Nitric Oxide-Protein Tyrosine Phosphatase 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PTP1B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建议。NO在血管损伤后新生内膜的形成中起重要的抑制作用。在培养的细胞中，特别是在活体中，解释这种效应的机制尚不清楚。我们发现，NO可增加培养的大鼠主动脉平滑肌细胞PTP1B的活性，但不增加其蛋白水平。此外，我们还发现PDGF和成纤维细胞生长因子增加了培养细胞中PTP1B的蛋白水平，血管损伤也同样导致损伤的大鼠颈动脉中PTP1B蛋白水平的增加。我们还发现，NO靶向IGF1受体，诱导受体酪氨酸去磷酸化，并阻断IGF1诱导的培养细胞信号转导。最后，我们已经证明，NO降低胞浆内钙并减弱IGF1诱导的过氧化氢生成，并且这种作用是通过钙螯合剂独立降低细胞内钙来模拟的。这些结果支持了钙还原可能参与了PTP1B的激活。IGF1在血管损伤中的作用目前尚不清楚。一方面，血管损伤导致IGF1水平上调，但另一方面，IGF1受体mRNA水平和IGF1受体结合力降低。与这些发现一致的是，我们发现血管损伤使IGF1受体蛋白水平下降了约30%，这是通过Western印迹分析确定的；此外，我们还发现，通过特异性受体酪氨酸磷酸化来衡量受体活性，降低了80%以上。这些新的和令人兴奋的发现首次描述了NO和涉及蛋白质酪氨酸磷酸酶的酪氨酸激酶受体去磷酸化之间的机制联系。综上所述，我们的结果提出了在PDGF或成纤维细胞生长因子与IGF1信号转导之间通过PTP1B升高的中介而产生负向串扰的可能性。在此基础上，我们提出了在培养的大鼠主动脉平滑肌细胞或大鼠或小鼠中进行的以下特定目标：确定胞浆内钙的减少是否必要和/或足以诱导PTP1B活性上调。确定PTP1B的上调是否必要和/或足以解释NO诱导的细胞增殖抑制和诱导培养细胞的凋亡。确定在血管损伤中，PDGF、成纤维细胞生长因子是否诱导PTP1B蛋白或活性水平上调。确定PTP1B是否在NO诱导的大鼠或小鼠血管损伤模型中细胞增殖、活力、细胞凋亡和新生内膜形成的减少中发挥作用。确定PTP1B是否在体内发挥减弱IGF受体激活的作用。

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.