The mTOR/p70 S6K1 pathway regulates vascular smooth muscle cell differentiation.
Vascular smooth muscle cells (VSMC) in mature, normal blood vessels exhibit a differentiated, quiescent, contractile morphology, but injury induces a phenotypic modulation toward a proliferative, dedifferentiated, migratory phenotype with upregulated extracellular matrix protein synthesis (synthetic phenotype), which contributes to intimal hyperplasia. The mTOR (the mammalian target of rapamycin) pathway inhibitor rapamycin inhibits intimal hyperplasia in animal models and in human clinical trials. We report that rapamycin treatment induces differentiation in cultured synthetic phenotype VSMC from multiple species. VSMC treated with rapamycin assumed a contractile morphology, quantitatively reflected by a 67% decrease in cell area. Total protein and collagen synthesis were also inhibited by rapamycin. Rapamycin induced expression of the VSMC differentiation marker contractile proteins smooth muscle (SM) alpha-actin, calponin, and SM myosin heavy chain (SM-MHC), as observed by immunoblotting and immunohistochemistry. Notably, we detected a striking rapamycin induction of calponin and SM-MHC mRNA, suggesting a role for mTOR in transcriptional control of VSMC gene expression. Rapamycin also induced expression of the cyclin-dependent kinase inhibitors p21(cip) and p27(kip), consistent with cell cycle withdrawal. Rapamycin inhibits mTOR, a signaling protein that regulates protein synthesis effectors, including p70 S6K1. Overexpression of p70 S6K1 inhibited rapamycin-induced contractile protein and p21(cip) expression, suggesting that this kinase opposes VSMC differentiation. In conclusion, we report that regulation of VSMC differentiation is a novel function of the rapamycin-sensitive mTOR signaling pathway.
Department of Surgery, Section of Vascular Surgery, Dartmouth Medical School, Lebanon, New Hampshire 03756, USA. Kathleen.A.Martin@Hitchcock.org
Kathleen A
KA
Martin
Eva M
EM
Rzucidlo
Bethany L
BL
Merenick
Diane C
DC
Fingar
David J
DJ
Brown
Robert J
RJ
Wagner
Richard J
RJ
Powell
D000818:Animals; D001013:Aorta, Thoracic / Q000166:cytology; D015415:Biomarkers; D002417:Cattle; D018797:Cell Cycle Proteins / Q000378:metabolism; D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D050759:Cyclin-Dependent Kinase Inhibitor p21; D050760:Cyclin-Dependent Kinase Inhibitor p27; D016213:Cyclins / Q000378:metabolism; D004730:Endothelium, Vascular / Q000166:cytology; D016326:Extracellular Matrix Proteins / Q000378:metabolism; D007166:Immunosuppressive Agents / Q000494:pharmacology; D009119:Muscle Contraction / Q000187:drug effects; D009131:Muscle, Smooth, Vascular / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism*; D010641:Phenotype; D011494:Protein Kinases / Q000378:metabolism*; D038762:Ribosomal Protein S6 Kinases, 70-kDa / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D020123:Sirolimus / Q000494:pharmacology; D058570:TOR Serine-Threonine Kinases; D025521:Tumor Suppressor Proteins / Q000378:metabolism
D015415:Biomarkers; D018797:Cell Cycle Proteins; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D016326:Extracellular Matrix Proteins; D007166:Immunosuppressive Agents; D025521:Tumor Suppressor Proteins; D050760:Cyclin-Dependent Kinase Inhibitor p27; D011494:Protein Kinases; C546842:MTOR protein, human; D038762:Ribosomal Protein S6 Kinases, 70-kDa; D058570:TOR Serine-Threonine Kinases; D020123:Sirolimus