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

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-alpha in primary cultures of VSMCs from rat aortas under different conditions of growth and differentiation. PKC-alpha 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-alpha 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 alpha-actin and desmin. PKC-alpha 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-alpha expression together with a more differentiated phenotype. Subcultured, rapidly growing VSMCs from SHR showed a decreased PKC-alpha expression compared with cells from WKY rats. To establish cause and effect, we next microinjected either PKC-alpha or inactivated material directly into dedifferentiated cells. We found that cells injected with active PKC-alpha expressed increased amounts of actin compared with control cells.(ABSTRACT TRUNCATED AT 250 WORDS)

The Proliferative Effect of Vascular Endothelial Growth Factor Requires Protein Kinase C-α and Protein Kinase C-ζ

“…This observation could indicate that VEGF induces a translocation of PKC-␣ into the nucleus. Nuclear translocation of this isoform by tyrosine kinase receptor signaling has been reported previously by us 21,30,31 and others. [32][33][34] Which signal directs PKC isoforms to the nucleus is presently unclear.…”

“…37 We recently showed that retinoic acid-induced differentiation of vascular smooth muscle cells is associated with increased expression of PKC-␣ and that this isoform can induce differentiation in these cells. 30 Others have suggested that PKC-␣ may be among the most likely mediators of G1/S inhibition in endothelial cells. 38 -40 VEGF also induced a translocation of PKC-, and the confocal micrographs show an increased immunoreactivity in the cytosol and in the nucleus.…”

Phenotypic diversity and molecular mechanisms of airway smooth muscle proliferation in asthma

“…Expression of PKCb was not detected in either freshly isolated or proliferating smooth muscle cells. In another study, PKCa was downregulated when rat vascular smooth muscle cells assumed a proliferative/secretory phenotype [117]. When the cells reassumed their contractile state, PKCa expression reverted to normal levels.…”

Neonatal pulmonary hypertension prevents reorganisation of the pulmonary arterial smooth muscle cytoskeleton after birth

“…The actin content of cultured smooth muscle cells decreases when the cells change shape and move (Haller et al 1995). Actin assembly is partly controlled by extracellular signals such as stretch, adhesion to the extracellular matrix and growth factors, many of which are sequestered in the matrix (Wilson et al 1995 ;ChrzanowskaWodnicka & Burridge, 1996 ;Dinbergs et al 1996 ;O'Reilly et al 1997).…”