DIFFERENTIATED PHENOTYPE OF SMOOTH-MUSCLE CELLS DEPENDS ON SIGNALING PATHWAYS THROUGH INSULIN-LIKE GROWTH-FACTORS AND PHOSPHATIDYLINOSITOL 3-KINASE

Under conventional culture conditions, smooth muscle cells display the ir phenotypic modulation from a differentiated to a dedifferentiated s tate. Here, we established a primary culture system of smooth muscle c ells maintaining a differentiated phenotype, as characterized by expre ssion of smooth muscle-specific marker genes such as h-caldesmon and c alponin, cell morphology, and ligand-induced contractility. Laminin re tarded the progression of dedifferentiation of smooth muscle cells. In sulin-like growth factors (IGF-I and IGF-II) and insulin markedly prol onged the differentiated phenotype, with IGF-I being the more potent, In contrast, serum, epidermal growth factor, transforming growth facto rs, and platelet-derived growth factors potently induced dedifferentia tion compared with angiotensin II, arginine-vasopressin, and basic fib roblast growth factor. Using the present culture system, we investigat ed signaling pathways regulating a phenotype of smooth muscle cells. I n cultured cells, IGF-I specifically activated phosphatidylinositol 3- kinase (PI3-kinase) and its downstream target, protein kinase B, but n ot mitogen-activated protein kinases. Specific inhibitors of PIS-kinas e (wortmannin and LY294002) induced dedifferentiation of smooth muscle cells even when they were cultured on laminin under IGF-I-stimulated conditions. The sole effect of laminin to retard the dedifferentiation was completely blocked by anti-IGF-I antibody, and laminin promoted t he endogenous expression of IGF-I in cultured cells. The reduced promo ter activity of the caldesmon gene induced by platelet-derived growth factor BE was overcome by the forced expression of the constitutive ac tive form of PI3-kinase p110 alpha catalytic subunit. These findings s uggest that an IGF-I signaling pathway through PI3-kinase plays a crit ical role in maintaining a differentiated phenotype of smooth muscle c ells.