Anisomycin induces COX-2 mRNA expression through p38(MAPK) and CREB independent of small GTPases in intestinal epithelial cells

Cyclooxygenase (COX)-2 expression in intestinal epithelial cells is associated with colorectal carcinogenesis. COX-2 expression is induced by numerous growth factors and gastrointestinal hormones through multiple protein kinase cascades. Here, the role of mitogen activated protein kinases (MAPKs) and small GTPases in COX-2 expression was investigated. Anisomycin and sorbitol induced COX-2 expression in non-transformed, intestinal epithelial IEC-18 cells. Both anisomycin and sorbitol activated p38(MAPK) followed by phosphorylation of CREB. SB202190 and PD169316 but neither PD98059 nor U0126 blocked COX-2 expression and CREB phosphorylation by anisomycin or sorbitol. Clostridium difficile toxin B inhibition of small GTPases did not affect anisomycin-induced COX-2 mRNA expression or phosphorylation of p38MAPK and CREB but did inhibit sorbitol-dependent COX-2 expression and phosphorylation of p38MAPK and CREB. Angiotensin (Ang) II-dependent induction of COX-2 mRNA and induced phosphorylation of p38MAPK and CREB were inhibited by toxin B. Reduction of CREB protein in cells transfected with CREB siRNAs inhibited anisomycin-induced COX-2 expression. These results indicate that activation of p38MAPK signaling is sufficient for COX-2 expression in IEC-18 cells. Ang II and sorbitol require small GTPase activity for COX-2 expression via p38MAPK while anisomycin-induced COX-2 expression by p38MAPK does not require small GTPases. This places small GTPase activity down-stream of the AT1 receptor and hyperosmotic stress and up-stream of p38MAPK and CREB.     

The induction of cell death in human osteoarthritis chondrocytes by nitric oxide is related to the production of prostaglandin E2 via the induction of cyclooxygenase-2

There is increasing evidence suggesting that chondrocyte death may contribute to the progression of osteoarthritis (OA). This study focused on the characterization of signaling cascade during NO-induced cell death in human OA chondrocytes. The NO generator, sodium nitroprusside (SNP), promoted chondrocyte death in association with DNA fragmentation, caspase-3 activation, and down-regulation of Bcl-2. Both caspase-3 inhibitor Z-Asp(OCH3)-Glu(OCH3)-Val-Asp(OCH3)-CH2F and caspase-9 inhibitor Z-Leu-Glu(OCH3)-His-Asp(OCH3)-CH2F prevented the chondrocyte death. Blocking the mitogen-activated protein kinase pathway by the mitogen-activated protein kinase kinase 1/2 inhibitor PD98059 or p38 kinase inhibitor SB202190 also inhibited the SNP-mediated cell death, suggesting possible requirements of both extracellular signal-related protein kinase 1/2 and p38 kinase for the NO-induced cell death. Furthermore, the selective inhibition of cyclooxygenase (COX)-2 by NS-398 or the inhibition of COX-1/COX-2 by indomethacin blocked the SNP-induced cell death. The chondrocyte death induced by SNP was associated with an overexpression of COX-2 protein (as determined by Western blotting) and an increase in PGE2 release. PD98059 and SB202190, but neither Z-DEVD FMK nor Z-LEHD FMK completely inhibited the SNP-mediated PGE2 production. Analysis of interactions between PGE2 and the cell death showed that PGE2 enhanced the SNP-mediated cell death, whereas PGE2 alone did not induce the chondrocyte death. These data indicate that NO-induced chondrocyte death signaling includes PGE2 production via COX-2 induction and suggest that both extracellular signal-related protein kinase 1/2 and p38 kinase pathways are upstream signaling of the PGE2 production. The results also demonstrate that exogenous PGE2 may sensitize human OA chondrocytes to the cell death induced by NO.     

PI 3-kinase and MAP kinase regulate bradykinin induced prostaglandin E(2) release in human pulmonary artery by modulating COX-2 activity

Here we studied the role of phosphoinositide 3-kinase (PI 3-kinase) and mitogen activated protein (MAP) kinase in regulating bradykinin (BK) induced prostaglandin E₂ (PGE₂) production in human pulmonary artery smooth muscle cells (HPASMC). BK increased PGE₂ in a three step process involving phospholipase A₂ (PLA₂), cyclooxygenase (COX) and PGE synthase (PGES). BK stimulated PGE₂ release in cultured HPASMC was inhibited by the PI 3-kinase inhibitor LY294002 and the p38 MAP kinase inhibitor SB202190. The inhibitory mechanism used by LY294002 did not involve cytosolic PLA₂ activation or COX-1, COX-2 and PGES protein expression but rather a novel effect on COX enzymatic activity. SB202190 also inhibited COX activity.     

TNF transactivation of EGFR stimulates cytoprotective COX-2 expression in gastrointestinal epithelial cells

TNF and epidermal growth factor (EGF) are well-known stimuli of cyclooxygenase (COX)-2 expression, and TNF stimulates transactivation of EGF receptor (EGFR) signaling to promote survival in colon epithelial cells. We hypothesized that COX-2 induction and cell survival signaling downstream of TNF are mediated by EGFR transactivation. TNF treatment was more cytotoxic to COX-2(-/-) mouse colon epithelial (MCE) cells than wild-type (WT) young adult mouse colon (YAMC) epithelial cells or COX-1(-/-) cells. TNF also induced COX-2 protein and mRNA expression in YAMC cells, but blockade of EGFR kinase activity or expression inhibited COX-2 upregulation. TNF-induced COX-2 expression was reduced and absent in EGFR(-/-) and TNF receptor-1 (TNFR1) knockout MCE cells, respectively, but was restored upon expression of the WT receptors. Inhibition of mediators of EGFR transactivation, Src family kinases and p38 MAPK, blocked TNF-induced COX-2 protein and mRNA expression. Finally, TNF injection increased COX-2 expression in colon epithelium of WT, but not kinase-defective EGFR(wa2) and EGFR(wa5), mice. These data indicate that TNFR1-dependent transactivation of EGFR through a p38- and/or an Src-dependent mechanism stimulates COX-2 expression to promote cell survival. This highlights an EGFR-dependent cell signaling pathway and response that may be significant in colitis-associated carcinoma.     

Elevated levels of cyclooxygenase-2 in antigen-stimulated mast cells is associated with minimal activation of p38 mitogen-activated protein kinase

We have investigated possible factors that underlie changes in the production of eicosanoids after prolonged exposure of mast cells to Ag. Ag stimulation of cultured RBL-2H3 mast cells resulted in increased expression of cyclooxygenase (COX-2) protein and message. Other eicosanoid-related enzymes, namely COX-1, 5-lipoxygenase, and cytosolic phospholipase A(2) were not induced. Activation of extracellular signal-regulated kinase, c-Jun N-terminal kinase, and p38 mitogen-activated protein (MAP) kinase preceded the induction of COX-2, whereas phosphatidylinositol 3' kinase and its substrate, Akt, were constitutively activated in RBL-2H3 cells. Studies with pharmacologic inhibitors indicated that of these kinases, only p38 MAP kinase regulated expression of COX-2. The induction of COX-2 was blocked by the p38 MAP kinase inhibitor SB202190, even when added 12-16 h after stimulation with Ag when p38 MAP kinase activity had returned to near basal, but still minimally elevated, levels. Interestingly, expression of COX-2 as well as cytosolic phospholipase A(2) and 5-lipoxygenase were markedly reduced by SB202190 in unstimulated cells. Collectively, the results imply that p38 MAP kinase regulates expression of eicosanoid-related enzymes, passively or actively, at very low levels of activity in RBL-2H3 cells. Also, comparison with published data suggest that different MAP kinases regulate induction of COX-2 in inflammatory cells of different and even similar phenotype and suggest caution in extrapolating results from one type of cell to another.     

CD28 signaling augments Elk-1-dependent transcription at the c-fos gene during antigen stimulation.

Untransformed CD4(+) Th1 cells stimulated with Ag and APC demonstrated a dependence on B7- and CD28-mediated costimulatory signals for the expression and function of AP-1 proteins. The induction of transactivation by the c-fos gene regulator Elk-1 mirrored this requirement for TCR and CD28 signal integration. c-Jun N-terminal kinase (JNK) (but not extracellular signal-regulated kinase or p38) protein kinase activity was similarly inhibited by neutralizing anti-B7 mAbs. Blockade of JNK protein kinase activity with SB 202190 prevented both Elk-1 transactivation and c-Fos induction. These results identify a unique role for B7 costimulatory molecules and CD28 in the activation of JNK during Ag stimulation in Th1 cells, and suggest that JNK regulates Elk-1 transactivation at the c-fos gene to promote the formation of AP-1 complexes important to IL-2 gene expression.     

Ca2+ stimulates COX-2 expression through calcium-sensing receptor in fibroblasts

Fibroblasts isolated from jaw cysts expressed calcium-sensing receptor (CasR). In the fibroblasts elevated extracellular Ca(2+) ([Ca(2+)](o)) increased fluo-3 fluorescence intensity, and the production of inositol(1,4,5)trisphosphate and active protein kinase C. Phospholipase C inhibitor U-73122 attenuated the Ca(2+)-induced increase in fluo-3 fluorescence intensity. Elevated [Ca(2+)](o) enhanced the expression of cyclooxygenase-2 (COX-2) mRNA and protein, and the secretion of prostaglandin E(2) in the fibroblasts. CasR activator neomycin also increased the expression of COX-2 mRNA, and U-73122 attenuated the Ca(2+)-induced expression of COX-2 mRNA. Elevated [Ca(2+)](o)-induced phosphorylation of extracellular signal-regulated protein kinase-1/2 (ERK1/2), p38 mitogen-activated protein kinase (MAPK), and c-Jun N-terminal kinase (JNK), and U-73122 inhibited the Ca(2+)-induced phosphorylation. The inhibitors for each kinase, PD98059, SB203580, and SP600125, attenuated the Ca(2+)-induced expression of COX-2 mRNA. These results suggest that in jaw cyst fibroblasts elevated extracellular Ca(2+) may enhance COX-2 expression via the activation of ERK1/2, p38 MAPK, and JNK through CasR.