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J Biol Chem, Vol. 273, Issue 44, 28860-28867, October 30, 1998
From the Department of Neurochemistry and Neuropharmacology,
Biomedical Research Center, Osaka University Medical School, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan
Under conventional culture conditions, smooth
muscle cells display their phenotypic modulation from a differentiated
to a dedifferentiated state. Here, we established a primary culture system of smooth muscle cells maintaining a differentiated phenotype, as characterized by expression of smooth muscle-specific marker genes
such as h-caldesmon and calponin, cell
morphology, and ligand-induced contractility. Laminin retarded the
progression of dedifferentiation of smooth muscle cells. Insulin-like
growth factors (IGF-I and IGF-II) and insulin markedly prolonged the
differentiated phenotype, with IGF-I being the more potent. In
contrast, serum, epidermal growth factor, transforming growth factors,
and platelet-derived growth factors potently induced dedifferentiation
compared with angiotensin II, arginine-vasopressin, and basic
fibroblast growth factor. Using the present culture system, we
investigated signaling pathways regulating a phenotype of smooth muscle
cells. In cultured cells, IGF-I specifically activated
phosphatidylinositol 3-kinase (PI3-kinase) and its downstream target,
protein kinase B, but not mitogen-activated protein kinases. Specific
inhibitors of PI3-kinase (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 the endogenous expression of
IGF-I in cultured cells. The reduced promoter activity of the caldesmon gene induced by platelet-derived growth factor BB was overcome by the
forced expression of the constitutive active form of PI3-kinase p110
catalytic subunit. These findings suggest that an IGF-I signaling
pathway through PI3-kinase plays a critical role in maintaining a
differentiated phenotype of smooth muscle cells.
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