Abstract
Caveolin-1 is the primary structural component of endothelial caveolae that is essential for transcellular trafficking of albumin and is also a critical scaffolding protein that regulates the activity of signaling molecules in caveolae. Phosphorylation of caveolin-1 plays a fundamental role in the mechanism of oxidant-induced vascular hyper permeability. However, the regulatory mechanism of caveolin-1 phosphorylation remains unclear. Here we identify a previously unexpected role for AMPK in inhibition of caveolin-1 phosphorylation under oxidative stress. A pharmacological activator of AMPK, 5-amino-4-imidazole carboxamide riboside (AICAR), inhibited oxidative stress-induced phosphorylation of both caveolin-1 and c-Abl, which is the major kinase of caveolin-1, and endocytosis of albumin in human umbilical vein endothelial cell. These effects were abolished by treatment with two specific inhibitors of AICAR, dipyridamole, and 5-iodotubericidin. Consistently, knockdown of the catalytic AMPKα subunit by siRNA abolished the inhibitory effect of AICAR on oxidant-induced phosphorylation of both caveolin-1 and c-Abl. Pretreatment with specific c-Abl inhibitor, imatinib mesylate, and knock down of c-Abl significantly decreased the caveolin-1 phosphorylation after H2O2 exposure and abolished the inhibitory effect of AICAR on the caveolin-1 phosphorylation. Interestingly, knockdown of Prdx-1, an antioxidant enzyme associated with c-Abl, increased phosphorylation of both caveolin-1 and c-Abl and abolished the inhibitory effect of AICAR on the caveolin-1 phosphorylation. Furthermore, co-immunoprecipitation experiment showed that AICAR suppressed the oxidant-induced dissociation between c-Abl and Prdx1. Overall, our results suggest that activation of AMPK inhibits oxidative stress-induced caveolin-1 phosphorylation and endocytosis, and this effect is mediated in part by stabilizing the interaction between c-Abl and Prdx-1.
Highlights
Oxidative stress increases vascular permeability though caveolin-1 phosphorylation
amino-4imidazole carboxamide riboside (AICAR) Suppresses Oxidative Stress-induced Phosphorylation of Caveolin-1 and c-Abl—It has already been reported that caveolin-1 is phosphorylated on tyrosine 14 under hyperosmotic shock and oxidative stress [17, 18] and that c-Abl, which is an upstream kinase of caveolin-1, is required for oxidative stress-induced phosphorylation of caveolin-1 [19]
To determine the effect of H2O2 and AICAR on the endocytosis, we evaluated the amount of fluorescein-conjugated albumin endocytosed by HUVEC
Summary
Oxidative stress increases vascular permeability though caveolin-1 phosphorylation. The exact role of AMPK is unknown. A pharmacological activator of AMPK, 5-amino-4imidazole carboxamide riboside (AICAR), inhibited oxidative stress-induced phosphorylation of both caveolin-1 and c-Abl, which is the major kinase of caveolin-1, and endocytosis of albumin in human umbilical vein endothelial cell. These effects were abolished by treatment with two specific inhibitors of AICAR, dipyridamole, and 5-iodotubericidin. We have recently demonstrated that activation of AMPK inhibits retinoblastoma cell proliferation, tumor growth, angiogenesis, ocular inflammation, and MMP-9 expression [11,12,13,14,15] Because these functions of AMPK are closely linked to the vascular hyper-permeability induced by oxidative stress, we hypothesized that AMPK has a protective role for vascular permeability. In the present study we examined the role of AMPK in the transcellular pathway, the effect of AMPK activation on the phosphorylation of caveolin-1 under oxidative stress
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