© 2000 American Heart Association, Inc.
Vascular Biology |
Doxazosin Inhibits Retinoblastoma Protein Phosphorylation and G1
S Transition in Human Coronary Smooth Muscle Cells
From the Department of Medicine (U.K., S.W., S.K., S.M.J., W.A.H., R.E.L.), Division of Endocrinology, Diabetes and Hypertension, School of Medicine, University of California, Los Angeles, and the Department of Medicine/Cardiology (U.K., E.F.), Virchowklinikum, Humboldt University Berlin, and German Heart Institute Berlin, Berlin, Germany.
Correspondence to Ronald E. Law, PhD, UCLA School of Medicine, Division of Endocrinology, Diabetes and Hypertension, Warren Hall, Second Floor, Suite 24-130, 900 Veteran Ave, Box 957073, Los Angeles, CA 90095. E-mail rlaw{at}med1.medsch.ucla.edu
Abstract—Previous studies have
demonstrated that the 
1-adrenergic receptor
antagonist doxazosin (Dox) inhibits multiple
mitogenic signaling pathways in human vascular smooth
muscle cells. This broad antiproliferative activity of Dox occurs
through a novel mechanism unrelated to its blocking the
1-adrenergic receptor. Flow cytometry demonstrated that
Dox prevents mitogen-induced G1
S progression of human
coronary artery smooth muscle cells (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
µmol/L insulin (P+I)–stimulated cells (P<0.01 for
10 µmol/L Dox versus P+I alone) and 52±18.7% for 10%
FBS-induced mitogenesis (P<0.05 for 10 µmol/L
Dox versus 10% FBS alone). Inhibition of G1 exit by Dox
was accompanied by a significant blockade of retinoblastoma protein
(Rb) phosphorylation.
Hypophosphorylated Rb sequesters the E2F transcription
factor, leading to G1 arrest. Adenoviral overexpression of
E2F-1 stimulated quiescent CASMCs to progress through G1
and enter the S phase. E2F-mediated G1 exit was not
affected by Dox, suggesting that it targets events upstream from Rb
hyperphosphorylation. Downregulation of the
cyclin-dependent kinase inhibitory protein p27 is important
for maximal activation of G1 cyclin/cyclin-dependent kinase
holoenzymes to overcome the cell cycle inhibitory activity
of Rb. In Western blot analysis, p27 levels decreased after
mitogenic stimulation (after P+I, 43±1.8% of quiescent
cells [P<0.01 versus quiescent cells]; after 10%
FBS, 55±7.7% of quiescent cells [P<0.05 versus
quiescent cells]), whereas the addition of Dox (10 µmol/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 present study demonstrates that Dox inhibits CASMC
proliferation by blocking cell cycle progression from the
G0/G1 phase to the S phase. This
G1S blockade likely results from an inhibition of
mitogen-induced Rb hyperphosphorylation through
prevention of p27 downregulation.
Key Words: vascular smooth muscle cells • proliferation • cell cycle • retinoblastoma • doxazosin
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