LRH-1 Research Articles

Dexamethasone downregulates expression of several genes encoding orphan nuclear receptors that are important to steroidogenesis in stallion testes

Glucocorticoids impair testosterone synthesis by an unknown mechanism. Stallions treated with the synthetic glucocorticoid dexamethasone had testes collected at 6 or 12 hours postinjection. The testicular expression of selected genes encoding nuclear receptors and steroidogenic enzymes was measured. At 6 hours, dexamethasone treatment decreased levels of NR0B2, NR4A1, NR5A1, and NR5A2 messenger RNAs (mRNAs) and NR5A2 mRNA levels remained depressed at 12 hours. In contrast, dexamethasone increased levels of NFKBIA mRNA at both time points. At 6 hours, dexamethasone did not alter levels of NR0B1, NR2F1, NR2F2, NR3C1, CYP11A1, CYP17A1, CYP19A1, DHCR24, GSTA3, HSD3B2, HSD17B3, LHCGR, or STAR mRNAs. In primary cultures of Leydig cells, 10 -9 and 10 -7 M dexamethasone decreased levels of NR4A1 and NR5A1 mRNAs and increased those of NFKBIA mRNA. Our discovery that dexamethasone downregulates NR4A1, NR5A1, and NR5A2 genes, known to be important for testicular functions, may be part of the mechanism by which glucocorticoids acutely decreases testosterone.

Signaling through non-membrane nuclear phosphoinositide binding proteins in human health and disease

Phosphoinositide membrane signaling is critical for normal physiology, playing well-known roles in diverse human pathologies. The basic mechanisms governing phosphoinositide signaling within the nucleus, however, have remained deeply enigmatic owing to their presence outside the nuclear membranes. Over 40% of nuclear phosphoinositides can exist in this non-membrane state, held soluble in the nucleoplasm by nuclear proteins that remain largely unidentified. Recently, two nuclear proteins responsible for solubilizing phosphoinositides were identified, steroidogenic factor-1 (SF-1; NR5A1) and liver receptor homolog-1 (LRH-1; NR5A2), along with two enzymes that directly remodel these phosphoinositide/protein complexes, phosphatase and tensin homolog (PTEN; MMAC) and inositol polyphosphate multikinase (IPMK; ipk2). These new footholds now permit the assignment of physiological functions for nuclear phosphoinositides in human diseases, such as endometriosis, nonalcoholic fatty liver disease/steatohepatitis, glioblastoma, and hepatocellular carcinoma. The unique nature of nuclear phosphoinositide signaling affords extraordinary clinical opportunities for new biomarkers, diagnostics, and therapeutics. Thus, phosphoinositide biology within the nucleus may represent the next generation of low-hanging fruit for new drugs, not unlike what has occurred for membrane phosphatidylinositol 3-kinase drug development. This review connects recent basic science discoveries in nuclear phosphoinositide signaling to clinical pathologies, with the hope of inspiring development of new therapies.

Intestinal glucocorticoid synthesis enzymes in pediatric inflammatory bowel disease patients.

Inflammatory bowel disease (IBD), such as Crohn's disease and ulcerative colitis are devastating chronic immunopathologies of the intestinal mucosa, which are frequently treated by immunosuppressive glucocorticoids. Endogenous glucocorticoids are not only produced by the adrenal glands, but also by the intestinal epithelium. Local glucocorticoid synthesis critically contributes to the immune homeostasis of the intestinal mucosa. As defective intestinal glucocorticoid synthesis has been associated with the development of IBD, we investigated the expression of steroidogenic enzymes and the key transcriptional regulator Liver Receptor Homolog-1 (LRH-1/NR5A2) in ileal and colonic biopsies human pediatric IBD and control patients. Furthermore, the induction of steroidogenic enzymes and their transcriptional regulation by LRH-1 was investigated in a mouse model of experimental colitis. These analyses revealed that colitis-induced expression of steroidogenic enzymes in the murine colon is dependent on the presence of LRH-1, as intestinal deletion of LRH-1 strongly reduced their colitis-induced expression. Similarly, a strong correlation between the expression of LRH-1 and different steroidogenic enzymes was seen in intestinal biopsies of human pediatric patients. Importantly, reduced expression of hydroxysteroid dehydrogenase 11B1 (HSD11B1) was observed in IBD patients compared to control patients, suggesting that defective local reactivation of glucocorticoids could contribute to the pathogenesis of IBD.

N-3 Fatty Acids Abrogate Dyslipidemia-Induced Changes in Bile Acid Uptake, Synthesis, and Transport in Young and Aged Dyslipidemic Rats.

In this study, the effect of n-3 fatty acids (FA) [α-linolenic acid (ALA) and eicosapentaenoic acid (EPA) + docosahexaenoic acid (DHA)] on the intestinal bile acid (BA) uptake, hepatic BA synthesis, and enterohepatic bile acid transporters (BAT) was assessed in young and aged dyslipidemic rats. Dyslipidemia was induced in young and aged rats by feeding a high-fat (HF) diet. Experimental groups received diets containing canola oil (HF + CNO) and fish oil (HF + FO) as a source of ALA and EPA + DHA, respectively. After 60 days of feeding, intestinal BA uptake and expression of apical sodium-dependent bile acid transporter (Asbt), organic solute transporter-alpha/beta (Osta/b) messenger RNA (mRNA), and hepatic expression of Na+ taurocholate cotransporting polypeptide (Ntcp), bile salt export pump (Bsep), cholesterol 7-α hydroxylase A1 (Cyp7a1), Farnesoid X receptor (Fxr), small heterodimer partner-1 (Shp), liver receptor homolog-1 (Lrh-1), and hepatic nuclear factor-4 alpha (Hnf4a) mRNA were measured. Hepatic 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA) reductase activity and total BA in serum, liver, and feces were assessed. The dyslipidemic HF group had: (1) increased intestinal BA uptake and Asbt and Osta/b mRNA expression, (2) increased BA in serum, (3) decreased hepatic expression of Ntcp, Bsep, and Cyp7a1 mRNA, (4) increased activity of HMG-CoA reductase, (5) increased hepatic expression of Fxr and Shp mRNA, (6) decreased hepatic expression of Lrh-1 and Hnf4a mRNA, and (7) decreased BA in feces, when compared to control, HF + CNO, and HF + FO groups. Immunostaining revealed increased expression of intestinal Asbt and hepatic Ntcp protein in the HF group when compared to control, HF + CNO, and HF + FO groups. n-3 FA abrogated dyslipidemia-induced changes in the intestinal uptake, hepatic synthesis, and enterohepatic transporters of BA in both young and aged rats. EPA + DHA was more effective than ALA in modulating dyslipidemia-induced changes.

Regulation of Hepatic Long Noncoding RNAs by Pregnane X Receptor and Constitutive Androstane Receptor Agonists in Mouse Liver.

Altered expression of long noncoding RNAs (lncRNAs) by environmental chemicals modulates the expression of xenobiotic biotransformation-related genes and may serve as therapeutic targets and novel biomarkers of exposure. The pregnane X receptor (PXR/NR1I2) is a critical xenobiotic-sensing nuclear receptor that regulates the expression of many drug-processing genes, and it has similar target-gene profiles and DNA-binding motifs with another xenobiotic-sensing nuclear receptor, namely, constitutive andronstrane receptor (CAR/Nr1i3). To test our hypothesis that lncRNAs are regulated by PXR in concert with protein-coding genes (PCGs) and to compare the PXR-targeted lncRNAs with CAR-targeted lncRNAs, RNA-Seq was performed from livers of adult male C57BL/6 mice treated with corn oil, the PXR agonist PCN, or the CAR agonist 1, 4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP). Among 125,680 known lncRNAs, 3843 were expressed in liver, and 193 were differentially regulated by PXR (among which 40% were also regulated by CAR). Most PXR- or CAR-regulated lncRNAs were mapped to the introns and 3'-untranslated regions (UTRs) of PCGs, as well as intergenic regions. Combining the RNA-Seq data with a published PXR chromatin immunoprecipitation coupled with high-throughput sequencing; cytochrome P450 (P450; ChIP-Seq) data set, we identified 774 expressed lncRNAs with direct PXR-DNA binding sites, and 26.8% of differentially expressed lncRNAs had changes in PXR-DNA binding after PCN exposure. De novo motif analysis identified colocalization of PXR with liver receptor homolog (LRH-1), which regulates bile acid synthesis after PCN exposure. There was limited overlap of PXR binding with an epigenetic mark for transcriptional activation (histone-H3K4-di-methylation, H3K4me2) but no overlap with epigenetic marks for transcriptional silencing [H3 lysine 27 tri-methylation (H3K27me3) and DNA methylation]. Among differentially expressed lncRNAs, 264 were in proximity of PCGs, and the lncRNA-PCG pairs displayed a high coregulatory pattern by PXR and CAR activation. This study was among the first to demonstrate that lncRNAs are regulated by PXR and CAR activation and that they may be important regulators of PCGs involved in xenobiotic metabolism.

Targeting LRH‑1 in hepatoblastoma cell lines causes decreased proliferation.

Hepatoblastoma is the most common malignant liver tumor in children. Since it is often unresectable and exhibits drug resistance, the treatment of advanced hepatoblastoma is challenging. The orphan nuclear receptor liver receptor homolog-1 (LRH-1) serves prominent roles in malignancy; however, to the best of our knowledge, the role of LRH-1 in hepatoblastoma remains unknown. In the present study, human hepatoblastoma cell lines were analyzed; the mRNA and protein expression levels of LRH-1 were significantly higher in HepG2 and HuH6 cells compared with those in HepT1 cells and control THLE-2 cells. Knockdown of LRH-1 resulted in decreased HepG2 and HuH6 cell proliferation via downregulation of cyclin D1 (CCND1) and c-Myc. Furthermore, treatment with an LRH-1 antagonist (LRA) inhibited the proliferation and colony formation of cell lines in a dose-dependent manner, and induced cell cycle arrest at G1 phase through inhibition of CCND1 expression. Finally, LRA treatment enhanced the cytotoxic effects of doxorubicin on hepatoblastoma cells. Collectively, these findings suggested that LRH-1 may have an important role in the progression of hepatoblastoma and implicated LRA as a novel, potential therapeutic agent for the treatment of hepatoblastoma.

Development of Hybrid Phospholipid Mimics as Effective Agonists for Liver Receptor Homologue-1.

The orphan nuclear receptor Liver Receptor Homologue-1 (LRH-1) is an emerging drug target for metabolic disorders. The most effective known LRH-1 modulators are phospholipids or synthetic hexahydropentalene compounds. While both classes have micromolar efficacy, they target different portions of the ligand binding pocket and activate LRH-1 through different mechanisms. Guided by crystallographic data, we combined aspects of both ligand classes into a single scaffold, resulting in the most potent and efficacious LRH-1 agonists to date.

Local synthesis of immunosuppressive glucocorticoids in the intestinal epithelium regulates anti-viral immune responses

The nuclear receptor Small Heterodimer Partner (SHP) is a transcriptional target and inhibitor of Liver Receptor Homolog 1 (LRH-1), the transcriptional regulator of intestinal glucocorticoid (GC) synthesis. The role of SHP in the regulation of intestinal GC synthesis and its impact on T cell-mediated anti-viral immune responses in the intestinal mucosa are currently not understood. Lymphocytic choriomeningitis virus (LCMV) infection promoted intestinal GC synthesis, which was enhanced in SHP-deficient mice. Intestinal GC suppressed the expansion and altered the activation of virus-specific T cells. In contrast, deletion of LRH-1 reduced intestinal GC synthesis and accelerated the expansion of cytotoxic T cells post LCMV infection. These findings show that virus-induced intestinal GC synthesis is controlled by LRH-1 and SHP, and that local steroidogenesis contributes to the maintenance of intestinal immune homeostasis. Thus, LRH-1-regulated intestinal GC synthesis could represent an interesting therapeutic target in the treatment of inflammatory disorders.

LRH-1 drives hepatocellular carcinoma partially through induction of c-myc and cyclin E1, and suppression of p21.

BackgroundTo explore potential therapeutic target is one of the areas of great interest in both clinical and basic hepatocellular carcinoma (HCC) studies. Nuclear receptor liver receptor homolog-1 (LRH-1, NR5A2) is proved to play a positive role in several cancers including breast cancer, pancreatic cancer and intestinal cancer in recent years. However, the exact role of LRH-1 in the development and progression of HCC is not fully elucidated.MethodsThe LRH-1 expression level in HCC clinical samples was examined by immunohis-tochemistry (IHC). Stable LRH-1-suppressed HepG2 clones (HepG2LRH-1/-) were generated by transcription activator-like effector nucleases (TALENs) and both in vitro and in vivo experiments were conducted.ResultsWe confirmed that LRH-1 showed an increased expression pattern in HCC clinical samples. Our in vitro and in vivo results indicated that suppression of LRH-1 in HepG2 significantly attenuated its proliferation rate and tumorigenic capacity. Gene expression microarray analysis indicated that LRH-1mostly regulated gene expression involved in cell cycle. In addition, our gain-of-function experiments indicated that ectopic expression of LRH-1 dramatically induced the mRNA and protein levels of c-myc and cyclin E1, while attenuating the expression of p21.ConclusionOur results suggest that LRH-1 might be a potential therapeutic target for clinical HCC treatment.

Liver receptor homologue 1, a novel prognostic marker in colon cancer patients.

Liver receptor homologue 1 (LRH-1) is an orphan nuclear receptor that is highly expressed in a variety of cancer tissues, promotes tumor cell proliferation and metastasis, and is involved in the tumor cell cycle and apoptosis. The aim of the present study was to assess the association between the expression of LRH-1 and the prognosis of patients with colon cancer. Immunohistochemistry was used to detect the expression of LRH1 in 128 cases of colon cancer and adjacent tissues. The 5-year survival rate was obtained from telephone follow-up data, outpatient review and through access to medical records. Positive expression of LRH-1 was found in 108/128 colon cancer samples, compared with 17/128 normal tissues. Statistical analysis showed that positive LRH-1 expression was significantly associated with clinical pathological stage, depth of invasion and lymph node metastasis. The overall survival (OS) rate of patients with positive LRH-1 expression was significantly lower than that of patients with low expression. Multivariate analysis showed that LRH-1 expression could be used as an independent predictor of OS. In conclusion, the present findings suggest that LRH-1 may serve an important role in the development and progression of colon cancer, with potential value as a prognostic molecular marker that could be used to assist in the diagnosis and evaluation of colon cancer. LRH-1 may become a target for novel therapies for patients with colon cancer.

Effects of Dietary Daidzein Supplementation on Reproductive Performance, Serum Hormones, and Reproductive-Related Genes in Rats.

The aim of this study was to investigate the effect of dietary daidzein supplementation on reproductive performance in rats. A total of twenty-four female Sprague–Dawley (SD) rats were randomly allocated to two groups and fed either with a basal diet (CON) or basal diet containing 50 mg/kg daidzein (DAI) from gestation until delivery stage. The results show that daidzein supplementation significantly increased the total litter weight and the total viable newborn weight (p < 0.05). Interestingly, daidzein supplementation acutely elevated the concentrations of serum estrogen, progesterone and insulin-like growth factor-1 (p < 0.01) after the maternal rats’ delivery. The concentrations of serum immunoglobulin A (IgA) and immunoglobulin G (IgG) were also significantly higher in the DAI maternal rats than in the CON maternal rats (p < 0.05). Moreover, daidzein significantly increased the total antioxidant capacity (T-AOC) in maternal rats’ sera and in newborns (p < 0.05) and elevated the concentration of superoxide dismutase (SOD) in both the maternal rats’ sera and their ovaries (p < 0.05). Importantly, daidzein supplementation significantly elevated the expression levels of estrogen receptor β (ERβ) and NR5A2 genes in maternal rats’ ovaries (p < 0.05) and downregulated the expression level of prolactin receptor (PRLR) in newborns (p < 0.05). These results suggest that dietary daidzein supplementation improves reproductive performance and fetal development in rats, which is associated with changes in serum hormones, tissue antioxidant capacity, and expression levels of reproductive-related genes, both in maternal rats and their offspring.

Targeting LRH-1/NR5A2 to treat type 1 diabetes mellitus.

Type 1 diabetes mellitus (T1DM) is defined as an autoimmune disease that targets the selective destruction of islet insulin-producing beta cells by infiltrating immune cells (insulitis). As a result, the organism is no longer able to produce insulin and develops hyperglycaemia and, if untreated, death. Despite advances in medical device technology and insulin analogues as well as strives in generating in vitro insulin-producing cells, there is still no robust therapy to substitute and protect beta cells that are lost in T1DM. Clinical trials aimed at blocking the immune-mediated beta cell destruction have had moderate success leaving a gap in our understanding of disease aetiology. Such breach in knowledge may stem from the oversight that inhibiting the immune attack likely impairs beta cell regeneration and emphasizes a fundamental paradigm in the approach to treat the disease: A non-mutually exclusive strategy in which the uncontrolled self-directed inflammatory immune response (and not the global immune system) as well as beta cell regeneration are exquisitely fine tuned in order to successfully regain immunological tolerance and restoration of a functional beta cell mass. As such, defining factors that can guide a pro-inflammatory immune cell destructive environment towards an anti-inflammatory environment facilitating beta cell survival and stimulate regeneration would define an unprecedented class of immune-regenerative therapeutic agents for T1DM. In our recent study we identify the liver receptor homolog 1 (LRH-1, also known as NR5A2) as a ‘druggable’ target that fulfills these criteria restoring glycemic control in various mouse models of T1DM as well as improving human islet survival and function both in vitro and in vivo (Nat Comms, 9:1488).

Significant role of microRNA‑219‑5p in diabetic retinopathy and its mechanism of action.

Diabetic retinopathy (DR) is the leading cause of blindness and visual impairment. The role of microRNA (miRNA) in DR remains largely unknown. The present study aimed to investigate the role of miR‑219‑5p in the progression of DR. Human retinal pigment epithelial (RPE) cells were treated with a high concentration of glucose (50mM D‑glucose) for 24h and the miR‑219‑5p level was detected using reverse transcription‑quantitative polymerase chain reaction. The results revealed that miR‑219‑5p was significantly upregulated by high glucose (HG) treatment. To explore the role and mechanism of miR‑219‑5p in DR progression, miR‑219‑5p was downregulated in ARPE‑19 cells. An MTT assay and flow cytometry were used to determine the level of viability and apoptosis of ARPE‑19 cells, respectively. MicroRNA.org was used to predict the targets of miR‑219‑5p and the prediction was investigated using a dual‑luciferase reporter assay. In addition, the level of associated proteins were measured using western blot analysis. It was observed that liver receptor homolog‑1 (LRH‑1) was a direct target of miR‑219‑5p. LRH‑1 was significantly downregulated in ARPE‑19 cells following HG treatment and negatively regulated by miR‑219‑5p in ARPE‑19 cells. MiR‑219‑5p inhibitor significantly prevented ARPE‑19 cell apoptosis induced by HG treatment and cell viability was markedly promoted. The results also suggested that the LRH‑1/Wnt/β‑Catenin signaling pathway was activated by miR‑219‑5p inhibition. In addition, it was revealed that LRH‑1 inhibition eliminated the effects of miR‑219‑5p inhibitor on ARPE‑19 cells. In conclusion, the results indicated that miR‑219‑5p was involved in the progression of DR through regulating human RPE cell apoptosis by modulation of the LRH‑1/Wnt/β‑Catenin signaling pathway.

Nuclear Receptor LRH-1 Functions to Promote Castration-Resistant Growth of Prostate Cancer via Its Promotion of Intratumoral Androgen Biosynthesis.

Targeting of steroidogenic enzymes (e.g., abiraterone acetate targeting CYP17A1) has been developed as a novel therapeutic strategy against metastatic castration-resistant prostate cancer (CRPC). However, resistance to steroidal inhibitors inevitably develops in patients, the mechanisms of which remain largely unknown. Liver receptor homolog-1 (LRH-1, NR5A2) is a nuclear receptor, originally characterized as an important regulator of some liver-specific metabolic genes. Here, we report that LRH-1, which exhibited an increased expression pattern in high-grade prostate cancer and CRPC xenograft models, functions to promote de novo androgen biosynthesis via its direct transactivation of several key steroidogenic enzyme genes, elevating intratumoral androgen levels and reactivating AR signaling in CRPC xenografts as well as abiraterone-treated CRPC tumors. Pharmacologic inhibition of LRH-1 activity attenuated LRH-1-mediated androgen deprivation and anti-androgen resistance of prostate cancer cells. Our findings not only demonstrate the significant role of LRH-1 in the promotion of intratumoral androgen biosynthesis in CRPC via its direct transcriptional control of steroidogenesis, but also suggest targeting LRH-1 could be a potential therapeutic strategy for CRPC management.Significance: These findings not only demonstrate the significant role of the nuclear receptor LRH-1 in the promotion of intratumoral androgen biosynthesis in CRPC via its direct transcriptional control of steroidogenesis, but also suggest targeting LRH-1 could be a potential therapeutic strategy for CRPC management. Cancer Res; 78(9); 2205-18. ©2018 AACR.

Liver Receptor Homolog-1 Regulates Organic Anion Transporter 2 and Docetaxel Pharmacokinetics.

Organic anion transporter 2 (OAT2/SLC22A7) is an uptake transporter that plays an important role in drug disposition. Here, we investigate a potential role of liver receptor homolog-1 (Lrh-1) in regulation of Oat2 and docetaxel pharmacokinetics. Hepatoma cells (Hepa1-6 and HepG2 cells) were transfected with Lrh-1/LRH-1 expression vector or siRNA. The relative mRNA and protein levels of Oat2/OAT2 in the cells or livers of Lrh-1hep-/- mice were determined by qPCR and Western blotting, respectively. Transcriptional regulation of Oat2/OAT2 by Lrh-1/LRH-1 was investigated using luciferase reporter, mobility shift, and chromatin immunoprecipitation (ChIP) assays. Pharmacokinetic studies were performed with wild-type (Lrh-1fl/fl) and Lrh-1hep-/- mice after intraperitoneal injection of docetaxel. Overexpression of Lrh-1 in Hepa1-6 cells led to significant increases in Oat2 mRNA and protein. Consistently, Lrh-1 knockdown caused decreases in Oat2 mRNA and protein, as well as reduced cellular uptake of PGE2, a prototypical substrate of Oat2. Similarly, an activation effect of LRH-1 on OAT2 expression was observed in HepG2 cells. In addition, the levels of Oat2 mRNA and protein were markedly reduced in Lrh-1hep-/- mice. Lrh-1/LRH-1 induced the transcription of Oat2/OAT2 in luciferase reporter assays. Truncation analysis revealed a potential Lrh-1 response element (-716- to -702-bp) in Oat2 promoter. Direct binding of Lrh-1 to this response element was confirmed by mobility shift and ChIP assays. Furthermore, systemic exposure of docetaxel was upregulated in Lrh-1hep-/- mice due to reduced hepatic uptake. In conclusion, Lrh-1 transcriptionally regulates Oat2, thereby impacting tissue uptake and pharmacokinetics of Oat2 substrates.

LRH-1 agonism favours an immune-islet dialogue which protects against diabetes mellitus

Type 1 diabetes mellitus (T1DM) is due to the selective destruction of islet beta cells by immune cells. Current therapies focused on repressing the immune attack or stimulating beta cell regeneration still have limited clinical efficacy. Therefore, it is timely to identify innovative targets to dampen the immune process, while promoting beta cell survival and function. Liver receptor homologue-1 (LRH-1) is a nuclear receptor that represses inflammation in digestive organs, and protects pancreatic islets against apoptosis. Here, we show that BL001, a small LRH-1 agonist, impedes hyperglycemia progression and the immune-dependent inflammation of pancreas in murine models of T1DM, and beta cell apoptosis in islets of type 2 diabetic patients, while increasing beta cell mass and insulin secretion. Thus, we suggest that LRH-1 agonism favors a dialogue between immune and islet cells, which could be druggable to protect against diabetes mellitus.

A novel estrogen receptor, G protein‐coupled receptor 30 (GPR30) plays a critical role, through a non‐transcriptional regulatory mode, in promoting the formation of estrogen (E2)‐induced cholesterol (Ch) gallstones in female mice

BackgroundCh gallstones are more common in women than in men in every population studied. We have found that the classical estrogen receptor α (ERα), but not ERβ, in the liver plays a key role in E2‐induced gallstones in female mice. We recently discovered that GPR30 is a new gallstone gene, Lith18, and is expressed in the endoplasmic reticulum, but not the nucleus, of hepatocytes. Thus, the molecular mechanisms of elucidating the lithogenic role of E2 in gallstone formation became more complicated. Our aim is to explore whether GPR30 produces an independent lithogenic action to promote hepatic Ch hypersecretion and gallstone formation in female mice. We also investigated whether the epidermal growth factor receptor (EGFR) is required for GPR30 to produce a non‐transcriptional regulatory role in inhibiting hepatic Ch 7α‐hydroxylase (CYP7A1) and the classical pathway of bile acid (BA) synthesis, leading to the availability of excess amounts of Ch for hepatic hypersecretion and gallstone formation.MethodsThe role of GPR30 in Ch crystallization and gallstone formation were investigated in ovariectomized (OVX) GPR30(−/−)/ERα(−/−) and GPR30(+/+)/ERα(−/−) mice implanted subcutaneously with pellets releasing E2 at 6 μg/day and fed a lithogenic diet for 8 wk. To study whether the metabolic abnormalities underlying the major source of the excess Ch molecules lead to Ch‐supersaturated bile as activated by GPR30, we examined the role of GPR30 in regulating CYP7A1 and the classical pathway of BA synthesis through the EGFR signaling.ResultsE2 induced rapid Ch crystallization through the liquid crystalline pathway and promoted gallstone formation in OVX GPR30(+/+)/ERα(−/−) mice fed the lithogenic diet. In contrast, the prevalence of gallstones was drastically reduced to 10% in OVX GPR30(−/−)/ERα(−/−) mice. Hepatic secretion of biliary BA was significantly reduced in mice expressing GPR30, leading to an increased ratio of Ch to BA. After E2 treatment, there was a significant increase in mRNA and protein levels of EGFR and a significant reduction in expression of liver receptor homolog‐1 (LRH‐1), coupled with reduced CYP7A1 mRNA and protein levels in OVX GPR30(+/+)/ERα(−/−), but not GPR30(−/−)/ERα(−/−), mice. In vitro studies of primary hepatocytes isolated from GPR30(+/+)/ERα(−/−) mice found that when EGFR was inhibited by AG1478, a highly potent EGFR kinase inhibitor, the mRNA and protein levels of CYP7A1 were unchanged even though GPR30 was activated by E2. However, this was not the case in GPR30(−/−)/ERα(−/−) hepatocytes.ConclusionsGPR30 regulates Cyp7a1 expression through the EGFR. In the lithogenic state, reduced hepatic synthesis of BA from the classical pathway inhibits the conversion of Ch to BA due to GPR30 repression of CYP7A1 in E2‐treated mice. As a result, a significant increase in the ratio of Ch to BA leads to the imbalance of biliary lipids for keeping Ch solubilized in bile, contributing to the formation of E2‐induced gallstones.Support or Funding InformationThis work was supported in part by research grants DK101793, DK106249 and AA03557, all from the National Institutes of Health (US Public Health Service).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Regulation by FSH of the dynamic expression of retinol-binding protein 4 in the mouse ovary

BackgroundOvarian retinoid homeostasis plays an important role in the physiological function of the ovary. Retinol-binding protein 4 (RBP4) acts as the mediator for the systemic and intercellular transport of retinol and is heavily involved in cellular retinol influx, efflux, and exchange. However, the expression patterns and regulatory mechanisms of Rbp4 in the ovary remain unclear.MethodsThe expression pattern of ovarian Rbp4 was examined in immature mice during different developmental stages and in adult mice during different stages of the estrous cycle. The potential regulation and mechanisms of ovarian Rbp4 expression by estrogen and related gonadotropins in mouse ovaries were also investigated.ResultsThe present study demonstrated that the ovarian expression of Rbp4 remained constant before puberty and increased significantly in the peripubertal period. In adult female mice, the expression of Rbp4 increased at proestrus and peaked at estrus at both the mRNA and protein levels. The protein distribution of RBP4 was mainly localized in the granulosa cell and theca cell layer in follicles. In addition, the expression of Rbp4 was significantly induced by follicle-stimulating hormone (FSH) or FSH + luteinizing hormone (LH) in combination in immature mouse (3 weeks old) ovaries in vivo and in granulosa cells cultured in vitro, both at the mRNA and protein levels. In contrast, treatment with LH or 17β-estradiol did not exhibit any observable effects on ovarian Rbp4 expression. Transcription factors high-mobility group AT-hook 1 (HMGA1), steroidogenic factor 1 (SF-1), and liver receptor homolog 1 (LRH-1) (which have been previously shown to be involved in activation of Rbp4 transcription), also responded to FSH stimulation. In addition, H-89, an inhibitor of protein kinase A (PKA), and the depletion of HMGA1, SF-1, and LRH-1 by small interfering RNAs (siRNAs), resulted in a dramatic loss of the induction of Rbp4 expression by FSH at both the mRNA and protein levels.ConclusionsThese data indicate that the dynamic expression of Rbp4 is mainly regulated by FSH through the cAMP-PKA pathway, involving transcriptional factors HMGA1, SF-1, and LRH-1, in the mouse ovary during different stages of development and the estrous cycle.

LRH1 enhances cell resistance to chemotherapy by transcriptionally activating MDC1 expression and attenuating DNA damage in human breast cancer.

Liver receptor homolog-1 (LRH1) has been shown to promote tumor proliferation and development. However, the functions of LRH1 in mediating cancer cells chemoresistance are still not clear. Here, we found LRH1 levels were significantly elevated in primary breast cancer tissues in patients who developed early recurrence. Similarly, adriamycin (ADR)-resistant breast cancer cell lines also exerted high LRH1 expression. Indeed, overexpression of LRH1 attenuated cytotoxicity of chemotherapeutic drugs ADR and cisplatin (DDP) in breast cancer cells in vitro and in nude mice tumor model. Comet and BrdU assays showed overexpression of LRH1 blocked breast cancer cells DNA damage by chemotherapeutic drug, whereas depletion of LRH1 enhanced DNA damage. Remarkably, knockdown of LRH1 decreased the levels and foci of DNA damage marker γH2AX induced by ADR and DDP. Furthermore, plasmid end-joining assay indicated that knockdown of LRH1 significantly decreased non-homologous end-joining (NHEJ)-mediated double-strand break (DSB) repair efficiencies. Afterwards, we provided evidences that LRH1 promoted MDC1 transcription by directly activating MDC1 promoter and therefore increased γH2AX levels. Importantly, a LRH1-binding site mapped between -1812 and -1804 bp of the proximal MDC1 promoter was identified. Moreover, LRH1 and MDC1 mRNA levels were positively correlated in recurrent breast cancer samples. These results implied LRH1 enhanced breast cancer cell chemoresistance by upregulating MDC1 and attenuating DNA damage. Additionally, we elucidated the coactivator NCOA3 acted synergistically with LRH1 to promote MDC1 expression and chemoresistance. Altogether, LRH1-MDC1 signaling might be considered as a novel molecular target for designing novel therapeutic regimen in chemotherapy resistance breast cancer.

LRH-1 regulates hepatic lipid homeostasis and maintains arachidonoyl phospholipid pools critical for phospholipid diversity.

Excess lipid accumulation is an early signature of nonalcoholic fatty liver disease (NAFLD). Although liver receptor homolog 1 (LRH-1) (encoded by NR5A2) is suppressed in human NAFLD, evidence linking this phospholipid-bound nuclear receptor to hepatic lipid metabolism is lacking. Here, we report an essential role for LRH-1 in hepatic lipid storage and phospholipid composition based on an acute hepatic KO of LRH-1 in adult mice (LRH-1AAV8-Cre mice). Indeed, LRH-1-deficient hepatocytes exhibited large cytosolic lipid droplets and increased triglycerides (TGs). LRH-1-deficient mice fed high-fat diet displayed macrovesicular steatosis, liver injury, and glucose intolerance, all of which were reversed or improved by expressing wild-type human LRH-1. While hepatic lipid synthesis decreased and lipid export remained unchanged in mutants, elevated circulating free fatty acid helped explain the lipid imbalance in LRH-1AAV8-Cre mice. Lipidomic and genomic analyses revealed that loss of LRH-1 disrupts hepatic phospholipid composition, leading to lowered arachidonoyl (AA) phospholipids due to repression of Elovl5 and Fads2, two critical genes in AA biosynthesis. Our findings reveal a role for the phospholipid sensor LRH-1 in maintaining adequate pools of hepatic AA phospholipids, further supporting the idea that phospholipid diversity is an important contributor to healthy hepatic lipid storage.