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.     

Clostridium difficile toxin A regulates inducible cyclooxygenase-2 and prostaglandin E2 synthesis in colonocytes via reactive oxygen species and activation of p38 MAPK

Clostridium difficile toxin A induces acute colitis with neutrophil infiltration and up-regulation of numerous pro-inflammatory mediators, but the contribution of cyclooxygenase-2 (COX-2) induction in this infection is unknown. We report here that toxin A induces expression of COX-2 and secretion of prostaglandin E2 (PGE2) in a dose- and time-dependent manner in cultured NCM460 human colonocytes and in human intestinal xenografts. This induction was blocked by SB203580, a p38 MAPK inhibitor, which also decreased the phosphorylation of MSK-1, CREB/ATF-1, and COX-2 promoter activity following toxin A stimulation. Gel shift assays indicated that CREB/ATF-1 was the major proteins binding to the COX-2-CRE. Moreover, colonocytes exposed to toxin A produced reactive oxygen species (ROS), which activated p38 MAPK, MSK-1, and CREB/ATF-1, leading to subsequent COX-2 induction and PGE2 secretion. In intact mice, blockage of p38 MAPK inhibited toxin A-mediated induction of COX-2 in enterocytes as well as lamina propria cells, and significantly blocked the toxin A-induced ileal secretion of fluid and PGE2. Furthermore, a selective COX-2 inhibitor also diminished toxin A-associated ileal fluid and PGE2 secretion. The main signaling pathway for toxin A induction of human COX-2 involves ROS-mediated activation of p38 MAPK, MSK-1, CREB, and ATF-1. Toxin A triggers ileal inflammation and secretion of fluid via COX-2 induction and release of PGE2.     

Critical role of cAMP-response element-binding protein for angiotensin II-induced hypertrophy of vascular smooth muscle cells

We reported previously an important role of cyclic AMP-response element (CRE) for the induction of interleukin-6 gene expression by angiotensin II (AngII). We examined signaling pathways that are responsible for AngII-induced phosphorylation of CRE-binding protein (CREB) at serine 133 that is a critical marker for the activation in rat vascular smooth muscle cells (VSMC). AngII time dependently induced phosphorylation of CREB with a peak at 5 min. The AngII-induced phosphorylation of CREB was blocked by CV11974, an AngII type I receptor antagonist, suggesting that AngII type I receptor may mediate the phosphorylation of CREB. Inhibition of extracellular signal-regulated protein kinase (ERK) by PD98059 or inhibition of p38 mitogen-activated protein kinase (MAPK) by SB203580 partially inhibited AngII-induced CREB phosphorylation. A protein kinase A inhibitor, H89, also partially suppressed AngII-induced CREB phosphorylation. Inhibition of epidermal growth factor-receptor by AG1478 suppressed the AngII-induced CREB phosphorylation as well as activation of ERK and p38MAPK. Overexpression of the dominant negative form of CREB by an adenovirus vector suppressed AngII-induced c-fos expression and incorporation of [(3)H]leucine to VSMC. These findings suggest that AngII may activate multiple signaling pathways involving two MAPK pathways and protein kinase A, all of which contribute to the activation of CREB. Transactivation of epidermal growth factor-receptor is also critical for AngII-induced CREB phosphorylation. Activation of CREB may be important for the regulation of gene expression and hypertrophy of VSMC induced by AngII.     

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.     

Induction of autophagy in hepatocellular carcinoma cells by SB203580 requires activation of AMPK and DAPK but not p38 MAPK

SB203580 is a well-known inhibitor of p38 mitogen-activated protein kinase (MAPK). However, it can suppress cell proliferation in a p38 MAPK independent manner. The inhibitory mechanism remains unknown. Here, we showed that SB203580 induced autophagy in human hepatocellular carcinoma (HCC) cells. SB203580 increased GFP-LC3-positive cells with GFP-LC3 dots, induced accumulation of autophagosomes, and elevated the levels of microtubule-associated protein light chain 3 and Beclin 1. It stimulated the phosphorylation of adenosine monophosphate-activated protein kinase (AMPK) and p53, but inhibited the phosphorylation of death-associated protein kinase (DAPK). Inhibition of AMPK, p53, or DAPK attenuated SB203580-induced autophagy. AMPK activation appeared to predate the DAPK signal. The activation of both AMPK and DAPK prompted the phosphorylation of p53 and enhanced Beclin 1 expression. Neither the downregulation of p38 MAPK by its siRNA or chemical inhibitor nor the upregulation of p38 MAPK by p38 MAPK DNA transfection affected B203580-induced autophagy. Collectively, the findings demonstrate a novel function of SB203580 to induce autophagy via activating AMPK and DAPK but independent of p38 MAPK. The induction of autophagy can thus account for the antiproliferative effect of SB203580 in HCC cells.     

Inhibition of p38 mitogen-activated protein kinase and phosphatidylinositol 3-kinase decreases UVB-induced activator protein-1 and cyclooxygenase-2 in a SKH-1 hairless mouse model

Activation of activator protein-1 (AP-1) and increased expression of cyclooxygenase-2 (COX-2) have been clearly shown to play a functional role in UVB-induced skin tumor promotion. In this study, we examined UVB-induced signal transduction pathways in SKH-1 mouse epidermis leading to increases in COX-2 expression and AP-1 activity. We observed rapid increases in p38 mitogen-activated protein kinase (MAPK) signaling through activation of p38 MAPK and its downstream target, MAPK activated protein kinase-2. UVB also increased phosphatidylinositol 3-kinase (PI3K) signaling as observed through increases in AKT and GSK-3beta phosphorylation. Activation of the p38 MAPK and PI3K pathways results in the phosphorylation of cyclic AMP-responsive element binding protein, which was also observed in UVB-irradiated SKH-1 mice. Topical treatment with SB202190 (a specific inhibitor of p38 MAPK) or LY294002 (a specific inhibitor of PI3K) significantly decreased UVB-induced AP-1 activation by 84% and 68%, respectively, as well as COX-2 expression. Our data show that in mouse epidermis, UVB activation of the p38 MAPK and PI3K pathways leads to AP-1 activation and COX-2 expression.