Testosterone regulation of androgen receptor protein expression in cultured vascular smooth muscle cells

Abstract

To investigate the effects of testosterone exposure on androgen receptor (AR) protein expression in vascular smooth muscle cells (VSMC) and the possible mechanisms mediating the effects. VSMC were cultured from thoracic aorta of male Sprague-Dawley rats by using the explant method. Cytoplasmic and nuclear extracts were prepared by means of cell lysis and high salt extraction respectively, and subjected to western blotting analysis for determination of AR protein level. Treatment of synchronized VSMC with testosterone increased both cytoplasmic and nuclear AR protein expression in a dose (0 - 4 micro mol/L) and time (1 - 48 h)-dependent fashion, whereas exposure of VSMC to testosterone at a physiologically relevant concentration of 40 nmol/L for 10 min induced a transient down-regulation of AR protein. Pretreatment with transcription inhibitor actinomycin D and translation inhibitor cycloheximide repressed cytoplasmic AR protein leves to 46% and 12% (means) of the androgen treatment control level respectively. Furthermore, androgen up-regulation of intracellular AR protein was partially inhibited (50%) by nonsteroidal androgen antagonist, flutamide. Homologous up-regulation of AR protein expression exist in synchronized VSMC, and the auto-regulation is time and testosterone dose dependent, accompanied by nuclear translocation of AR protein, and requires functional AR protein. In addition, our present data suggest that testosterone increases AR protein expression in VSMC at the level of both transcription and translation.