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Background: Although the lack of estrogen receptor β (ERβ) is a risk factor for the development of inflammatory bowel disease (IBD) and psychiatric disorders, the underlying cellular and molecular mechanisms are not fully understood. Herein, we revealed the role of gut microbiota in the development of IBD and related anxiety-like behavior in ERβ-deficient mice., Results: In response to dextran sodium sulfate (DSS) insult, the ERβ knockout mice displayed significant shift in α and β diversity in the fecal microbiota composition and demonstrated worsening of colitis and anxiety-like behaviors. In addition, DSS-induced colitis also induced hypothalamic-pituitary-adrenal (HPA) axis hyperactivity in ERβ-deficient mice, which was associated with colitis and anxiety-like behaviors. In addition, RNA sequencing data suggested that ErbB4 might be the target of ERβ that is involved in regulating the HPA axis hyperactivity caused by DSS insult. Gut microbiota remodeling by co-housing showed that both the colitis and anxiety-like behaviors were aggravated in co-housed wild-type mice compared to single-housed wild-type mice. These findings suggest that gut microbiota play a critical role in mediating colitis disease activity and anxiety-like behaviors via aberrant neural processing within the gut-brain axis., Conclusions: ERβ has the potential to inhibit colitis development and anxiety-like behaviors via remodeling of the gut microbiota, which suggests that ERβ is a promising therapeutic target for the treatment of IBD and related anxiety-like behaviors. Video Abstract., (© 2022. The Author(s).)

Summary: After the discovery of ERβ, a novel role for dihydrotestosterone (DHT) in estrogen signaling was revealed. Instead of just being a better androgen, DHT was found to be a precursor of the ERβ agonist 5α-androstane-3β, 17β-diol (3βAdiol), an estrogen which does not require aromatase for its synthesis. ERβ was found to oppose androgen signaling and thus is a potential target for treatment of prostate cancer. ERβ was also found to have effects that were independent of androgen signaling, particularly in the CNS. Although in rodent models of neurodegenerative diseases (Parkinson's disease, multiple sclerosis, and Alzheimer's disease), ERβ agonists are very effective in relieving symptoms and improving pathologies, this has not proven to be the case in humans. In this review we will focus on the main differences in ERβ signaling between rodents and humans and will make the point that a very important difference between the two species is in the splice variants which are expressed in humans and not rodents. The main conclusion at this point is that before we think of using ERβ agonists clinically, much more work on ERβ signaling in the human or in primates needs to be done.

Epilepsy is a highly prevalent and drug-refractory neurological disorder characterized by spontaneous recurrent seizures. Estrogen is identified to be proconvulsant and lowers the seizure threshold of female epilepsy. Estrogen receptor β (ERβ) has been proposed to mediate neuroprotection in epilepsy, although the underlying mechanism remains unknown. Rationale: In this study, we investigated the role of ERβ in the epileptogenesis of female temporal lobe epilepsy (TLE). Methods: Immunohistochemistry, immunofluorescence, western blots, Golgi staining, 1 H MRS and whole-cell patch-clamp were used to evaluate ERβ expression, pathological changes, and synaptic excitation /inhibition (E/I) balance in female TLE patients and ovariectomized (OVX) chronic epileptic mice. Electroencephalogram (EEG) recordings were recorded to evaluate the epileptic susceptibility in OVX WT and ERβ -/- mice. And high-throughput RNA-sequence was performed to identify differential expression genes (DEGs) which can elucidate the potential mechanism of ERβ regulating the seizure susceptibility. Results: ERβ expression was decreased in the brains of female TLE patients and OVX chronic epileptic mice. ERβ deletion enhanced seizure susceptibility and exacerbated the imbalance of synaptic E/I in hippocampal CA1 area of OVX epileptic mice. In line with these observations, RNA-sequence data further identified glutamine ligase (GLUL) as the target of ERβ involved in regulating synaptic E/I in CA1. Furthermore, ERβ agonist WAY-200070 markedly suppressed epileptic phenotypes and normalized GLUL expression in CA1 region of kainic acid (KA) induced OVX chronic epileptic model. Conclusions: Our data provide novel insight into the pathogenesis of female TLE, and indicate ERβ provides a new therapeutic strategy for female TLE patients., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Background: Male estrogen receptor beta (ERβ) knockout (BERKO) mice display anxiety and aggression linked to, among others, altered serotonergic signaling in the basolateral amygdala and dorsal raphe, impaired cortical radial glia migration, and reduced GABAergic signaling. The effects on primary motor cortex (M1 cortex) and locomotor activity as a consequence of ERβ loss have not been investigated., Objective: The aim of this study was to determine whether locomotor activity is altered as a consequence of the changes in the M1 cortex., Methods: The locomotor activity of male wild-type (WT) and BERKO mice was evaluated using the open-field and rotarod tests. Molecular changes in the M1 cortex were analyzed by RNA sequencing, electron microscopy, electrophysiology, and immunohistological techniques. In addition, we established oligodendrocyte (OL) cultures from WT and BERKO mouse embryonic stem cells to evaluate OL function., Results: Locomotor profiling revealed that BERKO mice were more active than WT mice but had impaired motor coordination. Analysis of the M1 cortex pointed out differences in synapse function and myelination. There was a reduction in GABAergic signaling resulting in imbalanced excitatory and inhibitory neurotransmission as well as a defective OL differentiation accompanied by myelin defects. The effects of ERβ loss on OL differentiation were confirmed in vitro., Conclusion: ERβ is an important regulator of GABAergic interneurons and OL differentiation, which impacts on adult M1 cortex function and may be linked to increased locomotor activity and decreased motor coordination in BERKO mice., (© 2020 The Author(s) Published by S. Karger AG, Basel.)

Achilles tendon healing (ATH) remains an unanswered question in the field of sports medicine because it does not produce tissue with homology to the previously uninjured tissue. Oestrogen receptor β (ERβ) is involved in the injury and repair processes of tendons. Our previous study confirmed that ERβ plays a role in the early stage of ATH by affecting adipogenesis, but its role in extracellular matrix (ECM) remodelling is unknown. We established a 4-week Achilles tendon repair model to investigate the mechanism through which ERβ affects ATH at the very beginning of ECM remodelling phase. In vitro studies were performed using tendon-derived stem cells (TDSCs) due to their promising role in tendon healing. Behavioural and biomechanical tests revealed that ERβ-deficient mice exhibit weaker mobility and inferior biomechanical properties, and immunofluorescence staining and qRT-PCR showed that these mice exhibited an erroneous ECM composition, as mainly characterized by decreased collagen type I (Col I) deposition. The changes in gene expression profiles between ERβ-knockout and WT mice at 1 week were analysed by RNA sequencing to identify factors affecting Col I deposition. The results highlighted the IRF5-CCL3 axis, and this finding was verified with CCL3-treated TDSCs. These findings revealed that ERβ regulates Col I deposition during ATH via the IRF5-CCL3 axis., (© 2020 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

Achilles tendon injury is one of the challenges of sports medicine, the aetiology of which remains unknown. For a long time, estrogen receptor β (ERβ) has been known as a regulating factor of the metabolism in many connective tissues, such as bone, muscle and cartilage, but little is known about its role in tendon. Recent studies have implicated ERβ as involved in the process of tendon healing. Tendon-derived stem cells (TDSCs) are getting more and more attention in tendon physiological and pathological process. In this study, we investigated how ERβ played a role in Achilles tendon healing. Achilles tendon injury model was established to analyse how ERβ affected on healing process in vivo. Cell proliferation assay, Western blots, qRT-PCR and immunocytochemistry were performed to investigate the effect of ERβ on TDSCs. Here, we showed that ERβ deletion in mice resulted in inferior gross appearance, histological scores and, most importantly, increased accumulation of adipocytes during the early tendon healing which involved activation of peroxisome proliferator-activated receptor γ (PPARγ) signalling. Furthermore, in vitro results of ours confirmed that the abnormity might be the result of abnormal TDSC adipogenic differentiation which could be partially reversed by the treatment of ERβ agonist LY3201. These data revealed a role of ERβ in Achilles tendon healing for the first time, thereby providing a new target for clinical treatment of Achilles tendon injury., (© 2019 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

Laminar organization of neurons in cerebral cortex is essential for normal brain function. Radial glial cells (RGCs), are highly polarized cells that serve as neuronal progenitors and as scaffolds for neuronal migration during construction of the cerebral cortex. Cadherins (E-cadherin and N-cadherin)-based adherins junctions, which anchor apical end-feet of adjacent RGCs to each other at the ventricular surface contribute to sustain the polarity and adhesion of RGCs, therefore affect production of RGCs and radial migration. Estrogen is a steroid hormone and contributes to the organizational sexual differentiation of the brain. We have previously demonstrated that ERβ expression in the cerebral cortex during corticogenesis and contribute to cerebral cortex development. This has been further confirmed by studies from estrogen receptor β knockout (ERβKO) mice, in which lack of ERβ in mice induced abnormal development of cerebral cortical structure, retarded migration of the neurons, and abnormal morphology of RGCs with truncated or less organized radial processes. These indicate that estrogen via ERβ affects RGCs development. Moreover, phenotype analysis in the ERβKO mice has confirmed that estrogen activation ERβ influence the polarity of epithelial tissue and structure integrity by modulating the level of cadherins (E-cadherin and N-cadherin). Thus, we propose that ERβ maybe affect the maintaining the polarity of RGCs through cadherins., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Context: Clinical studies suggest that an imbalance in the actions of estrogen receptor (ER)-α and ERβ is associated with invasion of tumors of epithelial origin. Both ER have been detected in the pituitary adenomas (PA). Our previous study together with other reports suggests that an imbalance between ERα and ERβ contributes to the pathogenesis and biological behavior of PA including invasion. However, the roles of the two ER in invasiveness of PA have not been clarified., Objective: The expression of ER, Slug, and E-cadherin in 41 cases of nonfunctional PA (NFPA) were determined to evaluate whether ER were related to the invasiveness of NFPA. Furthermore, we aimed by analysis of the correlation between ER and E-cadherin or Slug to understand molecular mechanisms related to invasiveness., Methods: Immunohistochemistry, RT-PCR, and Western blot were performed., Results: Nuclear ERα staining was significantly stronger in invasive NFPA than noninvasive ones (P < 0.01). In contrast, nuclear ERβ staining was significantly weaker in invasive NFPA than in noninvasive ones (P < 0.01). Both E-cadherin mRNA and protein were decreased in invasive NFPA compared with noninvasive ones. Moreover, Slug, a repressor of E-cadherin, was significantly increased in invasive over noninvasive NFPA (P < 0.01). There were significant correlations between ER and Slug or E-cadherin in NFPA, in which Slug was positively correlated with ERα and inversely correlated with ERβ, whereas E-cadherin was positively correlated with ERβ and inversely correlated with ERα., Conclusions: ERα and ERβ may act in opposite directions to regulate the Slug-E-cadherin pathway and to affect invasiveness of NFPA.

At 2 years of age, 100% (23/23) of ERbeta(-/-) female mice have developed large pituitary and ovarian tumors. The pituitary tumors are gonadotropin-positive and the ovarian tumors are sex cord (less differentiated) and granulosa cell tumors (differentiated and estrogen secreting). No male mice had pituitary tumors and no pituitary or ovarian tumors developed in ERalpha(-/-) mice or in ERalphabeta(-/-) double knockout mice. The tumors have high proliferation indices, are ERalpha-positive, ERbeta-negative, and express high levels of nuclear phospho-SMAD3. Mice with granulosa cell tumors also had hyperproliferative endometria. The cause of the pituitary tumors appeared to be excessive secretion of gonadotropin releasing hormone (GnRH) from the hypothalamus resulting from high expression of NPY. The ovarian phenotype is similar to that seen in mice where inhibin is ablated. The data indicate that ERbeta plays an important role in regulating GnRH secretion. We suggest that in the absence of ERbeta, the proliferative action of FSH/SMAD3 is unopposed and the high proliferation leads to the development of ovarian tumors. The absence of tumors in the ERalphabeta(-/-) mice suggests that tumor development requires the presence of ERalpha.

Estrogen acting through two estrogen receptors (ERs), ERalpha and ERbeta, regulates multiple functions in the central nervous system. Studies in rodent brains have revealed that ERalpha is the predominant ER in the hypothalamus and controls reproduction. ERbeta influences on non-reproductive processes and appears to be the main ER subtype expressed in the cerebral cortex, the hippocampus, the cerebellum and the dorsal raphe. During embryogenesis, estrogen plays an important role in brain development regulating maturation of distinct brain structures, thereby contributing to modulation of the function of these structures. Studies on the brains of ERbeta knockout mice revealed that, during embryonic brain development, ERbeta affects cortical layering and interneuron migration, thus playing a key role in brain morphogenesis. This chapter will focus on the roles of ERbeta in several aspects of the development and function of the mammalian central nervous system.

We examined nociceptive responses to mechanical stimulation in mice of both sexes lacking the estrogen receptor alpha or beta and in respective wild types under normal conditions, after inflammation of a hindpaw or peripheral nerve injury. In normal wild-type mice, females had significantly lower paw withdrawal threshold than males. There was no significant difference between wild-type mice and knock-outs of either estrogen receptor alpha or beta in mechanical response threshold in male mice. However, significantly elevated response threshold was observed in both knock-out female mice, which eliminated sex differences in nociception. After carrageenan-induced inflammation of a hindpaw, all wild-type and knock-out mice exhibited similar local edema with no difference between the sexes. Wild-type mice developed hypersensitivity (allodynia) to mechanical stimulation, which was more profound in the females than in males. Again, such sex difference was not observed in the knock-outs of either estrogen receptor. Photochemically induced partial sciatic nerve injury caused similar persistent mechanical hypersensitivity in the wild types and both estrogen receptor knock-outs with no difference between the sexes. These results suggest that the sex difference in basal mechanical pain threshold and inflammatory hypersensitivity is eliminated in mice lacking either the estrogen alpha receptors or beta receptors. However, these receptors do not seem to be directly involved in mediating pain sensitivity in general or in the development of neuropathic pain. It is unclear whether the elimination of sex differences observed in the knock-outs reflects an ongoing effect of estrogen acting through its receptors in females or the developmental changes that predominantly affect females.

Estrogen is known to influence pain, but the specific roles of the two estrogen receptors (ERs) in the spinal cord are unknown. In the present study, we have examined the expression of ERalpha and ERbeta in the spinal cord and have looked for defects in pain pathways in ERbeta knockout (ERbeta(-/-)) mice. In the spinal cords of 10-month-old WT mice, ERbeta-positive cells were localized in lamina II, whereas ERalpha-positive cells were mainly localized in lamina I. In ERbeta(-/-) mice, there were higher levels of calcitonin gene-regulated peptide and substance P in spinal cord dorsal horn and isolectin B4 in the dorsal root ganglion. In the superficial layers of the spinal cord, there was a decrease in the number of calretinin (CR)-positive neurons, and in the outer layer II, there was a loss of calbindin-positive interneurons. During embryogenesis, ERbeta was first detectable in the spinal cord at embryonic day 13.5 (E13.5), and ERalpha was first detectable at E15.5. During middle and later embryonic stages, ERbeta was abundantly expressed in the superficial layers of the dorsal horn. ERalpha was also expressed in the dorsal horn but was limited to fewer neurons. Double staining for ERbeta and CR showed that, in the superficial dorsal horn of WT neonates [postnatal day 0 (P0)], most CR neurons also expressed ERbeta. At this stage, few CR-positive cells were detected in the dorsal horn of ERbeta(-/-) mice. Taken together, these findings suggest that, early in embryogenesis, ERbeta is involved in dorsal horn morphogenesis and in sensory afferent fiber projections to the dorsal horn and that ERbeta is essential for survival of dorsal horn interneurons throughout life.

Our previous studies with estrogen receptor beta knockout (ERbeta(-/-)) mice demonstrated that ERbeta is necessary for embryonic development of the brain as early as embryonic day 14.5 (E14.5) and is involved in neuronal migration. Such early effects of ER were unexpected because estradiol synthesis and action in the brain occur at E18.5. In the present study, we examined the distribution of ERbeta in the developing brain and identified a population of ERbeta-regulated interneurons. ERbeta appears in the brain at E12.5, mainly localized in the wall of the midbrain, neuromere, hypothalamus, thalamus, and basal plate of pons. At E15.5 and E16.5, ERbeta expression increased in the hypothalamus, thalamus, and midbrain and appeared in the limbic forebrain. At E18.5, ERbeta expression was strongly expressed throughout the brain, including the cerebellum and striatum, whereas there were very few positive cells in the ventricular region. In the paraventricular thalamic nucleus and parafascicular nucleus, most of the calretinin-immunopositive interneurons expressed ERbeta. In ERbeta(-/-) mice, calretinin expression was markedly lower than in WT mice in the hippocampus, thalamus, and amygdala both at E16.5 and at E18.5. Epidermal growth factor receptor expression was lower in the cortex of ERbeta(-/-) than in WT mice at E15.5 and, unlike WT mice, was absent from the superficial marginal zone. These findings suggest that ERbeta in the embryonic brain is necessary for the development of calretinin-immunoreactive GABAergic interneurons and for neuronal migration in the cortex through modulating epidermal growth factor receptor expression at middle and later embryonic stages.