EFFECTS OF PROTEIN-KINASE-C-ALPHA OVEREXPRESSION ON A7R5 SMOOTH-MUSCLE CELL-PROLIFERATION AND DIFFERENTIATION

Smooth muscle cell differentiation and proliferation are increasingly seen to be intimately tied to the etiology of atherosclerosis and hype rtension. To determine the role of PKC alpha in the regulation of smoo th muscle cell differentiation and proliferation, the rat embryonic sm ooth muscle cell line A7r5 was transfected with an expression vector c ontaining the full-length PKC alpha cDNA. Neomycin-resistant clones wh ich exhibited increased PKC alpha levels compared to wild-type cells w ere selected. The A7r5 cells overexpressing PKC alpha had altered morp hology and decreased growth rates compared to wild-type cells and cell s transfected only with the neomycin resistance gene. Electrophoretic mobility shift assays showed that nuclear extracts from overexpressing clones gave a different pattern of protein-DNA binding to an AP-1 con sensus oligonucleotide compared to wild-type cells. In contrast to the growth characteristics of these clones, their levels of cell differen tiation marker proteins such as vinculin and desmin were not affected by PKC alpha overexpression. Moreover, the smooth muscle-specific diff erentiation marker alpha-actin was markedly reduced, while beta-actin levels were found to remain unchanged. Northern blot analysis confirme d that alpha-actin downregulation occurred at the RNA level. Western b lot analysis revealed that A7r5 cells have five different PKC isoforms and that these isoform protein levels were not changed by PKC alpha o verexpression. These findings suggest that PKC alpha regulates growth and differentiation of A7r5 smooth muscle cells and that these changes might result from altered expression/function of AP-1 transcription f actors. (C) 1997 Academic Press.