TGF-beta inhibits vascular smooth muscle cell proliferation through down regulation of cyclin A

Background: Smooth muscle cell (SMC) proliferation contributes to the development of intimal hyperplasia. In this study, we explore the mechanism by which TGF-β, a potent endogenous growth inhibitor of SMCs, affects cell cycle progression. Method: (Cells-A10 vascular SMCs) (SMC proliferation - 3H-thymidine) (protein levels of cyclin kinase inhibitors, P21,P27 and cyclin A - Western Blotting) (cyclin A mRNA - Northern blotting) (cyclin A gene expression - luciferase reporter containing cyclin A promoter) Results: TGF-β (5 ng/ml), which produced a 30% decrease in A10 SMC DNA synthesis, has been shown in other cellular systems to control proliferation by upregulating P21 and P27, two proteins that inhibit cell-cycle progression. Thus, we examined the effect of TGF-β on P21 and P27 in vascular SMC. We found no significant alteration in P21/P27 protein levels following stimulation of SMC with TGF-β for 5, 12 and 24 hrs. We then evaluated the effect of TGF-β on protein levels of cyclin A, a critical cell cycle protein down stream from P21/27 required for G1/S transition and completion of S. TGF-β (5 ng/ml) produced a 48.4 ± 17.5% decrease in cyclin A protein that was maximal at 17 hrs (p < 0.05) (Fig. 1). To determine if this reduction is the result of a decrease in cyclin A expression, we examined the level of cyclin A mRNA in A10 cells. We found a 50.2 ± 19.6% (p < 0.05) reduction in cyclin A mRNA following 17 hrs stimulation with TGF-β (5 ng/ml) (Fig. 2). To test whether TGF-β acts directly on the cyclin A promoter to reduce transcription, we transfected A10 cells with a luciferase reporter plasmid which contains the -516 to +245 region of the human cyclin A promoter. After transfection (3 hrs), cells were treated with TGF-β (5 ng/ml) for 17hrs. Lucifease activity in the TGF-β treated cells was decreased by 63.0 ± 12.0% (p < 0.05), suggesting that the cyclin A promoter activity is suppressed by TGF-β. Conclusion: Our data suggest that TGF-β directly inhibits cyclin A expression in SMC through an interaction with its proximal promoter. TGF-β appears to have no effect on the cell cycle inhibitors P21 and P27. Strategies that enhance the inhibitory effect of TGF-β on SMC proliferation and block its effect on matrix protein production will diminish the formation of intimal hyperplasia.