Transforming growth factor-β1 enhances Ha-ras-induced expression of cyclooxygenase-2 in intestinal epithelial cells via stabilization of mRNA

Oncogenic ras induces the expression of cycloogygenase-2 (COX-2) in a variety of cells. Here we investigated the role of transforming growth factor-beta (TGF-beta) in the Res-mediated induction of COX-2 in intestinal epithelial cells (RIE-1). RIE-1 cells were transfected with an inducible Ha.Ras(Val12) cDNA and are referred as RIE-iRas cells. the addition of 5 mM isopropyl-1-thio-beta-D-galactopyranoside (IPTG) induced the expression of Ha-Ras-(val12) closely followed by an increase in the expression of COX-2. Neutralizing anti-TGF-beta antibody partially blocked the Ras-induced increase In COX-2. Combined treatment with IPTG and TGF-beta 1 resulted in a 20-50-fold increase in the levels of COX-2 mRNA The t(1/2) of COX-2 mRNA was increased from 13 to 24 min by Ha-Res induction alone. The addition of TGF-beta 1 further stabilized the COX-2 mRNA (t(1/2) > 50 min). Stable transfection of a luciferase reporter construct containing the COX-2 3'-untranslated region (3'-UTR) revealed that TGF-beta 1 treatment and Ras induction each stabilized the COX-2 3'-UTR. Combined treatment with IPTG and TGF-beta 1 synergistically increased the luciferase activity. Furthermore, a conserved AU-rich region located in the proximal COX-2 3'-UTR is required for maximal stabilization of COX-2 3'-UTR by Res or TGF-beta 1 and is necessary for the synergistic stabilization of COX-2 3'-UTR by oncogenic Res and TGF-beta 1.