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Inhibitor U0126 (inhibitor + u0126)
Kinds of Inhibitor U0126 Selected Abstracts7-Ketocholesterol-induced apoptosisFEBS JOURNAL, Issue 12 2005Involvement of several pro-apoptotic but also anti-apoptotic calcium-dependent transduction pathways Oxysterols, and particularly 7-ketocholesterol, appear to be strongly involved in the physiopathology of atherosclerosis. These molecules are suspected to be cytotoxic to the cells of the vascular wall and monocytes/macrophages, particularly by inducing apoptosis. Previous studies have demonstrated that 7-ketocholesterol-induced apoptosis is triggered by a sustained increase of cytosolic-free Ca2+, which elicits the mitochondrial pathway of apoptosis by activation of the calcium-dependent phosphatase calcineurin, leading to dephosphorylation of the ,BH3 only' protein BAD. However, thorough study of the results suggests that other pathways are implicated in 7-ketocholesterol-induced cytotoxicity. In this study, we demonstrate the involvement of two other calcium-dependent pathways during 7-ketocholesterol-induced apoptosis. The activation of the MEK,ERK pathway by the calcium-dependent tyrosine kinase PYK 2, a survival pathway which delays apoptosis as shown by the use of the MEK inhibitor U0126, and a pathway involving another pro-apoptotic BH3 only protein, Bim. Indeed, 7-ketocholesterol treatment of human monocytic THP-1 cells induces the release of Bim-LC8 from the microtubule-associated dynein motor complex, and its association with Bcl-2. Therefore, it appears that 7-ketocholesterol-induced apoptosis is a complex phenomenon resulting from calcium-dependent activation of several pro-apoptotic pathways and also one survival pathway. [source] Down-regulation of the PI3-kinase/Akt pathway by ERK MAP kinase in growth factor signalingGENES TO CELLS, Issue 9 2008Hideko Hayashi The ERK MAP kinase and PI3-kinase/Akt pathways are major intracellular signaling modules, which are known to regulate diverse cellular processes including cell proliferation, survival and malignant transformation. However, it has not been fully understood how these two pathways interact with each other. Here, we demonstrate that inhibition of the ERK pathway by the MEK inhibitor U0126 or PD98059 significantly potentiates EGF- and FGF-induced Akt phosphorylation at both Thr308 and Ser473. We also show that hyperactivation of the ERK pathway greatly attenuates EGF- and FGF-induced Akt phosphorylation. Furthermore, the enhanced Akt phosphorylation induced by U0126 is inhibited by the PI3-kinase inhibitor LY294002, and is accompanied by the up-regulation of Ras activity. These results suggest that the ERK pathway inhibition enhances Akt phosphorylation through the Ras/PI3-kinase pathway. Thus, our results demonstrate that the ERK pathway negatively modulates the PI3-kinase/Akt pathway in response to growth factor stimulation. [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] Signalling responses linked to betulinic acid-induced apoptosis are antagonized by MEK inhibitor U0126 in adherent or 3D spheroid melanoma irrespective of p53 statusINTERNATIONAL JOURNAL OF CANCER, Issue 5 2006Manuel Rieber Abstract MEK1/2 inhibitors like U0126 can potentiate or antagonize the antitumor activity of cytotoxic agents such as cisplatin, paclitaxel or vinblastine, depending on the drug or the target cells. We now investigated whether U0126, differentially regulates melanoma signaling in response to UV radiation or betulinic acid, a drug lethal against melanoma. This report shows that U0126 inhibits early response (ERK) kinase activation and cyclin A expression in wt p53 C8161 melanoma exposed to either UV radiation or betulinic acid. However, U0126 does not protect from UV damage, but counteracts betulinic acid-mediated apoptosis in the same cells. Protection from the latter drug by joint treatment with U0126 was also evident in wt p53 MelJuso melanoma and mutant p53 WM164 melanoma. The latter cells were the most responsive to betulinic acid, showing a selective decline in the cdk4 protein, without a comparable change in other key cell cycle proteins like cdc2, cdk2, cdk7 or cyclin A, prior to apoptosis-associated PARP fragmentation. Laser scanning cytometry also showed that betulinic acid induced a significant increase in chromatin condensation in WM164 melanoma irrespective of whether they were in adherent form or as multicellular spheroids. All these betulinic acid-induced changes were counteracted by U0126. Our data show for the first time that (a) cdk4 protein is an early target of betulinic acid-induced apoptosis and (b) unrestricted ERK signaling favours betulinic acid-induced apoptosis, but this is counteracted by U0126, partly through counteracting chromatin condensation and restoring Akt activation decreased by betulinic acid treatment. © 2005 Wiley-Liss, Inc. [source] Enhanced cytotoxicity induced by gefitinib and specific inhibitors of the Ras or phosphatidyl inositol-3 kinase pathways in non-small cell lung cancer cellsINTERNATIONAL JOURNAL OF CANCER, Issue 1 2006Maarten L. Janmaat Abstract In this study, we have characterized a panel of NSCLC cell lines with differential sensitivity to gefitinib for activating mutations in egfr, pik3ca, and k-ras, and basal protein expression levels of PTEN. The egfr mutant NSCLC cell line H1650 as well as the egfr wild type cell lines H292 and A431 were highly sensitive to gefitinib treatment, indicating that other factors determine gefitinib-sensitivity in egfr wild type cells. Activating k-ras mutations were specifically detected in gefitinib-resistant cells, suggesting that the occurrence of k-ras mutations is correlated with resistance to EGFR antagonists. No pik3ca mutations were detected within the panel of cell lines, and PTEN protein expression levels did not correlate with gefitinib sensitivity. Gefitinib effectively blocked Akt and Erk phosphorylation in two gefitinib-sensitive NSCLC cell lines, further supporting our previous findings that persistent activity of the PI3K/Akt and/or Ras/Erk pathways is associated with gefitinib-resistance of NSCLC cell lines. Gefitinib-resistant NSCLC cell lines, showing EGFR-independent activity of the PI3K/Akt or Ras/Erk pathways, were treated with gefitinib in combination with specific inhibitors of mTOR, P13K, Ras, and MEK. Additive cytotoxicity was observed in A549 cells co-treated with gefitinib and the MEK inhibitor U0126 or the farnesyl transferase inhibitor SCH66336 and in H460 cells treated with gefitinib and the PI3K inhibitor LY294002, but not in H460 cells treated with gefitinib and rapamycin. These data suggest that combination treatment of NSCLC cells with gefitinib and specific inhibitors of the PI3K/Akt and Ras/Erk pathways may provide a successful strategy. © 2005 Wiley-Liss, Inc. [source] Adrenomedullin regulates expressions of transforming growth factor-,1 and ,1-induced matrix metalloproteinase-2 in hepatic stellate cellsINTERNATIONAL JOURNAL OF EXPERIMENTAL PATHOLOGY, Issue 3 2006Yi Wang Summary Adrenomedullin (AM), a peptide isolated from human pheochromocytoma, can be produced and secreted by various types of cells including hepatic stellate cells (HSCs), and its possible role in HSCs is not clear now. In the present study, the interactive regulation between transforming growth factor (TGF)-,1 and AM and the effect of AM on TGF-,1-induced matrix metalloproteinase (MMP)-2 expression in HSCs were investigated. TGF-,1 and AM inhibited gene transcript level mutually (real-time reverse transcription-polymerase chain reaction). AM suppressed the protein expression level of TGF-,1 (Western blot), but TGF-,1 might have no effect on AM secretion level. MMP-2 protein expression in HSCs was increased in response to TGF-,1, and upregulation of MMP-2 expression stimulated with TGF-,1 was suppressed by AM in dose-dependent manner (Western blot). AM decreased the phosphorylation level of extracellular signal-regulated kinase (ERK) in HSCs treated with TGF-,1, and TGF-,1-induced MMP-2 expression was suppressed by adding Mitogen-activated protein Kinase/ERK (MEK) inhibitor U0126 (Western blot). Our results suggest that AM may intervene the activation of HSCs by inhibiting TGF-,1 production and TGF-,1-induced MMP-2 expression; AM may suppress the upregulation of MMP-2 expression induced by TGF-,1 partially through ERK pathway. [source] Interleukin-1, enhances nucleotide-induced and ,-secretase-dependent amyloid precursor protein processing in rat primary cortical neurons via up-regulation of the P2Y2 receptorJOURNAL OF NEUROCHEMISTRY, Issue 5 2009Qiongman Kong Abstract The heterologous expression and activation of the human P2Y2 nucleotide receptor (P2Y2R) in human 1321N1 astrocytoma cells stimulates ,-secretase-dependent cleavage of the amyloid precursor protein (APP), causing extracellular release of the non-amyloidogenic protein secreted amyloid precursor protein (sAPP,). To determine whether a similar response occurs in a neuronal cell, we analyzed whether P2Y2R-mediated production of sAPP, occurs in rat primary cortical neurons (rPCNs). In rPCNs, P2Y2R mRNA and receptor activity were virtually absent in quiescent cells, whereas overnight treatment with the pro-inflammatory cytokine interleukin-1, (IL-1,) up-regulated both P2Y2R mRNA expression and receptor activity by four-fold. The up-regulation of the P2Y2R was abrogated by pre-incubation with Bay 11-7085, an I,B-, phosphorylation inhibitor, which suggests that P2Y2R mRNA transcript levels are regulated through nuclear factor-,-B (NF,B) signaling. Furthermore, the P2Y2R agonist Uridine-5,-triphosphate (UTP) enhanced the release of sAPP, in rPCNs treated with IL-1, or transfected with P2Y2R cDNA. UTP-induced release of sAPP, from rPCNs was completely inhibited by pre-treatment of the cells with the metalloproteinase inhibitor TACE inhibitor (TAPI-2) or the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002, and was partially inhibited by the MAPK/extracellular signal-regulated kinase inhibitor U0126 and the protein kinase C inhibitor GF109203. These data suggest that P2Y2R-mediated release of sAPP, from cortical neurons is directly dependent on a disintegrin and metalloproteinase (ADAM) 10/17 and PI3K activity, whereas extracellular signal-regulated kinase 1/2 and PI3K activity may indirectly regulate APP processing. These results demonstrate that elevated levels of pro-inflammatory cytokines associated with neurodegenerative diseases, such as IL-1,, can enhance non-amyloidogenic APP processing through up-regulation of the P2Y2R in neurons. [source] Mitogen-activated protein kinase pathway mediates N -(4-hydroxyphenyl)retinamide-induced neuronal differentiation in the ARPE-19 human retinal pigment epithelial cell lineJOURNAL OF NEUROCHEMISTRY, Issue 2 2008William Samuel Abstract We have shown previously that N -(4-hydroxyphenyl)retinamide (4HPR, fenretinide), a retinoic acid derivative, induces neuronal differentiation in cultured human retinal pigment epithelial (RPE) cells [Chen et al., J. Neurochem., 84 (2003), 972]. We asked the question whether the mitogen-activated protein kinase (MAPK) pathway is involved in the regulation of the 4HPR-induced neuronal differentiation of RPE (ARPE-19) cells. When we treated ARPE-19 cells with 4HPR, c-Raf and MEK1/2 kinase were activated resulting in activation of the downstream effector ERK1/2 and of SAPK/JNK. By blocking the upstream kinase MEK1/2 with specific inhibitor U0126 we abrogated the 4HPR-induced phosphorylation of ERK1/2 and SAPK/JNK, indicating that the neuronal differentiation occurs through a positive cross-talk between the ERK and the SAPK/JNK pathways. Both U0126 and the suppression of ERK1/2 expression with small interfering RNA effectively blocked the 4HPR-induced neuronal differentiation of RPE cells and the expression of calretinin. The activated ERK1/2 then induced a sequential activation of p90RSK, and increase in phosphorylation of transcription factors c- fos and c- jun leading to transcriptional activation of AP-1. Taken together, our results clearly demonstrate that c-Raf/MEK1/2 signaling cascade involving ERK1/2 plays a central role in mediating the 4HPR-induced neuronal differentiation and calretinin expression in the human ARPE-19 retinal pigment epithelial cell line. [source] Angiotensin II promotes the phosphorylation of cyclic AMP-responsive element binding protein (CREB) at Ser133 through an ERK1/2-dependent mechanismJOURNAL OF NEUROCHEMISTRY, Issue 6 2001Martín Cammarota In cells from the adrenal medulla, angiotensin II (AII) regulates both the activity and mRNA levels of catecholamine biosynthetic enzymes whose expression is thought to be under the control of cAMP-responsive element (CRE) binding protein (CREB). In this study, we evaluated the effect of AII stimulation on CREB phosphorylation at Ser133 (pCREB) in bovine adrenal chromaffin cells (BACC). We found that AII produces a rapid and AII type-1 receptor (AT1)-dependent increase in pCREB levels, which is blocked by the MEK1/2 inhibitor U0126 but not by H-89, SB203580 or KN-93, suggesting that it is mediated by the extracellular-regulated protein kinases 1 and 2 (ERK1/2) and not by cAMP-dependent protein kinase (PKA), p38 mitogen-activated protein kinase (p38MAPK) or Ca2+/calmodulin-dependent protein kinases (CaMKs) dependent pathways. Gel-shift experiments showed that the increase in pCREB levels is accompanied by an ERK1/2-dependent upregulation of CRE-binding activity. We also found that AII promotes a rapid and reversible increase in the activity of the non-receptor tyrosine kinase Src and that the inhibition of this enzyme completely blocks the AII-induced phosphorylation of ERK1/2, the CREB kinase p90RSK and CREB. Our data support the hypothesis that in BACC, AII upregulates CREB functionality through a mechanism that requires Src-mediated activation of ERK 1/2 and p90RSK. [source] Hyperosmotic mannitol induces Src kinase-dependent phosphorylation of ,-catenin in cerebral endothelial cellsJOURNAL OF NEUROSCIENCE RESEARCH, Issue 6 2005Attila Farkas Abstract Mannitol, which is a cell-impermeable and nontoxic polyalcohol, has been shown to be a useful tool for reversible opening of the blood,brain barrier (BBB). Despite successful clinical trials, the molecular mechanism of the mannitol-induced changes in cerebral endothelial cells (CECs) are poorly understood. For our experiments, we used CECs in culture, which were treated with different, clinically relevant concentrations of mannitol. We found that mannitol induced a rapid, concentration-dependent, and reversible tyrosine phosphorylation of a broad range of proteins between 50 and 190 kDa. One of the targets of tyrosine phosphorylation turned out to be the adherens junction protein ,-catenin. Phosphorylation of ,-catenin on tyrosine residues caused its subcellular redistribution and its dissociation from cadherin and ,-catenin as shown by coimmunoprecipitation studies. All these effects could be inhibited by the Src kinase inhibitor PP-1 but not by the Erk inhibitor U0126, the Rho kinase inhibitor Y27632, or the calcium channel blocker verapamil. Because ,-catenin is a key component of the junctional complex, its Src-mediated phpsphorylation may play an important role in the mannitol induced reversible opening of the BBB. © 2005 Wiley-Liss, Inc. [source] The upregulation of heat shock protein 47 in human gingival fibroblasts stimulated with cyclosporine AJOURNAL OF PERIODONTAL RESEARCH, Issue 3 2010T.-Y. Chang Chang T-Y, Tsai C-H, Chang Y-C. The upregulation of heat shock protein 47 in human gingival fibroblasts stimulated with cyclosporine A. J Periodont Res 2010; 45: 317,322. © 2009 John Wiley & Sons A/S Background and Objective:, Heat shock protein 47 (Hsp47), a collagen-specific molecular chaperone, is involved in the processing and/or secretion of procollagen. Heat shock protein 47 is consistently and dramatically upregulated in a variety of fibrotic diseases. The aim of this study was to compare Hsp47 expression in normal gingival tissues and cyclosporine A-induced gingival overgrowth specimens and further explore the potential mechanisms that may lead to induction of Hsp47 expression. Material and Methods:, Fifteen cyclosporine A-induced gingival overgrowth specimens and five normal gingival tissues were examined by immunohistochemistry. Western blot was used to investigate the effects of cyclosporine A on the expression of Hsp47 in human gingival fibroblasts. In addition, Aggregatibacter actinomycetemcomitans, interleukin-1, (IL-1,) and mitogen-activated protein kinase kinase (MEK) inhibitor U0126 were added to seek the possible regulatory mechanisms of Hsp47 expression. Results:, A significantly higher percentage of cells positively stained for Hsp47 was noted in the cyclosporine A-induced gingival overgrowth group than in the normal gingival group (p < 0.05). Expression of Hsp47 was observed mainly in the cytoplasm of fibroblasts, endothelial cells, epithelial cells and inflammatory cells. Expression of Hsp47 was significantly higher in cyclosporine A-induced gingival overgrowth specimens with higher levels of inflammatory infiltrates (p < 0.05). Cyclosporine A upregulated Hsp47 expression in human gingival fibroblasts in a dose-dependent manner (p < 0.05). The addition of A. actinomycetemcomitans or interlukin-1, significantly increased Hsp47 expression compared with cyclosporine A alone (p < 0.05). The MEK inhibitor U0126 was found to inhibit cyclosporine A-induced Hsp47 expression (p < 0.05). Conclusion:, Expression of Hsp47 is significantly upregulated in cyclosporine A-induced gingival overgrowth specimens, and Hsp47 expression induced by cyclosporine A in fibroblasts may be mediated by the MEK signal transduction pathway. The expression of Hsp47 could be significantly enhanced by A. actinomycetemcomitans and interlukin-1,. [source] Signal transduction pathways involved in the stimulation of tissue type plasminogen activator by interleukin-1, and Porphyromonas gingivalis in human osteosarcoma cellsJOURNAL OF PERIODONTAL RESEARCH, Issue 5 2006Yu-Chao Chang Background:, Recently, evidences have shown that tissue type plasminogen activator (t-PA) may play an important role in the pathogenesis of periodontal diseases. However, the mechanisms and signal transduction pathways involved in the production of t-PA in human osteosarcoma cells are not fully understood. Objectives:, The purpose of this study was to investigate the caseinolytic activity in human osteosarcoma cell line U2OS cells stimulated with interleukin-1, (IL-1,) or Porphyromonas gingivalis in the absence or presence of p38 inhibitor SB203580, mitogen-activated protein kinase kinase (MEK) inhibitor U0126, and phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002. Methods:, IL-1, and the supernatants of P. gingivalis were used to evaluate the caseinolytic activity in U2OS cells by using casein zymography and enzyme-linked immunosorbent assay (ELISA). Furthermore, to search possible signal transduction pathways, SB203580, U0126, and LY294002 were added to test how they modulated the caseinolytic activity. Results:, Casein zymography exhibited a caseinolytic band with a molecular weight of approximately 70 kDa, suggestive of the presence of t-PA. Secretion of t-PA was found to be stimulated with IL-1, and P. gingivalis during a 2-day culture period (p < 0.05). From the results of casein zymography and ELISA, SB203580, U0126, and LY294002 significantly reduced the IL-1, or P. gingivalis -stimulated t-PA production, respectively (p < 0.05). Conclusions:, Our findings demonstrated that IL-1, and P. gingivalis enhance t-PA production in human osteosarcoma cells, and that the signal transduction pathways p38, MEK, and PI3K are involved in the inhibition of t-PA. SB203580, U0126, and LY294002 suppress t-PA production and/or activity and may therefore be valuable therapeutics in t-PA-mediated periodontal destruction, and might be proved clinically useful agents, in combination with standard treatment modalities, in the treatment of periodontitis. [source] Prostaglandin E2 induces vascular endothelial growth factor secretion in prostate cancer cells through EP2 receptor-mediated cAMP pathwayMOLECULAR CARCINOGENESIS, Issue 11 2007Xingya Wang Abstract Prostaglandin E2 (PGE2) has been shown to induce expression of vascular endothelial growth factor (VEGF) and other signaling molecules in several cancers. PGE2 elicits its functions though four G-protein coupled membrane receptors (EP1,4). In this study, we investigated the role of EP receptors in PGE2 -induced molecular events in prostate cancer cells. qRT-PCR analysis revealed that PC-3 cells express a substantially higher level of EP2 and moderately higher EP4 than DU145 and LNCaP cells. LNCaP cells had virtually no detectable EP2 mRNA. EP1 and EP3 mRNAs were not detected in these cells. Treatment of prostate cancer cells with PGE2 (1 nM,10 µM) increased both VEGF secretion and cyclic adenosine monophosphate (cAMP) production. Levels of induction in PC-3 cells were greater than in DU145 and LNCaP cells. The selective EP2 agonist CAY10399 also significantly increased VEGF secretion and cAMP production in PC-3 cells, but not in DU145 and LNCaP cells. Moreover, PGE2 and CAY10399 increased mitogen activated protein kinase/extracellular signal regulated kinase (MAPK/Erk) and Akt phosphorylation in PC-3 and DU145 cells, but not in LNCaP cells. However, neither the MAPK/Erk inhibitor U0126 nor the PI3K/Akt inhibitor LY294002 abolished PGE2 -induced VEGF secretion in PC-3 cells. We further demonstrated that the adenylate cyclase activator forskolin and the cAMP anologue 8-bromo-cAMP mimicked the effects of PGE2 on VEGF secretion in PC-3 cells. Meanwhile, the adenylate cyclase inhibitor 2,5,-dideoxyadenosine, at concentrations that inhibited PGE2 -induced cAMP, significantly blocked PGE2 -induced VEGF secretion in PC-3 cells. We conclude that PGE2 -induced VEGF secretion in prostate cancer cells is mediated through EP2-, and possibly EP4-, dependent cAMP signaling pathways. © 2007 Wiley-Liss, Inc. [source] Inactivation of MAPK affects centrosome assembly, but not actin filament assembly, in mouse oocytes maturing in vitroMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 7 2007Seung-Eun Lee Abstract Mitogen-activated protein kinase (MAPK) plays a crucial role in meiotic maturation of mouse oocytes. In order to understand the mechanism by which MAPK regulates meiotic maturation, we examined the effects of the MAPK pathway inhibitor U0126 on microtubule organization, ,-tubulin and nuclear mitotic apparatus protein (NuMA) distribution, and actin filament assembly in mouse oocytes maturing in vitro. Western blotting with antibodies that detect active, phosphorylated MAPK revealed that MAPK was inactive in fully grown germinal vesicle (GV) oocytes. Phosphorylated MAPK was first detected 3 hr after the initiation of maturation cultures, was fully active at 6 hr, and remained active until metaphase II. Treatment of GV stage oocytes with 20 µM U0126 completely blocked MAPK phosphorylation, but did not affect GV breakdown (GVBD). However, the oocytes did not progress to the Metaphase I stage, which would normally occur after 9 hr in the maturation cultures. The inhibition of MAPK resulted in abnormal spindles and abnormal distributions of ,-tubulin and NuMA, but did not affect actin filament assembly. In oocytes treated with U0126 after GVBD, polar body extrusion was normal, but the organization of the metaphase plate and chromosome segregation were abnormal. In conclusion, the meiotic abnormalities caused by U0126, a specific inhibitor of MAPK signaling, indicate that MAPK plays an important regulatory role in microtubule and centrosome assembly, but not actin filament assembly. Mol. Reprod. Dev. © 2006 Wiley-Liss, Inc. Mol. Reprod. Dev. 74: 904,911, 2007. © 2007 Wiley-Liss, Inc. [source] Regulation of type I plasminogen activator inhibitor in human gingival fibroblasts with cyclosporine AORAL DISEASES, Issue 4 2010Y-C Ho Oral Diseases (2010) 16, 396,401 Objectives:, Cyclosporine A (CsA) is used as an immunosuppressive agent and its prominent side effect is the induction of gingival overgrowth. Type I plasminogen activator inhibitor (PAI-1) has shown to play an important role in CsA-induced gingival overgrowth. However, little is known about whether factors can modulate CsA-induced PAI-1 expression. Methods:, Cytotoxicity, reverse transcriptase-polymerase chain reaction, and enzyme-linked immunosorbent assay were used to investigate the effects of Human gingival fibroblasts (HGFs) exposed to CsA. In addition, Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, interlukin-1,, tumor necrosis factor-,, mitogen-activated protein kinase kinase (MEK) inhibitor U0126, signal-regulated protein kinase (ERK) inhibitor PD98059 and cell-permeable glutathione precursor N -acetyl- L -cysteine (NAC) were added to test how they modulated the effects of CsA-induced PAI-1 expression. Results:, The concentration of CsA higher than 500 ng ml,1 demonstrated cytotoxicity to HGFs (P < 0.05). Periodontal pathogens as well as proinflammatory cytokines were found to increase the CsA-induced PAI-1 mRNA and protein expression (P < 0.05). Pharmacological agents NAC, U0126, and PD98059 were found to decrease the CsA-induced PAI-1 mRNA and protein expression (P < 0.05). Conclusions:, Cyclosporine A (CsA) may predispose to gingival overgrowth under inflammatory environments. The regulation of PAI-1 expression induced by CsA might be critically related with the intracellular glutathione and the ERK-MAPK pathway. [source] Cyclooxygenase-2 Expression Induced by Photofrin Photodynamic Therapy Involves the p38 MAPK Pathway,PHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 2 2008Marian Luna Photodynamic therapy (PDT), using the porphyrin photosensitizer Photofrin (PH), is approved for the clinical treatment of solid tumors. In addition to the direct cytotoxic responses of PH,PDT-mediated oxidative stress, this procedure also induces expression of angiogenic and prosurvival molecules including cyclooxygenase-2 (COX-2). In vivo treatment efficacy is improved when PH-PDT is combined with inhibitors of COX-2. In the current study we evaluated the signaling pathways involved with PH,PDT-mediated COX-2 expression in a mouse fibrosarcoma cell line. COX-2 promoter reporter constructs with mutated transcription elements documented that the nuclear factor kappa B (NF,B) element, cyclic-AMP response element 2 (CRE-2), CCAAT/enhancer binding protein (C/EBP) element and activator binding protein-1 (AP-1) element were responsive to PH-PDT. Transcription factor binding assays demonstrated that nuclear protein binding to NF,B, CRE-2, c-fos and c-jun elements were elevated following PH-PDT. Kinase phosphorylation upstream of COX-2 expression was also examined following PH-PDT. Stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) and c-Jun were phosphorylated following PH-PDT but the SAPK/JNK inhibitor SP600125 failed to attenuate COX-2 expression. In contrast, p38 mitogen-activated protein kinase (MAPK), which activates CRE-2 binding, was phosphorylated following PH-PDT and inhibitors of p38 MAPK, SB203580 and SB202190, decreased PH,PDT-induced COX-2 expression at both the mRNA and protein levels. Extracellular signal-regulated kinase (ERK1/2) phosphorylation, which also increases CRE-2 binding activity, was initially high in untreated cells, decreased immediately following PH-PDT and then rapidly increased. MEK1/2 is immediately upstream of ERK1/2 and the MEK1 inhibitor PD98059 failed to attenuate COX-2 expression while the MEK1/2 inhibitor U0126 induced a slight decrease in COX-2 expression. The NF,B inhibitor SN50 failed to reduce COX-2 expression. These results demonstrate that multiple protein kinase cascades can be activated by oxidative stress and that the p38 MAPK signaling pathway and CRE-2 binding are involved in COX-2 expression following PH-PDT. [source] Activation of MAP Kinase in Lumbar Spinothalamic Cells Is Required for EjaculationTHE JOURNAL OF SEXUAL MEDICINE, Issue 7 2010Michael D. Staudt MSc ABSTRACT Introduction., Ejaculation is a reflex controlled by a spinal ejaculation generator located in the lumbosacral spinal cord responsible for the coordination of genital sensory with autonomic and motor outputs that regulate ejaculation. In the male rat, a population of lumbar spinothalamic cells (LSt cells) comprises an essential component of the spinal ejaculation generator. LSt cells are activated with ejaculation, but the nature of the signal transduction pathways involved in this activation is unknown. Moreover, it is unknown if LSt cell activation is required for expression of ejaculation. Aim., The current study tested the hypothesis that ejaculatory reflexes are triggered via activation of the mitogen-activated protein (MAP) kinase signaling pathway in the LSt cells. Methods., Expression of phosphorylated extracellular signal-related kinases 1 and 2 (pERK) was investigated following mating behavior, or following ejaculation induced by electrical stimulation of the dorsal penile nerve (DPN) in anesthetized, spinalized male rats. Next, the effects of intrathecal or intraspinal delivery of Mitogen-activated protein/extracellular signal-regulated kinase (MEK) inhibitor U0126 on DPN stimulation-induced ejaculation was examined. Main Outcome Measures., Expression of pERK in LSt cells and associated areas was analyzed. Electromyographic recordings of the bulbocavernosus muscle were recorded in anesthetized, spinalized rats. Results., Results indicate that the MAP kinase signaling pathway is activated in LSt cells following ejaculation in mating animals or induced by DPN stimulation in anesthetized, spinalized animals. Moreover, ERK activation in LSt cells is an essential trigger for ejaculation, as DPN stimulation-induced reflexes were absent following administration of MEK inhibitor in the L3-L4 spinal area. Conclusion., These data provide insight into the nature of the signal transduction pathways involved in the activation of ejaculation through LSt cells. The data demonstrate that ERK activation in LSt cells is essential for ejaculation and contribute to a more detailed understanding of the spinal generation of ejaculation. Staudt MD, de Oliveira CVR, Lehman MN, McKenna KE, and Coolen LM. Activation of MAP kinase in lumbar spinothalamic cells is required for ejaculation. J Sex Med 2010;7:2445,2457. [source] Sphingosine kinase 1 is critically involved in nitric oxide-mediated human endothelial cell migration and tube formationBRITISH JOURNAL OF PHARMACOLOGY, Issue 7 2010Stephanie Schwalm Background and purpose:, Sphingosine kinases (SKs) convert sphingosine to sphingosine 1-phosphate (S1P), which is a bioactive lipid that regulates a variety of cellular processes including proliferation, differentiation and migration. Experimental approach:, We used the human endothelial cell line EA.hy926 to investigate the effect of nitric oxide (NO) donors on SK-1 expression, and on cell migration and tube formation. Key results:, We showed that exposure of EA.hy926 cells to Deta-NO (125,1000 µM) resulted in a time- and concentration-dependent up-regulation of SK-1 mRNA and protein expression, and activity with a first significant effect at 250 µM of Deta-NO. The increased SK-1 mRNA expression resulted from an enhanced SK-1 promoter activity. A similar effect was also seen with various other NO donors. In mechanistic terms, the NO-triggered effect occurred independently of cGMP, but involved the classical mitogen-activated protein kinase cascade because the MEK inhibitor U0126 abolished the NO-induced SK-1 expression. The effect of NO was also markedly reduced by the thiol-reducing agent N -acetylcysteine, suggesting a redox-dependent mechanism. Functionally, Deta-NO triggered an increase in the migration of endothelial cells in an adapted Boyden chamber assay, and also increased endothelial tube formation in a Matrigel assay. These responses were both abolished in cells depleted of SK-1. Conclusions and implications:, These data show that NO donors up-regulate specifically SK-1 expression and activity in human endothelial cells, and SK-1 in turn critically contributes to the migratory capability and tube formation of endothelial cells. Thus, SK-1 may be considered an attractive novel target to interfere with pathological processes involving angiogenesis. [source] | |||||||||||||||||||||||||