Down-regulation of protein kinase C inhibits insulin-like growth factor I-induced vascular smooth muscle cell proliferation, migration, and gene expression.
Insulin-like growth factor-I (IGF-I) plays an important role in regulating vascular smooth muscle cell (VSMC) proliferation, directed migration, differentiation, and apoptosis. The signaling mechanisms used by IGF-I to elicit these actions, however, are not well defined. In this study, we examined the role(s) of protein kinase C (PKC) in mediating the IGF-I actions in cultured porcine VSMCs. Out of the eleven known members of PKC family, PKC-alpha, -betaI, -epsilon, -eta, -lambda, -theta, and -zeta, were detectable by Western immunoblot analysis in these cells. Further analysis indicated that the subcellular distribution of several PKC isoforms is regulated by IGF-I. While IGF-I stimulated membrane translocation of PKC-eta, -epsilon, and -zeta and regulated the cytosolic levels of PKC-betaI, it had no such effect on PKC-alpha and -lambda. To examine whether PKC activation is required for the IGF-I-regulated biological responses, phorbol myristate acetate (PMA) and GF109203X were used to down-regulate or inhibit PKC activity. Both PMA (1 microM) and GF109203X (20 microM) nearly completely suppressed the total PKC activity after a 30-min incubation (> 90%), and this inhibition lasted for at least 24 h. Down-regulation or inhibition of PKC activity abolished the IGF-I-induced DNA synthesis, migration and IGFBP-5 gene expression. In contrast, the IGFBP-5 expression induced by forskolin was unaffected by PKC down-regulation or inhibition, suggesting that PKC activation is required for the IGF-regulated but not the cAMP-regulated events. Because the actions of IGF-I on DNA synthesis and IGFBP-5 gene expression in VSMCs have been shown to be mediated through the phosphatidylinositol 3-kinase (PI3 kinase) signaling pathway in porcine VSMCs, the potential role of PKC in IGF-I-induced activation of PI3 kinase and PKB/Akt were examined. Treatment with either PMA or GF109203X did not significantly affect the effects of IGF-I on PI3 kinase activation or PKB/Akt phosphorylation. These results indicated that PKC-betaI, -eta, -epsilon, and -zeta may play an essential role(s) in IGF-I regulation of VSMC migration, DNA synthesis and gene expression, and that these PKC isoforms may either act independently of the PI3 kinase pathway or act further downstream of PKB/Akt in the IGF signaling network.
Yano
K
K
Department of Biology, University of Michigan, Ann Arbor 48109-1048, USA.
Bauchat
J R
JR
Liimatta
M B
MB
Clemmons
D R
DR
Duan
C
C
D000818:Animals; D002455:Cell Division / Q000187:drug effects; D002465:Cell Movement / Q000187:drug effects; D002478:Cells, Cultured; D004789:Enzyme Activation / Q000502:physiology; D015870:Gene Expression / Q000187:drug effects*; D018975:Insulin-Like Growth Factor Binding Protein 5 / Q000235:genetics; D007334:Insulin-Like Growth Factor I / Q000494:pharmacology / Q000502:physiology*; D007527:Isoenzymes / Q000378:metabolism; D009131:Muscle, Smooth, Vascular / Q000166:cytology* / Q000201:enzymology / Q000502:physiology*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000502:physiology*; D013347:Subcellular Fractions / Q000201:enzymology; D013552:Swine
D018975:Insulin-Like Growth Factor Binding Protein 5; D007527:Isoenzymes; D007334:Insulin-Like Growth Factor I; D019869:Phosphatidylinositol 3-Kinases; D011493:Protein Kinase C