Abstract
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.
Highlights
Grants HL56580 and AG02331. □S The on-line version of this article contains supplemental Figs. 1–3. 1 To whom correspondence should be addressed: Division of Endocrinology, University of North Carolina, CB 7170, 8024 Burnett-Womack, Chapel Hill, NC 27599-7170
Our studies have shown that SHPS-1 phosphorylation and the subsequent assembly of a signaling complex that includes SHP-2, Src, Shc (Src homology 2 domain-containing protein), and Grb2 is essential for these cells to respond to hyperglycemic stress and that it enhances the ability of insulin-like growth factor-I (IGF-I) to activate both MAPK and phosphatidylinositol 3-kinase pathways, leading to increased proliferation and migration [2, 3, 20]
Tyrosine phosphorylation of p52shc was significantly increased in the IRS-FF cells exposed to peptide 301 (2.9 Ϯ 0.2-fold, n ϭ 3, p Ͻ 0.001) compared with cells not exposed to the peptide in response to IGF-I (Fig. 5D). These results suggest that disruption of association IGF-IR/IRS-1 association and inhibition of SHP-2 binding to IRS-1 appears to be required for optimal SHP-2 transfer to SHPS-1 and Shc activation
Summary
Grants HL56580 and AG02331. □S The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. 1–3. 1 To whom correspondence should be addressed: Division of Endocrinology, University of North Carolina, CB 7170, 8024 Burnett-Womack, Chapel Hill, NC 27599-7170. Our studies have shown that SHPS-1 phosphorylation and the subsequent assembly of a signaling complex that includes SHP-2, Src, Shc (Src homology 2 domain-containing protein), and Grb (growth factor receptor-bound protein 2) is essential for these cells to respond to hyperglycemic stress and that it enhances the ability of IGF-I to activate both MAPK and phosphatidylinositol 3-kinase pathways, leading to increased proliferation and migration [2, 3, 20]. Following their tyrosine phosphorylation, IRS-1 and Shc bind. These studies were undertaken to determine the significance of the reduction in IRS-1 and whether failure to reduce the IRS-1 concentration alters assembly of the SHPS-1 complex, leading to altered cell proliferation and migration in response to IGF-I
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