ResearchPad - g-protein-coupled-receptor-signaling-in-endocrine-systems:-novel-mechanisms-in-health-and-disease https://www.researchpad.co Default RSS Feed en-us © 2020 Newgen KnowledgeWorks <![CDATA[OR24-07 Fetal Sex Impacts First Trimester Maternal-Fetal Communication in Humans]]> https://www.researchpad.co/article/elastic_article_8549 The placenta serves as a regulator of fetal growth throughout pregnancy. Signaling at the maternal-fetal interface is critical during placentation and lays the groundwork for placenta function, affecting pregnancy outcomes. Fetal growth is impacted by fetal sex, with males larger than females, and maternal gestational diabetes and obesity independently increase the risk of macrosomia in male fetuses only. We previously demonstrated differentially expressed genes (DEGs) among sexes involves ancient canonical pathways and metabolic functions in placenta tissue. As these are likely impacted by signaling at the maternal-fetal interface, our aim here was to identify sex differences in signaling at the maternal-fetal interface and among individual cell types within the placenta to explain these differences. RNA-sequencing of first trimester placenta and maternal decidua as well as single cell RNA-sequencing in first trimester placenta was performed in ongoing pregnancies. We identified 91 sexually dimorphic receptor-ligand pairs across the maternal-fetal interface. From these, 35 of 115 receptors and/or ligand genes were also found to be upstream regulators of pathways critical in sexually dimorphic placentation which may define regulation. Single cell analysis identified five major cell types (trophoblasts, stromal cells, hofbauer cells, antigen presenting cells, and endothelial cells), and all had sexually dimorphic genes. Among individual cell types, ligands from the CC-family of cytokines were most highly representative in females, with their corresponding receptors present on the maternal surface. Furthermore, upstream regulator analysis of sexually dimorphic genes demonstrated TGFβ1 and estradiol to significantly affect all cell types. Dihydrotestosterone, which is produced by the male fetus, was an upstream regulator that was most significant for the trophoblast population. In addition, gene ontology enrichment analysis identified distinctive enriched functions between male and female trophoblasts, with cytokine mediated signaling pathways most representative. MUC15 and NOTUM were the most highly expressed sexually dimorphic autosomal genes found in distinct cell types of the trophoblast population, cell types critical for placentation and nutrient exchange. Thus, differences in hormone and immune signaling pathways may account for differential gene expression and differences in trophoblast function during placentation, which may in turn explain developmental differences, including fetal size, well-being, and overall outcomes.

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<![CDATA[OR24-04 Ovarian Follicle Survival Is Determined by Follicle-Stimulating Hormone Receptor (FSHR) and Estrogen Receptor (GPER) Heteromers]]> https://www.researchpad.co/article/elastic_article_7191 Mechanisms regulating the selection of antral ovarian follicles are poorly understood and supposed to rely on low estrogen levels, decline of follicle-stimulating hormone (FSH) levels and receptor (FSHR) expression. These concepts are challenged in vitro, where apoptosis of human granulosa cells (hGLC) and transfected cell lines is induced by high doses of FSH or FSHR overexpression, while estrogens induce anti-apoptotic signals via nuclears and a G protein-coupled estrogen receptor (GPER). Therefore, in vitro data suggest that antral follicle selection may be driven by underestimated, FSH/FSHR-dependent apoptotic signals due to transiently maximized FSHR expression and overload of cAMP signalling, prevailing on estrogen-dependent signals. Here we demonstrate how FSHR/GPER physical interaction rescue ovarian follicles from FSH-mediated death. 10 nM FSH induces high intracellular levels of cAMP, measured by bioluminescence resonance energy transfer (BRET), and apoptosis in cultured hGLC under conditions where GPER levels are depleted by siRNA. This result was confirmed in transfected HEK293 cells overexpressing FSHR. Using BRET, photo-activated localization microscopy (PALM) and bioinformatics prodiction, we also demonstrate FSHR/GPER heteromers at the cell surface. The role of FSHR/GPER heteromers may be relevant to inhibit FSH-induced death signals, since increasing GPER expression levels in HEK293 cells co-expressing FSHR results in displacement of the Gαs-protein to FSHR, blockade of FSH-induced cAMP production and inhibition of apoptosis. However, in HEK293 cells coexpressing GPER/FSHR, FSH-induced activation of the anti-apoptotic AKT-pathway via a Gβγ-dependent mechanism, as demonstrated by Western blotting in cells treated using the inhibitor gallein. Inhibition of both FSH-induced cAMP production and apoptosis was lost when FSHR is coexpressed together with a mutant GPER, unable to heteromerize with FSHR, as well as in KO HEK293 cells unable to produce a molecular complex associated with GPER inhibiting cAMP. GPER/FSHR coexpression is confirmed in secondary follicles from paraffin-embedded tissues of human ovary by immunohistochemistry, suggesting that FSHR-GPER heterodimers could be physiologically relevant in vivo for inhibiting cAMP-linked apoptosis. Most importantly, FSHR and GPER co-expression correlates in hGLC from FSH-normo-responder women undergoing assisted reproduction, while it is not in hGLC from FSH-poor-responders, where increasing FSHR mRNA levels do not correspond to increasing GPER mRNA levels. We demonstrate that death signals in atretic follicles are delivered through overexpressed FSHR and inhibited by FSHR/GPER heteromerization, activating anti-apoptotic pathways. This finding unveils a novel working model of the physiology of dominant follicle selection and the relationship between FSH and estrogens.

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<![CDATA[OR24-06 USP8 Genetic Variants May Contribute to the Development of Bilateral Adrenal Hyperplasia and ACTH-Independent Cushing Syndrome]]> https://www.researchpad.co/article/elastic_article_6510 Background: Bilateral adrenocortical hyperplasias (BAHs), including primary pigmented nodular adrenocortical disease (PPNAD), isolated micronodular adrenocortical disease (iMAD) and primary macronodular adrenocortical hyperplasia (PMAH), are rare causes of ACTH-independent Cushing syndrome (CS). PPNAD and iMAD usually present in children or adolescents as multiple small (<1cm), cortisol-producing adrenocortical nodules. On the other hand, PMAH is most frequently identified in older patients with multiple large adrenal nodules. Most patients with PPNAD have PRKAR1A mutations whereas patients with PMAH may harbor variants in other genes (ARMC5, MC2R, GNAS, APC, MEN1). Even though several genes have been associated with ACTH-independent CS, there are still cases that the genetic cause has not been elucidated.

Clinical cases: Herein, we present two unrelated patients with ACTH-independent CS that harbor USP8 gene variants. USP8 is mainly known for being mutated in Cushing disease but as a deubiquitinase it may be involved into the Wnt/β-Catenin signaling pathway.

The first patient was diagnosed with BAH on prenatal ultrasound (26 gestational week) and subsequently required bilateral adrenalectomy for CS as she had virilization, hirsutism, hypertension and cardiac hypertrophy 9 weeks old. Adrenalectomy revealed that she had iMAD. She also presented with hemihypertrophy of the right leg, labia and mild newborn hypoglycemia, however she was negative for Beckwith-Wiedemann mutation. Gene analysis of PRKAR1A did not reveal any mutations. After whole exome sequencing (WES), we found a novel heterozygous USP8 variant (c.1387_1393delinsT, p.Ala463_Ile465delinsPhe) at germline level and loss of heterozygosity (LOH) at tumor level. Immunohistochemistry showed significantly lower expression of USP8 protein in both of her adrenals compared to a control tissue.

The second case is a 59-year old female with osteoporosis who failed to suppress cortisol levels after low dose dexamethasone administration. MRI revealed an adenoma on the right adrenal (2.6cm). She underwent right adrenalectomy and was found to have PMAH. We performed WES in germline level and we detected a novel heterozygous missense USP8 variant (c.287A>G, p.Lys96Arg) that is present also at tumor level. Immunohistochemistry showed significantly lower expression of USP8 protein in her adrenal tumor compared to the control tissue. No LOH was identified.

Conclusion: This is the first report of the association of USP8 in ACTH-independent CS and the preliminary findings support UPS8 involvement in the development of adrenocortical disease. We are currently performing further in vitro studies to evaluate the effect of these two USP8 variants into the canonical Wnt pathway which is commonly involved in adrenocortical disorders.

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<![CDATA[OR24-03 GRK2 Mediates Beta-Adrenergic Receptor Crosstalk to Enhanced Adrenocortical AngII-Dependent Aldosterone Production]]> https://www.researchpad.co/article/elastic_article_6115 Aldosterone is produced by adrenocortical zona glomerulosa (AZG) cells in response to hyperkalemia or angiotensin II (AngII) acting through its type I receptors (AT1Rs). AT1R is a G protein-coupled receptor (GPCR) that induces aldosterone synthesis and secretion via both G proteins and the GPCR adapter proteins βarrestins. AZG cells express all three subtypes of β-adrenergic receptor (AR) and respond to catecholamines by producing aldosterone. Being GPCRs, both activated βARs and AT1Rs are phosphorylated by GPCR-kinases (GRKs), followed by βarrestin binding to initiate G protein-independent signaling. Herein, we investigated whether the major adrenal GRKs, GRK2 and GRK5, are involved in catecholaminergic regulation of AngII-dependent aldosterone production. We used the human AZG cell line H295R, in which we measured aldosterone secretion via ELISA and synthesis via real-time PCR for steroidogenic acute regulatory (StAR) protein and CYP11B2 (aldosterone synthase) mRNA levels. Isoproterenol (Iso, a βAR full agonist) treatment significantly augmented AngII-dependent aldosterone synthesis (2.2+0.8-fold CYP11B2 & 1.6+0.5-fold StAR mRNA inductions over AngII alone; p<0.05, n=4), as well as secretion (2.3+0.8-fold of vehicle with Iso; 3.2+1.1-fold of vehicle with AngII; 7.4+1.1-fold of vehicle with Iso+AngII, p<0.05 vs. either agent alone; n=5) in H295R cells. Importantly, GRK2, but not the other major GRK isoform expressed in human adrenals GRK5, was indispensable for the catecholamine-mediated enhancement of aldosterone production in response to AngII in H295R cells. Specifically, GRK2 inhibition with the small molecule Cmpd101 abolished Iso effects on AngII-induced aldosterone synthesis and secretion (Iso+AngII-induced aldosterone secretion: 8.1+2.3-fold of vehicle without Cmpd101; 2.8+0.8-fold of vehicle with Cmpd101; p<0.05, n=5). In contrast, GRK5 knockout via CRISPR/Cas9 did not affect the synergism between isoproterenol and AngII in stimulating aldosterone production. Mechanistically, βAR-activated GRK2, but not GRK5, phosphorylated and activated the Ca2+-activated chloride channel anoctamine-1 (ANO1), also known as transmembrane member (TMEM)16A, ultimately increasing aldosterone production in H295R cells (Iso+10–6 M [Ca2+]-induced ANO1 activity of Cmpd101-pretreated cells: 55+15 % of non-Cmpd101-pretreated cells; p<0.05, n=5). AngII alone failed to stimulate GRK2 in H295R cells. In conclusion, GRK2 mediates a βAR-AT1R signaling crosstalk at the level of ANO1 activation, which results in enhanced aldosterone production in H295R cells. This finding suggests that adrenal GRK2 may be a molecular link connecting the sympathetic nervous and renin-angiotensin systems in the adrenal cortex and that GRK2 inhibition might be therapeutically advantageous for aldosterone suppression.

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<![CDATA[OR24-05 Identifying Regulatory Elements Within a Novel Enhancer of FSHB Containing Two PCOS-Associated Single Nucleotide Polymorphisms]]> https://www.researchpad.co/article/N76f98966-d7d9-432d-baa5-2788f1edbdcb <![CDATA[OR24-01 Mutational Study of the GPR119 Receptor Binding Site]]> https://www.researchpad.co/article/N12f9ad64-e20f-4656-975e-908acf4a6d38 <![CDATA[OR24-02 Identification of Membrane Proteins That Enhance the Responsiveness of the Ghrelin Receptor]]> https://www.researchpad.co/article/Nb208dcdf-cc56-4cbf-bf62-fcd988c79baf