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.

Increased vascular 20-hydroxyeicosatetraenoic acid (20-HETE), a cytochrome P450 arachidonic acid metabolite, promotes vascular dysfunction, injury, and hypertension that is dependent, in part, on the renin angiotensin system (RAS). We have shown that, in human microvascular endothelial cells, 20-HETE increases angiotensin-converting enzyme (ACE) mRNA, protein, and ACE activity via an epidermal growth factor receptor (EGFR)/ tyrosine kinase/mitogen-activated protein kinase (MAPK)/ inhibitor of kB kinase (IKK)b-mediated signaling pathway. In this work, we show that, similar to epidermal growth factor (EGF), 20-HETE (10 nM) activates EGFR by stimulating tyrosine phosphorylation; however, unlike 20-HETE, EGF does not induce ACE expression, and pretreatment with a neutralizing antibody against EGF does not prevent the 20-HETE-mediated ACE induction. Inhibition of nuclear factor kB (NF-kB) activation prevented the 4.58-fold (60.78; P , 0.05) 20-HETE-mediated induction of ACE. The 20-HETE increased NF-kB-binding activity in nuclear extracts and the activity of both the somatic and germinal ACE promoters by 4.37-fold (60.18; P , 0.05) and 2.53-fold (6 0.24; P , 0.05), respectively. The 20-HETE-stimulated ACE promoter activity was abrogated by the 20-HETE antagonist 20-hydroxy-6,15-eicosadienoic acid and by inhibitors of EGFR, MAPK, IKKb, and NF-kB activation. Sequence analysis demonstrated the presence of two and one putative NF-kB binding sites on the human somatic and germinal ACE promoters, respectively. Chromatin immunoprecipitation assay indicated that 20-HETE stimulates the translocation and subsequent binding of NF-kB to each of the putative binding sites (S1, 3.43 6 0.3-fold enrichment versus vehicle; S2, 3.72 6 0.68-fold enrichment versus vehicle; S3, 3.20 6 0.18-fold enrichment versus vehicle; P , 0.05). This is the first study to identify NF-kB as a transcriptional factor for ACE and to implicate a distinct EGFR/MAPK/IKK/NF-kB signaling cascade underlying 20-HETE-mediated transcriptional activation of ACE mRNA and stimulation of ACE activity.

Increased vascular 20-HETE is associated with hypertension and activation of the reninangiotensin system (RAS) through induction of vascular angiotensinconverting enzyme (ACE) expression. Cyp4a12tg mice, whose Cyp4a12-20-HETE synthase expression is under the control of a tetracycline (doxycycline, DOX) promoter, were used to assess the contribution of ACE/RAS to microvascular remodeling in 20-HETEdependent hypertension. Treatment of Cyp4a12tg mice with DOX increased systolic blood pressure (SBP; 136 Ϯ 2 vs. 102 Ϯ 1 mmHg; P Ͻ 0.05), and this increase was prevented by administration of 20-HEDGE, lisinopril, or losartan. DOX-induced hypertension was associated with microvascular dysfunction and remodeling of preglomerular microvessels, which was prevented by 20-HEDGE, a 20-HETE antagonist, yet only lessened, but not prevented, by lisinopril or losartan. In ACE 3/3 mice, which lack vascular endothelial ACE, administration of 5␣-dihydrotestosterone (DHT), a known inducer of 20-HETE production, increased SBP; however, the increase was about 50% of that in wild-type (WT) mice (151 Ϯ 1 vs. 126 Ϯ 1 mmHg). Losartan and 20-HEDGE prevented the DHT-induced increase in SBP in WT and ACE 3/3 mice. DHT treatment increased 20-HETE production and microvascular remodeling in WT and ACE 3/3 mice; however, remodeling was attenuated in the ACE 3/3 mice as opposed to WT mice (15.83 Ϯ 1.11 vs. 22.17 Ϯ 0.92 m; P Ͻ 0.05). 20-HEDGE prevented microvascular remodeling in WT and ACE 3/3 mice, while losartan had no effect on microvascular remodeling in ACE 3/3. Taken together, these results suggest that RAS contributes to 20-HETE-mediated microvascular remodeling in hypertension and that 20-HETE-driven microvascular remodeling independent of blood pressure elevation does not fully rely on ACE activity in the vascular endothelium.20-HETE; angiotensin II; ACE; vascular remodeling; hypertension; angiotensin-converting enzyme 20-HETE IS THE -HYDROXYLATION product of arachidonic acid metabolism by enzymes of the cytochrome P-450 (CYP) 4A and 4F families. It has been recognized as an eicosanoid of the microcirculation with renal, cerebral, cardiac, and mesenteric arteries having been shown to be rich sources of 20-HETE. Its effects on vascular function are multifaceted and include stimulation of smooth muscle contractility, migration, and proliferation, as well as activation of endothelial cell dysfunction, angiogenesis, and inflammation (1, 2, 4, 21, 34, 36). Such effects could have significant implications with regard to the development of hypertension and its cardiovascular complications. Indeed, numerous studies in experimental models of hypertension have documented a close relationship between increased vascular production of 20-HETE and blood pressure elevation. Models of 20-HETE-driven hypertension also exhibit vascular injury that is exemplified by endothelial and vascular dysfunction (11,15,17,33,35).Vascular remodeling is both a product of and contributor to the development of hypertension. This process is promoted by a variety of stimuli...

Here, we report that GPR75, a G protein-coupled receptor of the Gq rhodopsin subfamily, selectively binds 20-hydroxyeicosatetraenoic acid (20-HETE), a cytochrome P450-derived bioactive arachidonic acid metabolite implicated in the pathogenesis of hypertension and cardiovascular diseases. In endothelial cells, 20-HETE binding to GPR75 stimulates β-arrestin recruitment and GIT1-GPR75 association, which further facilitates a c-Src-mediated transactivation of EGFR. This results in downstream signaling pathways which induce ACE expression and decrease NO bioavailability. Knockdown of GPR75 prevents 20-HETE-mediated downstream effects in endothelial cells including EGFR activation and ACE induction. In vascular smooth muscle cells, GPR75-20-HETE pairing is associated with Gα q/11 -and GIT1-mediated PKC-stimulated phosphorylation of MaxiKβ, linking GPR75 activation to 20-HETE-mediated vasoconstriction. We used the conditional Cyp4a12tg mice, which display doxycycline (DOX)-mediated hypertension along with vascular dysfunction and remodeling in a 20-HETE-dependent manner, to assess whether GPR75 is a necessary component of 20-HETE pro-hypertensive actions. Administration of GPR75-targeted shRNA lentiviral particles to DOX-treated Cyp4a12tg mice, which resulted in 80% knockdown of GPR75 knockdown, prevented blood pressure elevation (100±3 vs 135±2 mmHg) and 20-HETE-mediated increases in ACE expression, endothelial dysfunction, smooth muscle contractility and vascular remodeling when compared to DOX-treated Cyp4a12tg mice receiving non-targeted shRNA. The discovery of 20-HETE-GPR75 pairing provides the molecular basis for the signaling and pathophysiological bioactions mediated by 20-HETE in hypertension. These results clearly place GPR75 as a novel target in the control of blood pressure and vascular function.