Rationale 20-Hydroxyeicosatetraenoic acid (20-HETE), one of the principle cytochrome P450 (CYP) eicosanoids, is a potent vasoactive lipid whose vascular effects include stimulation of smooth muscle contractility, migration and proliferation, as well as endothelial cell dysfunction and inflammation. Increased levels of 20-HETE in experimental animals and in humans are associated with hypertension, stroke, myocardial infarction and vascular diseases. Objective To date, a receptor/binding site for 20-HETE has been implicated based on the use of specific agonists and antagonists. The present study was undertaken to identify a receptor to which 20-HETE binds and through which it activates a signaling cascade that culminates in many of the functional outcomes attributed to 20-HETE in vitro and in vivo. Methods and Results Using crosslinking analogs, click chemistry, binding assays, and functional assays, we identified GPR75, currently an orphan G-protein coupled receptor (GPCR), as a specific target of 20-HETE. In cultured human endothelial cells, 20-HETE binding to GPR75 stimulated Gαq/11 protein dissociation and increased inositol phosphate (IP-1) accumulation as well as GPCR-kinase interacting protein-1 (GIT1)-GPR75 binding, which further facilitated the c-Src-mediated transactivation of endothelial EGFR. This results in downstream signaling pathways which induce angiotensin-converting enzyme (ACE) expression and endothelial dysfunction. Knockdown of GPR75 or GIT1 prevented 20-HETE-mediated endothelial growth factor receptor (EGFR) phosphorylation and ACE induction. In vascular smooth muscle cells, GPR75-20-HETE pairing is associated with Gαq/11- and GIT1-mediated protein kinase C (PKC)-stimulated phosphorylation of MaxiKβ, λinking GPR75 activation to 20-HETE-mediated vasoconstriction. GPR75 knockdown in a mouse model of 20-HETE-dependent hypertension prevented blood pressure elevation and 20-HETE-mediated increases in ACE expression, endothelial dysfunction, smooth muscle contractility and vascular remodeling. Conclusions This is the first report to identify a GPCR target for an eicosanoid of this class. The discovery of 20-HETE-GPR75 pairing presented here provides the molecular basis for the signaling and pathophysiological functions mediated by 20-HETE in hypertension and cardiovascular diseases.
20-Hydroxyeicosatetraenoic acid (20-HETE) has been shown to positively correlate with body mass index, hyperglycemia, and plasma insulin levels. This study seeks to identify a causal relationship between 20-HETE and obesity-driven insulin resistance. Cyp4a14−/− male mice, a model of 20-HETE overproduction, were fed a regular or high-fat diet (HFD) for 15 wk. 20-SOLA [2,5,8,11,14,17-hexaoxanonadecan-19-yl 20-hydroxyeicosa-6( Z),15( Z)-dienoate], a 20-HETE antagonist, was administered from week 0 or week 7 of HFD. HFD-fed mice gained significant weight (16.7 ± 3.2 vs. 3.8 ± 0.35 g, P < 0.05) and developed hyperglycemia (157 ± 3 vs. 121 ± 7 mg/dl, P < 0.05) and hyperinsulinemia (2.3 ± 0.4 vs. 0.5 ± 0.1 ng/ml, P < 0.05) compared with regular diet-fed mice. 20-SOLA attenuated HFD-induced weight gain (9.4 ± 1 vs. 16.7 ± 3 g, P < 0.05) and normalized the hyperglycemia (157 ± 7 vs. 102 ± 5 mg/dl, P < 0.05) and hyperinsulinemia (1.1 ± 0.1 vs. 2.3 ± 0.4 ng/ml, P < 0.05). The impaired glucose homeostasis and insulin resistance in HFD-fed mice evidenced by reduced insulin and glucose tolerance were also ameliorated by 20-SOLA. Circulatory and adipose tissue 20-HETE levels significantly increased in HFD-fed mice correlating with impaired insulin signaling, including reduction in insulin receptor tyrosine (Y972) phosphorylation and increased serine (S307) phosphorylation of the insulin receptor substrate-1 (IRS-1). 20-SOLA treatments prevented changes in insulin signaling. These findings indicate that 20-HETE contributes to HFD-induced obesity, insulin resistance, and impaired insulin signaling.
Hypertension is a serious risk factor for cardiovascular disease, and the angiotensinogen (AGT) gene locus is associated with human essential hypertension. The human AGT (hAGT) gene has an A/G polymorphism at ؊6, and the ؊6A allele is associated with increased blood pressure. However, transgenic mice containing 1.2 kb of the promoter with ؊6A of the hAGT gene show neither increased plasma AGT level nor increased blood pressure compared with ؊6G. We have found that the hAGT gene has three additional SNPs (A/G at ؊1670, C/G at ؊1562, and T/G at ؊1561). Variants ؊1670A, ؊1562C, and ؊1561T almost always occur with ؊6A, and variants ؊1670G, ؊1562G, and ؊1561G almost always occur with ؊6G. Therefore, the hAGT gene may be subdivided into either ؊6A or ؊6G haplotypes. We show that these polymorphisms affect the binding of HNF-1␣ and glucocorticoid receptor to the promoter, and a reporter construct containing a 1.8-kb hAGT gene promoter with ؊6A haplotype has 4-fold increased glucocorticoid-induced promoter activity as compared with ؊6G haplotype. In order to understand the physiological significance of these haplotypes in an in vivo situation, we have generated double transgenic mice containing either the ؊6A or ؊6G haplotype of the hAGT gene and the human renin gene. Our ChIP assay shows that HNF-1␣ and glucocorticoid receptor have stronger affinity for the chromatin obtained from the liver of transgenic mice containing ؊6A haplotype. Our studies also show that transgenic mice containing ؊6A haplotype have increased plasma AGT level and increased blood pressure as compared with ؊6G haplotype. Our studies explain the molecular mechanism involved in association of the ؊6A allele of the hAGT gene with hypertension.
Background: AT 1 R activation induces oxidative stress, promotes inflammation, and increases blood pressure. Results: SNPs in AT 1 R promoter occur in linkage disequilibrium, forming two haplotypes. Transgenic mice with haplotype I have USF2-dependent AT 1 R overexpression, increased oxidative stress, and increased blood pressure. Conclusion: Haplotype I leads to enhanced expression and pathophysiological effects of AT 1 R. Significance: Polymorphisms in AT 1 R provide for genetic predisposition to hypertension.
Background: Glucocorticoids modulate the RAS and cause hypertension. Results: SNPs in the hAGT promoter form two haplotypes, Ϫ6A and Ϫ6G. Transgenic mice with haplotype Ϫ6A respond to dexamethasone with tissue-specific up-regulation of hAGT, increased plasma AngII, and hypertension. Conclusion: Haplotypes of the hAGT gene govern transcriptional response to dexamethasone. Significance: Polymorphisms in hAGT provide for genetic predisposition to glucocorticoid-induced hypertension.
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