Transcriptional Regulation of HumanRev-erbαGene Expression by the Orphan Nuclear Receptor Retinoic Acid-related Orphan Receptor α

The Rev-erb and retinoic acid-related orphan receptors (ROR) are two related families of orphan nuclear receptors that recognize similar response elements but have opposite effects on transcription. Recently, theRev-erbαgene promoter has been characterized and shown to harbor a functional Rev-erbα-binding site known as Rev-DR2, responsible for negative feedback down-regulation of promoter activity by Rev-erbα itself. The present study aimed to investigate whetherRev-erbαgene expression is regulated by RORα. Gel shift analysis demonstrated thatin vitrotranslated hRORα1 protein binds to the Rev-DR2 site, both as monomer and dimer. Chromatin immunoprecipitation assays demonstrated that binding of RORα to this site also occurredin vivoin human hepatoma HepG2 cells. The Rev-DR2 site was further shown to be functional as it conferred hRORα1 responsiveness to a heterologous promoter and to the natural humanRev-erbαgene promoter in these cells. Mutation of this site in the context of the naturalRev-erbαgene promoter abolished its activation by RORα, indicating that this site plays a key role in hRORα1 action. Finally, adenoviral overexpression of hRORα1 in HepG2 cells led to enhanced hRev-erbα mRNA accumulation, further confirming the physiological importance of RORα1 in the regulation of Rev-erbα expression.

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Heterodimerization between Members of the Nur Subfamily of Orphan Nuclear Receptors as a Novel Mechanism for Gene Activation

Molecular and Cellular Biology ◽

10.1128/mcb.19.11.7549 ◽

1999 ◽

Vol 19 (11) ◽

pp. 7549-7557 ◽

Cited By ~ 166

Author(s):

Mario Maira ◽

Christine Martens ◽

Alexandre Philips ◽

Jacques Drouin

Keyword(s):

Mammalian Cells ◽

Target Genes ◽

Lymphoid Cells ◽

Orphan Receptor ◽

Target Sequence ◽

Related Factor ◽

Orphan Nuclear Receptor ◽

Orphan Nuclear Receptors

ABSTRACT We have recently shown that the orphan nuclear receptor Nur77 (NGFI-B) is most active in transcription when it is interacting with a cognate DNA sequence as a homodimer. Further, we have shown that the target for Nur77 dimers, the Nur response element (NurRE), is responsive to physiological stimuli in both endocrine and lymphoid cells, whereas other DNA targets of Nur77 action are not. The Nur77 subfamily also includes two related receptors, Nur-related factor 1 (Nurr1) and neuron-derived orphan receptor 1 (NOR-1). Often, more than one member of this subfamily is induced in response to extracellular signals. We now show that Nur77 and Nurr1 form heterodimers in vitro in the presence or absence of NurRE, and we have documented interactions between these proteins in vivo by using a two-hybrid system in mammalian cells. These heterodimers synergistically enhance transcription from NurRE reporters in comparison to that seen with homodimers. The naturally occurring NurRE from the pro-opiomelanocortin gene preferentially binds and activates transcription in the presence of Nur77 homo- or heterodimers, while a consensus NurRE sequence does not show this preference. Taken together, the data indicate that members of the Nur77 subfamily are most potent as heterodimers and that different dimers exhibit target sequence preference. Thus, we propose that a combinatorial code relying on specific NurRE sequences might be responsible for the activation of subsets of target genes by one of the members of the Nur77 subfamily of transcription factors.

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Spermatogenesis and Testis Development Are Normal in Mice Lacking Testicular Orphan Nuclear Receptor 2

Molecular and Cellular Biology ◽

10.1128/mcb.22.13.4661-4666.2002 ◽

2002 ◽

Vol 22 (13) ◽

pp. 4661-4666 ◽

Cited By ~ 30

Author(s):

Chih-Rong Shyr ◽

Loretta L. Collins ◽

Xiao-Min Mu ◽

Kenneth A. Platt ◽

Chawnshang Chang

Keyword(s):

Protein Expression ◽

Nuclear Receptor ◽

Expression Patterns ◽

Orphan Receptor ◽

Orphan Nuclear Receptor ◽

Male Mice ◽

Orphan Nuclear Receptors ◽

Nuclear Receptor Superfamily ◽

Testis Development

ABSTRACT Early in vitro cell culture studies suggested that testicular orphan nuclear receptor 2 (TR2), a member of the nuclear receptor superfamily, may play important roles in the control of several pathways including retinoic acids, vitamin D, thyroid hormones, and ciliary neurotrophic factor. Here we report the surprising results showing that mice lacking TR2 are viable and have no serious developmental defects. Male mice lacking TR2 have functional testes, including normal sperm number and motility, and both male and female mice lacking TR2 are fertile. In heterozygous TR2+/− male mice we found that β-galactosidase, the indicator of TR2 protein expression, was first detected at the age of 3 weeks and its expression pattern was restricted mainly in the spermatocytes and round spermatids. These protein expression patterns were further confirmed with Northern blot analysis of TR2 mRNA expression. Together, results from TR2-knockout mice suggest that TR2 may not play essential roles in spermatogenesis and normal testis development, function, and maintenance. Alternatively, the roles of TR2 may be redundant and could be played by other close members of the nuclear receptor superfamily such as testicular orphan receptor 4 (TR4) or unidentified orphan receptors that share many similar functions with TR2. Further studies with double knockouts of both orphan nuclear receptors, TR2 and TR4, may reveal their real physiological roles.

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Discovery and Optimization of a Series of Sulfonamide Inverse Agonists for the Retinoic Acid Receptor-Related Orphan Receptor-α

Medicinal Chemistry ◽

10.2174/1573406415666190222124745 ◽

2019 ◽

Vol 15 (6) ◽

pp. 676-684

Author(s):

Christelle Doebelin ◽

Yuanjun He ◽

Sean Campbell ◽

Philippe Nuhant ◽

Naresh Kumar ◽

...

Keyword(s):

Retinoic Acid Receptor ◽

Research Effort ◽

Pharmacokinetic Profile ◽

Orphan Receptor ◽

Orphan Nuclear Receptor ◽

Inverse Agonists ◽

Close Family Member ◽

Per Oral

Background: Despite a massive industry endeavor to develop RORγ-modulators for autoimmune disorders, there has been no indication of efforts to target the close family member RORα for similar indications. This may be due to the misconception that RORα is redundant to RORγ, or the inherent difficulty in cultivating tractable starting points for RORα. RORα-selective modulators would be useful tools to interrogate the biology of this understudied orphan nuclear receptor. Objectives: he goal of this research effort was to identify and optimize synthetic ligands for RORα starting from the known LXR agonist T0901317. Methods: Fourty-five analogs of the sulfonamide lead (1) were synthesized and evaluated for their ability to suppress the transcriptional activity of RORα, RORγ, and LXRα in cell-based assays. Analogs were characterized by 1H-NMR, 13C-NMR, and LC-MS analysis. The pharmacokinetic profile of the most selective RORα inverse agonist was evaluated in rats with intraperitoneal (i.p.) and per oral (p.o.)dosing. Results: Structure-activity relationship studies led to potent dual RORα/RORγ inverse agonists as well as RORα-selective inverse agonists (20, 28). LXR activity could be reduced by removing the sulfonamide nitrogen substituent. Attempts to improve the potency of these selective leads by varying substitution patterns throughout the molecule proved challenging. Conclusion: The synthetic RORα-selective inverse agonists identified (20, 28) can be utilized as chemical tools to probe the function of RORα in vitro and in vivo.

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Regulation of the Human Secretin Gene Is Controlled by the Combined Effects of CpG Methylation, Sp1/Sp3 Ratio, and the E-Box Element

Molecular Endocrinology ◽

10.1210/me.2003-0461 ◽

2004 ◽

Vol 18 (7) ◽

pp. 1740-1755 ◽

Cited By ~ 37

Author(s):

Leo Tsz-On Lee ◽

Kian-Cheng Tan-Un ◽

Ronald Ting-Kai Pang ◽

David Tai-Wai Lam ◽

Billy Kwok-Chong Chow

Keyword(s):

Gene Expression ◽

Hepg2 Cells ◽

Cpg Island ◽

Methylation Status ◽

Gene Promoter ◽

Mobility Shift ◽

Specific Pcr ◽

E Box

Abstract To unravel the mechanisms that regulate the human secretin gene expression, in this study, we have used secretin-expressing (HuTu-80 cells, human duodenal adenocarcinoma) and non-secretin-expressing [PANC-1 (human pancreatic ductile carcinoma) and HepG2 (human hepatocellular carcinoma) cells] cell models for in vitro and in vivo analyses. By transient transfection assays, within the promoter region (−11 to −341 from ATG, relative to the ATG initiation codon), we have initially identified several functional motifs including an E-box and 2 GC-boxes. Results from gel mobility shift and chromatin immunoprecipitation assays confirmed further that NeuroD, E2A, Sp1, and Sp3 bind to these E- and GC-boxes in HuTu-80 cells in vitro and in vivo, whereas only high levels of Sp3 is observed to bind the promoter in HepG2 cells. In addition, overexpression of Sp3 resulted in a dose-dependent repression of the Sp1-mediated transactivation. Collectively, these data suggest that the Sp1/Sp3 ratio is instrumental to controlling secretin gene expression in secretin-producing and non-secretin-producing cells. The functions of GC-box and Sp proteins prompted us to investigate the possible involvement of DNA methylation in regulating this gene. Consistent with this idea, we found a putative CpG island (−336 to 262 from ATG) that overlaps with the human secretin gene promoter. By methylation-specific PCR, all the CpG dinucleo-tides (26 of them) within the CpG island in HuTu-80 cells are unmethylated, whereas all these sites are methylated in PANC-1 and HepG2 cells. The expressions of secretin in PANC-1 and HepG2 cells were subsequently found to be significantly activated by a demethylation agent, 5′-Aza-2′ deoxycytidine. Taken together, our data indicate that the human secretin gene is controlled by the in vivo Sp1/Sp3 ratio and the methylation status of the promoter.

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Nobiletin Alleviates Non-alcoholic Steatohepatitis in MCD-Induced Mice by Regulating Macrophage Polarization

Frontiers in Physiology ◽

10.3389/fphys.2021.687744 ◽

2021 ◽

Vol 12 ◽

Author(s):

Si-wei Wang ◽

Tian Lan ◽

Hao Sheng ◽

Fang Zheng ◽

Mei-kang Lei ◽

...

Keyword(s):

Macrophage Polarization ◽

Orphan Receptor ◽

Inflammatory Factors ◽

Inflammatory Disorder ◽

Orphan Nuclear Receptor ◽

Alcoholic Steatohepatitis ◽

Klf4 Expression ◽

Hepatic Macrophages

Non-alcoholic steatohepatitis (NASH) is an inflammatory disorder that is characterized by chronic activation of the hepatic inflammatory response and subsequent liver damage. The regulation of macrophage polarization in liver is closely related to the progression of NASH. The orphan nuclear receptor retinoic-acid-related orphan receptor α (RORα) and Krüppel-like factor 4 (KLF4) are key regulators which promote hepatic macrophages toward M2 phenotype and protect against NASH in mice. Nobiletin (NOB), a natural polymethoxylated flavone, is previously reported as a RORα regulator in diet-induced obese mice. However, it is still unclear whether NOB has the protective effect on NASH. In this study, we investigated the role of NOB in NASH using a methionine and choline deficient (MCD)-induced NASH mouse model. Our results showed that NOB ameliorated hepatic damage and fibrosis in MCD fed mice. NOB treatment reduced the infiltration of macrophages and neutrophils in the liver in MCD-fed mice. Of importance, NOB significantly increased the proportion of M2 macrophages and the expression of anti-inflammatory factors in vivo and in vitro. Meanwhile, NOB also decreased the population of M1 macrophages and the expression of proinflammatory cytokines. Mechanistically, NOB elevated KLF4 expression in macrophages. Inhibition of KLF4 abolished NOB regulated macrophage polarization. Furthermore, the regulation of NOB in KLF4 expression was dependent on RORα.

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All-Trans Retinoic Acid Increases DRP1 Levels and Promotes Mitochondrial Fission

Cells ◽

10.3390/cells10051202 ◽

2021 ◽

Vol 10 (5) ◽

pp. 1202

Author(s):

Bojjibabu Chidipi ◽

Syed Islamuddin Shah ◽

Michelle Reiser ◽

Manasa Kanithi ◽

Amanda Garces ◽

...

Keyword(s):

Retinoic Acid ◽

Mitochondrial Fission ◽

Normal Function ◽

Mitochondrial Network ◽

Protein Levels ◽

Trans Retinoic Acid ◽

All Trans Retinoic Acid ◽

Area And Perimeter

In the heart, mitochondrial homeostasis is critical for sustaining normal function and optimal responses to metabolic and environmental stressors. Mitochondrial fusion and fission are thought to be necessary for maintaining a robust population of mitochondria, and disruptions in mitochondrial fission and/or fusion can lead to cellular dysfunction. The dynamin-related protein (DRP1) is an important mediator of mitochondrial fission. In this study, we investigated the direct effects of the micronutrient retinoid all-trans retinoic acid (ATRA) on the mitochondrial structure in vivo and in vitro using Western blot, confocal, and transmission electron microscopy, as well as mitochondrial network quantification using stochastic modeling. Our results showed that ATRA increases DRP1 protein levels, increases the localization of DRP1 to mitochondria in isolated mitochondrial preparations. Our results also suggested that ATRA remodels the mitochondrial ultrastructure where the mitochondrial area and perimeter were decreased and the circularity was increased. Microscopically, mitochondrial network remodeling is driven by an increased rate of fission over fusion events in ATRA, as suggested by our numerical modeling. In conclusion, ATRA results in a pharmacologically mediated increase in the DRP1 protein. It also results in the modulation of cardiac mitochondria by promoting fission events, altering the mitochondrial network, and modifying the ultrastructure of mitochondria in the heart.

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Biochemical, Ameliorative and Cytotoxic Effects of Newly Synthesized Curcumin Microemulsions: Evidence from In Vitro and In Vivo Studies

Nanomaterials ◽

10.3390/nano11030817 ◽

2021 ◽

Vol 11 (3) ◽

pp. 817

Author(s):

Abbas Rahdar ◽

Mohammad Reza Hajinezhad ◽

Saman Sargazi ◽

Maryam Zaboli ◽

Mahmood Barani ◽

...

Keyword(s):

Hepg2 Cells ◽

Density Functional ◽

Liver Enzymes ◽

Elevated Serum ◽

Ethyl Butyrate ◽

Polymeric Chain ◽

Prolonged Release ◽

Advanced Therapies

Curcumin is known to exhibit antioxidant and tissue-healing properties and has recently attracted the attention of the biomedical community for potential use in advanced therapies. This work reports the formulation and characterization of oil-in-water F127 microemulsions to enhance the bioavailability of curcumin Microemulsions showed a high encapsulation efficiency and prolonged release. To investigate the interactions of curcumin with one unit of the polymeric chain of surfactant F127, ethyl butyrate, and sodium octanoate, as well as the interaction between ethyl butyrate and one unit of the F127 polymer chain, the Density Functional Theory (DFT) calculations at the M06-2X level of theory, were performed in water solution. The MTT assay was used to assess the cytotoxicity of free and encapsulated curcumin on non-malignant and malignant cell lines. Combination effects were calculated according to Chou-Talalay’s principles. Results of in vitro studies indicated that MCF7 and HepG2 cells were more sensitive to curcumin microemulsions. Moreover, a synergistic relationship was observed between curcumin microemulsions and cisplatin in all affected fractions of MCF7 and HepG2 cells (CI < 0.9). For in vivo investigation, thioacetamide-intoxicated rats received thioacetamide (100 mg/kg Sc) followed by curcumin microemulsions (30 mg/kg Ip). Thioacetamide-intoxicated rats showed elevated serum liver enzymes, blood urea nitrogen (BUN), and creatinine levels, and a significant reduction in liver superoxide dismutase (SOD) and catalase (CAT) activities (p < 0.05). Curcumin microemulsions reduced liver enzymes and serum creatinine and increased the activity of antioxidant enzymes in thioacetamide-treated rats in comparison to the untreated thioacetamide-intoxicated group. Histopathological investigations confirmed the biochemical findings. Overall, the current results showed the desirable hepatoprotective, nephroprotective, and anti-cancer effects of curcumin microemulsions.

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Integrin α3β1 Promotes Invasive and Metastatic Properties of Breast Cancer Cells through Induction of the Brn-2 Transcription Factor

Cancers ◽

10.3390/cancers13030480 ◽

2021 ◽

Vol 13 (3) ◽

pp. 480

Author(s):

Rakshitha Pandulal Miskin ◽

Janine S. A. Warren ◽

Abibatou Ndoye ◽

Lei Wu ◽

John M. Lamar ◽

...

Keyword(s):

Breast Cancer ◽

Transcription Factor ◽

Cancer Cells ◽

Cell Invasion ◽

Breast Cancer Cells ◽

Gene Promoter ◽

Akt Inhibitor ◽

Integrin Α3β1

In the current study, we demonstrate that integrin α3β1 promotes invasive and metastatic traits of triple-negative breast cancer (TNBC) cells through induction of the transcription factor, Brain-2 (Brn-2). We show that RNAi-mediated suppression of α3β1 in MDA-MB-231 cells caused reduced expression of Brn-2 mRNA and protein and reduced activity of the BRN2 gene promoter. In addition, RNAi-targeting of Brn-2 in MDA-MB-231 cells decreased invasion in vitro and lung colonization in vivo, and exogenous Brn-2 expression partially restored invasion to cells in which α3β1 was suppressed. α3β1 promoted phosphorylation of Akt in MDA-MB-231 cells, and treatment of these cells with a pharmacological Akt inhibitor (MK-2206) reduced both Brn-2 expression and cell invasion, indicating that α3β1-Akt signaling contributes to Brn-2 induction. Analysis of RNAseq data from patients with invasive breast carcinoma revealed that high BRN2 expression correlates with poor survival. Moreover, high BRN2 expression positively correlates with high ITGA3 expression in basal-like breast cancer, which is consistent with our experimental findings that α3β1 induces Brn-2 in TNBC cells. Together, our study demonstrates a pro-invasive/pro-metastatic role for Brn-2 in breast cancer cells and identifies a role for integrin α3β1 in regulating Brn-2 expression, thereby revealing a novel mechanism of integrin-dependent breast cancer cell invasion.

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