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）：本提案旨在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的抑制作用的介质的作用。 NO 在血管损伤后的新内膜形成中起主要抑制作用。缺乏解释培养细胞尤其是体内这种效应的机制。我们发现，NO 会增加培养的大鼠主动脉平滑肌细胞中 PTP1B 的活性，但不会增加其蛋白质水平。此外，我们发现PDGF和FGF会增加培养细胞中的PTP1B蛋白水平，并且血管损伤同样会导致受伤的大鼠颈动脉中PTP1B蛋白水平增加。我们还表明，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，我们发现 NO 会降低细胞质 Ca 并减弱 IGF1 诱导的过氧化氢生成，并且这种效应可以通过 Ca 螯合剂独立降低细胞内 Ca 来模拟。这些结果支持减少的 Ca 可能参与激活 PTP1B。 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。与这些发现一致的是，根据蛋白质印迹分析确定，血管损伤使 IGF1 受体蛋白水平降低约 30%；此外，我们发现，通过特定受体酪氨酸磷酸化测量，受体激活降低了 80% 以上。这些新颖且令人兴奋的发现首次描述了 NO 和涉及蛋白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，我们的结果提出了一方面 PDGF 或 FGF 与另一方面 IGF1 信号转导之间通过升高的 PTP1B 介导的负串扰的可能性。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 的减少是否是诱导 PTP1B 活性上调所必需和/或充分的。确定 PTP1B 的上调是否是必要和/或足以解释 NO 诱导的细胞增殖抑制和培养细胞凋亡的诱导。确定 PDGF、FGF 或 NO 是否会诱导血管损伤中 PTP1B 蛋白或活性水平的上调。确定 PTP1B 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、凋亡和新内膜形成减少中发挥作用。确定 PTP1B 是否在体内减弱 IGF 受体激活中发挥作用。