Doxazosin inhibits retinoblastoma protein phosphorylation and G(1)-> S transition in human coronary smooth muscle cells

Previous studies have demonstrated that the alpha(1)-adrenergic receptor an tagonist doxazosin (Dox) inhibits multiple mitogenic signaling pathways in human vascular smooth muscle cells. This broad antiproliferative activity o f Dox occurs through a novel mechanism unrelated to its blocking the alpha( 1)-adrenergic receptor. Flow cytometry demonstrated that Dox prevents mitog en-induced G(1)-->S progression of human coronary artery smooth muscle cell s (CASMCs) in a dose-dependent manner, with a maximal reduction of S-phase transition by 88+/-10.5% in 20 ng/mL platelet-derived growth factor and 1 m u mol/L insulin (P+I)-stimulated cells (P<0.01 for 10 mu mol/L Dox versus P +I alone) and 52+/-18.7% for 10% FBS-induced mitogenesis (P<0.05 for 10 mu mol/L Dox versus 10% FBS alone). Inhibition of G(1) exit by Dox was accompa nied by a significant blockade of retinoblastoma protein (Rb) phosphorylati on. Hypophosphorylated Rb sequesters the E2F transcription factor, leading to G(1) arrest. Adenoviral overexpression of E2F-1 stimulated quiescent CAS MCs to progress through G(1) and enter the S phase. E2F-mediated G(1) exit was not affected by Dox, suggesting that it targets events upstream from Rb hyperphosphorylation. Downregulation of the cyclin-dependent kinase inhibi tory protein p27 is important for maximal activation of G(1) cyclin/cyclin- dependent kinase holoenzymes to overcome the cell cycle inhibitory activity of Rb, In Western blot analysis, p27 levels decreased after mitogenic stim ulation (after P+I, 43+/-1.8% of quiescent cells [P<0.01 versus quiescent c ells]; after 10% FBS, 55+/-7.7% of quiescent cells [P<0.05 versus quiescent cells]), whereas the addition of Dox (10 mu mol/L) markedly attenuated its downregulation (after P+I, 90+/-8.3% of quiescent cells [P<0.05 versus P+I alone]; after 10% FBS, 78+/-8.3% of quiescent cells [P<0.05 versus 10% FBS alone]). Furthermore, Dox inhibited cyclin A expression, an E2F regulated gene that is essential for cell cycle progression into the S phase. The pre sent study demonstrates that Dox inhibits CASMC proliferation by blocking c ell cycle progression from the G(0)/G(1) phase to the S phase. This G(1)--> S blockade likely results from an inhibition of mitogen-induced Rb hyperpho sphorylation through prevention of p27 downregulation.