Demonstration of follicle-stimulating hormone receptor and G protein-coupled estrogen receptor heteromers in vitro via BRET and super-resolution imaging

Secoisolariciresinol diglucoside (SDG), a lignan extracted from flaxseed, has been shown to suppress benign prostatic hyperplasia (BPH). However, little is known about the mechanistic basis for its anti-BPH activity. The present study showed that enterolactone (ENL), the mammalian metabolite of SDG, shared the similar binding site of G1 on a new type of membranous estrogen receptor, G-protein-coupled estrogen eceptor 1 (GPER), by docking simulations method. ENL and G1 (the specific agonist of GPER) inhibited the proliferation of human prostate stromal cell line WPMY-1 as shown by MTT assay and arrested cell cycle at the G0/G1 phase, which was displayed by propidium iodide staining following flow cytometer examination. Silencing GPER by short interfering RNA attenuated the inhibitory effect of ENL on WPMY-1 cells. The therapeutic potential of SDG in the treatment of BPH was confirmed in a testosterone propionate-induced BPH rat model. SDG significantly reduced the enlargement of the rat prostate and the number of papillary projections of prostatic alveolus and thickness of the pseudostratified epithelial and stromal cells when comparing with the model group. Mechanistic studies showed that SDG and ENL increased the expression of GPER both in vitro and in vivo. Furthermore, ENL-induced cell cycle arrest may be mediated by the activation of GPER/ERK pathway and subsequent upregulation of p53 and p21 and downregulation of cyclin D1. This work, in tandem with previous studies, will enhance our knowledge regarding the mechanism(s) of dietary phytochemicals on BPH prevention and ultimately expand the scope of adopting alternative approaches in BPH treatment.

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Discovery and Preclinical Development of Orally Active Small Molecules that Exhibit Highly Selective Follicle Stimulating Hormone Receptor Agonism

Frontiers in Pharmacology ◽

10.3389/fphar.2020.602593 ◽

2021 ◽

Vol 11 ◽

Author(s):

Selva Nataraja ◽

Henry Yu ◽

Joie Guner ◽

Stephen Palmer

Keyword(s):

Ovarian Stimulation ◽

Hormone Receptor ◽

Ovulation Induction ◽

Low Dose ◽

Intrauterine Insemination ◽

Follicle Stimulating Hormone ◽

Controlled Ovarian Stimulation ◽

Orally Active ◽

Stimulating Hormone

An orally active follicle stimulating hormone receptor allosteric agonist would provide a preferred treatment for over 16 million infertile women of reproductive age in low complexity methods (ovulation induction-intrauterine insemination) or in high complexity methods (controlled ovarian stimulation-in vitro fertilization). We present two oral follicle stimulating hormone receptor allosteric agonist compounds that have the desired pharmacology, drug metabolism, pharmacokinetics, and safety profile for clinical use. These molecules provide a single agent suitable for ovulation induction-intrauterine insemination or controlled ovarian stimulation-in vitro fertilization that is more convenient for patients and achieves similar preclinical efficacy as rec-hFSH. TOP5668, TOP5300 were evaluated in vitro in Chinese hamster ovary cells transfected with individual glycoprotein receptors measuring cAMP (FSHR, LH/CGR, thyroid stimulating hormone receptor). TOP5668 was found to have solely follicle stimulating hormone receptor allosteric agonist activity while TOP5300 was found to have mixed follicle stimulating hormone receptor allosteric agonist and LHR-AA activity. Both compounds stimulated concentration-dependent increases in estradiol production from cultured rat granulosa cells in the presence or absence of low dose rec-hFSH, while only TOP5300 stimulated testosterone production from rat primary Leydig cells. In pooled human granulosa cells obtained from patients undergoing controlled ovarian stimulation-in vitro fertilization, TOP5300 stimulated 7-fold greater maximal estradiol response than rec-hFSH and TOP5668 was 10-fold more potent than TOP5300. Both TOP5300 and TOP5668 stimulated follicular development in immature rat to the same efficacy as recombinant follicle stimulating hormone. In mice treated with TOP5300, in the presence of low dose of follicle stimulating hormone, there were no differences in oocyte number, fertilization rate, and hatched blastocyst rate in mice with TOP5300 and low dose follicle stimulating hormone vs. reference proteins pregnant mare serum gonadotropin or high dose rec-hFSH. ADME/PK and safety profiles were favorable. In addition, there was no appreciable activity on thyroid hormones by TOP5300 in 14-days toxicological study in rat or dog. The selected lead compound, TOP5300 stimulated a more robust increase in estradiol production from granulosa-lutein cells from women with polycystic ovarian syndrome patient compared to rec-hFSH. Conclusions: Two novel oral FSHR allosteric agonist, TOP5668 and TOP5300, were found to mimic the biological activity of rec hFSH in preclinical studies. Both compounds led to folliculogenesis and superovulation in rat and mice. Specifically, TOP5300 led to a similar number of ovulated oocytes that fertilized and developed into hatched blastocysts in mice when compared to rec-hFSH. The safety profile demonstrated lack of toxicity.

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Association of estrogen, progesterone and follicle stimulating hormone receptor polymorphisms with in vitro fertilization outcomes

Systems Biology in Reproductive Medicine ◽

10.1080/19396368.2018.1482030 ◽

2018 ◽

Vol 64 (4) ◽

pp. 260-265 ◽

Cited By ~ 3

Author(s):

Vijaya Ganesh ◽

Vettriselvi Venkatesan ◽

Teena Koshy ◽

Sanjeeva Nellapalli Reddy ◽

Suruli Muthumuthiah ◽

...

Keyword(s):

In Vitro Fertilization ◽

Hormone Receptor ◽

Follicle Stimulating Hormone ◽

Vitro Fertilization ◽

Stimulating Hormone

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G Protein-Coupled Estrogen Receptor 1 Regulates Human Neutrophil Functions

Biomedicine Hub ◽

10.1159/000454981 ◽

2017 ◽

Vol 2 (1) ◽

pp. 1-13 ◽

Cited By ~ 12

Author(s):

M. Carmen Rodenas ◽

Nicola Tamassia ◽

Isabel Cabas ◽

Federica Calzetti ◽

José Meseguer ◽

...

Keyword(s):

Gene Expression ◽

Estrogen Receptor ◽

Protein Kinase ◽

G Protein ◽

Respiratory Burst ◽

Human Neutrophils ◽

G Protein Coupled ◽

Estrogen Receptor 1

Background: The role of estrogens in immune functioning is relatively well known under both physiological and pathological conditions. Neutrophils are the most abundant circulating leukocytes in humans, and their abundance and function are regulated by estrogens, since they express estrogen receptors (ERs). Traditionally, estrogens were thought to act via classical nuclear ERs, namely ERα and ERβ. However, it was observed that some estrogens induced biological effects only minutes after their application. This rapid, “nongenomic” effect of estrogens is mediated by a membrane-anchored receptor called G protein-coupled estrogen receptor 1 (GPER1). Nevertheless, the expression and role of GPER1 in the immune system has not been exhaustively studied, and its relevance in neutrophil functions remains unknown. Methods: Human neutrophils were incubated in vitro with 10-100 µM of the GPER1-specific agonist G1 alone or in combination with lipopolysaccharide. GPER1 expression and subcellular localization, respiratory burst, life span, gene expression profile, and cell signaling pathways involved were then analyzed in stimulated neutrophils. Results: Human neutrophils express a functional GPER1 which regulates their functions through cAMP/protein kinase A/cAMP response element-binding protein, p38 mitogen-activated protein kinase, and extracellular regulated MAPK signaling pathways. Thus, GPER1 activation in vitro increases the respiratory burst of neutrophils, extends their life span, and drastically alters their gene expression proﬁle. Conclusions: Our results demonstrate that GPER1 activation promotes the polarization of human neutrophils towards a proinflammatory phenotype and point to GPER1 as a potential therapeutic target in immune diseases where neutrophils play a key role.

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A common Asn680Ser polymorphism in the follicle-stimulating hormone receptor gene is not associated with ovarian response to gonadotropin stimulation in patients undergoing in vitro fertilization

Fertility and Sterility ◽

10.1016/j.fertnstert.2012.08.037 ◽

2013 ◽

Vol 99 (1) ◽

pp. 149-155 ◽

Cited By ~ 23

Author(s):

Lamiya Mohiyiddeen ◽

William G. Newman ◽

Christian Cerra ◽

Helen McBurney ◽

Betselot Mulugeta ◽

...

Keyword(s):

In Vitro Fertilization ◽

Hormone Receptor ◽

Receptor Gene ◽

Follicle Stimulating Hormone ◽

Ovarian Response ◽

Vitro Fertilization ◽

Gonadotropin Stimulation ◽

Hormone Receptor Gene ◽

Stimulating Hormone

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Membrane estrogen receptor (GPER) and follicle-stimulating hormone receptor heteromeric complexes promote human ovarian follicle survival

10.1101/2020.04.21.053348 ◽

2020 ◽

Author(s):

Livio Casarini ◽

Clara Lazzaretti ◽

Elia Paradiso ◽

Silvia Limoncella ◽

Laura Riccetti ◽

...

Keyword(s):

Estrogen Receptor ◽

Cell Viability ◽

Oocyte Maturation ◽

Granulosa Cells ◽

Hormone Receptor ◽

Ovarian Follicle ◽

Follicle Stimulating Hormone ◽

Oocyte Growth ◽

Expression Levels ◽

Stimulating Hormone

AbstractClassically, follicle stimulating hormone receptor (FSHR) driven cAMP-mediated signaling boosts human ovarian follicle growth and would be essential for oocyte maturation. However, contradicting in vitro suggest a different view on physiological and clinical significance of FSHR-mediated cAMP signaling. We found that the G protein coupled estrogen receptor (GPER) heteromerizes with FSHR, reprogramming cAMP/death signals into proliferative stimuli fundamental for sustaining oocyte survival. In human granulosa cells, survival signals are effectively delivered upon equal expression levels of both receptors, while they are missing at high FSHR:GPER ratio, which negatively impacts follicle maturation and strongly correlates with FSH responsiveness of patients undergoing controlled ovarian stimulation. Consistent with high FSHR expression levels during follicular selection, cell viability is dramatically reduced in FSHR overexpressing cells due to preferential coupling to the Gαs protein/cAMP pathway. In contrast, FSHR/GPER heteromer formation resulted in FSH-triggered anti-apoptotic/proliferative signaling delivered via the Gβγ dimer while heteromer impairment or GPER-associated Gαs inhibitory protein complexes resulted in cell death. GPER-depleted granulosa cells have an amplified FSH-dependent decrease in cell viability and steroidogenesis, consistent with the requirement of estrogen signaling for successful oocyte growth. Therefore, our findings indicate how oocyte maturation depends on the capability of GPER to shape FSHR selective signals, indicating hormone receptor heteromers may be a marker of cell proliferation.One Sentence SummaryFSHR/GPER heteromers block cAMP-dependent selection of ovarian follicles and target tumor growth and poor FSH-response in women.

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