Transcription factor early growth response gene-1 (Egr-1) is rapidly expressed in various cell types in response to extracellular stimuli such as growth factors, cytokines, and injury. Increased Egr-1 expression may then regulate vascular response to injury. Both oxidative stress and insulin resistance play an important role in atherogenesis, possibly by affecting vascular smooth muscle cell (VSMC) function and stimulating Egr-1 expression. Insulin resistance is characterized by phosphatidylinositol 3-kinase (PI 3-K) pathway impairment with intact extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) signaling in the context of hyperinsulinemia. We studied intracellular signaling pathways mediating the effects of insulin and oxidative stress on VSMC Egr-1.
Methods Quiescent rat VSMC were treated with insulin (I) and/or H2O2 (OX) with or without inhibitors of the PI 3-K-dependent pathway or the ERK MAPK-dependent pathway. Wortmannin (WT) or adenoviral infection with P85-NSH2 (blocks P85 interaction with insulin receptor substrate-1), was used to inhibit the PI 3-K pathway. ERK 1/2 MAPK kinase inhibitors PD98059 (PD) or U0126 (also an ERK 5 inhibitor), or DN Ras, a dominant negative Ras adenoviral construct, was used to inhibit the ERK MAPK-dependent pathway. Egr-1 protein expression was evaluated by Western blotting.
Results Both insulin and OX caused a dose- and time-dependent increase in Egr-1 expression. At 1 hr, PI 3-K pathway inhibition with either WT or P85-NSH2 did not alter insulin effect on Egr-1 expression. However, PD and U0126 significantly blocked insulin effect on Egr-1 expression (I vs I+PD, p≤0.01; I vs I+U0126, p≤0.05) in VSMC. Similarly, DN Ras infection of VSMC significantly blocked the effect of insulin on Egr-1 expression (I vs DN Ras+I, p≤0.01). In contrast, OX effects on Egr-1 expression were not affected by PI 3-kinase or ERK MAPK pathway inhibition. However, U0126 significantly suppressed Egr-1 response to OX (OX vs OX+U0126, p≤0.01), possibly via inhibition of ERK 5.
Conclusions Insulin and oxidative stress each cause marked increases in VSMC transcription factor Egr-1 protein expression. Furthermore, the ERK 1/2 pathway mediates insulin effect on Egr-1 protein levels, while the PI 3-K pathway does not. OX effect on Egr-1 protein levels appears to be independent of either the PI 3-K or ERK 1/2-dependent pathways and may be mediated by ERK 5. These data support the possibility that Egr-1 may be preferentially increased in insulin resistance, particularly in the presence of oxidative stress, contributing to accelerated atherosclerosis.
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