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 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 p110alpha 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.
Hayashi
K
K
Department of Neurochemistry and Neuropharmacology, Biomedical Research Center, Osaka University Medical School, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan.
Saga
H
H
Chimori
Y
Y
Kimura
K
K
Yamanaka
Y
Y
Sobue
K
K
D000818:Animals; D002478:Cells, Cultured; D002642:Chick Embryo; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D004791:Enzyme Inhibitors / Q000494:pharmacology; D016326:Extracellular Matrix Proteins / Q000378:metabolism; D009130:Muscle, Smooth / Q000201:enzymology / Q000378:metabolism*; D010641:Phenotype; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D000081082:Phosphoinositide-3 Kinase Inhibitors; D015398:Signal Transduction; D013002:Somatomedins / Q000378:metabolism*; D014158:Transcription, Genetic
D015500:Chloramphenicol O-Acetyltransferase; D004791:Enzyme Inhibitors; D016326:Extracellular Matrix Proteins; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D013002:Somatomedins