Regulation of smooth muscle cell proliferation by β-catenin/T-cell factor signaling involves modulation of cyclin D1 and p21 expression

We previously observed that stimulation of vascular smooth muscle cell (VSMC) proliferation with growth factors is associated with dismantling of cadherin junctions and nuclear translocation of beta-catenin. In this study we demonstrate directly that growth factors stimulate beta-catenin/T-cell factor (TCF) signaling in primary VSMCs. To determine whether beta-catenin/TCF signaling regulates VSMC proliferation via modulation of the beta-catenin/TCF responsive cell cycle genes, cyclin D1 and p21, we inhibited beta-catenin/TCF signaling by adenoviral-mediated over-expression of N-Cadherin, ICAT (an endogenous inhibitor of beta-catenin/TCF signaling), or a dominant negative (dn) mutant of TCF-4. N-cadherin, ICAT or dnTCF-4 over-expression significantly reduced proliferation of isolated human VSMCs by approximately 55%, 80%, and 45% respectively. Similar effects were observed in human saphenous vein medial segments where proliferation was reduced by approximately 55%. Transfection of dnTCF-4 in the ISS10 human VSMC line significantly lowered TCF and cyclin D1 reporter activity but significantly elevated p21 reporter activity, indicating regulation of these genes by beta-catenin/TCF signaling. In support of this, over-expression of N-cadherin, ICAT or dnTCF-4 in isolated human VSMCs significantly lowered levels of cyclin D1 mRNA and protein levels. In contrast, over-expression of N-Cadherin, ICAT or dnTCF4 significantly elevated p21 mRNA and protein levels. In summary, we have demonstrated that increasing N-cadherin and inhibiting beta-catenin/TCF signaling reduces VSMC proliferation, decreases the expression of cyclin D1 and increases levels of the cell cycle inhibitor, p21. We therefore suggest that the N-cadherin and beta-catenin/TCF signaling pathway is a key modulator of VSMC proliferation via regulation of these 2 beta-catenin/TCF responsive genes.