Member Of Nuclear Receptor Family Research Articles

Targeting liver X receptors in cancer therapeutics.

Members of the nuclear receptor superfamily of ligand-dependent transcription factors carry out vital cellular functions and are highly druggable therapeutic targets. Liver X receptors (LXRs) are nuclear receptor family members that function in cholesterol transport, glucose metabolism and the modulation of inflammatory responses. There is now accumulating evidence to support the involvement of LXRs in a variety of malignancies and the potential efficacy of their ligands in these diseases. This Review summarizes the discovery and characterization of LXRs and their ligands, their effects and mechanisms in preclinical cancer models, and the future directions of basic and translational LXR research in cancer therapeutics.

The nuclear receptor nr4a1 controls CD8 T cell development through transcriptional suppression of runx3.

The NR4A nuclear receptor family member Nr4a1 is strongly induced in thymocytes undergoing selection, and has been shown to control the development of Treg cells; however the role of Nr4a1 in CD8+ T cells remains undefined. Here we report a novel role for Nr4a1 in regulating the development and frequency of CD8+ T cells through direct transcriptional control of Runx3. We discovered that Nr4a1 recruits the corepressor, CoREST to suppress Runx3 expression in CD8+ T cells. Loss of Nr4a1 results in increased Runx3 expression in thymocytes which consequently causes a 2-fold increase in the frequency and total number of intrathymic and peripheral CD8+ T cells. Our findings establish Nr4a1 as a novel and critical player in the regulation of CD8 T cell development through the direct suppression of Runx3.

Identification of modulators of the nuclear receptor peroxisome proliferator-activated receptor α (PPARα) in a mouse liver gene expression compendium.

The nuclear receptor family member peroxisome proliferator-activated receptor α (PPARα) is activated by therapeutic hypolipidemic drugs and environmentally-relevant chemicals to regulate genes involved in lipid transport and catabolism. Chronic activation of PPARα in rodents increases liver cancer incidence, whereas suppression of PPARα activity leads to hepatocellular steatosis. Analytical approaches were developed to identify biosets (i.e., gene expression differences between two conditions) in a genomic database in which PPARα activity was altered. A gene expression signature of 131 PPARα-dependent genes was built using microarray profiles from the livers of wild-type and PPARα-null mice after exposure to three structurally diverse PPARα activators (WY-14,643, fenofibrate and perfluorohexane sulfonate). A fold-change rank-based test (Running Fisher’s test (p-value ≤ 10-4)) was used to evaluate the similarity between the PPARα signature and a test set of 48 and 31 biosets positive or negative, respectively for PPARα activation; the test resulted in a balanced accuracy of 98%. The signature was then used to identify factors that activate or suppress PPARα in an annotated mouse liver/primary hepatocyte gene expression compendium of ~1850 biosets. In addition to the expected activation of PPARα by fibrate drugs, di(2-ethylhexyl) phthalate, and perfluorinated compounds, PPARα was activated by benzofuran, galactosamine, and TCDD and suppressed by hepatotoxins acetaminophen, lipopolysaccharide, silicon dioxide nanoparticles, and trovafloxacin. Additional factors that activate (fasting, caloric restriction) or suppress (infections) PPARα were also identified. This study 1) developed methods useful for future screening of environmental chemicals, 2) identified chemicals that activate or suppress PPARα, and 3) identified factors including diets and infections that modulate PPARα activity and would be hypothesized to affect chemical-induced PPARα activity.

In vitro metabolism of an estrogen-related receptor γ modulator, GSK5182, by human liver microsomes and recombinant cytochrome P450s.

GSK5182 (4-[(Z)-1-[4-(2-dimethylaminoethyloxy)phenyl]-hydroxy-2-phenylpent-1-enyl]phenol) is a specific inverse agonist for estrogen-related receptor γ, a member of the orphan nuclear receptor family that has important functions in development and homeostasis. This study was performed to elucidate the metabolites of GSK5182 and to characterize the enzymes involved in its metabolism. Incubation of human liver microsomes with GSK5182 in the presence of NADPH resulted in the formation of three metabolites, M1, M2 and M3. M1 and M3 were identified as N-desmethyl-GSK5182 and GSK5182 N-oxide, respectively, on the basis of liquid chromatography-tandem mass spectrometric (LC-MS/MS) analysis. M2 was suggested to be hydroxy-GSK5182 through interpretation of its MS/MS fragmentation pattern. In addition, the specific cytochrome P450 (P450) and flavin-containing monooxygenase (FMO) isoforms responsible for GSK5182 oxidation to the three metabolites were identified using a combination of correlation analysis, chemical inhibition in human liver microsomes and metabolism by expressed recombinant P450 and FMO isoforms. GSK5182 N-demethylation and hydroxylation is mainly mediated by CYP3A4, whereas FMO1 and FMO3 contribute to the formation of GSK5182 N-oxide from GSK5182. The present data will be useful for understanding the pharmacokinetics and drug interactions of GSK5182 in vivo.

Identification of chemical modulators of the constitutive activated receptor (CAR) in a gene expression compendium.

The nuclear receptor family member constitutive activated receptor (CAR) is activated by structurally diverse drugs and environmentally-relevant chemicals leading to transcriptional regulation of genes involved in xenobiotic metabolism and transport. Chronic activation of CAR increases liver cancer incidence in rodents, whereas suppression of CAR can lead to steatosis and insulin insensitivity. Here, analytical methods were developed to screen for chemical treatments in a gene expression compendium that lead to alteration of CAR activity. A gene expression biomarker signature of 83 CAR-dependent genes was identified using microarray profiles from the livers of wild-type and CAR-null mice after exposure to three structurally-diverse CAR activators (CITCO, phenobarbital, TCPOBOP). A rank-based algorithm (Running Fisher’s algorithm (p-value ≤ 10-4)) was used to evaluate the similarity between the CAR biomarker signature and a test set of 28 and 32 comparisons positive or negative, respectively, for CAR activation; the test resulted in a balanced accuracy of 97%. The biomarker signature was used to identify chemicals that activate or suppress CAR in an annotated mouse liver/primary hepatocyte gene expression database of ~1850 comparisons. CAR was activated by 1) activators of the aryl hydrocarbon receptor (AhR) in wild-type but not AhR-null mice, 2) pregnane X receptor (PXR) activators in wild-type and to lesser extents in PXR-null mice, and 3) activators of PPARα in wild-type and PPARα-null mice. CAR was consistently activated by five conazole fungicides and four perfluorinated compounds. Comparison of effects in wild-type and CAR-null mice showed that the fungicide propiconazole increased liver weight and hepatocyte proliferation in a CAR-dependent manner, whereas the perfluorinated compound perfluorooctanoic acid (PFOA) increased these endpoints in a CAR-independent manner. A number of compounds suppressed CAR coincident with increases in markers of inflammation including acetaminophen, concanavalin A, lipopolysaccharide, and 300 nm silica particles. In conclusion, we have shown that a CAR biomarker signature coupled with a rank-based similarity method accurately predicts CAR activation. This analytical approach, when applied to a gene expression compendium, increased the universe of known chemicals that directly or indirectly activate CAR, highlighting the promiscuous nature of CAR activation and signaling through activation of other xenobiotic-activated receptors.

Abstract 2366: Transcriptome analysis of human metastasis prostate cancer cells deciphers new function of Esrrb

Abstract Orphan nuclear receptor family member, Estrogen Related Receptor β (esrrb) became a research hot topic in recent years. Although the function of esrrb is relatively well studied in stem cells and early embryonic development, little is known about its function in cancer. Esrrb is reported to arrest prostate cancer cell cell-cycle by upregulating p21. Here we investigate the transcriptome alteration induced by esrrb expression and its synthetic ligand DY131 in a human prostate cancer cell line. We distinguished 205 qualified differentially expressed (DE) genes regulated by esrrb alone, and 3202 genes with DY131 treatment with esrrb expression, indicating esrrb has both ligand independent and ligand dependent activity. From the esrrb-regulated transcriptome, we established a hypothetical esrrb transcription regulation model in prostate cancer cells by matching the DNA motif recognized by transcriptional factors (TFs) to the promoter sequences of differentially expressed genes. We found esrrb-regulated gene expression is mediated by TAGLN, HOXB2 and HOXB8 from our model. Our findings potentially provide multiple markers to characterize esrrb's function in prostate cancer. Citation Format: Yuan Lu, Jilong Li, Jianlin Cheng, Dennis B. Lubahn. Transcriptome analysis of human metastasis prostate cancer cells deciphers new function of Esrrb. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2366. doi:10.1158/1538-7445.AM2014-2366

Transcriptional regulation of urate transportosome member SLC2A9 by nuclear receptor HNF4α.

Renal tubular handling of urate is realized by a network of uptake and efflux transporters, including members of drug transporter families such as solute carrier proteins and ATP-binding cassette transporters. Solute carrier family 2, member 9 (SLC2A9), is one key factor of this so called "urate transportosome." The aim of the present study was to understand the transcriptional regulation of SLC2A9 and to test whether identified factors might contribute to a coordinated transcriptional regulation of the transporters involved in urate handling. In silico analysis and cell-based reporter gene assays identified a hepatocyte nuclear factor (HNF)4α-binding site in the promoter of SLC2A9 isoform 1, whose activity was enhanced by transient HNF4α overexpression, whereas mutation of the binding site diminished activation. HNF4α overexpression induced endogenous SLC2A9 expression in vitro. The in vivo role of HNF4α in the modulation of renal SLC2A9 gene expression was supported by findings of quantitative real-time RT-PCR analyses and chromatin immunoprecipitation assays. Indeed, mRNA expression of SLC2A9 and HNF4α in human kidney samples was significantly correlated. We also showed that in renal clear cell carcinoma, downregulation of HNF4α mRNA and protein expression was associated with a significant decline in expression of the transporter. Taken together, our data suggest that nuclear receptor family member HNF4α contributes to the transcriptional regulation of SLC2A9 isoform 1. Since HNF4α has previously been assumed to be a modulator of several urate transporters, our findings support the notion that there could be a transcriptional network providing synchronized regulation of the functional network of the urate transportosome.

The nuclear hormone receptor family member NR5A2 controls aspects of multipotent progenitor cell formation and acinar differentiation during pancreatic organogenesis.

The orphan nuclear receptor NR5A2 is necessary for the stem-like properties of the epiblast of the pre-gastrulation embryo and for cellular and physiological homeostasis of endoderm-derived organs postnatally. Using conditional gene inactivation, we show that Nr5a2 also plays crucial regulatory roles during organogenesis. During the formation of the pancreas, Nr5a2 is necessary for the expansion of the nascent pancreatic epithelium, for the subsequent formation of the multipotent progenitor cell (MPC) population that gives rise to pre-acinar cells and bipotent cells with ductal and islet endocrine potential, and for the formation and differentiation of acinar cells. At birth, the NR5A2-deficient pancreas has defects in all three epithelial tissues: a partial loss of endocrine cells, a disrupted ductal tree and a >90% deficit of acini. The acinar defects are due to a combination of fewer MPCs, deficient allocation of those MPCs to pre-acinar fate, disruption of acinar morphogenesis and incomplete acinar cell differentiation. NR5A2 controls these developmental processes directly as well as through regulatory interactions with other pancreatic transcriptional regulators, including PTF1A, MYC, GATA4, FOXA2, RBPJL and MIST1 (BHLHA15). In particular, Nr5a2 and Ptf1a establish mutually reinforcing regulatory interactions and collaborate to control developmentally regulated pancreatic genes by binding to shared transcriptional regulatory regions. At the final stage of acinar cell development, the absence of NR5A2 affects the expression of Ptf1a and its acinar specific partner Rbpjl, so that the few acinar cells that form do not complete differentiation. Nr5a2 controls several temporally distinct stages of pancreatic development that involve regulatory mechanisms relevant to pancreatic oncogenesis and the maintenance of the exocrine phenotype.

Bazedoxifene-scaffold-based mimetics of solomonsterols A and B as novel pregnane X receptor antagonists.

Pregnane X receptor (PXR), a member of the NR1I nuclear receptor family, acts as a xenobiotic sensor and a paramount transcriptional regulator of drug-metabolizing enzymes and transporters. The overexpression of PXR in various cancer cells indicates the importance of PXR as a drug target for countering multidrug resistance in anticancer treatments. We describe the discovery of novel bazedoxifene-scaffold-based PXR antagonists inspired by the marine sulfated steroids solomonsterol A and B as natural leads. A luciferase reporter assay on a PXR-transfected HepG2 cell line identified compounds 19-24 as promising PXR antagonists. Further structure-activity relationship studies of the most active PXR antagonist from the series (compound 20, IC50 = 11 μM) revealed the importance of hydroxyl groups as hydrogen-bond donors for PXR antagonistic activity. PXR antagonists 20 and 24 (IC50 = 14 μM), in addition to the downregulation of PXR expression, exhibited inhibition of PXR-induced CYP3A4 expression, which illustrates their potential to suppress PXR-regulated phase-I drug metabolism.

Novel ligands for human pregnane X receptor differentially regulate CYP3A4 promoter activity (579.1)

Human pregnane X receptor (hPXR) also known as xenobiotic sensing nuclear receptor (SXR) is a member of nuclear receptor family and a critical regulator of the expression of genes involved in drug metabolism and clearance. The pregnane X receptor (PXR) regulates drug metabolism by regulating the expression of drug‐metabolizing enzymes such as cytochrome P450 3A4 (CYP3A4), which is involved in the metabolism of >50% of clinically prescribed drugs. It also plays an important role in cancer drug resistance by regulating the expression of MDR1. Activity of PXR can be modulated by agonists or antagonists. There are several agonists for PXR have been reported but the reported antagonists for PXR are limited. Therefore, there is still need to develop novel ligands for PXR. Here, we report the virtual screening of ~25,000 natural product derivatives from the ZINC database using the MOE docking software tool against the PXR‐Rifampicin complex X‐ray crystal structure. Our screening resulted in identification of compounds based on the lowest S scores which measures binding energy. All of these ligands were docked within the ligand binding pocket forming interactions with several hydrophobic amino acid residues. Further, all these main compounds and analogs were synthesized to verify with biological assays. We found that the compounds that bind directly to PXR, as revealed in an intrinsic tryptophan fluorescence assay, modulate CYP3A4 promoter activity differentially in HepG2 cells. Mutational analysis and docking studies showed that these compounds bind broadly in the ligand binding pocket but interact with different amino acid residues. The mechanism of binding and the functional group of the ligands that are important for distinguishing agonists/antagonists were also investigated.Grant Funding Source: Supported by the ALSAC, St. Jude CRH, NIH, NCI.

Inverse agonist of estrogen-related receptor γ controls Salmonella typhimurium infection by modulating host iron homeostasis

In response to microbial infection, expression of the defensin-like peptide hepcidin (encoded by Hamp) is induced in hepatocytes to decrease iron release from macrophages. To elucidate the mechanism by which Salmonella enterica var. Typhimurium (S. typhimurium), an intramacrophage bacterium, alters host iron metabolism for its own survival, we examined the role of nuclear receptor family members belonging to the NR3B subfamily in mouse hepatocytes. Here, we report that estrogen-related receptor γ (ERRγ, encoded by Esrrg) modulates the intramacrophage proliferation of S. typhimurium by altering host iron homeostasis, and we demonstrate an antimicrobial effect of an ERRγ inverse agonist. Hepatic ERRγ expression was induced by S. typhimurium-stimulated interleukin-6 signaling, resulting in an induction of hepcidin and eventual hypoferremia in mice. Conversely, ablation of ERRγ mRNA expression in liver attenuated the S. typhimurium-mediated induction of hepcidin and normalized the hypoferremia caused by S. typhimurium infection. An inverse agonist of ERRγ ameliorated S. typhimurium-mediated hypoferremia through reduction of ERRγ-mediated hepcidin mRNA expression and exerted a potent antimicrobial effect on the S. typhimurium infection, thereby improving host survival. Taken together, these findings suggest an alternative approach to control multidrug-resistant intracellular bacteria by modulating host iron homeostasis.

JAZF1 can regulate the expression of lipid metabolic genes and inhibit lipid accumulation in adipocytes

JAZF1 is a newly identified gene with unknown functions. A recent genome-wide association study showed that JAZF1 is associated with type 2 diabetes and is highly expressed in liver and adipose tissue. Studies have demonstrated that JAZF1 is the co-repressor for nuclear orphan receptor TAK1, whereas most nuclear orphan receptor family members are involved in the regulation of lipid metabolism. Therefore, JAZF1 could be closely related to glycolipid metabolism. In this study, JAZF1 was significantly upregulated during the induced differentiation process of 3T3-L1 preadipocytes. The overexpression of JAZF1 inhibited lipid accumulation in differentiated mature 3T3-L1 adipocytes and significantly inhibited the expression of SREBPl, ACC, and FAS, which were important in lipid synthesis, while upregulating the expression of key enzyme hormone-sensitive lipase in lipoclasis. Moreover, SREBPl exhibited an inhibitory function on the expression of JAZF1. SREBP1 reversed the inhibitory action on lipid accumulation of JAZF1. SREBP1 and JAZF1 were observed to regulate each other in adipocytes. Therefore, JAZF1 could regulate the expression of particular genes related to lipid metabolism and inhibit lipid accumulation in adipocytes. This result suggests that JAZF1 may be a potential target for the treatment of diseases, such as obesity and lipid metabolism disorders.

Mix ‘n’ match estrogens

Female sex hormones such as estrogen and progesterone are not only essential regulators of reproductive functions but play numerous physiological roles. Loss of endogenous hormone production after menopause can cause uncomfortable symptoms such as hot flashes and lead to the development of osteoporosis or cardiovascular disease. In order to maintain these beneficial anti-aging effects, various estrogen receptor ligands have been clinically tested for their use as postmenopausal hormone replacement therapy. To avoid potentially harmful stimulation of breast or endometrial cell proliferation, so-called selective estrogen receptor modulators or SERMs, have been developed. These compounds do not display the same profile as pure agonists or antagonists but can have differential effects in different tissues [1]. In this issue of Molecular Metabolism, Kim et al. investigate the use of the SERM bazedoxifene (BZA) in combination with estrogens to counter the weight gain and glucose intolerance associated with menopause [2]. The authors use ovariectomized mice fed a high fat diet and treated with various combinations of estrogen receptor (ER) ligands to study their effects on energy homeostasis. They demonstrate that a tissue-selective estrogen complex (TSEC) consisting of BZA plus conjugated equine estrogen, improves glucose homeostasis and insulin sensitivity without affecting the reproductive system. This therapy (BZA+CE) has been successful in the prevention of postmenopausal symptoms in phase III clinical trials, but its metabolic effects have not yet been studied [3,4]. Estrogen therapy for metabolic dysfunction is not a new concept [5,6], but, interestingly, the data presented by Kim et al. suggest that estrogens and BZA act via different pathways to increase hepatic lipid oxidation. As shown by ELISA and qRT-PCR assays on liver homogenates, only treatment with classical estrogens increases FGF21 expression levels, whereas BZA-containing complexes induce Sirtuin-1, PPARα and AMPK. FGF21 is known for its insulin-sensitizing potential and significant beneficial effects on several metabolic parameters [7]. Sirtuin-1 is the founder of the famous sirtuin deacetylase family, whose members play important roles in the regulation of lifespan and insulin sensitivity [8]. Likewise, the energy-sensing kinase adenosine monophosphate (AMP)-activated protein kinase (AMPK) has emerged as a central mediator of metabolism. AMPK responds to changes in the AMP:ATP ratio by promoting glucose and fatty acid catabolism or by inhibiting anabolic pathways. Linked to these pathways is the peroxisome proliferator-activated receptor PPARα, another nuclear receptor family member known to regulate fatty acid oxidation [9]. Activation of all of these pathways serves to enhance hepatic lipid oxidation and to elevate overall metabolic rate, and could explain the prevention of weight gain and insulin resistance observed during the experiments. How FGF21 activation in response to estrogens can achieve these effects to a similar extent as BZA-mediated SIRT1/PPARα/AMPK induction remains an open question. In the future, it will be revealing to determine the molecular mechanism for these observations. One might speculate how these ligands induce distinct target genes in the liver, for example by differential binding of ERα to target sites in the enhancer regions of Fgf21 versus Sirt1/PPARα. That would require BZA-bound ERα to bind to a different set of cis-regulatory elements than estrogen-ligand receptor. Another possibility would involve the activation of signaling pathways that indirectly influence hormone responses. It would also be interesting to test whether different receptor conformations lead to differential recruitment of coregulators. Finally, it is still unknown if these differential effects are only achieved via liver-specific pathways, or if systemic TSEC administration affects other systems such as adipose tissue, skeletal muscle or neuronal networks. Taken together, these findings provide mechanistic insight into ER-regulated metabolic gene programs and will hopefully provide a useful weapon in the battle against metabolic syndrome.

MINOR (NR4A3) Overexpression in Mouse Skeletal Muscle Enhances Insulin Action

MINOR (Mitogen-Inducible Nuclear Orphan Receptor) is one member of the NR4A3 nuclear orphan receptor family which are immediate early gene products involved in neuroendocrine regulation, neural differentiation, liver regeneration, cell apoptosis, and mitogenic and inflammatory stimulation in different cell types. We have found that MINOR can modulate insulin action and the glucose transport system in 3T3-L1 adipocytes; however, MINOR is highly expressed in skeletal muscle and its function in vivo is not well understood. To determine the role of MINOR in vivo, we have generated a mouse model that has the MINOR gene specifically expressed in the skeletal muscle using a muscle creatine kinase (MCK) promoter, and investigated whether the gene functions of MINOR would be linked to insulin action in vivo since skeletal muscle is one of the primary target tissues for insulin action. We demonstrate that these MCK-MINOR transgenic mice have reduced body weight due to a reduction of fat mass inside the body. Mice with MINOR overexpression also have improved insulin and glucose tolerances, reduced plasma levels of triglyceride, cholesterol and free fatty acid as well as enhanced expression of genes which are related to insulin action and its signaling pathways. Thus, MINOR functions in skeletal muscle act to improve insulin sensitivity and glucose intolerances and regulate insulin action and lipid and energy expenditure process.

Skp2 Regulates Subcellular Localization of PPARγ by MEK Signaling Pathways in Human Breast Cancer

Nuclear hormone receptor family member PPARγ plays an important role in mammary gland tumorigenesis. Previous studies have shown PPARγ has cytoplasmic activities upon tetradecanoyl phorbol acetate (TPA) stimulation. However, the clinical pathological significance of cytoplasmic PPARγ is not completely understood in human breast cancer. Skp2 is oncogenic, and its frequent amplification and overexpression correlated with the grade of malignancy. In this study, the role of cytoplasmic PPARγ and Skp2 expression was investigated in human breast cancer progression. Therefore, immunohistochemical analysis was performed on formalin-fixed paraffin sections of 70 specimens. Furthermore, Western blot and immunofluorescence microscopy analysis were used to study the relationship between expression of cytoplasmic PPARγ and Skp2 expression in human breast cancer cells in vitro. Results showed that the expression of cytoplasmic PPARγ was positively correlated with Skp2 expression (p < 0.05), and correlated significantly with estrogen receptor (p = 0.026) and pathological grade (p = 0.029), respectively. In addition, Skp2 overexpression can provoke cytoplasmic localization of PPARγ upon MEK1-dependent mechanisms in human breast cancer cells by nuclear-cytosolic fractionation technology and immunofluorescence microscopy analysis. Using RNA interference technology, we also found that down-regulated Skp2 reduced the phosphorylation level of MEK1 and significantly reversed TPA-induced nuclear export of PPARγ in MDA-MB-231 cells. The changes in the subcellular localization of PPARγ may represent a novel target for selective interference in patients with breast cancer.

Mineralocorticoid receptor activation as an etiological factor in kidney diseases

The mineralocorticoid receptor (MR) is a member of the steroid-responsive nuclear receptor family. Currently, in addition to its classical role in fluid homeostasis, attention has been focused on the pro-proteinuric and pro-inflammatory effects of MR in renal and cardiovascular diseases. Since proteinuria has been shown to be an important factor in the prognosis of patients with chronic kidney disease (CKD) [according to the newest Japanese Society of Nephrology and Kidney Disease: Improving Global Outcomes (KDIGO) guidelines for the treatment of CKD], it is worth discussing the role of MR in the progression of proteinuric CKD and the possible association with podocyte injury. Rac1, a Rho-GTPase family protein, is known for its role in the regulation of the cytoskeleton. We discovered the role of active Rac1 in amplifying MR activation in one of our studies and then continued to study how the Rac1-MR pathway contributes to the progression of kidney diseases. We then discovered the harmful effects of the activation of the Rac1-MR pathway in response to salt loading in the kidney for proteinuric kidney diseases of various animal models with salt-sensitive hypertension, such as Dahl salt-sensitive rats, RhoGDIα-knockout mice, angiotensin II-overproducing mice, and aldosterone-infused rats. In this review, we have introduced recent findings that suggest the contribution of MR activation to kidney diseases and the role of the Rac1-MR pathway in kidney injury associated with salt-sensitive hypertension and proteinuria. Thus, the Rac1-MR pathway is a potential therapeutic target in patients with proteinuric CKD.

Liver receptor homologue-1 expression in ovarian epithelial and granulosa cell tumours

Granulosa cell tumours of the ovary (GCT) express aromatase and produce oestrogens. The ovarian-specific aromatase promoter (pII) is regulated by members of the group 5A nuclear receptor family, SF-1 and LRH-1. Since both SF-1 and LRH-1 are implicated in proliferation and cancer, we hypothesised that alteration in the expression of either or both receptors may be associated with GCT. We therefore determined the expression of LRH-1, SF-1 and aromatase in a cohort of GCT, mucinous and serous cystadenocarcinomas, and normal ovaries. LRH-1 mRNA was present at low level in normal ovary and serous cystadenocarcinoma, but was elevated approximately 30-fold in GCT, and 8-fold in mucinous cystadenocarcinoma, compared to normal ovary. LRH-1 protein expression was confirmed in GCT by immunohistochemistry. SF-1 mRNA was significantly lower that of LRH-1 in all samples and not significantly altered in GCT, compared to normal ovary. Aromatase mRNA was present at low level in normal ovary and serous and mucinous cystadenocarcinoma, and significantly elevated (18-fold) in GCT compared to normal ovary. Despite the coordinate over-expression of both LRH-1 and aromatase in GCT versus normal ovary, their levels did not correlate in individual patients; rather, aromatase expression correlated with that of SF-1. Finally, although both LRH-1 and SF-1 activated aromatase promoter activity in transient transfection studies, gel-shift and chromatin immunoprecipitation data indicated that SF-1, but not LRH-1, bound to the aromatase promoter. We conclude that SF-1 regulates aromatase expression in GCT; over-expression of LRH-1 suggests that this receptor may be involved in the pathogenesis of GCT by mechanisms other than the regulation of aromatase. Its role in this disease therefore warrants further investigation.

Identification and Characterization of Novel Alternative Splice Variants of Human Constitutive Androstane Receptor in Liver Samples of Koreans and Caucasians

Human constitutive androstane receptor (hCAR, NR1I3) is a member of the orphan nuclear receptor family and regulates the transcription of many drug-metabolizing enzymes and drug transporters. Previous studies have shown that the hCAR gene produces a number of different kinds of mRNA splicing variants (SVs) in non-Asian ethnicities. In the present study, we identified 18 hCAR SVs (SV1-SV18), including four novel SVs in Korean human livers. Among the four novel SVs, SV2 showed enhanced transactivation activity when cotransfected with CYP2B6 reporter gene, whereas other SVs were nonfunctional. When profiles of major hCAR SVs were compared among 30 livers from Korean patients and 20 livers from Caucasian patients, the relative composition of each SV showed interethnic variation as well as interindividual variation. The most predominant form of hCAR SV was not wild type, but either SV4 or SV7. The summed relative amounts of SV4 and SV7 ranged from 34.5 to 57.6% in the 30 Korean livers and from 47.2 to 82.6% in the 20 Caucasian livers, suggesting large interindividual variation. The mean relative amount of nonfunctional SV9 was significantly higher in Koreans (29.8%) than in Caucasians (12.8%). The mean relative amount of novel SV2 was 9.7% in Korean livers and 3.5% in Caucasian livers. Expression profiling of hCAR proteins in human livers also supported large interindividual variation in the expressional ratio of wild-type and SVs. Our results describe for the first time the direct comparison of hCAR SV profiles between Koreans and Caucasians. The functional relevance of these interindividual and interethnic variations of hCAR mRNA expression needs to be further characterized.

The Nuclear Orphan Receptors NR4A as Therapeutic Target in Cancer Therapy

NR4A1 (Nur77), NR4A2 (Nurr1) and NR4A3 (Nor-1) are three members of the orphan nuclear receptor (NR) family referred to as NR4A family. This subgroup activates gene expression in a constitutive ligand-independent manner. These nuclear receptors are classified as early response genes that are induced by a diverse range of signals. These orphan NRs have been implicated in cell cycle regulation, apoptosis, inflammation, metabolism and more recently in carcinogenesis. The ultimate growth of a tumor depends not only on the rate of tumor cell proliferation, but also the rate of apoptosis and NR4A1 controls both, survival and death of cancer cells. It has been demonstrated that NR4A1 activities are regulated through its subcellular localisation. In the nucleus, NR4A1 can function in a context dependent manner either as an oncogenic survival factor, promoting cancer cell growth or as the opposite through the activation of apoptosis. Additionally, in an atypical fashion, it is a potent killer when migrating to the mitochondria, where it binds to Bcl-2 and converts its survival phenotype, triggering cytochrome c release and apoptosis. The most convincing evidence that nuclear orphan receptors function as critical tumor suppressors is the observation that the NR4A1 and NR4A3 double knock out mouse develops rapidly acute myeloid leukemia. Down regulation of NR4A1 and NR4A3 was a common feature in leukemic blasts from human AML patients. In particular, the recent identification of pro-apoptotic agents inducing NR4A expression or acting as agonists suggests that these members could serve as potential targets for cancer therapy.

The Orphan Nuclear Receptor DAX-1 Functions as a Potent Corepressor of the Constitutive Androstane Receptor (NR1I3)

Regulation of gene transcription is controlled in part by nuclear receptors that function coordinately with coregulator proteins. The human constitutive androstane receptor (CAR; NR1I3) is expressed primarily in liver and regulates the expression of genes involved in xenobiotic metabolism as well as hormone, energy, and lipid homeostasis. In this report, DAX-1, a nuclear receptor family member with corepressor properties, was identified as a potent CAR regulator. Results of transaction and mutational studies demonstrated that both DAX-1's downstream LXXLL and its PCFQVLP motifs were critical contributors to DAX-1's corepression activities, although two other LXXM/LL motifs located nearer the N terminus had no impact on the CAR functional interaction. Deletion of DAX-1's C-terminal transcription silencing domain restored CAR1 transactivation activity in reporter assays to approximately 90% of control, demonstrating its critical function in mediating the CAR repression activities. Furthermore, results obtained from mammalian two-hybrid experiments assessing various domain configurations of the respective receptors showed that full-length DAX-1 inhibited the CAR-SRC1 interaction by approximately 50%, whereas the same interaction was restored to 90% of control when the DAX-1 transcription silencing domain was deleted. Direct interaction between CAR and DAX-1 was demonstrated with both alpha-screen and coimmunoprecipitation experiments, and this interaction was enhanced in the presence of the CAR activator 6-(4-chlorophenyl)imidazo[2,1-b]thiazole-5-carbaldehyde O-(3,4-dichlorobenzyl)oxime (CITCO). Results obtained in primary human hepatocytes further demonstrated DAX-1 inhibition of CAR-mediated CITCO induction of the CYP2B6 target gene. The results of this investigation identify DAX-1 as a novel and potent CAR corepressor and suggest that DAX-1 functions as a coordinate hepatic regulator of CAR's biological function.