Thromboxane A2 Receptor Activates a Rho-associated Kinase/LKB1/PTEN Pathway to Attenuate Endothelium Insulin Signaling

This study was conducted to elucidate the molecular mechanisms of thromboxane A2 receptor (TP)-induced insulin resistance in endothelial cells. Exposure of human umbilical vein endothelial cells (HUVECs) or mouse aortic endothelial cells to either IBOP or U46619, two structurally related thromboxane A₂ mimetics, significantly reduced insulin-stimulated phosphorylation of endothelial nitric-oxide synthase (eNOS) at Ser¹¹⁷⁷ and Akt at Ser⁴⁷³. These effects were abolished by pharmacological or genetic inhibitors of TP. TP-induced suppression of both eNOS and Akt phosphorylation was accompanied by up-regulation of PTEN (phosphatase and tension homolog deleted on chromosome 10), Ser³⁸⁰/Thr^382/383 PTEN phosphorylation, and PTEN lipid phosphatase activity. PTEN-specific small interference RNA restored insulin signaling in the face of TP activation. The small GTPase, Rho, was also activated by TP stimulation, and pretreatment of HUVECs with Y27632, a Rho-associated kinase inhibitor, rescued TP-impaired insulin signaling. Consistent with this result, pertussis toxin abrogated IBOP-induced dephosphorylation of both Akt and eNOS, implicating the G_i family of G proteins in the suppressive effects of TP. In mice, high fat diet-induced diabetes was associated with aortic PTEN up-regulation, PTEN-Ser³⁸⁰/Thr^382/383 phosphorylation, and dephosphorylation of both Akt (at Ser⁴⁷³) and eNOS (at Ser¹¹⁷⁷). Importantly, administration of TP antagonist blocked these changes. We conclude that TP stimulation impairs insulin signaling in vascular endothelial cells by selectively activating the Rho/Rho-associated kinase/LKB1/PTEN pathway.Insulin exerts multiple biological actions relating to not only metabolism but also to endothelial functions (1, 2). Insulin has beneficial effects on the vasculature, primarily because of its ability to enhance endothelial nitric-oxide synthase (eNOS)² activation and expression. These effects, in turn, enhance the bioavailability of nitric oxide (3), which engenders a wide array of antiatherogenic effects. Global insulin resistance is a key feature of the metabolic syndrome leading to cardiovascular disease. In an insulin-resistant state, a systemic deregulation of the insulin signal leads to a combined deregulation of insulin-regulated metabolism and endothelial functions (4), resulting in glucose intolerance and cardiovascular disease. Insulin resistance is associated with endothelial dysfunction (5), a hallmark of atherosclerosis, and predicts adverse cardiovascular events (6). Therefore, endothelium-specific insulin resistance (impaired insulin-stimulated phosphorylation of Akt and eNOS) may play an important role in the development of cardiovascular diseases.Prostanoids have critical roles in the development of endothelial dysfunction (7). Thromboxane A₂ (TXA₂) is believed to be a prime mediator of a variety of cardiovascular and pulmonary diseases such as atherosclerosis, myocardial infarction, and primary pulmonary hypertension. TXA₂ perturbs the normal quiescent phenotype of endothelial cells (ECs). This results in leukocyte adhesion to the vessel wall as well as increased vascular permeability and expression of adhesion molecules on ECs, all important components of the inflammatory response. In smooth muscle cells, TXA₂ promotes proliferation (8) and migration, contributing to neointima formation (9). TXA₂ binds to the thromboxane A₂ receptor (TP), which has two isoforms TPα and TPβ in human (10–12), activation of which is implicated in atherosclerosis and inflammation (13–16). Atherosclerosis is accelerated by diabetes and is associated with increased levels of TXA₂ and other eicosanoids that stimulate TP (14). TP expression and plasma levels of TP ligands are elevated, both locally and systemically, in several vascular and thrombotic diseases (17). Importantly, TP activation induces EC apoptosis (15, 18) and prevents tube formation (19) by inhibiting Akt phosphorylation (18). TP activation also inhibits vascular endothelial growth factor-induced EC migration and angiogenesis by decreasing Akt and eNOS phosphorylation (20). However, the regulatory mechanisms underlying Akt inhibition by TP stimulation remain largely undefined. Moreover, whether TP activation impairs endothelial insulin signaling is also unclear.Here, we investigated whether TP ligands interfere with insulin signaling. Our results reveal that activation of TP using a potent and stable ligand (IBOP) abrogates insulin signaling in ECs. We also show that Rho/ROCK/LKB1-mediated PTEN (phosphatase and tensin homolog deleted on chromosome ten) up-regulation is required for TP-induced inhibition of insulin signaling in ECs.