Nitric Oxide-PTP Interactions In Aortic Smooth Muscle

主动脉平滑肌中一氧化氮-PTP 相互作用

• 批准号: 6824708
• 负责人: AVIV HASSID
• 金额: $ 36.07万
• 依托单位: UNIVERSITY OF TENNESSEE HEALTH SCI CTR
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-07-01 至 2008-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6824708
• 关键词: aorta; apoptosis; biological signal transduction; cardiovascular injury; cell motility; cell proliferation; enzyme activity; fibroblast growth factor; genetically modified animals; growth factor receptors; in situ hybridization; insulinlike growth factor; intermolecular interaction; laboratory mouse; laboratory rat; nitric oxide; phosphorylation; platelet derived growth factor; protein tyrosine phosphatase; receptor expression; tissue /cell culture; vascular smooth muscle

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.

描述（由申请人提供）：这是研究蛋白酪氨酸磷酸酶PTP1B作为血管平滑肌和血管重塑中一氧化氮（NO）抑制作用的介体的作用的提议。 NO在血管损伤后在新内膜形成中起着主要的抑制作用。缺乏在培养细胞（尤其是体内）中解释这种作用的机制。我们发现，没有增加PTP1B在培养的大鼠主动脉平滑肌细胞中的活性，而不会增加其蛋白质水平。此外，我们发现PDGF和FGF在培养细胞中提高了PTP1B蛋白水平，并且血管损伤类似地诱导了受伤的大鼠颈动脉的PTP1B蛋白水平升高。我们还表明，没有靶向IGF1受体，可以诱导受体酪氨酸去磷酸化并中断培养细胞中IGF1诱导的信号转导。最后，我们已经表明，没有降低细胞质Ca并减少了IGF1诱导的过氧化氢的产生，并且通过通过CA螯合剂独立降低细胞内Ca来模仿这种作用。这些结果支持降低CA参与激活PTP1B的可能参与。 IGF1在血管损伤中的作用目前尚不清楚。一方面，血管损伤诱导IGF1水平上调，但另一方面，IGF1受体mRNA水平和IGF1受体结合降低。与这些发现一致，我们发现血管损伤将IGF1受体蛋白水平降低了约30％，这是通过蛋白质印迹分析确定的。此外，我们发现，通过特定受体酪氨酸磷酸化测量的受体激活降低了80％以上。这些新颖而令人兴奋的发现首次描述了NO和酪氨酸激酶受体去磷酸化涉及蛋白酪氨酸磷酸酶的机械联系。综上所述，我们的结果通过PTP1B升高的Intermediacy，一方面是PDGF或FGF以及另一个IGF1信号转导的可能性。基于上述，我们提出以下特定目的，将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的减少是否需要和/或足以诱导PTP1B活性上调。确定PTP1B的上调是否需要和/或足以说明未诱导的细胞增殖和诱导培养细胞凋亡的诱导。确定PDGF，FGF还是不诱导PTP1B蛋白的上调或血管损伤的活性水平。确定PTP1B在大鼠或小鼠血管损伤模型中未诱导的细胞增殖，运动，凋亡和新内膜形成中是否起作用。确定PTP1B是否在减弱体内IGF受体激活中起作用。