Suppression of smooth-muscle alpha-actin expression by platelet-derived growth factor in vascular smooth-muscle cells involves Ras and cytosolic phospholipase A2.

Platelet-derived growth factor (PDGF), which is a potent mitogen for vascular smooth-muscle cells (VSMC), also inhibits the expression of specific smooth-muscle proteins, including smooth-muscle alpha-actin (SM-alpha-actin), in these cells. The goal of this study was to identify signalling pathways mediating these distinct effects. In rat aortic VSMC, PDGF caused a rapid activation of Ras and Raf, leading to the activation of mitogen-activated protein kinases (ERKs). Cells stably transfected with constitutively active Ras (H-Ras) expressed low levels of SM-alpha-actin protein. Arginine vasopressin, which stimulated SM-alpha-actin promoter activity in wild-type cells or controls (Neo; transfected with a plasmid lacking an insert), failed to do so in cells transiently expressing H-Ras. The effects of Ras on suppression of SM-alpha-actin expression were not mediated by the Raf/ERK pathway, since cells stably expressing constitutively active Raf (BxB-Raf) had normal levels of SM-alpha-actin protein, and stimulation of SM-alpha-actin promoter activity by vasopressin was unaffected in cells transiently expressing BxB-Raf. Furthermore a specific inhibitor of ERK activation had no effect on SM-alpha-actin expression. Exposure of wild-type VSMC to PDGF, or stable expression of Ras but not Raf, also resulted in constitutive increases in prostaglandin E2 production and cytosolic phospholipase A2 (cPLA2) activity, which was mediated by an increased expression of cPLA2 protein. Transient expression of cPLA2 in wild-type VSMC inhibited the stimulation of SM-alpha-actin promoter activity by vasopressin. These results suggest that PDGF-induced inhibition of SM-alpha-actin expression is mediated through a Ras-dependent/Raf independent pathway involving the induction of cPLA2 and eicosanoid production.

Abnormal function of protein kinase C in cells having phosphatidylethanolamine-deficient and phosphatidylcholine-excess membranes.

When rat mammary carcinoma 64-24 cells are grown in the absence of ethanolamine, their membrane phospholipid composition changes significantly, becoming phosphatidylethanolamine-deficient and phosphatidylcholine-excess due to a reduced de novo rate of phosphatidylethanolamine synthesis, and growth stops. We have assumed that this membrane phospholipid environment is not suitable for membrane-associated functions. We have previously demonstrated that functions normally stimulated by tumor-promoting phorbol ester, phorbol 12,13-dibutyrate, are not stimulated in ethanolamine-deprived cells, suggesting that function of protein kinase C may be abnormal under the altered membrane environment. In the present study, the behavior of protein kinase C in 64-24 cells grown in the presence and absence of ethanolamine (having normal and phosphatidylethanolamine-deficient/phosphatidylcholine-excess phospholipid) was compared by enzyme assay as well as Western blotting. The results show that the nature of association of protein kinase C to the membrane, which is induced by phorbol ester, is abnormal when cells have the altered membrane phospholipid, and thus argue that membrane phospholipid environment is important in the function of protein kinase C.