NR4A orphan nuclear receptors influence retinoic acid and docosahexaenoic acid signaling via up-regulation of fatty acid binding protein 5

The orphan nuclear receptor (NR) Nurr1 is expressed in the developing and adult nervous system and is also induced as an immediate early gene in a variety of cell types. In silico analysis of human promoters identified fatty acid binding protein 5 (FABP5), a protein shown to enhance retinoic acid-mediated PPARβ/δ signaling, as a potential Nurr1 target gene. Nurr1 has previously been implicated in retinoid signaling via its heterodimerization partner RXR. Since NRs are commonly involved in cross-regulatory control we decided to further investigate the regulatory relationship between Nurr1 and FABP5. FABP5 expression was up-regulated by Nurr1 and other NR4A NRs in HEK293 cells, and Nurr1 was shown to activate and bind to the FABP5 promoter, supporting that FABP5 is a direct downstream target of NR4A NRs. We also show that the RXR ligand docosahexaenoic acid (DHA) can induce nuclear translocation of FABP5. Moreover, via up-regulation of FABP5 Nurr1 can enhance retinoic acid-induced signaling of PPARβ/δ and DHA-induced activation of RXR. We also found that other members of the NR4A orphan NRs can up-regulate FABP5. Thus, our findings suggest that NR4A orphan NRs can influence signaling events of other NRs via control of FABP5 expression levels.     

A method for the separation and partial purification of the three forms of nitrate reductase present in wild-type soybean leaves

A rapid and simple purification method was used to separate and purify nitrate reductases (NR) from Williams soybean leaves. Blue Sepharose columns were sequentially eluted with 50 millimolar NADPH and 50 millimolar NADH, thus separating NAD(P)H:NR from NADH:NRs. Subsequent purification of the collected peaks on a fast protein liquid chromatography-Mono Q column enabled separation of two NADH:NRs. Sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed that the subunit relative molecular mass for all three NR forms (constitutive NAD(P)H:NR [pH 6.5], EC 1.6.6.2; constitutive NADH:NR [pH 6.5], EC not assigned; and inducible NADH:NR [pH 7.5], EC 1.6.6.1) was approximately 107 to 109 kilodaltons. All three NRs showed similar spectra with absorption maxima at 413 and 273 nanometers in the oxidized state, and with the characteristics of a cytochrome b type heme upon reduction with NADH (absorption maxima at 556, 527, and 424 nanometers). The technique developed provides an improved separation of the three NR forms from soybean leaves. The similarity of the NRs with regard to their cytochrome b₅₅₆ type heme content and in relative molecular mass indicated that other differences must exist to account for the different kinetic and physical properties previously reported.     

Evolutionary selection across the nuclear hormone receptor superfamily with a focus on the NR1I subfamily (vitamin D,pregnane X,and constitutive androstane receptors)

Background

The nuclear hormone receptor (NR) superfamily complement in humans is composed of 48 genes with diverse roles in metabolic homeostasis, development, and detoxification. In general, NRs are strongly conserved between vertebrate species, and few examples of molecular adaptation (positive selection) within this superfamily have been demonstrated. Previous studies utilizing two-species comparisons reveal strong purifying (negative) selection of most NR genes, with two possible exceptions being the ligand-binding domains (LBDs) of the pregnane X receptor (PXR, NR1I2) and the constitutive androstane receptor (CAR, NR1I3), two proteins involved in the regulation of toxic compound metabolism and elimination. The aim of this study was to apply detailed phylogenetic analysis using maximum likelihood methods to the entire complement of genes in the vertebrate NR superfamily. Analyses were carried out both across all vertebrates and limited to mammals and also separately for the two major domains of NRs, the DNA-binding domain (DBD) and LBD, in addition to the full-length sequences. Additional functional data is also reported for activation of PXR and the vitamin D receptor (VDR; NR1I1) to gain further insight into the evolution of the NR1I subfamily.

Results

The NR genes appear to be subject to strong purifying selection, particularly in the DBDs. Estimates of the ratio of the non-synonymous to synonymous nucleotide substitution rates (the ω ratio) revealed that only the PXR LBD had a sub-population of codons with an estimated ω ratio greater than 1. CAR was also unusual in showing high relative ω ratios in both the DBD and LBD, a finding that may relate to the recent appearance of the CAR gene (presumably by duplication of a pre-mammalian PXR gene) just prior to the evolution of mammals. Functional analyses of the NR1I subfamily show that human and zebrafish PXRs show similar activation by steroid hormones and early bile salts, properties not shared by sea lamprey, mouse, or human VDRs, or by Xenopus laevis PXRs.

Conclusion

NR genes generally show strong sequence conservation and little evidence for positive selection. The main exceptions are PXR and CAR, genes that may have adapted to cross-species differences in toxic compound exposure.

     

The mammalian orphan nuclear receptors: orphans as cellular guardians

In classical endocrinology, receptors are molecules that bind a hormone or a ligand to transduce signal within a target cell. Later, however, many intracellular receptors have been discovered in mammals, which have not been shown to bind endogenous ligands and are now are referred as “orphan receptors.” The orphan receptors share high degree of structural and functional homology with the classical nuclear receptors (NRs) and are now part of the NR superfamily and therefore referred as orphan nuclear receptors (ONRs). Interestingly, however, ONR members are not evolutionarily or functionally linked and they form a highly diverse group within the NR superfamily. In mammals, ONRs exhibit great functional diversity and majority of them are expressed in a tissue-specific fashion. In the past one decade, functional studies have revealed that they are mediators of multitude of crucial metabolic, developmental, reproductive, and immunological functions in mammals. Emerging studies also indicate the role of ONRs in the onset of several complex human diseases and hence they may be potential candidates for therapeutic drug targeting in the future.     

The mammalian orphan nuclear receptors: orphans as cellular guardians

In classical endocrinology, receptors are molecules that bind a hormone or a ligand to transduce signal within a target cell. Later, however, many intracellular receptors have been discovered in mammals, which have not been shown to bind endogenous ligands and are now are referred as "orphan receptors." The orphan receptors share high degree of structural and functional homology with the classical nuclear receptors (NRs) and are now part of the NR superfamily and therefore referred as orphan nuclear receptors (ONRs). Interestingly, however, ONR members are not evolutionarily or functionally linked and they form a highly diverse group within the NR superfamily. In mammals, ONRs exhibit great functional diversity and majority of them are expressed in a tissue-specific fashion. In the past one decade, functional studies have revealed that they are mediators of multitude of crucial metabolic, developmental, reproductive, and immunological functions in mammals. Emerging studies also indicate the role of ONRs in the onset of several complex human diseases and hence they may be potential candidates for therapeutic drug targeting in the future.     

Minireview: nuclear receptors and breast cancer

Until recently, the study of nuclear receptor (NR) function in breast cancer biology has been largely limited to estrogen and progesterone receptors. The development of reliable gene expression arrays, real-time quantitative RT-PCR, and immunohistochemical techniques for studying NR superfamily members in primary human breast cancers has now revealed the presence and potential importance of several additional NRs in the biology of breast cancer. These include receptors for steroid hormones (including androgens and corticosteroids), fat-soluble vitamins A and D, fatty acids, and xenobiotic lipids derived from diet. It is now clear that after NR activation, both genomic and nongenomic NR pathways can coordinately activate growth factor signaling pathways. Advances in our understanding of both NR functional networks and epithelial cell growth factor signaling pathways have revealed a frequent interplay between NR and epithelial cell growth factor family signaling that is clinically relevant to breast cancer. Understanding how growth factor receptors and their downstream kinases are activated by NRs (and vice-versa) is a central goal for maximizing treatment opportunities in breast cancer. In addition to the estrogen receptor, it is predicted that modulating the activity of other NRs will soon provide novel prevention and treatment approaches for breast cancer patients.     

Enhanced steatosis by nuclear receptor ligands: A study in cultured human hepatocytes and hepatoma cells with a characterized nuclear receptor expression profile

Steatosis is the first step in the development of non-alcoholic fatty liver disease (NAFLD). However, the mechanisms involved in its pathogenesis are not fully understood. Many nuclear receptors (NRs) involved in energy homeostasis and biotransformation constitute a network connecting fatty acids, cholesterol and xenobiotic metabolisms; therefore, multiple NRs and their ligands may play a prominent role in liver fat metabolism and accumulation. In this study we have attempted to gain insight into the relevance of the NR superfamily in NAFLD by investigating the steatogenic potential of 76 different NR ligands in fatty acid overloaded human hepatocytes and hepatoma cells. Moreover, we have determined the mRNA expression level of 24 NRs to correlate the steatogenic potential of the ligands with the expression of their associated NRs in the cultured cells. Our results demonstrate that 18% of the examined NR ligands enhanced lipid accumulation in human hepatocytes and/or hepatoma cells. Among them, ligands of PPARγ (e.g., thiazolidinediones), LXR (paxilline and 24(S),25-epoxycholesterol), PXR (hyperforin), CAR (3α,5α-androstenol), ERα (tamoxifen), FXR (Z-guggulsterone), VDR (25-hydroxyvitamin D3) and particular retinoids and farnesoids showed a significant pro-steatotic effect. The mRNA level of most of the NRs examined was well preserved in human hepatocytes, but HepG2 showed a deranged profile, where many of the receptors had a marginal or negligible level of expression in comparison with the human liver. By comparing the steatogenic effect of NR ligands with the NR expression levels, we conclude that LXR, PXR, RAR and PPARγ ligands likely induce fat accumulation by a NR-dependent mechanism. Indeed, over-expression of PXR in HepG2 cells enhanced the steatogenic effect of hyperforin and rifampicin. However, the accumulation of fat induced by other ligands did not correlate with the expression of their associated NR. Our results also suggest that human hepatocytes cultured with free fatty acids offer a highly valuable in vitro system to investigate the pathogenesis and therapeutics of the human fatty liver.     

Comparison of complete nuclear receptor sets from the human, Caenorhabditis elegans and Drosophila genomes

Background

The availability of complete genome sequences enables all the members of a gene family to be identified without limitations imposed by temporal, spatial or quantitative aspects of mRNA expression. Using the nearly completed human genome sequence, we combined in silico and experimental approaches to define the complete human nuclear receptor (NR) set. This information was used to carry out a comparative genomic study of the NR superfamily.

Results

Our analysis of the human genome identified two novel NR sequences. Both these contained stop codons within the coding regions, indicating that both are pseudogenes. One (HNF4 γ-related) contained no introns and expressed no detectable mRNA, whereas the other (FXR-related) produced mRNA at relatively high levels in testis. If translated, the latter is predicted to encode a short, non-functional protein. Our analysis indicates that there are fewer than 50 functional human NRs, dramatically fewer than in Caenorhabditis elegans and about twice as many as in Drosophila. Using the complete human NR set we made comparisons with the NR sets of C. elegans and Drosophila. Searches for the >200 NRs unique to C. elegans revealed no human homologs. The comparative analysis also revealed a Drosophila member of NR subfamily NR3, confirming an ancient metazoan origin for this subfamily.

Conclusions

This work provides the basis for new insights into the evolution and functional relationships of NR superfamily members.     

Changes in Nuclear Receptor and Vitellogenin Gene Expression in Response to Steroids and Heavy Metal in Caenorhabditis elegans

To gain basic understanding of the reproductive and developmental effects of endocrine disrupting chemicals in invertebrates, we have used C. elegans as an animal model. The completion of the C. elegans genome sequence brings to bear microarray analysis as a tool for these studies. We previously showed that the C. elegans genome was responsive to vertebrate steroid hormones, and changes in gene expression of traditional biomarkers used in environmental studies were detected; i.e., vitellogenin (vtg), cytochrome P450 (cyp450), glutathione-S-transferase (gst) and heat shock proteins (hsp). The data were interpreted to suggest that exogenous lipophilic compounds can be metabolized via cytochrome P450 proteins, and that the resulting metabolites can bind to members of the Nuclear Receptor (NR) class of proteins and regulate gene expression. In the present study, using DNA microarrays, we examined the pattern of gene expression after progesterone (10(-5), 10(-7) M), estradiol (10(-5) M), cholesterol (10(-9) M) and cadmium (0.1, 1 and 10 μM) exposure, with special attention to the members of NRs. Of approximately 284 NRs in C. elegans, expression of 25 NR genes (representing 9% of the total NRs in C. elegans) was altered after exposure to steroids. Of note, each steroid activated or inhibited different subsets of NR genes, and only estradiol regulated NR genes implicated in neurogenesis. These results suggest that NRs respond to a variety of exogenous steroids, which regulate important metabolic and developmental pathways. The response of the C. elegans genome to cholesterol and cadmium was analyzed in more detail. Cholesterol is a probable precursor to signaling molecules that may interact with NRs and we focused on expression of genes related to lipid metabolism (cyp450), transport and storage (i.e., vitellogenin). Worms exposed to cadmium respond principally by activating the expression of genes encoding stress-responsive proteins, such as mtl-2 and cdr-1, and no significant changes in expression of NRs or vtg genes were observed. The possible implications of these results with regard to the evolution of steroid receptors, endocrine disruption and the role of vitellogenin as a lipid transporter are discussed.     

Systems-level analysis of gene expression data revealed NR0B2/SHP as potential tumor suppressor in human liver cancer

Nuclear receptors (NRs) play pivotal roles in cell growth, proliferation, differentiation and homeostasis. Recent progress demonstrates that NR is tightly linked to human disease such as cancer, diabetes and obesity. Here we explore NR expression profiles in human tissue using systematic approaches. NR gene profiles reveal that individual NR has its own gene expression signature depending on tissue type. Of many organs, NRs expression is enriched in liver. Expression of many NRs was significantly changed in liver cancer. Notably, NR0B2/SHP expression level was significantly decreased in human liver cancer but not in normal liver. In addition, expression of SHP is well associated with good prognosis. SHP gene network analysis based on microarray data in liver cancer shows that SHP regulates cell proliferation and metabolism related gene sets. Our systematic approaches suggest that loss of SHP expression in liver might be key genetic events during hepatocarcinogenesis.