opa- and OpaD+ Gc in strain background FA1090 to show that Opa+ Gc is more sensitive to killing inside adherent, chemokine-treated primary human neutrophils due to increased bacterial residence in mature, degradative phagolysosomes that contain primary and secondary granule antimicrobial contents. Although Opa+ Gc stimulates a potent oxidative burst, neutrophil killing of Opa+ Gc was instead attributable to non-oxidative components, particularly neutrophil proteases and the bactericidal/permeability-increasing protein. Blocking interaction of Opa+ Gc with carcinoembryonic antigen-related cell adhesion molecules (CEACAMs) or inhibiting Src family kinase signalling, which is downstream of CEACAM activation, enhanced the survival of Opa+ Gc in neutrophils. Src family kinase signalling was required for fusion of Gc phagosomes with primary granules to generate mature phagolysosomes. Conversely, ectopic activation of Src family kinases or coinfection with Opa+ Gc resulted in decreased survival of Opa- Gc in neutrophils. From these results, we conclude that Opa protein expression is an important modulator of Gc survival characteristics in neutrophils by influencing phagosome dynamics and thus bacterial exposure to neutrophils' full antimicrobial arsenal. © 2014 John Wiley & Sons Ltd.

  6. Nitric oxide mediates the indole acetic acid induction activation of a mitogen-activated protein kinase cascade involved in adventitious root development.

    Science.gov (United States)

    Pagnussat, Gabriela Carolina; Lanteri, María Luciana; Lombardo, María Cristina; Lamattina, Lorenzo

    2004-05-01

    Recently, it was demonstrated that nitric oxide (NO) and cGMP are involved in the auxin response during the adventitious rooting process in cucumber (Cucumis sativus; Pagnussat et al., 2002, 2003). However, not much is known about the complex molecular network operating during the cell proliferation and morphogenesis triggered by auxins and NO in that process. Anatomical studies showed that formation of adventitious root primordia was clearly detected in indole acetic acid (IAA)- and NO-treated cucumber explants, while neither cell proliferation nor differentiation into root primordia could be observed in control explants 3 d after primary root was removed. In order to go further with signal transduction mechanisms that operate during IAA- and NO-induced adventitious root formation, experiments were designed to test the involvement of a mitogen-activated protein kinase (MAPK) cascade in that process. Cucumber explants were treated with the NO-donor sodium nitroprusside (SNP) or with SNP plus the specific NO-scavenger cPTIO. Protein extracts from those explants were assayed for protein kinase (PK) activity by using myelin basic protein (MBP) as substrate in both in vitro and in-gel assays. The activation of a PK of approximately 48 kD could be detected 1 d after NO treatment with a maximal activation after 3 d of treatment. In control explants, a PK activity was detected only after 4 d of treatment. The MBP-kinase activity was also detected in extracts from IAA-treated explants, while no signal was observed in IAA + cPTIO treatments. The PK activity could be inhibited by the cell-permeable MAPK kinase inhibitor PD098059, suggesting that the NO-dependent MBP-kinase activity is a MAPK. Furthermore, when PD098059 was administered to explants treated with SNP or IAA, it produced a delay in root emergence and a dose-dependent reduction in root number. Altogether, our results suggest that a MAPK signaling cascade is activated during the adventitious rooting process

  7. Thromboxane A2 Receptor Inhibition Suppresses Multiple Myeloma Cell Proliferation by Inducing p38/c-Jun N-terminal Kinase (JNK) Mitogen-activated Protein Kinase (MAPK)-mediated G2/M Progression Delay and Cell Apoptosis*

    Science.gov (United States)

    Liu, Qian; Tao, Bo; Liu, Guizhu; Chen, Guilin; Zhu, Qian; Yu, Ying; Yu, Yu; Xiong, Hong

    2016-01-01

    Multiple myeloma (MM) is a plasma cell malignancy without effective therapeutics. Thromboxane A2 (TxA2)/TxA2 receptor (T prostanoid receptor (TP)) modulates the progression of some carcinomas; however, its effects on MM cell proliferation remain unclear. In this study, we evaluated cyclooxygenase (COX) enzymes and downstream prostaglandin profiles in human myeloma cell lines RPMI-8226 and U-266 and analyzed the effects of COX-1/-2 inhibitors SC-560 and NS-398 on MM cell proliferation. Our observations implicate COX-2 as being involved in modulating cell proliferation. We further incubated MM cells with prostaglandin receptor antagonists or agonists and found that only the TP antagonist, SQ29548, suppressed MM cell proliferation. TP silencing and the TP agonist, U46619, further confirmed this finding. Moreover, SQ29548 and TP silencing promoted MM cell G2/M phase delay accompanied by reducing cyclin B1/cyclin-dependent kinase-1 (CDK1) mRNA and protein expression. Notably, cyclin B1 overexpression rescued MM cells from G2/M arrest. We also found that the TP agonist activated JNK and p38 MAPK phosphorylation, and inhibitors of JNK and p38 MAPK depressed U46619-induced proliferation and cyclin B1/CDK1 protein expression. In addition, SQ29548 and TP silencing led to the MM cell apoptotic rate increasing with improving caspase 3 activity. The knockdown of caspase 3 reversed the apoptotic rate. Taken together, our results suggest that TxA2/TP promotes MM cell proliferation by reducing cell delay at G2/M phase via elevating p38 MAPK/JNK-mediated cyclin B1/CDK1 expression and hindering cell apoptosis. The TP inhibitor has potential as a novel agent to target kinase cascades for MM therapy. PMID:26724804

  8. A Genetic Screen Identifies a Requirement for Cysteine-Rich-Receptor-Like Kinases in Rice NH1 (OsNPR1-Mediated Immunity.

    Directory of Open Access Journals (Sweden)

    Mawsheng Chern

    2016-05-01

    Full Text Available Systemic acquired resistance, mediated by the Arabidopsis NPR1 gene and the rice NH1 gene, confers broad-spectrum immunity to diverse pathogens. NPR1 and NH1 interact with TGA transcription factors to activate downstream defense genes. Despite the importance of this defense response, the signaling components downstream of NPR1/NH1 and TGA proteins are poorly defined. Here we report the identification of a rice mutant, snim1, which suppresses NH1-mediated immunity and demonstrate that two genes encoding previously uncharacterized cysteine-rich-receptor-like kinases (CRK6 and CRK10, complement the snim1 mutant phenotype. Silencing of CRK6 and CRK10 genes individually in the parental genetic background recreates the snim1 phenotype. We identified a rice mutant in the Kitaake genetic background with a frameshift mutation in crk10; this mutant also displays a compromised immune response highlighting the important role of crk10. We also show that elevated levels of NH1 expression lead to enhanced CRK10 expression and that the rice TGA2.1 protein binds to the CRK10 promoter. These experiments demonstrate a requirement for CRKs in NH1-mediated immunity and establish a molecular link between NH1 and induction of CRK10 expression.

  9. Effects of lentiviral RNA interference-mediated downregulation of integrin-linked kinase on biological behaviors of human lens epithelial cells

    Directory of Open Access Journals (Sweden)

    Yu-Ping Zheng

    2016-01-01

    Full Text Available AIM: To investigate the effects of lentivirus (LV mediated integrin-linked kinase (ILK RNA interference (RNAi on biological behaviors of human lens epithelial cells (LECs. METHODS: Human cataract LECs and immortalized human LEC line, human lens epithelial (HLE B-3 cells were transfected by lentiviral vector expressing ILK-specific short hairpin RNA (shRNA and then stimulated by transforming growth factor- (TGF-, the silencing of ILK gene and protein was identified by reverse transcription-polymerase chain reaction (RT-PCR and Western blot methods; biological behaviors including cell cycle and apoptosis, cell morphology, -smooth muscle actin (SMA stress fiber formation and cell migration were examined. RESULTS: Remarkable decreases of ILK protein expression were detected in LECs carrying lentiviral ILK-shRNA vector; flow cytometry revealed arresting of cell cycle progression through the G1/S transition and higher apoptosis rate in ILK-RNAi-LV transfected cells. Less -SMA stress fiber formation and migration was observed in ILK-RNAi-LV transfected LECs. CONCLUSION: The present study demonstrated that ILK was an important regulator for LECs proliferation and migration. LV mediated ILK RNAi is an effective way to decrease ILK-regulated cell growth by arresting cell cycle progression and increasing cell apoptosis, as well as, to prevent cell migration by inhibiting TGF- induced -SMA stress fiber formation. Thus, LV mediated ILK RNAi might be useful to prevent posterior capsular opacification.

  10. Chronological Lifespan in Yeast Is Dependent on the Accumulation of Storage Carbohydrates Mediated by Yak1, Mck1 and Rim15 Kinases

    Science.gov (United States)

    Tang, Yingzhi; Quan, Zhenzhen; Zhang, Zhe; Oliver, Stephen G.; Zhang, Nianshu

    2016-01-01

    Upon starvation for glucose or any other macronutrient, yeast cells exit from the mitotic cell cycle and acquire a set of characteristics that are specific to quiescent cells to ensure longevity. Little is known about the molecular determinants that orchestrate quiescence entry and lifespan extension. Using starvation-specific gene reporters, we screened a subset of the yeast deletion library representing the genes encoding ‘signaling’ proteins. Apart from the previously characterised Rim15, Mck1 and Yak1 kinases, the SNF1/AMPK complex, the cell wall integrity pathway and a number of cell cycle regulators were shown to be necessary for proper quiescence establishment and for extension of chronological lifespan (CLS), suggesting that entry into quiescence requires the integration of starvation signals transmitted via multiple signaling pathways. The CLS of these signaling mutants, and those of the single, double and triple mutants of RIM15, YAK1 and MCK1 correlates well with the amount of storage carbohydrates but poorly with transition-phase cell cycle status. Combined removal of the glycogen and trehalose biosynthetic genes, especially GSY2 and TPS1, nearly abolishes the accumulation of storage carbohydrates and severely reduces CLS. Concurrent overexpression of GSY2 and TSL1 or supplementation of trehalose to the growth medium ameliorates the severe CLS defects displayed by the signaling mutants (rim15Δyak1Δ or rim15Δmck1Δ). Furthermore, we reveal that the levels of intracellular reactive oxygen species are cooperatively controlled by Yak1, Rim15 and Mck1, and the three kinases mediate the TOR1-regulated accumulation of storage carbohydrates and CLS extension. Our data support the hypothesis that metabolic reprogramming to accumulate energy stores and the activation of anti-oxidant defence systems are coordinated by Yak1, Rim15 and Mck1 kinases to ensure quiescence entry and lifespan extension in yeast. PMID:27923067

  11. UVB-mediated activation of p38 mitogen-activated protein kinase enhances resistance of normal human keratinocytes to apoptosis by stabilizing cytoplasmic p53.

    Science.gov (United States)

    Chouinard, Nadine; Valerie, Kristoffer; Rouabhia, Mahmoud; Huot, Jacques

    2002-07-01

    Human keratinocytes respond to UV rays by developing a fast adaptive response that contributes to maintaining their functions and survival. We investigated the role of the mitogen-activated protein kinase pathways in transducing the UV signals in normal human keratinocytes. We found that UVA, UVB or UVC induced a marked and persistent activation of p38, whereas c-Jun N-terminal kinase or extracellular signal-regulated kinase were less or not activated respectively. Inhibition of p38 activity by expression of a dominant-negative mutant of p38 or with SB203580 impaired cell viability and led to an increase in UVB-induced apoptosis. This sensitization to apoptosis was independent of caspase activities. Inhibition of p38 did not sensitize transformed HaCaT keratinocytes to UVB-induced apoptosis. In normal keratinocytes, expression of a dominant-negative mutant of p53 increased UVB-induced cell death, pointing to a role for p53. In these cells, UVB triggered a p38-dependent phosphorylation of p53 on Ser-15. This phosphorylation was associated with an SB203580-sensitive accumulation of p53, even in the presence of a serine phosphatase inhibitor. Accumulated p53 was localized mainly in the cytoplasm, independently of CRM1 nuclear export. In HaCaT cells, p53 was localized exclusively in the nucleus and its distribution and level were not affected by UVB or p38 inhibition. However, UVB induced an SB203580-insensitive phosphorylation on Ser-15 of mutated p53. Overall, our results suggest that, in normal human keratinocytes, protection against UVB depends on p38-mediated phosphorylation and stabilization of p53 and is tightly associated with the cytoplasmic sequestration of wild-type p53. We conclude that the p38/p53 pathway plays a key role in the adaptive response of normal human keratinocytes against UV stress.

  12. The transcription factor Ste12 mediates the regulatory role of the Tmk1 MAP kinase in mycoparasitism and vegetative hyphal fusion in the filamentous fungus Trichoderma atroviride.

    Directory of Open Access Journals (Sweden)

    Sabine Gruber

    Full Text Available Mycoparasitic species of the fungal genus Trichoderma are potent antagonists able to combat plant pathogenic fungi by direct parasitism. An essential step in this mycoparasitic fungus-fungus interaction is the detection of the fungal host followed by activation of molecular weapons in the mycoparasite by host-derived signals. The Trichoderma atroviride MAP kinase Tmk1, a homolog of yeast Fus3/Kss1, plays an essential role in regulating the mycoparasitic host attack, aerial hyphae formation and conidiation. However, the transcription factors acting downstream of Tmk1 are hitherto unknown. Here we analyzed the functions of the T. atroviride Ste12 transcription factor whose orthologue in yeast is targeted by the Fus3 and Kss1 MAP kinases. Deletion of the ste12 gene in T. atroviride not only resulted in reduced mycoparasitic overgrowth and lysis of host fungi but also led to loss of hyphal avoidance in the colony periphery and a severe reduction in conidial anastomosis tube formation and vegetative hyphal fusion events. The transcription of several orthologues of Neurospora crassa hyphal fusion genes was reduced upon ste12 deletion; however, the Δste12 mutant showed enhanced expression of mycoparasitism-relevant chitinolytic and proteolytic enzymes and of the cell wall integrity MAP kinase Tmk2. Based on the comparative analyses of Δste12 and Δtmk1 mutants, an essential role of the Ste12 transcriptional regulator in mediating outcomes of the Tmk1 MAPK pathway such as regulation of the mycoparasitic activity, hyphal fusion and carbon source-dependent vegetative growth is suggested. Aerial hyphae formation and conidiation, in contrast, were found to be independent of Ste12.

  13. The Essential WalK Histidine Kinase and WalR Regulator Differentially Mediate Autolysis of Staphylococcus aureus RN4220.

    Science.gov (United States)

    Zheng, Li; Yan, Meiying; Fan, Frank; Ji, Yinduo

    2015-06-01

    The two-component regulatory system, WalR/WalK is necessary for growth of different gram-positive bacteria, including Staphylococcus aureus . In present study, we confirmed the essentiality of both the histidine kinase protein WalK and the response regulator WalR for growth using S. aureus RN4220 strain and demonstrated that the histidine kinase protein WalK and the response regulator WalR function differently in regulation of staphylococcal autolysis. The down-regulation of walR expression effectively inhibited Triton X-100-induced lysis and had a weak impact on bacterial tolerance to penicillin induced cell lysis. In contrast, the down-regulation of walK expression had no influence on either Triton X-100- or penicillin-caused autolysis. Moreover, we determined the effect of WalR and WalK on bacterial hydrolase activity using a zymogram analysis. The results showed that the cell lysate of down-regulated walR expression mutant displayed several bands of decreased cell wall hydrolytic activities; however, the down-regulation of WalK had no dramatic impact on the hydrolytic activities. Furthermore, we examined the impact of WalR on the transcription of cidA associated with staphylococcal autolysis, and the results showed that the down-regulation of WalR led to decreased transcription of cidA in the log phase of growth. Taken together, the above results suggest that the essential WalR response regulator and the essential WalK histidine kinase might differently control bacterial lysis in RN4220 strain.

  14. 14-3-3 proteins mediate inhibitory effects of cAMP on salt-inducible kinases (SIKs).

    Science.gov (United States)

    Sonntag, Tim; Vaughan, Joan M; Montminy, Marc

    2018-02-01

    The salt-inducible kinase (SIK) family regulates cellular gene expression via the phosphorylation of cAMP-regulated transcriptional coactivators (CRTCs) and class IIA histone deacetylases, which are sequestered in the cytoplasm by phosphorylation-dependent 14-3-3 interactions. SIK activity toward these substrates is inhibited by increases in cAMP signaling, although the underlying mechanism is unclear. Here, we show that the protein kinase A (PKA)-dependent phosphorylation of SIKs inhibits their catalytic activity by inducing 14-3-3 protein binding. SIK1 and SIK3 contain two functional PKA/14-3-3 sites, while SIK2 has four. In keeping with the dimeric nature of 14-3-3s, the presence of multiple binding sites within target proteins dramatically increases binding affinity. As a result, loss of a single 14-3-3-binding site in SIK1 and SIK3 abolished 14-3-3 association and rendered them insensitive to cAMP. In contrast, mutation of three sites in SIK2 was necessary to fully block cAMP regulation. Superimposed on the effects of PKA phosphorylation and 14-3-3 association, an evolutionary conserved domain in SIK1 and SIK2 (the so called RK-rich region; 595-624 in hSIK2) is also required for the inhibition of SIK2 activity. Collectively, these results point to a dual role for 14-3-3 proteins in repressing a family of Ser/Thr kinases as well as their substrates. © 2017 Federation of European Biochemical Societies.

  15. Activation of Transcription Factor Nrf2 Signalling by the Sphingosine Kinase Inhibitor SKI-II Is Mediated by the Formation of Keap1 Dimers

    Science.gov (United States)

    Mercado, Nicolas; Kizawa, Yasuo; Ueda, Keitaro; Xiong, Yeping; Kimura, Genki; Moses, Audric; Curtis, Jonathan M.; Ito, Kazuhiro; Barnes, Peter J.

    2014-01-01

    Background Anti-oxidant capacity is crucial defence against environmental or endogenous oxidative stress. Nuclear factor erythroid 2-related factor 2 (Nrf2) is a redox-sensitive transcription factor that plays a key defensive role against oxidative and cytotoxic stress and cellular senescence. However, Nrf2 signalling is impaired in several aging-related diseases, such as chronic pulmonary obstructive disease (COPD), cancer, and neurodegenerative diseases. Thus, novel therapeutics that enhance Nrf2 signalling are an attractive approach to treat these diseases. Methodology/Principal Findings Nrf2 was stabilized by SKI-II (2-(p-hydroxyanilino)-4-(p-chlorophenyl) thiazole), which is a known sphingosine kinase inhibitor, in human bronchial epithelial cell line, BEAS2B, and in primary human bronchial epithelial cells, leading to enhancement of anti-oxidant proteins, such as HO-1, NQO1 and GCLM. The activation of Nrf2 was achieved by the generation of inactive dimerized form of Keap1, a negative regulator of Nrf2 expression, which was independent of sphingosine kinase inhibition. Using mice that were exposed to cigarette smoke, SKI-II induced Nrf2 expression together with HO-1 in their lungs. In addition, SKI-II reduced cigarette smoke mediated oxidative stress, macrophages and neutrophil infiltration and markers of inflammation in mice. Conclusions/Significance SKI-II appears to be a novel activator of Nrf2 signalling via the inactivation of Keap1. PMID:24505412

  16. α-Terpineol induces fatty liver in mice mediated by the AMP-activated kinase and sterol response element binding protein pathway.

    Science.gov (United States)

    Choi, You-Jin; Sim, Woo-Cheol; Choi, Hyun Kyu; Lee, Seung-Ho; Lee, Byung-Hoon

    2013-05-01

    The use of herbal medicines in disease prevention and treatment is growing rapidly worldwide, without careful consideration of safety issues. α-Terpineol is a monoterpene alcoholic component of Melaleuca alternifolia, Salvia officinalis and Carthamus tinctorius that is used widely as a flavor and essential oil in food. The present study showed that α-terpineol induces fatty liver via the AMP-activated protein kinase (AMPK)-mTOR-sterol regulatory element-binding protein-1 (SREBP-1) pathway. α-Terpineol-treated hepatocytes had significantly increased neutral lipid accumulation. α-Terpineol suppressed AMPK phosphorylation, and increased p70S6 kinase (p70S6K) phosphorylation and SREBP-1 activation. It also increased luciferase activity in cells transfected with LXRE-tk-Luc and SRE-tk-Luc. Inhibition of mTOR signaling by co-treatment with rapamycin or co-transfection with dominant negative p70S6K blocked completely the effects of α-terpineol. α-Terpineol oral administration to mice for 2weeks led to decreased AMPK phosphorylation and increased SREBP-1 activation in the liver, followed by hepatic lipid accumulation. Conversely, rapamycin co-treatment reversed α-terpineol-induced SREBP-1 activation and fatty liver in mice. These data provide evidence that α-terpineol causes fatty liver, an effect mediated by the AMPK/mTOR/SREBP-1 pathway. Copyright © 2013 Elsevier Ltd. All rights reserved.

  17. Heterologous activation of protein kinase C stimulates phosphorylation of delta-opioid receptor at serine 344, resulting in beta-arrestin- and clathrin-mediated receptor internalization

    DEFF Research Database (Denmark)

    Xiang, B; Yu, G H; Guo, J

    2001-01-01

    The purpose of the current study is to investigate the effect of opioid-independent, heterologous activation of protein kinase C (PKC) on the responsiveness of opioid receptor and the underlying molecular mechanisms. Our result showed that removing the C terminus of delta opioid receptor (DOR......) containing six Ser/Thr residues abolished both DPDPE- and phorbol 12-myristate 13-acetate (PMA)-induced DOR phosphorylation. The phosphorylation levels of DOR mutants T352A, T353A, and T358A/T361A/S363S were comparable to that of the wild-type DOR, whereas S344G substitution blocked PMA-induced receptor......, and ionomycin resulted in DOR internalization that required phosphorylation of Ser-344. Expression of dominant negative beta-arrestin and hypertonic sucrose treatment blocked PMA-induced DOR internalization, suggesting that PKC mediates DOR internalization via a beta-arrestin- and clathrin-dependent mechanism...

  18. Role of MAP kinases in regulating expression of antioxidants and inflammatory mediators in mouse keratinocytes following exposure to the half mustard, 2-chloroethyl ethyl sulfide

    International Nuclear Information System (INIS)

    Black, Adrienne T.; Joseph, Laurie B.; Casillas, Robert P.; Heck, Diane E.; Gerecke, Donald R.; Sinko, Patrick J.; Laskin, Debra L.; Laskin, Jeffrey D.

    2010-01-01

    Dermal exposure to sulfur mustard causes inflammation and tissue injury. This is associated with changes in expression of antioxidants and eicosanoids which contribute to oxidative stress and toxicity. In the present studies we analyzed mechanisms regulating expression of these mediators using an in vitro skin construct model in which mouse keratinocytes were grown at an air-liquid interface and exposed directly to 2-chloroethyl ethyl sulfide (CEES), a model sulfur mustard vesicant. CEES (100-1000 μM) was found to cause marked increases in keratinocyte protein carbonyls, a marker of oxidative stress. This was correlated with increases in expression of Cu,Zn superoxide dismutase, catalase, thioredoxin reductase and the glutathione S-transferases, GSTA1-2, GSTP1 and mGST2. CEES also upregulated several enzymes important in the synthesis of prostaglandins and leukotrienes including cyclooxygenase-2 (COX-2), microsomal prostaglandin E synthase-2 (mPGES-2), prostaglandin D synthase (PGDS), 5-lipoxygenase (5-LOX), leukotriene A 4 (LTA 4 ) hydrolase and leukotriene C 4 (LTC 4 ) synthase. CEES readily activated keratinocyte JNK and p38 MAP kinases, signaling pathways which are known to regulate expression of antioxidants, as well as prostaglandin and leukotriene synthases. Inhibition of p38 MAP kinase suppressed CEES-induced expression of GSTA1-2, COX-2, mPGES-2, PGDS, 5-LOX, LTA 4 hydrolase and LTC 4 synthase, while JNK inhibition blocked PGDS and GSTP1. These data indicate that CEES modulates expression of antioxidants and enzymes producing inflammatory mediators by distinct mechanisms. Increases in antioxidants may be an adaptive process to limit tissue damage. Inhibiting the capacity of keratinocytes to generate eicosanoids may be important in limiting inflammation and protecting the skin from vesicant-induced oxidative stress and injury.

  19. NF-κB inducing kinase, NIK mediates cigarette smoke/TNFα-induced histone acetylation and inflammation through differential activation of IKKs.

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    Sangwoon Chung

    Full Text Available BACKGROUND: Nuclear factor (NF-κB inducing kinase (NIK is a central player in the non-canonical NF κB pathway, which phosphorylates IκB kinase α (IKKα resulting in enhancement of target gene expression. We have recently shown that IKKα responds to a variety of stimuli including oxidants and cigarette smoke (CS regulating the histone modification in addition to its role in NF-κB activation. However, the primary signaling mechanism linking CS-mediated oxidative stress and TNFα with histone acetylation and pro-inflammatory gene transcription is not well understood. We hypothesized that CS and TNFα increase NIK levels causing phosphorylation of IKKα, which leads to histone acetylation. METHODOLOGY: To test this hypothesis, we investigated whether NIK mediates effects of CS and TNFα on histone acetylation in human lung epithelial cells in vitro and in lungs of mouse exposed to CS in vivo. CS increased the phosphorylation levels of IKKα/NIK in lung epithelial cells and mouse lungs. NIK is accumulated in the nuclear compartment, and is recruited to the promoters of pro-inflammatory genes, to induce posttranslational acetylation of histones in response to CS and TNFα. Cells in which NIK is knocked down using siRNA showed partial attenuation of CSE- and TNFα-induced acetylation of histone H3 on pro-inflammatory gene promoters. Additional study to determine the role of IKKβ/NF-κB pathway in CS-induced histone acetylation suggests that the canonical pathway does not play a role in histone acetylation particularly in response to CS in mouse lungs. CONCLUSIONS: Overall, our findings provide a novel role for NIK in CS- and TNFα-induced histone acetylation, especially on histone H3K9.

  20. Accumulation of specific sterol precursors targets a MAP kinase cascade mediating cell–cell recognition and fusion

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    Weichert, Martin; Lichius, Alexander; Priegnitz, Bert-Ewald; Brandt, Ulrike; Gottschalk, Johannes; Nawrath, Thorben; Groenhagen, Ulrike; Read, Nick D.; Schulz, Stefan; Fleißner, André

    2016-01-01

    Sterols are vital components of eukaryotic cell membranes. Defects in sterol biosynthesis, which result in the accumulation of precursor molecules, are commonly associated with cellular disorders and disease. However, the effects of these sterol precursors on the metabolism, signaling, and behavior of cells are only poorly understood. In this study, we show that the accumulation of only ergosterol precursors with a conjugated double bond in their aliphatic side chain specifically disrupts cell–cell communication and fusion in the fungus Neurospora crassa. Genetically identical germinating spores of this fungus undergo cell–cell fusion, thereby forming a highly interconnected supracellular network during colony initiation. Before fusion, the cells use an unusual signaling mechanism that involves the coordinated and alternating switching between signal sending and receiving states of the two fusion partners. Accumulation of only ergosterol precursors with a conjugated double bond in their aliphatic side chain disrupts this coordinated cell–cell communication and suppresses cell fusion. These specific sterol precursors target a single ERK-like mitogen-activated protein (MAP) kinase (MAK-1)-signaling cascade, whereas a second MAP kinase pathway (MAK-2), which is also involved in cell fusion, is unaffected. These observations indicate that a minor specific change in sterol structure can exert a strong detrimental effect on a key signaling pathway of the cell, resulting in the absence of cell fusion. PMID:27708165

  1. A critical role of Src family kinase in SDF-1/CXCR4-mediated bone-marrow progenitor cell recruitment to the ischemic heart.

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    Cheng, Min; Huang, Kai; Zhou, Junlan; Yan, Dewen; Tang, Yao-Liang; Zhao, Ting C; Miller, Richard J; Kishore, Raj; Losordo, Douglas W; Qin, Gangjian

    2015-04-01

    The G protein-coupled receptor CXCR4 and its ligand stromal-cell derived factor 1 (SDF-1) play a crucial role in directing progenitor cell (PC) homing to ischemic tissue. The Src family protein kinases (SFK) can be activated by, and serve as effectors of, G proteins. In this study we sought to determine whether SFK play a role in SDF-1/CXCR4-mediated PC homing. First, we investigated whether SDF-1/CXCR4 signaling activates SFK. Bone-marrow mononuclear cells (BM MNCs) were isolated from WT and BM-specific CXCR4-KO mice and treated with SDF-1 and/or CXCR4 antagonist AMD3100. SDF-1 treatment rapidly induced phosphorylation (activation) of hematopoietic Src (i.e., Lyn, Fgr, and Hck) in WT cells but not in AMD3100-treated cells or CXCR4-KO cells. Then, we investigated whether SFK are involved in SDF-1/CXCR4-mediated PC chemotaxis. In a combined chemotaxis and endothelial-progenitor-cell (EPC) colony assay, Src inhibitor SU6656 dose-dependently inhibited the SDF-1-induced migration of colony-forming EPCs. Next, we investigated whether SFK play a role in SDF-1/CXCR4-mediated BM PC homing to the ischemic heart. BM MNCs from CXCR4BAC:eGFP reporter mice were i.v. injected into WT and SDF-1BAC:SDF1-RFP transgenic mice following surgically-induced myocardial infarction (MI). eGFP(+) MNCs and eGFP(+)c-kit(+) PCs that were recruited in the infarct border zone in SDF-1BAC:SDF1-RFP recipients were significantly more than that in WT recipients. Treatments of mice with SU6656 significantly reduced eGFP(+) and eGFP(+)c-kit(+) cell recruitment in both WT and SDF-1BAC:RFP recipients and abrogated the difference between the two groups. Remarkably, PCs isolated from BM-specific C-terminal Src kinase (CSK)-KO (Src activated) mice were recruited more efficiently than PCs from WT PCs in the WT recipients. In conclusion, SFK are activated by SDF-1/CXCR4 signaling and play an essential role in SDF-1/CXCR4-mediated BM PC chemotactic response and ischemic cardiac recruitment. Copyright © 2015

  2. Lipopolysaccharide inhibits colonic biotin uptake via interference with membrane expression of its transporter: a role for a casein kinase 2-mediated pathway.

    Science.gov (United States)

    Lakhan, Ram; Said, Hamid M

    2017-04-01

    Biotin (vitamin B7), an essential micronutrient for normal cellular functions, is obtained from both dietary sources as well as gut microbiota. Absorption of biotin in both the small and large intestine is via a carrier-mediated process that involves the sodium-dependent multivitamin transporter (SMVT). Although different physiological and molecular aspects of intestinal biotin uptake have been delineated, nothing is known about the effect of LPS on the process. We addressed this issue using in vitro (human colonic epithelial NCM460 cells) and in vivo (mice) models of LPS exposure. Treating NCM460 cells with LPS was found to lead to a significant inhibition in carrier-mediated biotin uptake. Similarly, administration of LPS to mice led to a significant inhibition in biotin uptake by native colonic tissue. Although no changes in total cellular SMVT protein and mRNA levels were observed, LPS caused a decrease in the fraction of SMVT expressed at the cell surface. A role for casein kinase 2 (CK2) (whose activity was also inhibited by LPS) in mediating the endotoxin effects on biotin uptake and on membrane expression of SMVT was suggested by findings that specific inhibitors of CK2, as well as mutating the putative CK2 phosphorylation site (Thr 78 Ala) in the SMVT protein, led to inhibition in biotin uptake and membrane expression of SMVT. This study shows for the first time that LPS inhibits colonic biotin uptake via decreasing membrane expression of its transporter and that these effects likely involve a CK2-mediated pathway.

  3. Sesquiterpene dimmer (DSF-27) inhibits the release of neuroinflammatory mediators from microglia by targeting spleen tyrosine kinase (Syk) and Janus kinase 2 (Jak2): Two major non-receptor tyrosine signaling proteins involved in inflammatory events

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    Zeng, Ke-Wu [State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University Health Science Center, Beijing 100191 (China); Wang, Shu [State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University Health Science Center, Beijing 100191 (China); Department of Medicinal Chemistry and Pharmaceutical Analysis, Logistics College of Chinese People' s Armed Police Forces, Tianjin 300162 (China); Dong, Xin; Jiang, Yong; Jin, Hong-Wei [State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University Health Science Center, Beijing 100191 (China); Tu, Peng-Fei, E-mail: pengfeitu@vip.163.com [State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University Health Science Center, Beijing 100191 (China)

    2014-03-15

    Non-receptor protein tyrosine kinases (NRPTKs)-dependent inflammatory signal transduction cascades play key roles in immunoregulation. However, drug intervention through NRPTKs-involved immunoregulation mechanism in microglia (the major immune cells of the central nervous system) has not been widely investigated. A main aim of the present study is to elucidate the contribution of two major NRPTKs (Syk and Jak2) in neuroinflammation suppression by a bioactive sesquiterpene dimmer (DSF-27). We found that LPS-stimulated BV-2 cells activated Syk and further initiated Akt/NF-κB inflammatory pathway. This Syk-dependent Akt/NF-κB inflammatory pathway can be effectively ameliorated by DSF-27. Moreover, Jak2 was activated by LPS, which was followed by transcriptional factor Stat3 activation. The Jak2/Stat3 signal was suppressed by DSF-27 through inhibition of Jak2 and Stat3 phosphorylation, promotion of Jak/Stat3 inhibitory factors PIAS3 expression, and down-regulation of ERK and p38 MAPK phosphorylation. Furthermore, DSF-27 protected cortical and mesencephalic dopaminergic neurons against neuroinflammatory injury. Taken together, our findings indicate NRPTK signaling pathways including Syk/NF-κB and Jak2/Stat3 cascades are potential anti-neuroinflammatory targets in microglia, and may also set the basis for the use of sesquiterpene dimmer as a therapeutic approach for neuroinflammation via interruption of these pathways. - Highlights: • Sesquiterpene dimmer DSF-27 inhibits inflammatory mediators' production in microglia. • Syk-dependent Akt/NF-κB pathway is important for DSF-27's anti-inflammation activity. • Jak2/Stat3 pathway is important for DSF-27's anti-inflammation activity. • Jak2/Stat3 signaling pathway is partly regulated by ERK and p38 MAPKs and PIAS3. • DSF-27 protects neurons against microglia-mediated neuroinflammatory injury.

  4. Sesquiterpene dimmer (DSF-27) inhibits the release of neuroinflammatory mediators from microglia by targeting spleen tyrosine kinase (Syk) and Janus kinase 2 (Jak2): Two major non-receptor tyrosine signaling proteins involved in inflammatory events

    International Nuclear Information System (INIS)

    Zeng, Ke-Wu; Wang, Shu; Dong, Xin; Jiang, Yong; Jin, Hong-Wei; Tu, Peng-Fei

    2014-01-01

    Non-receptor protein tyrosine kinases (NRPTKs)-dependent inflammatory signal transduction cascades play key roles in immunoregulation. However, drug intervention through NRPTKs-involved immunoregulation mechanism in microglia (the major immune cells of the central nervous system) has not been widely investigated. A main aim of the present study is to elucidate the contribution of two major NRPTKs (Syk and Jak2) in neuroinflammation suppression by a bioactive sesquiterpene dimmer (DSF-27). We found that LPS-stimulated BV-2 cells activated Syk and further initiated Akt/NF-κB inflammatory pathway. This Syk-dependent Akt/NF-κB inflammatory pathway can be effectively ameliorated by DSF-27. Moreover, Jak2 was activated by LPS, which was followed by transcriptional factor Stat3 activation. The Jak2/Stat3 signal was suppressed by DSF-27 through inhibition of Jak2 and Stat3 phosphorylation, promotion of Jak/Stat3 inhibitory factors PIAS3 expression, and down-regulation of ERK and p38 MAPK phosphorylation. Furthermore, DSF-27 protected cortical and mesencephalic dopaminergic neurons against neuroinflammatory injury. Taken together, our findings indicate NRPTK signaling pathways including Syk/NF-κB and Jak2/Stat3 cascades are potential anti-neuroinflammatory targets in microglia, and may also set the basis for the use of sesquiterpene dimmer as a therapeutic approach for neuroinflammation via interruption of these pathways. - Highlights: • Sesquiterpene dimmer DSF-27 inhibits inflammatory mediators' production in microglia. • Syk-dependent Akt/NF-κB pathway is important for DSF-27's anti-inflammation activity. • Jak2/Stat3 pathway is important for DSF-27's anti-inflammation activity. • Jak2/Stat3 signaling pathway is partly regulated by ERK and p38 MAPKs and PIAS3. • DSF-27 protects neurons against microglia-mediated neuroinflammatory injury

  5. Protein kinase A signaling and calcium ions are major players in PAF mediated toxicity against Aspergillus niger.

    Science.gov (United States)

    Binder, Ulrike; Benčina, Mojca; Fizil, Ádám; Batta, Gyula; Chhillar, Anil K; Marx, Florentine

    2015-05-08

    The Penicillium chrysogenum antifungal protein PAF is toxic against potentially pathogenic Ascomycetes. We used the highly sensitive aequorin-expressing model Aspergillus niger to identify a defined change in cytoplasmic free Ca(2+) dynamics in response to PAF. This Ca(2+) signature depended on an intact positively charged lysine-rich PAF motif. By combining Ca(2+) measurements in A. niger mutants with deregulated cAMP/protein kinase A (PKA) signaling, we proved the interconnection of Ca(2+) perturbation and cAMP/PKA signaling in the mechanistic function of PAF. A deep understanding of the mode of action of PAF is an invaluable prerequisite for its future application as new antifungal drug. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

  6. Bruton's tyrosine kinase mediates the synergistic signalling between TLR9 and the B cell receptor by regulating calcium and calmodulin.

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    Elaine F Kenny

    Full Text Available B cells signal through both the B cell receptor (BCR which binds antigens and Toll-like receptors (TLRs including TLR9 which recognises CpG DNA. Activation of TLR9 synergises with BCR signalling when the BCR and TLR9 co-localise within an auto-phagosome-like compartment. Here we report that Bruton's tyrosine kinase (BTK is required for synergistic IL6 production and up-regulation of surface expression of MHC-class-II, CD69 and CD86 in primary murine and human B cells. We show that BTK is essential for co-localisation of the BCR and TLR9 within a potential auto-phagosome-like compartment in the Namalwa human B cell line. Downstream of BTK we find that calcium acting via calmodulin is required for this process. These data provide new insights into the role of BTK, an important target for autoimmune diseases, in B cell activation.

  7. The apoptotic effects of toosendanin are partially mediated by activation of deoxycytidine kinase in HL-60 cells.

    Science.gov (United States)

    Ju, Jianming; Qi, Zhichao; Cai, Xueting; Cao, Peng; Huang, Yan; Wang, Shuzhen; Liu, Nan; Chen, Yijun

    2012-01-01

    Triterpenoid toosendanin (TSN) exhibits potent cytotoxic activity through inducing apoptosis in a variety of cancer cell lines. However, the target and mechanism of the apoptotic effects by TSN remain unknown. In this study, we captured a specific binding protein of TSN in HL-60 cells by serial affinity chromatography and further identified it as deoxycytidine kinase (dCK). Combination of direct activation of dCK and inhibition of TSN-induced apoptosis by a dCK inhibitor confirmed that dCK is a target for TSN partially responsible for the apoptosis in HL-60 cells. Moreover, the activation of dCK by TSN was a result of conformational change, rather than auto-phosphorylation. Our results further imply that, in addition to the dATP increase by dCK activation in tumor cells, dCK may also involve in the apoptotic regulation.

  8. The apoptotic effects of toosendanin are partially mediated by activation of deoxycytidine kinase in HL-60 cells.

    Directory of Open Access Journals (Sweden)

    Jianming Ju

    Full Text Available Triterpenoid toosendanin (TSN exhibits potent cytotoxic activity through inducing apoptosis in a variety of cancer cell lines. However, the target and mechanism of the apoptotic effects by TSN remain unknown. In this study, we captured a specific binding protein of TSN in HL-60 cells by serial affinity chromatography and further identified it as deoxycytidine kinase (dCK. Combination of direct activation of dCK and inhibition of TSN-induced apoptosis by a dCK inhibitor confirmed that dCK is a target for TSN partially responsible for the apoptosis in HL-60 cells. Moreover, the activation of dCK by TSN was a result of conformational change, rather than auto-phosphorylation. Our results further imply that, in addition to the dATP increase by dCK activation in tumor cells, dCK may also involve in the apoptotic regulation.

  9. The Rho Kinases: Critical Mediators of Multiple Profibrotic Processes and Rational Targets for New Therapies for Pulmonary Fibrosis

    Science.gov (United States)

    Knipe, Rachel S.; Tager, Andrew M.

    2015-01-01

    Idiopathic pulmonary fibrosis (IPF) is characterized by progressive lung scarring, short median survival, and limited therapeutic options, creating great need for new pharmacologic therapies. IPF is thought to result from repetitive environmental injury to the lung epithelium, in the context of aberrant host wound healing responses. Tissue responses to injury fundamentally involve reorganization of the actin cytoskeleton of participating cells, including epithelial cells, fibroblasts, endothelial cells, and macrophages. Actin filament assembly and actomyosin contraction are directed by the Rho-associated coiled-coil forming protein kinase (ROCK) family of serine/threonine kinases (ROCK1 and ROCK2). As would therefore be expected, lung ROCK activation has been demonstrated in humans with IPF and in animal models of this disease. ROCK inhibitors can prevent fibrosis in these models, and more importantly, induce the regression of already established fibrosis. Here we review ROCK structure and function, upstream activators and downstream targets of ROCKs in pulmonary fibrosis, contributions of ROCKs to profibrotic cellular responses to lung injury, ROCK inhibitors and their efficacy in animal models of pulmonary fibrosis, and potential toxicities of ROCK inhibitors in humans, as well as involvement of ROCKs in fibrosis in other organs. As we discuss, ROCK activation is required for multiple profibrotic responses, in the lung and multiple other organs, suggesting ROCK participation in fundamental pathways that contribute to the pathogenesis of a broad array of fibrotic diseases. Multiple lines of evidence therefore indicate that ROCK inhibition has great potential to be a powerful therapeutic tool in the treatment of fibrosis, both in the lung and beyond. PMID:25395505

  10. The vascular endothelial specific IL-4 receptor alpha-ABL1 kinase signaling axis regulates the severity of IgE-mediated anaphylactic reactions.

    Science.gov (United States)

    Yamani, Amnah; Wu, David; Waggoner, Lisa; Noah, Taeko; Koleske, Anthony J; Finkelman, Fred; Hogan, Simon P

    2017-11-17

    Severe IgE-mediated, food-induced anaphylactic reactions are characterized by pulmonary venous vasodilatation and fluid extravasation, which are thought to lead to the life-threatening anaphylactic phenotype. The underlying immunologic and cellular processes involved in driving fluid extravasation and the severe anaphylactic phenotype are not fully elucidated. We sought to define the interaction and requirement of IL-4 and vascular endothelial (VE) IL-4 receptor α chain (IL-4Rα) signaling in histamine-abelson murine leukemia viral oncogene homology 1 (ABL1)-mediated VE dysfunction and fluid extravasation in the severity of IgE-mediated anaphylactic reactions in mice. Mice deficient in VE IL-4Rα and models of passive and active oral antigen- and IgE-induced anaphylaxis were used to define the requirements of the VE IL-4Rα and ABL1 pathway in severe anaphylactic reactions. The human VE cell line (EA.hy926 cells) and pharmacologic (imatinib) and genetic (short hairpin RNA knockdown of IL4RA and ABL1) approaches were used to define the requirement of this pathway in VE barrier dysfunction. IL-4 exacerbation of histamine-induced hypovolemic shock in mice was dependent on VE expression of IL-4Rα. IL-4- and histamine-induced ABL1 activation in human VE cells and VE barrier dysfunction was ABL1-dependent. Development of severe IgE-mediated hypovolemia and shock required VE-restricted ABL1 expression. Treatment of mice with a history of food-induced anaphylaxis with the ABL kinase inhibitor imatinib protected the mice from severe IgE-mediated anaphylaxis. IL-4 amplifies IgE- and histamine-induced VE dysfunction, fluid extravasation, and the severity of anaphylaxis through a VE IL-4Rα/ABL1-dependent mechanism. These studies implicate an important contribution by the VE compartment in the severity of anaphylaxis and identify a new pathway for therapeutic intervention of IgE-mediated reactions. Copyright © 2017 American Academy of Allergy, Asthma & Immunology

  11. Acetyl-l-carnitine (ALCAR) prevents hypobaric hypoxia-induced spatial memory impairment through extracellular related kinase-mediated nuclear factor erythroid 2-related factor 2 phosphorylation.

    Science.gov (United States)

    Barhwal, K; Hota, S K; Jain, V; Prasad, D; Singh, S B; Ilavazhagan, G

    2009-06-30

    Exposure to hypobaric hypoxia, a condition involving decreased availability of oxygen is known to be associated with oxidative stress, neurodegeneration and memory impairment. The multifactorial response of the brain and the complex signaling pathways involved therewith limits the therapeutic efficacy of several antioxidants in ameliorating hypobaric hypoxia-induced memory impairment. The present study was therefore aimed at investigating the potential of acetyl-l-carnitine (ALCAR), a known antioxidant that has been reported to augment neurotrophin-mediated survival mechanisms, in ameliorating hypoxia-induced neurodegeneration and memory impairment. Nuclear factor erythroid 2-related factor 2 (Nrf2) is a key transcription factor involved in the cellular defense mechanism against oxidative stress related to brain injury and neurological disorders. The study was designed to understand the mechanisms involving Nrf2 stabilization following exposure to hypobaric hypoxia. The results displayed reference memory impairment in Sprague-Dawley rats exposed to hypobaric hypoxia (7620 m) for 14 consecutive days which however improved on administration of ALCAR during hypoxic exposure. The study also revealed Nrf2 regulated augmented antioxidant response on administration of ALCAR which was through a novel tyrosine kinase A (TrkA) receptor-mediated mechanism. A decrease in free radical generation, lipid peroxidation and protein oxidation was also observed along with a concomitant increase in thioredoxin and reduced glutathione levels on administration of ALCAR during exposure to hypobaric hypoxia. The present study therefore reveals the therapeutic potential of ALCAR under conditions of hypobaric hypoxia and elucidates a novel mechanism of action of the drug.

  12. The PI3-kinase delta inhibitor idelalisib (GS-1101 targets integrin-mediated adhesion of chronic lymphocytic leukemia (CLL cell to endothelial and marrow stromal cells.

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    Stefania Fiorcari

    Full Text Available CLL cell trafficking between blood and tissue compartments is an integral part of the disease process. Idelalisib, a phosphoinositide 3-kinase delta (PI3Kδ inhibitor causes rapid lymph node shrinkage, along with an increase in lymphocytosis, prior to inducing objective responses in CLL patients. This characteristic activity presumably is due to CLL cell redistribution from tissues into the blood, but the underlying mechanisms are not fully understood. We therefore analyzed idelalisib effects on CLL cell adhesion to endothelial and bone marrow stromal cells (EC, BMSC. We found that idelalisib inhibited CLL cell adhesion to EC and BMSC under static and shear flow conditions. TNFα-induced VCAM-1 (CD106 expression in supporting layers increased CLL cell adhesion and accentuated the inhibitory effect of idelalisib. Co-culture with EC and BMSC also protected CLL from undergoing apoptosis, and this EC- and BMSC-mediated protection was antagonized by idelalisib. Furthermore, we demonstrate that CLL cell adhesion to EC and VLA-4 (CD49d resulted in the phosphorylation of Akt, which was sensitive to inhibition by idelalisib. These findings demonstrate that idelalisib interferes with integrin-mediated CLL cell adhesion to EC and BMSC, providing a novel mechanism to explain idelalisib-induced redistribution of CLL cells from tissues into the blood.

  13. Stress-induced nuclear import of apoptosis signal-regulating kinase 1 is mediated by karyopherin α2/β1 heterodimer.

    Science.gov (United States)

    Sturchler, Emmanuel; Feurstein, Daniel; Chen, Weimin; McDonald, Patricia; Duckett, Derek

    2013-03-01

    The apoptosis signal-regulating kinase 1 (ASK1) is activated in response to a wide variety of extracellular stressors. Consequently, dysregulation of ASK1 is associated with multiple pathologies. Here, we show that ASK1 translocates from the cytoplasm to the nucleus in HEK293 cells and human cardiomyocytes in response to hydrogen peroxide (H(2)O(2)) or angiotensin respectively. Immunoprecipitation and mass spectrometry experiments reveal that ASK1 physically interacts with the karyopherin α2/β1 heterodimer in response to stress and genetic knockdown experiments confirm that this association mediates H(2)O(2)-induced ASK1 nuclear translocation. In addition, we have identified a nuclear localization signal (NLS)-like motif within the primary amino acid sequence of ASK1 composed of two clusters of basic amino acids separated by an intervening 16 amino acid spacer, KR[ACANDLLVDEFLKVSS]KKKK. Mutation of the downstream lysine cluster markedly reduces the H(2)O(2)-induced ASK1-karyopherin α2/β1 interaction and inhibits ASK1 nuclear translocation. Furthermore, we demonstrate that nuclear ASK1 is active and participates in H(2)O(2)-induced ASK1-mediated cell death. Collectively, our findings have identified a functional interaction between ASK1 and the karyopherin α2/β1 heterodimer and have also revealed a novel mechanism by which nuclear trafficking regulates the apoptotic function of ASK1 in response to stress. Copyright © 2012 Elsevier B.V. All rights reserved.

  14. An LRR/malectin receptor-like kinase mediates resistance to non-adapted and adapted powdery mildew fungi in barley and wheat

    Directory of Open Access Journals (Sweden)

    Jeyaraman Rajaraman

    2016-12-01

    Full Text Available Pattern recognition receptors (PRRs belonging to the multigene family of receptor-like kinases (RLKs are the sensing devices of plants for microbe- or pathogen-associated molecular patterns released from microbial organisms. Here we describe Rnr8 (for required for nonhost resistance 8 encoding HvLEMK1, a LRR-malectin domain-containing transmembrane RLK that mediates nonhost resistance of barley to the non-adapted wheat powdery mildew fungus Blumeria graminis f.sp. tritici. Transgenic barley lines with silenced HvLEMK1 allow entry and colony growth of the non-adapted pathogen, although sporulation was reduced and final colony size did not reach that of the adapted barley powdery mildew fungus Blumeria graminis f.sp. hordei. Transient expression of the barley or wheat LEMK1 genes enhanced resistance in wheat to the adapted wheat powdery mildew fungus while expression of the same genes did not protect barley from attack by the barley powdery mildew fungus. The results suggest that HvLEMK1 is a factor mediating nonhost resistance in barley and quantitative host resistance in wheat to the wheat powdery mildew fungus.

  15. Functional involvement of protein kinase C, Rho-kinase and TRPC3 decreases while PLC increases with advancement of pregnancy in mediating oxytocin-induced myometrial contractions in water buffaloes (Bubalus bubalis).

    Science.gov (United States)

    Sharma, Abhishek; Nakade, Udayraj P; Choudhury, Soumen; Garg, Satish Kumar

    2017-04-01

    Present study unravels the involvement of different calcium signaling pathways in oxytocin-induced contractions in myometrium of non-pregnant and pregnant buffaloes during early and mid-pregnancy stages. Uteri of pregnant animals were more sensitive than of non-pregnant buffaloes. Phasic contractions and frequency of contraction significantly increased with advancement of pregnancy, while tonic contractions non-significantly and amplitude significantly decreased from six months pregnancy onward. Oxytocin produced concentration-dependent-contraction on isolated myometrial strips of pregnant and non-pregnant buffaloes and the dose response curves (DRCs) of oxytocin were significantly (P < 0.05) shifted to right in the presence of nifedipine (1 μM), in Ca 2+ -free Ringer Locke solution (RLS), ruthenium red (30 μM), ruthenium red + nifedipine, cyclopiazonic acid (CPA; Ca 2+ free RLS as well as RLS), CPA (10 μM)+nifedipine, U-73122 (1 μM) + nifedipine and SKF96365 (25 μM) on uteri of non-pregnant and pregnant (early and mid) animals. The DRCs were also significantly shifted towards right in the presence of Y-27632 (10 μM), GF109203X (5 μM) and Pyr3 (10 μM) on uteri of non-pregnant and early pregnancy stage buffaloes while only in the presence of U-73122 (1 μM) on uteri of mid-pregnancy stage buffaloes. Our finding suggest that and L-type Ca 2+ channels, IP3-RyR-gated, and store-operated calcium channels including transient receptor potential channel (TRPC) pathways play significant role in mediating oxytocin-induced contractions in myometrium of pregnant and non-pregnant buffaloes. SERCA plays major role only during early-pregnancy while functional role of protein kinase C (PKC), Rho-kinase and TRPC3 pathways decreased and role of G-protein coupled receptor-phospholipase C (GPCR-PLC) pathway increased with advancement of pregnancy. Copyright © 2016 Elsevier Inc. All rights reserved.

  16. Expansion of Sphingosine Kinase and Sphingosine-1-Phosphate Receptor Function in Normal and Cancer Cells: From Membrane Restructuring to Mediation of Estrogen Signaling and Stem Cell Programming.

    Science.gov (United States)

    Sukocheva, Olga A

    2018-01-31

    Sphingolipids, sphingolipid metabolizing enzymes, and their receptors network are being recognized as part of the signaling mechanisms, which govern breast cancer cell growth, migration, and survival during chemotherapy treatment. Approximately 70% of breast cancers are estrogen receptor (ER) positive and, thus, rely on estrogen signaling. Estrogen activates an intracellular network composed of many cytoplasmic and nuclear mediators. Some estrogen effects can be mediated by sphingolipids. Estrogen activates sphingosine kinase 1 (SphK1) and amplifies the intracellular concentration of sphingosine-1-phosphate (S1P) in breast cancer cells during stimulation of proliferation and survival. Specifically, Estrogen activates S1P receptors (S1PR) and induces growth factor receptor transactivation. SphK, S1P, and S1PR expression are causally associated with endocrine resistance and progression to advanced tumor stages in ER-positive breast cancers in vivo . Recently, the network of SphK/S1PR was shown to promote the development of ER-negative cancers and breast cancer stem cells, as well as stimulating angiogenesis. Novel findings confirm and broaden our knowledge about the cross-talk between sphingolipids and estrogen network in normal and malignant cells. Current S1PRs therapeutic inhibition was indicated as a promising chemotherapy approach in non-responsive and advanced malignancies. Considering that sphingolipid signaling has a prominent role in terminally differentiated cells, the impact should be considered when designing specific SphK/S1PR inhibitors. This study analyzes the dynamic of the transformation of sphingolipid axis during a transition from normal to pathological condition on the level of the whole organism. The sphingolipid-based mediation and facilitation of global effects of estrogen were critically accented as a bridging mechanism that should be explored in cancer prevention.

  17. Expansion of Sphingosine Kinase and Sphingosine-1-Phosphate Receptor Function in Normal and Cancer Cells: From Membrane Restructuring to Mediation of Estrogen Signaling and Stem Cell Programming

    Directory of Open Access Journals (Sweden)

    Olga A. Sukocheva

    2018-01-01

    Full Text Available Sphingolipids, sphingolipid metabolizing enzymes, and their receptors network are being recognized as part of the signaling mechanisms, which govern breast cancer cell growth, migration, and survival during chemotherapy treatment. Approximately 70% of breast cancers are estrogen receptor (ER positive and, thus, rely on estrogen signaling. Estrogen activates an intracellular network composed of many cytoplasmic and nuclear mediators. Some estrogen effects can be mediated by sphingolipids. Estrogen activates sphingosine kinase 1 (SphK1 and amplifies the intracellular concentration of sphingosine-1-phosphate (S1P in breast cancer cells during stimulation of proliferation and survival. Specifically, Estrogen activates S1P receptors (S1PR and induces growth factor receptor transactivation. SphK, S1P, and S1PR expression are causally associated with endocrine resistance and progression to advanced tumor stages in ER-positive breast cancers in vivo. Recently, the network of SphK/S1PR was shown to promote the development of ER-negative cancers and breast cancer stem cells, as well as stimulating angiogenesis. Novel findings confirm and broaden our knowledge about the cross-talk between sphingolipids and estrogen network in normal and malignant cells. Current S1PRs therapeutic inhibition was indicated as a promising chemotherapy approach in non-responsive and advanced malignancies. Considering that sphingolipid signaling has a prominent role in terminally differentiated cells, the impact should be considered when designing specific SphK/S1PR inhibitors. This study analyzes the dynamic of the transformation of sphingolipid axis during a transition from normal to pathological condition on the level of the whole organism. The sphingolipid-based mediation and facilitation of global effects of estrogen were critically accented as a bridging mechanism that should be explored in cancer prevention.

  18. Nicotinamide riboside kinases display redundancy in mediating nicotinamide mononucleotide and nicotinamide riboside metabolism in skeletal muscle cells.

    Science.gov (United States)

    Fletcher, Rachel S; Ratajczak, Joanna; Doig, Craig L; Oakey, Lucy A; Callingham, Rebecca; Da Silva Xavier, Gabriella; Garten, Antje; Elhassan, Yasir S; Redpath, Philip; Migaud, Marie E; Philp, Andrew; Brenner, Charles; Canto, Carles; Lavery, Gareth G

    2017-08-01

    Augmenting nicotinamide adenine dinucleotide (NAD + ) availability may protect skeletal muscle from age-related metabolic decline. Dietary supplementation of NAD + precursors nicotinamide mononucleotide (NMN) and nicotinamide riboside (NR) appear efficacious in elevating muscle NAD + . Here we sought to identify the pathways skeletal muscle cells utilize to synthesize NAD + from NMN and NR and provide insight into mechanisms of muscle metabolic homeostasis. We exploited expression profiling of muscle NAD + biosynthetic pathways, single and double nicotinamide riboside kinase 1/2 (NRK1/2) loss-of-function mice, and pharmacological inhibition of muscle NAD + recycling to evaluate NMN and NR utilization. Skeletal muscle cells primarily rely on nicotinamide phosphoribosyltransferase (NAMPT), NRK1, and NRK2 for salvage biosynthesis of NAD + . NAMPT inhibition depletes muscle NAD + availability and can be rescued by NR and NMN as the preferred precursors for elevating muscle cell NAD + in a pathway that depends on NRK1 and NRK2. Nrk2 knockout mice develop normally and show subtle alterations to their NAD+ metabolome and expression of related genes. NRK1, NRK2, and double KO myotubes revealed redundancy in the NRK dependent metabolism of NR to NAD + . Significantly, these models revealed that NMN supplementation is also dependent upon NRK activity to enhance NAD + availability. These results identify skeletal muscle cells as requiring NAMPT to maintain NAD + availability and reveal that NRK1 and 2 display overlapping function in salvage of exogenous NR and NMN to augment intracellular NAD + availability.

  19. Autonomous and nonautonomous regulation of Wnt-mediated neuronal polarity by the C. elegans Ror kinase CAM-1.

    Science.gov (United States)

    Chien, Shih-Chieh Jason; Gurling, Mark; Kim, Changsung; Craft, Teresa; Forrester, Wayne; Garriga, Gian

    2015-08-01

    Wnts are a conserved family of secreted glycoproteins that regulate various developmental processes in metazoans. Three of the five Caenorhabditis elegans Wnts, CWN-1, CWN-2 and EGL-20, and the sole Wnt receptor of the Ror kinase family, CAM-1, are known to regulate the anterior polarization of the mechanosensory neuron ALM. Here we show that CAM-1 and the Frizzled receptor MOM-5 act in parallel pathways to control ALM polarity. We also show that CAM-1 has two functions in this process: an autonomous signaling function that promotes anterior polarization and a nonautonomous Wnt-antagonistic function that inhibits anterior polarization. These antagonistic activities can account for the weak ALM phenotypes displayed by cam-1 mutants. Our observations suggest that CAM-1 could function as a Wnt receptor in many developmental processes, but the analysis of cam-1 mutants may fail to reveal CAM-1's role as a receptor in these processes because of its Wnt-antagonistic activity. In this model, loss of CAM-1 results in increased levels of Wnts that act through other Wnt receptors, masking CAM-1's autonomous role as a Wnt receptor. Copyright © 2015 Elsevier Inc. All rights reserved.

  20. Increased expression of interleukin-1β in triglyceride-induced macrophage cell death is mediated by p38 MAP kinase.

    Science.gov (United States)

    Sung, Ho Joong; Son, Sin Jee; Yang, Seung-ju; Rhee, Ki-Jong; Kim, Yoon Suk

    2012-07-01

    Triglycerides (TG) are implicated in the development of atherosclerosis through formation of foam cells and induction of macrophage cell death. In this study, we report that addition of exogenous TG induced cell death in phorbol 12-myristate 13-acetate-differentiated THP-1 human macrophages. TG treatment induced a dramatic decrease in interleukin-1β (IL-1β) mRNA expression in a dose- and time-dependent manner. The expression of granulocyte macrophage colony-stimulating factor and platelet endothelial cell adhesion molecule remained unchanged. To identify signaling pathways involved in TG-induced downregulation of IL-1β, we added p38 MAPK, protein kinase C (PKC) or c-Raf1 specific inhibitors. We found that inhibition of p38 MAPK alleviated the TG-induced downregulation of IL-1β, whereas inhibition of PKC and c-Raf1 had no effect. This is the first report showing decreased IL-1β expression during TG-induced cell death in a human macrophage line. Our results suggest that downregulation of IL-1β expression by TG-treated macrophages may play a role during atherogenesis.

  1. Effect of Antrodia camphorata on Inflammatory Arterial Thrombosis-Mediated Platelet Activation: The Pivotal Role of Protein Kinase C

    Directory of Open Access Journals (Sweden)

    Wan-Jung Lu

    2014-01-01

    Full Text Available Antrodia camphorata is a rare Taiwanese medicinal mushroom. Antrodia camphorata extract has been reported to exhibit antioxidant, anti-inflammation, antimetastasis, and anticancer activities and plays a role in liver fibrosis, vasorelaxation, and immunomodulation. Critical vascular inflammation leads to vascular dysfunction and cardiovascular diseases, including abdominal aortic aneurysms, hypertension, and atherosclerosis. Platelet activation plays a crucial role in intravascular thrombosis, which is involved in a wide variety of cardiovascular diseases. However, the effect of Antrodia camphorata on platelet activation remains unclear. We examined the effects of Antrodia camphorata on platelet activation. In the present study, Antrodia camphorata treatment (56–224 μg/mL inhibited platelet aggregation induced by collagen, but not U46619, an analogue of thromboxane A2, thrombin, and arachidonic acid. Antrodia camphorata inhibited collagen-induced calcium (Ca2+ mobilization and phosphorylation of protein kinase C (PKC and Akt. In addition, Antrodia camphorata significantly reduced the aggregation and phosphorylation of PKC in phorbol-12, 13-dibutyrate (PDBu activated platelets. In conclusion, Antrodia camphorata may inhibit platelet activation by inhibiting of Ca2+ and PKC cascade and the Akt pathway. Our study suggests that Antrodia camphorata may be a potential therapeutic agent for preventing or treating thromboembolic disorders.

  2. c-Jun N-terminal kinase mediates AML1-ETO protein-induced connexin-43 expression

    International Nuclear Information System (INIS)

    Gao Fenghou; Wang Qiong; Wu Yingli; Li Xi; Zhao Kewen; Chen Guoqiang

    2007-01-01

    AML1-ETO fusion protein, a product of leukemia-related chromosomal translocation t(8;21), was reported to upregulate expression of connexin-43 (Cx43), a member of gap junction-constituted connexin family. However, its mechanism(s) remains unclear. By bioinformatic analysis, here we showed that there are two putative AML1-binding consensus sequences followed by two activated protein (AP)1 sites in the 5'-flanking region upstream to Cx43 gene. AML1-ETO could directly bind to these two AML1-binding sites in electrophoretic mobility shift assay, but luciferase reporter assay revealed that the AML1 binding sites were not indispensable for Cx43 induction by AML1-ETO protein. Conversely, AP1 sites exerted an important role in this event. In agreement, AML1-ETO overexpression in leukemic U937 cells activated c-Jun N-terminal kinase (JNK), while its specific inhibitor SP600125 effectively abrogated AML1-ETO-induced Cx43 expression, indicating that JNK signaling pathway contributes to AML1-ETO induced Cx43 expression. These results would shed new insights for understanding mechanisms of AML1-ETO-associated leukemogenesis

  3. The receptor-like cytoplasmic kinase BSR1 mediates chitin-induced defense signaling in rice cells.

    Science.gov (United States)

    Kanda, Yasukazu; Yokotani, Naoki; Maeda, Satoru; Nishizawa, Yoko; Kamakura, Takashi; Mori, Masaki

    2017-08-01

    Broad-Spectrum Resistance 1 (BSR1) encodes a rice receptor-like cytoplasmic kinase, and enhances disease resistance when overexpressed. Rice plants overexpressing BSR1 are highly resistant to diverse pathogens, including rice blast fungus. However, the mechanism responsible for this resistance has not been fully characterized. To analyze the BSR1 function, BSR1-knockout (BSR1-KO) plants were generated using a clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system. Experiments using suspension-cultured cells revealed that defense responses including H 2 O 2 production (i.e. oxidative burst) and expression of defense-related genes induced by autoclaved conidia of the rice blast fungus significantly decreased in BSR1-KO cells. Furthermore, a treatment with chitin oligomers which function as microbe-associated molecular patterns (MAMPs) of the rice blast fungus resulted in considerably suppressed defense responses in BSR1-KO cells. These results suggest that BSR1 is important for the rice innate immunity triggered by the perception of chitin.

  4. Phospholipid mediated activation of calcium dependent protein kinase 1 (CaCDPK1 from chickpea: a new paradigm of regulation.

    Directory of Open Access Journals (Sweden)

    Ajay Kumar Dixit

    Full Text Available Phospholipids, the major structural components of membranes, can also have functions in regulating signaling pathways in plants under biotic and abiotic stress. The effects of adding phospholipids on the activity of stress-induced calcium dependent protein kinase (CaCDPK1 from chickpea are reported here. Both autophosphorylation as well as phosphorylation of the added substrate were enhanced specifically by phosphatidylcholine and to a lesser extent by phosphatidic acid, but not by phosphatidylethanolamine. Diacylgylerol, the neutral lipid known to activate mammalian PKC, stimulated CaCDPK1 but at higher concentrations. Increase in V(max of the enzyme activity by these phospholipids significantly decreased the K(m indicating that phospholipids enhance the affinity towards its substrate. In the absence of calcium, addition of phospholipids had no effect on the negligible activity of the enzyme. Intrinsic fluorescence intensity of the CaCDPK1 protein was quenched on adding PA and PC. Higher binding affinity was found with PC (K(½ = 114 nM compared to PA (K(½ = 335 nM. We also found that the concentration of PA increased in chickpea plants under salt stress. The stimulation by PA and PC suggests regulation of CaCDPK1 by these phospholipids during stress response.

  5. Adrenaline is a critical mediator of acute exercise-induced AMP-activated protein kinase activation in adipocytes

    Science.gov (United States)

    Koh, Ho-Jin; Hirshman, Michael F.; He, Huamei; Li, Yangfeng; Manabe, Yasuko; Balschi, James A.; Goodyear, Laurie J.

    2007-01-01

    Exercise increases AMPK (AMP-activated protein kinase) activity in human and rat adipocytes, but the underlying molecular mechanisms and functional consequences of this activation are not known. Since adrenaline (epinephrine) concentrations increase with exercise, in the present study we hypothesized that adrenaline activates AMPK in adipocytes. We show that a single bout of exercise increases AMPKα1 and α2 activities and ACC (acetyl-CoA carboxylase) Ser79 phosphorylation in rat adipocytes. Similarly to exercise, adrenaline treatment in vivo increased AMPK activities and ACC phosphorylation. Pre-treatment of rats with the β-blocker propranolol fully blocked exercise-induced AMPK activation. Increased AMPK activity with exercise and adrenaline treatment in vivo was accompanied by an increased AMP/ATP ratio. Adrenaline incubation of isolated adipocytes also increased the AMP/ATP ratio and AMPK activities, an effect blocked by propranolol. Adrenaline incubation increased lipolysis in isolated adipocytes, and Compound C, an AMPK inhibitor, attenuated this effect. Finally, a potential role for AMPK in the decreased adiposity associated with chronic exercise was suggested by marked increases in AMPKα1 and α2 activities in adipocytes from rats trained for 6 weeks. In conclusion, both acute and chronic exercise are significant regulators of AMPK activity in rat adipocytes. Our findings suggest that adrenaline plays a critical role in exercise-stimulated AMPKα1 and α2 activities in adipocytes, and that AMPK can function in the regulation of lipolysis. PMID:17253964

  6. Heterogeneous nuclear ribonucleoprotein B1 protein impairs DNA repair mediated through the inhibition of DNA-dependent protein kinase activity

    International Nuclear Information System (INIS)

    Iwanaga, Kentaro; Sueoka, Naoko; Sato, Akemi; Hayashi, Shinichiro; Sueoka, Eisaburo

    2005-01-01

    Heterogeneous nuclear ribonucleoprotein B1, an RNA binding protein, is overexpressed from the early stage of lung cancers; it is evident even in bronchial dysplasia, a premalignant lesion. We evaluated the proteins bound with hnRNP B1 and found that hnRNP B1 interacted with DNA-dependent protein kinase (DNA-PK) complex, and recombinant hnRNP B1 protein dose-dependently inhibited DNA-PK activity in vitro. To test the effect of hnRNP B1 on DNA repair, we performed comet assay after irradiation, using normal human bronchial epithelial (HBE) cells treated with siRNA for hnRNP A2/B1: reduction of hnRNP B1 treated with siRNA for hnRNP A2/B1 induced faster DNA repair in normal HBE cells. Considering these results, we assume that overexpression of hnRNP B1 occurring in the early stage of carcinogenesis inhibits DNA-PK activity, resulting in subsequent accumulation of erroneous rejoining of DNA double-strand breaks, causing tumor progression

  7. Inhibitor of Tec kinase, LFM-A13, decreases pro-inflammatory mediators production in LPS-stimulated RAW264.7 macrophages via NF-κB pathway.

    Science.gov (United States)

    Wang, Fei; Zhang, Wei; Wang, Chao; Fang, Xu; Cheng, Hao; Liu, Sheng; Chen, Xu-Lin

    2017-05-23

    Tec kinase, a prototypical member of the Tec tyrosine kinases family, was shown to mainly govern lymphocyte proliferation. In the present study, we investigated the role of Tec kinase in acute inflammatory response in lipopolysaccharide (LPS) challenge. First, we demonstrate that Tec kinase activity was observed in RAW264.7 macrophages exposed to LPS. Tec and phosphorylated Tec expression were upregulated in a dose- and time-dependent manner after LPS stimulation. LPS increased monocyte chemotactic protein (MCP)-1 secretion and intercellular adhesion molecule (ICAM)-1 expression, and increasing mRNA expression was consistently observed. LPS also induced IκBα phoshporylaytion and its degradation, increased NF-κB p65 phoshporylaytion and translocation to nuclei in RAW264.7 cells. Pretreatment with LFM-A13 decreased LPS-induced cytokines and chemokines production and mRNA levels, blocked NF-κB transactivation. These effects of LPS were also prevented by Tec-siRNA. Additionally, LFM-A13 or Tec-siRNA obviously inhibited LPS-induced TGFβ-activated kinase 1(TAK1) phosphorylation. Taken together, our results suggest that Tec kinase involves in acute inflammation process in LPS-stimulated RAW264.7 cells, at least mediated by activating TAK1/ NF-κB signal pathway.

  8. Effect of a peptide inhibitor of protein kinase C on G-protein-mediated increase in myofilament Ca(2+)-sensitivity in rabbit arterial skinned muscle.

    Science.gov (United States)

    Itoh, T.; Suzuki, A.; Watanabe, Y.

    1994-01-01

    1. To investigate the role of protein kinase C in the increase mediated by guanosine 5'-triphosphate (GTP)-binding proteins (G-proteins) in the sensitivity of the contractile proteins to Ca2+ in vascular smooth muscle, the effect of a novel peptide inhibitor of protein kinase C (PKC19-36) on Ca(2+)-induced contraction and myosin light chain (MLC) phosphorylation was studied in the presence and absence of guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) in beta-escin-skinned smooth muscle strips of rabbit mesenteric artery. For comparison, the effects were also observed of PKC19-36 on the action of phorbol 12,13-dibutylate (PDBu, an activator of PKC) on the two Ca(2+)-induced responses. 2. In beta-escin-skinned strips treated with ionomycin, Ca2+ (0.1-3 microM) concentration-dependently produced contraction in parallel with an increase in MLC-phosphorylation. GTP gamma S (10 microM) and PDBu (0.1 microM) each shifted both the Ca(2+)-force and Ca(2+)-MLC-phosphorylation relationships to the left without a significant change in either maximum response. The relationship between force and MLC-phosphorylation was not modified by either GTP gamma S or PDBu, indicating that the sensitivity of MLC-phosphorylation to Ca2+ is enhanced by both GTP gamma S and PDBu. 3. PKC19-36 itself modified neither the contraction nor MLC-phosphorylation induced by Ca2+ but it did block the PDBu-induced enhancement of these two Ca(2+)-induced responses. By contrast, PKC19-36 did not modify the GTP gamma S-induced enhancement of the two Ca(2+)-induced responses.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:8012712

  9. G protein-coupled receptor (GPR)40-dependent potentiation of insulin secretion in mouse islets is mediated by protein kinase D1.

    Science.gov (United States)

    Ferdaoussi, M; Bergeron, V; Zarrouki, B; Kolic, J; Cantley, J; Fielitz, J; Olson, E N; Prentki, M; Biden, T; MacDonald, P E; Poitout, V

    2012-10-01

    Activation of the G protein-coupled receptor (GPR)40 by long-chain fatty acids potentiates glucose-stimulated insulin secretion (GSIS) from pancreatic beta cells, and GPR40 agonists are in clinical development for type 2 diabetes therapy. GPR40 couples to the G protein subunit Gα(q/11) but the signalling cascade activated downstream is unknown. This study aimed to determine the mechanisms of GPR40-dependent potentiation of GSIS by fatty acids. Insulin secretion in response to glucose, oleate or diacylglycerol (DAG) was assessed in dynamic perifusions and static incubations in islets from wild-type (WT) and Gpr40 (-/-) mice. Depolymerisation of filamentous actin (F-actin) was visualised by phalloidin staining and epifluorescence. Pharmacological and molecular approaches were used to ascertain the roles of protein kinase D (PKD) and protein kinase C delta in GPR40-mediated potentiation of GSIS. Oleate potentiates the second phase of GSIS, and this effect is largely dependent upon GPR40. Accordingly, oleate induces rapid F-actin remodelling in WT but not in Gpr40 (-/-) islets. Exogenous DAG potentiates GSIS in both WT and Gpr40 (-/-) islets. Oleate induces PKD phosphorylation at residues Ser-744/748 and Ser-916 in WT but not Gpr40 (-/-) islets. Importantly, oleate-induced F-actin depolymerisation and potentiation of GSIS are lost upon pharmacological inhibition of PKD1 or deletion of Prkd1. We conclude that the signalling cascade downstream of GPR40 activation by fatty acids involves activation of PKD1, F-actin depolymerisation and potentiation of second-phase insulin secretion. These results provide important information on the mechanisms of action of GPR40, a novel drug target for type 2 diabetes.

  10. Calcineurin inhibitors recruit protein kinases JAK2 and JNK, TLR signaling and the UPR to activate NF-κB-mediated inflammatory responses in kidney tubular cells

    International Nuclear Information System (INIS)

    González-Guerrero, Cristian; Ocaña-Salceda, Carlos; Berzal, Sergio; Carrasco, Susana; Fernández-Fernández, Beatriz

    2013-01-01

    The calcineurin inhibitors (CNIs) cyclosporine (CsA) and tacrolimus are key drugs in current immunosuppressive regimes for solid organ transplantation. However, they are nephrotoxic and promote death and profibrotic responses in tubular cells. Moreover, renal inflammation is observed in CNI nephrotoxicity but the mechanisms are poorly understood. We have now studied molecular pathways leading to inflammation elicited by the CNIs in cultured and kidney tubular cells. Both CsA and tacrolimus elicited a proinflammatory response in tubular cells as evidenced by a transcriptomics approach. Transcriptomics also suggested several potential pathways leading to expression of proinflammatory genes. Validation and functional studies disclosed that in tubular cells, CNIs activated protein kinases such as the JAK2/STAT3 and TAK1/JNK/AP-1 pathways, TLR4/Myd88/IRAK signaling and the Unfolded Protein Response (UPR) to promote NF-κB activation and proinflammatory gene expression. CNIs also activated an Nrf2/HO-1-dependent compensatory response and the Nrf2 activator sulforaphane inhibited JAK2 and JNK activation and inflammation. A murine model of CsA nephrotoxicity corroborated activation of the proinflammatory pathways identified in cell cultures. Human CNIs nephrotoxicity was also associated with NF-κB, STAT3 and IRE1α activation. In conclusion, CNIs recruit several intracellular pathways leading to previously non-described proinflammatory actions in renal tubular cells. Identification of these pathways provides novel clues for therapeutic intervention to limit CNIs nephrotoxicity. - Highlights: • Molecular mechanisms modulating CNI renal inflammation were investigated. • Kinases, immune receptors and ER stress mediate the inflammatory response to CNIs. • Several intracellular pathways activate NF-κB in CNIs-treated tubular cells. • A NF-κB-dependent cytokine profile characterizes CNIs-induced inflammation. • CNI nephrotoxicity was associated to inflammatory

  11. VaCPK20, a calcium-dependent protein kinase gene of wild grapevine Vitis amurensis Rupr., mediates cold and drought stress tolerance.

    Science.gov (United States)

    Dubrovina, Alexandra S; Kiselev, Konstantin V; Khristenko, Valeriya S; Aleynova, Olga A

    2015-08-01

    Abiotic stresses, such as drought, salinity, cold and heat, are major environmental factors that limit crop productivity. Vitis amurensis Rupr. is a wild grapevine species displaying a high level of abiotic and biotic stress resistance. Protein kinases, including Ca(2+)-dependent protein kinases (CDPKs), are known to mediate plant acclimation to various environmental changes. However, the functions of most grape CDPKs have not been clarified. A recent CDPK gene expression analysis revealed that 10 CDPK genes of V. amurensis were up-regulated under different abiotic stress treatments. The expression of the VaCPK20 gene was significantly up-regulated under low and high temperature stress in V. amurensis. In the current study, the effects of overexpressing the VaCPK20 gene in callus cell lines of V. amurensis and transgenic plants of A. thaliana on their responses to abiotic stresses were investigated. Transgenic Arabidopsis overexpressing the VaCPK20 gene showed higher tolerance to freezing and drought stresses, and transgenic grape cell cultures overexpressing the VaCPK20 gene showed higher resistance to cold stress in comparison with the controls transformed by the "empty" vector. Heat and salt stress resistance of the transgenic V. amurensis calli and A. thaliana was comparable to that of the wild type and vector controls. Overexpression of the VaCPK20 gene increased the expression of stress-responsive genes, such as COR47, NHX1, KIN1, or ABF3, in the transgenic Arabidopsis plants under non-stress conditions, after freezing, and under drought stress. The results imply that VaCPK20 may act as a regulatory factor involved in cold and drought stress response pathways. Copyright © 2015 Elsevier GmbH. All rights reserved.

  12. A physiological role of AMP-activated protein kinase in phenobarbital-mediated constitutive androstane receptor activation and CYP2B induction.

    Science.gov (United States)

    Shindo, Sawako; Numazawa, Satoshi; Yoshida, Takemi

    2007-02-01

    CAR (constitutive androstane receptor) is a nuclear receptor that regulates the transcription of target genes, including CYP (cytochrome P450) 2B and 3A. The transactivation by CAR is regulated by its subcellular localization; however, the mechanism that governs nuclear translocation has yet to be clarified. It has been reported recently that AMPK (AMP-activated protein kinase) is involved in phenobarbital-mediated CYP2B induction in a particular culture system. We therefore investigated in vivo whether AMPK is involved in the activation of CAR-dependent gene expression. Immunoblot analysis using an antibody which recognizes Thr-172-phosphorylated AMPKalpha1/2 revealed phenobarbital-induced AMPK activation in rat and mouse livers as well. Phenobarbital, however, failed to increase the liver phospho-AMPK level of tumour-bearing rats in which CAR nuclear translocation had been impaired. In in vivo reporter gene assays employing PBREM (phenobarbital-responsive enhancer module) from CYP2B1, an AMPK inhibitor 8-bromo-AMP abolished phenobarbital-induced transactivation. In addition, Cyp2b10 gene expression was attenuated by 8-bromo-AMP. Forced expression of a dominant-negative mutant and the wild-type of AMPKalpha2 in the mouse liver suppressed and further enhanced phenobarbital-induced PBREM-reporter activity respectively. Moreover, the AMPK activator AICAR (5-amino-4-imidazolecarboxamide riboside) induced PBREM transactivation and an accumulation of CAR in the nuclear fraction of the mouse liver. However, AICAR and metformin, another AMPK activator, failed to induce hepatic CYP2B in mice and rats. These observations suggest that AMPK is at least partly involved in phenobarbital-originated signalling, but the kinase activation by itself is not sufficient for CYP2B induction in vivo.

  13. Effects of an High-Fat Diet Enriched in Lard or in Fish Oil on the Hypothalamic Amp-Activated Protein Kinase and Inflammatory Mediators

    Science.gov (United States)

    Viggiano, Emanuela; Mollica, Maria Pina; Lionetti, Lillà; Cavaliere, Gina; Trinchese, Giovanna; De Filippo, Chiara; Chieffi, Sergio; Gaita, Marcello; Barletta, Antonio; De Luca, Bruno; Crispino, Marianna; Monda, Marcellino

    2016-01-01

    The high fat diet (HFD) rich in lard induces obesity, inflammation and oxidative stress, and the deregulation of hypothalamic nuclei plays an important role in this mechanism. One important factor involved in the food intake and inflammation is adenosine monophosphate-dependent kinase (AMPK), a serine/threonine kinase activated by phosphorylation. Omega (ω)3-polyunsaturated fatty acids (PUFA) are dietary compounds known to attenuate the obesity-related diseases, although the molecular mechanisms underlying their actions in the hypothalamus are not completely understood. We hypothesized that the beneficial effects of PUFA may be mediated by AMPK in the hypothalamus. To this aim, rats were fed a control diet (CD), or isocaloric HFD containing either fish oil (FD; rich in ω3-PUFA) or lard for 6 weeks, and the activation of AMPK, inflammatory state (IKKβ, TNF-α) and oxidative stress were analyzed in the hypothalamus. In addition, we also studied serum lipid profile, homeostatic model assessment (HOMA) index, and pro-inflammatory parameters. Our results showed, at the hypothalamic level of LD-fed rats, an increase of AMPK activation, inflammation and oxidative stress, while no modifications were detected in FD-fed animals compared to CD. In addition body weight gain, serum lipid profile, pro-inflammatory parameters and insulin resistance were reduced in FD animals compared to LD. In conclusion, our data indicate that the substitution of saturated by unsaturated fatty acids in the diet has beneficial effects on modulation of hypothalamic inflammation and function in obesity, underlying, at hypothalamic level, the interaction among insulin and/or leptin resistance, AMPK activation and hyperphagia. PMID:27375435

  14. Calcineurin inhibitors recruit protein kinases JAK2 and JNK, TLR signaling and the UPR to activate NF-κB-mediated inflammatory responses in kidney tubular cells

    Energy Technology Data Exchange (ETDEWEB)

    González-Guerrero, Cristian, E-mail: cristian.gonzalez@fjd.es [Renal and Vascular Pathology Laboratory, Instituto de Investigación Sanitaria-Fundación Jiménez Díaz (IIS-FJD), Av. Reyes Católicos 2, 28040 Madrid (Spain); Ocaña-Salceda, Carlos, E-mail: carlos.ocana@fjd.es [Renal and Vascular Pathology Laboratory, Instituto de Investigación Sanitaria-Fundación Jiménez Díaz (IIS-FJD), Av. Reyes Católicos 2, 28040 Madrid (Spain); Berzal, Sergio, E-mail: sberzal@fjd.es [Renal and Vascular Pathology Laboratory, Instituto de Investigación Sanitaria-Fundación Jiménez Díaz (IIS-FJD), Av. Reyes Católicos 2, 28040 Madrid (Spain); Carrasco, Susana, E-mail: scarrasco@fjd.es [Renal and Vascular Pathology Laboratory, Instituto de Investigación Sanitaria-Fundación Jiménez Díaz (IIS-FJD), Av. Reyes Católicos 2, 28040 Madrid (Spain); Fernández-Fernández, Beatriz, E-mail: bfernandez@fjd.es [Nephrology, Instituto de Investigación Sanitaria-Fundación Jiménez Díaz (IIS-FJD), Av. Reyes Católicos 2, 28040 Madrid (Spain); and others

    2013-11-01

    The calcineurin inhibitors (CNIs) cyclosporine (CsA) and tacrolimus are key drugs in current immunosuppressive regimes for solid organ transplantation. However, they are nephrotoxic and promote death and profibrotic responses in tubular cells. Moreover, renal inflammation is observed in CNI nephrotoxicity but the mechanisms are poorly understood. We have now studied molecular pathways leading to inflammation elicited by the CNIs in cultured and kidney tubular cells. Both CsA and tacrolimus elicited a proinflammatory response in tubular cells as evidenced by a transcriptomics approach. Transcriptomics also suggested several potential pathways leading to expression of proinflammatory genes. Validation and functional studies disclosed that in tubular cells, CNIs activated protein kinases such as the JAK2/STAT3 and TAK1/JNK/AP-1 pathways, TLR4/Myd88/IRAK signaling and the Unfolded Protein Response (UPR) to promote NF-κB activation and proinflammatory gene expression. CNIs also activated an Nrf2/HO-1-dependent compensatory response and the Nrf2 activator sulforaphane inhibited JAK2 and JNK activation and inflammation. A murine model of CsA nephrotoxicity corroborated activation of the proinflammatory pathways identified in cell cultures. Human CNIs nephrotoxicity was also associated with NF-κB, STAT3 and IRE1α activation. In conclusion, CNIs recruit several intracellular pathways leading to previously non-described proinflammatory actions in renal tubular cells. Identification of these pathways provides novel clues for therapeutic intervention to limit CNIs nephrotoxicity. - Highlights: • Molecular mechanisms modulating CNI renal inflammation were investigated. • Kinases, immune receptors and ER stress mediate the inflammatory response to CNIs. • Several intracellular pathways activate NF-κB in CNIs-treated tubular cells. • A NF-κB-dependent cytokine profile characterizes CNIs-induced inflammation. • CNI nephrotoxicity was associated to inflammatory

  15. Intravenous Administration Is an Effective and Safe Route for Cancer Gene Therapy Using the Bifidobacterium-Mediated Recombinant HSV-1 Thymidine Kinase and Ganciclovir

    Directory of Open Access Journals (Sweden)

    Huicong Zhou

    2016-06-01

    Full Text Available The herpes simplex virus thymidine kinase/ganciclovir (HSV TK/GCV system is one of the best studied cancer suicide gene therapy systems. Our previous study showed that caspase 3 expression was upregulated and bladder tumor growth was significantly reduced in rats treated with a combination of Bifidobacterium (BF and HSV TK/GCV (BF-rTK/GCV. However, it was raised whether the BF-mediated recombinant thymidine kinase combined with ganciclovir (BF-rTK/GCV was safe to administer via venous for cancer gene therapy. To answer this question, the antitumor effects of BF-rTK/GCV were mainly evaluated in a xenograft nude mouse model bearing MKN-45 gastric tumor cells. The immune response, including analysis of cytokine profiles, was analyzed to evaluate the safety of intramuscular and intravenous injection of BF-rTK in BALB/c mice. The results suggested that gastric tumor growth was significantly inhibited in vivo by BF-rTK/GCV. However, the BF-rTK/GCV had no effect on mouse body weight, indicating that the treatment was safe for the host. The results of cytokine profile analysis indicated that intravenous injection of a low dose of BF-rTK resulted in a weaker cytokine response than that obtained with intramuscular injection. Furthermore, immunohistochemical analysis showed that intravenous administration did not affect the expression of immune-associated TLR2 and TLR4. Finally, the BF-rTK/GCV inhibited vascular endothelial growth factor (VEGF expression in mouse model, which is helpful for inhibiting of tumor angiogenesis. That meant intravenous administration of BF-rTK/GCV was an effective and safe way for cancer gene therapy.

  16. The mechanism of action of ursolic acid as insulin secretagogue and insulinomimetic is mediated by cross-talk between calcium and kinases to regulate glucose balance.

    Science.gov (United States)

    Castro, Allisson Jhonatan Gomes; Frederico, Marisa Jádna Silva; Cazarolli, Luisa Helena; Mendes, Camila Pires; Bretanha, Lizandra Czermainski; Schmidt, Éder Carlos; Bouzon, Zenilda Laurita; de Medeiros Pinto, Veronica Aiceles; da Fonte Ramos, Cristiane; Pizzolatti, Moacir Geraldo; Silva, Fátima Regina Mena Barreto

    2015-01-01

    The effect of in vivo treatment with ursolic acid (UA) on glycemia in hyperglycemic rats and its mechanism of action on muscle were studied. The UA effects on glycemia, glycogen, LDH, calcium and on insulin levels were evaluated after glucose tolerance curve. The β-cells were evaluated through the transmission electron microscopy. UA mechanism of action was studied on muscles through the glucose uptake with/without specific insulin signaling inhibitors. The nuclear effect of UA and the GLUT4 expression on muscle were studied using thymidine, GLUT4 immunocontent, immunofluorescence and RT-PCR. UA presented a potent antihyperglycemic effect, increased insulin vesicle translocation, insulin secretion and augmented glycogen content. Also, UA stimulates the glucose uptake through the involvement of the classical insulin signaling related to the GLUT4 translocation to the plasma membrane as well as the GLUT4 synthesis. These were characterized by increasing the GLUT4 mRNA expression, the activation of DNA transcription, the expression of GLUT4 and its presence at plasma membrane. Also, the modulation of calcium, phospholipase C, protein kinase C and PKCaM II is mandatory for the full stimulatory effect of UA on glucose uptake. UA did not change the serum LDH and serum calcium balance. The antihyperglycemic role of UA is mediated through insulin secretion and insulinomimetic effect on glucose uptake, synthesis and translocation of GLUT4 by a mechanism of cross-talk between calcium and protein kinases. UA is a potential anti-diabetic agent with pharmacological properties for insulin resistance and diabetes therapy. Copyright © 2014 Elsevier B.V. All rights reserved.

  17. Feedback regulation on PTEN/AKT pathway by the ER stress kinase PERK mediated by interaction with the Vault complex

    DEFF Research Database (Denmark)

    Zhang, Wei; Neo, Suat Peng; Gunaratne, Jayantha

    2015-01-01

    inhibition of PERK has been reported to limit tumor growth in xenograft models. Here we provide evidence that inactive PERK interacts with the nuclear pore-associated Vault complex protein and that this compromises Vault-mediated nuclear transport of PTEN. Pharmacological inhibition of PERK under ER stress...... results is abnormal sequestration of the Vault complex, leading to increased cytoplasmic PTEN activity and lower AKT activation. As the PI3K/PTEN/AKT pathway is crucial for many aspects of cell growth and survival, this unexpected effect of PERK inhibitors on AKT activity may have implications...

  18. SPHK1/sphingosine kinase 1-mediated autophagy differs between neurons and SH-SY5Y neuroblastoma cells.

    Science.gov (United States)

    Moruno Manchon, Jose Felix; Uzor, Ndidi-Ese; Finkbeiner, Steven; Tsvetkov, Andrey S

    2016-08-02

    Although implicated in neurodegeneration, autophagy has been characterized mostly in yeast and mammalian non-neuronal cells. In a recent study, we sought to determine if SPHK1 (sphingosine kinase 1), implicated previously in macroautophagy/autophagy in cancer cells, regulates autophagy in neurons. SPHK1 synthesizes sphingosine-1-phosphate (S1P), a bioactive lipid involved in cell survival. In our study, we discovered that, when neuronal autophagy is pharmacologically stimulated, SPHK1 relocalizes to the endocytic and autophagic organelles. Interestingly, in non-neuronal cells stimulated with growth factors, SPHK1 translocates to the plasma membrane, where it phosphorylates sphingosine to produce S1P. Whether SPHK1 also binds to the endocytic and autophagic organelles in non-neuronal cells upon induction of autophagy has not been demonstrated. Here, we determined if the effect in neurons is operant in the SH-SY5Y neuroblastoma cell line. In both non-differentiated and differentiated SH-SY5Y cells, a short incubation of cells in amino acid-free medium stimulated the formation of SPHK1-positive puncta, as in neurons. We also found that, unlike neurons in which these puncta represent endosomes, autophagosomes, and amphisomes, in SH-SY5Y cells SPHK1 is bound only to the endosomes. In addition, a dominant negative form of SPHK1 was very toxic to SH-SY5Y cells, but cultured primary cortical neurons tolerated it significantly better. These results suggest that autophagy in neurons is regulated by mechanisms that differ, at least in part, from those in SH-SY5Y cells.

  19. Neuronal extracellular signal-regulated kinase (ERK activity as marker and mediator of alcohol and opioid dependence

    Directory of Open Access Journals (Sweden)

    Eva R. Zamora-Martinez

    2014-03-01

    Full Text Available Early pioneering work in the field of biochemistry identified phosphorylation as a crucial post-translational modification of proteins with the ability to both indicate and arbitrate complex physiological processes. More recent investigations have functionally linked phosphorylation of extracellular signal-regulated kinase (ERK to a variety of neurophysiological mechanisms ranging from acute neurotransmitter action to long-term gene expression. ERK phosphorylation serves as an intracellular bridging mechanism that facilitates neuronal communication and plasticity. Drugs of abuse, including alcohol and opioids, act as artificial yet powerful rewards that impinge upon natural reinforcement processes critical for survival. The graded progression from initial exposure to addiction (or substance dependence is believed to result from drug- and drug context-induced adaptations in neuronal signaling processes across brain reward and stress circuits following excessive drug use. In this regard, commonly abused drugs as well as drug-associated experiences are capable of modifying the phosphorylation of ERK within central reinforcement systems. In addition, chronic drug and alcohol exposure may drive ERK-regulated epigenetic and structural alterations that underlie a long-term propensity for escalating drug use. Under the influence of such a neurobiological vulnerability, encountering drug-associated cues and contexts can produce subsequent alterations in ERK signaling that drive relapse to drug and alcohol seeking. Current studies are determining precisely which molecular and regional ERK phosphorylation-associated events contribute to the addiction process, as well as which neuroadaptations need to be targeted in order to return dependent individuals to a healthy state.

  20. Rat vas deferens SERCA2 is modulated by Ca{sup 2+}/calmodulin protein kinase II-mediated phosphorylation

    Energy Technology Data Exchange (ETDEWEB)

    Rodriguez, J.B.R.; Muzi-Filho, H. [Programa de Farmacologia e Inflamação, Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ (Brazil); Valverde, R.H.F. [Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ (Brazil); Quintas, L.E.M. [Programa de Farmacologia e Inflamação, Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ (Brazil); Noel, F. [Programa de Desenvolvimento de Fármacos, Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ (Brazil); Einicker-Lamas, M. [Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ (Brazil); Instituto Nacional de Ciência e Tecnologia em Biologia Estrutural e Bioimagem, Rio de Janeiro, RJ (Brazil); Cunha, V.M.N. [Programa de Farmacologia e Inflamação, Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ (Brazil)

    2013-03-19

    Ca{sup 2+} pumps are important players in smooth muscle contraction. Nevertheless, little information is available about these pumps in the vas deferens. We have determined which subtype of sarco(endo)plasmic reticulum Ca{sup 2+}-ATPase isoform (SERCA) is expressed in rat vas deferens (RVD) and its modulation by calmodulin (CaM)-dependent mechanisms. The thapsigargin-sensitive Ca{sup 2+}-ATPase from a membrane fraction containing the highest SERCA levels in the RVD homogenate has the same molecular mass (∼115 kDa) as that of SERCA2 from the rat cerebellum. It has a very high affinity for Ca{sup 2+} (Ca{sub 0.5} = 780 nM) and a low sensitivity to vanadate (IC{sub 50} = 41 µM). These facts indicate that SERCA2 is present in the RVD. Immunoblotting for CaM and Ca{sup 2+}/calmodulin-dependent protein kinase II (CaMKII) showed the expression of these two regulatory proteins. Ca{sup 2+} and CaM increased serine-phosphorylated residues of the 115-kDa protein, indicating the involvement of CaMKII in the regulatory phosphorylation of SERCA2. Phosphorylation is accompanied by an 8-fold increase of thapsigargin-sensitive Ca{sup 2+} accumulation in the lumen of vesicles derived from these membranes. These data establish that SERCA2 in the RVD is modulated by Ca{sup 2+} and CaM, possibly via CaMKII, in a process that results in stimulation of Ca{sup 2+} pumping activity.

  1. Atorvastatin reduces lipid accumulation in the liver by activating protein kinase A-mediated phosphorylation of perilipin 5.

    Science.gov (United States)

    Gao, Xing; Nan, Yang; Zhao, Yuanlin; Yuan, Yuan; Ren, Bincheng; Sun, Chao; Cao, Kaiyu; Yu, Ming; Feng, Xuyang; Ye, Jing

    2017-12-01

    Statins have been proven to be effective in treating non-alcoholic fatty liver disease (NAFLD). Recently, it was reported that statins decreased the hepatic expression of perilipin 5 (Plin5), a lipid droplet (LD)-associated protein, which plays critical roles in regulating lipid accumulation and lipolysis in liver. However, the function and regulation mechanism of Plin5 have not yet been well-established in NAFLD treatment with statins. In this study, we observed that atorvastatin moderately reduced the expression of Plin5 in livers without changing the protein level of Plin5 in the hepatic LD fraction of mice fed with high-fat diet (HFD). Intriguingly, atorvastatin stimulated the PKA-mediated phosphorylation of Plin5 and reduced the triglyceride (TG) accumulation in hepatocytes with overexpression of wide type (Plin5-WT) compared to serine-155 mutant Plin5 (Plin5-S155A). Moreover, PKA-stimulated FA release of purified LDs carrying Plin5-WT but not Plin5-S155A. Glucagon, a PKA activator, stimulated the phosphorylation of Plin5-WT and inhibited its interaction with CGI-58. The results indicated that atorvastatin promoted lipolysis and reduced TG accumulation in the liver by increasing PKA-mediated phosphorylation of Plin5. This new mechanism of lipid-lowering effects of atorvastatin might provide a new strategy for NAFLD treatment. Copyright © 2017. Published by Elsevier B.V.

  2. Protein kinase A-dependent Neuronal Nitric Oxide Synthase Activation Mediates the Enhancement of Baroreflex Response by Adrenomedullin in the Nucleus Tractus Solitarii of Rats

    Directory of Open Access Journals (Sweden)

    Ho I-Chun

    2011-05-01

    Full Text Available Abstract Background Adrenomedullin (ADM exerts its biological functions through the receptor-mediated enzymatic mechanisms that involve protein kinase A (PKA, or neuronal nitric oxide synthase (nNOS. We previously demonstrated that the receptor-mediated cAMP/PKA pathway involves in ADM-enhanced baroreceptor reflex (BRR response. It remains unclear whether ADM may enhance BRR response via activation of nNOS-dependent mechanism in the nucleus tractus solitarii (NTS. Methods Intravenous injection of phenylephrine was administered to evoke the BRR before and at 10, 30, and 60 min after microinjection of the test agents into NTS of Sprague-Dawley rats. Western blotting analysis was used to measure the level and phosphorylation of proteins that involved in BRR-enhancing effects of ADM (0.2 pmol in NTS. The colocalization of PKA and nNOS was examined by immunohistochemical staining and observed with a laser confocal microscope. Results We found that ADM-induced enhancement of BRR response was blunted by microinjection of NPLA or Rp-8-Br-cGMP, a selective inhibitor of nNOS or protein kinase G (PKG respectively, into NTS. Western blot analysis further revealed that ADM induced an increase in the protein level of PKG-I which could be attenuated by co-microinjection with the ADM receptor antagonist ADM22-52 or NPLA. Moreover, we observed an increase in phosphorylation at Ser1416 of nNOS at 10, 30, and 60 min after intra-NTS administration of ADM. As such, nNOS/PKG signaling may also account for the enhancing effect of ADM on BRR response. Interestingly, biochemical evidence further showed that ADM-induced increase of nNOS phosphorylation was prevented by co-microinjection with Rp-8-Br-cAMP, a PKA inhibitor. The possibility of PKA-dependent nNOS activation was substantiated by immunohistochemical demonstration of co-localization of PKA and nNOS in putative NTS neurons. Conclusions The novel finding of this study is that the signal transduction cascade that

  3. Primate Torpor: Regulation of Stress-activated Protein Kinases During Daily Torpor in the Gray Mouse Lemur, Microcebus murinus

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    Kyle K. Biggar

    2015-04-01

    Full Text Available Very few selected species of primates are known to be capable of entering torpor. This exciting discovery means that the ability to enter a natural state of dormancy is an ancestral trait among primates and, in phylogenetic terms, is very close to the human lineage. To explore the regulatory mechanisms that underlie primate torpor, we analyzed signal transduction cascades to discover those involved in coordinating tissue responses during torpor. The responses of mitogen-activated protein kinase (MAPK family members to primate torpor were compared in six organs of control (aroused versus torpid gray mouse lemurs, Microcebus murinus. The proteins examined include extracellular signal-regulated kinases (ERKs, c-jun NH2-terminal kinases (JNKs, MAPK kinase (MEK, and p38, in addition to stress-related proteins p53 and heat shock protein 27 (HSP27. The activation of specific MAPK signal transduction pathways may provide a mechanism to regulate the expression of torpor-responsive genes or the regulation of selected downstream cellular processes. In response to torpor, each MAPK subfamily responded differently during torpor and each showed organ-specific patterns of response. For example, skeletal muscle displayed elevated relative phosphorylation of ERK1/2 during torpor. Interestingly, adipose tissues showed the highest degree of MAPK activation. Brown adipose tissue displayed an activation of ERK1/2 and p38, whereas white adipose tissue showed activation of ERK1/2, p38, MEK, and JNK during torpor. Importantly, both adipose tissues possess specialized functions that are critical for torpor, with brown adipose required for non-shivering thermogenesis and white adipose utilized as the primary source of lipid fuel for torpor. Overall, these data indicate crucial roles of MAPKs in the regulation of primate organs during torpor.

  4. Reduced Mechanical Stretch Induces Enhanced Endothelin B Receptor-mediated Contractility via Activation of Focal Adhesion Kinase and Extra Cellular-regulated Kinase 1/2 in Cerebral Arteries from Rat

    DEFF Research Database (Denmark)

    Spray, Stine; Rasmussen, Marianne N P; Skovsted, Gry F

    2016-01-01

    ETB receptor agonist sarafotoxin 6c. The involvement of extracellular regulated kinase (ERK) 1/2 and focal adhesion kinase (FAK) were studied by their specific inhibitors U0126 and PF-228, respectively. Compared to their stretched counterparts, un-stretched MCA segments showed a significantly...

  5. Salidroside attenuates inflammatory responses by suppressing nuclear factor-κB and mitogen activated protein kinases activation in lipopolysaccharide-induced mastitis in mice.

    Science.gov (United States)

    Li, Depeng; Fu, Yunhe; Zhang, Wen; Su, Gaoli; Liu, Bo; Guo, Mengyao; Li, Fengyang; Liang, Dejie; Liu, Zhicheng; Zhang, Xichen; Cao, Yongguo; Zhang, Naisheng; Yang, Zhengtao

    2013-01-01

    Mastitis is defined as inflammation of the mammary gland in domestic dairy animals and humans. Salidroside, a major component isolated from Rhodiola rosea L., has potent anti-inflammatory properties, but whether it can be used in mastitis treatment has not yet been investigated. The aim of this study was to assess the protective effects of salidroside against lipopolysaccharide (LPS)-induced mastitis in mice and the mechanism of action. We used a mouse mastitis model in which mammary gland inflammation was induced by LPS challenge. Salidroside administered 1 h before LPS infusion significantly attenuated inflammatory cell infiltration, reduced the activity of myeloperoxidase in mammary tissue, and decreased the concentration of tumor necrosis factor-α, interleukin (IL)-1β, and IL-6 in a dose-dependent manner. Further studies revealed that salidroside down-regulated phosphorylation of LPS-induced nuclear transcription factor-kappaB (NF-κB) p65 and inhibitor of NF-κB α (IκBα) in the NF-κB signal pathway, and suppressed phosphorylation of p38, extracellular signal-regulated kinase (ERK) and c-jun NH(2)-terminal kinase (JNK) in MAPKs signal pathways. This study demonstrates that salidroside is an effective suppressor of inflammation and may be a candidate for the prophylaxis of mastitis.

  6. Lead decreases cell survival, proliferation, and neuronal differentiation of primary cultured adult neural precursor cells through activation of the JNK and p38 MAP kinases

    Science.gov (United States)

    Engstrom, Anna; Wang, Hao; Xia, Zhengui

    2015-01-01

    Adult hippocampal neurogenesis is the process whereby adult neural precursor cells (aNPCs) in the subgranular zone (SGZ) of the dentate gyrus (DG) generate adult-born, functional neurons in the hippocampus. This process is modulated by various extracellular and intracellular stimuli, and the adult-born neurons have been implicated in hippocampus-dependent learning and memory. However, studies on how neurotoxic agents affect this process and the underlying mechanisms are limited. The goal of this study was to determine whether lead, a heavy metal, directly impairs critical processes in adult neurogenesis and to characterize the underlying signaling pathways using primary cultured SGZ-aNPCs isolated from adult mice. We report here that lead significantly increases apoptosis and inhibits proliferation in SGZ-aNPCs. In addition, lead significantly impairs spontaneous neuronal differentiation and maturation. Furthermore, we found that activation of the c-Jun NH2-terminal kinase (JNK) and p38 mitogen activated protein (MAP) kinase signaling pathways are important for lead cytotoxicity. Our data suggest that lead can directly act on adult neural stem cells and impair critical processes in adult hippocampal neurogenesis, which may contribute to its neurotoxicity and adverse effects on cognition in adults. PMID:25967738

  7. HIV-mediated phosphatidylinositol 3-kinase/serine-threonine kinase activation in APCs leads to programmed death-1 ligand upregulation and suppression of HIV-specific CD8 T cells.

    Science.gov (United States)

    Muthumani, Karuppiah; Shedlock, Devon J; Choo, Daniel K; Fagone, Paolo; Kawalekar, Omkar U; Goodman, Jonathan; Bian, Chaoran B; Ramanathan, Aarti A; Atman, Parikh; Tebas, Pablo; Chattergoon, Michael A; Choo, Andrew Y; Weiner, David B

    2011-09-15

    Recent evidence demonstrates that HIV-1 infection leads to the attenuation of cellular immune responses, which has been correlated with the increased expression of programmed death (PD)-1 on virus-specific CD8(+) T cells. PD-1 is induced upon T cell activation, and its prolonged expression facilitates CD8(+) T cell inhibitory signals when bound to its B7 family ligands, PD-ligand (L)1/2, which are expressed on APCs. Importantly, early reports demonstrated that blockade of the PD-1/PD-L interaction by Abs may help to counter the development of immune exhaustion driven by HIV viral persistence. To better understand the regulation of the PD-1 pathway during HIV infection, we examined the ability of the virus to induce PD-L expression on macrophages and dendritic cells. We found a direct relationship between the infection of APCs and the expression of PD-L1 in which virus-mediated upregulation induced a state of nonresponsiveness in uninfected HIV-specific T cells. Furthermore, this exhaustion phenotype was revitalized by the blockade of PD-L1, after which T cells regained their capacity for proliferation and the secretion of proinflammatory cytokines IFN-γ, IL-2, and IL-12 upon restimulation. In addition, we identify a critical role for the PI3K/serine-threonine kinase signaling pathway in PD-L1 upregulation of APCs by HIV, because inhibition of these intracellular signal transducer enzymes significantly reduced PD-L1 induction by infection. These data identify a novel mechanism by which HIV exploits the immunosuppressive PD-1 pathway and suggest a new role for virus-infected cells in the local corruption of immune responses required for viral suppression.

  8. Testin, a novel binding partner of the calcium-sensing receptor, enhances receptor-mediated Rho-kinase signalling

    International Nuclear Information System (INIS)

    Magno, Aaron L.; Ingley, Evan; Brown, Suzanne J.; Conigrave, Arthur D.; Ratajczak, Thomas; Ward, Bryan K.

    2011-01-01

    Highlights: → A yeast two-hybrid screen revealed testin bound to the calcium-sensing receptor. → The second zinc finger of LIM domain 1 of testin is critical for interaction. → Testin bound to a region of the receptor tail important for cell signalling. → Testin and receptor interaction was confirmed in mammalian (HEK293) cells. → Overexpression of testin enhanced receptor-mediated Rho signalling in HEK293 cells. -- Abstract: The calcium-sensing receptor (CaR) plays an integral role in calcium homeostasis and the regulation of other cellular functions including cell proliferation and cytoskeletal organisation. The multifunctional nature of the CaR is manifested through ligand-dependent stimulation of different signalling pathways that are also regulated by partner binding proteins. Following a yeast two-hybrid library screen using the intracellular tail of the CaR as bait, we identified several novel binding partners including the focal adhesion protein, testin. Testin has not previously been shown to interact with cell surface receptors. The sites of interaction between the CaR and testin were mapped to the membrane proximal region of the receptor tail and the second zinc-finger of LIM domain 1 of testin, the integrity of which was found to be critical for the CaR-testin interaction. The CaR-testin association was confirmed in HEK293 cells by coimmunoprecipitation and confocal microscopy studies. Ectopic expression of testin in HEK293 cells stably expressing the CaR enhanced CaR-stimulated Rho activity but had no effect on CaR-stimulated ERK signalling. These results suggest an interplay between the CaR and testin in the regulation of CaR-mediated Rho signalling with possible effects on the cytoskeleton.

  9. The mitogen-activated protein kinase pathway mediates growth arrest or E1A-dependent apoptosis in SKBR3 human breast cancer cells.

    Science.gov (United States)

    Blagosklonny, M V

    1998-11-09

    Previously, we have shown that phorbol ester (PMA) induces p21(WAF1/CIP1)-dependent growth arrest in SKBr3 breast cancer and LNCaP prostate cancer cells. Here, I demonstrate that inhibition of Raf-1 kinase by dominant-negative Raf-1 or pharmacological depletion of Raf-1 prevented PMA-mediated induction of p21(WAF1/CIP1). Similarly, PD98059, a specific inhibitor of MEK, abolished p21(WAF1/CIP1) induction and PMA-induced growth arrest. Like PMA, the H-ras oncogene, another activator of the Raf-1/MEK/MAPK pathway, transactivated p21(WAF1/CIP1) in SKBr3 cells. I further investigated PMA-induced growth arrest following infection of SKBr3 cells with 12S E1A-expressing adenovirus. Although high levels of E1A oncoprotein prevented both PMA-induced p21(WAF1/CIP1) and growth arrest, smaller amounts of E1A abrogated growth arrest without down-regulation of p21(WAF1/CIP1). Therefore, E1A can stimulate proliferation downstream of p21(WAF1/CIP1). Albeit less effective than full activity, either Rb- or p300-binding activity of E1A was sufficient for the abrogation of PMA-mediated growth arrest. E1A-driven proliferation of PMA-treated SKBr3 cells was accompanied by apoptosis. New therapeutic approaches can be envisioned that would utilize stimulation of the Raf-1/MEK/MAPK pathway to inhibit growth of PMA-sensitive cancer cells.

  10. Adenosine monophosphate activated protein kinase (AMPK), a mediator of estradiol-induced apoptosis in long-term estrogen deprived breast cancer cells.

    Science.gov (United States)

    Chen, Haiyan; Wang, Ji-Ping; Santen, Richard J; Yue, Wei

    2015-06-01

    Estrogens stimulate growth of hormone-dependent breast cancer but paradoxically induce tumor regress under certain circumstances. We have shown that long-term estrogen deprivation (LTED) enhances the sensitivity of hormone dependent breast cancer cells to estradiol (E2) so that physiological concentrations of estradiol induce apoptosis in these cells. E2-induced apoptosis involve both intrinsic and extrinsic pathways but precise mechanisms remain unclear. We found that exposure of LTED MCF-7 cells to E2 activated AMP activated protein kinase (AMPK). In contrast, E2 inhibited AMPK activation in wild type MCF-7 cells where E2 prevents apoptosis. As a result of AMPK activation, the transcriptional activity of FoxO3, a downstream factor of AMPK, was up-regulated in E2 treatment of LTED. Increased activity of FoxO3 was demonstrated by up-regulation of three FoxO3 target genes, Bim, Fas ligand (FasL), and Gadd45α. Among them, Bim and FasL mediate intrinsic and extrinsic apoptosis respectively and Gadd45α causes cell cycle arrest at the G2/M phase. To further confirm the role of AMPK in apoptosis, we used AMPK activator AICAR in wild type MCF-7 cells and examined apoptosis, proliferation and expression of Bim, FasL, and Gadd45α. The effects of AICAR on these parameters recapitulated those observed in E2-treated LTED cells. Activation of AMPK by AICAR also increased expression of Bax in MCF-7 cells and its localization to mitochondria, which is a required process for apoptosis. These results reveal that AMPK is an important factor mediating E2-induced apoptosis in LTED cells, which is implicative of therapeutic potential for relapsing breast cancer after hormone therapy.

  11. Follicle-stimulating hormone (FSH activates extracellular signal-regulated kinase phosphorylation independently of beta-arrestin- and dynamin-mediated FSH receptor internalization

    Directory of Open Access Journals (Sweden)

    Crepieux Pascale

    2006-06-01

    Full Text Available Abstract Background The follicle-stimulating hormone receptor (FSH-R is a seven transmembrane spanning receptor (7TMR which plays a crucial role in male and female reproduction. Upon FSH stimulation, the FSH-R activates the extracellular signal-regulated kinases (ERK. However, the mechanisms whereby the agonist-stimulated FSH-R activates ERK are poorly understood. In order to activate ERK, some 7 TMRs require beta-arrestin-and dynamin-dependent internalization to occur, whereas some others do not. In the present study, we examined the ability of the FSH-activated FSH-R to induce ERK phosphorylation, in conditions where its beta-arrestin- and dynamin-mediated internalization was impaired. Methods Human embryonic kidney (HEK 293 cells were transiently transfected with the rat FSH-R. Internalization of the FSH-R was manipulated by co-expression of either a beta-arrestin (319–418 dominant negative peptide, either an inactive dynamin K44A mutant or of wild-type beta-arrestin 1 or 2. The outcomes on the FSH-R internalization were assayed by measuring 125I-FSH binding at the cell surface when compared to internalized 125I-FSH binding. The resulting ERK phosphorylation level was visualized by Western blot analysis. Results In HEK 293 cells, FSH stimulated ERK phosphorylation in a dose-dependent manner. Co-transfection of the beta- arrestin (319–418 construct, or of the dynamin K44A mutant reduced FSH-R internalization in response to FSH, without affecting ERK phosphorylation. Likewise, overexpression of wild-type beta-arrestin 1 or 2 significantly increased the FSH-R internalization level in response to FSH, without altering FSH-induced ERK phosphorylation. Conclusion From these results, we conclude that the FSH-R does not require beta-arrestin- nor dynamin-mediated internalization to initiate ERK phosphorylation in response to FSH.

  12. Structure-based network analysis of activation mechanisms in the ErbB family of receptor tyrosine kinases: the regulatory spine residues are global mediators of structural stability and allosteric interactions.

    Directory of Open Access Journals (Sweden)

    Kevin A James

    Full Text Available The ErbB protein tyrosine kinases are among the most important cell signaling families and mutation-induced modulation of their activity is associated with diverse functions in biological networks and human disease. We have combined molecular dynamics simulations of the ErbB kinases with the protein structure network modeling to characterize the reorganization of the residue interaction networks during conformational equilibrium changes in the normal and oncogenic forms. Structural stability and network analyses have identified local communities integrated around high centrality sites that correspond to the regulatory spine residues. This analysis has provided a quantitative insight to the mechanism of mutation-induced "superacceptor" activity in oncogenic EGFR dimers. We have found that kinase activation may be determined by allosteric interactions between modules of structurally stable residues that synchronize the dynamics in the nucleotide binding site and the αC-helix with the collective motions of the integrating αF-helix and the substrate binding site. The results of this study have pointed to a central role of the conserved His-Arg-Asp (HRD motif in the catalytic loop and the Asp-Phe-Gly (DFG motif as key mediators of structural stability and allosteric communications in the ErbB kinases. We have determined that residues that are indispensable for kinase regulation and catalysis often corresponded to the high centrality nodes within the protein structure network and could be distinguished by their unique network signatures. The optimal communication pathways are also controlled by these nodes and may ensure efficient allosteric signaling in the functional kinase state. Structure-based network analysis has quantified subtle effects of ATP binding on conformational dynamics and stability of the EGFR structures. Consistent with the NMR studies, we have found that nucleotide-induced modulation of the residue interaction networks is not

  13. Leptin enhances NR2B-mediated N-methyl-D-aspartate responses via a mitogen-activated protein kinase-dependent process in cerebellar granule cells.

    Science.gov (United States)

    Irving, A J; Wallace, L; Durakoglugil, D; Harvey, J

    2006-01-01

    It is well documented that the hormone leptin regulates energy balance via its actions in the hypothalamus. However, evidence is accumulating that leptin plays a key role in numerous CNS functions. Indeed, leptin receptors are expressed in many extrahypothalamic brain regions, with high levels found in the hippocampus and cerebellum. In the hippocampus leptin has been shown to facilitate N-methyl-D-aspartate receptor function and modulate synaptic plasticity. A role for leptin in cerebellar function is also indicated as leptin-deficient rodents display reduced mobility that is unrelated to obesity. Here we show that leptin receptor immunolabeling can be detected in cultured cerebellar granule cells, being expressed at the somatic plasma membrane and also concentrated at synapses. Furthermore, leptin facilitated NR2B N-methyl-D-aspartate receptor-mediated Ca2+ influx in cerebellar granule cells via a mitogen-activated protein kinase-dependent pathway. These findings provide the first direct evidence for a cellular action of leptin in cerebellar neurons. In addition, given that N-methyl-D-aspartate receptor activity in the cerebellum is crucial for normal locomotor function, these data also have important implications for the potential role of leptin in the control of movement.

  14. Src Family Kinases Mediate Betel Quid-Induced Oral Cancer Cell Motility and Could Be a Biomarker for Early Invasion in Oral Squamous Cell Carcinoma

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    Jeff Yi-Fu Chen

    2008-12-01

    Full Text Available Betel quid (BQ-chewing oral cancer is a prevalent disease in many countries of Southeast Asia. Yet, the precise disease mechanism remains largely unknown. Here, we show that BQ extract-induced cell motility in three oral cancer cells (Ca9-22, SAS, and SCC9 presumably involves the Src family kinases (SFKs. Besides, BQ extract can markedly induce cell migration of wild type mouse embryonic fibroblasts (MEFs but not MEFs lacking three SFK members, namely, Src, Yes, and Fyn, indicating the requirement of SFKs for BQ-induced cell motility. Betel quid extract can also elevate cellular SFK activities because phosphorylation of tyrosine 416 at the catalytic domain is increased, which in turn promotes phosphorylation of an in vitro substrate, enolase. Furthermore, we identified that areca nut, a major component of BQ, is the key factor accounting for BQ-induced cell migration and invasion through SFKs-mediated signaling pathways. Immunohistochemistry revealed that, particularly in BQ-chewing cases, the activity of SFKs was significantly higher in tumor-adjacent mucosa than that in solid tumor areas (P < .01. These results suggest a possible role of SFKs in tumor-host interface and thus in early tumor invasion in vivo. Consistent with this is the observation that activation of SFKs is colocalized with invasive tumor fronts in oral squamous cell carcinoma. Together, we conclude that SFKs may represent a potential biomarker of invasion and therapeutic target in BQ-induced oral cancer.

  15. Adeno-associated virus (AAV-mediated suppression of Ca2+/calmodulin kinase IV activity in the nucleus accumbens modulates emotional behaviour in mice

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    Bading Hilmar

    2007-12-01

    Full Text Available Abstract Background Calcium/calmodulin-dependent protein kinase IV (CaMKIV controls activity-dependent gene transcription by regulating the activity of the cyclic AMP response element binding protein (CREB. This signaling pathway is involved in gating emotional responses in the CNS but previous studies did not address the potential roles of CaMKIV in discrete brain regions. In the present study, we aimed at specifically dissecting the role of CaMKIV in the nucleus accumbens of adult mice. Results We used recombinant adeno-associated virus (rAAV-mediated gene transfer of a dominant-negative CaMKIV variant (rAAV-dnCaMKIV to inhibit endogenous CaMKIV in the nucleus accumbens. rAAV-dnCaMKIV treated animals were subjected to a battery of tests including, prepulse inhibition of the acoustic startle response, open field, social interaction and anxiety-related behaviour. We found that basal locomotor activity in the open field, and prepulse inhibition or startle performance were unaltered in mice infected with rAAV-dnCaMKIV in the nucleus accumbens. However, anxiogenic effects were revealed in social interaction testing and the light/dark emergence test. Conclusion Our findings suggest a modulatory role of CaMKIV in the nucleus accumbens in anxiety-like behaviour but not sensorimotor gating.

  16. Eps15R is a tyrosine kinase substrate with characteristics of a docking protein possibly involved in coated pits-mediated internalization

    DEFF Research Database (Denmark)

    Coda, L; Salcini, A E; Confalonieri, S

    1998-01-01

    eps15R was identified because of its relatedness to eps15, a gene encoding a tyrosine kinase substrate bearing a novel protein-protein interaction domain, called EH. In this paper, we report a biochemical characterization of the eps15R gene product(s). In NIH-3T3 cells, three proteins of 125, 108......, and 76 kDa were specifically recognized by anti-eps15R sera. The 125-kDa species is a bona fide product of the eps15R gene, whereas p108 and p76 are most likely products of alternative splicing events. Eps15R protein(s) are tyrosine-phosphorylated following epidermal growth factor receptor activation...... in NIH-3T3 cells overexpressing the receptor, even at low levels of receptor occupancy, thus behaving as physiological substrates. A role for eps15R in clathrin-mediated endocytosis is suggested by its localization in plasma membrane-coated pits and in vivo association to the coated pits' adapter protein...

  17. AS160 associates with the Na+,K+-ATPase and mediates the adenosine monophosphate-stimulated protein kinase-dependent regulation of sodium pump surface expression.

    Science.gov (United States)

    Alves, Daiane S; Farr, Glen A; Seo-Mayer, Patricia; Caplan, Michael J

    2010-12-01

    The Na(+),K(+)-ATPase is the major active transport protein found in the plasma membranes of most epithelial cell types. The regulation of Na(+),K(+)-ATPase activity involves a variety of mechanisms, including regulated endocytosis and recycling. Our efforts to identify novel Na(+),K(+)-ATPase binding partners revealed a direct association between the Na(+),K(+)-ATPase and AS160, a Rab-GTPase-activating protein. In COS cells, coexpression of AS160 and Na(+),K(+)-ATPase led to the intracellular retention of the sodium pump. We find that AS160 interacts with the large cytoplasmic NP domain of the α-subunit of the Na(+),K(+)-ATPase. Inhibition of the activity of the adenosine monophosphate-stimulated protein kinase (AMPK) in Madin-Darby canine kidney cells through treatment with Compound C induces Na(+),K(+)-ATPase endocytosis. This effect of Compound C is prevented through the short hairpin RNA-mediated knockdown of AS160, demonstrating that AMPK and AS160 participate in a common pathway to modulate the cell surface expression of the Na(+),K(+)-ATPase.

  18. 2-Deoxy-D-glucose treatment of endothelial cells induces autophagy by reactive oxygen species-mediated activation of the AMP-activated protein kinase.

    Directory of Open Access Journals (Sweden)

    Qilong Wang

    2011-02-01

    Full Text Available Autophagy is a cellular self-digestion process activated in response to stresses such as energy deprivation and oxidative stress. However, the mechanisms by which energy deprivation and oxidative stress trigger autophagy remain undefined. Here, we report that activation of AMP-activated protein kinase (AMPK by mitochondria-derived reactive oxygen species (ROS is required for autophagy in cultured endothelial cells. AMPK activity, ROS levels, and the markers of autophagy were monitored in confluent bovine aortic endothelial cells (BAEC treated with the glycolysis blocker 2-deoxy-D-glucose (2-DG. Treatment of BAEC with 2-DG (5 mM for 24 hours or with low concentrations of H(2O(2 (100 µM induced autophagy, including increased conversion of microtubule-associated protein light chain 3 (LC3-I to LC3-II, accumulation of GFP-tagged LC3 positive intracellular vacuoles, and increased fusion of autophagosomes with lysosomes. 2-DG-treatment also induced AMPK phosphorylation, which was blocked by either co-administration of two potent anti-oxidants (Tempol and N-Acetyl-L-cysteine or overexpression of superoxide dismutase 1 or catalase in BAEC. Further, 2-DG-induced autophagy in BAEC was blocked by overexpressing catalase or siRNA-mediated knockdown of AMPK. Finally, pretreatment of BAEC with 2-DG increased endothelial cell viability after exposure to hypoxic stress. Thus, AMPK is required for ROS-triggered autophagy in endothelial cells, which increases endothelial cell survival in response to cell stress.

  19. α-thrombin-induced inositol phosphate formation in G0-arrested and cycling hamster lung fibroblasts: evidence for a protein kinase C-mediated desensitization response

    International Nuclear Information System (INIS)

    L'allemain, G.; Paris, S.; Magnaldo, I.; Pouyssegur, J.

    1986-01-01

    In resting Chinese hamster fibroblasts (CCL39) α-thrombin rapidly induces the breakdown of phosphoinositides. Accumulation of inositol phosphates (IP), measured in the presence of Li + , is detectable within 5s (seconds) of thrombin stimulation. Formation of inositol tris- and bisphosphates slightly precedes that of inositol monophosphate, indicating that thrombin activates primarily the phospholipase C-mediated generation of inositol trisphosphate from phosphatidylinositol 4,5-bisphosphate. Initial rates of IP production increase with thrombin concentration, with no apparent saturability over the range 10 -4 -10 U/ml. Thrombin-induced phosphoinositide hydrolysis rapidly desensitizes (t/sub 1/2/ > 5 min), but a residual activity, corresponding to about 10% of the initial stimulation is sustained for at least 9 h, in contrast with the undetectable activity of G0-arrested cells. This apparent desensitization may be due to a feedback regulation by protein kinase C, since pretreatment with the phorbol ester 12-O-tetradecanoyl phorbol 13-acetate (TPA) markedly inhibits (by up to 70%) subsequent thrombin-induced inositol phosphate formation. This up regulation was found maximal in A51, a very well growth-arrested CCL39 derivative,and reduced or virtually abolished in two tumoral and growth factor-relaxed derivatives of CCL39. Although preliminary, this observation suggests that a persistent activation of phosphatidyl inositol breakdown might operate in variants selected for autonomous growth

  20. Computational modeling reveals optimal strategy for kinase transport by microtubules to nerve terminals.

    Science.gov (United States)

    Koon, Yen Ling; Koh, Cheng Gee; Chiam, Keng-Hwee

    2014-01-01

    Intracellular transport of proteins by motors along cytoskeletal filaments is crucial to the proper functioning of many eukaryotic cells. Since most proteins are synthesized at the cell body, mechanisms are required to deliver them to the growing periphery. In this article, we use computational modeling to study the strategies of protein transport in the context of JNK (c-JUN NH2-terminal kinase) transport along microtubules to the terminals of neuronal cells. One such strategy for protein transport is for the proteins of the JNK signaling cascade to bind to scaffolds, and to have the whole protein-scaffold cargo transported by kinesin motors along microtubules. We show how this strategy outperforms protein transport by diffusion alone, using metrics such as signaling rate and signal amplification. We find that there exists a range of scaffold concentrations for which JNK transport is optimal. Increase in scaffold concentration increases signaling rate and signal amplification but an excess of scaffolds results in the dilution of reactants. Similarly, there exists a range of kinesin motor speeds for which JNK transport is optimal. Signaling rate and signal amplification increases with kinesin motor speed until the speed of motor translocation becomes faster than kinase/scaffold-motor binding. Finally, we suggest experiments that can be performed to validate whether, in physiological conditions, neuronal cells do indeed adopt such an optimal strategy. Understanding cytoskeletal-assisted protein transport is crucial since axonal and cell body accumulation of organelles and proteins is a histological feature in many human neurodegenerative diseases. In this paper, we have shown that axonal transport performance changes with altered transport component concentrations and transport speeds wherein these aspects can be modulated to improve axonal efficiency and prevent or slowdown axonal deterioration.

  1. Computational modeling reveals optimal strategy for kinase transport by microtubules to nerve terminals.

    Directory of Open Access Journals (Sweden)

    Yen Ling Koon

    Full Text Available Intracellular transport of proteins by motors along cytoskeletal filaments is crucial to the proper functioning of many eukaryotic cells. Since most proteins are synthesized at the cell body, mechanisms are required to deliver them to the growing periphery. In this article, we use computational modeling to study the strategies of protein transport in the context of JNK (c-JUN NH2-terminal kinase transport along microtubules to the terminals of neuronal cells. One such strategy for protein transport is for the proteins of the JNK signaling cascade to bind to scaffolds, and to have the whole protein-scaffold cargo transported by kinesin motors along microtubules. We show how this strategy outperforms protein transport by diffusion alone, using metrics such as signaling rate and signal amplification. We find that there exists a range of scaffold concentrations for which JNK transport is optimal. Increase in scaffold concentration increases signaling rate and signal amplification but an excess of scaffolds results in the dilution of reactants. Similarly, there exists a range of kinesin motor speeds for which JNK transport is optimal. Signaling rate and signal amplification increases with kinesin motor speed until the speed of motor translocation becomes faster than kinase/scaffold-motor binding. Finally, we suggest experiments that can be performed to validate whether, in physiological conditions, neuronal cells do indeed adopt such an optimal strategy. Understanding cytoskeletal-assisted protein transport is crucial since axonal and cell body accumulation of organelles and proteins is a histological feature in many human neurodegenerative diseases. In this paper, we have shown that axonal transport performance changes with altered transport component concentrations and transport speeds wherein these aspects can be modulated to improve axonal efficiency and prevent or slowdown axonal deterioration.

  2. GsAPK, an ABA-activated and calcium-independent SnRK2-type kinase from G. soja, mediates the regulation of plant tolerance to salinity and ABA stress.

    Science.gov (United States)

    Yang, Liang; Ji, Wei; Gao, Peng; Li, Yong; Cai, Hua; Bai, Xi; Chen, Qin; Zhu, Yanming

    2012-01-01

    Plant Snf1 (sucrose non-fermenting-1) related protein kinase (SnRK), a subfamily of serine/threonine kinases, has been implicated as a crucial upstream regulator of ABA and osmotic signaling as in many other signaling cascades. In this paper, we have isolated a novel plant specific ABA activated calcium independent protein kinase (GsAPK) from a highly salt tolerant plant, Glycine soja (50109), which is a member of the SnRK2 family. Subcellular localization studies using GFP fusion protein indicated that GsAPK is localized in the plasma membrane. We found that autophosphorylation and Myelin Basis Protein phosphorylation activity of GsAPK is only activated by ABA and the kinase activity also was observed when calcium was replaced by EGTA, suggesting its independence of calcium in enzyme activity. We also found that cold, salinity, drought, and ABA stress alter GsAPK gene transcripts and heterogonous overexpression of GsAPK in Arabidopsis alters plant tolerance to high salinity and ABA stress. In summary, we demonstrated that GsAPK is a Glycine soja ABA activated calcium independent SnRK-type kinase presumably involved in ABA mediated stress signal transduction.

  3. The monoclonal antibody Zt/f2 targeting RON receptor tyrosine kinase as potential therapeutics against tumor growth-mediated by colon cancer cells

    Directory of Open Access Journals (Sweden)

    Zhang Rui-Wen

    2011-07-01

    Full Text Available Abstract Background Overexpression of the RON receptor tyrosine kinase contributes to epithelial cell transformation, malignant progression, and acquired drug resistance. RON also has been considered as a potential target for therapeutic intervention. This study determines biochemical features and inhibitory activity of a mouse monoclonal antibody (mAb Zt/f2 in experimental cancer therapy. Results Zt/f2 is a mouse IgG2a mAb that is highly specific and sensitive to human RON and its oncogenic variants such as RON160 (ED50 = 2.3 nmol/L. Receptor binding studies revealed that Zt/f2 interacts with an epitope(s located in a 49 amino acid sequence coded by exon 11 in the RON β-chain extracellular sequences. This sequence is critical in regulating RON maturation and phosphorylation. Zt/f2 did not compete with ligand macrophage-stimulating protein for binding to RON; however, its engagement effectively induced RON internalization, which diminishes RON expression and impairs downstream signaling activation. These biochemical features provide the cellular basis for the use of Zt/f2 to inhibit tumor growth in animal model. Repeated administration of Zt/f2 as a single agent into Balb/c mice results in partial inhibition of tumor growth caused by transformed NIH-3T3 cells expressing oncogenic RON160. Colon cancer HT-29 cell-mediated tumor growth in athymic nude mice also was attenuated following Zt/f2 treatment. In both cases, ~50% inhibition of tumor growth as measured by tumor volume was achieved. Moreover, Zt/f2 in combination with 5-fluorouracil showed an enhanced inhibition effect of ~80% on HT-29 cell-mediated tumor growth in vivo. Conclusions Zt/f2 is a potential therapeutic mAb capable of inhibiting RON-mediated oncogenesis by colon cancer cells in animal models. The inhibitory effect of Zt/f2 in vivo in combination with chemoagent 5-fluorouracil could represent a novel strategy for future colon cancer therapy.

  4. G protein-coupled receptor kinase 5 mediates Tazarotene-induced gene 1-induced growth suppression of human colon cancer cells

    International Nuclear Information System (INIS)

    Wu, Chang-Chieh; Tsai, Fu-Ming; Shyu, Rong-Yaun; Tsai, Ya-Ming; Wang, Chun-Hua; Jiang, Shun-Yuan

    2011-01-01

    Tazarotene-induced gene 1 (TIG1) is a retinoid-inducible type II tumour suppressor gene. The B isoform of TIG1 (TIG1B) inhibits growth and invasion of cancer cells. Expression of TIG1B is frequently downregulated in various cancer tissues; however, the expression and activities of the TIG1A isoform are yet to be reported. Therefore, this study investigated the effects of the TIG1A and TIG1B isoforms on cell growth and gene expression profiles using colon cancer cells. TIG1A and TIG1B stable clones derived from HCT116 and SW620 colon cancer cells were established using the GeneSwitch system; TIG1 isoform expression was induced by mifepristone treatment. Cell growth was assessed using the WST-1 cell proliferation and colony formation assays. RNA interference was used to examine the TIG1 mediating changes in cell growth. Gene expression profiles were determined using microarray and validated using real-time polymerase chain reaction, and Western blot analyses. Both TIG1 isoforms were expressed at high levels in normal prostate and colon tissues and were downregulated in colon cancer cell lines. Both TIG1 isoforms significantly inhibited the growth of transiently transfected HCT116 cells and stably expressing TIG1A and TIG1B HCT116 and SW620 cells. Expression of 129 and 55 genes was altered upon induction of TIG1A and TIG1B expression, respectively, in stably expressing HCT116 cells. Of the genes analysed, 23 and 6 genes were upregulated and downregulated, respectively, in both TIG1A and TIG1B expressing cells. Upregulation of the G-protein-coupled receptor kinase 5 (GRK5) was confirmed using real-time polymerase chain reaction and Western blot analyses in both TIG1 stable cell lines. Silencing of TIG1A or GRK5 expression significantly decreased TIG1A-mediated cell growth suppression. Expression of both TIG1 isoforms was observed in normal prostate and colon tissues and was downregulated in colon cancer cell lines. Both TIG1 isoforms suppressed cell growth and

  5. A conditional form of Bruton's tyrosine kinase is sufficient to activate multiple downstream signaling pathways via PLC Gamma 2 in B cells

    Directory of Open Access Journals (Sweden)

    Witte Owen N

    2001-06-01

    Full Text Available Abstract Background Bruton's tyrosine kinase (Btk is essential for B cell development and function. Mutations of Btk elicit X-linked agammaglobulinemia in humans and X-linked immunodeficiency in the mouse. Btk has been proposed to participate in B cell antigen receptor-induced signaling events leading to activation of phospholipase C-γ2 (PLCγ2 and calcium mobilization. However it is unclear whether Btk activation is alone sufficient for these signaling events, and whether Btk can activate additional pathways that do not involve PLCγ2. To address such issues we have generated Btk:ER, a conditionally active form of the kinase, and expressed it in the PLCγ2-deficient DT40 B cell line. Results Activation of Btk:ER was sufficient to induce multiple B cell signaling pathways in PLCγ2-sufficient DT40 cells. These included tyrosine phosphorylation of PLCγ2, mobilization of intracellular calcium, activation of extracellular signal-regulated kinase (ERK and c-Jun NH2-terminal kinase (JNK mitogen-activated protein kinase (MAPK pathways, and apoptosis. In DT40 B cells deficient for PLCγ2, Btk:ER activation failed to induce the signaling events described above with the consequence that the cells failed to undergo apoptosis. Conclusions These data suggest that Btk:ER regulates downstream signaling pathways primarily via PLCγ2 in B cells. While it is not known whether activated Btk:ER precisely mimics activated Btk, this conditional system will likely facilitate the dissection of the role of Btk and its family members in a variety of biological processes in many different cell types.

  6. Nitric oxide protects the heart from ischemia-induced apoptosis and mitochondrial damage via protein kinase G mediated blockage of permeability transition and cytochrome c release

    Directory of Open Access Journals (Sweden)

    Jekabsone Aiste

    2009-08-01

    Full Text Available Abstract Background Heart ischemia can rapidly induce apoptosis and mitochondrial dysfunction via mitochondrial permeability transition-induced cytochrome c release. We tested whether nitric oxide (NO can block this damage in isolated rat heart, and, if so, by what mechanisms. Methods Hearts were perfused with 50 μM DETA/NO (NO donor, then subjected to 30 min stop-flow ischemia or ischemia/reperfusion. Isolated heart mitochondria were used to measure the rate of mitochondrial oxygen consumption and membrane potential using oxygen and tetraphenylphosphonium-selective electrodes. Mitochondrial and cytosolic cytochrome c levels were measured spectrophotometrically and by ELISA. The calcium retention capacity of isolated mitochondria was measured using the fluorescent dye Calcium Green-5N. Apoptosis and necrosis were evaluated by measuring the activity of caspase-3 in cytosolic extracts and the activity of lactate dehydrogenase in perfusate, respectively. Results 30 min ischemia caused release of mitochondrial cytochrome c to the cytoplasm, inhibition of the mitochondrial respiratory chain, and stimulation of mitochondrial proton permeability. 3 min perfusion with 50 μM DETA/NO of hearts prior to ischemia decreased this mitochondrial damage. The DETA/NO-induced blockage of mitochondrial cytochrome c release was reversed by a protein kinase G (PKG inhibitor KT5823, or soluble guanylate cyclase inhibitor ODQ or protein kinase C inhibitors (Ro 32-0432 and Ro 31-8220. Ischemia also stimulated caspase-3-like activity, and this was substantially reduced by pre-perfusion with DETA/NO. Reperfusion after 30 min of ischemia caused no further caspase activation, but was accompanied by necrosis, which was completely prevented by DETA/NO, and this protection was blocked by the PKG inhibitor. Incubation of isolated heart mitochondria with activated PKG blocked calcium-induced mitochondrial permeability transition and cytochrome c release. Perfusion of non

  7. UVB-mediated activation of p38 mitogen-activated protein kinase enhances resistance of normal human keratinocytes to apoptosis by stabilizing cytoplasmic p53.

    OpenAIRE

    Chouinard, Nadine; Valerie, Kristoffer; Rouabhia, Mahmoud; Huot, Jacques

    2002-01-01

    Human keratinocytes respond to UV rays by developing a fast adaptive response that contributes to maintaining their functions and survival. We investigated the role of the mitogen-activated protein kinase pathways in transducing the UV signals in normal human keratinocytes. We found that UVA, UVB or UVC induced a marked and persistent activation of p38, whereas c-Jun N-terminal kinase or extracellular signal-regulated kinase were less or not activated respectively. Inhibition of p38 activity ...

  8. Mediatization

    DEFF Research Database (Denmark)

    Hjarvard, Stig

    2017-01-01

    Mediatization research shares media effects studies' ambition of answering the difficult questions with regard to whether and how media matter and influence contemporary culture and society. The two approaches nevertheless differ fundamentally in that mediatization research seeks answers...... research is concerned with long-term structural changes involving media, culture, and society, i.e. the influences of the media are understood in relation to how media are implicated in social and cultural changes and how these processes come to create new conditions for human communication and interaction....... From the perspective of mediatization research, the most important effect of the media stems from their embeddedness in culture and society....

  9. RNAi-Mediated Downregulation of Inositol Pentakisphosphate Kinase (IPK1 in Wheat Grains Decreases Phytic Acid Levels and Increases Fe and Zn Accumulation

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    Sipla Aggarwal

    2018-03-01

    Full Text Available Enhancement of micronutrient bioavailability is crucial to address the malnutrition in the developing countries. Various approaches employed to address the micronutrient bioavailability are showing promising signs, especially in cereal crops. Phytic acid (PA is considered as a major antinutrient due to its ability to chelate important micronutrients and thereby restricting their bioavailability. Therefore, manipulating PA biosynthesis pathway has largely been explored to overcome the pleiotropic effect in different crop species. Recently, we reported that functional wheat inositol pentakisphosphate kinase (TaIPK1 is involved in PA biosynthesis, however, the functional roles of the IPK1 gene in wheat remains elusive. In this study, RNAi-mediated gene silencing was performed for IPK1 transcripts in hexaploid wheat. Four non-segregating RNAi lines of wheat were selected for detailed study (S3-D-6-1; S6-K-3-3; S6-K-6-10 and S16-D-9-5. Homozygous transgenic RNAi lines at T4 seeds with a decreased transcript of TaIPK1 showed 28–56% reduction of the PA. Silencing of IPK1 also resulted in increased free phosphate in mature grains. Although, no phenotypic changes in the spike was observed but, lowering of grain PA resulted in the reduced number of seeds per spikelet. The lowering of grain PA was also accompanied by a significant increase in iron (Fe and zinc (Zn content, thereby enhancing their molar ratios (Zn:PA and Fe:PA. Overall, this work suggests that IPK1 is a promising candidate for employing genome editing tools to address the mineral accumulation in wheat grains.

  10. Radiochemotherapy of hepatocarcinoma via lentivirus-mediated transfer of human sodium iodide symporter gene and herpes simplex virus thymidine kinase gene

    Energy Technology Data Exchange (ETDEWEB)

    Chen Libo, E-mail: libochen888@hotmail.com [Department of Nuclear Medicine, Shanghai Sixth People' s Hospital, Shanghai Jiao Tong University, Shanghai 200233 (China); Guo Guoying [Xinyuan Institute of Medicine and Biotechnology, School of Life Sciences, Zhejiang Sci-Tech University, Hangzhou 310018 (China); Liu Tianjing; Guo Lihe [Division of Biochemistry and Cell Biology, Shanghai Institute for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031 (China); Zhu Ruisen [Department of Nuclear Medicine, Shanghai Sixth People' s Hospital, Shanghai Jiao Tong University, Shanghai 200233 (China)

    2011-07-15

    Herpes simplex virus thymidine kinase (HSV-TK) gene/ganciclovir (GCV) system has been widely used as a traditional gene therapy modality, and the sodium/iodide symporter gene (NIS) has been found to be a novel therapeutic gene. Since the therapeutic effects of radioiodine therapy or prodrug chemotherapy on cancers following NIS or HSV-TK gene transfer need to be enhanced, this study was designed to investigate the feasibility of radiochemotherapy for hepatocarcinoma via coexpression of NIS gene and HSV-TK gene. Methods: HepG2 cells were stably transfected with NIS, TK and GFP gene via recombinant lentiviral vector and named HepG2/NTG. Gene expression was examined by reverse transcriptase polymerase chain reaction, fluorescence imaging and iodide uptake. The therapeutic effects were assessed by MTT assay and clonogenic assay. Results: HepG2/NTG cells concentrated {sup 125}I{sup -} up to 76-fold higher than the wild-type cells within 20 min, and the efflux happened with a T{sub 1/2eff} of less than 10 min. The iodide uptake in HepG2/NTG cells was specifically inhibited by sodium perchlorate. Dose-dependent toxicity to HepG2/NTG cells by either GCV or {sup 131}I was revealed by clonogenic assay and MTT assay, respectively. The survival rate of HepG2/NTG cells decreased to 49.7%{+-}2.5%, 43.4%{+-}2.8% and 8.6%{+-}1.2% after exposure to {sup 131}I, GCV and combined therapy, respectively. Conclusion: We demonstrate that radiochemotherapy of hepatocarcinoma via lentiviral-mediated coexpression of NIS gene and HSV-TK gene leads to stronger killing effect than single treatment, and in vivo studies are needed to verify these findings.

  11. Adiponectin induced AMP-activated protein kinase impairment mediates insulin resistance in Bama mini-pig fed high-fat and high-sucrose diet

    Directory of Open Access Journals (Sweden)

    Miaomiao Niu

    2017-08-01

    Full Text Available Objective Adipose tissue is no longer considered as an inert storage organ for lipid, but instead is thought to play an active role in regulating insulin effects via secretion adipokines. However, conflicting reports have emerged regarding the effects of adipokines. In this study, we investigated the role of adipokines in glucose metabolism and insulin sensitivity in obese Bama mini-pigs. Methods An obesity model was established in Bama mini-pigs, by feeding with high-fat and high-sucrose diet for 30 weeks. Plasma glucose and blood biochemistry levels were measured, and intravenous glucose tolerance test was performed. Adipokines, including adiponectin, interleukin-6 (IL-6, resistin and tumor necrosis factor alpha (TNF-α, and glucose-induced insulin secretion were also examined by radioimmunoassay. AMP-activated protein kinase (AMPK phosphorylation in skeletal muscle, which is a useful insulin resistance marker, was examined by immunoblotting. Additionally, associations of AMPK phosphorylation with plasma adipokines and homeostasis model assessment of insulin resistance (HOMA-IR index were assessed by Pearce’s correlation analysis. Results Obese pigs showed hyperglycemia, high triglycerides, and insulin resistance. Adiponectin levels were significantly decreased (p<0.05 and IL-6 amounts dramatically increased (p<0.05 in obese pigs both in serum and adipose tissue, corroborating data from obese mice and humans. However, circulating resistin and TNF-α showed no difference, while the values of TNF-α in adipose tissue were significantly higher in obese pigs, also in agreement with data from obese humans but not rodent models. Moreover, strong associations of skeletal muscle AMPK phosphorylation with plasma adiponectin and HOMA-IR index were obtained. Conclusion AMPK impairment induced by adiponectin decrease mediates insulin resistance in high-fat and high-sucrose diet induction. In addition, Bama mini-pig has the possibility of a conformable

  12. Inhibitory effect of 1,2,4,5-tetramethoxybenzene on mast cell-mediated allergic inflammation through suppression of IκB kinase complex

    Energy Technology Data Exchange (ETDEWEB)

    Je, In-Gyu [Department of Pharmacology, School of Medicine, Kyungpook National University, Daegu 700-422 (Korea, Republic of); Choi, Hyun Gyu [College of Pharmacy, Yeungnam University, Gyeongsan 712-749 (Korea, Republic of); Kim, Hui-Hun; Lee, Soyoung; Choi, Jin Kyeong [Department of Pharmacology, School of Medicine, Kyungpook National University, Daegu 700-422 (Korea, Republic of); Kim, Sung-Wan; Kim, Duk-Sil [Department of Thoracic and Cardiovascular Surgery, CHA Gumi Medical Center, CHA University, Gumi 730-040 (Korea, Republic of); Kwon, Taeg Kyu [Department of Immunology, School of Medicine, Keimyung University, Daegu 704-701 (Korea, Republic of); Shin, Tae-Yong [College of Pharmacy, Woosuk University, Jeonju 565-701 (Korea, Republic of); Park, Pil-Hoon [College of Pharmacy, Yeungnam University, Gyeongsan 712-749 (Korea, Republic of); Khang, Dongwoo, E-mail: dkhang@gachon.ac.kr [Department of Molecular Medicine, School of Medicine, Gachon University, Incheon 406-840 (Korea, Republic of); Kim, Sang-Hyun, E-mail: shkim72@knu.ac.kr [Department of Pharmacology, School of Medicine, Kyungpook National University, Daegu 700-422 (Korea, Republic of)

    2015-09-01

    As the importance of allergic disorders such as atopic dermatitis and allergic asthma, research on potential drug candidates becomes more necessary. Mast cells play an important role as initiators of allergic responses through the release of histamine; therefore, they should be the target of pharmaceutical development for the management of allergic inflammation. In our previous study, anti-allergic effect of extracts of Amomum xanthioides was demonstrated. To further investigate improved candidates, 1,2,4,5-tetramethoxybenzene (TMB) was isolated from methanol extracts of A. xanthioides. TMB dose-dependently attenuated the degranulation of mast cells without cytotoxicity by inhibiting calcium influx. TMB decreased the expression of pro-inflammatory cytokines such as tumor necrosis factor-α and interleukin (IL)-4 at both the transcriptional and translational levels. Increased expression of these cytokines was caused by translocation of nuclear factor-κB into the nucleus, and it was hindered by suppressing activation of IκB kinase complex. To confirm the effect of TMB in vivo, the ovalbumin (OVA)-induced active systemic anaphylaxis (ASA) and IgE-mediated passive cutaneous anaphylaxis (PCA) models were used. In the ASA model, hypothermia was decreased by oral administration of TMB, which attenuated serum histamine, OVA-specific IgE, and IL-4 levels. Increased pigmentation of Evans blue was reduced by TMB in a dose-dependent manner in the PCA model. Our results suggest that TMB is a possible therapeutic candidate for allergic inflammatory diseases that acts through the inhibition of mast cell degranulation and expression of pro-inflammatory cytokines. - Highlights: • TMB reduced the degranulation of mast cells. • TMB inhibited the production of pro-inflammatory cytokines. • TMB suppressed both active and passive anaphylaxis. • Anti-allergic inflammatory effects of TMB might be due to the blocking IKK complex. • TMB might be a candidate for the treatment of

  13. Glycogen synthase kinase-3β inhibition in the medial prefrontal cortex mediates paradoxical amphetamine action in a mouse model of ADHD

    Directory of Open Access Journals (Sweden)

    Yi-Chun eYen

    2015-03-01

    Full Text Available Psychostimulants show therapeutic efficacy in the treatment of attention-deficit hyperactivity disorder (ADHD. It is generally assumed that they ameliorate ADHD symptoms via interfering with monoaminergic signaling. We combined behavioral pharmacology, neurochemistry and molecular analyses to identify mechanisms underlying the paradoxical calming effect of amphetamine in low trait anxiety behavior (LAB mice, a novel multigenetic animal model of ADHD. Amphetamine (1 mg/kg and methylphenidate (10 mg/kg elicited similar dopamine and norepinephrine release in the medial prefrontal cortex (mPFC and in the striatum of LAB mice. In contrast, amphetamine decreased, while methylphenidate increased locomotor activity. This argues against changes in dopamine and/or norepinephrine release as mediators of amphetamine paradoxical effects. Instead, the calming activity of amphetamine corresponded to the inhibition of glycogen synthase kinase3β (GSK3β activity, specifically in the mPFC. Accordingly, not only systemic administration of the GSK3β inhibitor TDZD-8 (20 mg/kg, but also local microinjections of TDZD-8 and amphetamine into the mPFC, but not into the striatum, decreased locomotor activity in LAB mice. Amphetamine effects seem to depend on NMDA receptor signaling, since pre- or co-treatment with MK-801 (0.3 mg/kg abolished the effects of amphetamine (1 mg/kg on the locomotion and on the phosphorylation of GSK3β at the level of the mPFC. Taken together, the paradoxical calming effect of amphetamine in hyperactive LAB mice concurs with a decreased GSK3β activity in the mPFC. This effect appears to be independent of dopamine or norepinephrine release, but contingent on NMDA receptor signaling.

  14. MicroRNA-146a-5p Mediates High Glucose-Induced Endothelial Inflammation via Targeting Interleukin-1 Receptor-Associated Kinase 1 Expression

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    Wan-Yu Lo

    2017-08-01

    Full Text Available Background and Aims: Interleukin-1 receptor-associated kinase-1 (IRAK-1 is critical for mediating toll-like receptor and interleukin-1 receptor signaling. In this study, we have examined whether IRAK-1 expression is altered in high glucose (HG-stimulated human aortic endothelial cells (HAECs, and whether microRNAs (miRs target IRAK-1 to regulate HG-induced endothelial inflammation.Methods: HAECs were treated with HG for 24 and 48 h. Real-time PCR, Western blot, monocyte adhesion assay, bioinformatics analysis, TaqMan® arrays, microRNA mimic or inhibitor transfection, luciferase reporter assay and siRNA IRAK-1 transfection were performed. The aortic tissues from db/db type 2 diabetic mice were examined by immunohistochemistry staining.Results: HG time-dependently increased IRAK-1 mRNA and protein levels in HAECs, and was associated with increased VCAM-1/ICAM-1 gene expression and monocyte adhesion. Bioinformatic analysis, TaqMan® arrays, and real-time PCR were used to confirm that miR-146a-5p, miR-339-5p, and miR-874-3p were significantly downregulated in HG-stimulated HAECs, suggesting impaired feedback restraints on HG-induced endothelial inflammation via IRAK-1. However, only miR-146a-5p mimic transfection reduced the HG-induced upregulation of IRAK-1 expression, VCAM-1/ICAM-1 expression, and monocyte adhesion. Additionally, IRAK-1 depletion reduced HG-induced VCAM-1/ICAM-1 gene expression, and monocyte adhesion, indicating that HG-induced endothelial inflammation was mediated partially through IRAK-1. In vivo, intravenous injections of miR-146a-5p mimic prevented endothelial IRAK-1 and ICAM-1 expression in db/db mice.Conclusion: These results suggest that miR-146a-5p is involved in the regulation of HG-induced endothelial inflammation via modulation of IRAK-1; indicating that miR-146a-5p may be a novel target for the treatment of diabetic vascular complications.

  15. Bifidobacterial recombinant thymidine kinase-ganciclovir gene therapy system induces FasL and TNFR2 mediated antitumor apoptosis in solid tumors

    International Nuclear Information System (INIS)

    Wang, Changdong; Ma, Yongping; Hu, Qiongwen; Xie, Tingting; Wu, Jiayan; Zeng, Fan; Song, Fangzhou

    2016-01-01

    Directly targeting therapeutic suicide gene to a solid tumor is a hopeful approach for cancer gene therapy. Treatment of a solid tumor by an effective vector for a suicide gene remains a challenge. Given the lack of effective treatments, we constructed a bifidobacterial recombinant thymidine kinase (BF-rTK) -ganciclovir (GCV) targeting system (BKV) to meet this requirement and to explore antitumor mechanisms. Bifidobacterium (BF) or BF-rTK was injected intratumorally with or without ganciclovir in a human colo320 intestinal xenograft tumor model. The tumor tissues were analyzed using apoptosis antibody arrays, real time PCR and western blot. The colo320 cell was analyzed by the gene silencing method. Autophagy and necroptosis were also detected in colo320 cell. Meanwhile, three human digestive system xenograft tumor models (colorectal cancer colo320, gastric cancer MKN-45 and liver cancer SSMC-7721) and a breast cancer (MDA-MB-231) model were employed to validate the universality of BF-rTK + GCV in solid tumor gene therapy. The survival rate was evaluated in three human cancer models after the BF-rTK + GCV intratumor treatment. The analysis of inflammatory markers (TNF-α) in tumor indicated that BF-rTK + GCV significantly inhibited TNF-α expression. The results suggested that BF-rTK + GCV induced tumor apoptosis without autophagy and necroptosis occurrence. The apoptosis was transduced by multiple signaling pathways mediated by FasL and TNFR2 and mainly activated the mitochondrial control of apoptosis via Bid and Bim, which was rescued by silencing Bid or/and Bim. However, BF + GCV only induced apoptosis via Fas/FasL signal pathway accompanied with increased P53 expression. We further found that BF-rTK + GCV inhibited the expression of the inflammatory maker of TNF-α. However, BF-rTK + GCV did not result in necroptosis and autophagy. BF-rTK + GCV induced tumor apoptosis mediated by FasL and TNFR2 through the mitochondrial control of apoptosis via Bid and Bim

  16. EphA2 modulates radiosensitive of hepatocellular carcinoma cells via p38/mitogen-activated protein kinase-mediated signal pathways

    Directory of Open Access Journals (Sweden)

    Qiao Jin

    2015-10-01

    Full Text Available This experiment was conducted to investigate the role of EPH receptor A2 (EphA2 in the modulation of radiosensitivity of hepatic cellular cancer (HCC cells and to determine whether p38/mitogen-activated protein kinase (p38MAPK signaling mediated EphA2 function in this respect. The protein expressions of EphA2 and phosphorylated p38MAPK were tested in HCC and normal hepatic tissues. In HCC 97H cells, EphA2 was overexpressed and knocked out by transfection with EphA2 expression vector and EphA2-ShRNA, respectively, prior to cell exposure to low-dose irradiation. Significantly upregulated EphA2 and phosphorylated p38MAPK were observed in HCC tissues, compared with those in normal hepatic tissues. Low-dose irradiation (1 Gy only caused minor damage to HCC 97H cells, as assessed by alterations in cell viability, apoptosis rate, and cell healing capacity (p = 0.072, p = 0.078, and p = 0.069 respectively. However, EphA2 knock-out in HCC 97H cells induced significant reduction in cell viability and cell healing capacity after these cells were subjected to low-dose irradiation. Apoptosis rate underwent dramatic increase (p < 0.01. By contrast, EphA2 overexpression in HCC 97H cells reversed these effects and enhanced cell colony formation rate, thus displaying remarkable attenuation of radiosensitivity of HCC 97H cells. Further, SB203580, a specific inhibitor of p38MAPK, was added to HCC 97H cells over-expressing EphA2. The effect of EphA2 overexpression on the radiosensitivity of HCC 97H cells was abrogated. Thus, the present study indicates that EphA2 have the ability to negatively regulate the radiosensitivity of HCC 97H cells, which mainly depends on 38MAPK-mediated signal pathways.

  17. The Matrix Protein of Nipah Virus Targets the E3-Ubiquitin Ligase TRIM6 to Inhibit the IKKε Kinase-Mediated Type-I IFN Antiviral Response.

    Directory of Open Access Journals (Sweden)

    Preeti Bharaj

    2016-09-01

    Full Text Available For efficient replication, viruses have developed mechanisms to evade innate immune responses, including the antiviral type-I interferon (IFN-I system. Nipah virus (NiV, a highly pathogenic member of the Paramyxoviridae family (genus Henipavirus, is known to encode for four P gene-derived viral proteins (P/C/W/V with IFN-I antagonist functions. Here we report that NiV matrix protein (NiV-M, which is important for virus assembly and budding, can also inhibit IFN-I responses. IFN-I production requires activation of multiple signaling components including the IκB kinase epsilon (IKKε. We previously showed that the E3-ubiquitin ligase TRIM6 catalyzes the synthesis of unanchored K48-linked polyubiquitin chains, which are not covalently attached to any protein, and activate IKKε for induction of IFN-I mediated antiviral responses. Using co-immunoprecipitation assays and confocal microscopy we show here that the NiV-M protein interacts with TRIM6 and promotes TRIM6 degradation. Consequently, NiV-M expression results in reduced levels of unanchored K48-linked polyubiquitin chains associated with IKKε leading to impaired IKKε oligomerization, IKKε autophosphorylation and reduced IFN-mediated responses. This IFN antagonist function of NiV-M requires a conserved lysine residue (K258 in the bipartite nuclear localization signal that is found in divergent henipaviruses. Consistent with this, the matrix proteins of Ghana, Hendra and Cedar viruses were also able to inhibit IFNβ induction. Live NiV infection, but not a recombinant NiV lacking the M protein, reduced the levels of endogenous TRIM6 protein expression. To our knowledge, matrix proteins of paramyxoviruses have never been reported to be involved in innate immune antagonism. We report here a novel mechanism of viral innate immune evasion by targeting TRIM6, IKKε and unanchored polyubiquitin chains. These findings expand the universe of viral IFN antagonism strategies and provide a new

  18. An indoxyl compound 5-bromo-4-chloro-3-indolyl 1,3-diacetate, CAC-0982, suppresses activation of Fyn kinase in mast cells and IgE-mediated allergic responses in mice

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Jun Ho [Department of Immunology, School of Medicine, Konkuk University, Chungju 380-701 (Korea, Republic of); College of Medicine, Korea University, Seoul 136-701 (Korea, Republic of); Kim, Tae Hyung [College of Pharmacy, Sungkyunkwan University, Suwon 440-746 (Korea, Republic of); Kim, Hyuk Soon; Kim, A-Ram [Department of Immunology, School of Medicine, Konkuk University, Chungju 380-701 (Korea, Republic of); Kim, Do-Kyun [Department of Immunology, School of Medicine, Konkuk University, Chungju 380-701 (Korea, Republic of); Laboratory of Allergic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD (United States); Nam, Seung Taek; Kim, Hyun Woo; Park, Young Hwan; Her, Erk; Park, Yeong Min [Department of Immunology, School of Medicine, Konkuk University, Chungju 380-701 (Korea, Republic of); Kim, Hyung Sik [College of Pharmacy, Sungkyunkwan University, Suwon 440-746 (Korea, Republic of); Kim, Young Mi [College of Pharmacy, Duksung Women' s University, Seoul 132-714 (Korea, Republic of); Choi, Wahn Soo, E-mail: wahnchoi@kku.ac.kr [Department of Immunology, School of Medicine, Konkuk University, Chungju 380-701 (Korea, Republic of)

    2015-06-15

    Mast cells, constituents of virtually all organs and tissues, are critical cells in IgE-mediated allergic responses. The aim of this study was to investigate the effect and mechanism of an indoxyl chromogenic compound, 5-bromo-4-chloro-3-indolyl 1,3-diacetate, CAC-0982, on IgE-mediated mast cell activation and allergic responses in mice. CAC-0982 reversibly suppressed antigen-stimulated degranulation in murine mast cells (IC{sub 50}, ~ 3.8 μM) and human mast cells (IC{sub 50}, ~ 3.0 μM). CAC-0982 also inhibited the expression and secretion of IL-4 and TNF-α in mast cells. Furthermore, CAC-0982 suppressed the mast cell-mediated allergic responses in mice in a dose-dependent manner (ED{sub 50} 27.9 mg/kg). As for the mechanism, CAC-0982 largely suppressed the phosphorylation of Syk and its downstream signaling molecules, including LAT, Akt, Erk1/2, p38, and JNK. Notably, the tyrosine kinase assay of antigen-stimulated mast cells showed that CAC-0982 inhibited Fyn kinase, one of the upstream tyrosine kinases for Syk activation in mast cells. Taken together, these results suggest that CAC-0982 may be used as a new treatment for regulating IgE-mediated allergic diseases through the inhibition of the Fyn/Syk pathway in mast cells. - Highlights: • The anti-allergic effect of 5-bromo-4-chloro-3-indolyl 1,3-diacetate, CAC-0982, was measured. • CAC-0982 reversibly suppressed the activation of mast cells by IgE and antigen. • CAC-0982 inhibited passive cutaneous anaphylaxis in mice. • CAC-0982 suppresses mast cells through inhibition of Fyn activation in mast cells.

  19. Baicalein attenuates inflammatory responses by suppressing TLR4 mediated NF-κB and MAPK signaling pathways in LPS-induced mastitis in mice.

    Science.gov (United States)

    He, Xuexiu; Wei, Zhengkai; Zhou, Ershun; Chen, Libin; Kou, Jinhua; Wang, Jingjing; Yang, Zhengtao

    2015-09-01

    Baicalein is a phenolic flavonoid presented in the dry roots of Scutellaria baicalensis Georgi. It has been reported that baicalein possesses a number of biological properties, such as antiviral, antioxidative, anti-inflammatory, antithrombotic, and anticancer properties. However, the effect of baicalein on mastitis has not yet been reported. This research aims to detect the effect of baicalein on lipopolysaccharide (LPS)-induced mastitis in mice and to investigate the molecular mechanisms. Baicalein was administered intraperitoneally 1h before and 12h after LPS treatment. The results indicated that baicalein treatment markedly attenuated the damage of the mammary gland induced by LPS, suppressed the activity of myeloperoxidase (MPO) and the levels of tumor necrosis factor (TNF-α) and interleukin (IL-1β) in mice with LPS-induced mastitis. Besides, baicalein blocked the expression of Toll-like receptor 4 (TLR4) and then suppressed the phosphorylation of nuclear transcription factor-kappaB (NF-κB) p65 and degradation inhibitor of NF-κBα (IκBα) and, and inhibited the phosphorylation of p38, extracellular signal-regulated kinase (ERK) and c-jun NH2-terminal kinase (JNK) in mitogen-activated protein kinase (MAPK) signal pathway. These findings suggested that baicalein may have a potential prospect against mastitis. Copyright © 2015. Published by Elsevier B.V.

  20. Protein kinase mediated upregulation of endothelin A, endothelin B and 5-hydroxytryptamine 1B/1D receptors during organ culture in rat basilar artery

    DEFF Research Database (Denmark)

    Hansen-Schwartz, Jacob; Svensson, Carl-Lennart; Xu, Cang-Bao

    2002-01-01

    1. Organ culture has been shown to upregulate both endothelin (ET) and 5-hydroxytryptamine 1B/1D (5-HT(1B/1D)) receptors in rat cerebral arteries. The purpose of the present study was to investigate the involvement of protein kinases, especially protein kinases C (PKC) and A (PKA) in this process....... 2. The effect of inhibiting protein kinases during organ culture with staurosporine (unspecific protein kinase inhibitor), RO 31-7549 (specific inhibitor of classical PKC's) and H 89 (specific inhibitor of PKA) was examined using in vitro pharmacological examination of cultured vessel segments......-culture with RO 31-7549 abolished the contractile response (6.9 +/- 1.8%) and reduced the ET(B) receptor mRNA by 44 +/- 4% as compared to the cultured control. Correlation between decreased ET(B) receptor mRNA and abolished contractile function indicates upstream involvement of PKC. 4. Inhibition of PKA generally...

  1. Protein tyrosine kinases p53/56lyn and p72syk in MHC class I-mediated signal transduction in B lymphoma cells

    DEFF Research Database (Denmark)

    Pedersen, Anders Elm; Bregenholt, S; Skov, S

    1998-01-01

    Crosslinking of major histocompatibility complex class I (MHC-I) molecules on the surface of human B lymphoma cells was shown to induce protein tyrosine phosphorylation and mobilization of intracellular free calcium. Immunoprecipitations indicated that the protein tyrosine kinases p53/56lyn and p72......syk are among the tyrosine-phosphorylated proteins. The kinetics of phosphorylation of these kinases after MHC-I crosslinking differ from the kinetics observed after crosslinking of the B cell antigen receptor (BCR). Additional experiments were performed with chicken lyn- and syk-negative DT40 B cells...... and the results indicate that these two kinases have different substrate specificity and regulate intracellular free calcium differently in response to MHC-I crosslinking. In addition MHC-I crosslinking of a sIgM-negative DT40 chicken B cell variant results in less activity of tyrosine kinases and less...

  2. Insulin receptors mediate growth effects in cultured fetal neurons. II. Activation of a protein kinase that phosphorylates ribosomal protein S6

    International Nuclear Information System (INIS)

    Heidenreich, K.A.; Toledo, S.P.

    1989-01-01

    As an initial attempt to identify early steps in insulin action that may be involved in the growth responses of neurons to insulin, we investigated whether insulin receptor activation increases the phosphorylation of ribosomal protein S6 in cultured fetal neurons and whether activation of a protein kinase is involved in this process. When neurons were incubated for 2 h with 32Pi, the addition of insulin (100 ng/ml) for the final 30 min increased the incorporation of 32Pi into a 32K microsomal protein. The incorporation of 32Pi into the majority of other neuronal proteins was unaltered by the 30-min exposure to insulin. Cytosolic extracts from insulin-treated neurons incubated in the presence of exogenous rat liver 40S ribosomes and [gamma-32P]ATP displayed a 3- to 8-fold increase in the phosphorylation of ribosomal protein S6 compared to extracts from untreated cells. Inclusion of cycloheximide during exposure of the neurons to insulin did not inhibit the increased cytosolic kinase activity. Activation of S6 kinase activity by insulin was dose dependent (seen at insulin concentration as low as 0.1 ng/ml) and reached a maximum after 20 min of incubation. Addition of phosphatidylserine, diolein, and Ca2+ to the in vitro kinase reaction had no effect on the phosphorylation of ribosomal protein S6. Likewise, treatment of neurons with (Bu)2cAMP did not alter the phosphorylation of ribosomal protein S6 by neuronal cytosolic extracts. We conclude that insulin activates a cytosolic protein kinase that phosphorylates ribosomal S6 in neurons and is distinct from protein kinase-C and cAMP-dependent protein kinase. Stimulation of this kinase may play a role in insulin signal transduction in neurons

  3. Exposure to chronic hyperglycemic conditions results in Ras-related C3 botulinum toxin substrate 1 (Rac1)-mediated activation of p53 and ATM kinase in pancreatic β-cells.

    Science.gov (United States)

    Sidarala, Vaibhav; Kowluru, Anjaneyulu

    2017-05-01

    Chronic hyperglycemia (HG) promotes pancreatic islet dysfunction which leads to the onset of T2DM. This study is aimed at defining regulatory roles of Rac1, a small G-protein, in the activation of p53 and ATM kinase in pancreatic β-cells, under the duress of HG conditions. We report significant stimulatory effects of HG (20 mM; 24 h) on p53 activation in INS-1 832/13 cells, normal rodent and human islets. Pharmacological inhibition of Rac1 (EHT1864 or NSC23766) significantly suppressed HG-induced p53 activation in INS-1 832/13 cells and rat islets, suggesting novel roles for this small G-protein in the activation of p53. Inhibition of Rac1 geranylgeranylation with simvastatin or GGTI-2147, significantly attenuated HG-induced p53 activation, suggesting requisite roles for this signaling step in HG-mediated effects on β-cells. HG-induced p53 activation was also suppressed by SB203580, a known inhibitor of p38MAPK. Additionally, we observed increased activation of ATM kinase under HG conditions, which was blocked in presence of EHT1864. Furthermore, pharmacological inhibition of ATM kinase (KU55933) reduced activation of ATM kinase, but not p53, suggesting that HG-mediated activation of p53 and ATM could represent independent pro-apoptotic events. In conclusion, these data indicate that sustained activation of Rac1-p38MAPK signaling axis leads to activation of p53 leading to β-cell dysfunction under the duress of chronic hyperglycemic conditions.

  4. Cell-induced potentiation of the plasminogen activation system is abolished by a monoclonal antibody that recognizes the NH2-terminal domain of the urokinase receptor

    DEFF Research Database (Denmark)

    Rønne, E; Behrendt, N; Ellis, V

    1991-01-01

    We have raised four monoclonal antibodies recognizing different epitopes within the human cell-surface receptor for urokinase-type plasminogen activator (u-PA). One of these antibodies completely abolishes the potentiation of plasmin generation observed upon incubation of the zymogens pro...

  5. Acidic pH triggers conformational changes at the NH2-terminal propeptide of the precursor of pulmonary surfactant protein B to form a coiled coil structure.

    Science.gov (United States)

    Bañares-Hidalgo, A; Pérez-Gil, J; Estrada, P

    2014-07-01

    Pulmonary surfactant protein SP-B is synthesized as a larger precursor, proSP-B. We report that a recombinant form of human SP-BN forms a coiled coil structure at acidic pH. The protonation of a residue with pK=4.8±0.06 is the responsible of conformational changes detected by circular dichroism and intrinsic fluorescence emission. Sedimentation velocity analysis showed protein oligomerisation at any pH condition, with an enrichment of the species compatible with a tetramer at acidic pH. Low 2,2,2,-trifluoroethanol concentration promoted β-sheet structures in SP-BN, which bind Thioflavin T, at acidic pH, whereas it promoted coiled coil structures at neutral pH. The amino acid stretch predicted to form β-sheet parallel association in SP-BN overlaps with the sequence predicted by several programs to form coiled coil structure. A synthetic peptide ((60)W-E(85)) designed from the sequence of the amino acid stretch of SP-BN predicted to form coiled coil structure showed random coil conformation at neutral pH but concentration-dependent helical structure at acidic pH. Sedimentation velocity analysis of the peptide indicated monomeric state at neutral pH (s20, w=0.55S; Mr~3kDa) and peptide association (s20, w=1.735S; Mr=~14kDa) at acidic pH, with sedimentation equilibrium fitting to a Monomer-Nmer-Mmer model with N=6 and M=4 (Mr=14692Da). We propose that protein oligomerisation through coiled-coil motifs could then be a general feature in the assembly of functional units in saposin-like proteins in general and in the organization of SP-B in a functional surfactant, in particular. Copyright © 2014 Elsevier B.V. All rights reserved.

  6. Protein kinase C epsilon mediates the inhibition of angiotensin II on the slowly activating delayed-rectifier potassium current through channel phosphorylation.

    Science.gov (United States)

    Gou, Xiangbo; Wang, Wenying; Zou, Sihao; Qi, Yajuan; Xu, Yanfang

    2018-03-01

    The slowly activating delayed rectifier K + current (I Ks ) is one of the main repolarizing currents in the human heart. Evidence has shown that angiotensin II (Ang II) regulates I Ks through the protein kinase C (PKC) pathway, but the related results are controversial. This study was designed to identify PKC isoenzymes involved in the regulation of I Ks by Ang II and the underlying molecular mechanism. The whole-cell patch-clamp technique was used to record I Ks in isolated guinea pig ventricular cardiomyocytes and in human embryonic kidney (HEK) 293 cells co-transfected with human KCNQ1/KCNE1 genes and Ang II type 1 receptor genes. Ang II inhibited I Ks in a concentration-dependent manner in native cardiomyocytes. A broad PKC inhibitor Gö6983 (not inhibiting PKCε) and a selective cPKC inhibitor Gö6976 did not affect the inhibitory action of Ang II. In contrast, the inhibition was significantly attenuated by PKCε-selective peptide inhibitor εV1-2. However, direct activation of PKC by phorbol 12-myristate 13-acetate (PMA) increased the cloned human I Ks in HEK293 cells. Similarly, the cPKC peptide activator significantly enhanced the current. In contrast, the PKCε peptide activator inhibited the current. Further evidence showed that PKCε knockdown by siRNA antagonized the Ang II-induced inhibition on KCNQ1/KCNE1 current, whereas knockdown of cPKCs (PKCα and PKCβ) attenuated the potentiation of the current by PMA. Moreover, deletion of four putative phosphorylation sites in the C-terminus of KCNQ1 abolished the action of PMA. Mutation of two putative phosphorylation sites in the N-terminus of KCNQ1 and one site in KCNE1 (S102) blocked the inhibition of Ang II. Our results demonstrate that PKCε isoenzyme mediates the inhibitory action of Ang II on I Ks and by phosphorylating distinct sites in KCNQ1/KCNE1, cPKC and PKCε isoenzymes produce the contrary regulatory effects on the channel. These findings have provided new insight into the molecular mechanism

  7. Preliminary study of MR diffusion weighted imaging in nude mice models of hepatic Bel7402 tumors after adenovirus-mediated cytosine diaminase-thymidine kinase gene therapy

    International Nuclear Information System (INIS)

    Jiang Xinqing; Chen Liang; Wu Hongzhen; Huang Jingjun; Wei Xinhua; Mo Lei; Yang Ruimeng; Xiao Xiangsheng

    2012-01-01

    Objective: To study the characteristics of DWI in nude mice models of hepatic Bel7402 tumors after treatment with adenovirus-mediated cytosine diaminase-thymidine kinase (Ad. CD-TK) double suicide gene therapy, and then to identify whether DWI can be used for assessing curative effect of postoperative tumors. Methods: Thirty nude mice models of hepatic Bel7402 tumors were successfully created using cell suspension method, after the tumor grew to more than 1 cm in diameter, 20 tumor models were treated by intratumoral administration of Ad. CD-TK for 3 days plus intraperitonea (i.p.) treatment with 5-Fc and GCV for the duration of the study.Then they were randomly divided into three groups during 5-Fc and GCV treatment. The remaining 10 tumor models were used as controls. MR scanning were performed in 10 th day before and after tumor implantation in all models by using EPI-SE series and SENSE technology for treatment group. Tumor volumes and ADC values were calculated pretreatment and posttreatment. Cell apoptosis were determined by using TUNEL method. Analyze the change of ADC and apoptosis index (AI) in different times, t test was used for comparison the difference of AI and ADC values respectively. Results: After 10 days,the tumor volumes of the treatment groups and controls were respectively (724.16 ±57.45) mm 3 , (754.57 ± 66.84) mm 3 , with no significant difference (t=0.488, P >0.05). The ADC values of the treatment groups were (0.98 ±0.11) × 10 -3 mm 2 /s,the ones of the control groups were (0.68 ±0.04) × 10 -3 mm 2 /s; AI of the treatment groups were (23.25 ±6.57)%, the ones of the control groups were (2.57 ± 0.58)%. There were difference in both groups (t=4.473, 5.874; P<0.01). Conclusion: DWI can be effectively to monitor the early pathological changes of hepatic Bel7402 tumors after Ad. CD-TK double suicide gene therapy, and provide experimental evidences for clinical application. (authors)

  8. The human adenovirus E4-ORF1 protein subverts discs large 1 to mediate membrane recruitment and dysregulation of phosphatidylinositol 3-kinase.

    Directory of Open Access Journals (Sweden)

    Kathleen Kong

    2014-05-01

    Full Text Available Adenoviruses infect epithelial cells lining mucous membranes to cause acute diseases in people. They are also utilized as vectors for vaccination and for gene and cancer therapy, as well as tools to discover mechanisms of cancer due to their tumorigenic potential in experimental animals. The adenovirus E4-ORF1 gene encodes an oncoprotein that promotes viral replication, cell survival, and transformation by activating phosphatidylinositol 3-kinase (PI3K. While the mechanism of activation is not understood, this function depends on a complex formed between E4-ORF1 and the membrane-associated cellular PDZ protein Discs Large 1 (Dlg1, a common viral target having both tumor suppressor and oncogenic functions. Here, we report that in human epithelial cells, E4-ORF1 interacts with the regulatory and catalytic subunits of PI3K and elevates their levels. Like PI3K activation, PI3K protein elevation by E4-ORF1 requires Dlg1. We further show that Dlg1, E4-ORF1, and PI3K form a ternary complex at the plasma membrane. At this site, Dlg1 also co-localizes with the activated PI3K effector protein Akt, indicating that the ternary complex mediates PI3K signaling. Signifying the functional importance of the ternary complex, the capacity of E4-ORF1 to induce soft agar growth and focus formation in cells is ablated either by a mutation that prevents E4-ORF1 binding to Dlg1 or by a PI3K inhibitor drug. These results demonstrate that E4-ORF1 interacts with Dlg1 and PI3K to assemble a ternary complex where E4-ORF1 hijacks the Dlg1 oncogenic function to relocate cytoplasmic PI3K to the membrane for constitutive activation. This novel mechanism of Dlg1 subversion by adenovirus to dysregulate PI3K could be used by other pathogenic viruses, such as human papillomavirus, human T-cell leukemia virus type 1, and influenza A virus, which also target Dlg1 and activate PI3K in cells.

  9. Glycogen synthase kinase-3 inhibitors suppress the AR-V7-mediated transcription and selectively inhibit cell growth in AR-V7-positive prostate cancer cells.

    Science.gov (United States)

    Nakata, Daisuke; Koyama, Ryokichi; Nakayama, Kazuhide; Kitazawa, Satoshi; Watanabe, Tatsuya; Hara, Takahito

    2017-06-01

    Recent evidence suggests that androgen receptor (AR) splice variants, including AR-V7, play a pivotal role in resistance to androgen blockade in prostate cancer treatment. The development of new therapeutic agents that can suppress the transcriptional activities of AR splice variants has been anticipated as the next generation treatment of castration-resistant prostate cancer. High-throughput screening of AR-V7 signaling inhibitors was performed using an AR-V7 reporter system. The effects of a glycogen synthase kinase-3 (GSK3) inhibitor, LY-2090314, on endogenous AR-V7 signaling were evaluated in an AR-V7-positive cell line, JDCaP-hr, by quantitative reverse transcription polymerase chain reaction. The relationship between AR-V7 signaling and β-catenin signaling was assessed using RNA interference. The effect of LY-2090314 on cell growth in various prostate cancer cell lines was also evaluated. We identified GSK3 inhibitors as transcriptional suppressors of AR-V7 using a high-throughput screen with an AR-V7 reporter system. LY-2090314 suppressed the reporter activity and endogenous AR-V7 activity in JDCaP-hr cells. Because silencing of β-catenin partly rescued the suppression, it was evident that the suppression was mediated, at least partially, via the activation of β-catenin signaling. AR-V7 signaling and β-catenin signaling reciprocally regulate each other in JDCaP-hr cells, and therefore, GSK3 inhibition can repress AR-V7 transcriptional activity by accumulating intracellular β-catenin. Notably, LY-2090314 selectively inhibited the growth of AR-V7-positive prostate cancer cells in vitro. Our findings demonstrate the potential of GSK3 inhibitors in treating advanced prostate cancer driven by AR splice variants. In vivo evaluation of AR splice variant-positive prostate cancer models will help illustrate the overall significance of GSK3 inhibitors in treating prostate cancer. © 2017 Wiley Periodicals, Inc.

  10. Sphingosylphosphorylcholine induces α-smooth muscle actin expression in human lung fibroblasts and fibroblast-mediated gel contraction via S1P2 receptor and Rho/Rho-kinase pathway.

    Science.gov (United States)

    Wang, X Q; Mao, L J; Fang, Q H; Kobayashi, T; Kim, H J; Sugiura, H; Kawasaki, S; Togo, S; Kamio, K; Liu, X; Rennard, S I

    2014-01-01

    Chronic airway diseases like COPD and asthma are usually accompanied with airway fibrosis. Myofibroblasts, which are characterized by expression of smooth muscle actin (α-SMA), play an important role in a variety of developmental and pathological processes, including fibrosis and wound healing. Sphingosylphosphorylcholine (SPC), a sphingolipid metabolite, has been implicated in many physiological and pathological conditions. The current study tested the hypothesis that SPC may modulate tissue remodeling by affecting the expression of α-SMA in human fetal lung fibroblast (HFL-1) and fibroblast mediated gel contraction. The results show that SPC stimulates α-SMA expression in HFL-1 and augments HFL-1 mediated collagen gel contraction in a time- and concentration-dependent manner. The α-SMA protein expression and fibroblast gel contraction induced by SPC was not blocked by TGF-β1 neutralizing antibody. However, it was significantly blocked by S1P2 receptor antagonist JTE-013, the Rho-specific inhibitor C3 exoenzyme, and a Rho-kinase inhibitor Y-27632. These findings suggest that SPC stimulates α-SMA protein expression and HFL-1 mediated collagen gel contraction via S1P2 receptor and Rho/Rho kinase pathway, and by which mechanism, SPC may be involved in lung tissue remodeling. Copyright © 2014 Elsevier Inc. All rights reserved.

  11. Protein kinases mediate increment of the phosphorylation of cyclic AMP -responsive element binding protein in spinal cord of rats following capsaicin injection

    Directory of Open Access Journals (Sweden)

    Li Junfa

    2005-09-01

    Full Text Available Abstract Background Strong noxious stimuli cause plastic changes in spinal nociceptive neurons. Intracellular signal transduction pathways from cellular membrane to nucleus, which may further regulate gene expression by critical transcription factors, convey peripheral stimulation. Cyclic AMP-responsive element binding protein (CREB is a well-characterized stimulus-induced transcription factor whose activation requires phosphorylation of the Serine-133 residue. Phospho-CREB can further induce gene transcription and strengthen synaptic transmission by the activation of the protein kinase cascades. However, little is known about the mechanisms by which CREB phosphorylation is regulated by protein kinases during nociception. This study was designed to use Western blot analysis to investigate the role of mitogen-activated protein (MAP/extracellular signal-regulated kinase (ERK kinase (MEK 1/2, PKA and PKC in regulating the phosphorylation of CREB in the spinal cord of rats following intraplantar capsaicin injection. Results We found that capsaicin injection significantly increased the phosphorylation level of CREB in the ipsilateral side of the spinal cord. Pharmacological manipulation of MEK 1/2, PKA and PKC with their inhibitors (U0126, H89 and NPC 15473, respectively significantly blocked this increment of CREB phosphorylation. However, the expression of CREB itself showed no change in any group. Conclusion These findings suggest that the activation of intracellular MAP kinase, PKA and PKC cascades may contribute to the regulation of phospho-CREB in central nociceptive neurons following peripheral painful stimuli.

  12. Ablation of periostin inhibits post-infarction myocardial regeneration in neonatal mice mediated by the phosphatidylinositol 3 kinase/glycogen synthase kinase 3β/cyclin D1 signalling pathway.

    Science.gov (United States)

    Chen, Zhenhuan; Xie, Jiahe; Hao, Huixin; Lin, Hairuo; Wang, Long; Zhang, Yingxue; Chen, Lin; Cao, Shiping; Huang, Xiaobo; Liao, Wangjun; Bin, Jianping; Liao, Yulin

    2017-05-01

    To resolve the controversy as to whether periostin plays a role in myocardial regeneration after myocardial infarction (MI), we created a neonatal mouse model of MI to investigate the influence of periostin ablation on myocardial regeneration and clarify the underlying mechanisms. Neonatal periostin-knockout mice and their wildtype littermates were subjected to MI or sham surgery. In the wildtype mice after MI, fibrosis was detectable at 3 days and fibrotic tissue was completely replaced by regenerated myocardium at 21 days. In contrast, in the knockout mice, significant fibrosis in the infarcted area was present at even 3 weeks after MI. Levels of phosphorylated-histone 3 and aurora B in the myocardium, detected by immunofluorescence and western blotting, were significantly lower in knockout than in wildtype mice at 7 days after MI. Similarly, angiogenesis was decreased in the knockout mice after MI. Expression of both the endothelial marker CD-31 and α-smooth muscle actin was markedly lower in the knockout than in wildtype mice at 7 days after MI. The knockout MI group had elevated levels of glycogen synthase kinase (GSK) 3β and decreased phosphatidylinositol 3-kinase (PI3K), phosphorylated serine/threonine protein kinase B (p-Akt), and cyclin D1, compared with the wildtype MI group. Similar effects were observed in experiments using cultured cardiomyocytes from neonatal wildtype or periostin knockout mice. Administration of SB216763, a GSK3β inhibitor, to knockout neonatal mice decreased myocardial fibrosis and increased angiogenesis in the infarcted area after MI. Ablation of periostin suppresses post-infarction myocardial regeneration by inhibiting the PI3K/GSK3β/cyclin D1 signalling pathway, indicating that periostin is essential for myocardial regeneration. © The Author 2017. Published by Oxford University Press on behalf of the European Society of Cardiology

  13. Ser649 and Ser650 are the major determinants of protein kinase A-mediated activation of human hormone-sensitive lipase against lipid substrates

    DEFF Research Database (Denmark)

    Krintel, Christian; Osmark, Peter; Larsen, Martin Rask

    2008-01-01

    Hormone-sensitive lipase (HSL) is a key enzyme in the mobilization of fatty acids from stored triacylglycerols. Its activity is regulated by reversible protein phosphorylation. In rat HSL Ser563, Ser659 and Ser660 have been shown to be phosphorylated by protein kinase A (PKA) in vitro as well...

  14. Methanol extracts of Euphorbia cooperi inhibit the production of inflammatory mediators by inhibiting the activation of c‑Jun N‑terminal kinase and p38 in murine macrophages.

    Science.gov (United States)

    Cho, Young-Chang; Lee, In-Seon; Seo, Huiyun; Ju, Anna; Youn, Deokkyu; Kim, Younghyun; Choun, Jaehee; Cho, Sayeon

    2014-11-01

    Numerous Euphorbiaceae plants have been used for the treatment of diseases, including liver diseases, asthma and rheumatism. The present study evaluated the effect of methanol extracts from Euphorbia cooperi (MEC), a member of the Euphorbiaceae plant family, on the production of inflammatory cytokines interleukin (IL)‑6 and tumor necrosis factor (TNF)‑α, nitric oxide (NO) as well as the activation of mitogen‑activated protein kinase and nuclear factor (NF)‑κB signaling. Non‑cytotoxic concentrations of MEC significantly reduced the production of NO and IL‑6, but not TNF‑α, in lipopolysaccharide (LPS)‑stimulated RAW 264.7 macrophages. The decreased production of NO by MEC was due to alleviated expression of inducible NO synthase. Reporter assays with cells treated with MEC demonstrated reduced activator protein‑1 (AP-1) activity, while NF‑κB activity was not reduced. Furthermore, the phosphorylation levels of c‑Jun N‑terminal kinase (JNK) and p38 were suppressed by MEC while phosphorylation levels of inhibitor of κB were not reduced by MEC, suggesting that MEC‑mediated inactivation of JNK and p38 is the underlying regulatory mechanism for inflammatory mediators in LPS‑stimulated RAW 264.7 macrophages.

  15. Src mediates extracellular signal-regulated kinase 1/2 activation and autophagic cell death induced by cardiac glycosides in human non-small cell lung cancer cell lines.

    Science.gov (United States)

    Wang, Yan; Zhan, Yuechen; Xu, Rong; Shao, Rongguang; Jiang, Jiandong; Wang, Zhen

    2015-06-01

    Aberrant Na(+) /K(+) -ATPases (NKA) expression is closely related to the incidence and development of cancer, making NKA targeted cancer therapy more intriguing. Cardiac glycosides (CGs) belong to NKA inhibitors and possess potent anti-cancer properties in many cancers. Our previous work demonstrates that CGs family member digoxin or ouabain induces autophagic cell death in human non-small cell lung cancer (NSCLC) cell lines through regulation of both mammalian target of rapamycin and extracellular signal-regulated kinase 1/2 (ERK1/2) pathway. However, what acts as an upstream regulator of ERK1/2 activation during autophagy induction remains obscure. In the present study, the role of Src in the ERK1/2 signaling pathway as well as autophagic cell death induced by either digoxin or ouabain was examined in A549 and H460 cells. Src is significantly activated simultaneously with mitogen-activated protein kinase kinase 1/2 (MEK1/2) and ERK1/2 activation upon the drug treatment. Moreover, Src inhibitor PP2 could block either drug induced MEK1/2 and ERK1/2 phosphorylation, together with autophagic phenotypes in the cells. Knockdown of Src with siRNA causes the similar effect as PP2, both of which markedly alleviate the drugs' cytotoxicity. In addition, increased levels of intracellular reactive oxygen species (ROS) are found to be involved in Src mediated autophagy. Together, this work provides evidences showing that Src mediates MEK1/2 and ERK1/2 pathway as well as ROS generation, and regulates autophagic cell death induced by the cardiac glycosides. These observations may further help understand the molecular mechanisms of autophagy induced by NKA inhibitors in NSCLC cells. © 2014 Wiley Periodicals, Inc.

  16. Collagen-induced expression of collagenase-3 by primary chondrocytes is mediated by integrin α1 and discoidin domain receptor 2: a protein kinase C-dependent pathway.

    Science.gov (United States)

    Vonk, Lucienne A; Doulabi, Behrouz Z; Huang, ChunLing; Helder, Marco N; Everts, Vincent; Bank, Ruud A

    2011-03-01

    To investigate whether maintaining the chondrocyte's native pericellular matrix prevents collagen-induced up-regulation of collagenase-3 (MMP-13) and whether integrin α1 (ITGα1) and/or discoidin domain receptor 2 (DDR2) modulate MMP-13 expression and which signalling pathway plays a role in collagen-stimulated MMP-13 expression. Goat articular chondrocytes and chondrons were cultured on collagen coatings. Small interfering RNA (siRNA) oligonucleotides targeted against ITGα1 and DDR2 were transfected into primary chondrocytes. Chemical inhibitors for mitogen-activated protein kinase kinase (MEK1) (PD98059), focal adhesion kinase (FAK) (FAK inhibitor 14), mitogen-activated protein kinase 8 (JNK) (SP600125) and protein kinase C (PKC) (PKC412), and a calcium chelator (BAPTA-AM) were used in cell cultures. Real-time PCR was performed to examine gene expression levels of MMP-13, ITGα1 and DDR2 and collagenolytic activity was determined by measuring the amount of hydroxyproline released in the culture medium. Maintaining the chondrocyte's native pericellular matrix prevented MMP-13 up-regulation and collagenolytic activity when the cells were cultured on a collagen coating. Silencing of ITGα1 and DDR2 reduced MMP-13 gene expression and collagenolytic activity by primary chondrocytes cultured on collagen. Incubation with the PKC inhibitor strongly reduced MMP-13 gene expression levels. Gene expression levels of MMP-13 were also decreased by chondrocytes incubated with the MEK, FAK or JNK inhibitor. Maintaining the native pericellular matrix of chondrocytes prevents collagen-induced up-regulation of MMP-13. Both ITGα1 and DDR2 modulate MMP-13 expression after direct contact between chondrocytes and collagen. PKC, FAK, MEK and JNK are involved in collagen-stimulated expression of MMP-13.

  17. Changes in resting mitogen-activated protein kinases following resistance exercise overreaching and overtraining.

    Science.gov (United States)

    Nicoll, Justin X; Fry, Andrew C; Galpin, Andrew J; Sterczala, Adam J; Thomason, Donald B; Moore, Christopher A; Weiss, Lawrence W; Chiu, Loren Z F

    2016-12-01

    Many physiological maladaptations persist after overreaching and overtraining resistance exercise (RE). However, no studies have investigated changes in mitogen-activated protein kinases (MAPK) after overtraining in humans, despite their critical role regulating exercise-induced muscular adaptations. The purpose of this study was to describe the changes in total and resting phosphorylation status of extracellular signal-regulated kinase 1/2 (ERK1/2), c-Jun NH 2 -terminal kinase (JNK) and p38-MAPK following a period of RE overreaching or overtraining. Following 2-4 weeks of normal training (low volume/low intensity), two groups of males performed either a high-power overreaching protocol (HPOR n = 6, mean ± SD, age 23 ± 3.4 years, mass 86.5 ± 17.7 kg, height 1.77 ± 0.06 m) or high-intensity overtraining protocol (HIOT n = 8, age 19.8 ± 1.8 years, mass 76.8 ± 6.7 kg, height 1.8 ± 0.06 m). Resting muscle biopsies were obtained at baseline (BL; end of normal training period) and 24 h after the final session of stressful training (i.e., HPOR or HIOT programs). Total MAPK and ratio of phosphorylated/total (p-MAPK)- ERK1/2, JNK, and p38-MAPK were analyzed via western blotting. 2 × 2 (group × time) ANOVA determined differences in MAPK between BL and post-training protocols. Compared to BL, total-ERK increased after HPOR, but decreased after HIOT (p ≤ 0.05). p-ERK1/2/total-ERK increased after HIOT (p ≤ 0.05). The ratio of p-JNK/total-JNK and p-ERK1/2/total-ERK decreased after HPOR (p ≤ 0.05); however, this result was primarily due to increased total MAPK content. p-p38-MAPK decreased after HPOR (p ≤ 0.05). Total and p-MAPK are differentially expressed after HPOR and HIOT RE. These changes are likely involved in the maladaptation reported in overreaching and overtraining exercise. This is the first study describing altered MAPK in RE overtrained and overreached humans.

  18. c-Met Overexpression Contributes to the Acquired Apoptotic Resistance of Nonadherent Ovarian Cancer Cells through a Cross Talk Mediated by Phosphatidylinositol 3-Kinase and Extracellular Signal-Regulated Kinase 1/2

    Directory of Open Access Journals (Sweden)

    Maggie K.S. Tang

    2010-02-01

    Full Text Available Ovarian cancer is the most lethal gynecologic cancer mainly because of widespread peritoneal dissemination and malignant ascites. Key to this is the capacity of tumor cells to escape suspension-induced apoptosis (anoikis, which also underlies their resistance to chemotherapy. Here, we used a nonadherent cell culture model to investigate the molecular mechanisms of apoptotic resistance of ovarian cancer cells that may mimic the chemoresistance found in solid tumors. We found that ovarian cancer cells acquired a remarkable resistance to anoikis and apoptosis induced by exposure to clinically relevant doses of two front-line chemotherapeutic drugs cisplatin and paclitaxel when grown in three-dimensional than monolayer cultures. Inhibition of the hepatocyte growth factor (HGF receptor c-Met, which is frequently overexpressed in ovarian cancer, by a specific inhibitor or small interfering RNA blocked the acquired anoikis resistance and restored chemosensitivity in three-dimensional not in two-dimensional cultures. These effects were found to be dependent on both phosphatidylinositol 3-kinase (PI3K/Akt and extracellular signal-regulated kinase (ERK 1/2 signaling pathways. Inhibitors of PI3K/Akt abrogated ERK1/2 activation and its associated anoikis resistance in response to HGF, suggesting a signaling relay between these two pathways. Furthermore, we identified a central role of Ras as a mechanism of this cross talk. Interestingly, Ras did not lie upstream of PI3K/Akt, whereas PI3K/Akt signaling to ERK1/2 involved Ras. These findings shed new light on the apoptotic resistance mechanism of nonadherent ovarian cancer ascites cells and may have important clinical implications.

  19. Kinases and Cancer.

    Science.gov (United States)

    Cicenas, Jonas; Zalyte, Egle; Bairoch, Amos; Gaudet, Pascale

    2018-03-01

    Protein kinases are a large family of enzymes catalyzing protein phosphorylation. The human genome contains 518 protein kinase genes, 478 of which belong to the classical protein kinase family and 40 are atypical protein kinases [...].

  20. Kinases and Cancer

    OpenAIRE

    Jonas Cicenas; Egle Zalyte; Amos Bairoch; Pascale Gaudet

    2018-01-01

    Protein kinases are a large family of enzymes catalyzing protein phosphorylation. The human genome contains 518 protein kinase genes, 478 of which belong to the classical protein kinase family and 40 are atypical protein kinases [...

  1. Hexamethylenebisacetamide modulation of thyroglobulin and protein levels in thyroid cells is not mediated by phosphatidylinositol-3-kinase: a study with wortmannin.

    Science.gov (United States)

    Aouani, A; Samih, N; Amphoux-Fazekas, T; Hovsépian, S; Fayet, G

    1999-04-01

    Hexamethylenebisacetamide (HMBA) induces in murine erythroleukemia cells (MELC) the commitment to terminal differentiation leading to globin gene expression. In the thyroid, HMBA acts as a growth factor and also as a differentiating agent. In the present paper, we studied the effect of HMBA on the very specific thyroid marker thyroglobulin (Tg) in two different thyroid cell systems, i.e., porcine cells in primary culture and ovine cells in long term culture. Using wortmannin, a specific inhibitor of phosphatidylinositol-3-kinase, we investigated whether this enzyme is involved in HMBA mode of action. We found that HMBA is a positive modulator of Tg production in porcine cells, but a negative effector in the OVNIS cell line. As all HMBA effects studied in the present paper, i.e., Tg production and total protein levels, are not inhibited by wortmannin, we suggest the non-involvement of phosphatidylinositol-3-kinase in HMBA mode of action.

  2. Beneficial Effect of Intermedin 1-53 in Septic Shock Rats: Contributions of Rho Kinase and BKCA Pathway-Mediated Improvement in Cardiac Function.

    Science.gov (United States)

    Zhu, Yu; Wu, Huiling; Wu, Yue; Zhang, Jie; Peng, Xiaoyong; Zang, Jiatao; Xiang, Xinming; Liu, Liangming; Li, Tao

    2016-11-01

    Intermedin (IMD) is a calcitonin gene-related peptide shown to have a protective effect on myocardial function in ischemia-reperfusion injury. Whether IMD has beneficial effect in severe sepsis and septic shock (and its underlying mechanisms) is not known. We induced septic shock using cecal ligation and puncture (CLP). We focused on the potential beneficial effect of IMD1-53 on cardiac papillary muscle and cardiomyocytes against septic shock and its relationship with the protection of cardiac function. Early (immediately after CLP) and late (12 h after CLP) administration of IMD1-53 (0.5 μg/kg) improved animal survival significantly, increased cardiac contractility and function, and improved tissue perfusion and oxygen delivery. The effect of early administration of IMD1-53 was better than that of late administration. The Rho kinase/TnI and BKCa pathways participated in the protective effect of IMD1-53 on cardiac function in septic rats. An inhibitor of Rho kinase (Y-27632) or a BKCa opener (NS1619) abolished the protective effect of IMD1-53 on cardiac function. IMD1-53 increased expression of Rho kinase in cardiac muscle and inhibited TnI phosphorylation. IMD1-53 inhibited currents in BKCa channels and intracellular calcium concentration in cardiomyocytes. IMD1-53 is beneficial against severe sepsis/septic shock. IMD1-53 can improve cardiac contractility and cardiac function significantly, and then improve tissue perfusion and oxygen delivery. Rho kinase and the BKCa pathways have important roles in these effects. These findings provide a new treatment strategy for severe sepsis with cardiac dysfunction.

  3. Priming phosphorylation of Chk2 by polo-like kinase 3 (Plk3) mediates its full activation by ATM and a downstream checkpoint in response to DNA damage

    Energy Technology Data Exchange (ETDEWEB)

    Bahassi, El Mustapha [Department of Cell Biology, University of Cincinnati College of Medicine, 3125 Eden Avenue, Cincinnati, OH 45267 (United States); Myer, David L. [Department of Cell Biology, University of Cincinnati College of Medicine, 3125 Eden Avenue, Cincinnati, OH 45267 (United States); McKenney, Richard J. [Department of Cell Biology, University of Cincinnati College of Medicine, 3125 Eden Avenue, Cincinnati, OH 45267 (United States); Hennigan, Robert F. [Department of Cell Biology, University of Cincinnati College of Medicine, 3125 Eden Avenue, Cincinnati, OH 45267 (United States); Stambrook, Peter J. [Department of Cell Biology, University of Cincinnati College of Medicine, 3125 Eden Avenue, Cincinnati, OH 45267 (United States)]. E-mail: peter.stambrook@uc.edu

    2006-04-11

    The tumor suppressor gene Chk2 encodes a serine/threonine kinase that signals DNA damage to cell cycle checkpoints. In response to ionizing radiation, Chk2 is phosphorylated on threonine 68 (T68) by ataxia-telangiectasia mutated (ATM) protein leading to its activation. We have previously shown that polo-like kinase 3 (Plk3), a protein involved in DNA damage checkpoint and M-phase functions, interacts with and phosphorylates Chk2. When Chk2 was immunoprecipitated from Daudi cells (Plk3-deficient), it had weak kinase activity towards Cdc25C compared with Chk2 derived from T47D cells (Plk3-expressing cells). This activity was restored by addition of recombinant Plk3 in a dose-dependent manner. Plk3 phosphorylates Chk2 at two residues, serine 62 (S62) and serine 73 (S73) in vitro, and this phosphorylation facilitates subsequent phosphorylation of Chk2 on T68 by ATM in response to DNA damage. When the Chk2 mutant construct GFP-Chk2 S73A (serine 73 mutated to alanine) is transfected into cells, it no longer associates with a large complex in vivo, and manifests a significant reduction in kinase activity. It is also inefficiently activated by ATM by phosphorylation at T68 and, in turn, is unable to phosphorylate the Cdc25C peptide 200-256, which contains the inhibitory S216 target phosphorylation residue. As a consequence, tyrosine 15 (Y15) on Cdc2 remains hypophosphorylated, and there is a loss of the G2/M checkpoint. These data describe a functional role for Plk3 in a pathway linking ATM, Plk3, Chk2, Cdc25C and Cdc2 in cellular response to DNA damage.

  4. Coumestrol suppresses hypoxia inducible factor 1α by inhibiting ROS mediated sphingosine kinase 1 in hypoxic PC-3 prostate cancer cells.

    Science.gov (United States)

    Cho, Sung-Yun; Cho, Sunmi; Park, Eunkyung; Kim, Bonglee; Sohn, Eun Jung; Oh, Bumsuk; Lee, Eun-Ok; Lee, Hyo-Jeong; Kim, Sung-Hoon

    2014-06-01

    Among many signals to regulate hypoxia inducible factor 1α (HIF-1α), sphingosine kinase 1 (SPHK1) is also involved in various biological activities such as cell growth, survival, invasion, angiogenesis, and carcinogenesis. Thus, in the present study, molecular mechanisms of coumestrol were investigated on the SPHK1 and HIF-1α signaling pathway in hypoxic PC-3 prostate cancer cells. Coumestrol significantly suppressed SPHK1 activity and accumulation of HIF-1α in a time- and concentration-dependent manner in hypoxic PC-3 cells. In addition, coumestrol inhibited the phosphorylation status of AKT and glycogen synthase kinase-3β (GSK 3β) signaling involved in cancer metabolism. Furthermore, SPHK1 siRNA transfection, sphigosine kinase inhibitor (SKI), reactive oxygen species (ROS) enhanced the inhibitory effect of coumestrol on the accumulation of HIF-1α and the expression of pAKT and pGSK 3β in hypoxic PC-3 cells by combination index. Overall, our findings suggest that coumestrol suppresses the accumulation of HIF-1α via suppression of SPHK1 pathway in hypoxic PC-3 cells. Copyright © 2014. Published by Elsevier Ltd.

  5. Intracellular signaling of the Ufo/Axl receptor tyrosine kinase is mediated mainly by a multi-substrate docking-site.

    Science.gov (United States)

    Braunger, J; Schleithoff, L; Schulz, A S; Kessler, H; Lammers, R; Ullrich, A; Bartram, C R; Janssen, J W

    1997-06-05

    Ufo/Axl belongs to a new family of receptor tyrosine kinases with an extracellular structure similar to that of neural cell adhesion molecules. In order to elucidate intracellular signaling, the cytoplasmic moiety of Ufo/Axl was used to screen an expression library according to the CORT (cloning of receptor targets) method. Three putative Ufo substrates were identified: phospholipase Cgamma1 (PLCgamma), as well as p85alpha and p85beta subunits of phosphatidylinositol 3'-kinase (PI3-kinase). Subsequently, chimeric EGFR/Ufo receptors consisting of the extracellular domains of the epidermal growth factor receptor (EGFR) and the transmembrane and intracellular moiety of Ufo were engineered. Using different far-Western blot analyses and coimmunoprecipitation assays, receptor binding of PLCgamma and p85 proteins as well as GRB2, c-src and lck was examined in vitro and in vivo. Competitive inhibition of substrate binding and mutagenesis experiments with EGFR/Ufo constructs revealed C-terminal tyrosine 821 (EILpYVNMDEG) as a docking site for multiple effectors, namely PLCgamma, p85 proteins, GRB2, c-src and lck. Tyrosine 779 (DGLpYALMSRC) demonstrated an additional, but lower binding affinity for the p85 proteins in vitro. In addition, binding of PLCgamma occurred through tyrosine 866 (AGRpYVLCPST). Moreover, our in vivo data indicate that further direct or indirect binding sites for PLCgamma, GRB2, c-src and lck on the human Ufo receptor may exist.

  6. Ibuprofen abates cypermethrin-induced expression of pro-inflammatory mediators and mitogen-activated protein kinases and averts the nigrostriatal dopaminergic neurodegeneration.

    Science.gov (United States)

    Singh, Ashish; Tripathi, Pratibha; Prakash, Om; Singh, Mahendra Pratap

    2016-12-01

    Cypermethrin induces oxidative stress, microglial activation, inflammation and apoptosis leading to Parkinsonism in rats. While ibuprofen, a non-steroidal anti-inflammatory drug, relieves from inflammation, its efficacy against cypermethrin-induced Parkinsonism has not yet been investigated. The study aimed to explore the protective role of ibuprofen in cypermethrin-induced Parkinsonism, an environmentally relevant model of Parkinson's disease (PD), along with its underlying mechanism. Animals were treated with/without cypermethrin in the presence/absence of ibuprofen. Behavioural, immunohistochemical and biochemical parameters of Parkinsonism and expression of pro-inflammatory and pro-apoptotic proteins along with mitogen-activated protein kinases (MAPKs) were determined. Ibuprofen resisted cypermethrin-induced behavioural impairments, striatal dopamine depletion, oxidative stress in the nigrostriatal tissues and loss of the nigral dopamine producing cells and increase in microglial activation along with atypical expression of pro-inflammatory and apoptotic proteins that include cyclooxygenase-2, tumour necrosis factor-α, MAPKs (c-Jun N-terminal kinase, p38 and extracellular signal-regulated kinase), B cell lymphoma 2-associated protein X, tumour suppressor protein p53, cytochrome c and caspase-3 in the nigrostriatal tissue. The results obtained thus demonstrate that ibuprofen lessens inflammation and regulates MAPKs expression thereby averts cypermethrin-induced Parkinsonism.

  7. Rapid activation of c-Src kinase by dioxin is mediated by the Cdc37-HSP90 complex as part of Ah receptor signaling in MCF10A cells.

    Science.gov (United States)

    Park, Sujin; Dong, Bin; Matsumura, Fumio

    2007-01-23

    We investigated the mechanism by which activation of the Ah receptor by dioxin (TCDD) was accompanied by rapid activation of c-Src kinase activity. A Western blotting analysis showed that such action of TCDD in MCF10A cells could effectively be suppressed by treatment with a specific inhibitor of Src family kinase, PP-2, as judged by Western blot detection of the active form of Src protein, indicating that Src kinase is directly activated by TCDD. Such an event, occurring within 10-30 min of the addition of TCDD, is also accompanied by simultaneous translocation of both Src and cdc37 proteins from cytosol into the 100,000 x g membrane fraction containing the plasma membrane. By dissociating the cytosolic Src-cdc37-HSP90 complex with 17 nM geldanamycin, an optimum concentration for affecting this cytosolic cdc37 complex, but not the cytosolic Ah receptor complex, we could show that the action of TCDD in activating c-Src and cdc37 was abolished, but not its action on CYP1A1. The important role of cdc37 in the action of TCDD-induced activation of c-Src was also confirmed by blocking cdc37 gene translation with the antisense oligonucleotide treatment as well as the siRNA preparation designed to silence cdc37 expression. To understand the functional meaning of the disruption of the Src-cdc37-HSP90 complex by 17 nM geldanamycin at the cellular level, we investigated its effect on TCDD-induced anti-apoptotic action. The results showed that geldanamycin at this concentration could also abolish this cellular effect of TCDD. Interestingly, such a role of cdc37 in mediating the action of TCDD appears to be limited to activation of c-Src kinase, but not kinases associated with activation of NFkB, C/EBPalpha, or ERK. Together, these observations support the hypothesis that there is a specific coordination between the activation of the cytosolic Ah receptor and the c-Src- and cdc37-containing HSP90 complex.

  8. 4-Chlorotetrazolo[1,5-a]quinoxaline inhibits activation of Syk kinase to suppress mast cells in vitro and mast cell-mediated passive cutaneous anaphylaxis in mice

    Energy Technology Data Exchange (ETDEWEB)

    Park, Kui Lea [Center for Drug Development Assistance, National Institute of Food Drug Safety Evaluation (NIFDS), KFDA, Cheongwon-gun (Korea, Republic of); Ko, Na Young; Lee, Jun Ho; Kim, Do Kyun; Kim, Hyuk Soon; Kim, A-Ram; Her, Erk; Kim, Bokyung [Department of Immunology and physiology, College of Medicine, Konkuk University, Chungju (Korea, Republic of); Kim, Hyung Sik [College of Pharmacy, Pusan National University, Busan (Korea, Republic of); Moon, Eun-Yi [Department of Bioscience and Biotechnology, College of Biological Science, Sejong University, Seoul (Korea, Republic of); Kim, Young Mi [College of Pharmacy, Duksung Women' s University, Seoul (Korea, Republic of); Kim, Hang-Rae, E-mail: hangrae2@snu.ac.kr [Department of Anatomy, Seoul National University College of Medicine, Seoul (Korea, Republic of); Choi, Wahn Soo, E-mail: wahnchoi@kku.ac.kr [Department of Immunology and physiology, College of Medicine, Konkuk University, Chungju (Korea, Republic of)

    2011-12-15

    4-Chlorotetrazolo[1,5-a]quinoxaline is a quinoxaline derivative. We aimed to study the effects of 4-chlorotetrazolo[1,5-a]quinoxaline on activation of mast cells in vitro and in mice. 4-Chlorotetrazolo[1,5-a]quinoxaline reversibly inhibited degranulation of mast cells in a dose-dependent manner, and also suppressed the expression and secretion of TNF-{alpha} and IL-4 in mast cells. Mechanistically, 4-chlorotetrazolo[1,5-a]quinoxaline inhibited activating phosphorylation of Syk and LAT, which are crucial for early Fc{epsilon}RI-mediated signaling events, as well as Akt and MAP kinases, which play essential roles in the production of various pro-inflammatory cytokines in mast cells. Notably, although 4-chlorotetrazolo[1,5-a]quinoxaline inhibited the activation of Fyn and Syk, minimal inhibition was observed in mast cells in the case of Lyn. Furthermore, consistent with its in vitro activity, 4-chlorotetrazolo[1,5-a]quinoxaline significantly suppressed mast cell-mediated passive cutaneous anaphylaxis in mice. In summary, the results from this study demonstrate that 4-chlorotetrazolo[1,5-a]quinoxaline shows an inhibitory effect on mast cells in vitro and in vivo, and that this is mediated by inhibiting the activation of Syk in mast cells. Therefore, 4-chlorotetrazolo[1,5-a]quinoxaline could be useful in the treatment of mast cell-mediated allergic diseases. -- Highlights: Black-Right-Pointing-Pointer 4-chlorotetrazolo[1,5-a]quinoxaline is a quinoxaline derivative. Black-Right-Pointing-Pointer The effect of 4-chlorotetrazolo[1,5-a]quinoxaline on mast cells was investigated. Black-Right-Pointing-Pointer 4-chlorotetrazolo[1,5-a]quinoxaline reversibly inhibited Syk activation. Black-Right-Pointing-Pointer 4-chlorotetrazolo[1,5-a]quinoxaline could be useful for IgE-mediated allergy.

  9. 4-Chlorotetrazolo[1,5-a]quinoxaline inhibits activation of Syk kinase to suppress mast cells in vitro and mast cell-mediated passive cutaneous anaphylaxis in mice

    International Nuclear Information System (INIS)

    Park, Kui Lea; Ko, Na Young; Lee, Jun Ho; Kim, Do Kyun; Kim, Hyuk Soon; Kim, A-Ram; Her, Erk; Kim, Bokyung; Kim, Hyung Sik; Moon, Eun-Yi; Kim, Young Mi; Kim, Hang-Rae; Choi, Wahn Soo

    2011-01-01

    4-Chlorotetrazolo[1,5-a]quinoxaline is a quinoxaline derivative. We aimed to study the effects of 4-chlorotetrazolo[1,5-a]quinoxaline on activation of mast cells in vitro and in mice. 4-Chlorotetrazolo[1,5-a]quinoxaline reversibly inhibited degranulation of mast cells in a dose-dependent manner, and also suppressed the expression and secretion of TNF-α and IL-4 in mast cells. Mechanistically, 4-chlorotetrazolo[1,5-a]quinoxaline inhibited activating phosphorylation of Syk and LAT, which are crucial for early FcεRI-mediated signaling events, as well as Akt and MAP kinases, which play essential roles in the production of various pro-inflammatory cytokines in mast cells. Notably, although 4-chlorotetrazolo[1,5-a]quinoxaline inhibited the activation of Fyn and Syk, minimal inhibition was observed in mast cells in the case of Lyn. Furthermore, consistent with its in vitro activity, 4-chlorotetrazolo[1,5-a]quinoxaline significantly suppressed mast cell-mediated passive cutaneous anaphylaxis in mice. In summary, the results from this study demonstrate that 4-chlorotetrazolo[1,5-a]quinoxaline shows an inhibitory effect on mast cells in vitro and in vivo, and that this is mediated by inhibiting the activation of Syk in mast cells. Therefore, 4-chlorotetrazolo[1,5-a]quinoxaline could be useful in the treatment of mast cell-mediated allergic diseases. -- Highlights: ► 4-chlorotetrazolo[1,5-a]quinoxaline is a quinoxaline derivative. ► The effect of 4-chlorotetrazolo[1,5-a]quinoxaline on mast cells was investigated. ► 4-chlorotetrazolo[1,5-a]quinoxaline reversibly inhibited Syk activation. ► 4-chlorotetrazolo[1,5-a]quinoxaline could be useful for IgE-mediated allergy.

  10. Protein kinase C-mediated ATP stimulation of Na(+)-ATPase activity in LLC-PK1 cells involves a P2Y2 and/or P2Y4 receptor.

    Science.gov (United States)

    Wengert, M; Ribeiro, M C; Abreu, T P; Coutinho-Silva, R; Leão-Ferreira, L R; Pinheiro, A A S; Caruso-Neves, C

    2013-07-15

    ATP-activated P2Y receptors play an important role in renal sodium excretion. The aim of this study was to evaluate the modulation of ATPase-driven sodium reabsorption in the proximal tubule by ATP or adenosine (Ado). LLC-PK1 cells, a model of porcine proximal tubule cells, were used. ATP (10(-6)M) or Ado (10(-6)M) specifically stimulated Na(+)-ATPase activity without any changes in (Na(+)+K(+))-ATPase activity. Our results show that the Ado effect is mediated by its conversion to ATP. Furthermore, it was observed that the effect of ATP was mimicked by UTP, ATPγS and 2-thio-UTP, an agonist of P2Y2 and P2Y4 receptors. In addition, ATP-stimulated Na(+)-ATPase activity involves protein kinase C (PKC). Our results indicate that ATP-induced stimulation of proximal tubule Na(+)-ATPase activity is mediated by a PKC-dependent P2Y2 and/or P2Y4 pathway. These findings provide new perspectives on the role of the effect of P2Y-mediated extracellular ATP on renal sodium handling. Copyright © 2013 Elsevier Inc. All rights reserved.

  11. Diacylglycerol kinase α mediates 17-β-estradiol-induced proliferation, motility, and anchorage-independent growth of Hec-1A endometrial cancer cell line through the G protein-coupled estrogen receptor GPR30.

    Science.gov (United States)

    Filigheddu, Nicoletta; Sampietro, Sara; Chianale, Federica; Porporato, Paolo E; Gaggianesi, Miriam; Gregnanin, Ilaria; Rainero, Elena; Ferrara, Michele; Perego, Beatrice; Riboni, Francesca; Baldanzi, Gianluca; Graziani, Andrea; Surico, Nicola

    2011-12-01

    Increased levels of endogenous and/or exogenous estrogens are one of the well known risk factors of endometrial cancer. Diacylglycerol kinases (DGKs) are a family of enzymes which phosphorylate diacylglycerol (DAG) to produce phosphatidic acid (PA), thus turning off and on DAG-mediated and PA-mediated signaling pathways, respectively. DGK α activity is stimulated by growth factors and oncogenes and is required for chemotactic, proliferative, and angiogenic signaling in vitro. Herein, using either specific siRNAs or the pharmacological inhibitor R59949, we demonstrate that DGK α activity is required for 17-β-estradiol (E2)-induced proliferation, motility, and anchorage-independent growth of Hec-1A endometrial cancer cell line. Impairment of DGK α activity also influences basal cell proliferation and growth in soft agar of Hec-1A, while it has no effects on basal cell motility. Moreover, we show that DGK α activity induced by E2, as well as its observed effects, are mediated by the G protein-coupled estrogen receptor GPR30 (GPER). These findings suggest that DGK α may be a potential target in endometrial cancer therapy. Copyright © 2011 Elsevier Inc. All rights reserved.

  12. Cadmium induces apoptosis in pancreatic β-cells through a mitochondria-dependent pathway: the role of oxidative stress-mediated c-Jun N-terminal kinase activation.

    Directory of Open Access Journals (Sweden)

    Kai-Chih Chang

    Full Text Available Cadmium (Cd, one of well-known highly toxic environmental and industrial pollutants, causes a number of adverse health effects and diseases in humans. The growing epidemiological studies have suggested a possible link between Cd exposure and diabetes mellitus (DM. However, the toxicological effects and underlying mechanisms of Cd-induced pancreatic β-cell injury are still unknown. In this study, we found that Cd significantly decreased cell viability, and increased sub-G1 hypodiploid cells and annexin V-Cy3 binding in pancreatic β-cell-derived RIN-m5F cells. Cd also increased intracellular reactive oxygen species (ROS generation and malondialdehyde (MDA production and induced mitochondrial dysfunction (the loss of mitochondrial membrane potential (MMP and the increase of cytosolic cytochrome c release, the decreased Bcl-2 expression, increased p53 expression, poly (ADP-ribose polymerase (PARP cleavage, and caspase cascades, which accompanied with intracellular Cd accumulation. Pretreatment with the antioxidant N-acetylcysteine (NAC effectively reversed these Cd-induced events. Furthermore, exposure to Cd induced the phosphorylations of c-jun N-terminal kinases (JNK, extracellular signal-regulated kinases (ERK1/2, and p38-mitogen-activated protein kinase (MAPK, which was prevented by NAC. Additionally, the specific JNK inhibitor SP600125 or JNK-specific small interference RNA (si-RNA transfection suppressed Cd-induced β-cell apoptosis and related signals, but not ERK1/2 and p38-MAPK inhibitors (PD98059 and SB203580 did not. However, the JNK inhibitor or JNK-specific si-RNA did not suppress ROS generation in Cd-treated cells. These results indicate that Cd induces pancreatic β-cell death via an oxidative stress downstream-mediated JNK activation-triggered mitochondria-regulated apoptotic pathway.

  13. Angiotensin II–Induced MMP-2 Activity and MMP-14 and Basigin Protein Expression Are Mediated via the Angiotensin II Receptor Type 1–Mitogen-Activated Protein Kinase 1 Pathway in Retinal Pigment Epithelium

    Science.gov (United States)

    Pons, Marianne; Cousins, Scott W.; Alcazar, Oscar; Striker, Gary E.; Marin-Castaño, Maria E.

    2011-01-01

    Accumulation of various lipid-rich extracellular matrix (ECM) deposits under the retinal pigment epithelium (RPE) has been observed in eyes with age-related macular degeneration (AMD). RPE-derived matrix metalloproteinase (MMP)-2, MMP-14, and basigin (BSG) are major enzymes involved in the maintenance of ECM turnover. Hypertension (HTN) is a systemic risk factor for AMD. It has previously been reported that angiotensin II (Ang II), one of the most important hormones associated with HTN, increases MMP-2 activity and its key regulator, MMP-14, in RPE, inducing breakdown of the RPE basement membrane, which may lead to progression of sub-RPE deposits. Ang II exerts most of its actions by activating the mitogen-activated protein kinase (MAPK) signaling pathway. Herein is explored the MAPK signaling pathway as a potential key intracellular modulator of Ang II–induced increase in MMP-2 activity and MMP-14 and BSG protein expression. It was observed that Ang II stimulates phosphorylation of extracellular signal-regulated kinase (ERK) and p38 MAPK in RPE cells and ERK/p38 and Jun N-terminal kinase (JNK) in mice. These effects were mediated by Ang II type 1 receptors. Blockade of ERK or p38 MAPK abrogated the increase in MMP-2 activity and MMP-14 and BSG proteins in ARPE-19 cells. A better understanding of the molecular events by which Ang II induces ECM dysregulation is of critical importance to further define its contribution to the progression of sub-RPE deposits in AMD patients with HTN. PMID:21641389

  14. Anti-inflammatory effects of cordycepin in lipopolysaccharide-stimulated RAW 264.7 macrophages through Toll-like receptor 4-mediated suppression of mitogen-activated protein kinases and NF-κB signaling pathways

    Directory of Open Access Journals (Sweden)

    Choi YH

    2014-10-01

    Full Text Available Yung Hyun Choi,1,2 Gi-Young Kim,3 Hye Hyeon Lee4 1Department of Biochemistry, Dongeui University College of Korean Medicine, Busan, 2Anti-Aging Research Center and Blue-Bio Industry RIC, Dongeui University, Busan, 3Laboratory of Immunobiology, Department of Marine Life Sciences, Jeju National University, Jeju, 4Daegu Gyeongbuk Institute of Science and Technology, Daegu, Republic of Korea Abstract: Cordycepin is the main functional component of the Cordyceps species, which has been widely used in traditional Oriental medicine. This compound possesses many pharmacological properties, such as an ability to enhance immune function, as well as antioxidant, antiaging, and anticancer effects. In the present study, we investigated the anti-inflammatory effects of cordycepin using a murine macrophage RAW 264.7 cell model. Our data demonstrated that cordycepin suppressed production of proinflammatory mediators such as nitric oxide (NO and prostaglandin E2 by inhibiting inducible NO synthase and cyclooxygenase-2 gene expression. Cordycepin also inhibited the release of proinflammatory cytokines, including tumor necrosis factor-alpha and interleukin-1-beta, through downregulation of respective mRNA expression. In addition, pretreatment with cordycepin significantly inhibited lipopolysaccharide (LPS-induced phosphorylation of mitogen-activating protein kinases and attenuated nuclear translocation of NF-κB by LPS, which was associated with abrogation of inhibitor kappa B-alpha degradation. Furthermore, cordycepin potently inhibited the binding of LPS to macrophages and LPS-induced Toll-like receptor 4 and myeloid differentiation factor 88 expression. Taken together, the results suggest that the inhibitory effects of cordycepin on LPS-stimulated inflammatory responses in RAW 264.7 macrophages are associated with suppression of mitogen-activating protein kinases and activation of NF-κB by inhibition of the Toll-like receptor 4 signaling pathway. Keywords

  15. Spleen tyrosine kinase mediates high glucose-induced transforming growth factor-{beta}1 up-regulation in proximal tubular epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Won Seok; Chang, Jai Won [Division of Nephrology, Department of Internal Medicine, Asan Medical Center, College of Medicine, University of Ulsan, Seoul (Korea, Republic of); Han, Nam Jeong [Department of Cell Biology, Asan Institute for Life Sciences, Seoul (Korea, Republic of); Lee, Sang Koo [Division of Nephrology, Department of Internal Medicine, Asan Medical Center, College of Medicine, University of Ulsan, Seoul (Korea, Republic of); Park, Su-Kil, E-mail: skpark@amc.seoul.kr [Division of Nephrology, Department of Internal Medicine, Asan Medical Center, College of Medicine, University of Ulsan, Seoul (Korea, Republic of)

    2012-09-10

    The role of spleen tyrosine kinase (Syk) in high glucose-induced intracellular signal transduction has yet to be elucidated. We investigated whether Syk is implicated in high glucose-induced transforming growth factor-{beta}1 (TGF-{beta}1) up-regulation in cultured human proximal tubular epithelial cells (HK-2 cell). High glucose increased TGF-{beta}1 gene expression through Syk, extracellular signal-regulated kinase (ERK), AP-1 and NF-{kappa}B. High glucose-induced AP-1 DNA binding activity was decreased by Syk inhibitors and U0126 (an ERK inhibitor). Syk inhibitors suppressed high glucose-induced ERK activation, whereas U0126 had no effect on Syk activation. High glucose-induced NF-{kappa}B DNA binding activity was also decreased by Syk inhibitors. High glucose increased nuclear translocation of p65 without serine phosphorylation of I{kappa}B{alpha} and without degradation of I{kappa}B{alpha}, but with an increase in tyrosine phosphorylation of I{kappa}B{alpha} that may account for the activation of NF-{kappa}B. Both Syk inhibitors and Syk-siRNA attenuated high glucose-induced I{kappa}B{alpha} tyrosine phosphorylation and p65 nuclear translocation. Depletion of p21-activated kinase 2 (Pak2) by transfection of Pak2-siRNA abolished high glucose-induced Syk activation. In summary, high glucose-induced TGF-{beta}1 gene transcription occurred through Pak2, Syk and subsequent ERK/AP-1 and NF-{kappa}B pathways. This suggests that Syk might be implicated in the diabetic kidney disease.

  16. Xingshentongqiao Decoction Mediates Proliferation, Apoptosis, Orexin-A Receptor and Orexin-B Receptor Messenger Ribonucleic Acid Expression and Represses Mitogen-activated Protein Kinase Signaling

    Directory of Open Access Journals (Sweden)

    Yuanli Dong

    2015-01-01

    Full Text Available Background: Hypocretin (HCRT signaling plays an important role in the pathogenesis of narcolepsy and can be significantly influenced by Chinese herbal therapy. Our previous study showed that xingshentongqiao decoction (XSTQ is clinically effective for the treatment of narcolepsy. To determine whether XSTQ improves narcolepsy by modulating HCRT signaling, we investigated its effects on SH-SY5Y cell proliferation, apoptosis, and HCRT receptor 1/2 (orexin receptor 1 [OX1R] and orexin receptor 2 [OX2R] expression. The signaling pathways involved in these processes were also assessed. Methods: The effects of XSTQ on proliferation and apoptosis in SH-SY5Y cells were assessed using cell counting kit-8 and annexin V-fluorescein isothiocyanate assays. OX1R and OX2R expression was assessed by quantitative real-time polymerase chain reaction analysis. Western blotting for mitogen-activated protein kinase (MAPK pathway activation was performed to further assess the signaling mechanism of XSTQ. Results: XSTQ reduced the proliferation and induced apoptosis of SH-SY5Y cells. This effect was accompanied by the upregulation of OX1R and OX2R expression and the reduced phosphorylation of extracellular signal-regulated kinase (Erk 1/2, p38 MAPK and c-Jun N-terminal kinase (JNK. Conclusions: XSTQ inhibits proliferation and induces apoptosis in SH-SY5Y cells. XSTQ also promotes OX1R and OX2R expression. These effects are associated with the repression of the Erk1/2, p38 MAPK, and JNK signaling pathways. These results define a molecular mechanism for XSTQ in regulating HCRT and MAPK activation, which may explain its ability to treat narcolepsy.

  17. Protein tyrosine kinases p53/56lyn and p72syk in MHC class I-mediated signal transduction in B lymphoma cells

    DEFF Research Database (Denmark)

    Pedersen, Anders Elm; Bregenholt, S; Skov, S

    1998-01-01

    syk are among the tyrosine-phosphorylated proteins. The kinetics of phosphorylation of these kinases after MHC-I crosslinking differ from the kinetics observed after crosslinking of the B cell antigen receptor (BCR). Additional experiments were performed with chicken lyn- and syk-negative DT40 B cells...... mobilization of intracellular free calcium compared with MHC-I crosslinking of wild-type DT40 cells. Thus, expression of BCR at the cell surface is likely to be important for the signal cascade initiated by MHC-I crosslinking. Our data suggest that signal transduction initiated through ligation of the MHC...

  18. Myosins 1 and 6, myosin light chain kinase, actin and microtubules cooperate during antibody-mediated internalisation and trafficking of membrane-expressed viral antigens in feline infectious peritonitis virus infected monocytes.

    Science.gov (United States)

    Dewerchin, Hannah L; Desmarets, Lowiese M; Noppe, Ytse; Nauwynck, Hans J

    2014-02-12

    Monocytes infected with feline infectious peritonitis virus, a coronavirus, express viral proteins in their plasma membranes. Upon binding of antibodies, these proteins are quickly internalised through a new clathrin- and caveolae-independent internalisation pathway. By doing so, the infected monocytes can escape antibody-dependent cell lysis. In the present study, we investigated which kinases and cytoskeletal proteins are of importance during internalisation and subsequent intracellular transport. The experiments showed that myosin light chain kinase (MLCK) and myosin 1 are crucial for the initiation of the internalisation. With co-localisation stainings, it was found that MLCK and myosin 1 co-localise with antigens even before internalisation started. Myosin 6 co-localised with the internalising complexes during passage through the cortical actin, were it might play a role in moving or disintegrating actin filaments, to overcome the actin barrier. One minute after internalisation started, vesicles had passed the cortical actin, co-localised with microtubules and association with myosin 6 was lost. The vesicles were further transported over the microtubules and accumulated at the microtubule organising centre after 10 to 30 min. Intracellular trafficking over microtubules was mediated by MLCK, myosin 1 and a small actin tail. Since inhibiting MLCK with ML-7 was so efficient in blocking the internalisation pathway, this target can be used for the development of a new treatment for FIPV.

  19. Protein Kinase C-Mediated Phosphorylation of BCL11B at Serine 2 Negatively Regulates Its Interaction with NuRD Complexes during CD4+ T-Cell Activation.

    Science.gov (United States)

    Dubuissez, Marion; Loison, Ingrid; Paget, Sonia; Vorng, Han; Ait-Yahia, Saliha; Rohr, Olivier; Tsicopoulos, Anne; Leprince, Dominique

    2016-07-01

    The transcription factor BCL11B/CTIP2 is a major regulatory protein implicated in various aspects of development, function and survival of T cells. Mitogen-activated protein kinase (MAPK)-mediated phosphorylation and SUMOylation modulate BCL11B transcriptional activity, switching it from a repressor in naive murine thymocytes to a transcriptional activator in activated thymocytes. Here, we show that BCL11B interacts via its conserved N-terminal MSRRKQ motif with endogenous MTA1 and MTA3 proteins to recruit various NuRD complexes. Furthermore, we demonstrate that protein kinase C (PKC)-mediated phosphorylation of BCL11B Ser2 does not significantly impact BCL11B SUMOylation but negatively regulates NuRD recruitment by dampening the interaction with MTA1 or MTA3 (MTA1/3) and RbAp46 proteins. We detected increased phosphorylation of BCL11B Ser2 upon in vivo activation of transformed and primary human CD4(+) T cells. We show that following activation of CD4(+) T cells, BCL11B still binds to IL-2 and Id2 promoters but activates their transcription by recruiting P300 instead of MTA1. Prolonged stimulation results in the direct transcriptional repression of BCL11B by KLF4. Our results unveil Ser2 phosphorylation as a new BCL11B posttranslational modification linking PKC signaling pathway to T-cell receptor (TCR) activation and define a simple model for the functional switch of BCL11B from a transcriptional repressor to an activator during TCR activation of human CD4(+) T cells. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  20. Phosphoinositide 3-kinase/Akt Pathway Mediates Fip1-like1-platelet-derived Growth Factor Receptor α-induced Cell Infiltration and Activation: Possible Molecular Mechanism for the Malignant Phenotype of Chronic Eosinophilic Leukemia

    Directory of Open Access Journals (Sweden)

    Bin Li

    2015-01-01

    Full Text Available The fip1-like1/platelet-derived growth factor receptor-α fusion gene (F/P is responsible for 14-60% cases of hypereosinophilia syndrome (HES, also known as F/P-positive chronic eosinophilic leukemia (F/P(+ CEL. The major pathogenesis of F/P(+ CEL is known to involve migration and activation of mast cells and eosinophils, leading to severe multi-organ dysfunction, but the mechanism was still unclear. Phosphoinositide 3-kinase (PI3K and serine-threonine protein kinase Akt have been reported to be targets of F/P in the F/P-promoted cell proliferation. They are extensively involved in the migration and adhesion of hematopoietic stem/progenitor cells, and also control cell invasion in some leukemias. The PI3K/Akt pathway is involved in eosinophil/neutrophil activation and infiltration; its possible role in regulating F/P induced cytotoxicity and upregulation of A4-integrin in eosinophils, and inducing eosinophil activation through controlling F/P-induced Nuclear factor-kB activity. Akt was recently shown to be stimulated by F/P, synergistically with stem cell factor, resulting in the induction of MCs migration and excessive activation. PI3K/Akt pathway is also a principal mediator of interleukin-5 (IL-5-induced signal transduction promoting eosinophil trafficking and degranulation, whereas IL-5 is a necessary cytokine for F/P-mediated CEL development. We, therefore, propose the hypothesis that the PI3K/Akt pathway might be vital downstream of F/P to induce target cell activation and tissue infiltration, resulting in the malignant phenotype seen in F/P(+ CEL.

  1. Pro-survival Effects of 17β-Estradiol on Osteocytes Are Mediated by Nitric Oxide/cGMP via Differential Actions of cGMP-dependent Protein Kinases I and II*

    Science.gov (United States)

    Marathe, Nisha; Rangaswami, Hema; Zhuang, Shunhui; Boss, Gerry R.; Pilz, Renate B.

    2012-01-01

    Estrogens promote bone health in part by increasing osteocyte survival, an effect that requires activation of the protein kinases Akt and ERK1/2, but the molecular mechanisms involved are only partly understood. Because estrogens increase nitric oxide (NO) synthesis and NO can have anti-apoptotic effects, we examined the role of NO/cGMP signaling in estrogen regulation of osteocyte survival. Etoposide-induced death of MLO-Y4 osteocyte-like cells, assessed by trypan blue staining, caspase-3 cleavage, and TUNEL assays, was completely prevented when cells were pre-treated with 17β-estradiol. This protective effect was mimicked when cells were pre-treated with a membrane-permeable cGMP analog and blocked by pharmacological inhibitors of NO synthase, soluble guanylate cyclase, or cGMP-dependent protein kinases (PKGs), supporting a requirement for NO/cGMP/PKG signaling downstream of 17β-estradiol. siRNA-mediated knockdown and viral reconstitution of individual PKG isoforms demonstrated that the anti-apoptotic effects of estradiol and cGMP were mediated by PKG Iα and PKG II. Akt and ERK1/2 activation by 17β-estradiol required PKG II, and cGMP mimicked the effects of estradiol on Akt and ERK, including induction of ERK nuclear translocation. cGMP induced BAD phosphorylation on several sites, and experiments with phosphorylation-deficient BAD mutants demonstrated that the anti-apoptotic effects of cGMP and 17β-estradiol required BAD phosphorylation on Ser136 and Ser155; these sites were targeted by Akt and PKG I, respectively, and regulate BAD interaction with Bcl-2. In conclusion, 17β-estradiol protects osteocytes against apoptosis by activating the NO/cGMP/PKG cascade; PKG II is required for estradiol-induced activation of ERK and Akt, and PKG Iα contributes to pro-survival signaling by directly phosphorylating BAD. PMID:22117068

  2. Fas-Induced Apoptosis of Renal Cell Carcinoma is Mediated by Apoptosis Signal-Regulating Kinase 1 via Mitochondrial Damage-Dependent Caspase-8 Activation

    Directory of Open Access Journals (Sweden)

    Mohamed Hassan

    2009-01-01

    Full Text Available Renal cell carcinoma (RCC is a prototype of a chemo refractory tumour. It remains the most lethal of the common urologic cancers and is highly resistant to conventional therapy. Here, we confirmed the efficiency of anti-Fas monoclonal antibody (CH11 as alternative therapeutic approach for the treatment of RCC and investigated the molecular mechanism(s, whereby CH11 induces apoptosis of RCC cells. The present study shows an essential role for apoptosis signal-regulating kinase 1 (ASK1, together with both c-jun-N-terminal kinase (JNK and p38 pathways, and caspase-8 in this process. Furthermore, CH11-dependent induction of the ASK1–JNK/p38 pathways was found to activate the transcription factors AP-1 and ATF-2, and FADD-caspase-8-Bid signalling, resulting in the translocation of both Bax and Bak proteins, and subsequently mitochondrial dysregulation that is characterized by the loss of mitochondrial membrane potential (ΔΨm, cytochrome c release and cleavage of caspase-9, caspase-3 and PARP. Thus, the described molecular mechanisms of CH11-induced apoptosis suggest the reliability of Fas activation as an alternative therapeutic approach for the treatment of patients with advanced renal cell carcinoma.

  3. Torin1-mediated TOR kinase inhibition reduces Wee1 levels and advances mitotic commitment in fission yeast and HeLa cells.

    Science.gov (United States)

    Atkin, Jane; Halova, Lenka; Ferguson, Jennifer; Hitchin, James R; Lichawska-Cieslar, Agata; Jordan, Allan M; Pines, Jonathon; Wellbrock, Claudia; Petersen, Janni

    2014-03-15

    The target of rapamycin (TOR) kinase regulates cell growth and division. Rapamycin only inhibits a subset of TOR activities. Here we show that in contrast to the mild impact of rapamycin on cell division, blocking the catalytic site of TOR with the Torin1 inhibitor completely arrests growth without cell death in Schizosaccharomyces pombe. A mutation of the Tor2 glycine residue (G2040D) that lies adjacent to the key Torin-interacting tryptophan provides Torin1 resistance, confirming the specificity of Torin1 for TOR. Using this mutation, we show that Torin1 advanced mitotic onset before inducing growth arrest. In contrast to TOR inhibition with rapamycin, regulation by either Wee1 or Cdc25 was sufficient for this Torin1-induced advanced mitosis. Torin1 promoted a Polo and Cdr2 kinase-controlled drop in Wee1 levels. Experiments in human cell lines recapitulated these yeast observations: mammalian TOR (mTOR) was inhibited by Torin1, Wee1 levels declined and mitotic commitment was advanced in HeLa cells. Thus, the regulation of the mitotic inhibitor Wee1 by TOR signalling is a conserved mechanism that helps to couple cell cycle and growth controls.

  4. Danusertib, a potent pan-Aurora kinase and ABL kinase inhibitor, induces cell cycle arrest and programmed cell death and inhibits epithelial to mesenchymal transition involving the PI3K/Akt/mTOR-mediated signaling pathway in human gastric cancer AGS and NCI-N78 cells

    Directory of Open Access Journals (Sweden)

    Yuan CX

    2015-03-01

    autophagy-inducing effects on AGS and NCI-N78 cells. Danusertib arrested AGS and NCI-N78 cells in G2/M phase, with downregulation of expression of cyclin B1 and cyclin-dependent kinase 1 and upregulation of expression of p21 Waf1/Cip1, p27 Kip1, and p53. Danusertib induced mitochondria-mediated apoptosis, with an increase in expression of proapoptotic protein and a decrease in antiapoptotic proteins in both cell lines. Danusertib induced release of cytochrome c from the mitochondria to the cytosol and triggered activation of caspase 9 and caspase 3 in AGS and NCI-N78 cells. Further, danusertib induced autophagy, with an increase in expression of beclin 1 and conversion of microtubule-associated protein 1A/1B-light chain 3 (LC3-I to LC3-II in both cell lines. Inhibition of phosphatidylinositol 3-kinase (PI3K/protein kinase B (Akt/mammalian target of rapamycin (mTOR and p38 mitogen-activated protein kinase pathways as well as activation of 5' AMP-activated protein kinase contributed to the proautophagic effect of danusertib in AGS and NCI-N78 cells. SB202191 and wortmannin enhanced the autophagy-inducing effect of danusertib in AGS and NCI-N78 cells. In addition, danusertib inhibited epithelial to mesenchymal transition with an increase in expression of E-cadherin and a decrease in expression of N-cadherin in both cell lines. Taken together, danusertib has potent inducing effects on cell cycle arrest, apoptosis, and autophagy, but has an inhibitory effect on epithelial to mesenchymal transition, with involvement of signaling pathways mediated by PI3K/Akt/mTOR, p38 mitogen-activated protein kinase, and 5' AMP-activated protein kinase in AGS and NCI-N78 cells. Keywords: danusertib, gastric cancer, Aurora kinase, apoptosis, autophagy, epithelial to mesenchymal transition

  5. Far-infrared radiation acutely increases nitric oxide production by increasing Ca{sup 2+} mobilization and Ca{sup 2+}/calmodulin-dependent protein kinase II-mediated phosphorylation of endothelial nitric oxide synthase at serine 1179

    Energy Technology Data Exchange (ETDEWEB)

    Park, Jung-Hyun; Lee, Sangmi [Department of Molecular Medicine and Ewha Medical Research Institute, Ewha Womans University Medical School, Seoul 158-710 (Korea, Republic of); Cho, Du-Hyong [Department of Neuroscience, School of Medicine, Konkuk University, Seoul 143-701 (Korea, Republic of); Park, Young Mi [Department of Molecular Medicine and Ewha Medical Research Institute, Ewha Womans University Medical School, Seoul 158-710 (Korea, Republic of); Kang, Duk-Hee [Division of Nephrology, Department of Internal Medicine, Ewha Womans University Medical School, Seoul 158-710 (Korea, Republic of); Jo, Inho, E-mail: inhojo@ewha.ac.kr [Department of Molecular Medicine and Ewha Medical Research Institute, Ewha Womans University Medical School, Seoul 158-710 (Korea, Republic of)

    2013-07-12

    Highlights: •Far-infrared (FIR) radiation increases eNOS-Ser{sup 1179} phosphorylation and NO production in BAEC. •CaMKII and PKA mediate FIR-stimulated increases in eNOS-Ser{sup 1179} phosphorylation. •FIR increases intracellular Ca{sup 2+} levels. •Thermo-sensitive TRPV Ca{sup 2+} channels are unlikely to be involved in the FIR-mediated eNOS-Ser{sup 1179} phosphorylation pathway. -- Abstract: Repeated thermal therapy manifested by far-infrared (FIR) radiation improves vascular function in both patients and mouse model with coronary heart disease, but its underlying mechanism is not fully understood. Using FIR as a thermal therapy agent, we investigate the molecular mechanism of its effect on endothelial nitric oxide synthase (eNOS) activity and NO production. FIR increased the phosphorylation of eNOS at serine 1179 (eNOS-Ser{sup 1179}) in a time-dependent manner (up to 40 min of FIR radiation) in bovine aortic endothelial cells (BAEC) without alterations in eNOS expression. This increase was accompanied by increases in NO production and intracellular Ca{sup 2+} levels. Treatment with KN-93, a selective inhibitor of Ca{sup 2+}/calmodulin-dependent protein kinase II (CaMKII) and H-89, a protein kinase A inhibitor, inhibited FIR radiation-stimulated eNOS-Ser{sup 1179} phosphorylation. FIR radiation itself also increased the temperature of culture medium. As transient receptors potential vanilloid (TRPV) ion channels are known to be temperature-sensitive calcium channels, we explore whether TRPV channels mediate these observed effects. Reverse transcription-PCR assay revealed two TRPV isoforms in BAEC, TRPV2 and TRPV4. Although ruthenium red, a pan-TRPV inhibitor, completely reversed the observed effect of FIR radiation, a partial attenuation (∼20%) was found in cells treated with Tranilast, TRPV2 inhibitor. However, ectopic expression of siRNA of TRPV2 showed no significant alteration in FIR radiation-stimulated eNOS-Ser{sup 1179} phosphorylation. This

  6. Muscle A-Kinase Anchoring Protein-α is an Injury-Specific Signaling Scaffold Required for Neurotrophic- and Cyclic Adenosine Monophosphate-Mediated Survival

    Directory of Open Access Journals (Sweden)

    Yan Wang

    2015-12-01

    Full Text Available Neurotrophic factor and cAMP-dependent signaling promote the survival and neurite outgrowth of retinal ganglion cells (RGCs after injury. However, the mechanisms conferring neuroprotection and neuroregeneration downstream to these signals are unclear. We now reveal that the scaffold protein muscle A-kinase anchoring protein-α (mAKAPα is required for the survival and axon growth of cultured primary RGCs. Although genetic deletion of mAKAPα early in prenatal RGC development did not affect RGC survival into adulthood, nor promoted the death of RGCs in the uninjured adult retina, loss of mAKAPα in the adult increased RGC death after optic nerve crush. Importantly, mAKAPα was required for the neuroprotective effects of brain-derived neurotrophic factor and cyclic adenosine-monophosphate (cAMP after injury. These results identify mAKAPα as a scaffold for signaling in the stressed neuron that is required for RGC neuroprotection after optic nerve injury.

  7. Constitutive activation of extracellular signal-regulated kinase predisposes diffuse large B-cell lymphoma cell lines to CD40-mediated cell death

    DEFF Research Database (Denmark)

    Hollmann, C Annette; Owens, Trevor; Nalbantoglu, Josephine

    2006-01-01

    , including LCK and VAV. In addition, CD40-sensitive DLBCL cell lines also displayed constitutive activation of extracellular signal-regulated kinase (ERK) and failed to undergo apoptosis when ERK phosphorylation was inhibited. In contrast, CD40-resistant lines showed no constitutive activation of ERK......CD40 promotes survival, proliferation, and differentiation of normal B cells but can cause activation-induced cell death in malignant B lymphocytes. CD40 ligand and anti-CD40 antibodies have been used successfully to induce apoptosis in lymphoma lines both in vitro and in xenograft tumor models....... Although this makes CD40 an attractive target for antitumor therapies, the response of malignant B cells to CD40 signaling is variable, and CD40 stimulation can enhance proliferation and can increase chemoresistance in some cell lines. It would therefore be useful to identify markers that predict whether...

  8. Malignant transformation of CD4+ T lymphocytes mediated by oncogenic kinase NPM/ALK recapitulates IL-2-induced cell signaling and gene expression reprogramming

    DEFF Research Database (Denmark)

    Marzec, Michal; Halasa, Krzysztof; Liu, Xiaobin

    2013-01-01

    expressed in T cell lymphomas (TCLs), closely resembles cell activation induced by IL-2, the key cytokine supporting growth and survival of normal CD4(+) T lymphocytes. Direct comparison of gene expression by ALK(+) TCL cells treated with an ALK inhibitor and IL-2-dependent ALK(-) TCL cells stimulated......Anaplastic lymphoma kinase (ALK), physiologically expressed only by nervous system cells, displays a remarkable capacity to transform CD4(+) T lymphocytes and other types of nonneural cells. In this study, we report that activity of nucleophosmin (NPM)/ALK chimeric protein, the dominant form of ALK...... the target CD4(+) T lymphocytes, at least in part, by using the pre-existing, IL-2-dependent signaling pathways....

  9. Non-small cell lung cancer-derived soluble mediators enhance apoptosis in activated T lymphocytes through an I kappa B kinase-dependent mechanism.

    Science.gov (United States)

    Batra, Raj K; Lin, Ying; Sharma, Sherven; Dohadwala, Mariam; Luo, Jie; Pold, Mehis; Dubinett, Steven M

    2003-02-01

    T lymphocyte survival is critical for the development and maintenance of an effective host antitumor immune response; however, the tumor environment can negatively impact T-cell survival. Lymphocytes exposed to tumor supernatants (TSNs) were evaluated for apoptosis after mitogen stimulation. TSN was observed to significantly enhance phorbol 12-myristate 13-acetate/ionomycin- and anti-CD3-stimulated lymphocyte apoptosis. Enhanced lymphocyte apoptosis was associated with an impairment of nuclear factor kappa B nuclear translocation and diminished I kappa B alpha degradation. In lymphocytes stimulated after exposure to TSNs, cytoplasmic I kappa B alpha persisted as a result of alterations in I kappa B kinase (IKK) activity. Accordingly, although there were no apparent differences in IKK component concentrations, lymphocytes preexposed to TSNs exhibited markedly reduced IKK activity. We conclude that non-small cell lung cancer-derived soluble factors promote apoptosis in activated lymphocytes by an IKK-dependent pathway.

  10. Osthole Suppresses the Migratory Ability of Human Glioblastoma Multiforme Cells via Inhibition of Focal Adhesion Kinase-Mediated Matrix Metalloproteinase-13 Expression

    Directory of Open Access Journals (Sweden)

    Cheng-Fang Tsai

    2014-03-01

    Full Text Available Glioblastoma multiforme (GBM is the most common type of primary and malignant tumor occurring in the adult central nervous system. GBM often invades surrounding regions of the brain during its early stages, making successful treatment difficult. Osthole, an active constituent isolated from the dried C. monnieri fruit, has been shown to suppress tumor migration and invasion. However, the effects of osthole in human GBM are largely unknown. Focal adhesion kinase (FAK is important for the metastasis of cancer cells. Results from this study show that osthole can not only induce cell death but also inhibit phosphorylation of FAK in human GBM cells. Results from this study show that incubating GBM cells with osthole reduces matrix metalloproteinase (MMP-13 expression and cell motility, as assessed by cell transwell and wound healing assays. This study also provides evidence supporting the potential of osthole in reducing FAK activation, MMP-13 expression, and cell motility in human GBM cells.

  11. Tetrahydrocannabinol-induced neurotoxicity depends on CB1 receptor-mediated c-Jun N-terminal kinase activation in cultured cortical neurons

    Science.gov (United States)

    Downer, Eric J; Fogarty, Marie P; Campbell, Veronica A

    2003-01-01

    Δ9-Tetrahydrocannabinol (THC), the main psychoactive ingredient of marijuana, induces apoptosis in cultured cortical neurons. THC exerts its apoptotic effects in cortical neurons by binding to the CB1 cannabinoid receptor. The CB1 receptor has been shown to couple to the stress-activated protein kinase, c-Jun N-terminal kinase (JNK). However, the involvement of specific JNK isoforms in the neurotoxic properties of THC remains to be established. The present study involved treatment of rat cultured cortical neurons with THC (0.005–50 μM), and combinations of THC with the CB1 receptor antagonist, AM 251 (10 μM) and pertussis toxin (PTX; 200 ng ml−1). Antisense oligonucleotides (AS) were used to deplete neurons of JNK1 and JNK2 in order to elucidate their respective roles in THC signalling. Here we report that THC induces the activation of JNK via the CB1 receptor and its associated G-protein, Gi/o. Treatment of cultured cortical neurons with THC resulted in a differential timeframe of activation of the JNK1 and JNK2 isoforms. Use of specific JNK1 and JNK2 AS identified activation of caspase-3 and DNA fragmentation as downstream consequences of JNK1 and JNK2 activation. The results from this study demonstrate that activation of the CB1 receptor induces JNK and caspase-3 activation, an increase in Bax expression and DNA fragmentation. The data demonstrate that the activation of both JNK1 and JNK2 isoforms is central to the THC-induced activation of the apoptotic pathway in cortical neurons. PMID:14522843

  12. A Major Facilitator Superfamily Transporter-Mediated Resistance to Oxidative Stress and Fungicides Requires Yap1, Skn7, and MAP Kinases in the Citrus Fungal Pathogen Alternaria alternata.

    Directory of Open Access Journals (Sweden)

    Li-Hung Chen

    Full Text Available Major Facilitator Superfamily (MFS transporters play an important role in multidrug resistance in fungi. We report an AaMFS19 gene encoding a MFS transporter required for cellular resistance to oxidative stress and fungicides in the phytopathogenic fungus Alternaria alternata. AaMFS19, containing 12 transmembrane domains, displays activity toward a broad range of substrates. Fungal mutants lacking AaMFS19 display profound hypersensitivities to cumyl hydroperoxide, potassium superoxide, many singlet oxygen-generating compounds (eosin Y, rose Bengal, hematoporphyrin, methylene blue, and cercosporin, and the cell wall biosynthesis inhibitor, Congo red. AaMFS19 mutants also increase sensitivity to copper ions, clotrimazole, fludioxonil, and kocide fungicides, 2-chloro-5-hydroxypyridine (CHP, and 2,3,5-triiodobenzoic acid (TIBA. AaMFS19 mutants induce smaller necrotic lesions on leaves of a susceptible citrus cultivar. All observed phenotypes in the mutant are restored by introducing and expressing a wild-type copy of AaMFS19. The wild-type strain of A. alternata treated with either CHP or TIBA reduces radial growth and formation and germination of conidia, increases hyphal branching, and results in decreased expression of the AaMFS19 gene. The expression of AaMFS19 is regulated by the Yap1 transcription activator, the Hog1 and Fus3 mitogen-activated protein (MAP kinases, the 'two component' histidine kinase, and the Skn7 response regulator. Our results demonstrate that A. alternata confers resistance to different chemicals via a membrane-bound MFS transporter.

  13. Burst BMP triggered receptor kinase activity drives Smad1 mediated long-term target gene oscillation in C2C12 cells.

    Directory of Open Access Journals (Sweden)

    Daniela Schul

    Full Text Available Bone Morphogenetic Proteins (BMPs are important growth factors that regulate many cellular processes. During embryogenesis they act as morphogens and play a critical role during organ development. They influence cell fates via concentration-gradients in the embryos where cells transduce this extracellular information into gene expression profiles and cell fate decisions. How receiving cells decode and quantify BMP2/4 signals is hardly understood. There is little data on the quantitative relationships between signal input, transducing molecules, their states and location, and ultimately their ability to integrate graded systemic inputs and generate qualitative responses. Understanding this signaling network on a quantitative level should be considered a prerequisite for efficient pathway modulation, as the BMP pathway is a prime target for therapeutic invention. Hence, we quantified the spatial distribution of the main signal transducer of the BMP2/4 pathway in response to different types and levels of stimuli in c2c12 cells. We found that the subcellular localization of Smad1 is independent of ligand concentration. In contrast, Smad1 phosphorylation levels relate proportionally to BMP2 ligand concentrations and they are entirely located in the nucleus. Interestingly, we found that BMP2 stimulates target gene expression in non-linear, wave-like forms. Amplitudes showed a clear concentration-dependency, for sustained and transient stimulation. We found that even burst-stimulation triggers gene-expression wave-like modulations that are detectable for at least 30 h. Finally, we show here that target gene expression oscillations depend on receptor kinase activity, as the kinase drives further expression pulses without receptor reactivation and the target gene expression breaks off after inhibitor treatment in c2c12 cells.

  14. Burst BMP triggered receptor kinase activity drives Smad1 mediated long-term target gene oscillation in C2C12 cells.

    Science.gov (United States)

    Schul, Daniela; Schmitt, Alexandra; Regneri, Janine; Schartl, Manfred; Wagner, Toni Ulrich

    2013-01-01

    Bone Morphogenetic Proteins (BMPs) are important growth factors that regulate many cellular processes. During embryogenesis they act as morphogens and play a critical role during organ development. They influence cell fates via concentration-gradients in the embryos where cells transduce this extracellular information into gene expression profiles and cell fate decisions. How receiving cells decode and quantify BMP2/4 signals is hardly understood. There is little data on the quantitative relationships between signal input, transducing molecules, their states and location, and ultimately their ability to integrate graded systemic inputs and generate qualitative responses. Understanding this signaling network on a quantitative level should be considered a prerequisite for efficient pathway modulation, as the BMP pathway is a prime target for therapeutic invention. Hence, we quantified the spatial distribution of the main signal transducer of the BMP2/4 pathway in response to different types and levels of stimuli in c2c12 cells. We found that the subcellular localization of Smad1 is independent of ligand concentration. In contrast, Smad1 phosphorylation levels relate proportionally to BMP2 ligand concentrations and they are entirely located in the nucleus. Interestingly, we found that BMP2 stimulates target gene expression in non-linear, wave-like forms. Amplitudes showed a clear concentration-dependency, for sustained and transient stimulation. We found that even burst-stimulation triggers gene-expression wave-like modulations that are detectable for at least 30 h. Finally, we show here that target gene expression oscillations depend on receptor kinase activity, as the kinase drives further expression pulses without receptor reactivation and the target gene expression breaks off after inhibitor treatment in c2c12 cells.

  15. U-Bang-Haequi Tang: A Herbal Prescription that Prevents Acute Inflammation through Inhibition of NF-κB-Mediated Inducible Nitric Oxide Synthase

    Directory of Open Access Journals (Sweden)

    Min Hwangbo

    2014-01-01

    Full Text Available Since antiquity, medical herbs have been prescribed for both treatment and preventative purposes. Herbal formulas are used to reduce toxicity as well as increase efficacy in traditional Korean medicine. U-bang-haequi tang (UBT is a herbal prescription containing Arctii fructus and Forsythia suspensa as its main components and has treated many human diseases in traditional Korean medicine. This research investigated the effects of UBT against an acute phase of inflammation. For this, we measured induction of nitric oxide (NO and related proteins in macrophage cell line stimulated by lipopolysaccharide (LPS. Further, paw swelling was measured in carrageenan-treated rats. Carrageenan significantly induced activation of inflammatory cells and increases in paw volume, whereas oral administration of 0.3 or 1 g/kg/day of UBT inhibited the acute inflammatory response. In RAW264.7 cells, UBT inhibited mRNA and protein expression levels of iNOS. UBT treatment also blocked elevation of NO production, nuclear translocation of NF-κB, phosphorylation of Iκ-Bα induced by LPS. Moreover, UBT treatment significantly blocked the phosphorylation of p38 and c-Jun NH2-terminal kinases by LPS. In conclusion, UBT prevented both acute inflammation in rats as well as LPS-induced NO and iNOS gene expression through inhibition of NF-κB in RAW264.7 cells.

  16. Mitogen-activated protein kinase signaling pathways promote low-density lipoprotein receptor-related protein 1-mediated internalization of beta-amyloid protein in primary cortical neurons.

    Science.gov (United States)

    Yang, Wei-Na; Ma, Kai-Ge; Qian, Yi-Hua; Zhang, Jian-Shui; Feng, Gai-Feng; Shi, Li-Li; Zhang, Zhi-Chao; Liu, Zhao-Hui

    2015-07-01

    Mounting evidence suggests that the pathological hallmarks of Alzheimer's disease (AD) are caused by the intraneuronal accumulation of beta-amyloid protein (Aβ). Reuptake of extracellular Aβ is believed to contribute significantly to the intraneuronal Aβ pool in the early stages of AD. Published reports have claimed that the low-density lipoprotein receptor-related protein 1 (LRP1) mediates Aβ1-42 uptake and lysosomal trafficking in GT1-7 neuronal cells and mouse embryonic fibroblast non-neuronal cells. However, there is no direct evidence supporting the role of LRP1 in Aβ internalization in primary neurons. Our recent study indicated that p38 MAPK and ERK1/2 signaling pathways are involved in regulating α7 nicotinic acetylcholine receptor (α7nAChR)-mediated Aβ1-42 uptake in SH-SY5Y cells. This study was designed to explore the regulation of MAPK signaling pathways on LRP1-mediated Aβ internalization in neurons. We found that extracellular Aβ1-42 oligomers could be internalized into endosomes/lysosomes and mitochondria in cortical neurons. Aβ1-42 and LRP1 were also found co-localized in neurons during Aβ1-42 internalization, and they could form Aβ1-42-LRP1 complex. Knockdown of LRP1 expression significantly decreased neuronal Aβ1-42 internalization. Finally, we identified that p38 MAPK and ERK1/2 signaling pathways regulated the internalization of Aβ1-42 via LRP1. Therefore, these results demonstrated that LRP1, p38 MAPK and ERK1/2 mediated the internalization of Aβ1-42 in neurons and provided evidence that blockade of LRP1 or inhibitions of MAPK signaling pathways might be a potential approach to lowering brain Aβ levels and served a potential therapeutic target for AD. Copyright © 2015 Elsevier Ltd. All rights reserved.

  17. Acrolein-induced activation of mitogen-activated protein kinase signaling is mediated by alkylation of thioredoxin reductase and thioredoxin 1 ? ??

    OpenAIRE

    Randall, Matthew J.; Spiess, Page C.; Hristova, Milena; Hondal, Robert J.; van der Vliet, Albert

    2013-01-01

    Cigarette smoking remains a major health concern worldwide, and many of the adverse effects of cigarette smoke (CS) can be attributed to its abundant electrophilic aldehydes, such as acrolein (2-propenal). Previous studies indicate that acrolein readily reacts with thioredoxin reductase 1 (TrxR1), a critical enzyme involved in regulation of thioredoxin (Trx)-mediated redox signaling, by alkylation at its selenocysteine (Sec) residue. Because alkylation of Sec within TrxR1 has significant impl...

  18. Asarone from Acori Tatarinowii Rhizoma Potentiates the Nerve Growth Factor-Induced Neuronal Differentiation in Cultured PC12 Cells: A Signaling Mediated by Protein Kinase A.

    Directory of Open Access Journals (Sweden)

    Kelly Y C Lam

    Full Text Available Acori Tatarinowii Rhizoma (ATR, the rhizome of Acorus tatarinowii Schott, is being used clinically to treat neurological disorders. The volatile oil of ATR is being considered as an active ingredient. Here, α-asarone and β-asarone, accounting about 95% of ATR oil, were evaluated for its function in stimulating neurogenesis. In cultured PC12 cells, application of ATR volatile oil, α-asarone or β-asarone, stimulated the expression of neurofilaments, a bio-marker for neurite outgrowth, in a concentration-dependent manner. The co-treatment of ATR volatile oil, α-asarone or β-asarone, with low concentration of nerve growth factor (NGF potentiated the NGF-induced neuronal differentiation in cultured PC12 cells. In addition, application of protein kinase A inhibitors, H89 and KT5720, in cultures blocked the ATR-induced neurofilament expression, as well as the phosphorylation of cAMP-responsive element binding protein (CREB. In the potentiation of NGF-induced signaling in cultured PC12 cells, α-asarone and β-asarone showed synergistic effects. These results proposed the neurite-promoting asarone, or ATR volatile oil, could be useful in finding potential drugs for treating various neurodegenerative diseases, in which neurotrophin deficiency is normally involved.

  19. DAP-kinase-mediated phosphorylation on the BH3 domain of beclin 1 promotes dissociation of beclin 1 from Bcl-XL and induction of autophagy

    Science.gov (United States)

    Zalckvar, Einat; Berissi, Hanna; Mizrachy, Liat; Idelchuk, Yulia; Koren, Itay; Eisenstein, Miriam; Sabanay, Helena; Pinkas-Kramarski, Ronit; Kimchi, Adi

    2009-01-01

    Autophagy, an evolutionarily conserved process, has functions both in cytoprotective and programmed cell death mechanisms. Beclin 1, an essential autophagic protein, was recently identified as a BH3-domain-only protein that binds to Bcl-2 anti-apoptotic family members. The dissociation of beclin 1 from its Bcl-2 inhibitors is essential for its autophagic activity, and therefore should be tightly controlled. Here, we show that death-associated protein kinase (DAPK) regulates this process. The activated form of DAPK triggers autophagy in a beclin-1-dependent manner. DAPK phosphorylates beclin 1 on Thr 119 located at a crucial position within its BH3 domain, and thus promotes the dissociation of beclin 1 from Bcl-XL and the induction of autophagy. These results reveal a substrate for DAPK that acts as one of the core proteins of the autophagic machinery, and they provide a new phosphorylation-based mechanism that reduces the interaction of beclin 1 with its inhibitors to activate the autophagic machinery. PMID:19180116

  20. Phosphorylation of the von Hippel-Lindau protein (VHL) by protein kinase CK2 reduces its protein stability and affects p53 and HIF-1alpha mediated transcription.

    Science.gov (United States)

    Ampofo, Emmanuel; Kietzmann, Thomas; Zimmer, Andreas; Jakupovic, Mirza; Montenarh, Mathias; Götz, Claudia

    2010-10-01

    The von Hippel-Lindau tumour suppressor gene encodes a protein with 213 amino acids, which is known to be part of an E3-ubiquitin ligase targeting the HIF-1alpha transcription factor as well as to form a complex with p53. The VHL protein can be phosphorylated by protein kinase CK2 at serines 33, 38 and 43. However, the role of VHL phosphorylation in the context of p53 and HIF-1alpha regulation remained so far unknown. In the present study we investigated whether phosphorylation of VHL by CK2 might affect the function of p53 and HIF-1alpha. By using 4,5,6,7-tetrabromobenzotriazole (TBB), a CK2-specific inhibitor, as well as a mutant VHL where serines 33, 38 and 43 were replaced by alanines we found that CK2 phosphorylation affected the VHL protein half-life and increased VHL protein stability. Further, we found that inhibition of VHL phosphorylation by CK2 reduced p53 function. In addition, the enhanced levels of VHL due to CK2 inhibition contributed to the down-regulation of HIF-activity and degradation of HIF-1alpha. Thus, these results demonstrate that phosphorylation of VHL by CK2 plays an important role in the regulation of VHL protein stability and may contribute to the survival of tumour cells. Copyright 2010 Elsevier Ltd. All rights reserved.

  1. A Novel Occulta-Type Spina Bifida Mediated by Murine Double Heterozygotes EphA2 and EphA4 Receptor Tyrosine Kinases

    Directory of Open Access Journals (Sweden)

    Nor Linda Abdullah

    2017-12-01

    Full Text Available Members of the Eph receptor tyrosine kinase have previously been implicated in cranial neural tube development. Failure of neural tube closure leads to the devastating conditions known as anencephaly and spina bifida. EphA2 and EphA4 are expressed at the tips of the closing spinal neural folds prior and during neural tube closure. We investigated the possible role of murine EphA2 and EphA4 during the last step of primary neural tube closure, which is adhesion and fusion. The individual mouse knockouts of EphA2 and EphA4 per se do not exhibit neural tube defects (NTDs. The embryos generated by the crossing of double heterozygotes Epha2tm1Jrui/+Epha4rb-2J/+ displayed NTDs with a wide degree of severity including close exencephaly and close spina bifida (spina bifida occulta. Interestingly, mutants displaying NTDs had skin covering the underlying lesion. The tissue sections revealed the elevated neural folds had not adhered and fused. The phenotypes seen in Epha2tm1Jrui/+Epha4rb-2J/+ double heterozygous embryos suggest both genes play a compensatory role with each other in the adhesion and fusion of the neural tube. In this study, there exists a >50% penetrance of NTDs in the mouse mutants, which genetically have a single allele each of EphA2 and EphA4 absent.

  2. A Novel Occulta-Type Spina Bifida Mediated by Murine Double HeterozygotesEphA2andEphA4Receptor Tyrosine Kinases.

    Science.gov (United States)

    Abdullah, Nor Linda; Mohd-Zin, Siti W; Ahmad-Annuar, Azlina; Abdul-Aziz, Noraishah M

    2017-01-01

    Members of the Eph receptor tyrosine kinase have previously been implicated in cranial neural tube development. Failure of neural tube closure leads to the devastating conditions known as anencephaly and spina bifida. EphA2 and EphA4 are expressed at the tips of the closing spinal neural folds prior and during neural tube closure. We investigated the possible role of murine EphA2 and EphA4 during the last step of primary neural tube closure, which is adhesion and fusion. The individual mouse knockouts of EphA2 and EphA4 per se do not exhibit neural tube defects (NTDs). The embryos generated by the crossing of double heterozygotes Epha2 tm1Jrui/+ Epha4 rb-2J/+ displayed NTDs with a wide degree of severity including close exencephaly and close spina bifida (spina bifida occulta). Interestingly, mutants displaying NTDs had skin covering the underlying lesion. The tissue sections revealed the elevated neural folds had not adhered and fused. The phenotypes seen in Epha2 tm1Jrui/+ Epha4 rb-2J/+ double heterozygous embryos suggest both genes play a compensatory role with each other in the adhesion and fusion of the neural tube. In this study, there exists a >50% penetrance of NTDs in the mouse mutants, which genetically have a single allele each of EphA2 and EphA4 absent.

  3. Osabc1k8, an abc1-like kinase gene, mediates abscisic acid sensitivity and dehydration tolerance response in rice seedlings

    International Nuclear Information System (INIS)

    Liu, Y.; Li, T.; Yang, C.

    2015-01-01

    The activity of bc1 complex kinase (ABC1K) protein family, which widely exists in prokaryotes and eukaryotes, consists of 15 members in rice, and the role of this family in plants has not yet been studied in details. In this study, a novel function of OsABC1K8 (LOC-Os06g48770), a member of rice ABC1K family, was characterized. The transcript level of OsABC1K8 changes in response to salt, dehydration, cold, PEG, oxidative (H/sub 2/O/sub 2/) stresses, or abscisic acid (ABA) treatment. Overexpression of OsABC1K8 significantly increased sensitivity to dehydration and reduced sensitivity to ABA. In the contrast, RNAi transgenic lines displayed significantly reduced sensitivity to dehydration stress and increased sensitivity to ABA. Furthermore, the transcriptional levels of several ABA/stress-regulated responsive genes were suppressed in OsABC1K8 over-expressing plants under dehydration stress. In conclusion, our results suggested that OsABC1K8 is a negative regulator in response to dehydration stress through an ABA-dependent pathway. (author)

  4. Ketamine inhibits tumor necrosis factor-α and interleukin-6 gene expressions in lipopolysaccharide-stimulated macrophages through suppression of toll-like receptor 4-mediated c-Jun N-terminal kinase phosphorylation and activator protein-1 activation

    International Nuclear Information System (INIS)

    Wu, G.-J.; Chen, T.-L.; Ueng, Y.-F.; Chen, R.-M.

    2008-01-01

    Our previous study showed that ketamine, an intravenous anesthetic agent, has anti-inflammatory effects. In this study, we further evaluated the effects of ketamine on the regulation of tumor necrosis factor-α (TNF-α) and interlukin-6 (IL-6) gene expressions and its possible signal-transducing mechanisms in lipopolysaccharide (LPS)-activated macrophages. Exposure of macrophages to 1, 10, and 100 μM ketamine, 100 ng/ml LPS, or a combination of ketamine and LPS for 1, 6, and 24 h was not cytotoxic to macrophages. A concentration of 1000 μM of ketamine alone or in combined treatment with LPS caused significant cell death. Administration of LPS increased cellular TNF-α and IL-6 protein levels in concentration- and time-dependent manners. Meanwhile, treatment with ketamine concentration- and time-dependently alleviated the enhanced effects. LPS induced TNF-α and IL-6 mRNA syntheses. Administration of ketamine at a therapeutic concentration (100 μM) significantly inhibited LPS-induced TNF-α and IL-6 mRNA expressions. Application of toll-like receptor 4 (TLR4) small interfering (si)RNA into macrophages decreased cellular TLR4 levels. Co-treatment of macrophages with ketamine and TLR4 siRNA decreased the LPS-induced TNF-α and IL-6 productions more than alone administration of TLR4 siRNA. LPS stimulated phosphorylation of c-Jun N-terminal kinase and translocation of c-Jun and c-Fos from the cytoplasm to nuclei. However, administration of ketamine significantly decreased LPS-induced activation of c-Jun N-terminal kinase and translocation of c-Jun and c-Fos. LPS increased the binding of nuclear extracts to activator protein-1 consensus DNA oligonucleotides. Administration of ketamine significantly ameliorated LPS-induced DNA binding activity of activator protein-1. Therefore, a clinically relevant concentration of ketamine can inhibit TNF-α and IL-6 gene expressions in LPS-activated macrophages. The suppressive mechanisms occur through suppression of TLR4-mediated

  5. Momordica charantia polysaccharides could protect against cerebral ischemia/reperfusion injury through inhibiting oxidative stress mediated c-Jun N-terminal kinase 3 signaling pathway.

    Science.gov (United States)

    Gong, Juanjuan; Sun, Fumou; Li, Yihang; Zhou, Xiaoling; Duan, Zhenzhen; Duan, Fugang; Zhao, Lei; Chen, Hansen; Qi, Suhua; Shen, Jiangang

    2015-04-01

    Momordica charantia (MC) is a medicinal plant for stroke treatment in Traditional Chinese Medicine, but its active compounds and molecular targets are unknown yet. M. charantia polysaccharide (MCP) is one of the important bioactive components in MC. In the present study, we tested the hypothesis that MCP has neuroprotective effects against cerebral ischemia/reperfusion injury through scavenging superoxide (O2(-)), nitric oxide (NO) and peroxynitrite (ONOO(-)) and inhibiting c-Jun N-terminal protein kinase (JNK3) signaling cascades. We conducted experiments with in vivo global and focal cerebral ischemia/reperfusion rat models and in vitro oxygen glucose deprivation (OGD) neural cells. The effects of MCP on apoptotic cell death and infarction volume, the bioactivities of scavenging O2(-), NO and ONOO(-), inhibiting lipid peroxidation and modulating JNK3 signaling pathway were investigated. Major results are summarized as below: (1) MCP dose-dependently attenuated apoptotic cell death in neural cells under OGD condition in vitro and reduced infarction volume in ischemic brains in vivo; (2) MCP had directing scavenging effects on NO, O2(-) and ONOO(-) and inhibited lipid peroxidation; (3) MCP inhibited the activations of JNK3/c-Jun/Fas-L and JNK3/cytochrome C/caspases-3 signaling cascades in ischemic brains in vivo. Taken together, we conclude that MCP could be a promising neuroprotective ingredient of M. charantia and its mechanisms could be at least in part attributed to its antioxidant activities and inhibiting JNK3 signaling cascades during cerebral ischemia/reperfusion injury. Copyright © 2014 Elsevier Ltd. All rights reserved.

  6. Heroin Activates ATF3 and CytC via c-Jun N-Terminal Kinase Pathways to Mediate Neuronal Apoptosis

    Science.gov (United States)

    Pu, Hongwei; Wang, Xuemei; Su, Liping; Ma, Chuang; Zhang, Yan; Zhang, Liping; Chen, Xiao; Li, Xiujuan; Wang, Hua; Liu, Xiaoshan; Zhang, Jianlong

    2015-01-01

    Background Drug abuse and addiction has become a major public health problem that impacts all societies. The use of heroin may cause spongiform leukoencephalopathy (SLE). Material/Methods Cerebellar granule cells were derived from 7-day-old Sprague-Dawley rat pups. Neurons were dissociated from freshly dissected cerebella by mechanical disruption in the presence of 0.125% trypsin and DNaseI and then seeded at a density of 4×106 cells/ml in Dulbecco’s modified Eagle’s medium/nutrient mixture F-12 ham’s containing 10% fetal bovine serum and Arc-C(sigma) at concentrations to inhibit glial cell growth inoculated into 6-well plates and a small dish. Results We found that heroin induces the apoptosis of primary cultured cerebellar granule cells (CGCS) and that the c-Jun N-terminal kinase (JNK) pathway was activated under heroin treatment and stimulated obvious increases in the levels of C-jun, Cytc, and ATF3mRNA. CYTC and ATF3 were identified as candidate targets of the JNK/c-Jun pathway in this process because the specificity inhibitors SP600125 of JNK/C-jun pathways reduced the levels of C-jun, Cytc, and ATF3mRNA. The results suggested that SP600125 of JNK/C-jun can inhibit heroin-induced apoptosis of neurons. Conclusions The present study analyzes our understanding of the critical role of the JNK pathway in the process of neuronal apoptosis induced by heroin, and suggests a new and effective strategy to treat SLE. PMID:25848832

  7. Human p38δ MAP kinase mediates UV irradiation induced up-regulation of the gene expression of chemokine BRAK/CXCL14

    International Nuclear Information System (INIS)

    Ozawa, Shigeyuki; Ito, Shin; Kato, Yasumasa; Kubota, Eiro; Hata, Ryu-Ichiro

    2010-01-01

    The mitogen-activated protein kinase (MAPK) family comprises ERK, JNK, p38 and ERK5 (big-MAPK, BMK1). UV irradiation of squamous cell carcinoma cells induced up-regulation of gene expression of chemokine BRAK/CXCL14, stimulated p38 phosphorylation, and down-regulated the phosphorylation of ERK. Human p38 MAPKs exist in 4 isoforms: p38α, β, γ and δ. The UV stimulation of p38 phosphorylation was not inhibited by the presence of SB203580 or PD169316, inhibitors of p38α and β, suggesting p38 phosphorylation was not dependent on these 2 isoforms and that p38γ and/or δ was responsible for the phosphorylation. In fact, inhibition of each of these 4 p38 isoforms by the introduction of short hairpin (sh) RNAs for respective isoforms revealed that only shRNA for p38δ attenuated the UV-induced up-regulation of BRAK/CXCL14 gene expression. In addition, over-expression of p38 isoforms in the cells showed the association of p38δ with ERK1 and 2, concomitant with down-regulation of ERK phosphorylation. The usage of p38δ isoform by UV irradiation is not merely due to the abundance of this p38 isoform in the cells. Because serum deprivation of the cells also induced an increase in BRAK/CXCL14 gene expression, and in this case p38α and/or β isoform is responsible for up-regulation of BRAK/CXCL14 gene expression. Taken together, the data indicate that the respective stress-dependent action of p38 isoforms is responsible for the up-regulation of the gene expression of the chemokine BRAK/CXCL14.

  8. Neurotoxicity of developmental hypothyroxinemia and hypothyroidism in rats: Impairments of long-term potentiation are mediated by phosphatidylinositol 3-kinase signaling pathway.

    Science.gov (United States)

    Wang, Yi; Wei, Wei; Wang, Yuan; Dong, Jing; Song, Binbin; Min, Hui; Teng, Weiping; Chen, Jie

    2013-09-01

    Neurotoxicity of iodine deficiency-induced hypothyroidism during developmental period results in serious impairments of brain function, such as learning and memory. These impairments are largely irreversible, and the underlying mechanisms remain unclear. In addition to hypothyroidism, iodine deficiency may cause hypothyroxinemia, a relatively subtle form of thyroid hormone deficiency. Neurotoxicity of developmental hypothyroxinemia also potentially impairs learning and memory. However, more direct evidence of the associations between developmental hypothyroxinemia and impairments of learning and memory should be provided, and the underlying mechanisms remain to be elucidated. Thus, in the present study, we investigated the effects of developmental hypothyroxinemia and hypothyroidism on long-term potentiation (LTP), a widely accepted cellular model of learning and memory, in the hippocampal CA1 region. The activation of the phosphatidylinositol 3-kinase (PI3K) signaling pathway - a pathway closely associated with synaptic plasticity and learning and memory - was also investigated. Wistar rats were treated with iodine deficient diet or methimazole (MMZ) to induce developmental hypothyroxinemia or hypothyroidism. The results showed that developmental hypothyroxinemia caused by mild iodine deficiency and developmental hypothyroidism caused by severe iodine deficiency or MMZ significantly reduced the field-excitatory postsynaptic potential (f-EPSP) slope and the population spike (PS) amplitude. Decreased activation of the PI3K signaling pathway was also observed in rats subjected to developmental hypothyroxinemia or hypothyroidism. Our results may support the hypothesis that neurotoxicity of both developmental hypothyroxinemia and hypothyroidism causes damages to learning and memory. Our results also suggest that decreased activation of the PI3K signaling pathway may contribute to impairments of LTP caused by neurotoxicity of both developmental hypothyroxinemia and

  9. Protein kinase Cγ in colon cancer cells: Expression, Thr514 phophorylation and sensitivity to butyrate-mediated upregulation as related to the degree of differentiation

    Science.gov (United States)

    Garczarczyk, Dorota; Szeker, Krisztina; Galfi, Peter; Csordas, Adam; Hofmann, Johann

    2010-01-01

    Protein kinase C (PKC) isoenzymes are expressed and activated in a cell type-specific manner, and play an essential role in tissue-specific signal transduction. The presence of butyrate at millimolar concentrations in the colon raises the question of whether it affects the expression of PKC isoenzymes in the different cell types of the colonic epithelium. We investigated the protein expression levels of PKCγ, Thr514-phosphorylated PKCγ (pPKCγ-Thr514), and their subcellular distribution as affected by butyrate in a set of colon cancer cell lines. Thr514-phosphorylation of de novo synthesized PKCγ is the first step in priming of the inactive PKCγ before its release into the cytoplasm. For immunoblot analysis, we employed three antibodies, one against an unmodified sequence, mapping within 50 amino acids at its C-terminus, a second against pPKCγ-Thr514, and a third against pPKCγ-pan-Thr514. The antibody against an unmodified C-terminal peptide epitope did not recognize pPKCγ-Thr514, suggesting that phosphorylation at this site interferes with the binding of the antibody to the C-terminus. Marked butyrate-induced upregulation of PKCγ occurred in HT29 cells (colonocyte stem cells) and HT29-derived cell lines. However, in Caco2 and IEC-18 cells (differentiated intestinal epithelial cells), PKCγ was insensitive to upregulation, and present exclusively as pPKCγ-Thr514. Lovo and SW480 expressed higher levels of PKCγ. In HT29 cells, butyrate-induced upregulation of the non-phosphorylated PKCγ was observed in both the membrane and cytosolic fraction. In Caco2 cells, the Thr514-phosphorylated form was present at high levels in both fractions. The presence of unphosphorylated PKCγ in HT29 cells, and its complete absence in Caco2 cells demonstrates a cell type-dependent differential coupling of Thr514-phosphorylation with de novo synthesis of PKCγ in colon cancer cells. PMID:20188713

  10. Phosphoinositide 3-kinase participates in l-methionine sulfoximine-induced cell death via salicylic acid mediated signaling in Nicotiana benthamiana.

    Science.gov (United States)

    Sumida, Sayuri; Ito, Makoto; Galis, Ivan; Nakatani, Hiroko; Shinya, Tomonori; Ohnishi, Kouhei; Hikichi, Yasufumi; Kiba, Akinori

    2017-11-01

    Pseudomonas syringae pv. tabaci causes wildfire disease by the action of tabtoxinine-β-lactam (TβL), a non-specific bacterial toxin. To better understand the molecular mechanisms of wildfire disease and its development, we focused on the phosphoinositide 3-kinase in Nicotiana benthamiana (NbPI3K) and its potential role in the disease outbreak, using l-methionine sulfoximine (MSX) as an easily accessible mimic of the TβL action. The NbPI3K-silenced plants showed accelerated induction of cell death and necrotic lesion formation by MSX, and the expression of hin1, marker gene for the programmed cell death, was strongly induced in the plants. However, the accumulation of ammonium ions, caused by MSX inhibition of glutamine sythetase activity, was not affected by the NbPI3K-silencing. Interestingly, the expression of PR-1a, a marker gene for salicylic acid (SA) innate immunity signaling, and accumulation of SA were both enhanced in the NbPI3K-silenced plants. Accordingly, the acceleration of MSX-induced cell death by NbPI3K-silencing was reduced in NahG plants, and by double silencing of NbPI3K together with the NbICS1 encoding a SA-biosynthetic enzyme. As silencing of NbPI3K accelerated the TβL-induced necrotic lesions, and lesions of wildfire disease caused by P. syringae pv. tabaci, these results suggest that the NbPI3K-related pathway might act as a negative regulator of cell death during development of wildfire disease that involves SA-dependent signaling pathway downstream of TβL action in N. benthamiana. Copyright © 2017 Elsevier GmbH. All rights reserved.

  11. Atrial natriuretic peptide-mediated inhibition of microcirculatory endothelial Ca2+ and permeability response to histamine involves cGMP-dependent protein kinase I and TRPC6 channels.

    Science.gov (United States)

    Chen, Wen; Oberwinkler, Heike; Werner, Franziska; Gaßner, Birgit; Nakagawa, Hitoshi; Feil, Robert; Hofmann, Franz; Schlossmann, Jens; Dietrich, Alexander; Gudermann, Thomas; Nishida, Motohiro; Del Galdo, Sabrina; Wieland, Thomas; Kuhn, Michaela

    2013-09-01

    Histamine increases microvascular endothelial leakage by activation of complex calcium-dependent and -independent signaling pathways. Atrial natriuretic peptide (ANP) via its cGMP-forming guanylyl cyclase-A (GC-A) receptor counteracts this response. Here, we characterized the molecular mechanisms underlying this interaction, especially the role of cGMP-dependent protein kinase I (cGKI). We combined intravital microscopy studies of the mouse cremaster microcirculation with experiments in cultured microvascular human dermal endothelial cells. In wild-type mice, ANP had no direct effect on the extravasation of fluorescent dextran from postcapillary venules, but strongly reduced the histamine-provoked vascular leakage. This anti-inflammatory effect of ANP was abolished in mice with endothelial-restricted inactivation of GC-A or cGKI. Histamine-induced increases in endothelial [Ca(2+)]i in vitro and of vascular leakage in vivo were markedly attenuated by the Ca(2+)-entry inhibitor SKF96365 and in mice with ablated transient receptor potential canonical (TRPC) 6 channels. Conversely, direct activation of TRPC6 with hyperforin replicated the hyperpermeability responses to histamine. ANP, via cGKI, stimulated the inhibitory phosphorylation of TRPC6 at position Thr69 and prevented the hyperpermeability responses to hyperforin. Moreover, inhibition of cGMP degradation by the phosphodiesterase 5 inhibitor sildenafil prevented the edematic actions of histamine in wild types but not in mice with endothelial GC-A or cGKI deletion. ANP attenuates the inflammatory actions of histamine via endothelial GC-A/cGMP/cGKI signaling and inhibitory phosphorylation of TRPC6 channels. The therapeutic potential of this novel regulatory pathway is indicated by the observation that sildenafil improves systemic endothelial barrier functions by enhancing the endothelial effects of endogenous ANP.

  12. The small-molecule kinase inhibitor D11 counteracts 17-AAG-mediated up-regulation of HSP70 in brain cancer cells.

    Science.gov (United States)

    Schaefer, Susanne; Svenstrup, Tina H; Guerra, Barbara

    2017-01-01

    Many types of cancer express high levels of heat shock proteins (HSPs) that are molecular chaperones regulating protein folding and stability ensuring protection of cells from potentially lethal stress. HSPs in cancer cells promote survival, growth and spreading even in situations of growth factors deprivation by associating with oncogenic proteins responsible for cell transformation. Hence, it is not surprising that the identification of potent inhibitors of HSPs, notably HSP90, has been the primary research focus, in recent years. Exposure of cancer cells to HSP90 inhibitors, including 17-AAG, has been shown to cause resistance to chemotherapeutic treatment mostly attributable to induction of the heat shock response and increased cellular levels of pro-survival chaperones. In this study, we show that treatment of glioblastoma cells with 17-AAG leads to HSP90 inhibition indicated by loss of stability of the EGFR client protein, and significant increase in HSP70 expression. Conversely, co-treatment with the small-molecule kinase inhibitor D11 leads to suppression of the heat shock response and inhibition of HSF1 transcriptional activity. Beside HSP70, Western blot and differential mRNA expression analysis reveal that combination treatment causes strong down-regulation of the small chaperone protein HSP27. Finally, we demonstrate that incubation of cells with both agents leads to enhanced cytotoxicity and significantly high levels of LC3-II suggesting autophagy induction. Taken together, results reported here support the notion that including D11 in future treatment regimens based on HSP90 inhibition can potentially overcome acquired resistance induced by the heat shock response in brain cancer cells.

  13. Nitric oxide mediates stretch-induced Ca2+ release via activation of phosphatidylinositol 3-kinase-Akt pathway in smooth muscle.

    Science.gov (United States)

    Wei, Bin; Chen, Zheng; Zhang, Xu; Feldman, Morris; Dong, Xian-zhi; Doran, Robert; Zhao, Bao-Lu; Yin, Wen-xuan; Kotlikoff, Michael I; Ji, Guangju

    2008-06-25

    Hollow smooth muscle organs such as the bladder undergo significant changes in wall tension associated with filling and distension, with attendant changes in muscle tone. Our previous study indicated that stretch induces Ca(2+) release occurs in the form of Ca(2+) sparks and Ca(2+) waves in urinary bladder myocytes. While, the mechanism underlying stretch-induced Ca2+ release in smooth muscle is unknown. We examined the transduction mechanism linking cell stretch to Ca(2+) release. The probability and frequency of Ca(2+) sparks induced by stretch were closely related to the extent of cell extension and the time that the stretch was maintained. Experiments in tissues and single myocytes indicated that mechanical stretch significantly increases the production of nitric oxide (NO) and the amplitude and duration of muscle contraction. Stretch-induced Ca(2+) sparks and contractility increases were abrogated by the NO inhibitor L-NAME and were also absent in eNOS knockout mice. Furthermore, exposure of eNOS null mice to exogenously generated NO induced Ca(2+) sparks. The soluble guanylyl cyclase inhibitor ODQ did not inhibit SICR, but this process was effectively blocked by the PI3 kinase inhibitors LY494002 and wortmannin; the phosphorylation of Akt and eNOS were up-regulated by 204+/-28.6% and 258+/-36.8% by stretch, respectively. Moreover, stretch significantly increased the eNOS protein expression level. Taking together, these results suggest that stretch-induced Ca2+ release is NO dependent, resulting from the activation of PI3K/Akt pathway in smooth muscle.

  14. Nitric oxide mediates stretch-induced Ca2+ release via activation of phosphatidylinositol 3-kinase-Akt pathway in smooth muscle.

    Directory of Open Access Journals (Sweden)

    Bin Wei

    2008-06-01

    Full Text Available Hollow smooth muscle organs such as the bladder undergo significant changes in wall tension associated with filling and distension, with attendant changes in muscle tone. Our previous study indicated that stretch induces Ca(2+ release occurs in the form of Ca(2+ sparks and Ca(2+ waves in urinary bladder myocytes. While, the mechanism underlying stretch-induced Ca2+ release in smooth muscle is unknown.We examined the transduction mechanism linking cell stretch to Ca(2+ release. The probability and frequency of Ca(2+ sparks induced by stretch were closely related to the extent of cell extension and the time that the stretch was maintained. Experiments in tissues and single myocytes indicated that mechanical stretch significantly increases the production of nitric oxide (NO and the amplitude and duration of muscle contraction. Stretch-induced Ca(2+ sparks and contractility increases were abrogated by the NO inhibitor L-NAME and were also absent in eNOS knockout mice. Furthermore, exposure of eNOS null mice to exogenously generated NO induced Ca(2+ sparks. The soluble guanylyl cyclase inhibitor ODQ did not inhibit SICR, but this process was effectively blocked by the PI3 kinase inhibitors LY494002 and wortmannin; the phosphorylation of Akt and eNOS were up-regulated by 204+/-28.6% and 258+/-36.8% by stretch, respectively. Moreover, stretch significantly increased the eNOS protein expression level.Taking together, these results suggest that stretch-induced Ca2+ release is NO dependent, resulting from the activation of PI3K/Akt pathway in smooth muscle.

  15. Neurotoxicity of developmental hypothyroxinemia and hypothyroidism in rats: Impairments of long-term potentiation are mediated by phosphatidylinositol 3-kinase signaling pathway

    International Nuclear Information System (INIS)

    Wang, Yi; Wei, Wei; Wang, Yuan; Dong, Jing; Song, Binbin; Min, Hui; Teng, Weiping; Chen, Jie

    2013-01-01

    Neurotoxicity of iodine deficiency-induced hypothyroidism during developmental period results in serious impairments of brain function, such as learning and memory. These impairments are largely irreversible, and the underlying mechanisms remain unclear. In addition to hypothyroidism, iodine deficiency may cause hypothyroxinemia, a relatively subtle form of thyroid hormone deficiency. Neurotoxicity of developmental hypothyroxinemia also potentially impairs learning and memory. However, more direct evidence of the associations between developmental hypothyroxinemia and impairments of learning and memory should be provided, and the underlying mechanisms remain to be elucidated. Thus, in the present study, we investigated the effects of developmental hypothyroxinemia and hypothyroidism on long-term potentiation (LTP), a widely accepted cellular model of learning and memory, in the hippocampal CA1 region. The activation of the phosphatidylinositol 3-kinase (PI3K) signaling pathway – a pathway closely associated with synaptic plasticity and learning and memory – was also investigated. Wistar rats were treated with iodine deficient diet or methimazole (MMZ) to induce developmental hypothyroxinemia or hypothyroidism. The results showed that developmental hypothyroxinemia caused by mild iodine deficiency and developmental hypothyroidism caused by severe iodine deficiency or MMZ significantly reduced the field-excitatory postsynaptic potential (f-EPSP) slope and the population spike (PS) amplitude. Decreased activation of the PI3K signaling pathway was also observed in rats subjected to developmental hypothyroxinemia or hypothyroidism. Our results may support the hypothesis that neurotoxicity of both developmental hypothyroxinemia and hypothyroidism causes damages to learning and memory. Our results also suggest that decreased activation of the PI3K signaling pathway may contribute to impairments of LTP caused by neurotoxicity of both developmental hypothyroxinemia and

  16. Neurotoxicity of developmental hypothyroxinemia and hypothyroidism in rats: Impairments of long-term potentiation are mediated by phosphatidylinositol 3-kinase signaling pathway

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Yi; Wei, Wei; Wang, Yuan; Dong, Jing; Song, Binbin; Min, Hui [Department of Occupational and Environmental Health, School of Public Health, China Medical University, Shenyang (China); Teng, Weiping, E-mail: twpendocrine@yahoo.com.cn [Liaoning Provincial Key Laboratory of Endocrine Diseases, the First Hospital of China Medical University, Shenyang (China); Chen, Jie, E-mail: chenjie@mail.cmu.edu.cn [Department of Occupational and Environmental Health, School of Public Health, China Medical University, Shenyang (China)

    2013-09-01

    Neurotoxicity of iodine deficiency-induced hypothyroidism during developmental period results in serious impairments of brain function, such as learning and memory. These impairments are largely irreversible, and the underlying mechanisms remain unclear. In addition to hypothyroidism, iodine deficiency may cause hypothyroxinemia, a relatively subtle form of thyroid hormone deficiency. Neurotoxicity of developmental hypothyroxinemia also potentially impairs learning and memory. However, more direct evidence of the associations between developmental hypothyroxinemia and impairments of learning and memory should be provided, and the underlying mechanisms remain to be elucidated. Thus, in the present study, we investigated the effects of developmental hypothyroxinemia and hypothyroidism on long-term potentiation (LTP), a widely accepted cellular model of learning and memory, in the hippocampal CA1 region. The activation of the phosphatidylinositol 3-kinase (PI3K) signaling pathway – a pathway closely associated with synaptic plasticity and learning and memory – was also investigated. Wistar rats were treated with iodine deficient diet or methimazole (MMZ) to induce developmental hypothyroxinemia or hypothyroidism. The results showed that developmental hypothyroxinemia caused by mild iodine deficiency and developmental hypothyroidism caused by severe iodine deficiency or MMZ significantly reduced the field-excitatory postsynaptic potential (f-EPSP) slope and the population spike (PS) amplitude. Decreased activation of the PI3K signaling pathway was also observed in rats subjected to developmental hypothyroxinemia or hypothyroidism. Our results may support the hypothesis that neurotoxicity of both developmental hypothyroxinemia and hypothyroidism causes damages to learning and memory. Our results also suggest that decreased activation of the PI3K signaling pathway may contribute to impairments of LTP caused by neurotoxicity of both developmental hypothyroxinemia and

  17. GsSKP21, a Glycine soja S-phase kinase-associated protein, mediates the regulation of plant alkaline tolerance and ABA sensitivity.

    Science.gov (United States)

    Liu, Ailin; Yu, Yang; Duan, Xiangbo; Sun, Xiaoli; Duanmu, Huizi; Zhu, Yanming

    2015-01-01

    Plant SKP1-like family proteins, components of the SCF complex E3 ligases, are involved in the regulation of plant development and stress responses. Little is known about the precise function of SKP genes in plant responses to environmental stresses. GsSKP21 was initially identified as a potential stress-responsive gene based on the transcriptome sequencing of Glycine soja. In this study, we found that GsSKP21 protein contains highly conserved SKP domains in its N terminus and an extra unidentified domain in its C terminus. The transcript abundance of GsSKP21, detected by quantitative real-time PCR, was induced under the treatment of alkali and salt stresses. Overexpression of GsSKP21 in Arabidopsis dramatically increased plant tolerance to alkali stress. Furthermore, we found that overexpression of GsSKP21 resulted in decreased ABA sensitivity during both the seed germination and early seedling growth stages. GsSKP21 mediated ABA signaling by altering the expression levels of the ABA signaling-related and ABA-induced genes. We also investigated the tissue expression specificity and subcellular localization of GsSKP21. These results suggest that GsSKP21 is important for plant tolerance to alkali stress and plays a critical regulatory role in the ABA-mediated stress response.

  18. Cell-free methemoglobin drives platelets to apoptosis via mitochondrial ROS-mediated activation of JNK and p38 MAP kinase.

    Science.gov (United States)

    NaveenKumar, Somanathapura K; Hemshekhar, Mahadevappa; Sundaram, Mahalingam S; Kemparaju, Kempaiah; Girish, Kesturu S

    2017-09-09

    Cell-free hemoglobin (Hb), a well-known marker of intravascular hemolysis, is eventually oxidized to methemoglobin (MtHb). Elevated levels of MtHb have been noted, alongside depleted levels of platelets, in several hemolytic diseases. The current study aims to probe the possible role of MtHb in platelet death, based on the facts that it is a pro-inflammatory and pro-apoptotic agent, as well as the sensitive nature of platelets and their tendency to undergo apoptosis under oxidative stress. An attempt is made to establish the link between hemolysis and thrombocytopenia, by deciphering the underlying molecular signaling pathways. The results of this study demonstrate that MtHb, not Hb exerts oxidative stress on platelets, which triggers their death via ROS-mediated mitochondrial apoptotic pathway. It was further established that the MtHb-induced platelet apoptotic events mediate through JNK and p38 MAPK activation. Thus, the study presents a mechanistic insight into the previous studies that reported the incidence of thrombocytopenia in hemolytic diseases. This study highlights the fate of platelets in intravascular hemolytic conditions, which demands the need for a specific treatment strategy considering the risks associated with thrombocytopenia during severe hemolytic diseases. Copyright © 2017 Elsevier Inc. All rights reserved.

  19. Pro-life role for c-Jun N-terminal kinase and p38 mitogen-activated protein kinase at rostral ventrolateral medulla in experimental brain stem death.

    Science.gov (United States)

    Chang, Alice Y W

    2012-11-17

    Based on an experimental brain stem death model, we demonstrated previously that activation of the mitogen-activated protein kinase kinase 1/2 (MEK1/2)/extracellular signal-regulated kinase 1/2 (ERK1/2)/ mitogen-activated protein kinase signal-interacting kinase 1/2 (MNK1/2) cascade plays a pro-life role in the rostral ventrolateral medulla (RVLM), the origin of a life-and-death signal detected from systemic arterial pressure, which sequentially increases (pro-life) and decreases (pro-death) to reflect progressive dysfunction of central cardiovascular regulation during the advancement towards brain stem death in critically ill patients. The present study assessed the hypothesis that, in addition to ERK1/2, c-Jun NH2-terminal kinase (JNK) and p38 mitogen-activated protein kinase (p38MAPK), the other two mammalian members of MAPKs that are originally identified as stress-activated protein kinases, are activated specifically by MAPK kinase 4 (MAP2K4) or MAP2K6 and play a pro-life role in RVLM during experimental brain stem death. We further delineated the participation of phosphorylating activating transcriptional factor-2 (ATF-2) and c-Jun, the classical transcription factor activated by JNK or p38MAPK, in this process. An experimental model of brain stem death that employed microinjection of the organophosphate insecticide mevinphos (Mev; 10 nmol) bilaterally into RVLM of Sprague-Dawley rats was used, alongside cardiovascular, pharmacological and biochemical evaluations. Results from ELISA showed that whereas the total JNK, p38MAPK, MAP2K4 and MAP2K6 were not affected, augmented phosphorylation of JNK at Thr183 and Tyr185 and p38MAPK at Thr180 and Tyr182, accompanied by phosphorylation of their upstream activators MAP2K4 at Ser257 and Thr261 and MAP2K6 at Ser207 and Thr211 in RVLM occurred preferentially during the pro-life phase of experimental brain stem death. Moreover, the activity of transcription factors ATF-2 at Thr71 and c-Jun at Ser73, rather than Elk-1 at

  20. Ca2+-mediated potentiation of the swelling-induced taurine efflux from HeLa cells: On the role of calmodulin and novel protein kinase C isoforms

    DEFF Research Database (Denmark)

    Falktoft, Birgitte; Lambert, Ian H.

    2004-01-01

    The present work sets out to investigate how Ca2+ regulates the volume-sensitive taurine-release pathway in HeLa cells. Addition of Ca2+-mobilizing agonists at the time of exposure to hypotonic NaCl medium augments the swelling-induced taurine release and subsequently accelerates the inactivation...... of the release pathway. The accelerated inactivation is not observed in hypotonic Ca2+-free or high-K+ media. Addition of Ca2+-mobilizing agonists also accelerates the regulatory volume decrease, which probably reflects activation of Ca2+-activated K+ channels. The taurine release from control cells and cells...... exposed to Ca2+ agonists is equally affected by changes in cell volume, application of DIDS and arachidonic acid, indicating that the volume-sensitive taurine leak pathway mediates the Ca2+-augmented taurine release. Exposure to Ca2+-mobilizing agonists prior to a hypotonic challenge also augments...

  1. Human stem cell osteoblastogenesis mediated by novel glycogen synthase kinase 3 inhibitors induces bone formation and a unique bone turnover biomarker profile in rats

    International Nuclear Information System (INIS)

    Gilmour, Peter S.; O'Shea, Patrick J.; Fagura, Malbinder; Pilling, James E.; Sanganee, Hitesh; Wada, Hiroki; Courtney, Paul F.; Kavanagh, Stefan; Hall, Peter A.; Escott, K. Jane

    2013-01-01

    Wnt activation by inhibiting glycogen synthase kinase 3 (GSK-3) causes bone anabolism in rodents making GSK-3 a potential therapeutic target for osteoporotic and osteolytic metastatic bone disease. To understand the wnt pathway related to human disease translation, the ability of 3 potent inhibitors of GSK-3 (AZD2858, AR79, AZ13282107) to 1) drive osteoblast differentiation and mineralisation using human adipose-derived stem cells (hADSC) in vitro; and 2) stimulate rat bone formation in vivo was investigated. Bone anabolism/resorption was determined using clinically relevant serum biomarkers as indicators of bone turnover and bone formation assessed in femurs by histopathology and pQCT/μCT imaging. GSK-3 inhibitors caused β-catenin stabilisation in human and rat mesenchymal stem cells, stimulated hADSC commitment towards osteoblasts and osteogenic mineralisation in vitro. AZD2858 produced time-dependent changes in serum bone turnover biomarkers and increased bone mass over 28 days exposure in rats. After 7 days, AZD2858, AR79 or AZ13282107 exposure increased the bone formation biomarker P1NP, and reduced the resorption biomarker TRAcP-5b, indicating increased bone anabolism and reduced resorption in rats. This biomarker profile was differentiated from anabolic agent PTH 1–34 or the anti-resorptive Alendronate-induced changes. Increased bone formation in cortical and cancellous bone as assessed by femur histopathology supported biomarker changes. 14 day AR79 treatment increased bone mineral density and trabecular thickness, and decreased trabecular number and connectivity assessed by pQCT/μCT. GSK-3 inhibition caused hADSC osteoblastogenesis and mineralisation in vitro. Increased femur bone mass associated with changes in bone turnover biomarkers confirmed in vivo bone formation and indicated uncoupling of bone formation and resorption. - Highlights: • Wnt modulation with 3 novel GSK-3 inhibitors alters bone growth. • Human stem cell osteoblastogenesis and

  2. Human stem cell osteoblastogenesis mediated by novel glycogen synthase kinase 3 inhibitors induces bone formation and a unique bone turnover biomarker profile in rats

    Energy Technology Data Exchange (ETDEWEB)

    Gilmour, Peter S., E-mail: Peter.Gilmour@astrazeneca.com [New Opportunities Innovative Medicines group, AstraZeneca R and D, Alderley Park, Cheshire SK10 4TF (United Kingdom); O' Shea, Patrick J.; Fagura, Malbinder [New Opportunities Innovative Medicines group, AstraZeneca R and D, Alderley Park, Cheshire SK10 4TF (United Kingdom); Pilling, James E. [Discovery Sciences, AstraZeneca R and D, Alderley Park, Cheshire SK10 4TF (United Kingdom); Sanganee, Hitesh [New Opportunities Innovative Medicines group, AstraZeneca R and D, Alderley Park, Cheshire SK10 4TF (United Kingdom); Wada, Hiroki [R and I IMed, AstraZeneca R and D, Molndal (Sweden); Courtney, Paul F. [DMPK, AstraZeneca R and D, Alderley Park, Cheshire SK10 4TF (United Kingdom); Kavanagh, Stefan; Hall, Peter A. [Safety Assessment, AstraZeneca R and D, Alderley Park, Cheshire SK10 4TF (United Kingdom); Escott, K. Jane [New Opportunities Innovative Medicines group, AstraZeneca R and D, Alderley Park, Cheshire SK10 4TF (United Kingdom)

    2013-10-15

    Wnt activation by inhibiting glycogen synthase kinase 3 (GSK-3) causes bone anabolism in rodents making GSK-3 a potential therapeutic target for osteoporotic and osteolytic metastatic bone disease. To understand the wnt pathway related to human disease translation, the ability of 3 potent inhibitors of GSK-3 (AZD2858, AR79, AZ13282107) to 1) drive osteoblast differentiation and mineralisation using human adipose-derived stem cells (hADSC) in vitro; and 2) stimulate rat bone formation in vivo was investigated. Bone anabolism/resorption was determined using clinically relevant serum biomarkers as indicators of bone turnover and bone formation assessed in femurs by histopathology and pQCT/μCT imaging. GSK-3 inhibitors caused β-catenin stabilisation in human and rat mesenchymal stem cells, stimulated hADSC commitment towards osteoblasts and osteogenic mineralisation in vitro. AZD2858 produced time-dependent changes in serum bone turnover biomarkers and increased bone mass over 28 days exposure in rats. After 7 days, AZD2858, AR79 or AZ13282107 exposure increased the bone formation biomarker P1NP, and reduced the resorption biomarker TRAcP-5b, indicating increased bone anabolism and reduced resorption in rats. This biomarker profile was differentiated from anabolic agent PTH{sub 1–34} or the anti-resorptive Alendronate-induced changes. Increased bone formation in cortical and cancellous bone as assessed by femur histopathology supported biomarker changes. 14 day AR79 treatment increased bone mineral density and trabecular thickness, and decreased trabecular number and connectivity assessed by pQCT/μCT. GSK-3 inhibition caused hADSC osteoblastogenesis and mineralisation in vitro. Increased femur bone mass associated with changes in bone turnover biomarkers confirmed in vivo bone formation and indicated uncoupling of bone formation and resorption. - Highlights: • Wnt modulation with 3 novel GSK-3 inhibitors alters bone growth. • Human stem cell osteoblastogenesis

  3. Adenovirus-mediated delivery and expression of a cAMP-dependent protein kinase inhibitor gene to BEAS-2B epithelial cells abolishes the anti-inflammatory effects of rolipram, salbutamol, and prostaglandin E2: a comparison with H-89.

    Science.gov (United States)

    Meja, Koremu K; Catley, Matthew C; Cambridge, Lisa M; Barnes, Peter J; Lum, Hazel; Newton, Robert; Giembycz, Mark A

    2004-05-01

    cAMP-elevating drugs are thought to mediate their biological effects by activating the cAMP/cAMP-dependent protein kinase (PKA) cascade. However, this hypothesis is difficult to confirm due to a lack of selective inhibitors. Here, we have probed the role of PKA in mediating inhibitory effects of several cAMP-elevating drugs in BEAS-2B epithelial cells using an adenovirus vector encoding a PKA inhibitor protein (PKIalpha) and have compared it to H-89, a commonly used small molecule PKA inhibitor. Initial studies established efficient gene transfer and confirmed functionality of PKIalpha 48 h after virus infection. All cAMP-elevating drugs tested promoted the phosphorylation of cAMP response element-binding protein (CREB), activated a cAMP response element (CRE)-driven luciferase reporter gene, and suppressed both granulocyte/macrophage colony-stimulating factor (GM-CSF) generation and [(3)H]arachidonic acid (AA) release in response to interleukin-1beta and monocyte chemotactic protein (MCP)-1, respectively. These effects were abolished by PKIalpha. In contrast, H-89 behaved unpredictably under the same conditions. Thus, although CREB phosphorylation evoked by a range of cAMP-elevating drugs was abolished by H-89, neither activation of the CRE-dependent luciferase reporter gene construct nor the inhibition of GM-CSF generation was inhibited. Paradoxically, H-89 antagonized MCP-1-induced [(3)H]AA release and enhanced the inhibitory effect of submaximal concentrations of rolipram and 8-bromo-cAMP. We suggest that expression of PKIalpha in susceptible cells provides a simple and unambiguous way to assess the role of PKA in cAMP signaling and to probe the mechanism of action of other drugs and cAMP-dependent responses where the participation of PKA is equivocal. Furthermore, these data suggest that H-89 is not a selective inhibitor of PKA and should be avoided.

  4. Hyperglycemia-induced oxidative stress induces apoptosis by inhibiting PI3-kinase/Akt and ERK1/2 MAPK mediated signaling pathway causing downregulation of 8-oxoG-DNA glycosylase levels in glial cells.

    Science.gov (United States)

    Kumar, Premranjan; Rao, G Nageswar; Pal, Bibhuti Bhusan; Pal, Arttatrana

    2014-08-01

    Glial cells are very important for normal brain function and alterations in their activity due to hyperglycemia, could contribute to diabetes-related cognitive dysfunction. Oxidative insults often cause rapid changes in almost all cells including glial cells. However, pathophysiologic mechanisms that lead to diabetic complications are not completely elucidated. Therefore, we examined whether elevated glucose levels directly or indirectly disrupt antioxidant defense mechanisms causing alterations in signaling pathways, cell cycle dysregulation, and reactive oxygen/nitrogen species-mediated apoptosis in glial cells. Findings of this study demonstrated that exposure of glial cells to high glucose markedly induces cellular and molecular injuries, as evidenced by elevated levels of reactive oxygen/nitrogen species, biomolecules damage, cell cycle dysregulation, decrease in antioxidant enzymes, and decrease in cell viability. Pretreatment of cells with N-acetyl-L-cysteine reduced high glucose-induced cytotoxicity by increasing the levels of antioxidant enzymes, and decreasing the number of apoptotic cells. Further, at molecular level high glucose treatment resulted in a significant increase in phosphorylation of Akt, MAPKs, tuberin, down regulation of 8-oxoG-DNA glycosylase and increase in 8-hydroxydeoxyguanosine accumulations. Pretreatment of cells with N-acetyl-L-cysteine, phosphatidylinositol3-kinase/Akt and ERK1/2 inhibitors completely abolished the apoptotic effects of high glucose. Moreover, N-acetyl-L-cysteine significantly inhibited reactive oxygen/nitrogen species generation, elevated antioxidants levels, inhibited Akt, ERK1/2, tuberin phosphorylation, decreased 8-hydroxydeoxyguanosine accumulation and upregulated 8-oxoG-DNA glycosylase expression. Our results demonstrate that high glucose induces apoptosis and inhibits proliferation of glial cells, which may be mediated by the phosphorylation of tuberin, down regulation of 8-oxoG-DNA glycosylase and 8

  5. Function of Integrin-Linked Kinase in Modulating the Stemness of IL-6–Abundant Breast Cancer Cells by Regulating γ-Secretase–Mediated Notch1 Activation in Caveolae

    Directory of Open Access Journals (Sweden)

    En-Chi Hsu

    2015-06-01

    Full Text Available Interleukin-6 (IL-6 and Notch signaling are important regulators of breast cancer stem cells (CSCs, which drive the malignant phenotype through self-renewal, differentiation, and development of therapeutic resistance. We investigated the role of integrin-linked kinase (ILK in regulating IL-6–driven Notch1 activation and the ability to target breast CSCs through ILK inhibition. Ectopic expression/short hairpin RNA-mediated knockdown of ILK, pharmacological inhibition of ILK with the small molecule T315, Western blot analysis, immunofluorescence, and luciferase reporter assays were used to evaluate the regulation of IL-6–driven Notch1 activation by ILK in IL-6–producing triple-negative breast cancer cell lines (MDA-MB-231, SUM-159 and in MCF-7 and MCF-7IL-6 cells. The effects of ILK on γ-secretase complex assembly and cellular localization were determined by immunofluorescence, Western blots of membrane fractions, and immunoprecipitation. In vivo effects of T315-induced ILK inhibition on CSCs in SUM-159 xenograft models were assessed by mammosphere assays, flow cytometry, and tumorigenicity assays. Results show that the genetic knockdown or pharmacological inhibition of ILK suppressed Notch1 activation and the abundance of the γ-secretase components presenilin-1, nicastrin, and presenilin enhancer 2 at the posttranscriptional level via inhibition of caveolin-1-dependent membrane assembly of the γ-secretase complex. Accordingly, knockdown of ILK inhibited breast CSC-like properties in vitro and the breast CSC subpopulation in vivo in xenograft tumor models. Based on these findings, we propose a novel function of ILK in regulating γ-secretase–mediated Notch1 activation, which suggests the targeting of ILK as a therapeutic approach to suppress IL-6–induced breast CSCs.

  6. Synthesis and Physicochemical Characterization of D-Tagatose-1-phosphate: The Substrate of the Tagatose-1-Phosphate Kinase TagK in the PTS-mediated D-Tagatose Catabolic Pathway of Bacillus licheniformis

    Science.gov (United States)

    Van der Heiden, Edwige; Delmarcelle, Michaël; Simon, Patricia; Counson, Melody; Galleni, Moreno; Freedberg, Darón I.; Thompson, John; Joris, Bernard; Battistel, Marcos D.

    2015-01-01

    We report the first enzymatic synthesis of D-tagatose-1-phosphate (Tag-1P) by the multi-component PEP-dependent:tag-PTS present in tagatose-grown cells of Klebsiella pneumoniae. Physicochemical characterization by 31P and 1H NMR spectroscopy reveals that, in solution, this derivative is primarily in the pyranose form. Tag-1P was used to characterize the putative tagatose-1-phosphate kinase (TagK) of the Bacillus licheniformis PTS-mediated D-Tagatose catabolic Pathway (Bli-TagP). For this purpose, a soluble protein fusion was obtained with the 6 His-tagged trigger factor (TFHis6) of Escherichia coli. The active fusion enzyme was named TagK-TFHis6. Tag-1P and D-fructose-1-phosphate (Fru-1P) are substrates for the TagK-TFHis6 enzyme, whereas the isomeric derivatives D-tagatose-6-phosphate (Tag-6P) and D-fructose-6-phosphate (Fru-6P) are inhibitors. Studies of catalytic efficiency (kcat/Km) reveal that the enzyme specificity is markedly in favor of Tag-1P as substrate. Importantly, we show in vivo that the transfer of the phosphate moiety from PEP to the B. licheniformis tagatose-specific enzyme II (EIITag) in E.coli is inefficient. The capability of the PTS general cytoplasmic components of B. subtilis, HPr and EI, to restore the phosphate transfer is demonstrated. PMID:26159072

  7. Synthesis and Physicochemical Characterization of D-Tagatose-1-Phosphate: The Substrate of the Tagatose-1-Phosphate Kinase in the Phosphotransferase System-Mediated D-Tagatose Catabolic Pathway of Bacillus licheniformis.

    Science.gov (United States)

    Van der Heiden, Edwige; Delmarcelle, Michaël; Simon, Patricia; Counson, Melody; Galleni, Moreno; Freedberg, Darón I; Thompson, John; Joris, Bernard; Battistel, Marcos D

    2015-01-01

    We report the first enzymatic synthesis of D-tagatose-1-phosphate (Tag-1P) by the multicomponent phosphoenolpyruvate:sugar phosphotransferase system (PEP-PTS) present in tagatose-grown cells of Klebsiella pneumoniae. Physicochemical characterization by (31)P and (1)H nuclear magnetic resonance spectroscopy reveals that, in solution, this derivative is primarily in the pyranose form. Tag-1P was used to characterize the putative tagatose-1-phosphate kinase (TagK) of the Bacillus licheniformis PTS-mediated D-tagatose catabolic pathway (Bli-TagP). For this purpose, a soluble protein fusion was obtained with the 6 His-tagged trigger factor (TF(His6)) of Escherichia coli. The active fusion enzyme was named TagK-TF(His6). Tag-1P and D-fructose-1-phosphate are substrates for the TagK-TF(His6) enzyme, whereas the isomeric derivatives D-tagatose-6-phosphate and D-fructose-6-phosphate are inhibitors. Studies of catalytic efficiency (kcat/Km) reveal that the enzyme specificity is markedly in favor of Tag-1P as the substrate. Importantly, we show in vivo that the transfer of the phosphate moiety from PEP to the B. licheniformis tagatose-specific Enzyme II in E. coli is inefficient. The capability of the PTS general cytoplasmic components of B. subtilis, HPr and Enzyme I to restore the phosphate transfer is demonstrated. © 2015 S. Karger AG, Basel.

  8. MicroRNA-Mediated Down-Regulation of Apoptosis Signal-Regulating Kinase 1 (ASK1 Attenuates the Apoptosis of Human Mesenchymal Stem Cells (MSCs Transplanted into Infarcted Heart

    Directory of Open Access Journals (Sweden)

    Chang Youn Lee

    2016-10-01

    Full Text Available Stem cell therapy using adult stem cells, such as mesenchymal stem cells (MSCs has produced some promising results in treating the damaged heart. However, the low survival rate of MSCs after transplantation is still one of the crucial factors that limit the therapeutic effect of stem cells. In the damaged heart, oxidative stress due to reactive oxygen species (ROS production can cause the death of transplanted MSCs. Apoptosis signal-regulating kinase 1 (ASK1 has been implicated in the development of oxidative stress-related pathologic conditions. Thus, we hypothesized that down-regulation of ASK1 in human MSCs (hMSCs might attenuate the post-transplantation death of MSCs. To test this hypothesis, we screened microRNAs (miRNAs based on a miRNA-target prediction database and empirical data and investigated the anti-apoptotic effect of selected miRNAs on human adipose-derived stem cells (hASCs and on rat myocardial infarction (MI models. Our data indicated that miRNA-301a most significantly suppressed ASK1 expression in hASCs. Apoptosis-related genes were significantly down-regulated in miRNA-301a-enriched hASCs exposed to hypoxic conditions. Taken together, these data show that miRNA-mediated down-regulation of ASK1 protects MSCs during post-transplantation, leading to an increase in the efficacy of MSC-based cell therapy.

  9. Nanoparticle Mediated Drug Delivery of Rolipram to Tyrosine Kinase B Positive Cells in the Inner Ear with Targeting Peptides and Agonistic Antibodies

    Directory of Open Access Journals (Sweden)

    Rudolf eGlueckert

    2015-05-01

    Full Text Available AimSystemic pharmacotherapies have limitation due to blood-labyrinth barrier, so local delivery via the round window membrane opens a path for effective treatment. Multifunctional nanoparticle (NP mediated cell specific drug delivery may enhance efficacy and reduce side effects. Different NPs with ligands to target TrkB receptor were tested. Distribution, uptake mechanisms, trafficking, and bioefficacy of drug release of rolipram loaded NPs were evaluated.Methods We tested lipid based nanocapsules (LNCs, Quantum Dot, silica NPs with surface modification by peptides mimicking TrkB or TrkB activating antibodies. Bioefficacy of drug release was tested with rolipram loaded LNCs to prevent cisplatin induced apoptosis. We established different cell culture models with SH-SY-5Y and inner ear derived cell lines and used neonatal and adult mouse explants. Uptake and trafficking was evaluated with FACS and confocal as well as transmission electron microscopy. ResultsPlain NPs show some selectivity in uptake related to the in-vitro system properties, carrier material and NP size. Some peptide ligands provide enhanced targeted uptake to neuronal cells but failed to show this in cell cultures. Agonistic antibodies linked to silica NPs showed TrkB activation and enhanced binding to inner ear derived cells. Rolipram loaded LNCs proved as effective carriers to prevent cisplatin induced apoptosis.DiscussionMost NPs with targeting ligands showed limited effects to enhance uptake. NP aggregation and unspecific binding may change uptake mechanisms and impair endocytosis by an overload of NPs. This may affect survival signaling. NPs with antibodies activate survival signaling and show effective binding to TrkB positive cells but needs further optimization for specific internalization. Bioefficiacy of rolipram release confirms LNCs as encouraging vectors for drug delivery of lipophilic agents to the inner ear with ideal release characteristics independent of

  10. Low Level Pro-inflammatory Cytokines Decrease Connexin36 Gap Junction Coupling in Mouse and Human Islets through Nitric Oxide-mediated Protein Kinase Cδ*

    Science.gov (United States)

    Farnsworth, Nikki L.; Walter, Rachelle L.; Hemmati, Alireza; Westacott, Matthew J.; Benninger, Richard K. P.

    2016-01-01

    Pro-inflammatory cytokines contribute to the decline in islet function during the development of diabetes. Cytokines can disrupt insulin secretion and calcium dynamics; however, the mechanisms underlying this are poorly understood. Connexin36 gap junctions coordinate glucose-induced calcium oscillations and pulsatile insulin secretion across the islet. Loss of gap junction coupling disrupts these dynamics, similar to that observed during the development of diabetes. This study investigates the mechanisms by which pro-inflammatory cytokines mediate gap junction coupling. Specifically, as cytokine-induced NO can activate PKCδ, we aimed to understand the role of PKCδ in modulating cytokine-induced changes in gap junction coupling. Isolated mouse and human islets were treated with varying levels of a cytokine mixture containing TNF-α, IL-1β, and IFN-γ. Islet dysfunction was measured by insulin secretion, calcium dynamics, and gap junction coupling. Modulators of PKCδ and NO were applied to determine their respective roles in modulating gap junction coupling. High levels of cytokines caused cell death and decreased insulin secretion. Low levels of cytokine treatment disrupted calcium dynamics and decreased gap junction coupling, in the absence of disruptions to insulin secretion. Decreases in gap junction coupling were dependent on NO-regulated PKCδ, and altered membrane organization of connexin36. This study defines several mechanisms underlying the disruption to gap junction coupling under conditions associated with the development of diabetes. These mechanisms will allow for greater understanding of islet dysfunction and suggest ways to ameliorate this dysfunction during the development of diabetes. PMID:26668311

  11. The action of nitric oxide to enhance cell survival in chick cardiomyocytes is mediated through a cGMP and ERK1/2 pathway while p38 mitogen-activated protein kinase-dependent pathways do not alter cell death.

    Science.gov (United States)

    Rabkin, Simon W; Tsang, Michael Y C

    2008-07-01

    The objective of this study was to determine whether the dual action of nitric oxide (NO) on cardiomyocyte cell viability is mediated through p38 mitogen-activated protein kinase (MAPK)-induced cell death and extracellular signal-regulated kinase (ERK1/2)-mediated cell survival pathways, and whether either of these is mediated through a cGMP-protein kinase G (PKG) pathway. Cell viability of embryonic chick cardiomyocytes was assessed by the MTT assay, which is based on the ability of viable cells to reduce 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide. The NO donor sodium nitroprusside (SNP) produced a significant (P death. Sodium nitroprusside induced ERK1/2 phosphorylation, and the mitogen-activated protein kinase (MEK1/2) inhibitor PD 98059 significantly increased cell death. In contrast, SB202190, a relatively selective inhibitor of p38 MAPK, did not affect SNP-induced cell death. The cardioprotective effect of NO was prbably mediated in part via cGMP because 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, a selective inhibitor of NO-sensitive guanylyl cyclase, produced a significant enhancement of SNP-induced cell death. In contrast, the PKG inhibitor KT5823 did not affect cell viability. In summary, these data suggest that NO, via stimulation of soluble guanylyl cyclase, activates MEK1/2 whose product, ERK1/2, protects against cell death. In contrast, SNP-induced p38 MAPK activation does not modulate NO-induced cardiomyocyte cell death. Not all cGMP targets affect NO-induced cell death, since the PKG pathway does not enhance or suppress NO-induced cardiomyocyte cell death. Enhancement of the ERK1/2 responses to NO may permit the beneficial effects of NO to predominate.

  12. Activation of G protein-coupled estrogen receptor 1 induces coronary artery relaxation via Epac/Rap1-mediated inhibition of RhoA/Rho kinase pathway in parallel with PKA.

    Directory of Open Access Journals (Sweden)

    Xuan Yu

    Full Text Available Previously, we reported that cAMP/PKA signaling is involved in GPER-mediated coronary relaxation by activating MLCP via inhibition of RhoA pathway. In the current study, we tested the hypothesis that activation of GPER induces coronary artery relaxation via inhibition of RhoA/Rho kinase pathway by cAMP downstream targets, exchange proteins directly activated by cAMP (Epac as well as PKA. Our results show that Epac inhibitors, brefeldin A (BFA, 50 μM, or ESI-09 (20 μM, or CE3F4 (100 μM, all partially inhibited porcine coronary artery relaxation response to the selective GPER agonist, G-1 (0.3-3 μM; while concurrent administration of BFA and PKI (5 μM, a PKA inhibitor, almost completely blocked the relaxation effect of G-1. The Epac specific agonist, 8-CPT-2Me-cAMP (007, 1-100 μM, induced a concentration-dependent relaxation response. Furthermore, the activity of Ras-related protein 1 (Rap1 was up regulated by G-1 (1 μM treatment of porcine coronary artery smooth muscle cells (CASMCs. Phosphorylation of vasodilator-stimulated phosphoprotein (p-VASP was elevated by G-1 (1 μM treatment, but not by 007 (50 μM; and the effect of G-1 on p-VASP was blocked by PKI, but not by ESI-09, an Epac antagonist. RhoA activity was similarly down regulated by G-1 and 007, whereas ESI-09 restored most of the reduced RhoA activity by G-1 treatment. Furthermore, G-1 decreased PGF2α-induced p-MYPT1, which was partially reversed with either ESI-09 or PKI; whereas, concurrent administration of ESI-09 and PKI totally prevented the inhibitory effect of G-1. The inhibitory effects of G-1 on p- MLC levels in CASMCs were mostly restored by either ESI-09 or PKI. These results demonstrate that activation of GPER induces coronary artery relaxation via concurrent inhibition of RhoA/Rho kinase by Epac/Rap1 and PKA. GPER could be a potential drug target for preventing and treating cardiovascular diseases.

  13. Gi-mediated activation of the Ras/MAP kinase pathway involves a 100 kDa tyrosine-phosphorylated Grb2 SH3 binding protein, but not Src nor Shc

    NARCIS (Netherlands)

    Kranenburg, O.; Verlaan, I.; Hordijk, P. L.; Moolenaar, W. H.

    1997-01-01

    Mitogenic G protein-coupled receptors, such as those for lysophosphatidic acid (LPA) and thrombin, activate the Ras/MAP kinase pathway via pertussis toxin (PTX)-sensitive Gi, tyrosine kinase activity and recruitment of Grb2, which targets guanine nucleotide exchange activity to Ras. Little is known

  14. The Green Tea Component (-)-Epigallocatechin-3-Gallate Sensitizes Primary Endothelial Cells to Arsenite-Induced Apoptosis by Decreasing c-Jun N-Terminal Kinase-Mediated Catalase Activity.

    Science.gov (United States)

    Kim, Jee-Youn; Choi, Ji-Young; Lee, Hyeon-Ju; Byun, Catherine Jeonghae; Park, Jung-Hyun; Park, Jae Hoon; Cho, Ho-Seong; Cho, Sung-Jin; Jo, Sangmee Ahn; Jo, Inho

    2015-01-01

    The green tea component (-)-epigallocatechin-3-gallate (EGCG) has been shown to sensitize many different types of cancer cells to anticancer drug-induced apoptosis, although it protects against non-cancerous primary cells against toxicity from certain conditions such as exposure to arsenic (As) or ultraviolet irradiation. Here, we found that EGCG promotes As-induced toxicity of primary-cultured bovine aortic endothelial cells (BAEC) at doses in which treatment with each chemical alone had no such effect. Increased cell toxicity was accompanied by an increased condensed chromatin pattern and fragmented nuclei, cleaved poly(ADP-ribose) polymerase (PARP), activity of the pro-apoptotic enzymes caspases 3, 8 and 9, and Bax translocation into mitochondria, suggesting the involvement of an apoptotic signaling pathway. Fluorescence activated cell sorting analysis revealed that compared with EGCG or As alone, combined EGCG and As (EGCG/As) treatment significantly induced production of reactive oxygen species (ROS), which was accompanied by decreased catalase activity and increased lipid peroxidation. Pretreatment with N-acetyl-L-cysteine or catalase reversed EGCG/As-induced caspase activation and EC toxicity. EGCG/As also increased the phosphorylation of c-Jun N-terminal kinase (JNK), which was not reversed by catalase. However, pretreatment with the JNK inhibitor SP600125 reversed all of the observed effects of EGCG/As, suggesting that JNK may be the most upstream protein examined in this study. Finally, we also found that all the observed effects by EGCG/As are true for other types of EC tested. In conclusion, this is firstly to show that EGCG sensitizes non-cancerous EC to As-induced toxicity through ROS-mediated apoptosis, which was attributed at least in part to a JNK-activated decrease in catalase activity.

  15. The Green Tea Component (--Epigallocatechin-3-Gallate Sensitizes Primary Endothelial Cells to Arsenite-Induced Apoptosis by Decreasing c-Jun N-Terminal Kinase-Mediated Catalase Activity.

    Directory of Open Access Journals (Sweden)

    Jee-Youn Kim

    Full Text Available The green tea component (--epigallocatechin-3-gallate (EGCG has been shown to sensitize many different types of cancer cells to anticancer drug-induced apoptosis, although it protects against non-cancerous primary cells against toxicity from certain conditions such as exposure to arsenic (As or ultraviolet irradiation. Here, we found that EGCG promotes As-induced toxicity of primary-cultured bovine aortic endothelial cells (BAEC at doses in which treatment with each chemical alone had no such effect. Increased cell toxicity was accompanied by an increased condensed chromatin pattern and fragmented nuclei, cleaved poly(ADP-ribose polymerase (PARP, activity of the pro-apoptotic enzymes caspases 3, 8 and 9, and Bax translocation into mitochondria, suggesting the involvement of an apoptotic signaling pathway. Fluorescence activated cell sorting analysis revealed that compared with EGCG or As alone, combined EGCG and As (EGCG/As treatment significantly induced production of reactive oxygen species (ROS, which was accompanied by decreased catalase activity and increased lipid peroxidation. Pretreatment with N-acetyl-L-cysteine or catalase reversed EGCG/As-induced caspase activation and EC toxicity. EGCG/As also increased the phosphorylation of c-Jun N-terminal kinase (JNK, which was not reversed by catalase. However, pretreatment with the JNK inhibitor SP600125 reversed all of the observed effects of EGCG/As, suggesting that JNK may be the most upstream protein examined in this study. Finally, we also found that all the observed effects by EGCG/As are true for other types of EC tested. In conclusion, this is firstly to show that EGCG sensitizes non-cancerous EC to As-induced toxicity through ROS-mediated apoptosis, which was attributed at least in part to a JNK-activated decrease in catalase activity.

  16. Remote ischemic postconditioning protects against renal ischemia/reperfusion injury by activation of T-LAK-cell-originated protein kinase (TOPK)/PTEN/Akt signaling pathway mediated anti-oxidation and anti-inflammation.

    Science.gov (United States)

    Gao, Sumin; Zhu, Yi; Li, Haobo; Xia, Zhengyuan; Wu, Qingping; Yao, Shanglong; Wang, Tingting; Yuan, Shiying

    2016-09-01

    Recent clinical and animal studies suggested that remote limb ischemic postconditioning (RIPostC) can invoke potent cardioprotection or neuroprotection. However, the effect and mechanism of RIPostC against renal ischemia/reperfusion injury (IRI) are poorly understood. T-LAK-cell-originated protein kinase (TOPK) is crucial for the proliferation and migration of tumor cells. However, the function of TOPK and the molecular mechanism underlying renal protection remain unknown. Therefore, this study aimed to evaluate the role of TOPK in renoprotection induced by RIPostC. The renal IRI model was induced by left renal pedicle clamping for 45min followed by 24h reperfusion and right nephrectomy. All mice were intraperitoneally injected with vehicle, TOPK inhibitor HI-TOPK-032 or Akt inhibitor LY294002. After 24h reperfusion, renal histology, function, and inflammatory cytokines and oxidative stress were assessed. The proteins were measured by Western blotting. The results showed that RIPostC significantly protected the kidneys against IRI. The protective effects were accompanied by the attenuation of renal dysfunction, tubular damage, inflammation and oxidative stress. In addition, RIPostC increased the phosphorylation of TOPK, PTEN, Akt, GSK3β and the nuclear translocation of Nrf2 and decreased the nuclear translocation of NF-κB. However, all of the above renoprotective effects of RIPostC were eliminated either by the inhibition of TOPK or Akt with HI-TOPK-032 or LY294002. The current data reveal that RIPostC protects against renal IRI via activation of TOPK/PTEN/Akt signaling pathway mediated anti-oxidation and anti-inflammation. Copyright © 2016 Elsevier B.V. All rights reserved.

  17. Cholera toxin enhances interleukin-17A production in both CD4+ and CD8+ cells via a cAMP/protein kinase A-mediated interleukin-17A promoter activation.

    Science.gov (United States)

    Tsai, Hsing-Chuan; Velichko, Sharlene; Lee, Shanshan; Wu, Reen

    2018-01-27

    Cholera toxin (CT) is a bacterial component that increases intracellular cAMP levels in host cells and suppresses T-cell activation. Recently, CT was reported to induce T helper type 17-skewing dendritic cells and activate interleukin-17A (IL-17A) production in CD4 + T cells through a cAMP-dependent pathway. However, the underlying mechanism by which cAMP regulates IL-17A production in T cells is not completely defined. In this study, we took advantage of a small molecule protein kinase A (PKA) inhibitor (H89) and different cAMP analogues: a PKA-specific activator (N6-benzoyl-adenosine-cAMP), an exchange protein activated by cAMP-specific activator (Rp-8-chlorophenylthio-2'-O-methyl cAMP), and a PKA inhibitor (Rp-8-bromo-cAMP), to elucidate the signalling cascade of cAMP in IL-17A regulation in T cells. We found that CT induced IL-17A production and IL-17A promoter activity in activated CD4 + T cells through a cAMP/PKA pathway. Moreover, this regulation was via cAMP-response element binding protein (CREB) -mediated transcriptional activation by using the transfection of an IL-17A promoter-luciferase reporter construct and CREB small interfering RNA in Jurkat cells. Also, we showed that CREB bound to the CRE motif located at -183 of the IL-17A promoter in vitro. Most interestingly, not only in CD4 + T cells, CT also enhanced cAMP/PKA-dependent IL-17A production and CREB phosphorylation in CD8 + T cells. In conclusion, our data suggest that CT induces an IL-17A-dominated immune microenvironment through the cAMP/PKA/CREB signalling pathway. Our study also highlights the potentials of CT and cAMP in modulating T helper type 17 responses in vivo. © 2018 John Wiley & Sons Ltd.

  18. Outer membrane protein A (OmpA of Shigella flexneri 2a induces TLR2-mediated activation of B cells: involvement of protein tyrosine kinase, ERK and NF-κB.

    Directory of Open Access Journals (Sweden)

    Rajsekhar Bhowmick

    Full Text Available B cells are critically important in combating bacterial infections and their differentiation into plasma cells and memory cells aids bacterial clearance and long-lasting immunity conferred by essentially all vaccines. Outer membrane protein A (OmpA of Shigella flexneri 2a has been demonstrated to induce the production of IgG and IgA in vivo following immunization of mice through intranasal route, but the direct involvement of B cells in OmpA-mediated immune regulation was not determined. Consequently, we investigated whether OmpA can modulate B cell functions and identified the molecular events involved in OmpA-induced B cell immune response in vitro. We show that OmpA of S. flexneri 2a activates B cells to produce protective cytokines, IL-6 and IL-10 as well as facilitates their differentiation into antibody secreting cells (ASCs. The immunostimulatory properties of OmpA are attributed to the increased surface expression of MHCII and CD86 on B cells. We also report here that B cell activation by OmpA is mediated strictly through recognition by TLR2, resulting in initiation of cascades of signal transduction events, involving increased phosphorylation of protein tyrosine kinases (PTKs, ERK and IκBα, leading to nuclear translocation of NF-κB. Importantly, a TLR2 antibody diminishes OmpA-induced upregulation of MHCII and CD86 on B cell surface as well as significantly inhibits B cell differentiation and cytokine secretion. Furthermore, we illustrate that B cell differentiation into ASCs and induction of cytokine secretion by OmpA are dependent on PTKs activity. Moreover, we identify that OmpA-induced B cell differentiation is entirely dependent on ERK pathway, whereas both NF-κB and ERK are essential for cytokine secretion by B cells. Overall, our data demonstrate that OmpA of S. flexneri 2a amplifies TLR signaling in B cells and triggers B cell immune response, which is critical for the development of an effective adaptive immunity to an

  19. Cynatratoside-C from Cynanchum atratum displays anti-inflammatory effect via suppressing TLR4 mediated NF-κB and MAPK signaling pathways in LPS-induced mastitis in mice.

    Science.gov (United States)

    Hu, Ge; Hong, Dong; Zhang, Tao; Duan, Huiqin; Wei, Panying; Guo, Xinxin; Mu, Xiang

    2018-01-05

    The present study was conducted to isolate anti-inflammatory compound from Cynanchum atratum and investigate the molecular mechanisms of active compound against lipopolysaccharide (LPS)-induced mastitis in mice. Bioassay-guided fractionations and isolation (via ex vivo tests) of compounds with anti-inflammatory activity were performed on roots of C. atratum yielding a pure bioactive compound: Cynatratoside-C, identified by comparing spectral data (EI-MS, 1 H NMR and 13 C NMR) with literature values. Ex vivo tests showed that Cynatratoside-C inhibited the expression of TLR4 and pro-inflammatory cytokine (TNF-α, IL-6 and IL-1β) production in LPS-stimulated primary mouse mammary epithelial cells. In vivo results indicated that Cynatratoside-C markedly attenuated LPS-induced mammary histopathologic changes and mammary oxidative stress (MDA, SOD, GPx) activity. Besides, Cynatratoside-C blocked the expression of Toll-like receptor 4 (TLR4) and then suppressed the phosphorylation of nuclear transcription factor-kappa B (NF-κB) p65 and degradation inhibitor of NF-κBα (IκBα). Further study showed that Cynatratoside-C could suppress the phosphorylation of p38, extracellular signal-regulated kinase (ERK) and c-jun NH2-terminal kinase (JNK) in mitogen-activated protein kinase (MAPK) signal pathway. In conclusion, our results suggest that Cynatratoside-C played an anti-inflammatory role in LPS-induced mastitis by regulating TLR4 and the NF-κB and MAPK signaling pathways in mammary gland tissues. Cynatratoside-C may be a promising potential therapeutic reagent for the treatment of mastitis. Copyright © 2017. Published by Elsevier B.V.

  20. Gene regulation by MAP kinase cascades

    DEFF Research Database (Denmark)

    Fiil, Berthe Katrine; Petersen, Klaus; Petersen, Morten

    2009-01-01

    Mitogen-activated protein kinase (MAPK) cascades are signaling modules that transduce extracellular stimuli to a range of cellular responses. Research in yeast and metazoans has shown that MAPK-mediated phosphorylation directly or indirectly regulates the activity of transcription factors. Plant ...

  1. Mitogen-activated protein kinases mediate Mycobacterium ...

    Indian Academy of Sciences (India)

    2012-01-19

    Jan 19, 2012 ... heat shock, UV irradiation and also to inflammatory cytokines. ERK is mainly activated by growth factors and phorbol esters. (Lewis et al. 1998; Cowan and Storey 2003). The activation of some MAPK family members by. M. tuberculosis H37Rv in human monocytes has already been reported. Song et al.

  2. Mitogen-activated protein kinases mediate Mycobacterium ...

    Indian Academy of Sciences (India)

    2012-01-19

    Jan 19, 2012 ... to the site of infection (Leemans et al. 2003). Numerous other studies based on stimulation with several mycobacterial antigens (Ags) (Waters et al. 2003, 2009; Li et al. 2008;. Giri et al. 2009) also support the role of CD44 in protective immunity against tuberculosis. However, the signalling path- ways that ...

  3. Celastrol attenuates mitochondrial dysfunction and inflammation in palmitate-mediated insulin resistance in C3A hepatocytes.

    Science.gov (United States)

    Abu Bakar, Mohamad Hafizi; Sarmidi, Mohamad Roji; Tan, Joo Shun; Mohamad Rosdi, Mohamad Norisham

    2017-03-15

    Accumulating evidence indicates that mitochondrial dysfunction-induced inflammation is among the convergence points for the greatest hallmarks of hepatic insulin resistance. Celastrol, an anti-inflammatory compound from the root of Tripterygium Wilfordii has been reported to mitigate insulin resistance and inflammation in animal disease models. Nevertheless, the specific mechanistic actions of celastrol in modulating such improvements at the cellular level remain obscure. The present study sought to explore the mechanistic roles of celastrol upon insulin resistance induced by palmitate in C3A human hepatocytes. The hepatocytes exposed to palmitate (0.75mM) for 48h exhibited reduced both basal and insulin-stimulated glucose uptake, mitochondrial dysfunction, leading to increased mitochondrial oxidative stress with diminished fatty acid oxidation. Elevated expressions of nuclear factor-kappa B p65 (NF-κB p65), c-Jun NH(2)-terminal kinase (JNK) signaling pathways and the amplified release of pro-inflammatory cytokines including IL-8, IL-6, TNF-α and CRP were observed following palmitate treatment. Consistently, palmitate reduced and augmented phosphorylated Tyrosine-612 and Serine-307 of insulin receptor substrate-1 (IRS-1) proteins, respectively in hepatocytes. However, celastrol at the optimum concentration of 30nM was able to reverse these deleterious occasions and protected the cells from mitochondrial dysfunction and insulin resistance. Importantly, we presented evidence for the first time that celastrol efficiently prevented palmitate-induced insulin resistance in hepatocytes at least, via improved mitochondrial functions and insulin signaling pathways. In summary, the present investigation underlines a conceivable mechanism to elucidate the cytoprotective potential of celastrol in attenuating mitochondrial dysfunction and inflammation against the development of hepatic insulin resistance. Copyright © 2017 Elsevier B.V. All rights reserved.

  4. The PIM kinases in hematological cancers.

    Science.gov (United States)

    Alvarado, Yesid; Giles, Francis J; Swords, Ronan T

    2012-02-01

    The PIM genes represent a family of proto-oncogenes that encode three different serine/threonine protein kinases (PIM1, PIM2 and PIM3) with essential roles in the regulation of signal transduction cascades, which promote cell survival, proliferation and drug resistance. PIM kinases are overexpressed in several hematopoietic tumors and support in vitro and in vivo malignant cell growth and survival, through cell cycle regulation and inhibition of apoptosis. PIM kinases do not have an identified regulatory domain, which means that these proteins are constitutively active once transcribed. They appear to be critical downstream effectors of important oncoproteins and, when overexpressed, can mediate drug resistance to available agents, such as rapamycin. Recent crystallography studies reveal that, unlike other kinases, they possess a hinge region, which creates a unique binding pocket for ATP, offering a target for an increasing number of potent small-molecule PIM kinase inhibitors. Preclinical studies in models of various hematologic cancers indicate that these novel agents show promising activity and some of them are currently being evaluated in a clinical setting. In this review, we profile the PIM kinases as targets for therapeutics in hematologic malignancies.

  5. JNK Promotes Epithelial Cell Anoikis by Transcriptional and Post-translational Regulation of BH3-Only Proteins

    Directory of Open Access Journals (Sweden)

    Nomeda Girnius

    2017-11-01

    Full Text Available Summary: Developmental morphogenesis, tissue injury, and oncogenic transformation can cause the detachment of epithelial cells. These cells are eliminated by a specialized form of apoptosis (anoikis. While the processes that contribute to this form of cell death have been studied, the underlying mechanisms remain unclear. Here, we tested the role of the cJUN NH2-terminal kinase (JNK signaling pathway using murine models with compound JNK deficiency in mammary and kidney epithelial cells. These studies demonstrated that JNK is required for efficient anoikis in vitro and in vivo. Moreover, JNK-promoted anoikis required pro-apoptotic members of the BCL2 family of proteins. We show that JNK acts through a BAK/BAX-dependent apoptotic pathway by increasing BIM expression and phosphorylating BMF, leading to death of detached epithelial cells. : Developmental morphogenesis, tissue injury, and oncogenic transformation can cause epithelial cell detachment. These cells are eliminated by a specialized form of apoptosis termed anoikis. Girnius and Davis show that anoikis is mediated by the cJUN NH2-terminal kinase (JNK, which increases BIM expression and phosphorylates BMF to engage BAK/BAX-dependent apoptosis. Keywords: apoptosis, anoikis, epithelial cell, mammary gland, JNK, BAX, BAK, BH3-only protein, BIM, BMF

  6. Structural Bioinformatics and Protein Docking Analysis of the Molecular Chaperone-Kinase Interactions: Towards Allosteric Inhibition of Protein Kinases by Targeting the Hsp90-Cdc37 Chaperone Machinery

    Directory of Open Access Journals (Sweden)

    Gennady Verkhivker

    2013-11-01

    Full Text Available A fundamental role of the Hsp90-Cdc37 chaperone system in mediating maturation of protein kinase clients and supporting kinase functional activity is essential for the integrity and viability of signaling pathways involved in cell cycle control and organism development. Despite significant advances in understanding structure and function of molecular chaperones, the molecular mechanisms and guiding principles of kinase recruitment to the chaperone system are lacking quantitative characterization. Structural and thermodynamic characterization of Hsp90-Cdc37 binding with protein kinase clients by modern experimental techniques is highly challenging, owing to a transient nature of chaperone-mediated interactions. In this work, we used experimentally-guided protein docking to probe the allosteric nature of the Hsp90-Cdc37 binding with the cyclin-dependent kinase 4 (Cdk4 kinase clients. The results of docking simulations suggest that the kinase recognition and recruitment to the chaperone system may be primarily determined by Cdc37 targeting of the N-terminal kinase lobe. The interactions of Hsp90 with the C-terminal kinase lobe may provide additional “molecular brakes” that can lock (or unlock kinase from the system during client loading (release stages. The results of this study support a central role of the Cdc37 chaperone in recognition and recruitment of the kinase clients. Structural analysis may have useful implications in developing strategies for allosteric inhibition of protein kinases by targeting the Hsp90-Cdc37 chaperone machinery.

  7. Muscle phosphorylase kinase deficiency

    DEFF Research Database (Denmark)

    Preisler, N; Orngreen, M C; Echaniz-Laguna, A

    2012-01-01

    To examine metabolism during exercise in 2 patients with muscle phosphorylase kinase (PHK) deficiency and to further define the phenotype of this rare glycogen storage disease (GSD).......To examine metabolism during exercise in 2 patients with muscle phosphorylase kinase (PHK) deficiency and to further define the phenotype of this rare glycogen storage disease (GSD)....

  8. Pro-apoptotic and pro-autophagic effects of the Aurora kinase A inhibitor alisertib (MLN8237) on human osteosarcoma U-2 OS and MG-63 cells through the activation of mitochondria-mediated pathway and inhibition of p38 MAPK/PI3K/Akt/mTOR signaling pathway.

    Science.gov (United States)

    Niu, Ning-Kui; Wang, Zi-Li; Pan, Shu-Ting; Ding, Hui-Qiang; Au, Giang H T; He, Zhi-Xu; Zhou, Zhi-Wei; Xiao, Guozhi; Yang, Yin-Xue; Zhang, Xueji; Yang, Tianxin; Chen, Xiao-Wu; Qiu, Jia-Xuan; Zhou, Shu-Feng

    2015-01-01

    Osteosarcoma (OS) is the most common malignant bone tumor occurring mostly in children and adolescents between 10 and 20 years of age with poor response to current therapeutics. Alisertib (ALS, MLN8237) is a selective Aurora kinase A inhibitor that displays anticancer effects on several types of cancer. However, the role of ALS in the treatment of OS remains unknown. This study aimed to investigate the effects of ALS on the cell growth, apoptosis, autophagy, and epithelial to mesenchymal transition (EMT) and the underlying mechanisms in two human OS cell lines U-2 OS and MG-63. The results showed that ALS had potent growth inhibitory, pro-apoptotic, pro-autophagic, and EMT inhibitory effects on U-2 OS and MG-63 cells. ALS remarkably induced G2/M arrest and down-regulated the expression levels of cyclin-dependent kinases 1 and 2 and cyclin B1 in both U-2 OS and MG-63 cells. ALS markedly induced mitochondria-mediated apoptosis with a significant increase in the expression of key pro-apoptotic proteins and a decrease in main anti-apoptotic proteins. Furthermore, ALS promoted autophagic cell death via the inhibition of phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) and p38 mitogen-activated protein kinase (p38 MAPK) signaling pathways, and activation of 5'-AMP-dependent kinase (AMPK) signaling pathway. Inducers or inhibitors of apoptosis or autophagy simultaneously altered ALS-induced apoptotic and autophagic death in both U-2 OS and MG-63 cells, suggesting a crosstalk between these two primary modes of programmed cell death. Moreover, ALS suppressed EMT-like phenotypes with a marked increase in the expression of E-cadherin but a decrease in N-cadherin in U-2 OS and MG-63 cells. ALS treatment also induced reactive oxygen species (ROS) generation but inhibited the expression levels of sirtuin 1 and nuclear factor-erythroid-2-related factor 2 (Nrf2) in both cell lines. Taken together, these findings show that ALS promotes

  9. Structural analysis of phosphatidylinositol 4-kinase III beta (PI4KB) - 14-3-3 protein complex reveals internal flexibility and explains 14-3-3 mediated protection from degradation in vitro

    Czech Academy of Sciences Publication Activity Database

    Chalupská, Dominika; Eisenreichová, Andrea; Rozycki, B.; Řežábková, L.; Humpolíčková, Jana; Klíma, Martin; Bouřa, Evžen

    2017-01-01

    Roč. 200, č. 1 (2017), s. 36-44 ISSN 1047-8477 R&D Projects: GA ČR(CZ) GA17-05200S Institutional support: RVO:61388963 Keywords : lipid * kinase * PI4KB * 14-3-3 protein * phosphatidylinositol Subject RIV: EB - Genetics ; Molecular Biology OBOR OECD: Biochemistry and molecular biology Impact factor: 2.767, year: 2016

  10. A Mitogen-activated protein kinase kinase kinase mediates reactive oxygen species homeostasis in Arabidopsis

    Czech Academy of Sciences Publication Activity Database

    Nakagami, H.; Soukupová, Hana; Schikora, A.; Žárský, Viktor; Hirt, H.

    2006-01-01

    Roč. 281, č. 50 (2006), s. 38697-38704 ISSN 0021-9258 Grant - others:Marie Curie Training program(XE) EU-HPRN-CT-2002-00265 Institutional research plan: CEZ:AV0Z50380511 Keywords : OXIDATIVE STRESS * MAP * PATHWAYS Subject RIV: EF - Botanics Impact factor: 5.808, year: 2006

  11. Mitogen-activated protein kinase signaling in plants

    DEFF Research Database (Denmark)

    Rodriguez, Maria Cristina Suarez; Petersen, Morten; Mundy, John

    2010-01-01

    Eukaryotic mitogen-activated protein kinase (MAPK) cascades have evolved to transduce environmental and developmental signals into adaptive and programmed responses. MAPK cascades relay and amplify signals via three types of reversibly phosphorylated kinases leading to the phosphorylation of subs...... the Arabidopsis thaliana MAPKs MPK3, 4, and 6 and MAP2Ks MKK1, 2, 4, and 5. Future work needs to focus on identifying substrates of MAPKs, and on understanding how specificity is achieved among MAPK signaling pathways.......Eukaryotic mitogen-activated protein kinase (MAPK) cascades have evolved to transduce environmental and developmental signals into adaptive and programmed responses. MAPK cascades relay and amplify signals via three types of reversibly phosphorylated kinases leading to the phosphorylation...... of substrate proteins, whose altered activities mediate a wide array of responses, including changes in gene expression. Cascades may share kinase components, but their signaling specificity is maintained by spaciotemporal constraints and dynamic protein-protein interactions and by mechanisms that include...

  12. Protein kinase C signaling and cell cycle regulation

    OpenAIRE

    Black, Adrian R.; Black, Jennifer D.

    2013-01-01

    A link between T cell proliferation and the protein kinase C (PKC) family of serine/threonine kinases has been recognized for about thirty years. However, despite the wealth of information on PKC-mediated control of T cell activation, understanding of the effects of PKCs on the cell cycle machinery in this cell type remains limited. Studies in other systems have revealed important cell cycle-specific effects of PKC signaling that can either positively or negatively impact proliferation. Th...

  13. HSC-specific inhibition of Rho-kinase reduces portal pressure in cirrhotic rats without major systemic effects

    NARCIS (Netherlands)

    Klein, Sabine; Van Beuge, Marike Marjolijn; Granzow, Michaela; Beljaars, Leonie; Schierwagen, Robert; Kilic, Sibel; Heidari, Iren; Huss, Sebastian; Sauerbruch, Tilman; Poelstra, Klaas; Trebicka, Jonel

    2012-01-01

    Background & Aims: Rho-kinase activation mediates cell contraction and increases intrahepatic resistance and consequently portal pressure in liver cirrhosis. Systemic Rho-kinase inhibition decreases portal pressure in cirrhosis, but also arterial pressure. Thus, liver-specific Rho-kinase inhibition

  14. Diacylglycerol kinase ζ regulates RhoA activation via a kinase-independent scaffolding mechanism

    DEFF Research Database (Denmark)

    Ard, Ryan; Mulatz, Kirk; Abramovici, Hanan

    2012-01-01

    , but the underlying mechanisms are unclear. Diacylglycerol kinase ζ (DGKζ), which phosphorylates diacylglycerol to yield phosphatidic acid, selectively dissociates Rac1 by stimulating PAK1-mediated phosphorylation of RhoGDI on Ser-101/174. Similarly, phosphorylation of RhoGDI on Ser-34 by protein kinase Cα (PKCα...... DGKζ functions as a scaffold to assemble a signaling complex that functions as a RhoA-selective, GDI dissociation factor. As a regulator of Rac1 and RhoA activity, DGKζ is a critical factor linking changes in lipid signaling to actin reorganization....

  15. Pyruvate kinase blood test

    Science.gov (United States)

    ... medlineplus.gov/ency/article/003357.htm Pyruvate kinase blood test To use the sharing features on this page, ... energy when oxygen levels are low. How the Test is Performed A blood sample is needed. In the laboratory, white blood ...

  16. RhoA/Rho kinase in spinal cord injury

    Directory of Open Access Journals (Sweden)

    Xiangbing Wu

    2016-01-01

    Full Text Available A spinal cord injury refers to an injury to the spinal cord that is caused by a trauma instead of diseases. Spinal cord injury includes a primary mechanical injury and a much more complex secondary injury process involving inflammation, oxidation, excitotoxicity, and cell death. During the secondary injury, many signal pathways are activated and play important roles in mediating the pathogenesis of spinal cord injury. Among them, the RhoA/Rho kinase pathway plays a particular role in mediating spinal degeneration and regeneration. In this review, we will discuss the role and mechanism of RhoA/Rho kinase-mediated spinal cord pathogenesis, as well as the potential of targeting RhoA/Rho kinase as a strategy for promoting both neuroprotection and axonal regeneration.

  17. Phosphorylation of the regulatory beta-subunit of protein kinase CK2 by checkpoint kinase Chk1: identification of the in vitro CK2beta phosphorylation site

    DEFF Research Database (Denmark)

    Kristensen, Lars P; Larsen, Martin Røssel; Højrup, Peter

    2004-01-01

    The regulatory beta-subunit of protein kinase CK2 mediates the formation of the CK2 tetrameric form and it has functions independent of CK2 catalytic subunit through interaction with several intracellular proteins. Recently, we have shown that CK2beta associates with the human checkpoint kinase Chk...

  18. Molecular Mechanisms Behind the Chemopreventive Effects of Anthocyanidins

    Directory of Open Access Journals (Sweden)

    De-Xing Hou

    2004-01-01

    Full Text Available Anthocyanins are polyphenolic ring-based flavonoids, and are widespread in fruits and vegetables of red-blue color. Epidemiological investigations and animal experiments have indicated that anthocyanins may contribute to cancer chemoprevention. The studies on the mechanism have been done recently at molecular level. This review summarizes current molecular bases for anthocyanidins on several key steps involved in cancer chemoprevention: (i inhibition of anthocyanidins in cell transformation through targeting mitogen-activated protein kinase (MAPK pathway and activator protein 1 (AP-1 factor; (ii suppression of anthocyanidins in inflammation and carcinogenesis through targeting nuclear factor kappa B (NF-κB pathway and cyclooxygenase 2 (COX-2 gene; (iii apoptotic induction of cancer cells by anthocyanidins through reactive oxygen species (ROS / c-Jun NH2-terminal kinase (JNK-mediated caspase activation. These data provide a first molecular view of anthocyanidins contributing to cancer chemoprevention.

  19. Regulation of N-Formyl Peptide Receptor Signaling and Trafficking by Arrestin-Src Kinase Interaction.

    Directory of Open Access Journals (Sweden)

    Brant M Wagener

    Full Text Available Arrestins were originally described as proteins recruited to ligand-activated, phosphorylated G protein-coupled receptors (GPCRs to attenuate G protein-mediated signaling. It was later revealed that arrestins also mediate GPCR internalization and recruit a number of signaling proteins including, but not limited to, Src family kinases, ERK1/2, and JNK3. GPCR-arrestin binding and trafficking control the spatial and temporal activity of these multi-protein complexes. In previous reports, we concluded that N-formyl peptide receptor (FPR-mediated apoptosis, which occurs upon receptor stimulation in the absence of arrestins, is associated with FPR accumulation in perinuclear recycling endosomes. Under these conditions, inhibition of Src kinase and ERK1/2 prevented FPR-mediated apoptosis. To better understand the role of Src kinase in this process, in the current study we employed a previously described arrestin-2 (arr2 mutant deficient in Src kinase binding (arr2-P91G/P121E. Unlike wild type arrestin, arr2-P91G/P121E did not inhibit FPR-mediated apoptosis, suggesting that Src binding to arrestin-2 prevents apoptotic signaling. However, in cells expressing this mutant, FPR-mediated apoptosis was still blocked by inhibition of Src kinase activity, suggesting that activation of Src independent of arrestin-2 binding is involved in FPR-mediated apoptosis. Finally, while Src kinase inhibition prevented FPR-mediated-apoptosis in the presence of arr2-P91G/P121E, it did not prevent FPR-arr2-P91G/P121E accumulation in the perinuclear recycling endosome. On the contrary, inhibition of Src kinase activity mediated the accumulation of activated FPR-wild type arrestin-2 in recycling endosomes without initiating FPR-mediated apoptosis. Based on these observations, we conclude that Src kinase has two independent roles following FPR activation that regulate both FPR-arrestin-2 signaling and trafficking.

  20. Inhibition of estrogen receptor β-mediated human telomerase reverse transcriptase gene transcription via the suppression of mitogen-activated protein kinase signaling plays an important role in 15-deoxy-Δ12,14-prostaglandin J2-induced apoptosis in cancer cells

    International Nuclear Information System (INIS)

    Kondoh, Kei; Tsuji, Naoki; Asanuma, Koichi; Kobayashi, Daisuke; Watanabe, Naoki

    2007-01-01

    The nuclear hormone receptor peroxisome proliferator-activated receptor (PPAR)-γ plays a role in cancer development in addition to its role in glucose metabolism. The natural ligand of PPAR-γ, namely, 15-deoxy-Δ 12,14 -prostaglandin J 2 (15d-PGJ 2 ), has been shown to possess antineoplastic activity in cancer cells. However, the mechanism underlying its antineoplastic activity remains to be elucidated. Inhibition of the expression of human telomerase reverse transcriptase (hTERT), a major determinant of telomerase activity, reportedly induces rapid apoptosis in cancer cells. In this study, we investigated the effect of 15d-PGJ 2 on hTERT expression. We found that 15d-PGJ 2 induced apoptosis in the MIAPaCa-2 pancreatic cancer cells and dose-dependently decreased hTERT mRNA and protein expression. Down-regulation of hTERT expression by hTERT-specific small inhibitory RNA also induced apoptosis. Furthermore, 15d-PGJ 2 attenuated the DNA binding of estrogen receptor (ER). MIAPaCa-2 expressed only ERβ, and although its expression did not decrease due to 15d-PGJ 2 , its phosphorylation was suppressed. Additionally, a mitogen-activated protein kinase (MAPK) kinase inhibitor decreased ERβ phosphorylation, and 15d-PGJ 2 attenuated MAPK activity. We conclude that hTERT down-regulation by 15d-PGJ 2 plays an important role in the proapoptotic property of the latter. Furthermore, 15d-PGJ 2 inhibits ERβ-mediated hTERT gene transcription by suppressing ERβ phosphorylation via the inhibition of MAP kinase signaling

  1. Anomalous constitutive Src kinase activity promotes B lymphoma survival and growth

    OpenAIRE

    Ke, Jiyuan; Chelvarajan, R Lakshman; Sindhava, Vishal; Robertson, Darrell A; Lekakis, Lazaros; Jennings, C Darrell; Bondada, Subbarao

    2009-01-01

    Abstract Background Previously we have shown that B cell receptor (BCR) expression and B cell receptor signaling pathways are important for the basal growth of B lymphoma cells. In particular we have shown that the activation of Syk, a non-src family protein tyrosine kinase and the mitogen activated protein kinases (MAPK), ERK and JNK that mediate BCR signals are required for the constitutive growth of B lymphoma cells. Since src family protein tyrosine kinases (SFKs) like Lyn are known to be...

  2. The Drosophila rolled locus encodes a MAP kinase required in the sevenless signal transduction pathway.

    OpenAIRE

    Biggs, W H; Zavitz, K H; Dickson, B; van der Straten, A; Brunner, D; Hafen, E; Zipursky, S L

    1994-01-01

    Mitogen-activated protein (MAP) kinases have been proposed to play a critical role in receptor tyrosine kinase (RTK)-mediated signal transduction pathways. Although genetic and biochemical studies of RTK pathways in Caenorhabditis elegans, Drosophila melanogaster and mammals have revea