Role of Calcium-independent Phospholipase A2β in High Glucose-induced Activation of RhoA, Rho Kinase, and CPI-17 in Cultured Vascular Smooth Muscle Cells and Vascular Smooth Muscle Hypercontractility in Diabetic Animals

Zhongwen Xie,John Turk,Ming C Gong,Dongping Xie,Wen Su,Zhenheng Guo

doi:10.1074/jbc.m109.057711

Zhongwen Xie, John Turk + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m109.057711

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Abstract

Previous studies suggest that high glucose-induced RhoA/Rho kinase/CPI-17 activation is involved in diabetes-associated vascular smooth muscle hypercontractility. However, the upstream signaling that links high glucose and RhoA/Rho kinase/CPI-17 activation is unknown. Here we report that calcium-independent phospholipase A(2)beta (iPLA(2)beta) is required for high glucose-induced RhoA/Rho kinase/CPI-17 activation and thereby contributes to diabetes-associated vascular smooth muscle hypercontractility. We demonstrate that high glucose increases iPLA(2)beta mRNA, protein, and iPLA(2) activity in a time-dependent manner. Protein kinase C is involved in high glucose-induced iPLA(2)beta protein up-regulation. Inhibiting iPLA(2)beta activity with bromoenol lactone or preventing its expression by genetic deletion abolishes high glucose-induced RhoA/Rho kinase/CPI-17 activation, and restoring expression of iPLA(2)beta in iPLA(2)beta-deficient cells also restores high glucose-induced CPI-17 phosphorylation. Pharmacological and genetic inhibition of 12/15-lipoxygenases has effects on high glucose-induced CPI-17 phosphorylation similar to iPLA(2)beta inhibition. Moreover, increases in iPLA(2) activity and iPLA(2)beta protein expression are also observed in both type 1 and type 2 diabetic vasculature. Pharmacological and genetic inhibition of iPLA(2)beta, but not iPLA(2)gamma, diminishes diabetes-associated vascular smooth muscle hypercontractility. In summary, our results reveal a novel mechanism by which high glucose-induced, protein kinase C-mediated iPLA(2)beta up-regulation activates the RhoA/Rho kinase/CPI-17 via 12/15-lipoxygenases and thereby contributes to diabetes-associated vascular smooth muscle hypercontractility.

Highlights

Major causes of increased mortality and morbidity in diabetic patients [1]
IPLA2␤ Is Activated by High Glucose—To explore the potential role of independent PLA2 (iPLA2)␤ in diabetic vasculopathy, we first determined whether high concentrations of glucose activate and/or up-regulate iPLA2␤ in primary cultured rat vascular smooth muscle cells (VSMC)
We observed that high glucose significantly increased iPLA2␤ mRNA (Fig. 1, A and B), protein expression (Fig. 1, C and D), and iPLA2 activity (Fig. 1E) in a time-dependent manner

Summary

Introduction

Major causes of increased mortality and morbidity in diabetic patients [1]. It is recognized that abnormal vascular reactivity, including an increase in vasoconstrictive responses and/or a decrease in vasodilatory responses, are present early in diabetes, and this might contribute to the development of diabetes-associated vascular complications [2]. IPLA2␤ in Diabetic Vascular Complications obese and type 2 diabetic db/db mice, and ROCK is the kinase responsible for high glucose-induced CPI-17 phosphorylation and diabetes-associated hypercontractility in db/db mice [6]. It is unknown whether iPLA2␤ is involved in high glucose-induced RhoA/ROCK/CPI-17 activation in VSMC and in diabetic vascular smooth muscle hypercontractility.

Results

Conclusion