Elsevier

Pharmacological Research

Volume 71, May 2013, Pages 53-60
Pharmacological Research

Invited review
GPER: A novel target for non-genomic estrogen action in the cardiovascular system

https://doi.org/10.1016/j.phrs.2013.02.008Get rights and content

Abstract

A key to harnessing the enormous therapeutic potential of estrogens is understanding the diversity of estrogen receptors and their signaling mechanisms. In addition to the classic nuclear estrogen receptors (i.e., ERα and ERβ), over the past decade a novel G-protein-coupled estrogen receptor (GPER) has been discovered in cancer and other cell types. More recently, this non-genomic signaling mechanism has been found in blood vessels, and mediates vasodilatory responses to estrogen and estrogen-like agents; however, downstream signaling events involved acute estrogen action remain unclear. The purpose of this review is to discuss the latest knowledge concerning GPER modulation of cardiovascular function, with a particular emphasis upon how activation of this receptor could mediate acute estrogen effects in the heart and blood vessels (i.e., vascular tone, cell growth and differentiation, apoptosis, endothelial function, myocardial protection). Understanding the role of GPER in estrogen signaling may help resolve some of the controversies associated with estrogen and cardiovascular function. Moreover, a more thorough understanding of GPER function could also open significant opportunities for the development of new pharmacological strategies that would provide the cardiovascular benefits of estrogen while limiting the potentially dangerous side effects.

Introduction

Although early clinical studies demonstrated beneficial cardiovascular effects of estrogen (E2) in postmenopausal women, subsequent studies have failed to corroborate this evidence. At present the only consensus appears to be that estrogens exert complicated and poorly understood effects on cardiovascular health. What is well-known is that E2 is a potent vasodilatory hormone which relaxes blood vessels in intact organs [1], [2], [3], [4], [5] or isolated vessels in vitro [6], [7], [8], [9], [10], [11], [12], [13], [14], [15]. Most curiously, however, E2 can also exert a powerful and direct vasoconstrictor action on coronary arteries in vitro [16], demonstrating that this “well-known vasodilator” is actually a powerful, multifunctional vasoactive hormone whose vascular signaling mechanisms are heterogeneous and complicated. In light of these controversial (and sometimes contradictory) findings, it is apparent that there is still much to be learned of how E2 influences vascular function. An important first step in this process is to better understand the nature of the specific estrogen receptor molecules that bind E2 and thereby initiate the complicated process of E2 signaling in target cells.

Estrogens signal via both traditional genomic and more novel non-genomic transduction mechanisms, and it is now apparent that E2 signals via a diversity of estrogen receptors (ERs). In addition to the classic nuclear ER, some actions of E2 persist in presence of agents that inhibit transcription or protein synthesis. Further, binding sites for E2 on the extracellular surface of endometrial cells were discovered 35 years previously [17]; yet the function of such “membrane-associated” ERs has only recently come to light. In 1997 a novel, G-protein-coupled estrogen receptor (GPR30, now designated as GPER) was cloned from MCF7 [18] and other cells, including endothelial cells [19], [20], and the sequence revealed the heptahelical, transmembrane structure common to all G-protein-coupled receptors (GPCRs).

GPER expression has been detected in a variety of tissues and cell types, as recently reviewed [21], [22], [23]. For example, GPER is expressed in high density in human bone tissue, and may help mediate the ability of E2 to preserve bone density [24], [25]. These findings suggest a potential therapeutic role for GPER in preventing or alleviating menopausal osteoporosis, although such an action remains to be explored. GPER expression and function are now being investigated in cardiovascular tissues (i.e., myocardium, vascular endothelium, and vascular smooth muscle cells), where accumulating evidence indicates a protective effect of GPER activation on both cardiac and vascular function. For example, GPER activation has been shown to improve functional recovery and decrease myocardial inflammation after global ischemia-reperfusion injury [26] and to also inhibit tumor necrosis factor (TNF)-induced upregulation of inflammatory protein in endothelial cells nuclei [27], thus substantiating the potential role of GPER as a therapeutic target in the cardiovascular system. GPER is also highly expressed in renal tubules [28], and may help reduce proteinuria associated with renal dysfunction in salt-sensitive hypertension [29]. Exocrine cells on the gastrointestinal tract also express GPER [30]. Thus, an increasing number of cell types express GPER. However, we are only now beginning to unravel the complicated nature of E2 signaling in target cells, particularly the cross-talk between ERs and other receptor types as well as genomic vs. non-genomic E2 effect and their role in physiological regulation and pathophysiological conditions.

With the advent of compounds exhibiting selectivity for GPER (e.g., the selective agonist, G-1, and especially the “specific” antagonist, G15), it is becoming increasingly likely that GPER is a bona vide estrogen target protein. In contrast, the precise cellular localization of GPER remains somewhat controversial. GPER activation can initiate very rapid cellular responses that are highly consistent with generation of a plasma membrane-associated signal. For example, Funakoshi et al. [31] demonstrated a similar rapid calcium activation of pyramidal neurons via plasma membrane GPER (with no evidence of GPER in the endoplasmic reticulum or Golgi apparatus). In contrast, Revankar et al. [32] found that only cell-permeable GPER agonists could activate rapid calcium signaling in COS7 cells. More recently, intracellular injection of G-1 was found to produce a more rapid and greater increase in intracellular calcium in cultured spinal neurons compared to G-1 applied extracellularly [33]. These studies indicated that GPER-mediated rapid signaling responses need not be initiated at the plasma membrane, but could involve activation of a cytoplasmic GPER. There is evidence that GPER may localized to the endoplasmic reticulum [34], [35]. Further, E2 can stimulate nuclear translocation of GPER in breast cancer-associated fibroblasts [36]. In contrast, GPER has been found strongly localized to the plasma membrane in HEK293 cells [37], renal epithelial cells [38], and some neurons [31]. In sum, the distribution of GPER appears to be heterogeneous with regard to cellular localization and function, as evidence indicates that GPER can be localized to either the plasma membrane or specific intracellular sites. A potential explanation for this phenomenon has been proposed by Cheng et al. [38], who have observed that plasma membrane-associated GPER proteins are endocytosed via clathrin-coated vesicles in an arrestin-independent manner. GPER proteins are apparently trafficked from the plasma membrane to the perinuclear compartment for sorting and possible degradation in the Golgi or endoplasmic reticulum, thus preventing cells from being exposed to excessive estrogen signaling. Although this mechanism could possibly account for the controversy associated with GPER localization, more research is needed to clarify the exact nature of GPER cellular localization.

ICI182,780 (fulvestrant) is known to be a “pure” ERα/ERβ antagonist, and is typically considered a powerful anti-estrogen devoid of any E2 agonist activity [39]. Nonetheless, several studies now indicate that ICI182,780 does not attenuate the vasodilatory action of E2 in some vessels [40], [41], and that ICI182,780 itself can induce acute relaxation of porcine coronary [13], [42] and other arteries [43]. These findings imply expression and activity of a novel ER distinct from the classic nuclear ERα or ERβ. Interestingly, ICI182,780 exhibits agonistic effects on GPER [42], [44], and this action is likely to be responsible for its unexpected vasodilatory effect. These studies strongly suggest a potential role for GPER in mediating cardiovascular responses to estrogens.

GPER is expressed in the heart and blood vessels, and GPER function in cardiovascular regulation is now being explored. For example, female GPER knockout (KO) mice develop increased mean arterial pressure compared to age-matched controls [45]. Further, GPER activation induces a vasodilatory response that is abrogated in carotid arteries from GPER KO mice compared to arteries from control animals [46]. These studies indicate that GPER activation may exert a tonic vasodilatory influence to lower blood pressure, and can mediate agonist-induced vasodilation as well. In addition, G-1 (a GPER-selective agonist which exhibits nanomolar affinity for GPER, but does not bind ERα or ERβ significantly) [47] produced a dose-dependent, acute lowering of mean arterial pressure in normotensive rats [46] or in ovariectomized, hypertensive mRen2.Lewis rats [48]. Further, acute treatment with G-1 relaxes rat aorta [48], rat mesenteric arteries [46], human internal mammary arteries [46], rat carotid arteries [49], [50], and porcine coronary arteries [42], [51]. Recent studies employing G15, a selective GPER antagonist, indicate that pharmacological inhibition of GPER can attenuate vascular relaxation induced by E2 [52] or G-1 [51], [52]. Thus, there is increasingly consistent evidence obtained from both genetic and pharmacological studies that activation of GPER exerts a vasodilatory effect, and these findings raise the intriguing possibility that at least a portion of the normal vasodilatory response to E2 may be mediated via GPER.

Estrogens may also help protect against cardiac dysfunction by exhibiting an action similar to that associated with ischemic preconditioning [50], [53], [54]. Interestingly, G-1 reduces postischemic contractile dysfunction and infarct size, possibly through activation of the PI3 kinase/Akt/ERK 1/2 signaling cascade [55], [56]. In addition, GPER activation may also inhibit activity of the mitochondria permeability transition pore [57], and G-1 can reduce inflammatory damage associated with ischemia [26]. How GPER might reduce inflammation is not completely understood; however, G-1 has been reported to stimulate secretion of interleukin 10, an immune suppressor [58]. These findings are consistent with cellular experiments indicating that hypoxia upregulates GPER expression in cell lines derived from breast cancer or cardiac myocytes, and that knockdown of GPER expression attenuates the protective effect of E2 on hypoxia-induced apoptosis [59]. In summary, these studies provide evidence for a functional link between GPER and the protective effects of E2 (and G-1) in the heart. Nonetheless, further research is required to better understand the cellular signaling of GPER activation in the heart before the therapeutic potential of GPER may be unlocked. Lastly, GPER could indirectly promote cardiovascular health in diabetic patients by enhancing insulin secretion [60]. Thus, accumulating evidence strongly suggests a number of potential mechanisms whereby GPER activation could protect against or treat cardiovascular dysfunction.

Section snippets

GPER regulates both conduit artery and resistance artery tone

GPER activation relaxes both large and small arteries from various tissues in different species. For example, both E2 and G-1 decrease infarct volume in brain after focal stroke in ovariectomized female mice most likely by inducing relaxation of the middle cerebral artery (MCA) [61], [62]. In addition, both G-1 and ICI 182,780 exert a direct relaxation effect on porcine coronary arteries [13], [42], [51]. For example, 3 μM G-1 or ICI 182,780 relaxed precontracted epicardial coronary arteries by

The potential role of GPER in nitric oxide release

GPER was cloned from human umbilical vein endothelial cells (HUVECs) exposed to shear stress. Although GPER mRNA levels were very low or undetectable in control HUVECs, mRNA increased as fluid flow elevated shear stress [19]. These findings suggested that GPER is expressed on the endothelial cell surface under physiological conditions, and may function as a shear stress-sensing mechanism helping to regulate endothelial cell function. GPER protein expression has been detected in both endothelial

GPER induces calcium mobilization

The effect of GPER activation on intracellular calcium concentration has been investigated in VSM cells from humans and GPER-deficient mice. Haas et al. [46] observed that intracellular G-1 produced a fast, transient increase in calcium concentration in human aortic smooth muscle cells, whereas pretreatment with G-1 completely blunted the robust increase in intracellular calcium [Ca2+]i stimulated by 5-HT; however, extracellular G-1 produced a slow and sustained increase in calcium over a few

GPER action in endothelial cell proliferation

Estrogen receptors play an important role in endothelial cell growth and re-endothelialization [81]. A recent in vivo study employing balloon angioplasty demonstrated a protective effect of E2 delivered locally to the coronary endothelium [82]. In these studies improved endothelial function was evidenced four weeks after percutaneous transluminal coronary angioplasty by enhanced eNOS expression and reendothelialization. Holm et al. [83] investigated the effects of G-1 on endothelial cell

Conclusions

Acute, non-genomic estrogen signaling via GPER is a new and exciting development, particularly for its potential as a novel target for cardiovascular therapeutics. Estrogens clearly exert a protective effect on cardiovascular function during a woman's childbearing years, but the challenge has been to convey these beneficial effects without unwanted steroid side effects on other systems. Moreover, the apparently harmful effects of E2 reported in postmenopausal women would also have to be

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    College of Life Science, Henan Normal University, Xinxiang, Henan Province 453007, PR China.

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