PKCδ mediates anti-proliferative, pro-apoptic effects of testosterone on coronary smooth muscle

Abstract

Sex hormone status has emerged as an important modulator of coronary physiology and cardiovascular disease risk in both males and females. Our previous studies have demonstrated that testosterone increases protein kinase C (PKC) δ expression and activity in coronary smooth muscle (CSMC). Because PKCδ has been implicated in regulation of proliferation and apoptosis in other cell types, we sought to determine if testosterone modulates CSMC proliferation and/or apoptosis through PKCδ. Porcine CSMC cultures (passages 2–6) from castrated males were treated with testosterone for 24 h. Testosterone (20 and 100 nM) decreased [3H]thymidine incorporation in proliferating CSMC to 59 ± 5.3 and 33.1 ± 4.5% of control. Flow cytometric analysis demonstrated that testosterone induced G1arrest in CSMC with a concomitant reduction in the S phase cells. Testosterone reduced protein levels of cyclins D1and E and phosphorylation of retinoblastoma protein while elevating levels of p21cip1and p27kip1. There were no significant differences in the levels of cyclins D3, CDK2, CDK4, or CDK6. Testosterone significantly reduced kinase activity of CDK2 and -6, but not CDK4, -7, or -1. PKCδ small interfering RNA (siRNA) prevented testosterone-mediated G1arrest, p21cip1upregulation, and cyclin D1and E downregulation. Furthermore, testosterone increased CSMC apoptosis in a dose-dependent manner, which was blocked by either PKCδ siRNA or caspase 3 inhibition. These findings demonstrate that the anti-proliferative, pro-apoptotic effects of testosterone on CSMCs are substantially mediated by PKCδ.