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 cyclooxygenase-2 (COX-2) in a variety of cells. Here we investigated the role of transforming growth factor-β (TGF-β) in the Ras-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-β-D-galactopyranoside (IPTG) induced the expression of Ha- Ras(Val12), closely followed by an increase in the expression of COX-2. Neutralizing anti-TGF-β antibody partially blocked the Ras-induced increase in COX-2. Combined treatment with IPTG and TGF-β1 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-Ras induction alone. The addition of TGF-β1 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-β1 treatment and Ras induction each stabilized the COX-2 3'-UTR. Combined treatment with IPTG and TGF-β1 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 Ras or TGF-β1 and is necessary for the synergistic stabilization of COX-2 3'-UTR by oncogenic Ras and TGF-β1.