Nitric Oxide-Protein Tyrosine Phosphatase Interactions In Aortic Smooth Muscle

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DESCRIPTION (provided by applicant): This is a proposal to investigate the role of protein tyrosine phosphatase PTP1B as mediator of the inhibitory effects of nitric oxide (NO) in vascular smooth muscle and in vascular remodeling. NO plays a major inhibitory role in neointima formation after vascular injury. Mechanisms explaining this effect in cultured cells and especially in vivo are lacking. We have found that NO increases the activity of PTP1B in cultured rat aortic smooth muscle cells, without increasing its protein levels. Moreover, we have found that PDGF and FGF increase PTP1B protein levels in cultured cells and that vascular injury similarly induces increased PTP1B protein levels in injured rat carotid artery. We have also shown that NO targets the IGF1 receptor, inducing receptor tyrosine dephosphorylation and interrupting IGF1-induced signal transduction in cultured cells. Finally, we have shown that NO decreases cytoplasmic Ca and attenuates IGF1-induced hydrogen peroxide generation and that this effect is mimicked by independent lowering of intracellular Ca by a Ca chelator. These results support a possible involvement of reduced Ca in activating PTP1B. The role of IGF1 in vascular injury is currently unclear. On the one hand, vascular injury induces upregulation of IGF1 levels but on the other, IGF1 receptor mRNA levels and IGF1 receptor binding are decreased. Consistent with these findings, we have found that vascular injury decreases IGF1 receptor protein levels by about 30%, as determined by Western blot analysis; moreover, we have found that receptor activation, as measured by specific receptor tyrosine phosphorylation, is decreased by more than 80%. These novel and exciting findings describe for the first time a mechanistic link between NO and tyrosine kinase receptor dephosphorylation involving a protein tyrosine phosphatase. Taken together, our results raise the possibility of negative cross-talk between, on the one hand PDGF or FGF, and on the other IGF1 signal transduction, via the intermediacy of elevated PTP1B. Based on the above, we propose the following specific aims, to be performed in cultured rat aortic smooth muscle cells or in rats or mice: Determine whether reduction of cytoplasmic Ca is necessary and/or sufficient to induce upregulation of PTP1B activity. Determine whether upregulation of PTP1B is necessary and/or sufficient to account for NO-induced inhibition of cell proliferation and induction of apoptosis in cultured cells. Determine whether PDGF, FGF or NO induces upregulation of PTP1B protein or activity levels in vascular injury. Determine whether PTP1B plays a role in NO-induced decrease of cell proliferation, motility, apoptosis and neointima formation in models of rat or mouse vascular injury. Determine whether PTP1B plays a role in attenuating IGF receptor activation in vivo.