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

Hongmiao Sheng, Jinyi Shao, Dan A. Dixon, Christopher S. Williams, Stephen M. Prescott, Raymond N. DuBois, R. Daniel Beauchamp

Research output: Contribution to journalArticle

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Abstract

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.

Original languageEnglish (US)
Pages (from-to)6628-6635
Number of pages8
JournalJournal of Biological Chemistry
Volume275
Issue number9
DOIs
StatePublished - Mar 3 2000
Externally publishedYes

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Transforming Growth Factors
Cyclooxygenase 2
Stabilization
Epithelial Cells
Messenger RNA
3' Untranslated Regions
Reactive ion etching
Galactose
Luciferases
Transfection
Complementary DNA
cyclooxygenase-3

ASJC Scopus subject areas

  • Biochemistry

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Transforming growth factor-β1 enhances Ha-ras-induced expression of cyclooxygenase-2 in intestinal epithelial cells via stabilization of mRNA. / Sheng, Hongmiao; Shao, Jinyi; Dixon, Dan A.; Williams, Christopher S.; Prescott, Stephen M.; DuBois, Raymond N.; Beauchamp, R. Daniel.

In: Journal of Biological Chemistry, Vol. 275, No. 9, 03.03.2000, p. 6628-6635.

Research output: Contribution to journalArticle

Sheng, Hongmiao ; Shao, Jinyi ; Dixon, Dan A. ; Williams, Christopher S. ; Prescott, Stephen M. ; DuBois, Raymond N. ; Beauchamp, R. Daniel. / Transforming growth factor-β1 enhances Ha-ras-induced expression of cyclooxygenase-2 in intestinal epithelial cells via stabilization of mRNA. In: Journal of Biological Chemistry. 2000 ; Vol. 275, No. 9. pp. 6628-6635.
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abstract = "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.",
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AU - Sheng, Hongmiao

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AU - Williams, Christopher S.

AU - Prescott, Stephen M.

AU - DuBois, Raymond N.

AU - Beauchamp, R. Daniel

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