Abstract
Hyperglycemia and insulin resistance accelerate atherosclerosis by an unclear mechanism. The two factors down-regulate insulin receptor substrate-1 (IRS-1), an intermediary of the insulin/IGF-I signaling system. We previously reported that IRS-1 down-regulation leads to vascular smooth muscle cell (VSMC) dedifferentiation and that IRS-1 deletion from VSMCs in normoglycemic mice replicates this response. However, we did not determine IRS-1's role in mediating differentiation. Here, we sought to define the mechanism by which IRS-1 maintains VSMC differentiation. High glucose or IRS-1 knockdown decreased p53 levels by enhancing MDM2 proto-oncogene (MDM2)-mediated ubiquitination, resulting in decreased binding of p53 to Krüppel-like factor 4 (KLF4). Exposure to nutlin-3, which dissociates MDM2/p53, decreased p53 ubiquitination and enhanced the p53/KLF4 association and differentiation marker protein expression. IRS-1 overexpression in high glucose inhibited the MDM2/p53 association, leading to increased p53 and p53/KLF4 levels, thereby increasing differentiation. Nutlin-3 treatment of diabetic or Irs1-/- mice enhanced p53/KLF4 and the expression of p21, smooth muscle protein 22 (SM22), and myocardin and inhibited aortic VSMC proliferation. Injecting normoglycemic mice with a peptide disrupting the IRS-1/p53 association reduced p53, p53/KLF4, and differentiation. Analyzing atherosclerotic lesions in hypercholesterolemic, diabetic pigs, we found that p53, IRS-1, SM22, myocardin, and KLF4/p53 levels are significantly decreased compared with their expression in nondiabetic pigs. We conclude that IRS-1 is critical for maintaining VSMC differentiation. Hyperglycemia- or insulin resistance-induced IRS-1 down-regulation decreases the p53/KLF4 association and enhances dedifferentiation and proliferation. Our results suggest that enhancing IRS-1-dependent p53 stabilization could attenuate the progression of atherosclerotic lesions in hyperglycemia and insulin-resistance states.
Highlights
Hyperglycemia and insulin resistance accelerate atherosclerosis by an unclear mechanism
To investigate whether changes in p53 could be related to changes in Krüppel-like factor 4 (KLF4) function, we determined whether hyperglycemia regulated p53
Because p53 inhibits vascular smooth muscle cell (VSMC) proliferation and counteracts the deleterious effects of oxidative stress on VSMC function, we utilized p53 knockdown and overexpression to further delineate the role of p53 in regulating VSMC differentiation. p53 knockdown resulted in no significant change in KLF4 or insulin receptor substrate-1 (IRS-1) (Fig. 1E), but there was marked reduction in nuclear p53 and p53/KLF4 (Fig. 1F)
Summary
Of atherosclerotic lesions in hyperglycemia and insulin-resistance states. Insulin-like growth factor-I (IGF-I) and insulin coordinately regulate cellular growth and differentiation in response to changes in nutritional status and intermediary metabolism [1]. In cell types that are capable of undergoing dedifferentiation, such as vascular smooth muscle cells (VSMC), IRS-1 down-regulation is associated with up-regulation of a cell surface–associated scaffolding protein termed SHPS-1 [7] Under these conditions, stimulation of the IGF-I receptor leads to recruitment of the tyrosine kinase CTK to the plasma membrane, and CTK directly phosphorylates SHPS-1 [8]. SHPS-1 functions as a scaffold and recruits kinases that activate both the PI3K and mitogen-activated protein kinase pathways [6, 9] This signaling switch occurs in vivo in VSMCs of diabetic mice in response to hyperglycemia. Because dedifferentiation is an important component of the atherosclerotic process [14] and hyperglycemia and insulin resistance, which down-regulate IRS-1, are known to accelerate the development of atherosclerosis [15, 16], we undertook these studies to determine the mechanism by which IRS-1 functions to maintain VSMC differentiation
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