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Cyclooxygenase-2 induction and transforming growth factor beta growth inhibition in rat intestinal epithelial cells
1Department of Surgery, Vanderbilt Cancer Center, Vanderbilt University Medical Center, Nashville, Tennessee 37232-2279, USA.
Abstract:
Rat intestinal epithelial cells (RIE-1) permanently transfected with the prostaglandin endoperoxide synthase 2 (also referred to as cyclooxygenase-2; COX-2) gene exhibit decreased cyclin D1 levels, decreased cdk4-associated kinase activity, and delayed G1 cell cycle progression, which represents a phenotype similar to that which follows transforming growth factor beta (TGF-beta) treatment. In the current study, we have found that addition of TGF-beta 1 to the parental RIE-1 cells (designated RIE-P) caused a rapid induction of COX-2 mRNA and protein. COX-2 protein levels progressively increased and reached peak levels 6 h after TGF-beta 1 addition. Cyclin D1 was decreased by 74% at 6 h and was undetectable 24 h after addition of TGF-beta 1. In RIE cells transfected with the COX-2 antisense expression vector (RIE-AS cells), TGF-beta 1 induction of COX-2 protein was reduced greater than 90%. Addition of TGF-beta 1 did not reduce the abundant cyclin D1 protein expression in the RIE-AS cells, unlike the effect in RIE-P cells. TGF-beta 1 treatment reduced peak [3H]thymidine incorporation by 60% and delayed G1/S-phase transition by at least 4 h in the RIE-P cells. In contrast, S-phase entry occurred at 16 h in RIE-AS cells and was not altered by TGF-beta 1 treatment. Restoration of cyclin D1 expression by transfection of the cyclin D1 cDNA under transcriptional control of the cytomegalovirus promoter/enhancer in the COX-2-overexpressing (RIE-S) cells decreased the time required for S-phase entry by at least 4 h and increased the peak level of [3H]thymidine incorporation. Taken together, the results demonstrate that TGF-beta 1 strongly induces COX-2 at both the mRNA and protein levels and suggest that this induction of COX-2 is involved in the down-regulation of cyclin D1 and inhibition of cell growth caused by TGF-beta 1 in rat intestinal epithelial cells.
Insights
Transforming growth factor beta (TGF-beta) 1 induces cyclooxygenase-2 (COX-2) in rat intestinal cells, leading to reduced cyclin D1 and inhibited cell growth. This COX-2 induction is key to TGF-beta’s effects on cell cycle progression.
Area of Science:
- Cell Biology
- Molecular Biology
- Gastroenterology
Background:
- Cyclooxygenase-2 (COX-2) is implicated in cell proliferation.
- Transforming growth factor beta (TGF-beta) regulates cell growth and differentiation.
- Previous studies showed COX-2 overexpression phenocopies TGF-beta effects on cell cycle.
Purpose of the Study:
- To investigate the role of COX-2 induction in TGF-beta 1-mediated cell cycle inhibition in rat intestinal epithelial cells.
- To elucidate the relationship between TGF-beta 1, COX-2, and cyclin D1 expression.
Main Methods:
- Treatment of RIE-1 cells (parental and COX-2 antisense transfected) with TGF-beta 1.
- Analysis of COX-2 and cyclin D1 mRNA and protein levels via RT-PCR and Western blotting.
- Cell cycle progression assessed by [3H]thymidine incorporation and flow cytometry.
Main Results:
- TGF-beta 1 rapidly induced COX-2 mRNA and protein in parental RIE-1 cells.
- TGF-beta 1 treatment decreased cyclin D1 levels and delayed G1/S-phase transition.
- COX-2 induction was significantly reduced in antisense-transfected cells, which were resistant to TGF-beta 1-induced cyclin D1 down-regulation and cell cycle arrest.
- Restoring cyclin D1 expression in COX-2-overexpressing cells partially reversed TGF-beta 1's effects.
Conclusions:
- TGF-beta 1 strongly induces COX-2 expression in rat intestinal epithelial cells.
- COX-2 induction is a critical mediator of TGF-beta 1's inhibitory effects on cyclin D1 expression and cell proliferation.
- Targeting COX-2 may offer therapeutic strategies for conditions involving aberrant TGF-beta signaling in intestinal epithelium.