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SB203580
Kinds of SB203580 Selected AbstractsSimultaneous flow cytometric detection of basophil activation marker CD63 and intracellular phosphorylated p38 mitogen-activated protein kinase in birch pollen allergy,CYTOMETRY, Issue 1 2009Nicolaas E. Aerts Abstract Background: Phosphorylation of p38 MAPK is a crucial step in IgE-receptor signaling in basophils. The relation of p38 MAPK to the well-validated diagnostic cell surface marker CD63 has not been evaluated in a clinical allergy model. Methods: Expression of CD63 and phosphorylation of p38 MAPK were analyzed flow cytometrically in anti-IgE-gated basophils from 18 birch pollen allergic patients, five grass pollen allergic patients, and five healthy individuals, after 3 and 20 min of stimulation with recombinant major birch pollen allergen (rBet v 1). Additional time points and the influence of p38 MAPK inhibitor SB203580 were studied in birch pollen allergic patients. Results: Phospho-p38 MAPK and CD63 were expressed dose-dependently in birch pollen allergic patient basophils within 1 minute of rBet v 1 stimulation. P38 MAPK phosphorylation was fastest and subsided gradually while CD63 expression remained elevated for at least 20 min. Inhibition of p38 MAPK significantly inhibited CD63 upregulation. With optimal stimulation of the cells (1 ,g/mL), sensitivity and specificity for the discrimination between patients and a group of control individuals (grass pollen allergic patients and healthy controls) were 94% and 100% for CD63 at 3 and 20 min and for phospho-p38 MAPK at 3 min. Conclusion: Antigen-induced p38 MAPK phosphorylation in human basophils essentially contributes to CD63 upregulation. It is a sensitive and specific intracellular marker for allergy diagnosis and offers new insight into the mechanisms of basophil activation. © 2008 Clinical Cytometry Society [source] Independent signaling pathways in ATP-evoked secretion of plasminogen and cytokines from microgliaDRUG DEVELOPMENT RESEARCH, Issue 2-3 2001*Article first published online: 28 AUG 200, Kazuhide Inoue Abstract We investigated the action of ATP on the secretion of plasminogen, TNF-,, and IL-6 from microglia. ATP (10,100 ,M) stimulated the release of plasminogen from rat cultured microglia in a concentration-dependent manner with a peak response at 5,10 min after the stimulation. The release was dependent on extracellular Ca2+ and was blocked by pretreatment with oxidized ATP, a blocker of P2X7. UTP, an agonist of P2Y2, also stimulated the release of plasminogen from a subpopulation (about 20% of total cells) of cultured microglia. The release was also dependent on extracellular Ca2+, suggesting a role of stocker-operated calcium entry (SOC). ATP potently stimulated TNF-, release from 2 h after the stimulation with TNF-, mRNA expression in primary cultures of rat brain microglia. The TNF-, release was maximally elicited by 1 mM ATP and 2,- and 3,-O-(4-benzoylbenzoyl)-adenosine 5,-triphosphate (BzATP), a P2X7 selective agonist, suggesting the involvement of P2X7. This TNF-, release was correlated with a sustained Ca2+ influx. The release was inhibited by PD98059, an inhibitor of MEK1 which activates extracellular signal-regulated protein kinase (ERK), and SB203580, an inhibitor of p38 MAP kinase. However, both ERK and p38 were rapidly activated by ATP even in the absence of extracellular Ca2+. These results indicate that extracellular ATP triggers TNF-, release in rat microglia via P2X7 in a manner dependent on the sustained Ca2+ influx and via the ERK/p38 cascade independently of Ca2+ influx. ATP caused the mRNA expression and release of IL-6 in a concentration-dependent manner in MG-5. The physiological meaning of these independent release mechanisms is also discussed. Drug Dev. Res. 53:166,171, 2001. © 2001 Wiley-Liss, Inc. [source] Retinol binding protein isolated from acute renal failure patients inhibits polymorphonuclear leucocyte functionsEUROPEAN JOURNAL OF CLINICAL INVESTIGATION, Issue 11 2004G. Cohen Abstract Background, Protein factors accumulating in sera of patients with end-stage renal disease (ESRD) that interfere with the nonspecific immune response by inhibiting essential functions of polymorphonuclear leucocytes (PMNLs) have previously been described. No such factor has been isolated from acute renal failure (ARF) patients to date. Materials and methods, Using a three-step chromatographic procedure involving ion exchange, size exclusion and hydrophobic interaction chromatography we purified the apo- and holo-form of retinol binding protein (RBP) from high-flux dialyser (polyacrylonitrile; AN69) ultrafiltrates of patients with ARF. Their effect on the chemotaxis of PMNLs isolated from healthy donors was determined by the under-agarose method. Whole-blood assays applying flow cytometry were used to assess phagocytosis and the oxidative metabolism of PMNLs. Apoptosis was assessed by determining the DNA content using propidium iodide. Results, Isolated apo- and holo-forms of RBP were truncated on their C-terminus as determined by mass spectrometry. All isolates significantly inhibited the chemotactic movement of PMNLs obtained from healthy donors and the PMNL oxidative metabolism stimulated by E. coli. These effects were concentration dependent. Retinol binding protein had no influence on the PMNL oxidative metabolism stimulated by PMA and on PMNL phagocytosis. Commercially available RBP isolated from urine influenced PMNL functions in the same way. Inhibition of p38 mitogen-activated protein kinase (MAPK) by SB203580 significantly attenuated the phagocytosis-induced respiratory burst and RBP did not lead to a further decrease. Polymorphonuclear leucocyte apoptosis was significantly inhibited by RBP. Conclusions, The apo- and holo-forms of RBP isolated from the ultrafiltrate of ARF patients inhibit PMNL chemotaxis, oxidative metabolism and apoptosis. Therefore, RBP may be considered a uraemic toxin contributing to a disturbed immune defence. [source] Mitogen-activated protein kinases regulate Mycobacterium avium -induced tumor necrosis factor-, release from macrophagesFEMS IMMUNOLOGY & MEDICAL MICROBIOLOGY, Issue 1 2002Asima Bhattacharyya Abstract Tumor necrosis factor-, (TNF-,) is one of the key cytokines elicited by host macrophages upon challenge with pathogenic mycobacteria. Infection of human peripheral blood mononuclear cells or the murine macrophage cell line J774A,1 with Mycobacterium avium induced activation of the mitogen-activated protein kinases (MAPKs) ERK1/2, p38 and c-Jun N-terminal kinase. U0126, an MEK-specific inhibitor, abrogated M. avium -induced TNF-, secretion. Transfection of cells with dominant-negative MEK1 led to the suppression of TNF-, release in M. avium -challenged macrophages. M. avium activated p38 MAPK and use of the p38 MAPK inhibitor, SB203580, revealed that the p38 signaling pathway negatively regulates activation of ERK1/2 and release of TNF-,. Taken together, these results provide evidence that M. avium -induced TNF-, release from macrophages depends on an interplay between the ERK1/2 and the p38 MAPK signaling pathways. [source] Regulation of mitotic function of Chk1 through phosphorylation at novel sites by cyclin-dependent kinase 1 (Cdk1)GENES TO CELLS, Issue 5 2006Takashi Shiromizu Chk1 is phosphorylated at Ser317 and Ser345 by ATR in response to stalled replication and genotoxic stresses. This Chk1 activation is thought to play critical roles in the prevention of premature mitosis. However, the behavior of Chk1 in mitosis remains largely unknown. Here we reported that Chk1 was phosphorylated in mitosis. The reduction of this phosphorylation was observed at the metaphase-anaphase transition. Two-dimensional phosphopeptide mapping revealed that Chk1 phosphorylation sites in vivo were completely overlapped with the in vitro sites by cyclin-dependent protein kinase (Cdk) 1 or by p38 MAP kinase. Ser286 and Ser301 were identified as novel phosphorylation sites on Chk1. Treatment with Cdk inhibitor butyrolactone I induced the reduction of Chk1-S301 phosphorylation, although treatment with p38-specific inhibitor SB203580 or siRNA did not. In addition, ionizing radiation (IR) or ultraviolet (UV) light did not induce Chk1 phosphorylation at Ser317 and Ser345 in nocodazole-arrested mitotic cells. These observations imply the regulation of mitotic Chk1 function through Chk1 phosphorylation at novel sites by Cdk1. [source] Secretion of matrix metalloproteinase-9 by the proinflammatory cytokine, IL-1,: a role for the dual signalling pathways, Akt and ErkGENES TO CELLS, Issue 6 2003A. R. M. Ruhul Amin Background: Matrix metalloproteinases including MMP-9 mediate matrix destruction during chronic inflammatory diseases such as arthritis and atherosclerosis. MMP-9 up-regulation by inflammatory cytokines involve interactions between several transcription factors including activator protein-1 and NF,B. The upstream regulatory pathways are less well understood. Results: To search for the mechanism of tissue destruction in the process of inflammatory disorders, we investigated the signalling pathway critical for the activation of MMP-9 expression and secretion by IL-1,. Treatment of Balb 3T3 cells with IL-1, activated MMP-9 transcription and subsequent secretion in a time- and dose-dependent manner. Concomitantly, IL-1, treatment of cells activated phosphorylation of Akt, Erk and p38. Treatment of cells with either LY294002, a PI3K inhibitor, or expression of a dominant negative form of Akt drastically suppressed the IL-1,-dependent secretion of MMP-9. Pretreatment of cells with a MEK1 inhibitor, U0126, also strongly inhibited IL-1,-dependent secretion of MMP-9. In contrast, pre-treatment with a specific p38 kinase inhibitor, SB203580, had no effect on IL-1,-dependent secretion of MMP-9. In addition, cells expressing constitutively active form of Akt or MEK1 showed no clear activation of MMP-9 secretion, whereas these cells responded well to IL-1, treatment. However, co-transfection of cells with both active Akt and MEK1 was sufficient to induce MMP-9 secretion without stimulation with IL-1,. Conclusion: Taken together, our results suggest that IL-1, stimulation of cells activates MMP-9 secretion by the activation of the dual signalling pathways, the PI3K-Akt and MEK1-Erk and constitutive activation of these pathways were sufficient to induce MMP-9 secretion. [source] HSP27 mediates SPARC-induced changes in glioma morphology, migration, and invasionGLIA, Issue 10 2008William A. Golembieski Abstract Secreted protein acidic and rich in cysteine (SPARC) regulates cell,extracellular matrix interactions that influence cell adhesion and migration. We have demonstrated that SPARC is highly expressed in human gliomas, and it promotes brain tumor invasion in vitro and in vivo. To further our understanding regarding SPARC function in glioma migration, we transfected SPARC-green fluorescent protein (GFP) and control GFP vectors into U87MG cells, and assessed the effects of SPARC on cell morphology, migration, and invasion after 24 h. The expression of SPARC was associated with elongated cell morphology, and increased migration and invasion. The effects of SPARC on downstream signaling were assessed from 0 to 6 h and 24 h. SPARC increased the levels of total and phosphorylated HSP27; the latter was preceded by activation of p38 MAPK and inhibited by the p38 MAPK inhibitor SB203580. Augmented expression of SPARC was correlated with increased levels of HSP27 mRNA. In a panel of glioma cell lines, increasing levels of SPARC correlated with increasing total and phosphorylated HSP27. SPARC and HSP27 were colocalized to invading cells in vivo. Inhibition of HSP27 mRNA reversed the SPARC-induced changes in cell morphology, migration, and invasion in vitro. These data indicate that HSP27, a protein that regulates actin polymerization, cell contraction, and migration, is a novel downstream effector of SPARC-regulated cell morphology and migration. As such, it is a potential therapeutic target to inhibit SPARC-induced glioma invasion. © 2008 Wiley-Liss, Inc. [source] p38 mitogen-activated protein kinase is required for central nervous system myelinationGLIA, Issue 15 2007Gabriela Fragoso Abstract The p38 MAPKs are a family of kinases that regulate a number of cellular functions including cell migration, proliferation, and differentiation. Here, we report that p38 regulates oligodendrocyte differentiation. Inhibition of p38 with PD169316 and SB203580 prevented accumulation of protein and mRNA of cell-stage specific markers characteristic of differentiated oligodendrocytes, including myelin basic protein, myelin-associated glycoprotein, and the glycosphingolipids, galactosylceramide and sulfatide. In addition, the cell cycle regulator p27kip1 and the transcription factor Sox10 were also significantly reduced. Most significantly, p38 inhibitors completely and irreversibly blocked myelination of dorsal root ganglion neurons by oligodendrocytes and prevented the axolemmal organization of the axo-glial adhesion molecule Caspr. Our results suggest a role(s) for this kinase in key regulatory steps in the maturation of OLGs and initiation of myelination. © 2007 Wiley-Liss, Inc. [source] Induction of cellular resistance against Kupffer cell,derived oxidant stress: A novel concept of hepatoprotection by ischemic preconditioningHEPATOLOGY, Issue 2 2003Rolf J. Schauer Ischemic preconditioning (IP) triggers protection of the liver from prolonged subsequent ischemia. However, the underlying protective mechanisms are largely unknown. We investigated whether and how IP protects the liver against reperfusion injury caused by Kupffer cell (KC)-derived oxidants. IP before 90 minutes of warm ischemia of rat livers in vivo significantly reduced serum alanine aminotransferase (AST) levels and leukocyte adherence to sinusoids and postsinusoidal venules during reperfusion. This protective effect was mimicked by postischemic intravenous infusion of glutathione (GSH), an antioxidative strategy against KC-derived H2O2. Interestingly, no additional protection was achieved by infusion of GSH to preconditioned animals. These findings and several additional experiments strongly suggest IP mediated antioxidative effects: IP prevented oxidant cell injury in isolated perfused rat livers after selective KC activation by zymosan. Moreover, IP prevented cell injury and pertubations of the intracellular GSH/GSSG redox system caused by direct infusion of H2O2 (0.5 mmol/L). IP-mediated resistance against H2O2 could neither be blocked by the adenosine A2a antagonist DMPX nor mimicked by A2a agonist CGS21680. In contrast, H2O2 resistance was abolished by the p38 mitogen-activated protein kinase (p38 MAPK) inhibitor SB203580, but induced when p38 MAPK was directly activated by anisomycin. In conclusion, we propose a novel concept of hepatoprotection by IP: protection of liver cells by enhancing their resistance against KC-derived H2O2. Activation of p38 MAPK and preservation of the intracellular GSH/oxidized glutathione (GSSG) redox system, but not adenosine A2a receptor stimulation, seems to be pivotal for the development of H2O2 resistance in preconditioned livers. [source] A p38 MAP kinase regulates the expression of the Aedes aegypti defensin gene in mosquito cellsINSECT MOLECULAR BIOLOGY, Issue 4 2007R. Chen-Chih Wu Abstract An Aedes aegypti p38 (Aap38) mitogen-activated protein kinase was isolated and characterized in this study. The 1761 bp long full-length Aap38 cDNA encodes an open reading frame of 358 amino acids, exhibiting characteristics of Thr/Tyr dual kinase specificities. We showed that bacteria activate both the kinase activity of Aap38 and the expression of the Aedes aegypti defensin A (AaDefA) gene, which is inhibited by a p38 kinase inhibitor SB203580 and dsRNA interference of Aap38. A similar result was obtained by a reporter construct containing the AaDefA regulatory region linked to Ds-Red. The lipopolysaccharide-activated reporter gene was inhibited by SB203580. In addition, Aap38 translocated to the nucleus after lipopolysaccharide induction. Our findings suggest that the p38 protein kinase pathway is involved in the antibacterial peptide synthesis in mosquitoes. [source] Examination of the signal transduction pathways leading to upregulation of tissue type plasminogen activator by Porphyromonas endodontalis in human pulp cellsINTERNATIONAL ENDODONTIC JOURNAL, Issue 12 2005F.-M. Huang Abstract Aim, To investigate the tissue type plasminogen activator (t-PA) activity in human pulp cells stimulated with Porphyromonas endodontalis (P. endodontalis) in the absence or presence of p38 inhibitor SB203580, mitogen-activated protein kinase kinase (MEK) inhibitor U0126 and phosphatidylinositaol 3-kinase (PI3K) inhibitor LY294002. Methodology, The supernatants of P. endodontalis were used to evaluate t-PA activity in human pulp cells using casein zymography and enzyme-linked immunosorbent assay (ELISA). Furthermore, to search for possible signal transduction pathways, SB203580, U0126 and LY294002 were added to test how they modulated the t-PA activity. Results, The main casein secreted by human pulp cells migrated at 70 kDa and represented t-PA. Secretion of t-PA was found to be stimulated with P. endodontalis during 2-day cultured period (P < 0.05). From the results of casein zymography and ELISA, SB203580 and U0126 significantly reduced the P. endodontalis stimulated t-PA production respectively (P < 0.05). However, LY294002 lacked the ability to change the P. endodontalis stimulated t-PA production (P > 0.05). Conclusions,Porphyromonas endodontalis enhances t-PA production in human pulp cells, and the signal transduction pathways p38 and MEK are involved in the inhibition of t-PA. [source] Pml and TAp73 interacting at nuclear body mediate imatinib-induced p53-independent apoptosis of chronic myeloid leukemia cellsINTERNATIONAL JOURNAL OF CANCER, Issue 1 2009Jin-Hwang Liu Abstract Bcr-abl signals for leukemogenesis of chronic myeloid leukemia (CML) and activates ras. Since the function of promyelocytic leukemia protein (pml) is provoked by ras to promote apoptosis and senescence in untransformed cells, the function is probably masked in CML. Imatinib specifically inhibits bcr-abl and induces apoptosis of CML cells. As reported previously, p53wild CML was more resistant to imatinib than that lacking p53. Here, we searched for an imatinib-induced p53 independent proapoptotic mechanism. We found imatinib up-regulated phosphorylation of p38 mitogen-activated protein kinase (MAPK), checkpoint kinase 2 (chk2) and transactivation-competent (TA) p73; expression of pml and bax; formation of PML-nuclear body (NB); and co-localization of TAp73/PML-NB in p53-nonfunctioning K562 and p53mutant Meg-01 CML cells, but not in BCR-ABL - HL60 cells. In K562 cells, with short interfering RNAs (siRNAs), knockdown of pml led to dephosphorylation of TAp73. Knockdown of either pml or TAp73 abolished the imatinib-induced apoptosis. Inhibition of p38 MAPK with SB203580 led to dephosphorylation of TAp73, abolishment of TAp73/PML-NB co-localization, and the subsequent apoptosis. Conversely, interferon ,-2a (IFN,), which increased phosphrylated TAp73 and TAp73/PML-NB co-localization, increased additively apoptosis with imatinib. The imatinib-induced TAp73/PML-NB co-localization was accompanied by co-immpunoprecipitation of TAp73 with pml. The imatinib-induced co-localization was also found in primary CML cells from 3 of 6 patients, including 2 with p53mutant and one with p53wild. A novel p53-independent proapoptotic mechanism using p38 MAPK /pml/TAp73 axis with a step processing at PML-NB and probably with chk2 and bax being involved is hereby evident in some imatinib-treated CML cells. © 2009 UICC [source] Dominant-negative Rac increases both inherent and ionizing radiation-induced cell migration in C6 rat glioma cellsINTERNATIONAL JOURNAL OF CANCER, Issue 8 2006So-Young Hwang Abstract Rho-like GTPases, including Cdc42, Rac1 and RhoA, regulate distinct actin cytoskeleton changes required for cell adhesion, migration and invasion. In the present study, we examined the role of Rac signaling in inherent migration, as well as radiation-induced migration, of rat glioma cells. Stable overexpression of dominant-negative Rac1N17 in a C6 rat glioma cell line (C6-RacN17) promoted cell migration, and ionizing radiation further increased this migration. Migration was accompanied by decreased expression of the focal adhesion molecules FAK and paxillin. Focal contacts and actin stress fibers were also reduced in C6-RacN17 cells. Downstream effectors of Rac include JNK and p38 MAP kinases. Irradiation transiently activated p38, JNK and ERK1/2 MAP kinases in C6-RacN17 cells, while p38 and JNK were constitutively activated in C6 control cells. Blocking JNK activity with JNK inhibitor SP600125 inhibited migration, suggesting that the JNK pathway may regulate radiation-induced, as well as inherent, migration of C6-RacN17 cells. Additionally, the radiation-induced migration increase was also inhibited by SB203580, a specific inhibitor of p38 MAP kinase. However, PD98059, a MEK kinase 1 inhibitor, failed to influence migration. This is the first evidence that suppression of Rac signaling may be involved in invasion or metastasis of glioma cells before and/or after radiotherapy. These data further suggest that radiotherapy for malignant glioma needs to be used with caution because of the potential for therapy-induced cell migration or invasion and that pharmacological inhibition of cell migration and invasion through targeting the Rac signaling pathway may represent a new approach for improving the therapeutic efficacy of radiotherapy for malignant glioma. © 2005 Wiley-Liss, Inc. [source] Induction of Transcriptional Activity of the Cyclic Adenosine Monophosphate Response Element Binding Protein by Parathyroid Hormone and Epidermal Growth Factor in Osteoblastic Cells,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 8 2002John T. Swarthout Abstract Previously, we have shown that parathyroid hormone (PTH) transactivation of cyclic adenosine monophosphate (cAMP) response element binding protein (CREB) requires both serine 129 (S129) and serine 133 (S133) in rat osteosarcoma cells UMR 106-01 (UMR) cells. Furthermore, although protein kinase A (PKA) is responsible for phosphorylation at S133, glycogen synthase kinase 3, (GSK-3,) activity is required and may be responsible for phosphorylation of CREB at S129. Here, we show, using the GAL4-CREB reporter system, that epidermal growth factor (EGF) can transactivate CREB in UMR cells in addition to PTH. Additionally, treatment of UMR cells with both PTH and EGF results in greater than additive transactivation of CREB. Furthermore, using mutational analysis we show that S129 and S133 are required for EGF-induced transcriptional activity. EGF activates members of the MAPK family including p38 and extracellular signal,activated kinases (ERKs), and treatment of UMR cells with either the p38 inhibitor (SB203580) or the MEK inhibitor (PD98059) prevents phosphorylation of CREB at S133 by EGF but not by PTH. Treatment of cells with either SB203580 or PD98059 alone or together significantly inhibits transactivation of CREB by EGF but not by PTH, indicating that EGF regulates CREB phosphorylation and transactivation through p38 and ERKs and PTH does not. Finally, the greater than additive transactivation of CREB by PTH and EGF is significantly inhibited by the PKA inhibitor H-89 or by cotreatment with SB203580 and PD98059. Thus, several different signaling pathways in osteoblastic cells can converge on and regulate CREB activity. This suggests, in vivo, that circulating agents such as PTH and EGF are acting in concert to exert their effects. [source] Basic Fibroblast Growth Factor Stimulates Vascular Endothelial Growth Factor Release in Osteoblasts: Divergent Regulation by p42/p44 Mitogen-Activated Protein Kinase and p38 Mitogen-Activated Protein KinaseJOURNAL OF BONE AND MINERAL RESEARCH, Issue 12 2000Haruhiko Tokuda Abstract We previously showed that basic fibroblast growth factor (bFGF) activates p38 mitogen-activated protein (MAP) kinase via Ca2+ mobilization, resulting in interleukin-6 (IL-6) synthesis in osteoblast-like MC3T3-E1 cells. In the present study, we investigated the effect of bFGF on the release of vascular endothelial growth factor (VEGF) in these cells. bFGF stimulated VEGF release dose dependently in the range between 10 and 100 ng/ml. SB203580, an inhibitor of p38 MAP kinase, markedly enhanced the bFGF-induced VEGF release. bFGF induced the phosphorylation of both p42/p44 MAP kinase and p38 MAP kinase. PD98059, an inhibitor of upstream kinase of p42/p44 MAP kinase, reduced the VEGF release. SB203580 enhanced the phosphorylation of p42/p44 MAP kinase induced by bFGF. The enhancement by SB203580 of the bFGF-stimulated VEGF release was suppressed by PD98059. The depletion of extracellular Ca2+ by [ethylenebis-(oxyethylenenitrilo)]tetracetic acid (EGTA) or 1,2-bis-(O -aminophinoxy)-ethane- N,N,N,N -tetracetic acid tetracetoxymethyl ester (BAPTA/AM), a chelator of intracellular Ca2+, suppressed the bFGF-induced VEGF release. A23187, a Ca ionophore, or thapsigargin, known to induce Ca2+ release from intracellular Ca2+ store, stimulated the release of VEGF by itself. A23187 induced the phosphorylation of p42/p44 MAP kinase and p38 MAP kinase. PD98059 suppressed the VEGF release induced by A23187. SB203580 had little effect on either A23187-induced VEGF release or the phosphorylation of p42/p44 MAP kinase by A23187. These results strongly suggest that bFGF stimulates VEGF release through p42/p44 MAP kinase in osteoblasts and that the VEGF release is negatively regulated by bFGF-activated p38 MAP kinase. [source] Platelet lysate promotes in vitro wound scratch closure of human dermal fibroblasts: different roles of cell calcium, P38, ERK and PI3K/AKTJOURNAL OF CELLULAR AND MOLECULAR MEDICINE, Issue 8b 2009Elia Ranzato Abstract There is a growing interest for the clinical use of platelet derivates in wound dressing. Platelet beneficial effect is attributed to the release of growth factors and other bioactive substances, though mechanisms are mostly unknown. We studied wound-healing processes of human primary fibroblasts, by exposing cells to a platelet lysate (PL) obtained from blood samples. Crystal violet and tetrazolium salt (MTS) assays showed dose,response increase of cell proliferation and metabolism. In scratch wound and transwell assays, a dose of 20% PL induced a significant increase of wound closure rate at 6 and 24 hrs, and had a strong chemotactic effect. BAPTA-AM, SB203580 and PD98059 caused 100% inhibition of PL effects, whereas wortmannin reduced to about one third the effect of PL on wound healing and abolished the chemotactic response. Confocal imaging showed the induction by PL of serial Ca2+ oscillations in fibroblasts. Data indicate that cell Ca2+ plays a fundamental role in wound healing even without PL, p38 and ERK1/2 are essential for PL effects but are also activated by wounding per se, PI3K is essential for PL effects and its downstream effector Akt is activated only in the presence of PL. In conclusion, PL stimulates fibroblast wound healing through the activation of cell proliferation and motility with different patterns of involvement of different signalling pathways. [source] H2S-induced pancreatic acinar cell apoptosis is mediated via JNK and p38 MAP kinaseJOURNAL OF CELLULAR AND MOLECULAR MEDICINE, Issue 4 2008Sharmila Adhikari Abstract Treatment of pancreatic acinar cells by hydrogen sulphide has been shown to induce apoptosis. However, a potential role of mitogen-activated protein kinases (MAPKs) in this apoptotic pathway remains unknown. The present study examined the role of MAPKs in H2S-induced apoptosis in mouse pancreatic acinar cells. Pancreatic acinar cells were treated with 10 ,M NaHS (a donor of H2S) for 3 hrs. For the evaluation of the role of MAPKs, PD98059, SP600125 and SB203580 were used as MAPKs inhibitors for ERK1/2, JNK1/2 and p38 MAPK, respectively. We observed activation of ERK1/2, JNK1/2 and p38 when pancreatic acini were exposed to H2S. Moreover, H2S-induced ERK1/2, JNK1/2 and p38 activation were blocked by pre-treatment with their corresponding inhibitor in a dose-dependent manner. H2S-induced apoptosis led to an increase in caspase 3 activity and this activity was attenuated when caspase 3 inhibitor were used. Also, the cleavage of caspase 3 correlated with that of poly-(ADP-ribose)-polymerase (PARP) cleavage. H2S treatment induced the release of cytochrome c, smac from mitochondria into the cytoplasm, translocation of Bax into mitochondria and decreased the protein level of Bcl-2. Inhibition of ERK1/2 using PD98059 caused further enhancement of apoptosis as evidenced by annexin V staining, while SP600125 and SB203580 abrogated H2S-induced apoptosis. Taken together, the data suggest that activation of ERKs promotes cell survival, whereas activation of JNKs and p38 MAP kinase leads to H2S-induced apoptosis. [source] Differential regulation of platelet-derived growth factor stimulated migration and proliferation in osteoblastic cells,JOURNAL OF CELLULAR BIOCHEMISTRY, Issue 4 2004Meenal Mehrotra Abstract Osteoblastic migration and proliferation in response to growth factors are essential for skeletal development, bone remodeling, and fracture repair, as well as pathologic processes, such as metastasis. We studied migration in response to platelet-derived growth factor (PDGF, 10 ng/ml) in a wounding model. PDGF stimulated a twofold increase in migration of osteoblastic MC3T3-E1 cells and murine calvarial osteoblasts over 24,48 h. PDGF also stimulated a tenfold increase in 3H-thymidine (3H-TdR) incorporation in MC3T3-E1 cells. Migration and DNA replication, as measured by BrdU incorporation, could be stimulated in the same cell. Blocking DNA replication with aphidicolin did not reduce the distance migrated. To examine the role of mitogen-activated protein (MAP) kinases in migration and proliferation, we used specific inhibitors of p38 MAP kinase, extracellular signal regulated kinase (ERK), and c-Jun N-terminal kinase (JNK). For these signaling studies, proliferation was measured by carboxyfluorescein diacetate succinimidyl ester (CFSE) using flow cytometry. Inhibition of the p38 MAP kinase pathway by SB203580 and SB202190 blocked PDGF-stimulated migration but had no effect on proliferation. Inhibition of the ERK pathway by PD98059 and U0126 inhibited proliferation but did not inhibit migration. Inhibition of JNK activity by SP600125 inhibited both migration and proliferation. Hence, the stimulation of migration and proliferation by PDGF occurred by both overlapping and independent pathways. The JNK pathway was involved in both migration and proliferation, whereas the p38 pathway was predominantly involved in migration and the ERK pathway predominantly involved in proliferation. © 2004 Wiley-Liss, Inc. [source] Platelet-derived growth factor-BB phosphorylates heat shock protein 27 in cardiac myocytesJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 2 2004Motoki Takenaka Abstract It is recognized that heat shock protein 27 (HSP27) is highly expressed in heart. In the present study, we investigated whether platelet-derived growth factor (PDGF) phosphorylates HSP27 in mouse myocytes, and the mechanism underlying the HSP27 phosphorylation. Administration of PDGF-BB induced the phosphorylation of HSP27 at Ser-15 and -85 in mouse cardiac muscle in vivo. In primary cultured myocytes, PDGF-BB time dependently phosphorylated HSP27 at Ser-15 and -85. PDGF-BB stimulated the phosphorylation of p44/p42 mitogen-activated protein (MAP) kinase, p38 MAP kinase, and stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) among the MAP kinase superfamily. SB203580, a specific inhibitor of p38 MAP kinase, reduced the PDGF-BB-stimulated phosphorylation of HSP27 at both Ser-15 and -85, and phosphorylation of p38 MAP kinase. However, PD98059, a specific inhibitor of MEK, or SP600125, a specific inhibitor of SAPK/JNK, failed to affect the HSP27 phosphorylation. These results strongly suggest that PDGF-BB phosphorylates HSP27 at Ser-15 and -85 via p38 MAP kinase in cardiac myocytes. © 2003 Wiley-Liss, Inc. [source] JNK phosphorylates the HSF1 transcriptional activation domain: Role of JNK in the regulation of the heat shock responseJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 2 2001Jeonghyeon Park Abstract The role of c-Jun NH2 -terminal kinase (JNK) signaling cascade in the stress-inducible phosphorylation of heat shock factor 1 (HSF1) was investigated using known agonists and antagonists of JNK. We showed that treatment of HeLa cells with MG132, a proteasome inhibitor and known JNK activator, caused the transcriptional activation domain of HSF1 to be targeted and phosphorylated by JNK2 in vivo. Dose-response and time course studies of the effects of heat shock and anisomycin treatment showed a close correlation of the activation of JNK and hyperphosphorylation of HSF1. SB203580 inhibited JNK at the 100 ,M concentration and significantly reduced the amount of hyperphosphorylated HSF1 upon heat shock or anisomycin treatment. SB203580 and dominant-negative JNK suppress hsp70 promoter-driven reporter gene expression selectively at 45°C but not at 42°C heat stress, suggesting that JNK would be preferentially associated with the protective heat shock response against severe heat stress. The possibility that JNK-mediated phosphorylation of HSF1 may selectively stabilize the HSF1 protein and confers protection to cells under conditions of severe stress is discussed. J. Cell. Biochem. 82: 326,338, 2001. © 2001 Wiley-Liss, Inc. [source] Alterations in the temporal expression and function of cadherin-7 inhibit cell migration and condensation during chondrogenesis of chick limb mesenchymal cells in vitroJOURNAL OF CELLULAR PHYSIOLOGY, Issue 1 2009Dongkyun Kim Endochondral bone formation requires a complex interplay among immature mesenchymal progenitor cells to form the cartilaginous anlagen, which involves migration, aggregation and condensation. Even though condensation of chondrogenic progenitors is an essential step in this process, its mechanism(s) has not been well studied. Here, we show that cadherin-7 plays a central role in cellular condensation by modulating cell motility and migration. In this study, many mesenchymal cells failed to migrate, and precartilage condensation was inhibited, after knockdown of endogenous cadherin-7 levels. Exposure of mesenchymal cells to SB203580 (a specific inhibitor of p38MAPK), LiCl (an inhibitor of GSK-3,) or overexpression of ,-catenin resulted in inhibition of cadherin-7 levels and, subsequently, suppression of cell migration. Collectively, our results suggest that cadherin-7 controls cell migration in chick limb bud mesenchymal cells, and that p38MAPK and GSK signals are responsible for regulating cadherin-7-mediated cell migration. J. Cell. Physiol. 221: 161,170, 2009. © 2009 Wiley-Liss, Inc [source] Sox9, a key transcription factor of bone morphogenetic protein-2-induced chondrogenesis, is activated through BMP pathway and a CCAAT box in the proximal promoter,JOURNAL OF CELLULAR PHYSIOLOGY, Issue 1 2008Qiuhui Pan Mouse embryonic fibroblasts (MEFs) can be differentiated into fully functional chondrocytes in response to bone morphogenetic protein-2 (BMP-2). The expression of Sox9, a critical transcription factor for the multiple steps of chondrogenesis, has been reported to be upregulated during this process. But the molecular mechanisms by which BMP-2 promotes chondrogenesis still remain largely unknown. The aim of the present study was therefore to investigate the underlying mechanism. In the MEFs, BMP-2 efficiently induced Sox9 expression along with chondrogenic differentiation in a time- and dose-dependent manner. SB203580, a specific inhibitor for p38 pathway, blocked BMP-2-induced chondrogenic differentiation as well as Sox9 expression and its transactivation of downstream genes. Forced expression of Smad6, a natural antagonist for BMP/Smad pathway, only inhibited Sox9 protein function without rendering any effects on its mRNA expression. A CCAAT box was identified in Sox9 promoter as the cis -elements responsible for BMP-2 stimulation. This study provides insight into the mechanisms underlying BMP-2-regulated Sox9 expression and activity in MEFs, and suggests differential roles of BMP-2/p38 and BMP-2/Smad pathways in modulating the function of Sox9 during chondrogenesis. J. Cell. Physiol. 217: 228,241, 2008. © 2008 Wiley-Liss, Inc. [source] Osterix is a key target for mechanical signals in human thoracic ligament flavum cellsJOURNAL OF CELLULAR PHYSIOLOGY, Issue 3 2007Dongwei Fan Mechanical stress is considered to be an important factor in the progression of thoracic ossification of the ligament flavum (TOLF). To elucidate the mechanism underlying mechanical stress-induced TOLF, we investigated the effect of stretching on cultured flavum ligament cells derived from TOLF and non-TOLF patients. We found that the mRNA expression of alkaline phosphatase (ALP), osteocalcin, Runx2, and osterix, but not that of Dlx5 and Msx2, was significantly increased by stretching in TOLF cells. In addition, the effect seems to be finely tuned by stretching-triggered activation of distinct mitogen-activated protein kinase cascades. Specifically, a p38 specific inhibitor, SB203580, significantly inhibited stretching-induced osterix expression as well as ALP activity, whereas a specific inhibitor of ERK1/2, U0126, prevented stretching-induced Runx2 expression. We showed that overexpression of osterix resulted in a significant increase of ALP activity in TOLF cells, and osterix-specific RNAi completely abrogated the stretching-induced ALP activity, indicating that osterix plays a key role in stretching-stimulated osteogenic effect in TOLF cells. These results suggest that mechanical stress plays important roles in the progression of TOLF through induction of osteogenic differentiation of TOLF cells, and our findings support that osterix functions as a molecular link between mechanostressing and osteogenic differentiation. J. Cell. Physiol. 211: 577,584, 2007. © 2007 Wiley-Liss, Inc. [source] Hypoxia-induced apoptosis and tube breakdown are regulated by p38 MAPK but not by caspase cascade in an in vitro capillary model composed of human endothelial cellsJOURNAL OF CELLULAR PHYSIOLOGY, Issue 3 2007Toshiro Ohta In order to improve medical treatment of ischemic injury such as myocardial infarction, it is important to elucidate hypoxia-induced changes to endothelial cells. An in vitro blood vessel model, in which HUVECs are stimulated to form a network of capillary-like tubes, was used to analyze hypoxia-induced morphological and biochemical changes. When exposed to hypoxia, the network of capillary tubes broke down into small clusters. This tube breakdown was accompanied by chromatin condensation and cell nuclear fragmentation, morphological markers of apoptosis, and activation of two apoptotic signals, caspase-3 and p38. We investigated what roles caspase cascade and p38 play in hypoxia-induced apoptosis and tube breakdown by using zVAD-fmk and SB203580, specific inhibitors of these two apoptotic signals, respectively. Chromatin condensation and cell nuclear fragmentation and tube breakdown were effectively inhibited by SB203580, but not by zVAD-fmk. SB203580 caused dephosphorylation of p38, which indicates that p38 was autophosphorylated. Inhibition by zVAD-fmk caused slight MW increase in p17 and emergence of p19, which indicates that the inhibitor caused partial processing of caspase-3. Inhibition of p38 suppressed activation of caspase-3 but not vice versa. In addition, these two inhibitors were shown to differentially inhibit cleavage of so-called caspase substrates. SB203580 inhibited cleavage of PARP and lamin A/C, while zVAD-fmk inhibited cleavage of lamin A/C but not that of PARP. Taken together, these results show that p38 is located upstream of caspase cascade and that, although caspase-3 is activated, a p38-regulated caspase-independent pathway is crucial for the execution of hypoxia-induced apoptosis and tube breakdown. J. Cell. Physiol. 211: 673,681, 2007. © 2007 Wiley-Liss, Inc. [source] Rap1 and p38 MAPK mediate 8-chloro-cAMP-induced growth inhibition in mouse fibroblast DT cellsJOURNAL OF CELLULAR PHYSIOLOGY, Issue 3 2006Young-Ho Ahn 8-Cl-cAMP, which is known to induce differentiation, growth inhibition, and apoptosis in various cancer cells, has been investigated as a putative anti-cancer drug. Previously, we reported that 8-Cl-cAMP and its metabolite 8-Cl-adenosine induce growth inhibition and apoptosis through p38 mitogen-activated protein kinase (MAPK) activation. To further investigate the signal mechanisms that regulate the cellular effects of 8-Cl-cAMP, we focused on a small GTPase Rap1 that is known to be involved in growth inhibition and reverse-transformation. 8-Cl-cAMP and 8-Cl-adenosine could increase Rap1 activity, which was blocked by ABT702,an adenosine kinase inhibitor. This suggests that 8-Cl-cAMP-induced Rap1 activation is also dependent on the metabolic degradation of 8-Cl-cAMP. Overexpression of a constitutively active mutant form of Rap1 (Rap1V12) attenuated cellular growth and soft-agar colony formation, which was basically the same effect as that observed with the 8-Cl-cAMP treatment. Furthermore, the Rap1V12 transfectant showed a high level of p38 MAPK activation. However, 8-Cl-cAMP-induced Rap1 activation was not diminished by SB203580, a p38 MAPK inhibitor, suggesting that Rap1 activation might act upstream of p38 MAPK activation during 8-Cl-cAMP-induced growth inhibition. J. Cell. Physiol. 209: 1039,1045, 2006. © 2006 Wiley-Liss, Inc. [source] Inhibition of Cdk6 expression through p38 MAP kinase is involved in differentiation of mouse prechondrocyte ATDC5JOURNAL OF CELLULAR PHYSIOLOGY, Issue 3 2005Toru Moro Because a temporal arrest in the G1-phase of the cell cycle is a prerequisite for cell differentiation, this study investigated the involvement of cell cycle factors in the differentiation of cultured mouse prechondrocyte cell line ATDC5. Among the G1 cell cycle factors examined, both protein and mRNA levels of cyclin-dependent kinase (Cdk6) were downregulated during the culture in a differentiation medium. The protein degradation of Cdk6 was not involved in this downregulation because proteasome inhibitors did not reverse the protein level. When inhibitors of p38 MAPK, ERK-1/2, and PI3K/Akt were added to the culture, only a p38 MAPK inhibitor SB203580 blocked the decrease in the Cdk6 protein level by the differentiation medium, indicating that the Cdk6 inhibition was mediated by p38 MAPK pathway. In fact, p38 MAPK was confirmed to be phosphorylated during differentiation of ATDC5 cells. Enforced expression of Cdk6 in ATDC5 cells blocked the chondrocyte differentiation and inhibited Sox5 and Sox6 expressions. However, the Cdk6 overexpression did not affect the proliferation or the cell cycle progression, suggesting that the inhibitory effect of Cdk6 on the differentiation was exerted by a mechanism largely independent of its cell cycle regulation. These results indicate that Cdk6 may be a regulator of chondrocyte differentiation and that its p38-mediated downregulation is involved in the efficient differentiation. © 2005 Wiley-Liss, Inc. [source] Stress kinase p38 mediates EGFR transactivation by hyperosmolar concentrations of sorbitolJOURNAL OF CELLULAR PHYSIOLOGY, Issue 2 2002Hao Cheng Activation of the epidermal growth factor receptor (EGFR) has been shown to occur by ligand-dependent and ligand-independent mechanisms. Different molecular mechanisms have been found to be responsible for ligand-independent receptor transactivation. Here, we show that hyperosmolar concentrations of sorbitol activate the EGFR in human keratinocytes. Experiments using specific inhibitors of EGFR phosphorylation show that the increased amount of activated receptors is the result of a decreased rate of dephosphorylation. Furthermore, sorbitol treatment results in a strong activation of stress kinase p38. Treatment of the cells with SB203580, a known inhibitor of p38 , and , kinases, results in impairment of receptor activation, indicating that the stress kinase is involved in receptor activation modulation. This is further reinforced by experiments showing that addition of Toxin B, known to be an inhibitor of the small Rho GTPases rac1, cdc42, and Rho A/B, to the cells results in a strong induction of EGFR activation. Our results point, therefore, to a mechanism by which osmotic shock activates EGFR through the small Rho GTPases-p38 stress kinase pathway. © 2002 Wiley-Liss, Inc. [source] Characterization of a novel fibroblast-like cell line from rainbow trout and responses to sublethal anoxiaJOURNAL OF FISH BIOLOGY, Issue 4 2004C. G. Ossum A novel fibroblast-like cell line RTHDF was established from hypodermal connective tissue of rainbow trout Oncorhynchus mykiss and telomerase activity was demonstrated early and late in cell line development. When RTHDF cells were exposed to bioenergetic stress, i.e. anoxia, activation of the stress activated member of the mitogen-activated protein kinase family, p38MAPK and induction of heat shock protein (Hsp70) were evident. The time-course of the p38MAPK activation and the induction of Hsp70 expression in RTHDF were studied in response to chemically induced anoxia. p38MAPK was activated rapidly, with maximal activity after 10 min of anoxia. Hsp70 was induced after 30 min of anoxia, followed by overnight recovery in growth medium at 21° C. Using the p38MAPK -specific inhibitor SB203580, the enhanced expression of Hsp70 occurred independently of p38MAPK activation in RTHDF. These data suggests that RTHDF can be useful in studying biochemical responses of teleost cells to environmental stress. [source] Ischemia activates JNK/c-Jun/AP-1 pathway to up-regulate 14-3-3, in astrocyteJOURNAL OF NEUROCHEMISTRY, Issue 2009Yan Dong Abstract Ischemia occurs in the brain as the result of stroke and other related injuries and few therapies are effective. If more is understood then potential treatments could be investigated. It was previously reported that 14-3-3, could be up-regulated by ischemia in astrocyte to protect cells from ischemia-induced apoptosis. In this study, we attempted to uncover the mechanism responsible for this 14-3-3, up-regulation in primary culture of astrocytes under ischemic-like conditions. It was found that in vitro ischemia may activate PI3K/Akt and MAPK signaling pathways. Astrocyte cultures were treated with LY294002 (PI3K inhibitor), U0126 (ERK inhibitor), SB203580 (p38 inhibitor) and SP600125 (JNK inhibitor). Only SP600125 could inhibit the ischemia-induced 14-3-3, up-regulation in astrocytes. At the same time, we observed an ischemia-induced nuclear translocation of p-c-Jun, a major downstream component of JNK. Inhibition of AP-1 with curcumin also inhibited 14-3-3, up-regulation indicating that ischemia-induced up-regulation of 14-3-3, in astrocyte involves activation of the JNK/p-c-Jun/AP-1 pathway. [source] Participation of protein kinase C , isoform and extracellular signal-regulated kinase in neurite outgrowth of GT1 hypothalamic neuronsJOURNAL OF NEUROCHEMISTRY, Issue 6 2002Youngshik Choe Abstract In the present study, we investigated the selective role of protein kinase C (PKC) isoforms on neurite outgrowth of the GT1 hypothalamic neurons using several PKC isoform-selective inhibitors and transfection-based expression of enhanced green fluorescence protein (EGFP)-fused PKC isoforms. 12- O -Tetradecanoylphorbol-13-acetate (TPA) induced neurite outgrowth and growth cone formation, effects that were blocked by GF 109203X (a PKC inhibitor), safingolTM(a PKC,-selective inhibitor), but not by rottlerinTM (a PKC,-selective inhibitor), indicating that PKC, may be selectively involved in neurite outgrowth and cytoskeletal changes of filamentous actin and ,-tubulin. To define the differential localization of PKC isoforms, EGFP-tagged PKC,, PKC,, and PKC, were transfected into GT1 neuronal cells. TPA treatment induced relocalization of PKC,-EGFP to growth cones and cell,cell adhesion sites, PKC,-EGFP to the nucleus, and PKC,-EGFP to the membrane ruffle, respectively. An EGFP chimera of the catalytic domain of PKC, (PKC,-Cat-EGFP), the expression of which was inducible by doxycycline, was employed to directly ascertain the effect of PKC, enzymatic activity on neurite outgrowth of GT1 cells. Transient transfection of PKC,-Cat-EGFP alone increased the neurite-outgrowth and doxycycline treatment further augmented the number of neurite-containing cells. We also examined the involvement of the extracellular signal-regulated kinase (ERK) MAP kinase in TPA-induced neurite outgrowth. TPA treatment increased phosphorylated ERK MAP kinase, but not p38 MAP kinase. Specific inhibition of PKC, with safingol blocked the phosphorylation of ERK induced by TPA. More importantly, both neurite outgrowth and phosphorylation of ERK by TPA were blocked by PD 098059, a specific inhibitor of MEK (MAP kinase/ERK kinase-1), but not by SB203580, a specific inhibitor of p38 MAP kinase. These results demonstrate that PKC, isoform-specific activation is involved in neurite outgrowth of GT1 hypothalamic neuronal cells via ERK, but not the p38 MAP kinase signal pathway. [source] | |||||||||||||||||||||||||||||||