Department of Medicine, University of North Carolina School of Medicine, Chapel Hill, North Carolina 27599, USA.
J Biol Chem. 2010 May 21;285(21):15682-95. doi: 10.1074/jbc.M109.092270. Epub 2010 Mar 5.
Vascular smooth muscle cells maintained in normal (5.6 mm) glucose respond to insulin-like growth factor-I (IGF-I) with increased protein synthesis but do not proliferate. In contrast, hyperglycemia alters responsiveness to IGF-I, resulting in increased SHPS-1 phosphorylation and assembly of a signaling complex that enhances MAPK and phosphatidylinositol 3-kinase pathways. Hyperglycemia also reduces the basal IRS-1 concentration and IGF-I-stimulated IRS-1-linked signaling. To determine if failure to down-regulate IRS-1 alters vascular smooth muscle cell (VSMC) responses to IGF-I, we overexpressed IRS-1 in VSMCs maintained in high glucose. These cultures showed reduced SHPS-1 phosphorylation, transfer of SHP-2 to SHPS-1, and impaired Shc and MAPK phosphorylation and cell proliferation in response to IGF-I. In vitro studies demonstrated that SHPS-1 was a substrate for type I IGF receptor (IGF-IR) and that IRS-1 competitively inhibited SHPS-1 phosphorylation. Exposure of VSMC cultures to a peptide that inhibited IRS-1/IGF-IR interaction showed that IRS-1 binding to IGF-IR impairs SHPS-1 phosphorylation in vivo. IRS-1 also sequestered SHP-2. Expression of an IRS-1 mutant (Y1179F/Y1229F) reduced IRS-1/SHP-2 association, and exposure of cells expressing the mutant to the inhibitory peptide enhanced SHPS-1 phosphorylation and SHP-2 transfer. This result was confirmed by expressing an IRS-1 mutant that had both impaired binding to IGF-IR and to SHP-2 IGF-I increased SHPS-1 phosphorylation, SHP-2 association with SHPS-1, Shc MAPK phosphorylation, and proliferation in cells expressing the mutant. We conclude that IRS-1 is an important factor for maintaining VSMCs in the non-proliferative state and that its down-regulation is a component of the VSMC response to hyperglycemic stress that results in an enhanced response to IGF-I.
在正常（5.6mmol/L）葡萄糖环境中培养的血管平滑肌细胞对胰岛素样生长因子-I（IGF-I）的反应是增加蛋白质合成，但不增殖。相反，高血糖会改变对 IGF-I 的反应，导致 SHPS-1 磷酸化增加，并组装一个信号复合物，增强 MAPK 和磷脂酰肌醇 3-激酶途径。高血糖还会降低基础 IRS-1 浓度和 IGF-I 刺激的 IRS-1 相关信号。为了确定 IRS-1 下调失败是否改变血管平滑肌细胞（VSMC）对 IGF-I 的反应，我们在高葡萄糖环境中培养的 VSMC 中过表达 IRS-1。这些培养物显示 SHPS-1 磷酸化减少，SHP-2 向 SHPS-1 的转移，以及 Shc 和 MAPK 磷酸化和对 IGF-I 的细胞增殖受损。体外研究表明，SHPS-1 是 I 型 IGF 受体（IGF-IR）的底物，IRS-1 竞争性抑制 SHPS-1 磷酸化。将 VSMC 培养物暴露于抑制 IRS-1/IGF-IR 相互作用的肽中表明，IRS-1 与 IGF-IR 的结合会损害体内 SHPS-1 磷酸化。IRS-1 还隔离了 SHP-2。表达 IRS-1 突变体（Y1179F/Y1229F）减少 IRS-1/SHP-2 结合，将表达突变体的细胞暴露于抑制性肽中可增强 SHPS-1 磷酸化和 SHP-2 转移。通过表达一种既不能与 IGF-IR 结合又不能与 SHP-2 结合的 IRS-1 突变体证实了这一结果，IGF-I 增加了突变体表达细胞中的 SHPS-1 磷酸化、SHP-2 与 SHPS-1 的结合、Shc MAPK 磷酸化和增殖。我们得出结论，IRS-1 是维持 VSMC 处于非增殖状态的重要因素，其下调是 VSMC 对高血糖应激反应的一个组成部分，导致对 IGF-I 的反应增强。
