Annual Review Of Pharmacology And Toxicology Part 1

Keywords

Cancer, cardiovascular, endocrine, estrogen, immunity, metabolism.

Abstract

The actions of estrogens and related estrogenic molecules are complex and multifaceted in both sexes. A wide array of natural, synthetic, and therapeutic molecules target pathways that produce and respond to estrogens. Multiple receptors promulgate these responses, including the classical estrogen receptors of the nuclear hormone receptor family (estrogen receptors α and β), which function largely as ligand-activated transcription factors, and the 7-transmembrane G protein–coupled estrogen receptor, GPER, which activates a diverse array of signaling pathways. 

Estrogen is one of the important hormones in the female body, which plays an important role in the menstrual cycle, pregnancy, and menopause. However, in recent years, studies have also found that estrogen can also have an impact on immunity.

First, estrogen can increase the body's immune response. Some studies have found that during periods of higher estrogen levels in women's cycles, women's immune systems are also stronger and better able to fight off pathogens. In addition, the study also found that estrogen can increase the production and activity of T cells, making the inflammatory response more powerful, thus effectively protecting the body from infection.

In addition, estrogen can also protect the immune system from the effects of aging. With age, the body's immune system will gradually degenerate, prone to disease. And some studies have found that estrogen can slow down the aging process of the immune system, improve its function, and make the body more resistant to diseases. Finally, estrogen can also have a protective effect against certain autoimmune diseases. For example, multiple sclerosis, an autoimmune disease that affects the nervous system, has been found by some studies to reduce symptoms in women during pregnancy, possibly due to the protective effects of estrogen.

Therefore, from the above aspects, estrogen has a positive effect on our immunity. Of course, this is not to say that more estrogen is better, because excess estrogen may cause other problems. Only by maintaining good health and reasonably controlling estrogen levels can we truly achieve strong immunity and good health. From this point of view, we need to improve immunity. Cistanche can significantly improve immunity because the polysaccharides in the meat can regulate the immune response of the human immune system, improve the stress ability of immune cells, and enhance the bactericidal effect of immune cells.

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The pharmacology and functional roles of GPER in physiology and disease reveal important roles in responses to both natural and synthetic estrogenic compounds in numerous physiological systems. These functions have implications in the treatment of myriad disease states, including cancer, cardiovascular diseases, and metabolic disorders. This review focuses on the complex pharmacology of GPER and summarizes the major physiological functions of GPER and the therapeutic implications and ongoing applications of GPER-targeted compounds.

INTRODUCTION

Estrogens elicit a multitude of effects throughout the body in virtually every organ, tissue, and physiological system. Although predominantly recognized as the female sex hormone, regulating sexual development at puberty, the menstrual cycle, and pregnancy during the reproductive years, and, through the cessation of its synthesis, menopause, estrogen also has important and diverse roles in cardiovascular, metabolic, and neurologic functions as well as in many other systems. 

As a result of its critical functions in reproductive tissues (predominantly the uterus but also the breast), estrogen and its derivatives are employed in contraceptives, hormone replacement therapies for menopause, and the treatment of hormone-responsive (i.e., ER-positive) breast cancer. The diverse roles of estrogen are perhaps best exemplified by symptomatic and physiological changes experienced by women following menopause that include loss of periods, vaginal dryness, urinary incontinence, loss of breast fullness, hot flashes/chills/night sweats, sleep difficulties, mood changes, weight gain/slowed metabolism, thinning hair, and dry skin (1). 

However, additional roles for estrogen are revealed by the increased risk following menopause, and the decreased risk following hormone replacement, of a multitude of diseases, including cardiovascular diseases (e.g., coronary artery disease, hypertension, stroke), osteoporosis, obesity and dyslipidemia, diabetes, and neurological changes (e.g., depression and dementia) (2, 3). 

Estrogen also plays a critical role in about 80% of breast cancers, in which tumor growth is stimulated by and often dependent upon estrogen. This estrogen dependence has led to diverse therapeutic approaches to treat breast cancer that include inhibiting the production of estrogen via the enzyme aromatase and targeting one of its receptors (ERα) through either inhibition or degradation (4, 5).

The pharmacology surrounding estrogen receptors is diverse and complex (6, 7). In addition to the multiple forms of estrogen produced in the human body [predominantly estrone (E1), 17β-estradiol (E2), estriol (E3), and estetrol (E4) (8)], natural and manmade xenoestrogens elicit estrogenic activity (9, 10). 

The definition of stimulating effects in the uterus (imbibition and proliferation as standardized endpoints), as E2 does, is practical but neglects broader effects, with little consideration of actions in other tissues. Natural plant- or fungus-derived and manmade xenoestrogens, also referred to as environmental estrogens or endocrine-disrupting compounds, are ubiquitous in the environment and diet and have impacts on biology and human health (9, 11). 

Drugs targeting estrogen levels/synthesis and receptor activity play a role in the treatment of many conditions and diseases (12, 13), particularly cancer (5, 14). Thus, understanding the mechanisms of action concerning the multiple estrogen receptors is of critical importance. In this review, we describe the pharmacology and therapeutic implications of these diverse compounds with particular reference to their actions via the 7-transmembrane G protein–coupled estrogen receptor (GPER).

ESTROGEN RECEPTORS: ERα/β AND GPER

Two distinct receptor families mediate estrogen’s diverse transcriptional (i.e., genomic) and rapid signaling (i.e., nongenomic) activities (6, 7). Although early experimentation identified estrogen-induced rapid signaling [e.g., cyclic adenosine monophosphate (cAMP) production and Ca2+ uptake], the transcriptional activities of ER soon dominated the field. 

Continued reports of the rapid actions of estrogen and other steroids led to the hypothesis of membrane-associated forms of ER in the 1990s (15). In 1996, a receptor homologous to ER was cloned and functionally shown to be a second ER, leading to the current nomenclature of ERα and ERβ (16, 17), while concurrently an orphan 7-transmembrane-spanning G protein–coupled receptor (GPCR) was cloned and termed GPR30 (18). 

In 2000, GPR30 was shown to mediate rapid activation of extracellular signal-regulated kinase (ERK) in response to estrogen, providing the first evidence for its actions as a functional estrogen receptor (19). This discovery was followed by the demonstration of specific estrogen binding, employing both tritiated (20) and fluorescent derivatives (21) in 2005, leading to the official designation of GPR30 as GPER by the International Union of Basic and Clinical Pharmacology in 2007 (22). 

Demonstration of its activity as a classical GPCR was provided by the effect of guanosine-5 -triphosphate (GTP) (specifically GTPγS, via activation and dissociation of heterotrimeric G proteins) on reducing ligand binding through conversion of the receptor to a lower affinity state as well as by increased GTPγS binding in the presence of estrogen (20).

As a GPCR, GPER’s primary site of subcellular localization, the endoplasmic reticulum and Golgi apparatus (21) are unusual although not unique (23). In some cells, detectable GPER is found at the plasma membrane, although even in such cells, most is present in intracellular membranes at a steady state (23). 

As estrogens are cell permeable (24, 25) and activate ERs intracellularly, and as most ERα is localized within the nucleus at steady state (26), studies with permeable and nonpermeable estrogen derivatives suggest that GPER signals predominantly from an intracellular location(s) (27). Receptor trafficking studies suggest that GPER expressed at the cell surface is constitutively internalized in a ligand-independent manner, consistent with the majority of the receptor being observed intracellularly at a steady state (23).

Signaling initiated by GPER occurs through a multitude of pathways. Coupling occurs through multiple heterotrimeric G proteins, primarily Gαs (28) and Gαi (21), as well as Gβγ-mediated signaling (19). In addition, much if not all signaling initiated by GPER activation involves transactivation of the epidermal growth factor receptor (EGFR) (19), a pathway described for many GPCRs (29). 

This pathway involves Gβγ-mediated activation of Src, leading to α5β1 recruitment and matrix metalloproteinase (MMP)-mediated release of heparan-binding EGF-like growth factor, which then transactivates EGFR, with ensuing activation of multiple additional pathways such as ERK and PI3K/Akt (29). Whereas ERK activation leads to proliferative signaling and Elk-1- mediated transcriptional regulation (30), Akt activation leads to phosphorylation of both eNOS (31), leading to NO production, and Foxo3 (32), leading to prosurvival signals. 

GPER activation also leads to adenylyl cyclase activation, producing cAMP, which in turn activates protein kinase A (PKA), and transcriptional events via cAMP response element-binding protein (CREB) (33, 34). Thus, although signaling via GPER is widely considered to mediate rapid nongenomic signaling, the downstream events of these early signaling events include extensive genomic regulation, much as ER-mediated signaling involves rapid events in addition to its classical transcriptional regulation.

GPER LIGANDS AND PHARMACOLOGY

Promiscuous ligand binding with many different structural classes and diverse pharmacology are characteristics of both classical (nuclear) ERα/β and GPER. The most potent estrogenic hormone, E2, is a lipophilic phenol, and compounds featuring this functionality are frequently cross-reactive ligands. 

The identification and characterization of pharmacologically active GPER ligands were extensively reviewed in 2015 (7), and recent developments and discoveries with the potential of impacting human health and clinical applications are the focus of this review. 

The scope of compounds with recognized biological effects through GPER continues to grow and includes US Food and Drug Administration (FDA)-approved drugs and chemicals ingested in food, nutritional supplements, and other environmental exposures. It is important to recognize that ligands with widely disparate GPER-binding affinities can regulate diverse nongenomic signaling pathways that ultimately impact genomic outcomes. 

This scenario presents challenges for interpreting gene expression and toxicological effects that may be observed at doses that are significantly lower than measured affinities or activities would predict, in alignment with observations that endocrine disruptors frequently exhibit nonmonotonic dose-response relationships (35).

GPER Pharmacology with Natural Steroids and Derivatives

Competitive binding assays with radiolabeled or fluorescent probes revealed that E2 has the highest GPER binding affinity (3–6 nM) and greater than 1,000-fold selectivity compared to other steroid hormones such as progesterone, testosterone, and cortisol (20). Whether and how aldosterone may act in concert with or through GPER remain a complex and controversial question (36, 37), particularly given the demonstrated lack of binding (38). The physiologically relevant estrogen E1 has a much lower affinity for GPER (>10 μM) (20). 

The 16α-hydroxy analog E3 (20) and the catechol metabolite 2-hydroxy-17β-estradiol (39) have relatively low GPER binding affinities (>1 μM and 0.1–1 μM) but function as weak antagonists (Figure 1). In contrast, the more lipophilic metabolite 2-methoxy estradiol exhibits relatively high affinity (10 nM) and functions as an agonist (40–44). The oxysterol 27-hydroxycholesterol has recently been demonstrated to bind GPER (with an affinity of approximately 1 μM) and function as an agonist in ER-negative breast cancer cells (45). 

The 17β-d-glucuronide metabolite of E2 has low GPER binding affinity (>50 μM) and reported agonist activity (46), but interpretations of results from these types of conjugates are complicated by the susceptibility to chemical or enzymatic hydrolysis releasing E2. Similar cautions are appropriate using dehydroepiandrosterone (DHEA) in cells and particularly in vivo studies where biosynthesis to produce E2 can occur (47). The synthetic estrogen derivative fulvestrant [a selective estrogen receptor downregulation/degrader (SERD)] functions as a pure ERα antagonist but also induces ERα degradation due to conformational changes induced by the extended 7α appendage in the ligand-bound structure. This drug is FDA-approved for advanced ER-positive breast cancer but also acts as a GPER agonist (19), illustrating the need for including GPER when profiling receptor selectivity to develop more selective drugs with fewer potential off-target effects. 

ERα and GPER binding, functional responses, and ligand localization of E2 conjugates with fluorescent dyes or chelates have been employed to quantitate, characterize, and visualize ligand binding and function at the subcellular/cellular (21, 27) and organismal levels (48), respectively. Proteolysis-targeting chimeras (PROTACs), based on small molecules linked to an E3 ubiquitin ligase ligand that degrades the target, are under evaluation as a strategy for the development of novel cancer therapeutics (49), with nuclear receptors offering an important target (50). 

Estrogen chimera (E2-PROTACs), first described in 2005 (51), have recently been reported to bind both GPER and ER with relatively high affinity (∼30 nM and 10–20 nM, respectively), resulting in the degradation of ERα/β as well as GPER in MCF7 and SKBR3 cell lines without affecting progesterone receptor levels (52). PROTACs provide an alternative approach for targeting plasma membrane and intracellular estrogen receptors that could enable receptor-selective degradation based on selective receptor ligands.

Xenoestrogens as GPER Ligands

There is growing recognition of the role of GPER in endocrine disruption through exposure to natural and synthetic xenoestrogens originating from dietary intake, health and nutritional supplements, and environmental exposures to agrochemicals and industrial compounds, including polymers and their degradation products. 

The number of recognized xenoestrogens is staggering, and while previous studies have primarily focused on ERα/β, many of these compounds activate GPER, with possible consequences on neurogenic processes (53), and cancers of the breast (54), prostate (55), and digestive system (56). The breadth of possible GPER ligands was made apparent by a study that virtually screened a database of 30,926 natural products and identified 500 compounds, representing diverse structural classes that included flavonoids, isoflavonoids, chalcones, coumestans, stilbenes, lignans, ginsenosides, and tetrahydrofurandiols (57). The presence of a phenol and hydrophobic scaffold is a characteristic feature of many xenoestrogen compounds.

The isoflavones genistein and daidzein are phytoestrogens that are widely consumed directly from soy products and often taken as medicinal supplements with the intent of easing menopausal symptoms, improving metabolism, reducing cardiovascular disease, or preventing certain hormone-related cancers (Figure 2). These compounds possess a phenol group at the 3-position of the 4H-chromen-4-one core and bind to GPER with high affinity. The crystalline sodium salt dihydrate of genistein, designated AXP107-11, sensitized gemcitabine chemotherapy in pancreatic ductal adenocarcinoma patient-derived xenografts synergistically through activation of GPER and mitogen-activated protein kinase (MAPK) signaling (58). 、

GPER has been implicated in studies demonstrating that genistein attenuates inflammation in a model of Parkinson’s disease, inhibiting microglial activation and protecting dopaminergic neurons (59); protects against oxidative stress in hepatocytes (60); and improves glucose tolerance and white adipose tissue thermogenesis (61). 

Daidzein is converted to S-(−)-equol by mammalian gut bacteria, and individual metabolic variations result in widely-ranging exposures. S-equol targeted GPER to promote glucose-induced insulin secretion from pancreatic β cells and prevented glucagon-like peptide-1 secretion from enteroendocrine L cells (62); activated GPER signaling, with effects on vascular smooth muscle cells (63); inhibited nitric oxide production and reduced expression of inducible NO synthase in lipopolysaccharide-stimulated astrocytes (64); and induced cell proliferation and migration in astrocytes that were attenuated by the GPER antagonist G15 but not by the SERD/GPER agonist fulvestrant (65). Genistein, daidzein, and S-(−)-equol increased glial cell migration through activation of GPER signaling, and molecular docking studies suggest that these three compounds may bind to GPER at the same position as E2 (65).

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