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: 6899840
• 负责人: AVIV HASSID
• 金额: $ 36.5万
• 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/6899840
• 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）：这是一项研究蛋白酪氨酸磷酸酶PTP1B在血管平滑肌和血管重塑中作为一氧化氮（NO）抑制作用中介的作用的提案。NO在血管损伤后新生内膜形成中起主要抑制作用。在培养细胞中特别是在体内解释这种作用的机制尚不清楚。我们发现NO增加了培养大鼠主动脉平滑肌细胞中PTP1B的活性，但不增加其蛋白水平。此外，我们发现PDGF和FGF增加了培养细胞中PTP1B蛋白水平，血管损伤同样诱导损伤大鼠颈动脉中PTP1B蛋白水平升高。我们还发现NO靶向IGF1受体，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，我们已经证明NO可以降低胞质Ca并减弱igf1诱导的过氧化氢生成，并且这种作用可以通过Ca螯合剂独立降低细胞内Ca来模拟。这些结果支持Ca减少可能参与激活PTP1B。IGF1在血管损伤中的作用目前尚不清楚。血管损伤一方面诱导IGF1水平上调，另一方面抑制IGF1受体mRNA水平和IGF1受体结合。与这些发现一致，我们发现血管损伤使IGF1受体蛋白水平降低了约30%，这是通过Western blot分析确定的；此外，我们发现，通过特定受体酪氨酸磷酸化来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一氧化氮与酪氨酸激酶受体去磷酸化之间的机制联系，涉及蛋白质酪氨酸磷酸酶。综上所述，我们的研究结果提高了PDGF或FGF与IGF1信号转导之间的负交互作用的可能性，通过PTP1B的升高作为中介。基于上述，我们提出以下具体目标，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的减少是否必要和/或足以诱导PTP1B活性的上调。确定PTP1B的上调是否必要和/或充分解释no诱导的细胞增殖抑制和诱导培养细胞凋亡。确定PDGF、FGF或NO是否诱导血管损伤中PTP1B蛋白或活性水平上调。在大鼠或小鼠血管损伤模型中，确定PTP1B是否在no诱导的细胞增殖、运动、凋亡和新生内膜形成的降低中起作用。确定PTP1B在体内是否起到减弱IGF受体激活的作用。