Liver Receptor Research Articles

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.

Targeting transdifferentiated hepatic stellate cells and monitoring the hepatic fibrogenic process by means of IGF2R-specific peptides designed in silico.

The lack of accurate and easily applicable methods for the diagnosis of liver fibrosis, a disease characterized by an accumulation of the extracellular matrix released by activated hepatic stellate cells (HSCs), has been a major limitation for the clinical management of liver diseases. The identification of biomarkers specific to liver microstructure alterations, combined with a non-invasive optical imaging modality, could guide clinicians towards a therapeutic strategy. In this study, structural information of the insulin-like growth factor 2 receptor (IGF2R), an overexpressed protein on activated HSCs, was used for in silico screening of novel IGF2R-specific peptide ligands. Molecular dynamics simulations, followed by computational alanine scanning of the IGF2R/IGF2 complex, led to the identification of a putative peptide sequence containing the most relevant amino acids for the receptor-ligand interaction (IGF2 E12-C21). The Residue Scan tool, implemented in the MOE software, was then used to optimize the binding affinity of this sequence by amino acid mutations. The designed peptides and their associated scrambled sequences were fluorescently labelled and their binding affinity to LX-2 cells (model for activated human HSCs) was tested using flow cytometry and confocal microscopy. In vitro binding was verified for all sequences (KD ≤ 13.2 μM). With respect to the putative binding sequence, most mutations led to an increased affinity. All sequences have shown superior binding compared to their associated scrambled sequences. Using HPLC, all peptides were tested in vitro for their proteolytic resistance and showed a stability of ≥60% intact after 24 h at 37 °C in 50% v/v FBS. In view of their prospective diagnostic application, a comparison of binding affinity was performed in perpetuated and quiescent-like LX-2 cells. Furthermore, the IGF2R expression for different cell phenotypes was analysed by a quantitative mass spectrometric approach. Our peptides showed increased binding to the perpetuated cell state, indicating their good selectivity for the diagnostically relevant phenotype. In summary, the increased binding affinity of our peptides towards perpetuated LX-2 cells, as well as the satisfactory proteolytic stability, proves that the in silico designed sequences offer a new potential strategy for the targeting of hepatic fibrosis.

Role of coxsackie-adenovirus receptor in cardiac development and pathogenesis of congenital heart disease.

The coxsackie-adenovirus receptor (CAR) is a cell surface transmembrane protein originally recognized for its role as a binding site for coxsackie- and adeno-viruses. As such, it is believed to play an important role in pathogenesis of myocarditis. Other studies have suggested that CAR also plays an important role in embryonic development, which is not surprising given the strong expression of the receptor in heart, brain, liver, pancreas, kidney, small intestine, and various epithelia during development. A number of studies have looked at downregulation and upregulation of CAR and have confirmed the central role of CAR during critical periods of development. These studies all demonstrated embryonic lethality with variable phenotypes: electrophysiological abnormalities, cardiac structural deformations, and extracardiac abnormalities, such as lymphatic malformations. The purpose of this review is to summarize the existing literature about CAR and formulate some questions for future studies, with an emphasis on the role of CAR during embryonic heart development.

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.

Effect of transcutaneous auricular vagus nerve stimulation on hyperglycemia and insulin receptors expression in impaired glucose tolerance rats

To explore the effect of transcutaneous auricular vagus nerve stimulation (taVNS) on blood glucose regulation and the expression of insulin receptors (INR) of hypothalamus, liver and skeletal muscle tissues in impaired glucose tolerance (IGT) rats, so as to reveal its mechanisms underlying improvement of IGT. Thirty-six male Wistar rats were randomly divided into control, model, transcutaneous auricular none-vagus nerve stimulation (tnVNS), and taVNS groups (n=9 in each group). The IGT model was established by feeding the rats with high-fat and high-sugar diet for 5 weeks, and subsequent intraperitoneal injection of a dose of streptozotocin (20 mg/kg). Transcutaneous electrostimulation (2 mA, 2 Hz/15 Hz) was applied to auricular concha (taVNS) or auricular margin (tnVNS), respectively. The treatment was conducted for 30 min once daily for 4 weeks. The body weight, fasting plasma glucose (FPG), 2 h plasma glucose (2 h PG) were recorded every week. The contents of plasma insulin (INS), glucagon (GC), glycosylated hemoglobin (GHbA1c) were detected by using enzyme linked immunosorbent assay (ELISA). The expression levels of INR in hypothalamus, liver and skeletal muscle tissues were detected by Western blot. After modeling, the rats' body weight, the contents of FPG, 2 h PG, GC and GHbA1c were significantly up-regulated (P<0.001, P<0.05, P<0.01), and the content of INS and expression of INR in hypothalamus, liver and skeletal muscle tissues were significantly down-regulated in the model group compared with the control group (P<0.001, P<0.01, P<0.05). Following the treatment, the increased FPG, 2 h PG, GC, and the decreased INS and INR expression of hypothalamus, liver and skeletal muscle tissues were apparently reversed in the taVNS group relevant to the model group (P<0.001, P<0.01, P<0.05). Compared with the tnVNS group, the FPG and 2 h PG contents were considerable decreased, and the content of INS and INR expression of hypothalamus and liver were obviously increased in the taVNS group (P<0.001, P<0.05, P<0.01). taVNS can improve the blood glucose and insulin sensitivity in IGT rats, which may contribute to its effectiveness in up-regulating the expression of INR in hypothalamus, liver and skeletal muscle tissues.

Genes Induced by Panax Notoginseng in a Rodent Model of Ischemia-Reperfusion Injury

Stroke is a cerebrovascular disease that results in decreased blood flow. Although Panax notoginseng (PN), a Chinese herbal medicine, has been proven to promote stroke recovery, its molecular mechanism remains unclear. In this study, middle cerebral artery occlusion (MCAO) was induced in rats with thrombi generated by thread and subsequently treated with PN. After that, staining with 2,3,5-triphenyltetrazolium chloride was employed to evaluate the infarcted area, and electron microscopy was used to assess ultrastructural changes of the neurovascular unit. RNA-Seq was performed to determine the differential expressed genes (DEGs) which were then verified by qPCR. In total, 817 DEGs were identified to be related to the therapeutic effect of PN on stroke recovery. Further analysis by Gene Oncology analysis and Kyoto Encyclopedia of Genes and Genomes revealed that most of these genes were involved in the biological function of nerves and blood vessels through the regulation of neuroactive live receptor interactions of PI3K-Akt, Rap1, cAMP, and cGMP-PKG signaling, which included in the 18 pathways identified in our research, of which, 9 were reported firstly that related to PN's neuroprotective effect. This research sheds light on the potential molecular mechanisms underlying the effects of PN on stroke recovery.

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.

Cannabinoid receptors are differentially regulated in the pancreatic islets during the early development of metabolic syndrome

ABSTRACT The endocannabinoid system is found in tissues that regulate the glycemia, including adipose tissue, muscle, and pancreatic islets. Diet-induced metabolic syndrome changes the expression of the CB receptors in muscle, adipose tissue, and liver. However, it is poorly understood whether metabolic syndrome (MetS) affects the expression of CB receptors in pancreatic β cells. We analyzed the expression of CB receptors in pancreatic β cells under chronic high-sucrose diet (HSD)-induced MetS. Wistar rats fed an HSD as a model of MetS were used to investigate changes in cannabinoid receptors. After 8 weeks of treatment, we evaluated the appearance of the following MetS biomarkers: glucose intolerance, hyperinsulinemia, insulin resistance, hypertriglyceridemia, and an increase in visceral adiposity. To determine the presence of CB1 and CB2 receptors in pancreatic β cells, immunofluorescence of primary cell cultures and pancreatic sections was performed. For whole-islet quantification of membrane-bound CB1 and CB2 receptors, western-blotting following differential centrifugation was conducted. Our results revealed that an HSD treatment closely mimics the alterations seen in MetS. We observed that in primary cell culture, CB1 and CB2 receptors were expressed at a higher level in pancreatic β cells compared with non-β cells. MetS resulted in a reduction of CB1 in the islet, whereas abundant CB2 was observed after the treatment. CB1 and CB2 receptors are differentially expressed in pancreatic β cells during MetS development.

The Status of Bile Acids and Farnesoid X Receptor in Brain and Liver of Rats with Thioacetamide-Induced Acute Liver Failure.

Acute liver failure (ALF) leads to neurological symptoms defined as hepatic encephalopathy (HE). Although accumulation of ammonia and neuroinflammation are generally accepted as main contributors to HE pathomechanism, a buildup of bile acids (BA) in the blood is a frequent component of liver injury in HE patients. Recent studies have identified the nuclear farnesoid X receptor (FXR) acting via small heterodimer partner (SHP) as a mediator of BA-induced effects in the brain of ALF animals. The present study investigated the status of the BA–FXR axis in the brain and the liver, including selective changes in pertinent genes in thioacetamide (TAA)-induced ALF in Sprague–Dawley rats. FXR was found in rat neurons, confirming earlier reports for mouse and human brain. BA accumulated in blood but not in the brain tissue. Expression of mRNAs coding for Fxr and Shp was reduced in the hippocampus and of Fxr mRNA also in the cerebellum. Changes in Fxr mRNA levels were not followed by changes in FXR protein. The results leave open the possibility that mobilization of the BA–FXR axis in the brain may not be necessarily pathognomonic to HE but may depend upon ALF-related confounding factors.

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.

P2Y12 shRNA normalizes inflammatory dysfunctional hepatic glucokinase activity in type 2 diabetic rats

The celiac ganglion projects its postganglionic (including purinergic) fibers to the liver. P2Y12 receptor is one of the P2Y family members. We found that the expression levels of P2Y12 receptor in both celiac ganglia and liver were increased in type 2 diabetes mellitus (T2DM) rats which also displayed an enhanced activity of celiac sympathetic nerve discharge (SND). In addition, a marked decrease of hepatic glucokinase (GK) expression was accompanied by reduced hepatic glycogen synthesis in T2DM rats, whereas meanwhile the levels of NLRP3, active caspase-1, NF-κB, and interleukin-1β were elevated. All these abnormal alterations could be largely reversed after treatment of short hairpin RNA (shRNA) targeting P2Y12. Our results indicate that the silence of P2Y12 by shRNA may effectively correct the anomalous activity of celiac SND and improve the dysfunctional hepatic glucokinase by counteracting hepatocyte inflammation and likely pyroptosis due to activated NLRP3 inflammasome and caspase-1 signaling, thereby attenuating hyperglycemia in T2DM rats.

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.

β-catenin mediates the effect of GLP-1 receptor agonist on ameliorating hepatic steatosis induced by high fructose diet.

The hypoglycemic drug GLP-1 receptor agonist can ameliorate hepatic steatosis but the mechanism is not clear. Intake of high fructose leads to non-alcoholic fatty liver disease by stimulating lipid synthesis, and β-catenin is the key molecule for realizing GLP-1 function in extrahepatic tissues; with the discovery of GLP-1 receptor in liver, we speculate that β-catenin might mediate GLP-1 receptor agonist on ameliorating hepatic steatosis induced by high fructose. Wistar rats were fed with high fructose diet for 8 weeks and then treated with GLP-1 receptor agonist exenatide for 4 weeks; the changes of lipid synthesis pathway factors, the expression and nuclear translocation of β-catenin, and the hepatic steatosis of the rats were observed. After the intervention of exenatide, the hepatic steatosis induced by high fructose was improved, the nuclear translocation and expression of β-catenin were facilitated, and the mRNA and protein expression of the upstream regulator SREBP-1 and the downstream key enzymes ACC, FAS and SCD-1 of de novo lipogenesis were down-regulated. GLP-1 receptor agonist may ameliorate hepatic steatosis induced by high fructose by β-catenin regulating de novo lipogenesis pathway. GLP-1 receptor agonist may be a potential new drug for the treatment of non-alcoholic fatty liver disease, and the β-catenin may be an important target for the drug therapy.

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.

Abstract 3490: Investigation of 27-hydroxycholesterol as a biomarker for aggressive endometrial cancer

Abstract Background: Strong risk factors for the most common (endometrioid) type of endometrial cancer (EC) include obesity and exposure to high estrogen levels. While EC has a favorable prognosis overall, one third of women present with advanced stage disease at diagnosis which has high recurrence and mortality rates. Given the estrogen-mediated etiology of EC, the current study focuses on the selective estrogen receptor modulator (SERM) 27-hydroxycholesterol (27HC) pathway. SERMs like 27HC can mimic estrogenic actions, and experimental work indicates 27HC exposure leads to continuous estrogenic stimulation and increases proliferation in the endometrium. Objective: to identify biomarkers for more aggressive disease, particularly markers present in early stages of carcinogenesis, to help guide decisions regarding treatment options. Methods: We conducted immunohistochemical staining on two tissue microarrays (TMAs) representing EC and its precursor endometrial intraepithelial neoplasia (EIN) tissues to measure protein levels of the enzymes that metabolize cholesterol into 27HC (CYP27A1) and 27HC into bile acids (CYP7B1), the receptors that bind 27HC (Estrogen Receptor [ER])-alpha, ER-beta as well as Liver Receptor X [LXR]-alpha and LXR-beta) and progesterone receptor (PR). We will use Fisher's exact test to evaluate 27HC associated biomarkers (categories split at the median) in relation to the presence of myometrial invasion (MI) and final diagnosis (EC or not) in the EIN TMA, and to assess clinico-pathologic factors including disease stage and grade in the EC TMA. Results: We created a new EIN TMA using formalin-fixed paraffin-embedded endometrial curettage or Pipelle endometrial biopsies from 144 EIN cases of diverse racial/ethnic backgrounds (Japanese, Hawaiian, White, Filipino and Asian/Pacific Islander) who were treated between 2007-16 in the Hawaii Pacific Health System. The EIN TMA contains both abnormal EIN tissues and neighboring benign/non-atypical tissues. In total, 50% of the EIN cases had no EC, 30% had cancer with no myometrial invasion (MI) and 20% had cancer with myometrial invasion. We are concurrently evaluating 27HC pathway markers in a TMA representing EC tumors from 96 cases who were treated in the same hospitals between 2001-07. The EC TMA represents diverse racial/ethnic groups including 28% Japanese American, 25% Native Hawaiian and 23% white cases (24% other). The EC TMA contains representative tissues from 91% endometrioid histotype cases and 72% localized and 28% regional/distant stage cases. We will examine associations between 27HC pathway biomarkers and clinico-pathologic characteristics of EC and EIN and will present our findings at the meeting. Conclusions: This study provides new data to determine whether 27HC pathway biomarkers may relate to characteristics of aggressive EC, such as the presence of myometrial invasion and higher stage and grade at diagnosis. Citation Format: Danja Sarink, Lenora W. Loo, Jeffrey Killeen, Brenda Y. Hernandez, Owen T. Chan, Melissa A. Merrit. Investigation of 27-hydroxycholesterol as a biomarker for aggressive endometrial cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3490.