Nuclear Hormone Receptor Binding Research Articles

Human dyskerin binds to cytoplasmic H/ACA-box-containing transcripts affecting nuclear hormone receptor dependence

BackgroundDyskerin is a nuclear protein involved in H/ACA box snoRNA-guided uridine modification of RNA. In humans, its defective function is associated with cancer development and induces specific post-transcriptional alterations of gene expression. In this study, we seek to unbiasedly identify mRNAs regulated by dyskerin in human breast cancer-derived cells.ResultsWe find that dyskerin depletion affects the expression and the association with polysomes of selected mRNA isoforms characterized by the retention of H/ACA box snoRNA-containing introns. These snoRNA retaining transcripts (snoRTs) are bound by dyskerin in the cytoplasm in the form of shorter 3′ snoRT fragments. We then characterize the whole cytoplasmic dyskerin RNA interactome and find both H/ACA box snoRTs and protein-coding transcripts which may be targeted by the snoRTs’ guide properties. Since a fraction of these protein-coding transcripts is involved in the nuclear hormone receptor binding, we test to see if this specific activity is affected by dyskerin. Obtained results indicate that dyskerin dysregulation may alter the dependence on nuclear hormone receptor ligands in breast cancer cells. These results are paralleled by consistent observations on the outcome of primary breast cancer patients stratified according to their tumor hormonal status. Accordingly, experiments in nude mice show that the reduction of dyskerin levels in estrogen-dependent cells favors xenograft development in the absence of estrogen supplementation.ConclusionsOur work suggests a cytoplasmic function for dyskerin which could affect mRNA post-transcriptional networks relevant for nuclear hormone receptor functions.

Exploration of the Molecular Mechanism of Danzhi Xiaoyao Powder in Endometrial Cancer through Network Pharmacology.

Endometrial cancer (EC) is a common malignant tumor of the female reproductive system. Current treatments such as surgery and long-term hormone therapy are ineffective and have side effects. Danzhi Xiaoyao powder (DXP) can inhibit the growth of EC cells and induce apoptosis, but the pharmacological and molecular mechanisms of anticancer effects are still unclear. In this study, active components and potential targets of DXP were obtained from public databases. Protein effects and regulatory pathways of common targets were analyzed by protein-protein interaction (PPI), GO and KEGG. The results of network pharmacology showed that there are 87 common targets between EC and DXP. GO enrichment analysis showed that these targets were associated with response to oxidative stress, response to nutrient levels, hormone receptor binding and nuclear hormone receptor binding, etc. The results of KEGG analysis indicated that IL-17, TNF, PI3K/AKT, and RAS/RAF/MEK/ERK (ERK) signaling pathway were enriched in the anti-EC of DXP. Additionally, we cultured HEC-1B and KLE cells for validate experiments. DXP showed an inhibition of proliferation, migration, and cell cycle of both cells. Moreover, the expression of RAS, p-RAF, p-MEK, ERK, and p-ERK related proteins were downregulated. In conclusion, DXP might inhibit the proliferation of EC cells via apoptosis. Furthermore, DXP-induced inhibition of EC development might involve RAS/RAF/MEK/ERK pathway.

Low free triiodothyronineis predicts worsen neurological outcome of patients with acute ischemic stroke: a retrospective study with bioinformatics analysis

BackgroudPatients with acute ischemic stroke (AIS) often experience low serum free triiodothyronine (FT3), but the association of low FT3 with stroke severity, subtype and prognosis has not yet been thoroughly studied, and the molecular events underlying these clinical observation were also unclear.MethodsWe retrospectively collected 221 cases of AIS and 182 non-AIS cases with detailed clinical data from our department. FT3 concentrations were measured on admission to predict functional outcome within 3 months using multivariable models adjusted for other risk factors. Receiver operating characteristic (ROC) curves were calculated to define the best cutoff value of FT3 of stroke severity, subtypes and neurological outcome. Gene set enrichment, pathway mapping and network analyses of deferentially expressed genes (DEGs) were performed.ResultsFT3 was significantly decreased in AIS patients with National Institutes of Health Stroke Scale (NIHSS) > 3 and 3-months modified Rankin Scale (mRS) > 2. The cut-off value of FT3 for NIHSS on admission was 4.30 pmol/L. Also, FT3 level was significantly lower in large artery atherosclerosis (LAA) group and cardioembolism (CE) group than that in small vessel occlusion (SVO). FT3 value served as an independent predictor for neurological outcomes for which the cut-off value of FT3 was 4.38 pmol/l. Gene ontology (GO) analysis showed that the biological function of DEGs was mainly enriched in multicellur organism, neuron differentiation and cellular response to hypoxia. The cellular components were involved in extracelluar region, exosome and matrix, and the molecular functions were transcriptional activator activity, DNA binding and nuclear hormone receptor binding. Signal pathways analysis was indicative of neuroactive ligand-receptor interaction, thyroid hormone signaling pathway, and protein digestion and absorption these DEGs were involved in. Six related gene were identified as hubs from the protein-protein interaction (PPI) networks. Three modules were selected from PPI, of which MMP4, ADRA2C and EIF3E were recognized as the seed genes.ConclusionsLow FT3 value on admission was associated with stroke severity, subtype and prognosis. In addition, DEGs identified from bioinformatics analysis are likely to be candidates for elucidating clinical outcomes with low FT3, and provide us with therapeutic targets for improving stroke prognosis.

Mediation of antiinflammatory effects of Rg3-enriched red ginseng extract from Korean Red Ginseng via retinoid X receptor α–peroxisome-proliferating receptor γ nuclear receptors

BackgroundGinseng has a wide range of beneficial effects on health, such as the mitigation of minor and major inflammatory diseases, cancer, and cardiovascular diseases. There are abundant data regarding the health-enhancing properties of whole ginseng extracts and single ginsenosides; however, no study to date has determined the receptors that mediate the effects of ginseng extracts. In this study, for the first time, we explored whether the antiinflammatory effects of Rg3-enriched red ginseng extract (Rg3-RGE) are mediated by retinoid X receptor α–peroxisome-proliferating receptor γ (RXRα-PPARγ) heterodimer nuclear receptors. MethodsNitric oxide assay, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide assay, quantitative reverse transcription polymerase chain reaction, nuclear hormone receptor–binding assay, and molecular docking analyses were used for this study. ResultsRg3-RGE exerted antiinflammatory effects via nuclear receptor heterodimers between RXRα and PPARγ agonists and antagonists. ConclusionThese findings indicate that Rg3-RGE can be considered a potent antiinflammatory agent, and these effects are likely mediated by the nuclear receptor RXRα-PPARγ heterodimer.

Transcriptional Regulation of the Xenopus laevis Stromelysin-3 Gene by Thyroid Hormone Is Mediated by a DNA Element in the First Intron

The matrix metalloproteinase (MMP) stromelysin-3 (ST3) (MMP11) was first isolated as a breast cancer-associated gene and is expressed in diverse human carcinomas and various developmental processes involving apoptosis. The Xenopus laevis ST3 is highly up-regulated by thyroid hormone (T3) during amphibian metamorphosis, and its expression is spatially and temporally correlated with apoptosis in different tissues. Furthermore, it has been shown in vivo and in organ cultures to play a critical role in regulating T3-induced epithelial cell death during intestinal metamorphosis. Earlier studies suggest that ST3 is a direct T3 response gene, although a thyroid hormone response element (TRE) was not found in the initial analysis of the ST3 promoter. Here, we have identified a strong TRE consisting of two nearly perfect direct repeats of the consensus nuclear hormone receptor binding element AGGTCA separated by 4 bp in the first intron of the Xenopus ST3 gene. We show that the heterodimers of T3 receptor (TR) and 9-cis-retinoic acid receptor bind to the TRE both in vitro and in vivo in the context of chromatin. Furthermore, T3 induces strong activation of the promoter through the intronic TRE. Interestingly, although the unliganded TR/9-cis-retinoic acid receptor was able to recruit corepressors to the promoter, it had little repressive effect on the promoter in vivo. These results suggest that the intronic TRE mediates the inductive effect of T3 and that promoter context plays an important role in gene repression by unliganded TR.

Mutagenesis in mice of nuclear hormone receptor binding sites in the Igf2/H19 imprinting control region

The H19/Igf2 imprinting control region (ICR) is a DNA methylation-dependent chromatin insulator in somatic cells. The hypomethylated maternally inherited ICR binds the insulator protein CTCF at four sites, and blocks activity of the proximal Igf2 promoter by insulating it from the shared distal enhancers. The hypermethylated paternally inherited ICR lacks CTCF binding and insulator activity, but induces methylation-silencing of the paternal H19 promoter. The paternal-specific methylation of the ICR is established in the male germ cells, while the ICR emerges from the female germ line in an unmethylated form. Despite several attempts to find cis-regulatory elements, it is still unknown what determines these male and female germ cell-specific epigenetic modifications. We recently proposed that five in vivo footprints spanning fifteen half nuclear hormone receptor (NHR) binding sites within the ICR might be involved, and here we report on the effects of mutagenizing all of these half sites in mice. No effect was obtained – in the female and male germ lines the mutant ICR remained hypomethylated and hypermethylated, respectively. The ICR imprinting mechanism remains undefined.

16 Kallikrein 15 (KLK15) in prostate cancer: in silico analysis and single nucleotide polymorphism verification

Introduction: Prostate cancer is the most common cancer in Caucasian men and there is strong evidence that kallikreins are part of an enzymatic cascade pathway activated in this disease. Altered KLK15 expression has been associated with cancer progression and grade and we postulate that single nucleotide polymorphisms (SNPs) in the KLK15 gene, will alter gene expression and will be associated with prostate cancer susceptibility and prognosis.Materials and Methods: We have used in silico prediction of function of wildtype and variant promoter sequences through assessment of hormone receptor elements and transcription factor binding sites; as well as prediction of likely splice variants through genomic, splicing and EST databases and web sites, and multiple sequence alignment packages. We have also used PCR and sequence analysis to further characterise the promoter region of the gene.Results: In silico analysis of the KLK15 gene has identified the following: two putative promoter regions, two putative androgen response elements (AREs) and four putative estrogen response elements (EREs); two clusters of cis elements; and 109 SNPs. Forty‐seven SNPs alter transcription factor sites (22 gain sites), 20 gain/increase probability of an ERE and three alter nuclear hormone receptor binding sites. Three new EST clones have been identified by analysis of gene expression in CGAP databases and suggest a new KLK15 splice variant, with a different start site.Conclusion: We have identified a number of new SNPs in the KLK15 gene, which may be functionally important and, in collaboration with the Queensland Cancer Fund (ProsCan Study), we will further investigate the association of these SNPs with prostate cancer risk and prognosis.

The NR4A Subgroup: Immediate Early Response Genes with Pleiotropic Physiological Roles

The nuclear hormone receptor (NR) superfamily includes the orphan NR4A subgroup, comprised of Nur77 (NR4A1), Nurr1 (NR4A2) and NOR-1 (NR4A3). These NRs are classified as early response genes, are induced by a diverse range of signals, including fatty acids, stress, growth factors, cytokines, peptide hormones, phorbol esters, neurotransmitters, and physical stimuli (for example magnetic fields, shear stress). The ability to sense and rapidly respond to changes in the cellular environment thus appears to be a hallmark of this subfamily. The members of the NR4A subgroup are well conserved in the DNA binding domain (~91-95%) and the C-terminal ligand-binding domain (~60%), but are divergent in the N-terminal AB region. These receptors bind as monomers, homodimers and heterodimers with RXRs (to mediate retinoid signaling) to different permutations of the canonical NR binding motif. The NR4A subgroup activates gene expression in a constitutive ligand-independent manner. NR4A-mediated trans-activation (LBD) involves unusually active N-terminal AF-1 domains that mediate coactivator recruitment. Moreover, the NR4A receptors encode atypical LBDs and AF-2 domains. For example, the LBDs contain no cavity due to bulky hydrophobic residue side chains, and lack the classical coactivator-binding cleft constituted by helices 3, 4 and 12. However, a hydrophobic patch exists between helices 11 and 12, that encodes a novel cofactor interface that modulates transcriptional activity. In line with the pleiotropic physiological stimuli that induce the NR4A subgroup, these orphan NRs have been implicated in cell cycle regulation (and apoptosis), neurological disease, steroidogenesis, inflammation, carcinogenesis and atherogenesis.

Rapid Detection of Subtype-Selective Nuclear Hormone Receptor Binding with Bacterial Genetic Selection

Subtype-selective nuclear hormone receptor modulators could potentially allow the development of valuable tissue-specific therapeutics. A simple biosensor that allows subtype-specific nuclear hormone receptor binding to be reflected by the growth phenotype of Escherichia coli cells has been constructed. This system will potentially enable the facile detection or evolution of subtype-selective hormone analogues.

The Orphan Nuclear Receptor, Steroidogenic Factor 1, Regulates Neuronal Nitric Oxide Synthase Gene Expression in Pituitary Gonadotropes

Steroidogenic factor 1 (SF-1), an essential nuclear receptor, plays key roles in steroidogenic cell function within the adrenal cortex and gonads. It also contributes to reproductive function at all three levels of the hypothalamic-pituitary-gonadal axis. SF-1 regulates genes in the steroidogenic pathway, such as LHbeta, FSHbeta, and steroid hydroxylase. Abundant evidence suggests that nitric oxide (NO) has an important role in the control of reproduction due to its ability to control GnRH secretion from the hypothalamus and the preovulatory LH surge in pituitary gonadotropes. Recently, we cloned and characterized the promoter of mouse neuronal NO synthase (nNOS). nNOS is localized at all three levels of the hypothalamic-pituitary-gonadal axis to generate NO. We find that its major promoter resides at exon 2 in the pituitary gonadotrope alphaT3-1 cell line and that there is a nuclear hormone receptor binding site in this region, to which SF-1 can bind and regulate nNOS transcription. Mutation of the nuclear hormone receptor binding site dramatically decreases basal promoter activity and abolishes SF-1 responsiveness. A dominant negative of SF-1, in which the transactivation (AF-2) domain of SF-1 was deleted, inhibits nNOS exon 2 promoter activity. Dosage-sensitive reversal- adrenal hypoplasia congenita critical region on the X chromosome, gene 1 (DAX-1), which colocalizes and interferes with SF-1 actions in multiple cell lineages, negatively modulates SF-1 regulation of nNOS transcription. These findings demonstrate that mouse nNOS gene expression is regulated by the SF-1 gene family in pituitary gonadotropes. nNOS, a member of the cytochrome p450 gene family, could be one of the downstream effector genes, which mediates SF-1's reproductive function and developmental patterning.

Isolation and Characterization of a Novel Class II Histone Deacetylase, HDAC10

A novel histone deacetylase, HDAC10, was isolated from a mixed tissue human cDNA library. HDAC10 was classified as a class II subfamily member based upon similarity to HDAC6. The genomic structure of HDAC10 was found to consist of 20 exons. HDAC10 has two sequence variants, HDAC10v1 and HDAC10v2, and two transcripts were detectable by Northern blot analysis. HDAC10v1 and HDAC10v2 were found to be identical through exon 17 but diverged after this exon. HDAC10v2 has an 82-bp alternate exon that generates a frameshift and shortens the sequence by 11 amino acids. In this study, the characterization of HDAC10v1 was performed. HDAC10v1 has an N-terminal catalytic domain, two putative C-terminal retinoblastoma protein binding domains, and a nuclear hormone receptor binding motif. The HDAC10v1 enzyme was found to be catalytically active based upon its ability to deacetylate a (3)H-acetylated histone H4 N-terminal peptide. Immunofluorescence detection of transfected HDAC10v1-FLAG indicated that the enzyme is a nuclear protein. Furthermore, coimmunoprecipitation experiments indicated that HDAC10v1 associated with HDAC2 and SMRT (silencing mediator for retinoid and thyroid hormone receptors). In addition, based upon the public data base, a single nucleotide polymorphism was found in the C terminus of HDAC10 which changes a Gly residue to Cys, suggesting that HDAC10 molecules containing these single nucleotide polymorphisms may be folded improperly. HDAC10 extends the HDAC superfamily and adds to a growing number of HDACs that have been found to have splice variants, suggesting that RNA processing may play a role in mediating the activity of HDACs.

Bioaffinity Sensor for Endocrine Disruptor Using Receptor-modified Au Electrode

Recombinant human estrogen receptor ligand-binding domain was immobilized on gold disk electrode with Ni(II)-mediated chemisorption using the histidine tag and disulfide-derivatives nitrilotriacetic acid. Cyclic voltammograms of ferrocyanide/ferricyanide redox couple with the receptor-modified electrode gave the peak currents due to the reversible electrode reaction, which were significantly suppressed by the presence of estrogen in concentration dependent manner. This bioaffinity sensor is potentially worthy for the development of rapid screening system of environmental chemicals for nuclear hormone receptor binding activities.

Transcriptional control of intestinal cytochrome P-4503A by 1alpha,25-dihydroxy vitamin D3.

It was previously shown that CYP3A4 is induced in the human intestinal Caco-2 cell model by treatment with 1alpha,25-dihydroxy vitamin D3 (1,25-D3). We demonstrate the vitamin D analog, 19-nor-1alpha,25-dihydroxy vitamin D2, is also an effective inducer of CYP3A4 in Caco-2 cells, but with half the potency of 1,25-D3. We report that treatment of LS180 cells, a human intestinal cell line, with 1 to 10 nM 1,25-D3 dose dependently increased CYP3A4 protein and CYP3A4 mRNA expression. CYP3A4- and CYP3A23-promoter-Luciferase reporter constructs transiently transfected into LS180 cells were transcriptionally activated in a dose-dependent manner by 1,25-D3, whereas mutation of the nuclear hormone receptor binding motif (ER6) in the CYP3A4 promoter abrogated 1,25-D3 activation of CYP3A4. Although the CYP3A4 ER6 promoter element has been shown to bind the pregnane X receptor (PXR), this receptor does not mediate 1,25-D3 induction of CYP3A4 because a) PXR is not expressed in Caco-2 cells; b) PXR mRNA expression is not induced by 1,25-D3 treatment of LS180 cells; and c) the ligand binding domain of human PXR was not activated by 1,25-D3. 1,25-D3 uses the vitamin D receptor to induce CYP3A4 because a) the vitamin D receptor (VDR)-retinoid X receptor (RXR) heterodimer binds specifically to the CYP3A4 ER6; b) selective mutation of the CYP3A4 ER6 disrupted the binding of VDR-RXR; and c) reporter constructs containing only three copies of the CYP3A4 ER6 linked to a TK-CAT reporter were activated by 1,25-D3 only in cells cotransfected with a human VDR expression plasmid. These data support the hypothesis that 1,25-D3 and VDR induce expression of intestinal CYP3A by binding of the activated VDR-RXR heterodimer to the CYP3A PXR response element and promoting gene transcription.

Replication of the wild type and a natural hepatitis B virus nucleocapsid promoter variant is differentially regulated by nuclear hormone receptors in cell culture.

A natural hepatitis B virus (HBV) variant associated with seroconversion from HBeAg to anti-HBe antibody contains two nucleotide substitutions (A1764T and G1766A) in the proximal nuclear hormone receptor binding site in the nucleocapsid promoter. These nucleotide substitutions prevent the binding of the retinoid X receptor alpha (RXR alpha)-peroxisome proliferator-activated receptor alpha (PPAR alpha) heterodimer without greatly altering the efficiency of binding of hepatocyte nuclear factor 4 (HNF4) to this recognition sequence. In addition, these nucleotide substitutions create a new binding site for HNF1. Analysis of HBV transcription and replication in nonhepatoma cells indicates that RXR alpha-PPAR alpha heterodimers support higher levels of pregenomic RNA transcription from the wild-type than from the variant nucleocapsid promoter, producing higher levels of wild-type than of variant replication intermediates. In contrast, HNF4 supports higher levels of pregenomic RNA transcription from the variant than from the wild-type nucleocapsid promoter, producing higher levels of variant than of wild-type replication intermediates. HNF1 can support variant virus replication at a low level but is unable to support replication of the wild-type HBV genome. These observations indicate that the replication of wild-type and variant viruses can be differentially regulated by the liver-specific transcription factors that bind to the proximal nuclear hormone receptor binding site of the nucleocapsid promoter. Differential regulation of viral replication may be important in the selection of specific viral variants as a result of an antiviral immune response.

The COP9 complex is conserved between plants and mammals and is related to the 26S proteasome regulatory complex

The COP9 complex, genetically identified in Arabidopsis as a repressor of photomorphogenesis, is composed of multiple subunits including COP9, FUS6 (also known as COP11) and the Arabidopsis JAB1 homolog 1 (AJH1) ([1–3]; unpublished observations).We have previously demonstrated the existence of the mammalian counterpart of the COP9 complex and purified the complex by conventional biochemical and immunoaffinity procedures [4]. Here, we report the molecular identities of all eight subunits of the mammalian COP9 complex. We show that the COP9 complex is highly conserved between mammals and higher plants, and probably among most multicellular eukaryotes. It is not present in the single-cell eukaryote Saccharomyces cerevisiae, however. All of the subunits of the COP9 complex contain structural features that are also present in the components of the proteasome regulatory complex and the translation initiation factor eIF3 complex. Six subunits of the COP9 complex have overall similarity with six distinct non-ATPase regulatory subunits of the 26S proteasome, suggesting that the COP9 complex and the proteasome regulatory complex are closely related in their evolutionary origin. Subunits of the COP9 complex include regulators of the Jun N-terminal kinase (JNK) and c-Jun, a nuclear hormone receptor binding protein and a cell-cycle regulator. This suggests that the COP9 complex is an important cellular regulator modulating multiple signaling pathways.