Protein kinase Cδ inhibition of S-phase transition in capillary endothelial cells involves the cyclin-dependent kinase inhibitor p27^Kip1

Distinct protein kinase C (PKC) isoforms differentially regulate cellular proliferation in rat microvascular endothelial cells (EC). Overexpression of PKCα has little effect on proliferation, whereas PKCδ slows endothelial cell proliferation and induces S-phase arrest. Analyses were performed on EC overexpressing PKCα (PKCαEC) or PKCδ (PKCδEC) to determine the role of specific cell cycle regulatory proteins in the PKCδ- induced cell cycle arrest. Serum-induced stimulation of cyclins D1, E, and A- associated kinase activity was delayed by 12 h in the PKCδEC line in association with S-phase arrest. However, the protein levels for cyclins D1, E, and A were similar. Nuclear accumulation of cyclin D1 protein in response to serum was also delayed in PKCδEC. In the PKCδEC line, serum induced p27(Kip1) but not p16(Ink4a) or p21(Cip1). Serum did not affect p27(Kip1) levels in the control vascular endothelial cell line. Immunoprecipitation- Western blotting analysis of p27(Kip1) showed serum stimulation of the vascular endothelial cell line resulted in increased amounts of cyclin D1 bound to p27(Kip1). In the PKCδEC line, serum did not increase the amount of cyclin D1 bound to p27(Kip1). Transfection of full-length p27(Kip1) antisense into the PCKδEC line reversed the S-phase arrest and resulted in normal cell cycle progression, suggesting a critical role for p27(Kip1) in the PKCδ- mediated S-phase arrest.