Nitric Oxide-PTP Interactions In Aortic Smooth Muscle

描述（由申请人提供）：这是一项研究蛋白酪氨酸磷酸酶PTP 1B作为一氧化氮（NO）在血管平滑肌和血管重塑中抑制作用的介体的作用的提案。NO在血管损伤后新生内膜形成中起主要抑制作用。在培养的细胞中，特别是在体内，解释这种效应的机制是缺乏的。我们已经发现，NO在培养的大鼠主动脉平滑肌细胞中增加PTP 1B的活性，而不增加其蛋白水平。此外，我们发现PDGF和FGF增加培养细胞中的PTP 1B蛋白水平，并且血管损伤类似地诱导损伤大鼠颈动脉中PTP 1B蛋白水平增加。我们还表明，NO的目标IGF 1受体，诱导受体酪氨酸去磷酸化和中断IGF 1诱导的信号转导培养细胞。最后，我们已经表明，NO降低细胞质钙和减弱IGF 1诱导的过氧化氢的产生，这种效果是模仿由钙螯合剂的细胞内钙的独立降低。这些结果支持可能参与减少钙激活PTP 1B。IGF 1在血管损伤中的作用目前尚不清楚。一方面，血管损伤诱导IGF 1水平上调，但另一方面，IGF 1受体mRNA水平和IGF 1受体结合降低。与这些发现一致，我们发现血管损伤使IGF 1受体蛋白水平降低约30%，如通过Western印迹分析所确定的;此外，我们发现通过特异性受体酪氨酸磷酸化测量的受体活化降低超过80%。这些新的和令人兴奋的发现首次描述了一氧化氮和酪氨酸激酶受体脱磷酸化之间的机制联系，涉及蛋白酪氨酸磷酸酶。两者合计，我们的研究结果提出了负面的串扰之间的可能性，一方面PDGF或FGF，另一方面IGF 1信号转导，通过升高的PTP 1B的中介作用。基于上述情况，我们提出了以下具体目标，在培养的大鼠主动脉平滑肌细胞或大鼠或小鼠中进行：确定细胞质Ca的减少是否是必要的和/或足以诱导PTP 1B活性上调。确定PTP 1B的上调是否是必需的和/或足以解释培养细胞中NO诱导的细胞增殖抑制和细胞凋亡诱导。确定PDGF、FGF或NO是否诱导血管损伤中PTP 1B蛋白或活性水平的上调。确定PTP 1B是否在大鼠或小鼠血管损伤模型中NO诱导的细胞增殖、运动、凋亡和新生内膜形成减少中起作用。确定PTP 1B是否在体内减弱IGF受体活化中发挥作用。

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.