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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 p15^Ink4b and p27^kip1

Division of Endocrinology and Metabolism, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15213

Address all correspondence and requests for reprints to: Nathalie Fiaschi- Taesch, Ph.D., Assistant Professor, BST E-1140, Division of Endocrinology and Metabolism, University of Pittsburgh School of Medicine, 200 Lothrop Street, Pittsburgh, Pennsylvania 15213. E-mail: taeschn{at}dom.pitt.edu.

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 (NLS-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 NLS-PTH-related protein at angioplasty completely inhibits arterial restenosis in rats. In this study we explored the cellular mechanisms through which NLS-PTH-related protein arrests the cell cycle. In vivo, adenoviral delivery of NLS-PTH-related protein at angioplasty markedly inhibited VSMC proliferation as compared with angioplastied carotids infected with control adenovirus (Ad.LacZ). In vitro, NLS-PTH-related protein overexpression was associated with a decrease in phospho-pRb, and a G₀/G₁ 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 NLS-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 NLS-PTH-related protein in VSMCs. Finally, NLS-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 NLS-PTH-related protein actions, and suggest that NLS-PTH-related protein may have particular efficacy for the prevention of arterial restenosis.