Transforming growth factor-beta1 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-beta (TGF-beta) 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-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-beta1 resulted in a 20-50-fold increase in the levels of COX-2 mRNA. The t1/2 of COX-2 mRNA was increased from 13 to 24 min by Ha-Ras induction alone. The addition of TGF-beta1 further stabilized the COX-2 mRNA (t1/2 > 50 min). Stable transfection of a luciferase reporter construct containing the COX-2 3'-untranslated region (3'-UTR) revealed that TGF-beta1 treatment and Ras induction each stabilized the COX-2 3'-UTR. Combined treatment with IPTG and TGF-beta1 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-beta1 and is necessary for the synergistic stabilization of COX-2 3'-UTR by oncogenic Ras and TGF-beta1.
Sheng
H
H
Department of Surgery, The Vanderbilt-Ingram Cancer Center, Vanderbilt University Medical Center, Nashville, Tennessee 37232, USA. hongmiao.sheng@mcmail.vanderbilt.edu
Shao
J
J
Dixon
D A
DA
Williams
C S
CS
Prescott
S M
SM
DuBois
R N
RN
Beauchamp
R D
RD
D020413:3' Untranslated Regions; D000818:Animals; D002460:Cell Line; D051546:Cyclooxygenase 2; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects*; D017930:Genes, Reporter; D007413:Intestinal Mucosa / Q000378:metabolism; D007527:Isoenzymes / Q000235:genetics* / Q000378:metabolism; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D011451:Prostaglandin-Endoperoxide Synthases / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism*; D014162:Transfection; D016212:Transforming Growth Factor beta / Q000494:pharmacology*; D018631:ras Proteins / Q000378:metabolism*
D020413:3' Untranslated Regions; D007527:Isoenzymes; D012333:RNA, Messenger; D016212:Transforming Growth Factor beta; D007544:Isopropyl Thiogalactoside; D051546:Cyclooxygenase 2; D011451:Prostaglandin-Endoperoxide Synthases; D018631:ras Proteins