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(Circulation Research. 2009;104:288.)
© 2009 American Heart Association, Inc.

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Regulatory Role of G Protein–Coupled Estrogen Receptor for Vascular Function and Obesity

Elvira Haas^*, Indranil Bhattacharya^*, Eugen Brailoiu^*, Marlen Damjanovi, G. Cristina Brailoiu, Xin Gao, Laurence Mueller-Guerre, Nicole A. Marjon, André Gut, Roberta Minotti, Matthias R. Meyer, Kerstin Amann, Emerita Ammann, Ana Perez-Dominguez, Michele Genoni, Deborah J. Clegg, Nae J. Dun, Thomas C. Resta, Eric R. Prossnitz, Matthias Barton

From the Departement für Innere Medizin (E.H., I.B., M.D., L.M.-G., A.G., R.M., M.R.M., E.A., A.P.-D., M.B.), Klinik und Poliklinik für Innere Medizin; and Klinik für Herz- und Gefässchirurgie (M.G.), Universitätsspital Zürich, Switzerland; Department of Pharmacology (E.B., G.C.B., X.G., N.J.D.), Temple University School of Medicine, Philadelphia, Pa; Department of Cell Biology and Physiology (N.A.M., T.C.R., E.R.P.) and University of New Mexico Cancer Center (E.R.P.), University of New Mexico Health Sciences Center, Albuquerque, NM; Pathologisches Institut (K.A.), Universitätsklinikum Erlangen, Germany; Klinik für Herzchirurgie (M.G.), Stadtspital Triemli, Zürich, Switzerland; and Department of Internal Medicine (D.J.C.), Touchstone Diabetes Center, University of Texas, Southwestern Medical Center, Dallas, Tex.

Correspondence to Matthias Barton, Departement für Innere Medizin, Klinik und Poliklinik für Innere Medizin, Universitätsspital Zürich, Rämistrasse 100, CH-8091 Zürich, Switzerland. E-mail barton{at}usz.ch

	Abstract

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We found that the selective stimulation of the intracellular, transmembrane G protein–coupled estrogen receptor (GPER), also known as GPR30, acutely lowers blood pressure after infusion in normotensive rats and dilates both rodent and human arterial blood vessels. Stimulation of GPER blocks vasoconstrictor-induced changes in intracellular calcium concentrations and vascular tone, as well as serum-stimulated cell proliferation of human vascular smooth muscle cells. Deletion of the GPER gene in mice abrogates vascular effects of GPER activation and is associated with visceral obesity. These findings suggest novel roles for GPER in protecting from cardiovascular disease and obesity.

Key Words: adipocytes • atherosclerosis • sex differences • vascular disease • metabolic syndrome

Coronary artery disease and stroke remain the leading causes of death in both men and women. The lower cardiovascular risk for premenopausal women as compared to men has been linked to the protective effects of endogenous estrogens on vascular tone, cell growth, and risk factors such as obesity and hypertension.¹ Vascular estrogen binding sites include nuclear estrogen receptors ER and ERβ, as well as the novel G protein–coupled estrogen receptor (GPER), also known as GPR30,^2,3 which is localized to the endoplasmic reticulum and mediates nongenomic estrogen signaling.^2,4 The GPER gene maps to chromosome 7p22.3, a region implicated in arterial hypertension in genetic linkage studies in humans,⁵ suggesting a role of GPER in blood pressure control. Some studies have proposed that estrogen-dependent intracellular calcium signaling and cell contraction in the cardiovascular system are independent of classic estrogen receptors, ER and ERβ⁶; it has also been demonstrated that nongenomic actions of estrogens involve signaling pathways similar to G protein–coupled receptors.³ Whether GPER, which is highly expressed in human arteries,⁷ contributes to the regulation of cellular homeostasis or intracellular calcium signaling in the cardiovascular system is unknown.

In premenopausal women, endogenous estrogen causes tonic vasodilation and thereby counteracts blood pressure elevation.⁸ To test whether GPER affects blood pressure, we intravenously infused the selective GPER agonist G-1⁹ into normotensive Sprague–Dawley rats (expanded Materials and Methods section in the online data supplement, available at http://circres.ahajournals.org).

G-1 infusion resulted in an acute reduction in mean arterial blood pressure (Figure 1A); the response began within 2 minutes, and a maximum effect was typically present 8 minutes after infusion. Next, using pressurized rat mesenteric resistance arteries of the same model, we recorded changes in inner diameter over time and found that G-1 promotes acute dilation of the preconstricted arteries (Figure 1B). Because human internal mammary arteries dilate in response to 17β-estradiol,¹⁰ we next tested whether G-1 also affects vascular tone in these arteries. We found, that in human internal mammary arteries, the relaxant response to G-1 was even stronger than that to 17β-estradiol (P<0.05 versus E2, Figure 1C). Finally, we investigated effects of G-1 on vascular tone in precontracted murine arteries. 17β-Estradiol produced time-dependent relaxation that was only significant from vehicle control in the aorta but not in the carotid artery. In contrast, the same concentration of G-1 caused dilation in both arteries that was more potent than 17β-estradiol (Figure 1D and Figure Ia in the online data supplement).

View larger version (19K): [in this window] [in a new window]   Figure 1. A, Acute effect of intravenous infusion of acetylcholine (ACh) or increasing doses of G-1 (41.2 ng/kg, 412 ng/kg, 4.12 ng/kg, and 20.6 µg/kg) into normotensive Sprague–Dawley rats. The y-axis values are expressed as percentage changes of mean arterial pressure. G-1 produced a dose-dependent reduction in blood pressure of 2.6±2%, 10±1%, 13±1%, and 13.5±2.2%, respectively. *P<0.05 vs control. B, Acute vascular effects of solvent control (ethanol, 0.1%) (CTL) and GPER agonist G-1 (1 µmol/L) in pressurized rat mesenteric resistance arteries. Arteries were preconstricted with UTP to induce a stable contraction plateau and exposed to EtOH or G-1, and vasodilatory responses (percentage reversal of UTP-induced vasoconstriction) were recorded. At 40 minutes, G-1 produced a 29±4% vasodilatory response (n=3 to 4 per group). *P<0.05 vs CTL. C, Acute vascular effects of solvent control (CTL), 17β-estradiol, or GPER agonist G-1 on vascular tone in human internal mammary arteries. Arteries were preconstricted with prostaglandin F2 to induce a stable contraction plateau and exposed to solvent control (ethanol 0.3% CTL), 17β-estradiol (E2), or G-1 (both at 3 µmol/L), and changes in tone were recorded. Both 17β-estradiol and G-1 induced a relaxant response; however, the relaxation in response to G-1 was more potent. *P<0.05 vs control, **P<0.05 vs 17β-estradiol (unpaired t test; n=4 to 7 per group). D, Acute vascular effects of solvent control (ethanol, 0.3%) (CTL), 17β-estradiol (E2), or GPER agonist G-1 on vascular tone in mouse carotid arteries. Arteries were preconstricted with prostaglandin F2 to induce a stable contraction plateau and exposed to 17β-estradiol or G-1 (both at 3 µ mol/L), and changes in tone were recorded. G-1, but not 17β-estradiol, reduced vascular tone by 44±5%. *P<0.05 vs solvent (unpaired t test; n=4 to 7 per group). E, Acute vascular effects of solvent control (ethanol, 0.3%) (CTL), 17β-estradiol (E2), or GPER agonist G-1 on vascular tone in mouse carotid arteries. Arteries were preincubated with 17β-estradiol or G-1 (both at 3 µmol/L) for 45 minutes and then exposed to serotonin (5HT) (1 µmol/L). G-1 reduced contraction by 39%. *P<0.05 vs solvent (unpaired t test; n=4 to 8 per group). F, Left, Representative original recordings of the effects of intracellular ligand injection on intracellular calcium concentrations in Fura-2–loaded human aortic vascular smooth muscle cells. Solvent (ETOH) had only small effects on intracellular calcium concentrations. Serotonin (5-HT) produced a strong calcium mobilization response. Intracellular injection of G-1 produced a fast and transient increase in calcium concentration, and pretreatment with G-1 completely blunted the subsequent 5-HT–induced changes in intracellular calcium. Right, Averaged data of 4 experiments. *P<0.05 vs 5-HT alone (paired t test; n=4 per group). G, Effects of G-1 (10 and 100 nmol/L) on ERK-1/2 phosphorylation in human vascular smooth muscle cells expressing only GPER determined by Western blot. Left, Representative example. Right, Averaged (n=8 per group). *P<0.05 vs solvent (unpaired t test). H, Effects of GPER agonists G-1 and ICI 182,780 on serum-stimulated cell proliferation in human vascular smooth muscle cells expressing only GPER. Both agonists reduced cell proliferation between 60% and 80% at a concentration of 1000 nmol/L. *P<0.05 vs solvent (n=6 per group).

Estrogen also indirectly acts as a vasodilator by blocking the activity of vasoconstrictors, such as platelet-derived serotonin.¹¹ Whereas G-1 preincubation reduced serotonin-induced vascular tone, 17β-estradiol did not affect contractions in aorta or carotid arteries (Figure 1E and supplemental Figure Ib). Similar to murine blood vessels, G-1 preincubation also inhibited the contractile response to serotonin in human arteries (12±2 versus 22±5% of KCl; n=5 to 6 per group; P<0.05). Taken together, these data indicate that activation of GPER directly and indirectly mediates acute vasodilation, thus reducing blood pressure.

Calcium mobilization plays an important role in vascular smooth muscle cell relaxation. Activation of GPER leads to phospholipase C activation, resulting in intracellular calcium mobilization.² On the other hand, calcium channel–blocking effects of sex steroid hormones have been described.¹² Because estrogen-induced mechanisms independent of ER and ERβ have been recently proposed to regulate cardiac myocyte contraction,⁶ we next determined whether the inhibitory effects of GPER activation on vascular tone involves changes in intracellular calcium concentrations within vascular smooth muscle cells. G-1 effects on intracellular calcium concentrations in the absence and presence of serotonin were determined in single human aortic smooth muscle cells by intracellular injection. The serotonin-induced calcium increase was almost completely abrogated after intracellular injection of G-1 (Figure 1F). Interestingly, when applied extracellularly, G-1 yielded a slow and sustained increase in calcium over a few minutes (supplemental Figure II), compared with an instantaneous increase on intracellular injection, consistent with an intracellular localization of GPER.² RNA interference of GPER reduced GPER gene expression (supplemental Figure IIIa and IIIb) and almost completely abrogated G-1–induced increases in intracellular calcium (supplemental Figure IIIc). The observation that intracellular injection but not external application of G-1 produced a rapid yet transient calcium increase (Figure 1F) indicates that the dynamics of changes in intracellular calcium concentrations via GPER depend on whether stimulation occurs intra- or extracellularly. This would be consistent with the rather slow dilator response in isolated arterial blood vessels and effects on blood pressure both requiring several minutes.

We recently reported that 17β-estradiol mediates phosphorylation of extracellular signal-regulated kinase (ERK)-1/2 in human vascular smooth muscle cells⁷ and now tested whether GPER also mediates ERK-1/2 phosphorylation. We used human umbilical vein smooth muscle cells, which lose expression of ER and ERβ yet retain GPER expression on cell culture (supplemental Figure IV). At nanomolar concentrations of G-1, a robust increase in ERK-1/2 phosphorylation was observed (Figure 1G). In contrast, neither ER agonist PPT nor ERβ agonist DPN at equimolar concentrations affected ERK-1/2 phosphorylation (supplemental Figure V).

Because vascular smooth muscle cell proliferation is a prerequisite for the development of atherosclerosis,¹² and estrogen inhibits vascular smooth muscle cell growth,^1,12 we next determined if GPER activation plays a role in cell growth regulation using 2 agonists for GPER, G-1, and ICI182,780.^9,13 Both compounds had no effect on basal cell growth as determined by ³H-thymidine incorporation; however, serum-stimulated cell growth was inhibited by 60% to 80% (Figure 1H), in line with recently described growth-inhibitory effects of GPER activation in certain cancer cells.¹⁴

Finally, we examined the effects of GPER deletion on vascular responses in carotid arteries. Arteries from GPER^–/– animals showed normal vascular reactivity to contractile stimuli such as potassium chloride (wild-type: 19.94±0.95 mN; GPER^–/– 21.12±1.03 mN; P=NS, n=18 to 26 per group). However, genetic deletion of GPER was associated with increased body weight and visceral adiposity in both male and female animals (P<0.05, Figure 2A and 2B); in addition, the inhibitory effect of G-1 on serotonin-mediated contractions was equally effective in wild-type males (18±2 versus 27±3%) and females (20±3 versus 30±4%), suggesting sex-independent, GPER-mediated effects. The different aspects related to obesity in GPER^–/– mice, including responses to dietary interventions, have been extensively characterized in a separate study (DJ Clegg and colleagues, manuscript in preparation, 2009). Expression of GPER was detected in adipose tissue as well as in carotid artery and aorta of wild-type animals (data not shown). Markers of adipocyte differentiation, C/EBP and PPAR, were similarly expressed at the mRNA level in both wild-type and GPER^–/– mice (I Bhattacharya, M Barton, unpublished observations, 2009). As expected for a GPER-mediated response, G-1 had no dilator effects in carotid artery rings of GPER^–/– mice (Figure 2C) and attenuated the contractile response to serotonin in wild-type mice (10±1% versus 17±3%, n=7 to 8, P<0.05) but not in GPER^–/– mice (17±5% versus 17±2%, n=5 to 7; P=NS; Figure 2D).

View larger version (53K): [in this window] [in a new window]   Figure 2. A, Effect of GPER deficiency on body weight in male (left) and female (right) GPER+/+ (open bars) and GPER–/– (hatched bars) mice. *P<0.05 vs wild-type (Mann–Whitney U test). B, Representative examples of abdominal fat distribution in GPER+/+ (left) and GPER–/– (right) mice. Note the almost complete lack of abdominal fat in the wild-type animal compared to the GPER–/– animal. C, Acute vascular effects of solvent control (ethanol, 0.1%) (CTL) or GPER agonist G-1 on vascular tone in carotid arteries of GPER+/+ (left) and GPER–/– (right) mice. Arteries were preconstricted with prostaglandin F2 to induce a stable contraction plateau and exposed to G1 (1 µmol/L), and changes in tone were recorded. G-1 reduced vascular tone in GPER+/+ mice (*P<0.05 vs solvent; unpaired t test) (left) but not in GPER–/– mice (P=NS) (right) (n=6 to 9 per group). D, Contractions to serotonin (100 nmol/L) after preincubation with G-1 (1 µmol/L) in GPER+/+ (left) and GPER–/– (right) mice. *P<0.05 (Mann–Whitney U-test; n=5 to 8 per group); *P<0.05 vs solvent control.

The present findings indicate a novel role for GPER as an intracellular G protein–coupled estrogen receptor controlling vascular tone and blood pressure as well as body weight. Our findings suggest the possibility that, in contrast to its known direct effects on calcium mobilization via EGF receptor transactivation,^2,13 activation of GPER might also antagonize changes in intracellular calcium evoked by vasoconstrictor agonists such as serotonin, possibly involving ERK-1/2.

Our finding of increased body weight and abdominal obesity in male and female GPER-deficient mice is in contrast to a most recent publication using a different type of GPER^–/– mouse generated using a cre/lox approach.¹⁵ Whereas we found obesity in GPER^–/– mice, these investigators found slightly reduced body weight in female animals only. Moreover, the authors did not detect any expression of GPER in fat tissue,¹⁵ whereas we found expression of GPER in fat tissue of both male and female wild-type animals. The authors also claim slightly increased blood pressure levels; however, the difference presented was mainly attributable to a reduction of blood pressure in the wild-type animals, rather than an increase in the knockout animals, because mean arterial blood pressure was essentially normal at 75 mm Hg. The contrasting results between the study by Mårtensson et al¹⁵ and our study are currently unclear. We speculate that the cre/lox approach used by these authors may have involved cryptic or pseudo loxP sites, which may cause unwanted chromosomal translocations.¹⁶ Such factors would not be expected to play a role in the GPER^–/– used in the present study, which were created using homologous recombination of ES cells (online data supplement and the study by Wang et al¹⁷).

In summary, the present study demonstrates for the first time that GPER contributes to regulation of blood pressure, vascular tone, and obesity and suggests the possibility that some of the known vasculoprotective effects of estrogen involve GPER activation. Given that selective activation of GPER appears to be highly effective in blocking vasoconstriction and cell growth, GPER could represent a novel target to interfere with the development of vascular disease and obesity in humans.

	Sources of Funding

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Supported by Swiss National Science Foundation grants 3200-108258/1 and K-33KO122504/1 (to M.B.); NIH grants HL-90804 (to E.B.) and CA-116662 and CA-118743 (to E.R.P.); NS-18710 and HL-51314 (to N.J.D.); and HL-77876 and HL-88192 (to T.C.R.); and the Interdisziplinäres Zentrum für Klinische Forschung Erlangen, Project A11 (to K.A.).

Disclosures

The University of New Mexico has filed a patent application on compounds used in this study.

	Footnotes

 
*These authors contributed equally to this study.

Original received November 7, 2008; revision received January 12, 2009; accepted January 15, 2009.

	References

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This Article Free upon publication Abstract Full Text (PDF) Data Supplement All Versions of this Article: 104/3/288    most recent CIRCRESAHA.108.190892v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Haas, E. Articles by Barton, M. Search for Related Content PubMed PubMed Citation Articles by Haas, E. Articles by Barton, M. Related Collections Obesity Risk Factors Cell signalling/signal transduction Gene regulation Smooth muscle proliferation and differentiation Other Vascular biology