20-HETE Signals Through G-Protein-Coupled Receptor GPR75 (Gq) to Affect Vascular Function and Trigger Hypertension

Abstract

Rationale: 20-Hydroxyeicosatetraenoic acid (20-HETE), one of the principle cytochrome P450 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 G-protein receptor 75 (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 Gα_q/11 protein dissociation and increased inositol phosphate accumulation and GPCR-kinase interacting protein-1-GPR75 binding, which further facilitated the c-Src-mediated transactivation of epidermal growth factor receptor. This results in downstream signaling pathways that induce angiotensin-converting enzyme expression and endothelial dysfunction. Knockdown of GPR75 or GPCR-kinase interacting protein-1 prevented 20-HETE-mediated endothelial growth factor receptor phosphorylation and angiotensin-converting enzyme induction. In vascular smooth muscle cells, GPR75-20-HETE pairing is associated with Gα_q/11- and GPCR-kinase interacting protein-1-mediated protein kinase C-stimulated phosphorylation of MaxiKβ, linking 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 angiotensin-converting enzyme 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.

Keywords: cardiovascular diseases; cytochrome P-450 enzyme system; hypertension; receptor, epidermal growth factor; vascular remodeling.

Figure 1. Isolation of a 20-HETE-associated membrane protein complex by the click-in chemistry

A) Structures of the pharmacological probe 20-azido-N-4-(3-(4-benzoylphenylpropanamido)phenylsulfonyl)-19(S)-hydroxyeicosa-5(Z),14(Z)-dienamide (20-APheDa) and 20-HETE. B) Cumulative concentration-response curve to phenylephrine in renal interlobar arteries from C57BL/6 mice preincubated with the 20-HETE biosynthesis inhibitor DDMS (30 μmol/L) and cumulative concentration responses to phenylephrine (10-3 to 102 μmol/L) was constructed in the presence and absence of 20-HETE (10 μmol/L) or 20-HETE (10 μmol/L) +20-APheDa (10 μmol/L) (*p<0.05 vs DDMS; 3p<0.05 vs DDMS+20-HETE; mean±SEM, n=6). C) A representative (n=8) In-gel image band composition of 20-APheDa (10 nmol/L) bound to human microvascular endothelial cell membrane proteins (ECm, 20μg). D–E) Binding of 20-APheDa (0.1 nmol/L) to ECm (10 μg) is competed for by 20-HETE (100 μmol/L) but not by 12(S)-HETE (100 μmol/L) (representative image, n=5-6). The line between the marker lane (M) and the ECm lane in panels C-E reflects the fact that although the lanes are part of the same gel they are on a separate infrared channel.

Figure 2. 20-HETE binds to GPR75

A) Displacement of radiolabeled [³H₈] 20-HETE in membranes by unlabeled 20-HETE and 12(S)-HETE. B) Assessment of Kd. C) GPR75 protein levels in EC treated with control and GPR75-specific siRNA for 36 h. D) Displacement of radiolabeled [³H₈] 20-HETE (6.67 nmol/L) by increasing concentrations of unlabeled 20-HETE in membranes from EC treated with control and GPR75-specific siRNA for 36 h. E) Assessment of Kd from displacement bindings in 3D. F) Displacement of [³H₈] 20-HETE by 20-6,15-HEDGE (1 nmol/L). G–H) Effect of 20-HETE (10 nmol/L) on IP-1 accumulation in EC treated with control and GPR75-specific siRNAs. All results are mean±SEM (n=4–6, *p<0.05 vs. vehicle, ^#p<0.05 vs. 20-HETE)

Figure 3. Expression of GPR75 in the vascular endothelium and effect of 20-HETE on GPR75 association with GIT1, HIC-5 and Gα_q/11 in EC

A) A representative image of GPR75 (red) and CD31 (green) immunofluorescence of kidney sections from C57BL/6 mice (20X; scale bar, 10μm). B) A representative Western blot of GPR75 immunoprecipitate showing association with Gα_q/11, GIT1 and HIC-5 in EC. 20-HETE alters GPR75 association with (C) Gα_q/11, (D) GIT1 and (E) HIC-5. F) Effect of 20-6,15-HEDGE on 20-HETE-stimulated GPR75-GIT1 association. EC were incubated with 20-HETE (1 or 10 nmol/L) with and without 20-6,15-HEDGE (10 nmol/L) for 5 min and GPR75 was immunoprecipitated and immunoblot for the indicated proteins (mean±SEM, n=4, *p<0.05 vs Vehicle, ^#p<0.05 vs 20-HETE).

Figure 4. GPR75 and GIT1 are required for 20-HETE signaling in EC (A–C)

EC were transfected with control siRNA, GPR75- or GIT1-specific siRNA for 36 h. Cells were then incubated in a serum free media for 12 h prior to addition of 20-HETE (10 nmol/L) for 5 min. A) Representative western blot showing GPR75 and B) GIT1 knockdown. C) Effect of GPR75 and GIT1 knockdown on 20-HETE-stimulated EGFR phosphorylation (immunoblots are from separate experiments noted by dividing line; mean±SEM, n=4, *p<0.05 vs. Vehicle). 20-HETE stimulation of EGFR phosphorylation is mediated by GPR75-GIT1-cSrc activation in EC (D–F). The effects of 20-HETE (10 nmol/L) treatment (5 min) on: D) phosphorylated EGFR, E) c-Src bound to GIT and F) c-Src bound to EGFR, (mean±SEM, n=4, *p<0.05 vs Vehicle). G) GPR75 knockdown prevents 20-HETE-mediated ACE induction in EC. Endothelial ACE mRNA expression from control and GPR75 siRNA treated cells in the presence and absence of 20-HETE (10 nmol/L) for 2 h (mean±SEM, n=3, *p<0.05 vs Vehicle, ^#p<0.05 vs 20-HETE).

Figure 5. GPR75 Knockdown Prevents 20-HETE-Dependent Hypertension and ACE induction in Cyp4a12tg Mice

A) Systolic blood pressure monitoring in Cyp4a12tg mice receiving water, control shRNA+DOX and GPR75 shRNA+DOX (n=4, *p<0.05 vs water) for 14 days. B) Systolic blood pressure of Cyp4a12tg mice at day 14 of treatment with water, DOX, control shRNA, control shRNA+DOX or GPR75 shRNA+DOX. C) Vascular GPR75 expression in PGMVs from control and GPR75 shRNA-treated Cyp4a12tg mice at day 14. D) Vascular ACE expression in preglomerular arteries from Cyp4a12tg mice receiving water, DOX, control shRNA, control shRNA+DOX and GPR75 shRNA+DOX for 14 days. Blood pressure was measured by the tail cuff method. Results are mean±SEM, n=4, *p<0.05 vs water or control shRNA+DOX.

Figure 6. GPR75 knockdown prevent 20-HETE-dependent hypertension, vascular dysfunction and vascular remodeling (A–F)

A) Cumulative concentration-response curve to acetylcholine in renal interlobar arteries from Cyp4a12tg mice treated with water, Control shRNA, DOX, control shRNA+DOX, and GPR75 shRNA+DOX for 14 days (meaan±SEM, n=4, *p<0.05 vs water, ^#p<0.05 vs control shRNA+DOX). B) Cumulative concentration-response curve to phenylephrine in renal interlobar arteries from water, control and GPR75 shRNA treated mice receiving DOX after 12 days. EC50 and Rmax are given in the upper inset (n=6, *p<0.05 vs water). C) Systolic blood pressure measurements (tail cuff) in control and GPR75 shRNA-treated Cyp4a12tg mice receiving DOX for 35 days (n=3, *p<0.05 vs water, ^#p<0.05 vs control shRNA+DOX). Vascular remodeling measured by pressure myography as D) media thickness, E) media-to-lumen ratio and F) cross sectional area in renal interlobar arteries from mice treated with control and GPR75 shRNA after receiving DOX for 35 days as previously described (n=4, *p<0.05 vs water, ^#p<0.05 vs control shRNA+DOX).

Figure 7. GPR75-20-HETE pairing in vascular smooth muscle is linked to inhibition of MaxiKβ

Cultured aortic vascular smooth muscle cells were treated with 20-HETE (10 nmol/L) for 5 min. Immunoprecipitation followed by immunoblotting and densitometry analysis showed increases in: A) Gα_q/11 dissociation from GPR75, B) GPR75-GIT1 association, C) GIT1-PKCα dissociation, D) PKCα-MaxiKβ association, E) cSrc-MaxiKβ association and F) MaxiKβ tyrosine phosphorylation (mean±SEM, n=4, *p<0.05 vs Vehicle).

Figure 8. Proposed 20-HETE-GPR75-mediated signaling in endothelial cells

20-HETE-GPR75 pairing stimulates the dissociation of Gα_q/11 and the association of GIT1 to the receptor. The later facilitates c-Src-mediated EGFR transactivation. The 20-HETE-GPR75-mediated activation of EGFR results in the stimulation of downstream cascades that regulate vascular ACE expression and decreases in NO bioavailability.