COX-2 promoter activation by AT1R-Gq-PAK-p38beta signaling in intestinal epithelial cells.

Cyclooxygenase-2 (COX-2) is rapidly induced by angiotensin (Ang)-II in the non-tumorigenic, rat intestinal epithelial cell line, IEC-18, through its G-protein coupled receptor, AT1R. Here, we investigate the ability of Ang II to regulate transcription of the COX-2 promoter and a Gal4-CREB heterologous promoter in IEC-18 cells. Ang II and EGF induced similar levels of transcription from the COX-2 and Gal4-CREB promoters. Over-expression of constitutively active Galpha proteins q, 11, 12 and 13, showed induction by GalphaqQ209L and by Galpha11Q209L for both the COX-2 and Gal4-CREB promoters. Co-expression of RGS 2, 3 or 4 but not the RGS domain of p115RhoGEF inhibited Ang II-dependent induction of the COX-2 and Gal4-CREB promoters. Expression of constitutively active MKK6 EE but not MKK3 EE induced the COX-2 and Gal4-CREB promoters via p38MAPK. SB202190 but not PD98059 inhibited induction of the COX-2 promoter by over-expression of the constitutively active PAK1T423E. Expression of the kinase-inactive PAK1K299R inhibited both Ang II-dependent induction of the COX-2 promoter and induction of the COX-2 and Gal4-CREB promoters by GalphaqQ209L. These data demonstrate that in IEC-18 cells, Ang II-dependent activation of the COX-2 promoter is mediated primarily through Gq/11 signaling via a PAK/MKK6/p38beta/CREB signaling cascade.

Ang II and EGF synergistically induce COX-2 expression via CREB in intestinal epithelial cells.

Cyclooxygenase (COX)-2 derived prostaglandins (PGs) play a major role in intestinal inflammation and colorectal carcinogenesis. Because COX-2 is the rate-limiting step in the production of PGs, mechanisms that regulate COX-2 expression control PG production in the cell. Using the non-tumorigenic, rat intestinal epithelial cell, IEC-18, we demonstrate that co-activation of endogenously expressed AT(1) receptor and EGFR resulted in synergistic expression of COX-2 mRNA and protein involving transcriptional and post-transcriptional mechanisms. Ang II and EGF induced transient phosphorylation of ERK, p38(MAPK) and CREB. Co-stimulation with Ang II and EGF prolonged phosphorylation of ERK, p38(MAPK), and CREB. The p38(MAPK) selective inhibitor, SB202190, but not the MEK selective inhibitor, PD98059, or the EGFR kinase inhibitor, AG1478, inhibited Ang II-dependent COX-2 expression and CREB phosphorylation. EGF-dependent COX-2 expression and CREB phosphorylation were inhibited by SB202190, PD98059, and AG1478. Inhibition of CREB expression using two separate RNAi methods blocked COX-2 expression by Ang II and EGF. Expression of a dominant negative CREB mutant inhibited Ang II- and EGF-dependent induction of the COX-2 promoter. Ang II induced luciferase expression in cells transfected with the CRE-luc reporter vector and cells co-transfected with Gal4-luc reporter vector and a Gal4-CREB expression vector. Chromatin immunoprecipitation assays demonstrated CREB binding to the proximal rat COX-2 promoter region containing a CRE cis-acting element. These results indicate that co-stimulation with Ang II and EGF synergistically induced COX-2 expression in these intestinal epithelial cells through p38(MAPK) mediated signaling cascades that converge onto CREB.

15-Hydroxyprostaglandin dehydrogenase suppresses K-RasV12-dependent tumor formation in Nu/Nu mice.

Oncogenic Ras mutations are early genetic events in colorectal cancer that induce cyclooxygenase (COX)-2 expression and prostaglandin E(2) (PGE(2)) biosynthesis. PGE(2), a downstream product of COX-2, promotes cancer progression by modulating proliferation, apoptosis and angiogenesis. 15-hydroxyprostaglandin dehydrogenase (PGDH) degrades PGE(2) and is down-regulated in colorectal cancer, suggesting that PGDH plays a role in regulating PGE(2) levels and that PGDH over-expression could attenuate Ras-mediated tumorigenesis. Lentiviral transduction was used to express GFP (18.GFP), K-Ras(V12) (18.K-Ras(V12)), PGDH (18.PGDH) or both K-Ras(V12) and PGDH (18.K-Ras(V12).PGDH) in nontumorigenic rat intestinal epithelial (IEC-18) cells. 18.K-Ras(V12) cells exhibited increased phosphorylation of MAP kinases and CREB, proliferation rates, COX-2 and microsomal prostaglandin E synthase (mPGES)-1 expression and PGE(2) and PGI(2) levels. 18.PGDH and 18.K-Ras(V12).PGDH cells had 10(4)-fold increases in PGDH activity with decreased PGE(2) and PGI(2) levels, COX-2 and mPGES-1 expression and proliferation rates. 18.GFP, 18.PGDH, and 18.K-Ras(V12).PGDH cells were unable to grow in soft agar media whereas 18.K-Ras(V12) cells exhibited anchorage-independent cell growth. Xenografts of implanted 18.K-Ras(V12) cells in nu/nu mice produced rapid (2 wk) tumors with uniform antibody staining for COX-2 and mPGES-1 throughout the tumor and elevated PGE(2) levels. Xenografts of 18.K-Ras(V12).PGDH cells exhibited delayed (8 wk) tumor formation with negligible COX-2 and mPGES-1 expression and significantly decreased PGE(2) levels. 18.K-Ras(V12).PGDH tumors had decreased staining of the proliferative marker, Ki-67, and a significant increase in apoptosis in the central region of the tumor. Based on these data, we conclude that PGDH expression suppresses K-Ras(V12)-mediated tumorigenesis in intestinal epithelial cells.