N-cadherin and b-catenin signalling regulates vascular smooth muscle cell proliferation and cell cycle genes

Vascular smooth muscle cell (VSMC) proliferation is a key event in the pathogenesis of vascular diseases. Dismantling of cadherin junctions and translocation of b-catenin to the cytoplasm occurs in proliferating VSMCs. To determine whether N-cadherin and b-catenin signalling are essential in the regulation of VSMC proliferation we over-expressed, using adenoviral delivery, full length N-Cadherin, ICAT (an endogenous inhibitor of b-catenin signalling), or a dominant negative (dn) mutant of the b-catenin transcriptional co-factor, TCF-4. N-cadherin, ICAT and dnTCF-4 over-expression significantly reduced the proliferation of isolated human VSMCs, detected by BrdU incorporation, by 55 ± 6% (p < 0.003), 80 ± 5% (p < 0.001) and 46 ± 7% (p < 0.03), respectively. Similar effects were observed in medial saphenous vein segments infected with the ICAT and dnTCF-4 adenoviruses (51 ± 12% (p < 0.04) and 56 ± 9% (p < 0.02) of control, respectively). Co-transfection with a dnTCF4 construct in the ISS10 human SMC line significantly lowered TCF and cyclin D1 reporter activity by 73 ± 1% (p < 0.03) and 67 ± 4% (p < 0.001) respectively, and elevated p21 reporter activity by 108 ± 19% (p < 0.03). N-cadherin, ICAT and dnTCF-4 over-expression significantly lowered levels of cyclin D1 mRNA by 19 ± 2% (p < 0.01), 34 ± 6% (p < 0.02) and 36 ± 8% (p < 0.03), and lowered levels of cyclin D1 protein by 22 ± 4% (p < 0.04), 44 ± 6% (p < 0.03) and 48 ± 12% (p < 0.01), respectively. In contrast, over-expression of ICAT and dnTCF4 in isolated human VSMCs significantly elevated p21 mRNA levels by 49 ± 14% (p < 0.03) and 55 ± 15% (p < 0.04), and elevated p21 protein levels by 196 ± 45% (p < 0.03) and 454 ± 72% (p < 0.03) respectively. We have demonstrated that increasing N-cadherin mediated cell–cell contacts and inhibiting b-catenin signalling reduces VSMC proliferation, decreases the expression of cyclin D1 and increases levels of the cell cycle inhibitor, p21. We suggest that the N-cadherin and b-catenin signalling pathway is a key modulator of VSMC proliferation via the regulation of these two b-catenin responsive genes