Sheng H, Shao J, Dixon D A, Williams C S, Prescott S M, DuBois R N, Beauchamp R D
Department of Surgery, The Vanderbilt-Ingram Cancer Center, Vanderbilt University Medical Center, Nashville, Tennessee 37232, USA.
J Biol Chem. 2000 Mar 3;275(9):6628-35. doi: 10.1074/jbc.275.9.6628.
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.
致癌性Ras可在多种细胞中诱导环氧合酶-2（COX-2）的表达。在此，我们研究了转化生长因子-β（TGF-β）在Ras介导的肠上皮细胞（RIE-1）中COX-2诱导过程中的作用。RIE-1细胞用可诱导的Ha-Ras（Val12）cDNA进行转染，称为RIE-iRas细胞。添加5 mM异丙基-1-硫代-β-D-半乳糖苷（IPTG）可诱导Ha-Ras（Val12）的表达，随后COX-2的表达紧接着增加。中和性抗TGF-β抗体部分阻断了Ras诱导的COX-2增加。IPTG与TGF-β1联合处理导致COX-2 mRNA水平增加20至50倍。单独由Ha-Ras诱导使COX-2 mRNA的半衰期从13分钟增加到24分钟。添加TGF-β1进一步稳定了COX-2 mRNA（半衰期>50分钟）。稳定转染包含COX-2 3'-非翻译区（3'-UTR）的荧光素酶报告构建体显示，TGF-β1处理和Ras诱导均使COX-2 3'-UTR稳定。IPTG与TGF-β1联合处理协同增加了荧光素酶活性。此外，位于近端COX-2 3'-UTR的一个保守的富含AU区域对于Ras或TGF-β1使COX-2 3'-UTR最大程度稳定是必需的，并且对于致癌性Ras和TGF-β1协同稳定COX-2 3'-UTR也是必需的。
