Differentiation of Vascular Smooth Muscle Cells and the Regulation of Protein Kinase C-α

Abstract

Abstract Dedifferentiation and proliferation of vascular smooth muscle cells (VSMCs) are important features of atherosclerosis. The molecular mechanisms are largely unclear; however, protein kinase C (PKC) is a key enzyme in the intracellular signaling pathways that mediate this process. We studied the activity and immunoreactivity of PKC-α in primary cultures of VSMCs from rat aortas under different conditions of growth and differentiation. PKC-α was determined under the following conditions: (1) during the growth phase and after confluence of cultured (passages 1 through 3) VSMCs, (2) before and after induction of differentiation in VSMCs by retinoic acid, and (3) in primary cultures of VSMCs from spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats during early passages. PKC activity was measured by in vitro substrate phosphorylation. PKC-α immunoreactivity was assessed by Western blot using specific polyclonal antibodies and by immunostaining with confocal microscopy. Cell proliferation was measured by direct count. The cell phenotype was characterized by immunostaining and Western blot for α-actin and desmin. PKC-α expression and PKC activity during VSMC growth showed a decrease during rapid growth and an increase in confluent cells. This pattern was associated with the respective changes in cell differentiation. Retinoic acid induced an increase in PKC-α expression together with a more differentiated phenotype. Subcultured, rapidly growing VSMCs from SHR showed a decreased PKC-α expression compared with cells from WKY rats. To establish cause and effect, we next microinjected either PKC-α or inactivated material directly into dedifferentiated cells. We found that cells injected with active PKC-α expressed increased amounts of actin compared with control cells. We identified a close correlation between PKC-α and actin immunofluorescence. We conclude that PKC-α is downregulated in rapidly growing VSMCs. Our findings demonstrate an inverse association between PKC-α expression and VSMC differentiation. They suggest a role for downregulation of PKC-α in the proliferative response of these cells.