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

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 calciumindependent phospholipase A 2 ␤ (iPLA 2 ␤) 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 ␤ mRNA, protein, and iPLA 2 activity in a timedependent manner. Protein kinase C is involved in high glucoseinduced iPLA 2 ␤ protein up-regulation. Inhibiting iPLA 2 ␤ 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 ␤ in iPLA 2 ␤-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 ␤ inhibition. Moreover, increases in iPLA 2 activity and iPLA 2 ␤ protein expression are also observed in both type 1 and type 2 diabetic vasculature. Pharmacological and genetic inhibition of iPLA 2 ␤, but not iPLA 2 ␥, 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 ␤ up-regulation activates the RhoA/Rho kinase/CPI-17 via 12/15-lipoxygenases and thereby contributes to diabetes-associated vascular smooth muscle hypercontractility.The prevalence of diabetes has increased dramatically in the United States and worldwide. Diabetes-associated microvascular and macrovascular complications are the 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). Although diabetes-associated endothelial and vasodilatatory dysfunction has been studied extensively, much less is known about vascular smooth muscle dysfunction in diabetes, and the molecular mechanisms that underlie endothelium-independent, diabetes-associated vascular smooth muscle hypercontractility are largely unknown.G-protein-coupled receptor agonists can induce significant smooth muscle contraction by inhibiting myosin light chain phosphatase at a given Ca 2ϩ concentration by a mechanism designated "Ca 2ϩ sensitization" (3). A small GTP-binding protein RhoA, its downstream effector Rho kinase (ROCK), 2 protein kinase C (PKC), and CPI-17 (protein kinase C-potentiated phosphatase inhibitor of 17 kDa) are recognized as four key participants in Ca 2ϩ sensitization of vascular smooth muscle contraction (4). Importantly, abnormal act...

Identification of a cAMP-response Element in the Regulator of G-protein Signaling-2 (RGS2) Promoter as a Key Cis-regulatory Element for RGS2 Transcriptional Regulation by Angiotensin II in Cultured Vascular Smooth Muscles

“…Real-time PCR-Primers for RGS2 and 18S RNA and the procedure for real-time PCR were previously described (16,25,28,29).…”

“…arachidonic acid and/or its oxygenated metabolites, are involved in Ang II-induced cAMP accumulation, VSMCs were pretreated with pharmacologic inhibitors of relevant enzymes (16,25). These include BEL, an iPLA 2 inhibitor; nordihydroguaiaretic acid, an inhibitor of LO; indomethacin, an inhibitor of COX-1 and -2; and 17-octadecynoic acid, an inhibitor of cytochrome P450 epoxygenases.…”

Analysis of Two Major Intracellular Phospholipases A2 (PLA2) in Mast Cells Reveals Crucial Contribution of Cytosolic PLA2α, Not Ca2+-independent PLA2β, to Lipid Mobilization in Proximal Mast Cells and Distal Fibroblasts

“…Dissociation of iPLA 2 ␤ from phospholipid remodeling has also been demonstrated recently in testis, macrophages, and pancreatic ␤-cells (44, 76, 77), in which the enzyme appears to play signaling roles in processes such as Ca 2ϩ entry, lipid mediator synthesis, cell migration, exocytosis, and apoptosis (41)(42)(43)(44)(45)(46)(47)(48)(49). However, in BMMCs, iPLA 2 ␤ is apparently nonessential for stimulus-coupled AA mobilization and subsequent eicosanoid generation, granule exocytosis, cytokine secretion, and even upstream Ca 2ϩ entry (Figs.…”

“…For instance, thapsigargin-or A23187-stimulated AA release is dramatically attenuated in Pla2g6 Ϫ/Ϫ aortic smooth muscle cells, and these cells show decreased migration and proliferation due to reduced PGE 2 generation in a model of vascular injury (42). A high glucose-induced iPLA 2 ␤ activates RhoA/Rho kinase via 12/15-lipoxygenase metabolites, which contributes to vascular smooth muscle hypercontractility in diabetic animals (43). In lung endothelial cells, thrombin-or tryptase-stimulated synthesis of PGI 2 and platelet-activating factor is attenuated by iPLA 2 ␤ deficiency (83).…”

“…Mast cells also express iPLA 2 ␤, a prototypic iPLA 2 isoform, and the iPLA 2 inhibitor bromoenol lactone (BEL) can attenuate granule exocytosis by mast cells (39), leading to the suggestion that iPLA 2 ␤ participates in the regulation of mast cell activation. Although iPLA 2 ␤ has long been thought to play a role in phospholipid remodeling (40), recent studies using mice or cells that are devoid of iPLA 2 ␤ as a result of gene targeting or siRNA knockdown have defined its roles in various signaling events, such as Ca 2ϩ release-activated Ca 2ϩ (CRAC) channel opening (41), lipid mediator generation (42,43), exocytosis (44), cytokine secretion (45), cell migration (46), vascular contractility (47), apoptosis (48,49), cancer (50), and neuronal degeneration (51,52). Because of its roles in Ca 2ϩ gating and lipid mediator generation, the sequential action of iPLA 2 ␤ and cPLA 2 ␣ for the full operation of AA release has been proposed in vascular cells (42).…”