Protein Kinase A-dependent Translocation of Hsp90α Impairs Endothelial Nitric-oxide Synthase Activity in High Glucose and Diabetes

Abstract

Diabetes mellitus (DM) and high glucose (HG) are known to reduce the bioavailability of nitric oxide (NO) by modulating endothelial nitric-oxide synthase (eNOS) activity. eNOS is regulated by several mechanisms including its interaction with heat shock protein (Hsp) 90. We previously discovered that DM in vivo and HG in vitro induced the translocation of Hsp90␣ to the outside of aortic endothelial cells. In this report we tested the hypothesis that translocation of Hsp90␣ is responsible for the decline in NO production observed in HG-treated cells. We found that HG increased phosphorylation of Hsp90␣ in a cAMP-dependent protein kinase A-dependent manner, and that this event was required for translocation of Hsp90␣ in porcine aortic endothelial cells. Furthermore, preventing translocation of Hsp90␣ protected from the HG-induced decline in eNOS⅐Hsp90␣ complex and NO production. Notably, DM increased phosphorylation of Hsp90␣ and reduced its association with eNOS in the aortic endothelium of diabetic rats. These studies suggest that translocation of Hsp90␣ is a novel mechanism by which HG and DM impair eNOS activity and thereby reduce NO production. Endothelium-derived nitric oxide (NO)2 , a small, diffusible, lipophilic free radical gas, is a critical regulator of cardiovascular homeostasis (1, 2). Vascular NO dilates all types of blood vessels. It also inhibits platelet aggregation and adhesion, and leukocyte adhesion to the vessel wall (3-5). A deficiency of NO (decreased NO production, increased degradation of NO or decreased NO sensitivity) can promote atherogenesis (1, 6).Endothelial NO synthase (eNOS) is responsible for most of the vascular NO produced. A functional eNOS oxidizes its substrate L-arginine to L-citrulline and NO (4, 5). eNOS is highly regulated by post-translational modifications including myristoylation, palmitoylation, and phosphorylation (7-11). This enzyme is also tightly regulated by specific interactions with inhibitory and activating proteins such as caveolin-1 and Hsp90, respectively. The binding of heat shock protein 90 (Hsp90) to eNOS enhances eNOS activation, assists with the intracellular trafficking of eNOS, and helps activate eNOS by dissociating it from caveolin-1 (12, 13). Furthermore, Hsp90 provides a scaffold for eNOS and Akt, and thereby enhances eNOS activation (9, 14 -16).Several studies have demonstrated that hyperglycemia/diabetes mellitus (DM) causes a loss of endothelium-derived NO in both animals (17-19) and humans (20), but the underlining mechanism is poorly understood. DM does not influence the overall eNOS protein level or its mRNA level (21). One potential mechanism may be the reduced association of eNOS with Hsp90 (19).Hsp90 is an ATP-dependent chaperone that interacts with over 100 proteins (22,23), and has been implicated in many physiological and pathological processes (24 -27). DM induces an increase in the amount of Hsp90␣ at the luminal surface of the aorta, and this event could be mimicked by treating cultured vascular endothelial cells with high glucose ...

A Novel Epac-Rap-PP2A Signaling Module Controls cAMP-dependent Akt Regulation

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“…36,37,40,41 Further, PKA phosphorylation of Thr90 induced by 3-hydroxy-3-methylglutarylcoenzyme A reductase inhibitors has been reported to increase association of human Hsp90α with the client protein eNOS. 42 Lastly, a study reported that protein phosphatase 5 (Pp5/Ppt1) can dephosphorylate Hsp90 in vitro. 43 This study also showed that Ppt1 deletion in yeast compromised Hsp90 activity.…”

Role of Acetylation and Extracellular Location of Heat Shock Protein 90α in Tumor Cell Invasion

“…In addition to cochaperone association as well as ATP binding and hydrolysis, posttranslational modifications such as hyperphosphorylation (13)(14)(15), S-nitrosylation, and reversible hyperacetylation have also been shown to regulate the chaperone function of hsp90 (16)(17)(18). Several serine-threonine phosphorylation sites have been identified in hsp90.…”