Nonclassical Estrogen Receptor Research Articles

GPR30-mediated non-classic estrogen pathway in mast cells participates in endometriosis pain via the production of FGF2.

Pain is one of the main clinical symptoms of endometriosis, but its underlying mechanism is still not clear. Recent studies have shown that the secretory mediators of mast cells activated by estrogen are involved in the pathogenesis of endometriosis-related pain, but how estrogen-induced mast cell mediators are involved in endometriosis-related pain remains unclear. Here, mast cells were found to be increased in the ovarian endometriotic lesions of patients. They were also closely located closely to the nerve fibers in the ovarian endometriotic lesions from of patients with pain symptoms. Moreover, fibroblast growth factor 2 (FGF2)-positive mast cells were upregulated in endometriotic lesions. The concentration of FGF2 in ascites and the protein level of fibroblast growth factor receptor 1 (FGFR1) were higher in patients with endometriosis than in those without endometriosis, and they were correlated with pain symptoms. In vitro, estrogen could promote the secretion of FGF2 through G-protein-coupled estrogen receptor 30 (GPR30) via the MEK/ERK pathway in rodent mast cells. Estrogen-stimulated mast cells enhanced the concentration of FGF2 in endometriotic lesions and aggravated endometriosis-related pain in vivo. Targeted inhibition of the FGF2 receptor significantly restrained the neurite outgrowth and calcium influx in dorsal root ganglion (DRG) cells. Administration of FGFR1 inhibitor remarkably elevated the mechanical pain threshold (MPT) and prolonged the heat source latency (HSL) in a rat model of endometriosis. These results suggested that the up-regulated production of FGF2 by mast cells through non-classic estrogen receptor GPR30 plays a vital role in the pathogenesis of endometriosis-related pain.

Differential distribution of steroid hormone signaling networks in the human choroid-retinal pigment epithelial complex

BackgroundThe retinal pigment epithelium (RPE), a layer of pigmented cells that lies between the neurosensory retina and the underlying choroid, plays a critical role in maintaining the functional integrity of photoreceptor cells and in mediating communication between the neurosensory retina and choroid. Prior studies have demonstrated neurotrophic effects of select steroids that mitigate the development and progression of retinal degenerative diseases via an array of distinct mechanisms of action.MethodsHere, we identified major steroid hormone signaling pathways and their key functional protein constituents controlling steroid hormone signaling, which are potentially involved in the mitigation or propagation of retinal degenerative processes, from human proteome datasets with respect to their relative abundances in the retinal periphery, macula, and fovea.ResultsAndrogen, glucocorticoid, and progesterone signaling networks were identified and displayed differential distribution patterns within these three anatomically distinct regions of the choroid-retinal pigment epithelial complex. Classical and non-classical estrogen and mineralocorticoid receptors were not identified.ConclusionIdentified differential distribution patterns suggest both selective susceptibility to chronic neurodegenerative disease processes, as well as potential substrates for drug target discovery and novel drug development focused on steroid signaling pathways in the choroid-RPE.

Factors Influencing Skin Aging and the Important Role of Estrogens and Selective Estrogen Receptor Modulators (SERMs)

The narrative for this overview focuses on updating the factors that influence skin aging and the important role estrogens and selective estrogen receptor modulators (SERMs) play in this process (mainly utilizing journal reports and reviews from the last four years). Estrogens have been known and studied for over a century. For many years, it has been recognized that estrogens are important in the maintenance of human skin. Women seek cosmetic and medical treatments to improve dermal health and physical characteristics to enhance their self-perception and inhibit skin aging, particularly in highly visible body areas. The goal: to retain estrogen’s positive benefits while aging and especially at/after menopause where estrogen-deficient skin contributes to the dramatic decline in skin health. In this overview, both background information and recent novel findings are included that cover aging (general mechanisms), skin aging, and factors that influence skin aging (intrinsic, extrinsic, skin microbiome and gut microbiome.) Plus, estrogen’s general role in maintaining skin health is presented through the classical estrogen receptors alpha (α) and beta (β) and non-classical (or non-genomic) estrogen receptor (G protein-coupled seven transmembrane receptor). More importantly, the various benefits of 17β-estradiol in skin health are examined (ie, skin collagen and elastin profiles that follow 17β-estradiol levels during aging and at/after menopause). Finally, a revision of information for estrogenic skin topical applications involving isoflavonoid compounds that act as SERMs, but are classified as endocrine disruptors, and a topical estrogen analog are explored to update the known and unknown characteristics of these treatments. Further study is warranted to understand the biological and molecular mechanisms by which estrogens support and enhance dermal health and wellbeing.

The sex steroid precursor dehydroepiandrosterone prevents nonalcoholic steatohepatitis by activating the AMPK pathway mediated by GPR30

The prevalence of nonalcoholic steatohepatitis (NASH) caused by estrogen deficiency increased sharply in recent decades and has become a major threat to liver health in postmenopausal women. There is no effective strategy to control the incidence and development of NASH. Dehydroepiandrosterone (DHEA) is the most abundant circulating steroid with immune and metabolic regulatory properties, and its level markedly declines with increasing age in humans. Importantly, DHEA can convert into active sex hormones depending on the local needs of target tissues with little diffusion, which serves to avoid systemic side-effects from other tissues’ exposure to estrogen. Here, we found that DHEA prevented the incidence and development of NASH, which is characterized by the reduction of hepatic steatosis, fibrosis, and inflammation in female mice fed with high-fat/high-cholesterol diets and effectively attenuated lipid accumulation, inflammatory response, and oxidative stress in palmitic acid-challenged hepatocytes. Mechanistically, in vitro and in vivo studies showed that the anti-NASH function of DHEA depended on its biotransformation into estrogen rather than androgen, and which up-regulates the expression of G protein-coupled estrogen receptor (GPR30), a non-classical estrogen receptor. The activation of GPR30-mediated AMP-activated protein kinase signaling is a necessary prerequisite for the alleviative effects of DHEA on NASH. Collectively, our data show the mechanisms of DHEA treatment and its effects on NASH that were previously overlooked; the data also show that GPR30 can be used as a target for treating lipid metabolism disorders and related diseases, such as NASH. Furthermore, these findings have the potential to help researchers develop new strategies for preventing NASH in postmenopausal women.

Expression and function of GPR30 in human chondrocytes

Purpose: Estrogens play an important role in cartilage homeostasis. However, the role of a non-classical estrogen receptor, the G protein-coupled estrogen receptor (GPER or GPR30), as a mediator of the effects of estrogens in articular tissues is unknown. Thus, the purpose of this work was to evaluate the expression and function of GPR30 in primary adult human articular chondrocytes.

The Impact of Estrogen and Estrogen-Like Molecules in Neurogenesis and Neurodegeneration: Beneficial or Harmful?

Estrogens and estrogen-like molecules can modify the biology of several cell types. Estrogen receptors alpha (ERα) and beta (ERβ) belong to the so-called classical family of estrogen receptors, while the G protein-coupled estrogen receptor 1 (GPER-1) represents a non-classical estrogen receptor mainly located in the plasma membrane. As estrogen receptors are ubiquitously distributed, they can modulate cell proliferation, differentiation, and survival in several tissues and organs, including the central nervous system (CNS). Estrogens can exert neuroprotective roles by acting as anti-oxidants, promoting DNA repair, inducing the expression of growth factors, and modulating cerebral blood flow. Additionally, estrogen-dependent signaling pathways are involved in regulating the balance between proliferation and differentiation of neural stem/progenitor cells (NSPCs), thus influencing neurogenic processes. Since several estrogen-based therapies are used nowadays and estrogen-like molecules, including phytoestrogens and xenoestrogens, are omnipresent in our environment, estrogen-dependent changes in cell biology and tissue homeostasis have gained attention in human health and disease. This article provides a comprehensive literature review on the current knowledge of estrogen and estrogen-like molecules and their impact on cell survival and neurodegeneration, as well as their role in NSPCs proliferation/differentiation balance and neurogenesis.

The Hepatoprotective mechanisms of 17β-estradiol after traumatic brain injury in male rats: Classical and non-classical estrogen receptors

Protective effects of estrogen (E2) on traumatic brain injury (TBI) have been determined. In this study, the hepatoprotective effects of E2 after TBI through its receptors and oxidative stress regulation have been evaluated. Diffuse TBI induced by the Marmarou method in male rats. G15, PHTPP, MPP, and ICI182–780 as selective antagonists of E2 were injected before TBI. The results indicated that TBI induces a significant increase in liver enzymes [Alkaline phosphatase (ALP), Aspartate aminotransferase (AST), Alanine aminotransferase (ALT), Glutamyl transferase (GGT)], and oxidants levels [Malondialdehyde (MDA), Nitric oxide (NO)] and decreases in antioxidant biomarkers [Glutathione peroxidase (GPx) and Superoxide dismutase (SOD)] in the brain and liver, and plasma. We also found that E2 significantly preserved levels of these biomarkers and enzymatic activity. All antagonists inhibited the effects of E2 on increasing SOD and GPx. Also, the effects of E2 on brain MDA levels were inhibited by all antagonists, but in the liver, only ICI + G15 + E2 + TBI group was affected. The impacts of E2 on brain and liver and plasma NO levels were inhibited by all antagonists. The current findings demonstrated that E2 probably improved liver injury after TBI by modulating oxidative stress. Also, both classic (ERβ, ERα) and non-classic [G protein-coupled estrogen receptor (GPER)] receptors are affected in the protective effects of E2.

E2-BSA and G1 exert neuroprotective effects and improve behavioral abnormalities following traumatic brain injury: The role of classic and non-classic estrogen receptors

The role of classical and non-classical estrogen receptors (ERs) in mediating the neuroprotective effects of this hormone on brain edema and long-term behavioral disorders was evaluated after traumatic brain injury (TBI). Ovariectomized rats were divided as follows: E2 (17 β-estradiol), E2-BSA (E2 conjugated to bovine serum albumin), G1 [G-protein-coupled estrogen receptor agonist (GPER)] or their vehicle was injected following TBI, whereas ICI (classical estrogen receptor antagonist), G15 (GPER antagonist), ICI + G15, and their vehicle were injected before the induction of TBI and the injection of E2 and E2-BSA. Brain water (BWC) and Evans blue (EB) contents were measured 24 h and 5 h after TBI, respectively. Intracranial pressure (ICP) and cerebral perfusion pressure (CPP) were measured before and at different times after TBI. Locomotor activity, anxiety-like behavior, and spatial memory were assessed on days 3, 7, 14, and 21 after injury. E2, E2-BSA, and G1 prevented the increase of BWC and EB content after TBI, and these effects were inhibited by ICI and G15. ICI and G15 also inhibited the beneficial effects of E2, E2-BSA on ICP, as well as CPP, after trauma. E2, E2-BSA, and G1 prevented the cognitive deficiency and behavioral abnormalities induced by TBI. Similar to the above parameters, ICI and G15 also reversed this E2 and E2-BSA effects on days 3, 7, 14, and 21. Our findings indicated that the beneficial effects of E2-BSA and E2 were inhibited by both ICI and G15, suggesting that GPER and classic ERs were involved in mediating the long-term effects of E2.

NDV-D90 inhibits 17β-estradiol-mediated resistance to apoptosis by differentially modulating classic and nonclassic estrogen receptors in breast cancer cells.

Newcastle disease virus (NDV) is endowed with the oncolytic ability to kill tumor cells, while rarely causing side effects in normal cells. Both estrogen receptor α (ERα) and the G protein estrogen receptor (GPER) modulate multiple biological activities in response to estrogen, including apoptosis in breast cancer (BC) cells. Here, we investigated whether NDV-D90, a novel strain isolated from natural sources in China, promoted apoptosis by modulating the expression of ERα or the GPER in BC cells exposed to 17β-estradiol (E2). We found that NDV-D90 significantly killed the tumor cell lines MCF-7 and BT549 in a time- and dose-dependent manner. We also found that NDV-D90 exerted its effects on the two cell lines mainly by inducing apoptosis but not necrosis. NDV-D90 induced apoptosis via the intrinsic and extrinsic signaling pathways in MCF-7 cells (ER-positive cells) during E2 exposure not only by disrupting the E2/ERα axis and enhancing GPER expression but also by modulating the expression of several apoptosis-related proteins through ERα-and GPER-independent processes. NDV-D90 promoted apoptosis via the intrinsic signaling pathway in BT549 cells (ER-negative cells), possibly by impairing E2-mediated GPER expression. Furthermore, NDV-D90 exerted its antitumor effects in vivo by inducing apoptosis. Overall, these results demonstrated that NDV-D90 promotes apoptosis by differentially modulating the expression of ERα and the GPER in ER-positive and negative BC cells exposed to estrogen, respectively, and can be utilized as an effective approach to treating BC.

Expression and function of the nonclassical estrogen receptor, GPR30, in human cartilage and chondrocytes.

Estrogen hormones are important for cartilage homeostasis, but nothing is known regarding the expression and role of the membrane G protein-coupled estrogen receptor (GPER), G protein-coupled receptor 30 (GPR30), in adult articular chondrocytes. Using immunohistochemistry of cartilage sections, quantitative real-time polymerase chain reaction and Western blot of chondrocyte extracts, we found that these cells express GPR30. Nonetheless, the pattern of bands detected by two distinct antibodies does not overlap, suggesting that the proteins detected represent partially degraded forms of the receptor. Treatment with GPR30 agonists did not induce Akt or ERK1/2 phosphorylation, two known GPR30-activated signaling pathways, suggesting that GPR30 is not functional in human chondrocytes. Therefore, the protective anti-osteoarthritic role of estrogen hormones in cartilage homeostasis is likely independent of GPR30. This study was performed using human cartilage collected from the distal femoral condyles of multiorgan donors at the Bone and Tissue Bank of the University and Hospital Center of Coimbra.

The meaning of non-classical estrogen receptors and peroxisome proliferator-activated receptor for boar Leydig cell of immature testis

Communication in biological systems involves diverse-types of cell-cell interaction including cross-talk between receptors expressed by the target cells. Recently, novel sort of estrogen receptors (G protein - coupled estrogen receptor; GPER and estrogen-related receptor; ERR) that signal directly via estrogen binding and/or via mutual interaction-regulated estrogen signaling were reported in various organs including testis. Peroxisome proliferator - activated receptor (PPAR) is responsible for maintaining of lipid homeostasis that is critical for sex steroid production in the testis. Here, we investigated the role of interaction between GPER, ERRβ and PPARγ in steroidogenic Leydig cells of immature boar testis. Testicular fragments cultured ex vivo were treated with GPER or PPARγ antagonists. Then, cell ultrastructure, expression and localization of GPER, ERRβ, PPARγ together with the molecular receptor mechanism, through cyclic AMP and Raf/Ras/extracellular signal activated kinases (ERK), in the control of cholesterol concentration and estrogen production by Leydig cells were studied. In the ultrastructure of antagonist-treated Leydig cells, mitochondria were not branched and not bifurcated as they were found in control. Additionally, in PPARγ-blocked Leydig cells changes in the number of lipid droplets were revealed. Independent of used antagonist, western blot revealed decreased co-expression of GPER, ERRβ, PPARγ with exception of increased expression of ERRβ after PPARγ blockage. Immunohistochemistry confirmed presence of all receptors partially located in the nucleus or cytoplasm of Leydig cells of both control and treated testes. Changes in receptor expression, decreased cholesterol and increased estradiol tissue concentrations occurred through decreased cAMP level (with exception after GPER blockage) as well as Raf/Ras/ERK pathway expression. These all findings indicate that GPER-ERRβ-PPARγ interaction exists in immature boar testis and regulates Leydig cell function. Further detailed studies and considerations on GPER-ERRβ-PPARγ as possible diagnosis/therapy target in disturbances of testis steroidogenic function are needed.

Pharmacologic Activation of the G Protein-Coupled Estrogen Receptor Inhibits Pancreatic Ductal Adenocarcinoma.

Background & AimsFemale sex is associated with lower incidence and improved clinical outcomes for most cancer types including pancreatic ductal adenocarcinoma (PDAC). The mechanistic basis for this sex difference is unknown. We hypothesized that estrogen signaling may be responsible, despite the fact that PDAC lacks classic nuclear estrogen receptors.MethodsHere we used murine syngeneic tumor models and human xenografts to determine that signaling through the nonclassic estrogen receptor G protein-coupled estrogen receptor (GPER) on tumor cells inhibits PDAC.ResultsActivation of GPER with the specific, small molecule, synthetic agonist G-1 inhibited PDAC proliferation, depleted c-Myc and programmed death ligand 1 (PD-L1), and increased tumor cell immunogenicity. Systemically administered G-1 was well-tolerated in PDAC bearing mice, induced tumor regression, significantly prolonged survival, and markedly increased the efficacy of PD-1 targeted immune therapy. We detected GPER protein in a majority of spontaneous human PDAC tumors, independent of tumor stage.ConclusionsThese data, coupled with the wide tissue distribution of GPER and our previous work showing that G-1 inhibits melanoma, suggest that GPER agonists may be useful against a range of cancers that are not classically considered sex hormone responsive and that arise in tissues outside of the reproductive system.

Disruption of 17β-estradiol secretion by persistent organic pollutants present in human follicular fluid is dependent on the potential of ovarian granulosa tumor cell lines to metabolize estrogen

Endocrine-disrupting chemicals (EDCs), such as perfluorooctanoate, perfluorooctane sulfonate, 2,2-dichlorodiphenyldichloroethylene, hexachlorobenzene, and polychlorinated biphenyl 153 are persistent pollutants that are found in human follicular fluid (FF). These compounds may affect endocrine function, disrupt steroid secretion by granulosa cells, and play a role in granulosa cell tumor (GCT) development. GCTs demonstrate endocrine activity, expressing aromatase and secreting 17β-estradiol (E2). We aimed to determine the effects of a mixture of EDCs, similar to that found in human FF, on human granulosa tumor cell lines representing the juvenile (JGCT) and adult (AGCT) forms (COV434 and KGN cells, respectively). We found that all the individual compounds and mixtures tested altered granulosa tumor cell function by disrupting E2 secretion. In KGN cells, which possess significantly higher basal aromatase gene expression, and therefore secrete more E2 than JGCT cells, EDC mixtures activated estrogen receptors (ERs) and G protein-coupled receptor-30 signaling, thereby stimulating E2 secretion, without affecting aromatase expression. By contrast, in COV434 cells, which demonstrate higher CYP1A1 expression, a key mediator of estrogen metabolism, than KGN cells, EDC mixtures reduced E2 secretion in parallel with increases in the 2-hydroxyestrogen 1/E2 ratio and CYP1A1 expression, implying an upregulation of E2 metabolism. These results indicate that the EDC mixture present in FF disrupts E2 secretion in JGCT and AGCT cells according to the estrogen metabolic potential of the cell type, involving both classical and non-classical ER pathways.

Is Testicular Germ Cell Cancer Estrogen Dependent? The Role of Endocrine Disrupting Chemicals.

Testicular germ cell cancer (TGCC) is the most frequent cancer of the young male, with an increasing incidence worldwide. The pathogenesis and reasons for this increase remain unknown. However, epidemiological and experimental data have suggested that, similar to genital malformations and sperm impairment, it could result from the interaction of genetic and environmental factors including fetal exposure to endocrine-disrupting chemicals (EDCs) with estrogenic effects. In this review, we analyze the expression of classic and nonclassic estrogen receptors by TGCC cells, the way they may influence germ cell proliferation induced by EDCs, and discuss how this estrogen dependency supports the developmental and environmental hypothesis.

Protective Effects of G Protein-Coupled Estrogen Receptor 1 (GPER1) on β-Amyloid-Induced Neurotoxicity: Implications for Alzheimer’s Disease

Alzheimer’s disease (AD) is a problematic disease that has shown a significant increase in patient numbers worldwide. AD is identified pathologically by the accumulation of the toxic amyloid beta (Aβ) protein, neurofibrillary tangles and neuropil threads in postmortem brains of AD patients. Women are more prone to AD either due to their increased life expectancy or the decline in Estrogen hormone levels around menopause. Estrogens play a physiologically important role in the brain, but there is debate about the association between estrogen and Ad. The neuroprotective effects of estrogens are possibly mediated by estrogen receptors (ERs), which include classical nuclear estrogen receptors (ERα and ERβ) and nonclassical ER (G protein-coupled estrogen receptor 1, GPER1(GPR30)). The effect of GPER1 on Aβ-induced neurotoxicity is unclear. Here we studied the effect of GPER1 receptor agonists G-1 on rat neuronal cells. Rat neuronal cells were incubated with Aβ1–42, either alone or in combination with GPER1 agonist G-1 (10−7, 10−8 and 10−9 M). Cell viability was determined by MTT assays and apoptotic effects induced by Aβ1–42 were measured by Cell Death Detection kit. Oxidative stress parameters, including nitric oxide (NO) levels and total oxidant status (TOS) were measured by spectrophotometry. Approximately half of the cell death was observed with 10−6 M Aβ1–42 incubation for 48 hours. This is the first study that explores the effect of activation of GPER1 by its agonist G-1 on neuroprotection against Aβ1–42-toxicity in rat neuronal cells. GPER1 activation significantly reduced on rat neuronal cells. Aβ1–42 induced cell death was significantly reduced by co-incubating with G-1. Our results suggest that G-1 treatment protects neurons from Aβ1–42 induced neurotoxicity by changing the oxidative parameters on rat neuronal cells.

Function of G-Protein-Coupled Estrogen Receptor-1 in Reproductive System Tumors.

The G-protein-coupled estrogen receptor-1 (GPER-1), also known as GPR30, is a novel estrogen receptor mediating estrogen receptor signaling in multiple cell types. The progress of estrogen-related cancer is promoted by GPER-1 activation through mitogen-activated protein kinases (MAPK), phosphoinositide 3-kinase (PI3K), and phospholipase C (PLC) signaling pathways. However, this promoting effect of GPER-1 is nonclassic estrogen receptor (ER) dependent manner. In addition, clinical evidences revealed that GPER-1 is associated with estrogen resistance in estrogen-related cancer patients. These give a hint that GPER-1 may be a novel therapeutic target for the estrogen-related cancers. However, preclinical studies also found that GPER-1 activation of its special agonist G-1 inhibits cancer cell proliferation. This review aims to summarize the characteristics and complex functions of GPER-1 in cancers.

Selective Activation of the Non-Classical Estrogen Receptor Gper Elicits Potent Anti-Tumor Activity in Multiple Myeloma

Within the hematopoietic compartment, estrogens influence the differentiation, proliferation and survival of B cells and also increase the number of plasmacells (PCs) and their capacity to produce immunoglobulins. In addition to the classical estrogen receptors, an extensive body of literature has shed light on a 7-transmembrane spanning G-protein coupled estrogen receptor, GPER (also known as GPR30). The discovery of GPER-selective agonists and antagonists has led to the comprehension of the pathophysiological role of GPER and its involvement in cancer biology. However, the functional role of GPER in hematological malignancies has not been previously investigated.We detected GPER expression in 12 MM cell lines, including dexamethasone, bortezomib and carfilzomib-resistant cell lines, as well as in Waldestrom Macroglobulinemia (WM) cell lines either at mRNA and protein level, as assessed by qRT-PCR and western blotting, respectively. Importantly, MM patients' expression dataset analysis revealed a progressive decline of GPER mRNA levels during MM progression. Moreover, adhesion of MM cell lines (NCI-H929, U266) to bone marrow stromal cells (BMSCs) reduced GPER mRNA and protein levels even further, supporting a potential role of the BM microenvironment in regulating GPER expression.To address the role of GPER-mediated signaling on MM cell survival and cell death, first we tested the selective GPER agonist G-1 ((±)-1-[(3aR *,4S *,9bS *)-4-(6-Bromo-1,3-benzodioxol-5-yl) -3a,4,5,9b-tetrahydro-3H cyclopenta [c]quinolin-8-yl] ethanone) in vitro. G-1 inhibited, in a dose-dependent manner, the survival of WM (n=2) and MM cell lines (n=9), as well as of primary MM cells (n=3), with an IC50 at 48 h ranging from 2 to 3 μM, while it did not affect the viability of healthy donors' PBMCs (n=3); moreover, G-1 synergized with bortezomib in MM1S MM cells and with ibrutinib in BCMW1 WM cells. Both in MM and WM cell lines, G-1 treatment increased cells in G2/M phase and induced a potent and dose-dependent apoptotic cell death, as assessed by Annexin V/7AAD staining and western blot analysis of active caspases 3, 7 and 9. G-1-induced apoptosis was reverted by the pan-caspase inhibitor ZVAD-FMK and was not impaired by co-colture with BMSCs or by exogenous IL-6, IGF-1 or HGF. 17β-estradiol (10nM) and the selective GPER-antagonist G-15 (0.5µM) slightly increased the survival of MM and WM cells, and this effect was overcome by G-1 treatment. Moreover, G-1 induced the occurrence of a cytoprotective autophagy, as shown by the expression of autophagic markers, such as beclin-1 and LC3A/B, the cytosolic punctate pattern of LC3B and down-regulation of p62/SQSTM1 expression, and treatment of MM and WM cell lines with the autophagy inhibitor chloroquine potentiated G-1-triggered anti-survival effects. Importantly, intraperitoneal injection of G-1 (2mg/kg) in SCID mice significantly reduced the growth of subcutaneous MM1S and bortezomib-resistant AMO-abzb xenografts, and prolonged survival of treated animals.Since GPER-dependent molecular effects in solid tumors have been attributed to modulation of MAPK activity, by using an antibody array we analyzed the phosphorylation status of 24 protein kinases after GPER activation. Notably, treatment with G-1 (2µM) reduced the phosphorylation of ERK1/2, p38α-γ, AKT and GSK3α-β.Finally, on the light of the emerging role of miRNAs in MM pathobiology, we investigated by TaqMan qPCR the effects of G-1 on the miRNA profile and found that G-1 induced the expression of tumor suppressor miR-29b by down-regulating Sp1, the major miR-29b-negative regulator, thus disrupting the previously reported miR-29b/Sp1 negative feedback loop. Consistently, an inverse correlation between GPER and Sp1 mRNA levels emerged in MM patient PCs. In addition, miR-29b canonical targets, such as CDK6 and MCL-1, were downregulated in G-1-treated MM cells.Altogether, our results indicate that GPER is expressed in MM and WM and its selective activation via G-1 triggers potent anti-tumor activity through inhibition of oncogenic protein kinases and activation of miR-29b, providing the preclinical rationale for clinical investigation of GPER-agonists to treat these hematological malignancies. DisclosuresNo relevant conflicts of interest to declare.

Role of sex steroids and their receptors in human preterm infants: Impacts on future treatment strategies for cerebral development

Preterm birth is a major risk factor for cerebral complications, such as hemorrhage or periventricular leukomalacia, which lead to lifelong neurodevelopmental deficits. Hypoxia/ischemia, inflammation, hyperoxia, and prematurity itself contribute to the extent of impaired neurodevelopment. Preterm birth leads to disruption of the placental supply of estrogens and progesterone. Postnatally, the plasma levels of estrogens and progesterone drop 100-fold. Preterm infants are deprived of the placental supply of these hormones for up to sixteen weeks. Thus, supplementation of estradiol and progesterone to mimic intrauterine conditions may potentially improve a premature infant́s extrauterine development and help protect the brain against neurological complications. However, preliminary clinical studies did not find improved outcomes except for a trend towards less cerebral palsy. The decrease in estrogen and progesterone concentrations is accompanied by persistent, high postnatal production of fetal zone steroids, mainly dehydroepiandrosterone, which serve as precursors for maternal estrogen synthesis during pregnancy. This commentary will combine knowledge from endocrinology, pharmacology, and neonatology to explain the discrepancies between promising animal models and clinical findings. Most important targets will be classical and non-classical estrogen receptors, which interact differently—not only with estrogens but also with fetal zone steroids. The fetal zone is unique among humans and higher primates. Therefore, a clearly defined model is required to study the role of sex steroids and their receptors before further clinical studies begin.

Decreased Expression of GPER1 Gene and Protein in Goiter.

Goiter is more common in women, suggesting that estrogen could be involved in its physiopathology. The presence of classical estrogen receptors (ERα and ERβ) has been described in thyroid tissue, suggesting a direct effect of estrogen on the gland. A nonclassic estrogen receptor, the G-protein-coupled estrogen receptor (GPER1), has been described recently in several tissues. However, in goiter, the presence of this receptor has not been studied yet. We investigated GPER1 gene and protein expressions in normal thyroid and goiter using reverse transcription quantitative polymerase chain reaction (RT-qPCR) and Western blot, respectively. In normal thyroid (n = 16) and goiter (n = 19), GPER1 gene was expressed in all samples, while GPER1 protein was expressed in all samples of normal thyroid (n = 15) but in only 72% of goiter samples (n = 13). When comparing GPER1 gene and protein levels in both conditions, gene expression and protein levels were higher in normal thyroid than in goiter, suggesting a role of this receptor in this condition. Further studies are needed to elucidate the role of GPER1 in normal thyroid and goiter.

Phytoestrogen, tanshinone IIA diminishes collagen deposition and stimulates new elastogenesis in cultures of human cardiac fibroblasts

It has been previously reported that oral or intra-peritoneal administration of tanshinone IIA can alleviate the ventricular hypertrophy and fibrosis that develops in rats after experimental cardiac infarction. Our present studies, performed on cultures of human cardiac fibroblasts, investigated the mechanism by which tanshinone IIA produces these beneficial effects. We found that treatment of cardiac fibroblasts with 0.1–10µM tanshinone IIA significantly inhibited their deposition of collagen I, while enhancing production of new elastic fibers. Moreover, both anti-collagenogenic and pro-elastogenic effects of tanshinone IIA occurred only after selective activation of the G protein-coupled estrogen receptor (GPER). This subsequently leads to initiation of the PKA/CREB phosphorylation pathway that inversely modulated transcription of collagen I and elastin genes. Interestingly, treatment of human cardiac fibroblasts with tanshinone IIA additionally up-regulated the production of the 67-kDa elastin binding protein, which facilitates tropoelastin secretion, and increased synthesis of lysyl oxidase, catalyzing cross-linkings of tropoelastin. Moreover, tanshinone IIA also caused up-regulation in the synthesis of collagenolytic MMP-1, but down-regulated levels of elastolytic MMP-2 and MMP-9. In summary, our data validate a novel mechanism in which tanshinone IIA, interacting with a non-classic estrogen receptor, maintains the proper balance between the net deposition of collagen and elastin, allowing for optimal durability and resiliency of the newly deposited matrix.