Orphan Nuclear Receptor LRH-1 Research Articles

The orphan nuclear receptor LRH-1/NR5a2 critically regulates T cell functions.

LRH-1 (liver receptor homolog-1/NR5a2) is an orphan nuclear receptor, which regulates glucose and lipid metabolism, as well as intestinal inflammation via the transcriptional control of intestinal glucocorticoid synthesis. Predominantly expressed in epithelial cells, its expression and role in immune cells are presently enigmatic. LRH-1 was found to be induced in immature and mature T lymphocytes upon stimulation. T cell-specific deletion of LRH-1 causes a drastic loss of mature peripheral T cells. LRH-1-depleted CD4+ T cells exert strongly reduced activation-induced proliferation in vitro and in vivo and fail to mount immune responses against model antigens and to induce experimental intestinal inflammation. Similarly, LRH-1-deficient cytotoxic CD8+ T cells fail to control viral infections. This study describes a novel and critical role of LRH-1 in T cell maturation, functions, and immopathologies and proposes LRH-1 as an emerging pharmacological target in the treatment of T cell-mediated inflammatory diseases.

Transcriptional profiling of five isolated size-matched stages of human preantral follicles

Little is known of the early stages of human follicular development and the complex processes that regulate follicular growth. To identify genes of potential importance, we analysed follicle-related transcripts in five populations of isolated size-matched human preantral follicles by microarray analysis. Oocyte-specific genes were found to be the most abundant and differentially expressed transcripts and included germ cell transcription factors LHX8 and SOHLH2 which were significantly down-regulated during preantral follicle development. Differentially expressed genes also included transcription factors of NOTCH signalling, IGF2, orphan nuclear receptor LRH-1, and homeobox gene HOXA7, indicating potentially important regulatory roles for these genes during early human folliculogenesis. We also found that FSHR mRNA and protein were present in the earliest stages of preantral follicles, whereas LHR was absent. In conclusion, our data identify specific oocyte and somatic genes in small human follicles that impact early follicle growth, and provide foundation for further analysis of the signalling pathways involved in early human folliculogenesis.

The Orphan Nuclear Receptor LRH-1 and ERα Activate GREB1 Expression to Induce Breast Cancer Cell Proliferation

BackgroundLiver Receptor Homolog 1 (LRH-1, NR5A2) is an orphan nuclear receptor that is over-expressed in cancers in tissues such as the breast, colon and pancreas. LRH-1 plays important roles in embryonic development, steroidogenesis and cholesterol homeostasis. In tumor cells, LRH-1 induces proliferation and cell cycle progression. High LRH-1 expression is demonstrated in breast cancers, positively correlating with ERα status and aromatase activity. LRH-1 dependent cellular mechanisms in breast cancer epithelial cells are poorly defined. Hence in the present study we investigated the actions of LRH-1 in estrogen receptor α (ERα) positive breast cancer cells.ResultsThe study aimed to investigate LRH-1 dependent mechanisms that promote breast cancer proliferation. We identified that LRH-1 regulated the expression of Growth Regulation by Estrogen in Breast Cancer 1 (GREB1) in MCF-7 and MDA-MB-231 cells. Over-expression of LRH-1 increased GREB1 mRNA levels while knockdown of LRH-1 reduced its expression. GREB1 is a well characterised ERα target gene, with three estrogen response elements (ERE) located on its promoter. Chromatin immunoprecipitation studies provided evidence of the co-localisation of LRH-1 and ERα at all three EREs. With electrophoretic mobility shift assays, we demonstrated direct binding of LRH-1 to EREs located on GREB1 and Trefoil Factor 1 (TFF1, pS2) promoters. LRH-1 and ERα co-operatively activated transcription of ERE luciferase reporter constructs suggesting an overlap in regulation of target genes in breast cancer cells. Over-expression of LRH-1 resulted in an increase in cell proliferation. This effect was more pronounced with estradiol treatment. In the presence of ICI 182,780, an ERα antagonist, LRH-1 still induced proliferation.ConclusionsWe conclude that in ER-positive breast cancer cells, LRH-1 promotes cell proliferation by enhancing ERα mediated transcription of target genes such as GREB-1. Collectively these findings indicate the importance of LRH-1 in the progression of hormone-dependent breast cancer and implicate LRH-1 as a potential avenue for drug development.

Phosphatidylcholines as regulators of glucose and lipid homeostasis: Promises and potential risks

Nuclear hormone receptors regulate diverse metabolic pathways and the orphan nuclear receptor LRH-1 (also known as NR5A2) regulates bile acid biosynthesis. Structural studies have identified phospholipids as potential LRH-1 ligands, but their functional relevance is unclear. Here we show that an unusual phosphatidyl-choline species with two saturated 12 carbon fatty acid acyl side chains (dilauroyl phosphatidylcholine (DLPC)) is an LRH-1 agonist ligand in vitro. DLPC treatment induces bile acid biosynthetic enzymes in mouse liver, increases bile acid levels, and lowers hepatic triglycerides and serum glucose. DLPC treatment also decreases hepatic steatosis and improves glucose homeostasis in two mouse models of insulin resistance. Both the antidiabetic and lipotropic effects are lost in liver-specific Lrh-1 knockouts. These findings identify an LRH-1 dependent phosphatidylcholine signalling pathway that regulates bile acid metabolism and glucose homeostasis.

A lipid to treat non-alcoholic fatty liver disease – The dawn of ‘lipo-rehabilitation’?

Nuclear hormone receptors regulate diverse metabolic pathways and the orphan nuclear receptor LRH-1 (also known as NR5A2) regulates bile acid biosynthesis. Structural studies have identified phospholipids as potential LRH-1 ligands, but their functional relevance is unclear. Here we show that an unusual phosphatidylcholine species with two saturated 12 carbon fatty acid acyl sidechains (dilauroyl phosphatidylcholine (DLPC)) is an LRH-1 agonist ligand in vitro. DLPC treatment induces bile acid biosynthetic enzymes in mouse liver, increases bile acid levels, and lowers hepatictriglycerides and serum glucose. DLPC treatment also decreases hepatic steatosis and improves glucose homeostasis in two mouse models of insulin resistance. Both the antidiabetic and lipotropic effects are lost in liver-specific Lrh-1 knockouts. These findings identify an LRH-1 dependent phosphatidylcholine signalling pathway that regulates bile acid metabolism and glucose homeostasis.

A lipid for fat disorders

A high-fat diet often leads to metabolic disorders such as diabetes, fatty liver disease and obesity. One lipid, however, might mitigate these effects through an unexpected signalling role in the nucleus. See Letter p.506 Excess liver fat, or steatosis, is an important risk factor for obesity-associated diabetes. A natural product, dilauroyl phosphatidylcholine (DLPC), has now been identified as a possible antisteatosis agent, with therapeutic potential in pre-diabetic people. DLPC activates the orphan nuclear receptor LRH-1, loss of which decreases bile acid levels. Elevated bile acid is known to decrease steatosis. Acting as an LHR-1 agonist, DLPC decreases hepatic steatosis and improves glucose homeostasis in mouse models of insulin resistance.

A nuclear-receptor-dependent phosphatidylcholine pathway with antidiabetic effects

Nuclear hormone receptors regulate diverse metabolic pathways and the orphan nuclear receptor LRH-1 (NR5A2) regulates bile acid biosynthesis1,2. Structural studies have identified phospholipids as potential LRH-1 ligands3–5, but their functional relevance is unclear. Here we show that an unusual phosphatidylcholine species with two saturated 12 carbon fatty acid acyl side chains (dilauroyl phosphatidylcholine, DLPC) is an LRH-1 agonist ligand in vitro. DLPC treatment induces bile acid biosynthetic enzymes in mouse liver, increases bile acid levels, and lowers hepatic triglycerides and serum glucose. DLPC treatment also decreases hepatic steatosis and improves glucose homeostasis in two mouse models of insulin resistance. Both the antidiabetic and lipotropic effects are lost in liver specific Lrh-1 knockouts. These findings identify an LRH-1 dependent phosphatidylcholine signaling pathway that regulates bile acid metabolism and glucose homeostasis.

Tissue-specific regulation of aromatase promoter II by the orphan nuclear receptor LRH-1 in breast adipose stromal fibroblasts

In postmenopausal breast cancers, the increase in aromatase expression observed in tumour associated adipose stromal cells is mediated via the upregulation of promoter II (PII) transcription. Factors such as PGE 2 which are secreted from breast carcinomas induce PII expression. The orphan nuclear receptor LRH-1/NR5A2 is one of the critical downstream transcriptional mediators of this effect. The aim of the current study was to determine whether LRH-1 could bind directly to PII and whether the suppression of LRH-1 expression could inhibit aromatase expression in human adipose stromal fibroblasts. Chromatin immunoprecipitation demonstrated endogenous LRH-1 occupancy on PII under basal conditions and with treatment with forskolin and phorbol 12-myristate 13-acetate (PMA). To assess the impact of LRH-1 knockdown on FSK/PMA mediated PII expression, cells were transfected with shRNA targeted against LRH-1 (shLRH-1) and treated with forskolin and PMA. A decrease in LRH-1, PII and total aromatase mRNA transcripts was observed in shLRH-1 transfected cells compared to controls under basal and treatment conditions. The results of this study support the hypothesis that suppression of LRH-1 may potentially be beneficial in the tissue specific regulation of aromatase expression in post menopausal breast cancer.

PIASy inhibits LRH-1-dependent CYP11A1 expression by competing for SRC-1 binding

The orphan nuclear receptor LRH-1 (liver receptor homologue-1; NR5A2) plays a critical role in development, bile acid synthesis and cholesterol metabolism. LRH-1 is also expressed in the ovary where it is implicated in the regulation of steroidogenic genes for steroid hormone synthesis. In the present study, we investigated the molecular mechanisms of the transcriptional regulation of CYP11A1 by LRH-1 and found that LRH-1-mediated transactivation was markedly repressed by PIASy [protein inhibitor of activated STAT (signal transducer and activator of transcription) y], the shortest member of the PIAS family. The suppression of LRH-1 activity requires the N-terminal repression domain. Although PIAS proteins also function as E3 SUMO (small ubiquitin-related modifier) ligases and enhance SUMO conjugation, PIASy-mediated repression was independent of LRH-1 SUMOylation status. In addition, histone deacetylase activity was not involved in the inhibition of LRH-1 by PIASy. Immunoprecipitation and mammalian two-hybrid analyses indicated that PIASy interacted with LRH-1 through the C-terminal region, including the AF-2 (activation function-2) motif, which was also involved in the interaction between LRH-1 and the co-activator SRC-1 (steroid receptor co-activator-1). PIASy inhibited the binding of SRC-1 to LRH-1, although overexpression of SRC-1 partially overcame the PIASy inhibition of LRH-1 induction of the CYP11A1 promoter. The results of the present study suggest that competition with co-activators may be an important mechanism underlying the PIASy repression of LRH-1-mediated transactivation.

Regulation of Anti-atherogenic Apolipoprotein M Gene Expression by the Orphan Nuclear Receptor LRH-1

The orphan nuclear receptor liver receptor homolog-1 (LRH-1, NR5A2) has been reported to play a crucial role in early development, in the control of the hepatic inflammatory response, in intestinal cell crypt renewal as well as in bile acid biosynthesis and reverse cholesterol transport (RCT). Here, we report the identification of apolipoprotein M (APOM) as a novel target gene for LRH-1. Using gene-silencing experiments, adenovirus-mediated overexpression, transient transfection, and chromatin immunoprecipitation (ChIP) assays, it is shown that LRH-1 directly regulates human and mouse APOM transcription by binding to an LRH-1 response element located in the proximal APOM promoter region. In addition, we demonstrate that bile acids suppress APOM expression in a SHP-dependent manner in vitro and in vivo by inhibiting LRH-1 transcriptional activity on the APOM promoter as demonstrated by in vivo ChIP assay. Taken together, our results demonstrate that LRH-1 is a novel regulator of APOM transcription and further extend the role of this orphan nuclear receptor in lipoprotein metabolism and cholesterol homeostasis.

Tpt1 Activates Transcription of oct4 and nanog in Transplanted Somatic Nuclei

SummaryNuclear transfer to eggs or oocytes provides a potential route for cell-replacement therapies [1] because oocytes directly reprogram transplanted mammalian somatic-cell nuclei such that they have an embryo-like pattern of gene expression. This includes a large increase in the mRNA level of the stem-cell marker gene oct4[2]. We have developed a novel procedure to identify new proteins that greatly increase the level of oct4 mRNA upon nuclear transfer. We have isolated Xenopus oocyte proteins that bind to the regulatory region of the mouse oct4 gene and identified these by mass spectrometry. The proteins include the retinoic-acid-receptor γ, a known repressor of oct4 transcription, and Tpt1, a cancer-associated factor [3]. The depletion of transcripts of retinoic-acid receptor γ from oocytes increases oct4 and nanog transcription as expected, and depletion of tpt1 transcripts in oocytes reduces oct4 and nanog transcription in injected HeLa nuclei. An elevation of tpt1 transcripts in oocytes results in an earlier activation of oct4 transcription. Therefore, we identify a novel role for tpt1 in activating pluripotency genes upon nuclear transfer. Our results help to elucidate the mechanism by which somatic-cell nuclei are reprogrammed to have an embryo-like pattern of gene expression.

The stimulatory effect of LXRα is blocked by SHP despite the presence of a LXRα binding site in the rabbit CYP7A1 promoter

The transcription of the cholesterol 7alpha-hydroxylase gene (CYP7A1) is greatly decreased in cholesterol-fed rabbits. To determine whether the molecular structure of the promoter is responsible for this downregulation, we cloned the rabbit CYP7A1 promoter, identified the binding sites for alpha-fetoprotein transcription factor (FTF) and liver X receptor (LXRalpha), and studied the effects of FTF, LXRalpha, and SHP on its transcription. Adding LXRalpha/retinoid X receptor together with their ligands (L/R) to the promoter/reporter construct transfected into HepG2 cells greatly increased its activity. FTF did not increase promoter activity, nor did it enhance the stimulatory effect of L/R. Mutating the FTF binding site abolished the promoter baseline activity. Increasing amounts of SHP abolished the effect of L/R, and FTF enhanced the ability of SHP to decrease promoter activity below baseline levels. Thus, downregulation of CYP7A1 in cholesterol-fed rabbits is attributable secondarily to the activation of farnesoid X receptor, which increases SHP expression to override the positive effects of LXRalpha. Although FTF is a competent factor for maintaining baseline activity, it does not further enhance and may suppress CYP7A1 transcription.

SUMO-Dependent Compartmentalization in Promyelocytic Leukemia Protein Nuclear Bodies Prevents the Access of LRH-1 to Chromatin

Posttranslational modification by SUMO elicits a repressive effect on many transcription factors. In principle, sumoylation may either influence transcription factor activity on promoters, or it may act indirectly by targeting the modified factors to specific cellular compartments. To provide direct experimental evidence for the above, not necessarily mutually exclusive models, we analyzed the role of SUMO modification on the localization and the activity of the orphan nuclear receptor LRH-1. We demonstrate, by using fluorescence resonance energy transfer (FRET) and fluorescence recovery after photobleaching (FRAP) assays, that sumoylated LRH-1 is exclusively localized in promyelocytic leukemia protein (PML) nuclear bodies and that this association is a dynamic process. Release of LRH-1 from nuclear bodies correlated with its desumoylation, pointing to the pivotal role of SUMO conjugation in keeping LRH-1 in these locations. SUMO-dependent shuttling of LRH-1 into PML bodies defines two spatially separated pools of the protein, of which only the soluble, unmodified one is associated with actively transcribed target genes. The results suggest that SUMO-PML nuclear bodies may primarily function as dynamic molecular reservoirs, controlling the availability of certain transcription factors to active chromatin domains.

Orphan Nuclear Receptor LRH-1 Is Required To Maintain Oct4 Expression at the Epiblast Stage of Embryonic Development

Oct4 plays an essential role in maintaining the inner cell mass and pluripotence of embryonic stem (ES) cells. The expression of Oct4 is regulated by the proximal enhancer and promoter in the epiblast and by the distal enhancer and promoter at all other stages in the pluripotent cell lineage. Here we report that the orphan nuclear receptor LRH-1, which is expressed in undifferentiated ES cells, can bind to SF-1 response elements in the proximal promoter and proximal enhancer of the Oct4 gene and activate Oct4 reporter gene expression. LRH-1 is colocalized with Oct4 in the inner cell mass and the epiblast of embryos at early developmental stages. Disruption of the LRH-1 gene results in loss of Oct4 expression at the epiblast stage and early embryonic death. Using LRH-1(-/-) ES cells, we also show that LRH-1 is required to maintain Oct4 expression at early differentiation time points. In vitro and in vivo results show that LRH-1 plays an essential role in the maintenance of Oct4 expression in ES cells at the epiblast stage of embryonic development, thereby maintaining pluripotence at this crucial developmental stage prior to segregation of the primordial germ cell lineage at gastrulation.

Orphan Nuclear Receptor LRH-1 Is Required To Maintain Oct4 Expression at the Epiblast Stage of Embryonic Development

Orphan Nuclear Receptor LRH-1 Is Required To Maintain Oct4 Expression at the Epiblast Stage of Embryonic Development

Regulation of aromatase expression by the nuclear receptor LRH-1 in adipose tissue

Aromatase is the enzyme responsible for estrogen production, and is the product of the CYP19 gene. This gene is under the control of many tissue-specific promoters, each of which is regulated by different cohorts of factors. In normal breast adipose tissue, relatively low levels of aromatase are expressed via the action of the adipose specific promoter I.4. Breast tumor-derived factors such as prostaglandin E 2 (PGE 2) strongly stimulate aromatase expression via an alternative promoter, promoter II, leading to increased estrogenic drive and tumor growth. Understanding the mechanisms that regulate promoter II activity in tumorous breast may therefore identify new targets for breast cancer drug discovery. The current study describes the role of the orphan nuclear receptor LRH-1 and its co-regulators in modulating aromatase expression in breast adipose tissue.

LRH-1/hB1F and HNF1 synergistically up-regulate hepatitis B virus gene transcription and DNA replication.

Enhancer II (ENII) is one of the critical cis-elements in the Hepatitis B Virus (HBV) genome for the hepatic viral gene transcription and DNA replication. The liver-specific activity of ENII is regulated by multiple liver-enriched transcription factors, including LRH-1/hB1F, HNF1, HNF3b, HNF4 and C/EBP. Knowledge on the interplay of these important factors is still limited. In this study, we demonstrate a functional synergism between the orphan nuclear receptor LRH-1/hB1F and the homeoprotein HNF1 in up-regulating the liver-specific activity of ENII. This synergism is sufficient for initiating the viral gene transcription and DNA replication in non-hepatic cells. We have defined the activation domains in hB1F and HNF1 that contribute to the synergism. We further show that hB1F and HNF1 can interact directly in vitro and have mapped the domains required for this interaction.

The Orphan Nuclear Receptor LRH-1 Potentiates the Sterol-mediated Induction of the Human CETP Gene by Liver X Receptor

The human cholesteryl ester transfer protein (CETP) transfers cholesteryl esters from high density lipoproteins to triglyceride-rich lipoproteins, indirectly facilitating cholesteryl esters uptake by the liver. Hepatic CETP gene expression is increased in response to dietary hypercholesterolemia, an effect that is mediated by the activity of liver X receptor/retinoid X receptor (LXR/RXR) on a direct repeat 4 element in the CETP promoter. In this study we show that the orphan nuclear receptor LRH-1 also transactivates the CETP promoter by binding to a proximal promoter element distinct from the DR4 site. LRH-1 potentiates the sterol-dependent regulation of the wild type CETP promoter by LXR/RXR. Small heterodimer partner, a repressor of LRH-1, abolishes the potentiation effect of LRH-1 but not its basal transactivation of the CETP promoter. Since this mode of regulation of CETP is very similar to that recently reported for the bile salt-mediated repression of Cyp7a (encoding the rate-limiting enzyme for conversion of cholesterol into bile acid in the liver), we examined the effects of bile salt feeding on CETP mRNA expression in human CETP transgenic mice. Hepatic CETP mRNA expression was repressed by a diet containing 1% cholic acid in male mice but was induced by the same diet in female mice. Microarray analysis of hepatic mRNA showed that about 1.5% of genes were repressed, and 2.5% were induced by the bile acid diet. However, the sexually dimorphic regulatory pattern of the CETP gene was an unusual response. Our data provide further evidence for the regulation of CETP and Cyp7a genes by similar molecular mechanisms, consistent with coordinate transcriptional regulation of sequential steps of reverse cholesterol transport. However, differential effects of the bile salt diet indicate additional complexity in the response of these two genes.