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.     

Expression profiling of liver receptor homologue 1 (LRH-1) in mouse tissues using tissue microarray

Liver receptor homologue 1 (LRH-1) is a nuclear receptor that plays important roles in lipid homeostasis and embryogenesis. To elucidate systemic physiological functions of LRH-1, we used tissue microarray-based immunohistochemistry to examine the tissue distribution and localization of LRH-1 in adult mouse tissues. LRH-1 immunoreactivity was observed in the nucleus of multiple epithelial lineage cells in the digestive system (including absorptive epithelial cells in the small and large intestines, goblet cells, acinar cells of the exocrine glands, chief cells and mucus neck cells in the stomach, granular and prickle layer cells in the tongue and forestomach, and gall bladder epithelium); respiratory system (alveolar type II cells); and urinary system (transitional epithelium). Nuclear LRH-1 immunoreactivity was also localized in cells involved in fatty acid/glucose metabolism, including hepatocytes, brown adipocytes, and cardiomyocytes, and neurons involved in the regulation of food intake, including the arcuate nucleus in the hypothalamus and paraventricular nucleus of thalamus. Additionally, LRH-1 immunoreactivity was observed in testicular Leydig cells and ovarian follicular cells. These data suggest that LRH-1 functions in multiple organ systems to regulate epithelial cell physiology and differentiation, energy metabolism, and reproduction. Electronic Supplementary Material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

LRH-1在肿瘤及干细胞中的功能研究进展

核受体(nuclear receptors,NRs)是转录因子家族中最大的成员,多以配体依赖的方式特异性调节其靶基因的表达,参与机体代谢、发育和生殖功能的调控。LRH-1(liver receptor homolog-1),也称为NR5A2(nuclear receptor subfamily 5,group A,member 2),是核受体家族的成员,作为转录共激活子调控相关基因的表达。LRH-1调控多种重要的生理功能,包括调节脂肪酸和胆固醇的代谢,另外在胚胎发育和分化中也起了重要作用。LRH-1在促进多种癌症的发生过程中扮演重要的角色,如结肠癌、胰腺癌、卵巢癌和乳腺癌。随着对LRH-1研究的深入,其在疾病和胚胎干细胞中的功能作用已备受关注,这也使得LRH-1成为了许多疾病的潜在治疗靶点。     

Synergistic regulation of the mouse orphan nuclear receptor SHP gene promoter by CLOCK-BMAL1 and LRH-1

Small heterodimer partner (SHP; NR0B2) is an orphan nuclear receptor and acts as a repressor for wide variety of nuclear hormone receptors. We demonstrated here that mouse SHP mRNA showed a circadian expression pattern in the liver. Transient transfection of the mSHP promoter demonstrated that CLOCK-BMAL1, core circadian clock components, bound to E-box (CACGTG), and stimulated the promoter activity by 4-fold. Liver receptor homologue-1 (LRH-1; NR5A2) stimulated the mSHP promoter, and CLOCK-BMAL1 synergistically enhanced the LRH-1-mediated transactivation. Interestingly, SHP did not affect the CLOCK-BMAL1-mediated promoter activity, but strongly repressed the synergistic activation of CLOCK-BMAL1 and LRH-1. Furthermore, in vitro pull-down assays revealed the existence of direct protein-protein interaction between LRH-1 and CLOCK. In summary, this study shows that CLOCK-BMAL1, LRH-1 and SHP coordinately regulate the mSHP gene to generate the circadian oscillation. The cyclic expression of mSHP may affect daily activity of other nuclear receptors and contribute to circadian liver functions.     

Liver receptor homolog 1 controls the expression of the scavenger receptor class B type I

The scavenger receptor class B type I (SR-BI), which mediates selective cellular cholesterol uptake from high-density lipoproteins (HDLs), plays a key role in reverse cholesterol transport. The orphan nuclear receptor liver receptor homolog 1 (LRH-1) and SR-BI are co-expressed in liver and ovary, suggesting that LRH-1 might control the expression of SR-BI in these tissues. LRH-1 induces human and mouse SR-BI promoter activity by binding to an LRH-1 response element in the promoter. Retroviral expression of LRH-1 robustly induces SR-BI, an effect associated with histone H3 acetylation on the SR-BI promoter. The decrease in SR-BI mRNA levels in livers of LRH-1(+/-) animals provides in vivo evidence that LRH-1 regulates SR-BI expression. Our data demonstrate that SR-BI is an LRH-1 target gene and underscore the pivotal role of LRH-1 in reverse cholesterol transport.     

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-1^f/f and LRH-1 liver-specific knockout (Lrh-1^LKO) 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-1^LKO mice showed increased accumulation of triglycerides in the liver compared to that in Lrh-1^f/f mice. Interestingly, in the Lrh-1^LKO 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-1^f/f primary hepatocytes showed increased co-localization of PLIN5 in lipid droplets (LDs) compared to that in fasted Lrh-1^LKO 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.     

Mechanisms in the regulation of aromatase in developing ovary and placenta

During human gestation, the placental syncytiotrophoblast develops the capacity to synthesize large amounts of estrogen from C₁₉-steroids secreted by the fetal adrenals. The conversion of C₁₉-steroids to estrogens is catalyzed by aromatase P450 (P450arom), product of the CYP19 gene. The placenta-specific promoter of the hCYP19 gene lies 100,000 bp upstream of the translation initiation site in exon II. In studies using transgenic mice and transfected human trophoblast cells we have defined a 246-bp region upstream of placenta-specific exon I.1 that mediates placental cell-specific expression. Using transgenic mice, we also observed that as little as 278 bp of DNA flanking the 5′-end of ovary-specific hCYP19 exon IIa was sufficient to target ovary-specific expression. This ovary-specific promoter contains response elements that bind cAMP-response element-binding protein (CREB) and the orphan nuclear receptors SF-1 and LRH-1, which are required for cAMP-mediated stimulation of CYP19 expression in granulosa and luteal cells during the estrous cycle and pregnancy. In this article, we review our studies to define genomic regions and response elements that mediate placenta-specific expression of the hCYP19 gene. The temporal and spatial expression of LRH-1 versus SF-1 in the developing gonad during mouse embryogenesis and in the postnatal ovary also will be considered.     

Distal enhancer of the mouse FGF-4 gene and its human counterpart exhibit differential activity: critical role of a GT box

Previous studies have shown that there is a strict requirement for fibroblast growth factor-4 (FGF-4) during mammalian embryogenesis, and that FGF-4 expression in embryonic stem (ES) cells and embryonal carcinoma (EC) cells are controlled by a powerful downstream distal enhancer. More recently, mouse ES cells were shown to express significantly more FGF-4 mRNA than human ES cells. In the work reported here, we demonstrate that mouse EC cells also express far more FGF-4 mRNA than human EC cells. Using a panel of FGF-4 promoter/reporter gene constructs, we demonstrate that the enhancer of the mouse FGF-4 gene is approximately tenfold more active than its human counterpart. Moreover, we demonstrate that the critical difference between the mouse and the human FGF-4 enhancer is a 4 bp difference in the sequence of an essential GT box. Importantly, we demonstrate that changing 4 bp in the human enhancer to match the sequence of the mouse GT box elevates the activity of the human FGF-4 enhancer to the same level as that of the mouse enhancer. We extended these studies by examining the roles of Sp1 and Sp3 in FGF-4 expression. Although we demonstrate that Sp3, but not Sp1, can activate the FGF-4 promoter when artificially tethered to the FGF-4 enhancer, we show that Sp3 is not essential for expression of FGF-4 mRNA in mouse ES cells. Finally, our studies with human EC cells suggest that the factor responsible for mediating the effect of the mouse GT box is unlikely to be Sp1 or Sp3, and this factor is either not expressed in human EC cells or it is not sufficiently active in these cells.