Leptin stimulates tissue inhibitor of metalloproteinase-1 in human hepatic stellate cells: respective roles of the JAK/STAT and JAK-mediated H2O2-dependant MAPK pathways

Leptin is recognized as a profibrogenic hormone in the liver, but the mechanisms involved have not been clarified. The tissue inhibitor of metalloproteinase (TIMP)-1, which acts through inhibition of collagen degradation, is synthesized by activated hepatic stellate cells (HSC) in response to fibrogenic substances. The capacity of leptin to induce TIMP-1 and its signaling molecules were investigated in a human HSC cell line, LX-2. Leptin stimulated TIMP-1 protein, mRNA, and promoter activity. JAK1 and -2, as well as STAT3 and -5, were activated. After leptin, there was increased expression of tyrosine 1141-phosphorylated leptin receptor, which may contribute to STAT3 activation. AG 490, a JAK inhibitor, blocked JAK phosphorylation with concomitant inhibition of STAT activation, TIMP-1 mRNA expression, and promoter activity. Leptin also induced an oxidative stress, which was inhibited by AG 490, indicating a JAK mediation process. ERK1/2 MAPK and p38 were activated, which was prevented by catalase, indicating an H2O2-dependent mechanism. Catalase treatment resulted in total suppression of TIMP-1 mRNA expression and promoter activity. SB203580, a p38 inhibitor, prevented p38 activation and reduced TIMP-1 message half-life with down-regulation of TIMP-1 mRNA. These changes were reproduced by overexpression of the dominant negative p38alpha and p38beta mutants. PD098059, an ERK1/2 inhibitor, opposed ERK1/2 activation and TIMP-1 promoter activity, leading to TIMP-1 mRNA down-regulation. Thus, leptin has a direct action on liver fibrogenesis by stimulating TIMP-1 production in activated HSC. This process appears to be mediated by the JAK/STAT pathway via the leptin receptor long form and the H2O2-dependent p38 and ERK1/2 pathways via activated JAK.     

DLPC and SAMe prevent alpha1(I) collagen mRNA up-regulation in human hepatic stellate cells, whether caused by leptin or menadione

We previously reported that the combination of dilinoleoylphosphatidylcholine (DLPC) and S-adenosylmethionine (SAMe), which have antioxidant properties and antifibrogenic actions, prevented leptin-stimulated tissue inhibitor of metalloproteinase (TIMP)-1 production in hepatic stellate cells (HSCs) by inhibiting H2O2-mediated signal transduction. We now show that DLPC and SAMe inhibit alpha1(I) collagen mRNA expression induced by leptin or menadione in LX-2 human HSCs. We found that DLPC and SAMe prevent H2O2 generation and restore reduced glutathione (GSH) depletion whether caused by leptin or menadione. Blocking H2O2 signaling through ERK1/2 and p38 pathways resulted in a complete inhibition of leptin or menadione-induced alpha1(I) collagen mRNA. The inhibition of collagen mRNA by DLPC and SAMe combined is at least two times more effective than that by DLPC or SAMe alone. In conjunction with the prevention of TIMP-1 production, the ability of DLPC and SAMe to inhibit alpha1(I) collagen mRNA expression provides a mechanistic basis for these innocuous compounds in the prevention of hepatic fibrosis, because enhanced TIMP-1 and collagen productions are associated with hepatic fibrogenesis and their attenuation may diminish fibrosis.     

DLPC decreases TGF-beta1-induced collagen mRNA by inhibiting p38 MAPK in hepatic stellate cells

Dilinoleoylphosphatidylcholine (DLPC), the active component of polyenylphosphatidylcholine extracted from soybeans, decreases collagen accumulation induced by TGF-beta1 in cultured hepatic stellate cells (HSCs). Because DLPC exerts antioxidant effects and TGF-beta1 generates oxidative stress, we evaluated whether the antifibrogenic effect of DLPC is linked to its antioxidant action. In passage 1 culture of rat HSCs, TGF-beta1 induced a concentration-dependent increase in procollagen-alpha(1)(I) mRNA levels and enhanced intracellular H(2)O(2) and superoxide anion formation and lipid peroxidation but decreased GSH levels. These changes were prevented by DLPC. Upregulation of collagen mRNA by TGF-beta1 was likewise inhibited by catalase and p38 MAPK inhibitor SB-203580, suggesting involvement of H(2)O(2) and p38 MAPK signaling in this process. TGF-beta1 or addition of H(2)O(2) to HSCs activated p38 MAPK with a rise in procollagen mRNA level; these changes were blocked by catalase and SB-203580 and likewise by DLPC. alpha-Smooth muscle actin abundance in HSCs was not altered by TGF-beta1 treatment (with or without DLPC), indicating that downregulation of procollagen mRNA by DLPC was not due to alteration in HSC activation. These results demonstrate that DLPC prevents TGF-beta1-induced increase in collagen mRNA by inhibiting generation of oxidative stress and associated H(2)O(2)-dependent p38 MAPK activation, which explains its antifibrogenic effect. DLPC, an innocuous phospholipid, may be considered for prevention and treatment of liver fibrosis.     

Oxidative signaling in renal epithelium: Critical role of cytosolic phospholipase A2 and p38(SAPK)

Previous studies from this laboratory have demonstrated a critical role of cytosolic phospholipase A2 (cPLA2) and arachidonic acid in angiotensin II (Ang II) AT2 receptor-mediated signal transduction in renal epithelium. In primary proximal tubular epithelial cells exposed to hydrogen peroxide (H2O2), both the selective cPLA2 inhibitors and the cPLA2 antisense oligonucleotides significantly attenuated H2O2-induced arachidonic acid liberation and activation of p38(SAPK), ERK1/2, and Akt1. This H2O2-induced kinase activation was significantly attenuated by a Src kinase inhibitor PP2, or by transient transfection of carboxyl-terminal Src kinase (CSK) that maintained Src in the dormant form. Under basal conditions, Src coimmunoprecipitated with epidermal growth factor receptor (EGFR), while H2O2 increased EGFR phosphorylation in the complex. We observed that inhibition of EGFR kinase activity with AG1478 significantly attenuated H2O2-induced p38(SAPK) and ERK1/2 activation, but did not inhibit Akt1 activation. Furthermore, it seems that p38(SAPK) is upstream of ERK1/2 and Akt1, since a p38(SAPK) inhibitor SB203580 significantly blocked H2O2-induced activation of ERK1/2 and Akt1. Interestingly, overexpression of the dominant-negative p38(SAPK) isoform alpha inhibited ERK1/2 but not Akt1 activation. Our observations demonstrate that in these nontransformed cells, activation of cPLA2 is a converging point for oxidative stress and Ang II, which share common downstream signaling mechanisms including Src and EGFR. In addition, p38(SAPK) provides a positive input to both growth and antiapoptotic signaling pathways induced by acute oxidative stress.     

Activation of extracellular signal-regulated kinase 1/2 inhibits type I collagen expression by human skin fibroblasts

Treatment with the lipid second messenger, ceramide, activates extracellular signal-regulated kinase-1/2 (ERK1/2), c-Jun N-terminal kinase, and p38 in human skin fibroblasts and induces their collagenase-1 expression (Reunanen, N., Westermarck, J., H?kkinen, L., Holmstr?m, T. H., Elo, I., Eriksson, J. E., and K?h?ri, V.-M. (1998) J. Biol. Chem. 273, 5137-5145). Here we show that C(2)-ceramide inhibits expression of type I and III collagen mRNAs in dermal fibroblasts, suppresses proalpha2(I) collagen promoter activity, and reduces stability of type I collagen mRNAs. The down-regulatory effect of C(2)-ceramide on type I collagen mRNA levels was abrogated by protein kinase C inhibitors H7, staurosporine, and Ro-31-8220 and potently inhibited by a combination of MEK1,2 inhibitor PD98059 and p38 inhibitor SB203580. Activation of ERK1/2 by adenovirus-mediated expression of constitutively active MEK1 resulted in marked down-regulation of type I collagen mRNA levels and production in fibroblasts, whereas activation of p38 by constitutively active MAPK kinase-3b and MAPK kinase-6b slightly up-regulated type I collagen expression. These results identify the ERK1/2 signaling cascade as a potent negative regulatory pathway with respect to type I collagen expression in fibroblasts, suggesting that it mediates inhibition of collagen production in response to mitogenic stimulation and transformation.     

Endoglin modulation of TGF-beta1-induced collagen synthesis is dependent on ERK1/2 MAPK activation.

BACKGROUND/AIMS: Transforming growth factor-beta1 (TGF-beta1) plays a pivotal role in the extracellular matrix accumulation observed in fibrotic diseases. Endoglin is an important component of the TGF-beta receptor complex highly expressed in tissues undergoing fibrotic processes. Endoglin expression regulates the effect of TGF-beta on extracellular matrix synthesis. The purpose of our study has been to understand the molecular mechanism by which endoglin exerts its effects on fibrosis and the possible role of MAP kinases in these effects. METHODS: We have assessed in mock and in endoglin-transfected L6E9 myoblasts the effect of TGF-beta1 on collagen mRNA by Northern blot and effect of TGF-beta1 on collagen content in the cultured medium by [(3)H]-Proline incorporation into collagen proteins. Total and activated MAPK and their role on collagen synthesis were assessed by Western blot. RESULTS: TGF-beta1 induced an increase on alpha(2) (I) collagen mRNA expression and collagen accumulation in mock-transfected myoblasts, whereas the response was much lower in endoglintransfected cells. TGF-beta1 activated the ERK1/2 and p38 MAPK pathways but not the JNK pathway in L6E9 myoblasts. TGF-beta1-induced alpha(2) (I) collagen mRNA expression and collagen accumulation were completely inhibited by SB203580, in either mock or endoglintransfected myoblasts. PD98059 increased TGF-beta1 induced-collagen synthesis and accumulation in endoglin-transfected myoblasts but not in mock cells. CONCLUSION: Our studies demonstrate that TGF-beta1- induced collagen synthesis is mediated by p38 MAPK activation in L6E9 myoblasts. Furthermore, endoglin expression reduces basal and TGF-beta1 induced collagen synthesis when ERK1/2 pathway is operating.     

Angiotensin II suppresses growth arrest specific homeobox (Gax) expression via redox-sensitive mitogen-activated protein kinase (MAPK)

Oxidative stress is known to be involved in growth control of vascular smooth muscle cells (VSMCs). We and others have demonstrated that angiotensin II (Ang II) has an important role in vascular remodeling. Several reports suggested that VSMC growth induced by Ang II was elicited by oxidative stress. Gax, growth arrest-specific homeobox is a homeobox gene expressed in the cardiovascular system. Over expression of Gax is demonstrated to inhibit VSMC growth. We previously reported that Ang II down-regulated Gax expression. To address the regulatory mechanism of Gax, we investigated the significance of oxidative stress in Ang II-induced suppression of Gax expression. We further examined the involvement of mitogen-activated protein kinases (MAPKs), which is crucial for cell growth and has shown to be activated by oxidative stress, on the regulation of Gax expression by Ang II. Ang II markedly augmented intracellular H2O2 production which was decreased by pretreatment with N-acetylcystein (NAC), an anti-oxidant. Ang II and H2O2 decreased Gax expression dose-dependently and these effects were blocked by administration of both NAC and pyrrolidine dithiocarbamate (PDTC), another anti-oxidant. Ang II and H2O2 induced marked activation of extracellular signal-responsive kinase1/2 (ERK1/2), which was blocked by NAC. Ang II and H2O2 also activated p38MAPK, and they were blocked by pre-treatment with NAC. However, the level of activated p38MAPK was quite low in comparison with ERK1/2. Ang II- or H2O2 -induced Gax down-regulation was significantly inhibited by PD98059, an ERK1/2 inhibitor but not SB203580, a p38MAPK inhibitor. The present results demonstrated the significance of regulation of Gax expression by redox-sensitive ERK1/2 activation.     

Hepatitis C virus E2 protein induce reactive oxygen species (ROS)-related fibrogenesis in the HSC-T6 hepatic stellate cell line

Chronic infection of hepatitis C virus (HCV) leads to hepatic fibrosis and subsequently cirrhosis, although the underlying mechanisms have not been established. Previous studies have indicated that the binding of HCV E2 protein and CD81 on the surface of hepatic stellate cells (HSCs) lead to the increased protein level and activity of matrix metallopeptidase (MMP) 2, indicating that E2 may involve in the HCV-induced fibrosis. This study was designed to investigate the involvement of HCV E2 protein in the hepatic fibrogenesis. Results showed that E2 protein may promote the expression levels of α-smooth muscle actin (α-SMA) and collagen α(I). Furthermore, several pro-fibrosis or pro-inflammatory cytokines, including transforming growth factor (TGF)-β1, connective tissue growth factor (CTGF), interleukin (IL)-6 and IL-1β, were significantly increased in E2 transfected-HSC cell lines, while the expression of MMP-2 are also considerably increased. Moreover, the significant increases of CTGF and TGF-β1 in a stable E2-expressing Huh7 cell line were also observed the same results. Further molecular studies indicated that the impact of E2 protein on collagen production related to higher production of ROS and activated Janus kinase (JAK)1, JAK2 and also enhance the activation of ERK1/2 and p38, while catalase and inhibitors specific for JAK, ERK1/2, and p38 abolish E2-enhanced expression of collagen α(I). Taken together, this study indicated that E2 protein involve in the pathogenesis of HCV-mediated fibrosis via an up-regulation of collagen α(I) and oxidative stress, which is JAK pathway related.     

Globular adiponectin decreases leptin-induced tumor necrosis factor-alpha expression by murine macrophages: involvement of cAMP-PKA and MAPK pathways

Several lines of evidence have supported a link between obesity and inflammation. The present study investigated the capacity of leptin and globular adiponectin to affect tumor necrosis factor alpha (TNF-alpha) production in murine peritoneal macrophages. Leptin stimulated TNF-alpha production at mRNA as well as protein levels in a dose- and time-dependent manner. Intracellular cAMP concentration was increased and protein kinase A (PKA) was activated with the treatment of leptin, subsequently downstream MAPK signal proteins, ERK1/2 and p38, were phosphorylated. Specific inhibitors for the signal proteins, Rp cAMPS, H89, PD98059, and U0126, or SB203580, suppressed the signaling pathway and TNF-alpha expression. Although gAd partially increased cAMP concentration and PKA activity, it directly reduced leptin-induced ERK1/2 and p38 MAPK phosphorylation thus inhibiting TNF-alpha production. In conclusion, leptin promotes inflammation by stimulating TNF-alpha production, which is mediated by cAMP-PKA-ERK1/2 and p38 MAPK pathways. gAd inhibited leptin-induced TNF-alpha production through suppressing phosphorylation of ERK1/2 and p38 pathways.     

Enhancement of fibroblast collagenase-1 (MMP-1) gene expression by tumor promoter okadaic acid is mediated by stress-activated protein kinases jun N-terminal kinase and p38

Collagenase-1 (matrix metalloproteinase-1, MMP-1) is expressed by several types of cells, including fibroblasts, and apparently plays an important role in the remodeling of collagenous extracellular matrix in various physiologic and pathologic situations. Here, we have examined the molecular mechanisms of the activation of fibroblast MMP-1 gene expression by a naturally occurring non-phorbol ester type tumor promoter okadaic acid (OA), a potent inhibitor of serine/threonine protein phosphatase 2A. We show that in fibroblasts OA activates three distinct subgroups of mitogen activated protein kinases (MAPKs): extracellular signal-regulated kinase 1,2 (ERK 1,2), c-Jun N-terminal-kinase/stress-activated protein kinase (JNK/SAPK) and p38. Activation of MMP-1 promoter by OA is entirely blocked by overexpression of dual-specificity MAPK phosphatase CL100. In addition, expression of kinase-deficient forms of ERK 1,2, SAPKβ, p38, or JNK/SAPK kinase SEK1 strongly inhibited OA-elicited activation of MMP-1 promoter. OA-elicited enhancement of MMP-1 mRNA abundance was also strongly prevented by two chemical MAPK inhibitors: PD 98059, a specific inhibitor of the activation of ERK1,2 kinases MEK1,2; and SB 203580, a selective inhibitor of p38 activity. Results of this study show that MMP-1 gene expression in fibroblasts is coordinately regulated by ERK1,2, JNK/SAPK, and p38 MAPKs and suggest an important role for the stress-activated MAPKs JNK/SAPK and p38 in the activation of MMP-1 gene expression. Based on these observations, it is conceivable that specific inhibition of stress-activated MAPK pathways may serve as a novel therapeutic target for inhibiting degradation of collagenous extracellular matrix.     

Involvement of MAPK activation in chemokine or COX-2 productions by Toxoplasma gondii

This experiment focused on MAPK activation in host cell invasion and replication of T. gondii, as well as the expression of CC chemokines, MCP-1 and MIP-1 alpha , and enzyme, COX-2/prostaglandin E2 (PGE2) in infected cells via western blot, [3H]-uracil incorporation assay, ELISA and RT-PCR. The phosphorylation of ERK1/2 and p38 in infected HeLa cells was detected at 1 hr and/or 6 hr postinfection (PI). Tachyzoite proliferation was reduced by p38 or JNK MAPK inhibitors. MCP-1 secretion was enhanced in infected peritoneal macrophages at 6 hr PI. MIP-1 alpha mRNA was increased in macrophages at 18 hr PI. MCP-1 and MIP-1 alpha were reduced after treatment with inhibitors of ERK1/2 and JNK MAPKs. COX-2 mRNA gradually increased in infected RAW 264.7 cells and the secretion of COX-2 peaked at 6 hr PI. The inhibitor of JNK suppressed COX-2 expression. PGE2 from infected RAW 264.7 cells was increased and synthesis was suppressed by PD98059, SB203580, and SP600125. In this study, the activation of p38, JNK and/or ERK1/2 MAPKs occurred during the invasion and proliferation of T. gondii tachyzoites in HeLa cells. Also, increased secretion and expression of MCP-1, MIP-1 alpha , COX-2 and PGE2 were detected in infected macrophages, and appeared to occur via MAPK signaling pathways.     

Regulation of Indian hedgehog mRNA levels in chondrocytic cells by ERK1/2 and p38 mitogen-activated protein kinases

Indian hedgehog (Ihh) is produced by growth plate pre-hypertrophic chondrocytes, and is an important regulator of endochondral ossification. However, little is known about the regulation of Ihh in chondrocytes. We have examined the role of integrins and mitogen-activated protein (MAP) kinases in Ihh mRNA regulation in CFK-2 chondrocytic cells. Cells incubated with the beta1-integrin blocking antibody had decreased Ihh mRNA levels, which was accompanied by decreases of activated extracellular signal-regulated kinases (ERK1/2) and activated p38 MAPK. Ihh mRNA levels were also inhibited by U0126, a specific MEK1/2 inhibitor, or SB203580, a specific p38 MAPK inhibitor. Cells transfected with constitutively active MEK1 or MKK3 had increased Ihh mRNA levels, which were diminished by dominant-negative MEK1, p38alpha or p38beta. Stimulation of the PTH1R with 10(-8) M rPTH (1-34) resulted in dephosphorylation of ERK1/2 that was evident within 15 min and sustained for 1 h, as well as transient dephosphorylation of p38 MAPK that was maximal after 25 min. PTH stimulation decreased Ihh mRNA levels, and this effect was blocked by transfecting the cells with constitutively active MEK1 but not by MKK3. These studies demonstrated that activation of ERK1/2 or p38 MAPK increased Ihh mRNA levels. Stimulation of the PTH1R or blocking of beta1-integrin resulted in inhibition of ERK1/2 and p38 MAPK and decreased levels of Ihh mRNA. Our data demonstrate the central role of MAPK in the regulation of Ihh in CFK-2 cells.     

A common pathway in differentiation and inflammation: p38 mediates expression of the acute phase SIP24 iron binding lipocalin in chondrocytes

SIP24 is an acute phase iron binding lipocalin physiologically expressed in vivo in developing cartilage by prehypertrophic/hypertrophic chondrocytes. Taking advantage of the chondrocytic cell line MC615 and using SIP24 as a marker we investigated the pathways active in cartilage differentiation and inflammation. MC615 cells were cultured as: (i) proliferating prechondrogenic cells expressing type I collagen (ii) differentiated hyperconfluent cells expressing Sox9 and type II collagen. In proliferating cells the pathway PKC/ERK1, ERK2 was activated and SIP24 was not expressed while in differentiated cells the pathway p38/NF-kappaB was activated and SIP24 was expressed. Proliferating cells treated with inflammatory agents expressed a large amount of SIP24 and showed activation of p38/NF-kappaB pathway and inhibition of PKC/ERK1, ERK2 pathway indicating that in inflammation and differentiation the same factors are activated (p38, NF-kappaB) or inactivated (PKC, ERKs). Treatment of proliferating cells with the p38 specific inhibitor SB203580 inhibited the inflammation induced activation of p38 and the synthesis of SIP24. PMA treatment induced activation of PKC, inactivation of p38 and suppression of SIP24 synthesis, suggesting that PKC activation inhibits p38 activation. In differentiated hyperconfluent cells the same factors (p38/NF-kappaB/SIP24) are constitutively activated: treatment with inflammatory agents does not increase synthesis of SIP24 while treatment with SB203580 and with PMA does not repress activation of p38 nor synthesis of SIP24. We propose that the SIP24 stress related protein is expressed via p38 activation/NF-kappaB recruitment both in chondrocyte differentiation and inflammation and that a signaling pathway active in the acute phase response is physiologically activated in differentiation.     

Correlation between TGF‐β1 expression and proteomic profiling induced by severe acute respiratory syndrome coronavirus papain‐like protease

Severe acute respiratory syndrome (SARS) coronavirus (SARS‐CoV) papain‐like protease (PLpro), a deubiquitinating enzyme, demonstrates inactivation of interferon (IFN) regulatory factor 3 and NF‐κB, reduction of IFN induction, and suppression of type I IFN signaling pathway. This study investigates cytokine expression and proteomic change induced by SARS‐CoV PLpro in human promonocyte cells. PLpro significantly increased TGF‐β1 mRNA expression (greater than fourfold) and protein production (greater than threefold). Proteomic analysis, Western blot, and quantitative real‐time PCR assays indicated PLpro upregulating TGF‐β1‐associated genes: HSP27, protein disulfide isomerase A3 precursor, glial fibrillary acidic protein, vimentin, retinal dehydrogenase 2, and glutathione transferase omega‐1. PLpro‐activated ubiquitin proteasome pathway via upregulation of ubiquitin‐conjugating enzyme E2–25k and proteasome subunit alpha type 5. Proteasome inhibitor MG‐132 significantly reduced expression of TGF‐β1 and vimentin. PLpro upregulated HSP27, linking with activation of p38 MAPK and ERK1/2 signaling. Treatment with SB203580 and U0126 reduced PLpro‐induced expression of TGF‐β1, vimentin, and type I collagen. Results point to SARS‐CoV PLpro triggering TGF‐β1 production via ubiquitin proteasome, p38 MAPK, and ERK1/2‐mediated signaling.     

Oxidative stress-induced intestinal epithelial cell apoptosis is mediated by p38 MAPK

Free oxygen radicals are involved in the pathogenesis of necrotizing enterocolitis (NEC) in premature infants. The stress-activated p38 mitogen-activated protein kinase (MAPK) has been implicated in gut injury. Here, we found that phosphorylated p38 was detected primarily in the villus tips of normal intestine, whereas it was expressed in the entire mucosa in NEC. H(2)O(2) treatment resulted in a rapid phosphorylation of p38 MAPK and subsequent apoptosis of rat intestinal epithelial (RIE)-1 cells; this induction was attenuated by treatment with SB203580, a selective p38 MAPK inhibitor, or transfection with p38alpha siRNA. Moreover, SB203580 also blocked H(2)O(2)-induced PKC activation. In contrast, the PKC inhibitor (GF109203x) did not affect p38 activation, indicating that p38 MAPK activation occurs upstream of PKC activation in H(2)O(2)-induced apoptosis. H(2)O(2) treatment also decreased mitochondrial membrane potential; pretreatment with SB203580 attenuated this response. Our study demonstrates that the p38 MAPK/PKC pathway plays an important role as a pro-apoptotic cellular signaling during oxidative stress-induced intestinal epithelial cell injury.     

Cyclooxygenase-2 induction and prostacyclin release by protease-activated receptors in endothelial cells require cooperation between mitogen-activated protein kinase and NF-kappaB pathways

The functional significance of protease-activated receptors (PARs) in endothelial cells is largely undefined, and the intracellular consequences of their activation are poorly understood. Here, we show that the serine protease thrombin, a PAR-1-selective peptide (TFLLRN), and SLIGKV (PAR-2-selective peptide) induce cyclooxygenase-2 (COX-2) protein and mRNA expression in human endothelial cells without modifying COX-1 expression. COX-2 induction was accompanied by sustained production of 6-keto-PGF1alpha, the stable hydrolysis product of prostacyclin, and this was inhibited by indomethacin and the COX-2-selective inhibitor NS398. PAR-1 and PAR-2 stimulation rapidly activated both ERK1/2 and p38MAPK, and pharmacological blockade of MEK with either PD98059 or U0126 or of p38MAPK by SB203580 or SB202190 strongly inhibited thrombin- and SLIGKV-induced COX-2 expression and 6-keto-PGF1alpha formation. Thrombin and peptide agonists of PAR-1 and PAR-2 increased luciferase activity in human umbilical vein endothelial cells infected with an NF-kappaB-dependent luciferase reporter adenovirus, and this, as well as PAR-induced 6-keto-PGF1alpha synthesis, was inhibited by co-infection with adenovirus encoding wild-type or mutated (Y42F) IkappaBalpha. Thrombin- and SLIGKV-induced COX-2 expression and 6-keto-PGF1alpha generation were markedly attenuated by the NF-kappaB inhibitor PG490 and partially inhibited by the proteasome pathway inhibitor MG-132. Activation of PAR-1 or PAR-2 promoted nuclear translocation and phosphorylation of p65-NF-kappaB, and thrombin-induced but not PAR-2-induced p65-NF-kappaB phosphorylation was reduced by inhibition of MEK or p38MAPK. Activation of PAR-4 by AYPGKF increased phosphorylation of ERK1/2 and p38MAPK without modifying NF-kappaB activation or COX-2 induction. Our data show that PAR-1 and PAR-2, but not PAR-4, are coupled with COX-2 expression and sustained endothelial production of vasculoprotective prostacyclin by mechanisms that depend on ERK1/2, p38MAPK, and IkappaBalpha-dependent NF-kappaB activation.