G-36

Male exposure to bisphenol a impairs spermatogenesis and triggers histone hyperacetylation in zebrafish testes.[Pubmed:30818116]

Environ Pollut. 2019 Feb 8;248:368-379.

Bisphenol A (BPA) is an endocrine disruptor whose ubiquitous presence in the environment has been related with impairment of male reproduction. BPA can cause both transcriptomic and epigenetic changes during spermatogenesis. To evaluate the potential effects of male exposure to BPA, adult zebrafish males were exposed during spermatogenesis to doses of 100 and 2000mug/L, which were reported in contaminated water bodies and higher than those allowed for human consumption. Fertilization capacity and survival at hatching were analysed after mating with untreated females. Spermatogenic progress was analysed through a morphometrical study of testes and apoptosis was evaluated by TUNEL assay. Testicular gene expression was evaluated by RT-qPCR and epigenetics by using ELISA and immunocytochemistry. In vitro studies were performed to investigate the role of Gper. Chromatin fragmentation and the presence of transcripts were also evaluated in ejaculated sperm. Results on testes from males treated with the highest dose showed a significant decrease in spermatocytes, an increase in apoptosis, a downregulation of ccnb1 and sycp3, all of which point to an alteration of spermatogenesis and to meiotic arrest and an upregulation of gper1 and esrrga receptors. Additionally, BPA at 2000mug/L caused missregulation of epigenetic remodelling enzymes transcripts in testes and promoted DNA hypermethylation and H3K27me3 demethylation. BPA also triggered an increase in histone acetyltransferase activity, which led to hyperacetylation of histones (H3K9ac, H3K14ac, H4K12ac). In vitro reversion of histone acetylation changes using a specific GPER antagonist, G-36, suggested this receptor as mediator of histone hyperacetylation. Males treated with the lower dose only showed an increase in some histone acetylation marks (H3K14ac, H4K12ac) but their progeny displayed very limited survival at hatching, revealing the deleterious effects of unbalanced paternal epigenetic information. Furthermore, the highest dose of BPA led to chromatin fragmentation, promoting direct reproductive effects, which are incompatible with embryo development.

Selective activation of estrogen receptors, ERalpha and GPER-1, rapidly decreases food intake in female rats.[Pubmed:29807036]

Horm Behav. 2018 Jul;103:54-61.

Many of estradiol's behavioral effects are mediated, at least partially, via extra-nuclear estradiol signaling. Here, we investigated whether two estrogen receptor (ER) agonists, targeting ERalpha and G protein-coupled ER-1 (GPER-1), can promote rapid anorexigenic effects. Food intake was measured in ovariectomized (OVX) rats at 1, 2, 4, and 22h following subcutaneous (s.c.) injection of an ERalpha agonist (PPT; 0-200mug/kg), a GPER-1 agonist (G-1; 0-1600mug/kg), and a GPER-1 antagonist (G-36; 0-80mug/kg). To investigate possible cross-talk between ERalpha and GPER-1, we examined whether GPER-1 blockade affects the anorexigenic effect of PPT. Feeding was monitored in OVX rats that received s.c. injections of vehicle or 40mug/kg G-36 followed 30min later by s.c. injections of vehicle or 200mug/kg PPT. Selective activation of ERalpha and GPER-1 alone decreased food intake within 1h of drug treatment, and feeding remained suppressed for 22h following PPT treatment and 4h following G-1 treatment. Acute administration of G-36 alone did not suppress feeding at any time point. Blockade of GPER-1 attenuated PPT's rapid (within 1h) anorexigenic effect, but did not modulate PPT's ability to suppress food intake at 2, 4 and 22h. These findings demonstrate that selective activation of ERalpha produces a rapid (within 1h) decrease in food intake that is best explained by a non-genomic signaling pathway and thus implicates the involvement of extra-nuclear ERalpha. Our findings also provide evidence that activation of GPER-1 is both sufficient to suppress feeding and necessary for PPT's rapid anorexigenic effect.

The activation of G protein-coupled estrogen receptor induces relaxation via cAMP as well as potentiates contraction via EGFR transactivation in porcine coronary arteries.[Pubmed:29360846]

PLoS One. 2018 Jan 23;13(1):e0191418.

Estrogen exerts protective effects against cardiovascular diseases in premenopausal women, but is associated with an increased risk of both coronary heart disease and stroke in older postmenopausal women. Studies have shown that activation of the G-protein-coupled estrogen receptor 1 (GPER) can cause either relaxation or contraction of arteries. It is highly likely that these dual actions of GPER may contribute to the seemingly paradoxical effects of estrogen in regulating coronary artery function. The objective of this study was to test the hypothesis that activation of GPER enhances agonist-stimulated porcine coronary artery contraction via epidermal growth factor receptor (EGFR) transactivation and its downstream extracellular signal-regulated kinases (ERK1/2) pathway. Isometric tension studies and western blot were performed to determine the effect of GPER activation on coronary artery contraction. Our findings demonstrated that G-1 caused concentration-dependent relaxation of ET-1-induced contraction, while pretreatment of arterial rings with G-1 significantly enhanced ET-1-induced contraction. GPER antagonist, G-36, significantly inhibited both the G-1-induced relaxation effect and G-1-enhanced ET-1 contraction. Gallein, a Gbetagamma inhibitor, significantly increased G-1-induced relaxation, yet inhibited G-1-enhanced ET-1-mediated contraction. Similarly, inhibition of EGFR with AG1478 or inhibition of Src with phosphatase 2 further increased G-1-induced relaxation responses in coronary arteries, but decreased G-1-enhanced ET-1-induced contraction. Western blot experiments in porcine coronary artery smooth muscle cells (PCASMC) showed that G-1 increased tyrosine phosphorylation of EGFR, which was inhibited by AG-1478. Furthermore, enzyme-linked immunosorbent assays showed that the level of heparin-binding EGF (HB-EGF) released by ET-1 treatment increased two-fold; whereas pre-incubation with G-1 further increased ET-1-induced HB-EGF release to four-fold over control conditions. Lastly, the role of ERK1/2 was determined by applying the MEK inhibitor, PD98059, in isometric tension studies and detecting phospho-ERK1/2 in immunoblotting. PD98059 potentiated G-1-induced relaxation response, but blocked G-1-enhanced ET-1-induced contraction. By western blot, G-1 treatment decreased phospho-ERK1/2, however, in the presence of the adenylyl cyclase inhibitor, SQ22536, G-1 significantly increased ERK1/2 phosphorylation in PCASMC. These data demonstrate that activation of GPER induces relaxation via cAMP as well as contraction via a mechanism involving transactivation of EGFR and the phosphorylation of ERK1/2 in porcine coronary arteries.

Identification of a GPER/GPR30 antagonist with improved estrogen receptor counterselectivity.[Pubmed:21782022]

J Steroid Biochem Mol Biol. 2011 Nov;127(3-5):358-66.

GPER/GPR30 is a seven-transmembrane G protein-coupled estrogen receptor that regulates many aspects of mammalian biology and physiology. We have previously described both a GPER-selective agonist G-1 and antagonist G15 based on a tetrahydro-3H-cyclopenta[c]quinoline scaffold. The antagonist lacks an ethanone moiety that likely forms important hydrogen bonds involved in receptor activation. Computational docking studies suggested that the lack of the ethanone substituent in G15 could minimize key steric conflicts, present in G-1, that limit binding within the ERalpha ligand binding pocket. In this report, we identify low-affinity cross-reactivity of the GPER antagonist G15 to the classical estrogen receptor ERalpha. To generate an antagonist with enhanced selectivity, we therefore synthesized an isosteric G-1 derivative, G36, containing an isopropyl moiety in place of the ethanone moiety. We demonstrate that G36 shows decreased binding and activation of ERalpha, while maintaining its antagonist profile towards GPER. G36 selectively inhibits estrogen-mediated activation of PI3K by GPER but not ERalpha. It also inhibits estrogen- and G-1-mediated calcium mobilization as well as ERK1/2 activation, with no effect on EGF-mediated ERK1/2 activation. Similar to G15, G36 inhibits estrogen- and G-1-stimulated proliferation of uterine epithelial cells in vivo. The identification of G36 as a GPER antagonist with improved ER counterselectivity represents a significant step towards the development of new highly selective therapeutics for cancer and other diseases.

The G-protein-coupled estrogen receptor GPER in health and disease.[Pubmed:21844907]

Nat Rev Endocrinol. 2011 Aug 16;7(12):715-26.

Estrogens mediate profound effects throughout the body and regulate physiological and pathological processes in both women and men. The low prevalence of many diseases in premenopausal women is attributed to the presence of 17beta-estradiol, the predominant and most potent endogenous estrogen. In addition to endogenous estrogens, several man-made and plant-derived molecules, such as bisphenol A and genistein, also exhibit estrogenic activity. Traditionally, the actions of 17beta-estradiol are ascribed to two nuclear estrogen receptors (ERs), ERalpha and ERbeta, which function as ligand-activated transcription factors. However, 17beta-estradiol also mediates rapid signaling events via pathways that involve transmembrane ERs, such as G-protein-coupled ER 1 (GPER; formerly known as GPR30). In the past 10 years, GPER has been implicated in both rapid signaling and transcriptional regulation. With the discovery of GPER-selective ligands that can selectively modulate GPER function in vitro and in preclinical studies and with the use of Gper knockout mice, many more potential roles for GPER are being elucidated. This Review highlights the physiological roles of GPER in the reproductive, nervous, endocrine, immune and cardiovascular systems, as well as its pathological roles in a diverse array of disorders including cancer, for which GPER is emerging as a novel therapeutic target and prognostic indicator.