Mutant parathyroid hormone-related protein, devoid of the nuclear localization signal, markedly inhibits arterial smooth muscle cell cycle and neointima formation by coordinate up-regulation of p15Ink4b and p27kip1.
Arterial expression of PTH-related protein is markedly induced by angioplasty. PTH-related protein contains a nuclear localization signal (NLS). PTH-related protein mutants lacking the NLS (DeltaNLS-PTH-related protein) are potent inhibitors of arterial vascular smooth muscle cell (VSMC) proliferation in vitro. This is of clinical relevance because adenoviral delivery of DeltaNLS-PTH-related protein at angioplasty completely inhibits arterial restenosis in rats. In this study we explored the cellular mechanisms through which DeltaNLS-PTH-related protein arrests the cell cycle. In vivo, adenoviral delivery of DeltaNLS-PTH-related protein at angioplasty markedly inhibited VSMC proliferation as compared with angioplastied carotids infected with control adenovirus (Ad.LacZ). In vitro, DeltaNLS-PTH-related protein overexpression was associated with a decrease in phospho-pRb, and a G(0)/G(1) arrest. This pRb underphosphorylation was associated with stable levels of cdks 2, 4, and 6, the D and E cyclins, p16, p18, p19, and p21, but was associated with a dramatic decrease in cdk-2 and cdk4 kinase activities. Cyclin A was reduced, but restoring cyclin A adenovirally to normal did not promote cell cycle progression in DeltaNLS-PTH-related protein VSMC. More importantly, p15(INK4) and p27(kip1), two critical inhibitors of the G(1/S) progression, were markedly increased. Normalization of both p15(INK4b) and p27(kip1) by small interfering RNA knockdown normalized cell cycle progression. These data indicate that the changes in p15(INK4b) and p27(kip1) fully account for the marked cell cycle slowing induced by DeltaNLS-PTH-related protein in VSMCs. Finally, DeltaNLS-PTH-related protein is able to induce p15(INK4) and p27(kip1) expression when delivered adenovirally to primary murine VSMCs. These studies provide a mechanistic understanding of DeltaNLS-PTH-related protein actions, and suggest that DeltaNLS-PTH-related protein may have particular efficacy for the prevention of arterial restenosis.
Division of Endocrinology and Metabolism, University of Pittsburgh School of Medicine, Pennsylvania 15213, USA. taeschn@dom.pitt.edu
Nathalie
N
Fiaschi-Taesch
Brian
B
Sicari
Kiran
K
Ubriani
Irene
I
Cozar-Castellano
Karen K
KK
Takane
Andrew F
AF
Stewart
D000818:Animals; D001158:Arteries / Q000187:drug effects / Q000378:metabolism; D002453:Cell Cycle / Q000187:drug effects* / Q000235:genetics; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D023903:Coronary Restenosis / Q000517:prevention & control; D050762:Cyclin-Dependent Kinase Inhibitor p15 / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D050760:Cyclin-Dependent Kinase Inhibitor p27 / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D009131:Muscle, Smooth, Vascular / Q000187:drug effects* / Q000378:metabolism; D050505:Mutant Proteins / Q000737:chemistry / Q000494:pharmacology; D032389:Myocytes, Smooth Muscle / Q000187:drug effects / Q000378:metabolism; D018919:Neovascularization, Physiologic / Q000187:drug effects; D019913:Nuclear Localization Signals / Q000502:physiology; D044162:Parathyroid Hormone-Related Protein / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology*; D034741:RNA, Small Interfering / Q000494:pharmacology; D051381:Rats; D017539:Tunica Intima / Q000187:drug effects / Q000254:growth & development / Q000378:metabolism; D015854:Up-Regulation / Q000187:drug effects
D050762:Cyclin-Dependent Kinase Inhibitor p15; D050505:Mutant Proteins; D019913:Nuclear Localization Signals; D044162:Parathyroid Hormone-Related Protein; D034741:RNA, Small Interfering; D050760:Cyclin-Dependent Kinase Inhibitor p27