Migration of smooth muscle cells is of critical importance to vascular development, angiogenesis, neointima formation and tissue remodeling that occurs after vascular injury. Preliminary observations indicate that NO elicits reorganization of the actin cytoskeleton and that it increases the motility of rat aortic smooth muscle cells in primary culture, but not subculture. These effects are associated with increased levels of protein tyrosine phosphatase 1D (PTP1D) in primary culture but not subculture. We have also found that reduction of PTP1D protein levels with antisense oligodeoxynucleotide (ODN) attenuates NO- induced motility in primary cultures. The overall purpose of this project is to investigate the role of PTP1D in NO-induced cell motility in culture and in vivo. The proposal is divided into three interrelated parts. A.
Aims dealing with mechanisms of NO-stimulated PTP1D expression A.i.) Determine whether NO increases PTP1D enzyme activity, concomitantly with increased protein levels. A.ii.) Determine whether NO increases PTP1D mRNA levels and if so, whether this effect requires cGMP-dependent protein kinase (PKG) activity. If mRNA levels are increased, determine whether increased mRNA synthesis can explain this effect or whether increased mRNA stability may be involved. A.iii.) Determine whether NO increases the rate of PTP1D synthesis and/or decreases the rate of PTP1D metabolism. B.
Aims dealing with consequences of gain-of-function or loss-of-function of PKG or PTP1D in cultured cells. B.i.) Determine whether enforced expression of PKG in subcultured cells deficient in PKG restores the capacity of NO to increase PTP1D proteins levels and stimulate cell motility. B.ii.) Determine whether enforced expression of wild-type PTP1D, but not catalytically inactive mutant PTP1D, in subcultured cells deficient in PTP1D enhances cell motility. B.iii.) Determine whether NO-elicited actin cytoskeletal reorganization and increased cell motility in primary culture are blocked by agents that interfere with PTP1D function and whether overexpression of PTP1D mimics the effects of NO. C.
Aim dealing with expression of PTP1D and modulation of cell motility by PTP1D in vivo: Determine whether vascular injury increases PTP1D protein levels and whether expression of dominant-interfering mutant of PTP1D attenuates vascular injury- induced cell motility and neointima formation. We anticipate that this project will provide new information on mechanisms that are likely to explain the motogenic capacity of NO in aortic smooth muscle cells and in vivo.
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