LRH-1 Research Articles

Perilipin 5 is a novel target of nuclear receptor LRH-1 to regulate hepatic triglycerides metabolism

Liver receptor homolog-1 (LRH-1) has emerged as a regulator of hepatic glucose, bile acid, and mitochondrial metabolism. However, the functional mechanism underlying the effect of LRH-1 on lipid mobilization has not been addressed. This study investigated the regulatory function of LRH-1 in lipid metabolism in maintaining a normal liver physiological state during fasting. The Lrh-1f/f and LRH-1 liver-specific knockout (Lrh-1LKO) mice were either fed or fasted for 24 h, and the liver and serum were isolated. The livers were used for qPCR, western blot, and histological analysis. Primary hepatocytes were isolated for immunocytochemistry assessments of lipids. During fasting, the Lrh-1LKO mice showed increased accumulation of triglycerides in the liver compared to that in Lrh-1f/f mice. Interestingly, in the Lrh-1LKO liver, decreases in perilipin 5 (PLIN5) expression and genes involved in β-oxidation were observed. In addition, the LRH-1 agonist dialauroylphosphati-dylcholine also enhanced PLIN5 expression in human cultured HepG2 cells. To identify new target genes of LRH-1, these findings directed us to analyze the Plin5 promoter sequence, which revealed −1620/−1614 to be a putative binding site for LRH-1. This was confirmed by promoter activity and chromatin immuno-precipitation assays. Additionally, fasted Lrh-1f/f primary hepatocytes showed increased co-localization of PLIN5 in lipid droplets (LDs) compared to that in fasted Lrh-1LKO primary hepatocytes. Overall, these findings suggest that PLIN5 might be a novel target of LRH-1 to mobilize LDs, protect the liver from lipid overload, and manage the cellular needs during fasting.

NF-κB Regulation of LRH-1 and ABCG5/8 Potentiates Phytosterol Role in the Pathogenesis of Parenteral Nutrition-Associated Cholestasis.

Chronically administered parenteral nutrition (PN) in patients with intestinal failure carries the risk for developing PN-associated cholestasis (PNAC). We have demonstrated that farnesoid X receptor (FXR) and liver X receptor (LXR), proinflammatory interleukin-1 beta (IL-1β), and infused phytosterols are important in murine PNAC pathogenesis. In this study we examined the role of nuclear receptor liver receptor homolog 1 (LRH-1) and phytosterols in PNAC. In a C57BL/6 PNAC mouse model (dextran sulfate sodium [DSS] pretreatment followed by 14 days of PN; DSS-PN), hepatic nuclear receptor subfamily 5, group A, member 2/LRH-1 mRNA, LRH-1 protein expression, and binding of LRH-1 at the Abcg5/8 and Cyp7a1 promoter was reduced. Interleukin-1 receptor-deficient mice (Il-1r-/- /DSS-PN) were protected from PNAC and had significantly increased hepatic mRNA and protein expression of LRH-1. NF-κB activation and binding to the LRH-1 promoter were increased in DSS-PN PNAC mice and normalized in Il-1r-/- /DSS-PN mice. Knockdown of NF-κB in IL-1β-exposed HepG2 cells increased expression of LRH-1 and ABCG5. Treatment of HepG2 cells and primary mouse hepatocytes with an LRH-1 inverse agonist, ML179, significantly reduced mRNA expression of FXR targets ATP binding cassette subfamily C member 2/multidrug resistance associated protein 2 (ABCC2/MRP2), nuclear receptor subfamily 0, groupB, member 2/small heterodimer partner (NR0B2/SHP), and ATP binding cassette subfamily B member 11/bile salt export pump (ABCB11/BSEP). Co-incubation with phytosterols further reduced expression of these genes. Similar results were obtained by suppressing the LRH-1 targets ABCG5/8 by treatment with small interfering RNA, IL-1β, or LXR antagonist GSK2033. Liquid chromatography-mass spectrometry and chromatin immunoprecipitation experiments in HepG2 cells showed that ATP binding cassette subfamily G member 5/8 (ABCG5/8) suppression by GSK2033 increased the accumulation of phytosterols and reduced binding of FXR to the SHP promoter. Finally, treatment with LRH-1 agonist, dilauroyl phosphatidylcholine (DLPC) protected DSS-PN mice from PNAC. This study suggests that NF-κB regulation of LRH-1 and downstream genes may affect phytosterol-mediated antagonism of FXR signaling in the pathogenesis of PNAC. LRH-1 could be a potential therapeutic target for PNAC.

LRH-1 high expression in the ovarian granulosa cells of PCOS patients.

Polycystic ovary syndrome (PCOS) is considered one of the most common endocrine disorders with heterogeneity. There are also reports that liver receptor homolog 1 [LRH-1 or nuclear receptor subfamily 5 group A member 2] plays an important role in the reproductive system. But up to now, there are no reports related to the link with PCOS and LRH-1. In this study, we aimed to detect the LRH-1 expression in the ovarian granulosa cell (GC) of PCOS patients and explore the potential relationship between LRH-1 and PCOS. In all, 146 follicular fluid samples were collected in this study, including 72 from PCOS patients and 74 from control patients who underwent intracytoplasmic sperm injection or in vitro fertilization-embryo transfer. The ovarian GCs were extracted from the patient's follicular fluid by magnetic-activated cell sorting method, and real-time quantitative PCR was used to measure the expression of LRH-1 in ovarian GCs. Then we analyzed the correlation between the expression level of LRH-1 and the clinical characteristics of the patient by using Pearson Correlation analysis. The expression of LRH-1 was significantly higher in PCOS patients ovarian GCs than that in the control patients [(1.38 ± 0.47) vs (1.03 ± 0.32), t = 5.327, p < 0.0001], and it was positively correlated with antral follicles counting (r = 0.3607, p < 0.0001) and the serum anti-Mullerian hormone (r = 0.2662, p = 0.0012), luteotropic hormone (r = 0.2518, p = 0.0022), testosterone (r = 0.2794, p = 0.0006) in all patients. No statistical significance between LRH-1 and body mass index, follicle-stimulating hormone, homeostasis model assessment of insulin resistance, dehydroepiandrosterone sulfate, progesterone. Compared with the control group, we found that LRH-1 was highly expressed in the ovarian GCs of PCOS patients. Our study has revealed the relationship between the LRH-1 expression and PCOS, which suggested that LRH-1 may play an important role in ovulation disorders. While this finding provided new ideas for the study of pathogenesis, it also provided a theoretical basis for the clinical diagnosis and treatment for PCOS.

Uniting features from strongly activating small molecules to develop chimeric LRH‐1 agonist

Nuclear receptors are transcription factors that respond to external lipophilic signaling molecules to alter target gene expression. Liver receptor homolog-1 (LRH-1) is a nuclear receptor that operates as a master metabolic regulator in the liver, making it an attractive target for treating a variety of diseases characterized by dysregulated glucose and lipid homeostasis. Phospholipids (PLs) represent the putative endogenous ligands for LRH-1, but their insolubility, rapid turnover, and poor potency have made synthetic agonists more attractive tools for targeting this receptor. With structural data that defined the binding pose of the first class of synthetic LRH-1 agonists, we designed and characterized small molecules with enhanced solubility, potency, and efficacy. This work led to a compound (“6N”) that binds hydrophilic residues deep within the binding pocket, exhibiting nanomolar potency. We have also targeted residues contacted by the headgroups of activating PLs as a complementary strategy for activation. These modifications resulted in our most efficacious small molecule (“10CA”). To capture the characteristics of both compounds, we have synthesized a chimeric small molecule that includes the key moieties of 6N and 10CA responsible for high affinity and efficacy, respectively. Excitingly, this new hybrid compound exhibits the highest affinity, potency, and effect on receptor thermal stability. While the hybrid activates LRH-1 to an equivalent level as 10CA in reporter assays, it elicits LRH-1 target gene expression to a greater degree than either 6N or 10CA alone. Interestingly, RNA-seq studies have revealed that the hybrid compound captures a PL-mediated gene expression response more effectively that either 6N or 10CA alone. Further crystallography and molecular dynamics studies reveal that these characteristics are the result of the hybrid compound engaging with, and communicating from, both sites of agonist binding. Altogether, our studies demonstrate the differences between our leading agonists and suggest that the hybrid molecule is our most effective agonist to date, providing a critical tool for both studying LRH-1 biology and treating a range of metabolic disorders.

Emerging functions of the nuclear receptor LRH-1 in liver physiology and pathology

Nuclear receptors play pleiotropic roles in cell differentiation, development, proliferation, and metabolic processes to govern liver physiology and pathology. The nuclear receptor, liver receptor homolog-1 (LRH-1, NR5A2), originally identified in the liver as a regulator of bile acid and cholesterol homeostasis, was recently recognized to coordinate a multitude of other hepatic metabolic processes, including glucose and lipid processing, methyl group sensing, and cellular stress responses. In this review, we summarize the physiological and pathophysiological functions of LRH-1 in the liver, as well as the molecular mechanisms underlying these processes. This review also focuses on the recent advances highlighting LRH-1 as an attractive target for liver-associated diseases, such as non-alcoholic fatty liver disease (NAFLD) and hepatocellular carcinoma (HCC).

LRH1 Acts as an Oncogenic Driver in Human Osteosarcoma and Pan-Cancer.

Osteosarcoma (OS) that mainly occurs during childhood and adolescence is a devastating disease with poor prognosis presented by extreme metastases. Recent studies have revealed that liver receptor homolog 1 (LRH-1) plays a vital role in the metastasis of several human cancers, but its role is unknown in the metastasis of OS. In this study, Gene Ontology (GO) enrichment analyses based on high-throughput RNA-seq data revealed that LRH-1 acted a pivotal part in the positive regulation of cell migration, motility, and angiogenesis. Consistently, LRH-1 knockdown inhibited the migration of human OS cells, which was concurrent with the downregulation of mesenchymal markers and the upregulation of epithelial markers. In addition, short hairpin RNAs (shRNAs) targeting LRH-1 inactivated transforming growth factor beta (TGF-β) signaling pathway. LRH-1 knockdown inhibited human umbilical vein endothelial cell (HUVEC) proliferation, migration, and tube formation. Vascular endothelial growth factor A (VEGFA) expression was also downregulated after LRH-1 knockdown. Immunohistochemistry (IHC) revealed that the expression of LRH-1 protein was significantly higher in tumor tissues than in normal bone tissues. We found that high LRH-1 expression was associated with poor differentiation and advanced TNM stage in OS patients using IHC. Based on The Cancer Genome Atlas (TCGA) database, high LRH-1 expression predicts poor survival in lung squamous cell carcinoma (LUSC), kidney renal papillary cell carcinoma (KIRP), and pancreatic adenocarcinoma (PAAD). The downregulation of LRH-1 significantly hindered the migration and motility of LUSC cells. Using multi-omic bioinformatics, the positive correlation between LRH-1- and EMT-related genes was found across these three cancer types. GO analysis indicated that LRH-1 played a vital role in “blood vessel morphogenesis” or “vasculogenesis” in KIRP. Our results indicated that LRH-1 plays a tumor-promoting role in human OS, could predict the early metastatic potential, and may serve as a potential target for cancer therapy.

Another One Bites the Gut: Nuclear Receptor LRH-1 in Intestinal Regeneration and Cancer.

Simple SummaryThe nuclear receptor Liver Receptor Homolog-1 (LRH-1) is widely involved in the complex and balanced biology of the intestine, thus guaranteeing the several functions played by this organ. Alterations of LRH-1 pathways are involved in tumor formation. This review covers the main aspects related to LRH-1 contribution in both physiological and pathological aspects of the intestine.The process of self-renewal in normal intestinal epithelium is characterized by a fine balance between proliferation, differentiation, migration, and cell death. When even one of these aspects escapes the normal control, cellular proliferation and differentiation are impaired, with consequent onset of tumorigenesis. In humans, colorectal cancer (CRC) is the main pathological manifestation of this derangement. Nowadays, CRC is the world’s fourth most deadly cancer with a limited survival after treatment. Several conditions can predispose to CRC development, including dietary habits and pre-existing inflammatory bowel diseases. Given their extraordinary ability to interact with DNA, it is widely known that nuclear receptors play a key role in the regulation of intestinal epithelium, orchestrating the expression of a series of genes involved in developmental and homeostatic pathways. In particular, the nuclear receptor Liver Receptor Homolog-1 (LRH-1), highly expressed in the stem cells localized in the crypts, promotes intestine cell proliferation and renewal in both direct and indirect DNA-binding manner. Furthermore, LRH-1 is extensively correlated with diverse intestinal inflammatory pathways. These evidence shed a light in the dynamic intestinal microenvironment in which increased regenerative epithelial cell turnover, mutagenic insults, and chronic DNA damages triggered by factors within an inflammatory cell-rich microenvironment act synergistically to favor cancer onset and progression.

A role for orphan nuclear receptor liver receptor homolog-1 (LRH-1, NR5A2) in primordial follicle activation

Liver receptor homolog-1 (NR5A2) is expressed specifically in granulosa cells of developing ovarian follicles where it regulates the late stages of follicle development and ovulation. To establish its effects earlier in the trajectory of follicular development, NR5A2 was depleted from granulosa cells of murine primordial and primary follicles. Follicle populations were enumerated in neonates at postnatal day 4 (PND4) coinciding with the end of the formation of the primordial follicle pool. The frequency of primordial follicles in PND4 conditional knockout (cKO) ovaries was greater and primary follicles were substantially fewer relative to control (CON) counterparts. Ten-day in vitro culture of PND4 ovaries recapitulated in vivo findings and indicated that CON mice developed primary follicles in the ovarian medulla to a greater extent than did cKO animals. Two subsets of primordial follicles were observed in wildtype ovaries: one that expressed NR5A2 and the second in which the transcript was absent. Neither expressed the mitotic marker. KI-67, indicating their developmental quiescence. RNA sequencing on PND4 demonstrated that loss of NR5A2 induced changes in 432 transcripts, including quiescence markers, inhibitors of follicle activation, and regulators of cellular migration and epithelial-to-mesenchymal transition. These experiments suggest that NR5A2 expression poises primordial follicles for entry into the developing pool.

Nuclear receptors: Key regulators of somatic cell functions in the ovulatory process

The development of the ovarian follicle to its culmination by ovulation is an essential element of fertility. The final stages of ovarian follicular growth are characterized by granulosa cell proliferation and differentiation, and steroid synthesis under the influence of follicle-stimulating hormone (FSH). The result is a population of granulosa cells poised to respond to the ovulatory surge of luteinizing hormone (LH). Members of the nuclear receptor superfamily of transcription factors play indispensable roles in the regulation of these events. The key regulators of the final stages of follicular growth that precede ovulation from this family include the estrogen receptor beta (ESR2) and the androgen receptor (AR), with additional roles for others, including steroidogenic factor-1 (SF-1) and liver receptor homolog-1 (LRH-1). Following the LH surge, the mural and cumulus granulosa cells undergo rapid changes that result in expansion of the cumulus layer, and a shift in ovarian steroid hormone biosynthesis from estradiol to progesterone production. The nuclear receptor best associated with these events is LRH-1. Inadequate cumulus expansion is also observed in the absence of AR and ESR2, but not the progesterone receptor (PGR). The terminal stages of ovulation are regulated by PGR, which increases the abundance of the proteases that are directly responsible for rupture. It further regulates the prostaglandins and cytokines associated with the inflammatory-like characteristics of ovulation. LRH-1 regulates PGR, and is also a key regulator of steroidogenesis, cellular proliferation, and cellular migration, and cytoskeletal remodeling. In summary, nuclear receptors are among the panoply of transcriptional regulators with roles in ovulation, and several are necessary for normal ovarian function.

Enantiomer-specific activities of an LRH-1 and SF-1 dual agonist

Chirality is an important consideration in drug development: it can influence recognition of the intended target, pharmacokinetics, and off-target effects. Here, we investigate how chirality affects the activity and mechanism of action of RJW100, a racemic agonist of the nuclear receptors liver receptor homolog-1 (LRH-1) and steroidogenic factor-1 (SF-1). LRH-1 and SF-1 modulators are highly sought as treatments for metabolic and neoplastic diseases, and RJW100 has one of the few scaffolds shown to activate them. However, enantiomer-specific effects on receptor activation are poorly understood. We show that the enantiomers have similar binding affinities, but RR-RJW100 stabilizes both receptors and is 46% more active than SS-RJW100 in LRH-1 luciferase reporter assays. We present an LRH-1 crystal structure that illuminates striking mechanistic differences: SS-RJW100 adopts multiple configurations in the pocket and fails to make an interaction critical for activation by RR-RJW100. In molecular dynamics simulations, SS-RJW100 attenuates intramolecular signalling important for coregulator recruitment, consistent with previous observations that it weakly recruits coregulators in vitro. These studies provide a rationale for pursuing enantiomerically pure RJW100 derivatives: they establish RR-RJW100 as the stronger LRH-1 agonist and identify a potential for optimizing the SS-RJW100 scaffold for antagonist design.

LRH-1 (liver receptor homolog-1) derived affinity peptide ligand to inhibit interactions between β-catenin and LRH-1 in pancreatic cancer cells: from computational design to experimental validation

Poor prognosis, rapid progression and the lack of an effective treatment make pancreatic cancer one of the most lethal malignancies. Recent studies point to a role for liver receptor homolog-1 (LRH-1) in pathogenesis of pancreatic cancer and suggest prevention of the β-catenin/LRH-1 complex formation as a potential strategy for inhibition of the pancreas cancer cells progression. In the current investigation, we have followed a biomimetic strategy and designed an affinity peptide with sequence DEMEEPQQTE to inhibit formation of the β-catenin/LRH-1 complex. Quantitative real-time PCR experiments on the AsPC-1 pancreatic metastatic cells showed that the peptide has an inhibitory effect on the Wnt signaling proliferation line by reducing the expression levels of the CCND1, CCNE1, and MYC genes. Furthermore, the increased expression level of BAX gene showed that AsPC-1 cells were directed to the apoptosis pathway. At last, POU5F1, KLF4, and CD44 gene expression levels suggested that the peptide has an inhibitory effect on the stemness feature of the AsPC-1 cells. Here, we introduced a novel peptide inhibitor targeting an important protein–protein interaction, the β-catenin/LRH-1 complex, which may provide highly promising starting points for subsequent drug design. Communicated by Ramaswamy H. Sarma

MicroRNA-219-5p participates in cyanotic congenital heart disease progression by regulating cardiomyocyte apoptosis.

MicroRNAs (miRs) play important roles in the protection against and development of congenital heart disease (CHD). However, the role and potential mechanisms of miR-219-5p in cyanotic CHD remains unclear. Reverse transcription-quantitative PCR (RT-qPCR) was used to measure miR-219-5p levels in cyanotic CHD and hypoxia-induced H9C2 cells. Dual luciferase reporter gene assay was used to confirm whether liver receptor homolog-1 (LRH-1) was a direct target of miR-219-5p. miR-219-5p inhibitor and LRH-1-small interfering RNA were transfected into H9C2 cells under hypoxic conditions to investigate the role of miR-219-5p in hypoxia-induced H9C2 cells. Subsequently, cell viability was detected using an MTT assay and cell apoptosis was detected using flow cytometry. In addition, RT-qPCR and western blotting assays were performed to detect the mRNA and protein expression of LRH-1, cyclin D1 and β-catenin, respectively. The data showed that miR-219-5p expression was higher in patients with cyanotic CHD compared with patients with acyanotic CHD gradually increased in H9C2 cells with prolonged hypoxia time. Dual luciferase reporter assay results showed that LRH-1 was a direct target gene of miR-219-5p. Inhibition of miR-219-5p reversed hypoxia-induced cell viability reduction and attenuated hypoxia-induced cell apoptosis. In addition, hypoxia induction inhibited the expression of LRH-1, cyclin D1 and β-catenin, which was reversed by miR-219-5p inhibitor. However, LRH-1 downregulation reversed the miR-219-5p inhibitor enhanced cell viability, decreased cell apoptosis and increased expression of LRH-1, cyclin D1 and β-catenin in hypoxia-treated cardiomyocytes. The present results demonstrated that downregulation of miR-219-5p promoted the expression of the LRH-1/Wnt/β-catenin signaling pathway-associated components, reduced cardiomyocyte apoptosis and increased cell growth under hypoxic conditions. miR-219-5p may be a potential therapeutic target for cyanotic CHD therapy.

LRH1 Promotes Tumor Cell Proliferation and Migration and Is Correlated With Poor Prognosis in Ovarian Cancer.

BackgroundLiver receptor homolog 1 (LRH1) plays a vital role in several human cancers, but its role in ovarian cancer (OC) remains unclear. We aimed to explore the functions of LRH1 and its clinical relevance.MethodsLRH1 expression was evaluated by immunohistochemistry and reverse transcription quantitative polymerase chain reaction (RT-qPCR). The effects of LRH1 on tumor cell proliferation, migration and epithelial–mesenchymal transition (EMT) were evaluated in vitro. Furthermore, bioinformatics analysis was applied to predict the functions of LRH1.ResultsRT-qPCR showed that LRH1 mRNA expression was higher in the invasive lesions (P < 0.05). LRH1 overexpression was extremely related with elevated International Federation of Gynecology and Obstetrics (FIGO) stage (P = 0.001), lymph node metastasis (P = 0.011), peritoneal metastasis (P = 0.001), and platinum resistance (P = 0.037). Furthermore, LRH1 expression was an independent prognostic index for disease-free survival in patients with OC (P = 0.041). LRH1 overexpression (P = 0.011), FIGO stage (P < 0.001), and ascites (P = 0.015) independently affected peritoneal metastasis in patients with OC. LRH1 knockdown significantly inhibited the proliferation, migration, and EMT of human OC cells (P < 0.05); however, it reversed cisplatin resistance. Bioinformatics analysis indicated that the functions of LRH1 were associated with the PRC1 complex, nuclear ubiquitin ligase complex, and Polycomb-group (PcG) proteins.ConclusionsThis study provides evidence of the predictive value of LRH1 on peritoneal metastasis and poor outcome and highlights the potential role of LRH1 as a biomarker for the targeted therapy of OC. Furthermore, LRH1 promotes OC cell proliferation, migration, and EMT in vitro, and its functions may be associated with PRC1 complex, nuclear ubiquitin ligase complex, and PcG proteins.

Effect of selected bisphenol derivatives on nuclear receptor expression in ovarian cell line COV434.

Objectives. Bisphenol A (BPA), as an indispensable plastic additive, has also been proven as an endocrine disruptor associated with adverse health effects including impaired ovarian function and cancer. Due to the restrictions of its usage, several analogs have been employed to replace BPA. Although many studies revealed a harmfulness in the biological effects of BPA analogs, their specific targets remain largely unknown. Nuclear receptors (NRs) may be one of the most important targets of bisphenols. Therefore, in this study, our attention was directed to explore the effect of BPA and its analogs, AF and S, on the mRNA expression of selected NRs involved in the steroidogenic and carcinogenic pathways in the human granulosa cell line COV434. The NRs investigated included: thyroid hormone receptor α (THRA), peroxisome proliferator activating receptor β/δ (PPARD), retinoid X receptor α (RXRA), chicken ovalbumin upstream promoter-transcription factor II (COUPTFII), nuclear receptor-related protein 1 (NURR1), and liver receptor homolog-1 (LRH1).Methods. COV434 cells were treated with the bisphenols at the concentrations of 10-9 M, 10-7 M, and 10-5 M, and after 24 and 48 h, cell viability was monitored by the MTS assay and gene expressions were analyzed using RT-qPCR.Results. Bisphenol treatment did not alter the COV434 cell viability. After 24 h, the expression of neither of the NRs was changed. Likewise, after 48 h, the expression of the selected genes was not altered. However, both BPAF and BPS increased, at the highest concentration (10-5 M) used, the mRNA levels of both PPARD and NURR1 NRs after 48 h of the treatment. In the BPA-treated groups, no significant upregulation was observed.Conclusions. In the present study, the effect of bisphenols on COUP-TFII, Nurr1, and LRH-1 NRs was investigated for the first time. Although generally we did not observe that BPs provoked any alterations in the expression of the selected NRs in COV434 cells, at specific concentrations and time points they might alter mRNA expression of certain NRs (NURR1, PPARD).

Transcriptional Regulation of Müllerian Inhibiting Substance (MIS) and Establishment of a Gonadal Somatic Cell Line Using mis-GFP Transgenic Medaka (Oryzias latipes).

In vertebrate germ cell differentiation, gonadal somatic cells and germ cells are closely related. By analyzing this relationship, it has recently been reported in mammals that primordial germ cells (PGCs), induced from pluripotent stem cells and germline stem cells, can differentiate into functional gametes when co-cultured in vitro with fetal gonadal somatic cells. In some fish species, differentiation into functional sperm by reaggregation or co-culture of gonadal somatic cells and germ cells has also been reported; however, the relationship between gonadal somatic cells and germ cells in these species is not well-understood. Here, we report the transcriptional regulation of Müllerian inhibiting substance (MIS) and the establishment of a gonadal somatic cell line using mis-GFP transgenic fish, in medaka (Oryzias latipes)—a fish model which offers many advantages for molecular genetics. MIS is a glycoprotein belonging to the transforming growth factor β superfamily. In medaka, mis mRNA is expressed in gonadal somatic cells of both sexes before sex differentiation, and MIS regulates the proliferation of germ cells during this period. Using luciferase assays, we found that steroidogenic factor 1 (SF1) and liver receptor homolog 1 (LRH1) activate medaka mis gene transcription, probably by binding to the mis promoter. We also report that mis-GFP transgenic medaka emit GFP fluorescence specific to gonadal somatic cells in the gonads. By fusing Sertoli cells from transgenic medaka with a cell line derived from medaka hepatoma cancer, we produced a hybridoma cell line that expresses gonadal somatic cell-specific markers, including Sertoli and Leydig cell markers. Moreover, embryonic PGCs co-cultured with the established hybridoma, as feeder cells, proliferated and formed significant colonies after 1 week. PGCs cultured for 3 weeks expressed a germ cell marker dnd, as well as the meiotic markers sycp1 and sycp3. Thus, we here provide the first evidence in teleosts that we have successfully established a gonadal somatic cell-derived hybridoma that can induce both the proliferation and meiosis of germ cells.

Nuclear-mitochondrial crosstalk: On the role of the nuclear receptor liver receptor homolog-1 (NR5A2) in the regulation of mitochondrial metabolism, cell survival, and cancer.

Liver receptor homolog-1 (LRH-1, NR5A2) is an orphan nuclear receptor with widespread activities in the regulation of development, stemness, metabolism, steroidogenesis, and proliferation. Many of the LRH-1-regulated processes target the mitochondria and associated activities. While under physiological conditions, a balanced LRH-1 expression and regulation contribute to the maintenance of a physiological equilibrium, deregulation of LRH-1 has been associated with inflammation and cancer. In this review, we discuss the role and mechanism(s) of how LRH-1 regulates metabolic processes, cell survival, and cancer in a nuclear-mitochondrial crosstalk, and evaluate its potential as a pharmacological target.

Canonical Wnt Pathway Controls mESC Self-Renewal Through Inhibition of Spontaneous Differentiation via β-Catenin/TCF/LEF Functions.

SummaryThe Wnt/β-catenin signaling pathway is a key regulator of embryonic stem cell (ESC) self-renewal and differentiation. Constitutive activation of this pathway has been shown to increase mouse ESC (mESC) self-renewal and pluripotency gene expression. In this study, we generated a novel β-catenin knockout model in mESCs to delete putatively functional N-terminally truncated isoforms observed in previous knockout models. We showed that aberrant N-terminally truncated isoforms are not functional in mESCs. In the generated knockout line, we observed that canonical Wnt signaling is not active, as β-catenin ablation does not alter mESC transcriptional profile in serum/LIF culture conditions. In addition, we observed that Wnt signaling activation represses mESC spontaneous differentiation in a β-catenin-dependent manner. Finally, β-catenin (ΔC) isoforms can rescue β-catenin knockout self-renewal defects in mESCs cultured in serum-free medium and, albeit transcriptionally silent, cooperate with TCF1 and LEF1 to inhibit mESC spontaneous differentiation in a GSK3-dependent manner.

Temporal activation of LRH-1 and RAR-γ in human pluripotent stem cells induces a functional naïve-like state.

Naïve pluripotency can be established in human pluripotent stem cells (hPSCs) by manipulation of transcription factors, signaling pathways, or a combination thereof. However, differences exist in the molecular and functional properties of naïve hPSCs generated by different protocols, which include varying similarities with pre-implantation human embryos, differentiation potential, and maintenance of genomic integrity. We show here that short treatment with two chemical agonists (2a) of nuclear receptors, liver receptor homologue-1 (LRH-1) and retinoic acid receptor gamma (RAR-γ), along with 2i/LIF (2a2iL) induces naïve-like pluripotency in human cells during reprogramming of fibroblasts, conversion of pre-established hPSCs, and generation of new cell lines from blastocysts. 2a2iL-hPSCs match several defined criteria of naïve-like pluripotency and contribute to human-mouse interspecies chimeras. Activation of TGF-β signaling is instrumental for acquisition of naïve-like pluripotency by the 2a2iL induction procedure, and transient activation of TGF-β signaling substitutes for 2a to generate naïve-like hPSCs. We reason that 2a2iL-hPSCs are an easily attainable system to evaluate properties of naïve-like hPSCs and for various applications.

Development of a new class of liver receptor homolog-1 (LRH-1) agonists by photoredox conjugate addition

LRH-1 is a nuclear receptor that regulates lipid metabolism and homeostasis, making it an attractive target for the treatment of diabetes and non-alcoholic fatty liver disease. Building on recent structural information about ligand binding from our labs, we have designed a series of new LRH-1 agonists that further engage LRH-1 through added polar interactions. While the current synthetic approach to this scaffold has, in large part, allowed for decoration of the agonist core, significant variation of the bridgehead substituent is mechanistically precluded. We have developed a new synthetic approach to overcome this limitation, identified that bridgehead substitution is necessary for LRH-1 activation, and described an alternative class of bridgehead substituents for effective LRH-1 agonist development. We determined the crystal structure of LRH-1 bound to a bridgehead-modified compound, revealing a promising opportunity to target novel regions of the ligand binding pocket to alter LRH-1 target gene expression.

Integrated Structural Modeling of Full-Length LRH-1 Reveals Inter-domain Interactions Contribute to Receptor Structure and Function

Liver receptor homolog-1 (LRH-1; NR5A2) is a nuclear receptor that regulates a diverse array of biological processes. In contrast to dimeric nuclear receptors, LRH-1 is an obligate monomer and contains a subtype-specific helix at the C terminus of the DNA-binding domain (DBD), termed FTZ-F1. Although detailed structural information is available for individual domains of LRH-1, it is unknown how these domains exist in the intact nuclear receptor. Here, we developed an integrated structural model of human full-length LRH-1 using a combination of HDX-MS, XL-MS, Rosetta computational docking, and SAXS. The model predicts the DBD FTZ-F1 helix directly interacts with ligand binding domain helix 2. We confirmed several other predicted inter-domain interactions via structural and functional analyses. Comparison between the LRH-1/Dax-1 co-crystal structure and the integrated model predicted and confirmed Dax-1 co-repressor to modulate LRH-1 inter-domain dynamics. Together, these data support individual LRH-1 domains interacting to influence receptor structure and function.