32 research outputs found
Control of Gene Expression by the Retinoic Acid-Related Orphan Receptor Alpha in HepG2 Human Hepatoma Cells
Retinoic acid-related Orphan Receptor alpha (RORα; NR1F1) is a widely distributed nuclear receptor involved in several (patho)physiological functions including lipid metabolism, inflammation, angiogenesis, and circadian rhythm. To better understand the role of this nuclear receptor in liver, we aimed at displaying genes controlled by RORα in liver cells by generating HepG2 human hepatoma cells stably over-expressing RORα. Genes whose expression was altered in these cells versus control cells were displayed using micro-arrays followed by qRT-PCR analysis. Expression of these genes was also altered in cells in which RORα was transiently over-expressed after adenoviral infection. A number of the genes found were involved in known pathways controlled by RORα, for instance LPA, NR1D2 and ADIPOQ in lipid metabolism, ADIPOQ and PLG in inflammation, PLG in fibrinolysis and NR1D2 and NR1D1 in circadian rhythm. This study also revealed that genes such as G6PC, involved in glucose homeostasis, and AGRP, involved in the control of body weight, are also controlled by RORα. Lastly, SPARC, involved in cell growth and adhesion, and associated with liver carcinogenesis, was up-regulated by RORα. SPARC was found to be a new putative RORα target gene since it possesses, in its promoter, a functional RORE as evidenced by EMSAs and transfection experiments. Most of the other genes that we found regulated by RORα also contained putative ROREs in their regulatory regions. Chromatin immunoprecipitation (ChIP) confirmed that the ROREs present in the SPARC, PLG, G6PC, NR1D2 and AGRP genes were occupied by RORα in HepG2 cells. Therefore these genes must now be considered as direct RORα targets. Our results open new routes on the roles of RORα in glucose metabolism and carcinogenesis within cells of hepatic origin
Liver Receptor Homolog 1 Is a Negative Regulator of the Hepatic Acute-Phase Response
The orphan nuclear receptor liver receptor homolog 1 (LRH-1) has been reported to play an important role in bile acid biosynthesis and reverse cholesterol transport. Here, we show that LRH-1 is a key player in the control of the hepatic acute-phase response. Ectopic expression of LRH-1 with adenovirus resulted in strong inhibition of both interleukin-6 (IL-6)- and IL-1β-stimulated haptoglobin, serum amyloid A, and fibrinogen β gene expression in hepatocytes. Furthermore, induction of the hepatic inflammatory response was significantly exacerbated in HepG2 cells expressing short hairpin RNA targeting LRH-1 expression. Moreover, transient-transfection experiments and electrophoretic mobility shift and chromatin immunoprecipitation assays revealed that LRH-1 regulates this cytokine-elicited inflammatory response by, at least in part, antagonizing the CCAAT/enhancer binding protein β signaling pathway. Finally, we show, by using LRH-1 heterozygous mice, that LRH-1 is involved in the control of the inflammatory response at the hepatic level in vivo. Taken together, our results outline an unexpected role for LRH-1 in the modulation of the hepatic acute-phase response
The lipoprotein lipase inhibitor ANGPTL3 is negatively regulated by thyroid hormone
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Oxidized low-density lipoprotein and peroxisome-proliferator-activated receptor α down-regulate platelet-activating-factor receptor expression in human macrophages
MG53 is not a critical regulator of insulin signaling pathway in skeletal muscle.
In type 2 diabetes (T2D), both muscle and liver are severely resistant to insulin action. Muscle insulin resistance accounts for more than 80% of the impairment in total body glucose disposal in T2D patients and is often characterized by an impaired insulin signaling. Mitsugumin 53 (MG53), a muscle-specific TRIM family protein initially identified as a key regulator of cell membrane repair machinery has been suggested to be a critical regulator of muscle insulin signaling pathway by acting as ubiquitin E3 ligase targeting both the insulin receptor and insulin receptor substrate 1 (IRS1). Here, we show using in vitro and in vivo approaches that MG53 is not a critical regulator of insulin signaling and glucose homeostasis. First, MG53 expression is not consistently regulated in skeletal muscle from various preclinical models of insulin resistance. Second, MG53 gene knock-down in muscle cells does not lead to impaired insulin response as measured by Akt phosphorylation on Serine 473 and glucose uptake. Third, recombinant human MG53 does not alter insulin response in both differentiated C2C12 and human skeletal muscle cells. Fourth, ectopic expression of MG53 in HEK293 cells lacking endogenous MG53 expression fails to alter insulin response as measured by Akt phosphorylation. Finally, both male and female mg53 -/- mice were not resistant to high fat induced obesity and glucose intolerance compared to wild-type mice. Taken together, these results strongly suggest that MG53 is not a critical regulator of insulin signaling pathway in skeletal muscle
Identification of small molecule agonists of the orphan nuclear receptors liver receptor homolog-1 and steroidogenic factor-1
We report the identification of substituted cis-bicyclo[3.3.0]oct-2-enes as small molecule agonists of subfamily V orphan nuclear receptors (NR5A), liver receptor homolog-1 (LRH-1) and steroidogenic factor-1 (SF-1). Using fluorescence resonance energy transfer (FRET)-based biochemical assays, compound 5a (GSK8470) was identified as a high-affinity ligand for LRH-1 and SF-1. In liver cells, 5a increased the expression of the LRH-1 target gene small heterodimer partner (SHP). Synthesis of analogues modified at three positions led to the development of compounds with functional selectivity between LRH-1 and SF-1
Comparative genotypic and phenotypic analysis of human peripheral blood monocytes and surrogate monocyte-like cell lines commonly used in metabolic disease research
<div><p>Monocyte-like cell lines (MCLCs), including THP-1, HL-60 and U-937 cells, are used routinely as surrogates for isolated human peripheral blood mononuclear cells (PBMCs). To systematically evaluate these immortalised cells and PBMCs as model systems to study inflammation relevant to the pathogenesis of type II diabetes and immuno-metabolism, we compared mRNA expression of inflammation-relevant genes, cell surface expression of cluster of differentiation (CD) markers, and chemotactic responses to inflammatory stimuli. Messenger RNA expression analysis suggested most genes were present at similar levels across all undifferentiated cells, though notably, <i>IDO1</i>, which encodes for indoleamine 2,3-dioxygenase and catabolises tryptophan to kynureninase (shown to be elevated in serum from diabetic patients), was not expressed in any PMA-treated MCLC, but present in GM-CSF-treated PBMCs. There was little overall difference in the pattern of expression of CD markers across all cells, though absolute expression levels varied considerably and the correlation between MCLCs and PBMCs was improved upon MCLC differentiation. Functionally, THP-1 and PBMCs migrated in response to chemoattractants in a transwell assay, with varying sensitivity to MCP-1, MIP-1α and LTB-4. However, despite similar gene and CD expression profiles, U-937 cells were functionally impaired as no migration was observed to any chemoattractant. Our analysis reveals that the MCLCs examined only partly replicate the genotypic and phenotypic properties of human PBMCs. To overcome such issues a universal differentiation protocol should be implemented for these cell lines, similar to those already used with isolated monocytes. Although not perfect, in our hands the THP-1 cells represent the closest, simplified surrogate model of PBMCs for study of inflammatory cell migration.</p></div
PPARα activation differently affects microparticle content in atherosclerotic lesions and liver of a mouse model of atherosclerosis and NASH.
International audienceBACKGROUND: Atherosclerosis and non-alcoholic fatty liver disease (NAFLD) are complex pathologies characterized by lipid accumulation, chronic inflammation and extensive tissue remodelling. Microparticles (MPs), small membrane vesicles produced by activated and apoptotic cells, might not only be biomarkers, but also functional actors in these pathologies. The apoE2-KI mouse is a model of atherosclerosis and NAFLD. Activation of the nuclear receptor PPARα decreases atherosclerosis and components of non-alcoholic steatohepatitis (NASH) in the apoE2-KI mouse. OBJECTIVES: (1) To determine whether MPs are present in atherosclerotic lesions, liver and plasma during atherosclerosis and NASH progression in apoE2-KI mice, and (2) to study whether PPARα activation modulates MP concentrations. METHODS: ApoE2-KI mice were fed a Western diet to induce atherosclerosis and NASH. MPs were isolated from atherosclerotic lesions, liver and blood and quantified by flow cytometry. RESULTS: An increase of MPs was observed in the atherosclerotic lesions and in the liver of apoE2-KI mice upon Western diet feeding. PPARα activation with fenofibrate decreased MP levels in the atherosclerotic lesions in a PPARα-dependent manner, but did not influence MP concentrations in the liver. CONCLUSION: Here we report that MPs are present in atherosclerotic lesions and in the liver of apoE2-KI mice. Their concentration increased during atherosclerosis and NASH development. PPARα activation differentially modulates MP levels in a tissue-specific manner
The orphan nuclear receptor RORα is a negative regulator of the inflammatory response
Retinoid-related orphan receptor α (RORα) (NR1F1) is a member of the nuclear receptor superfamily whose biological functions are largely unknown. Since staggerer mice, which carry a deletion in the RORα gene, suffer from immune abnormalities, we generated an adenovirus encoding RORα1 to investigate its potential role in control of the inflammatory response. We demonstrated that RORα is expressed in human primary smooth-muscle cells and that ectopic expression of RORα1 inhibits TNFα-induced IL-6, IL-8 and COX-2 expression in these cells. RORα1 negatively interferes with the NF-κB signalling pathway by reducing p65 translocation as demonstrated by western blotting, immunostaining and electrophoretic mobility shift assays. This action of RORα1 on NF-κB is associated with the induction of IκBα, the major inhibitory protein of the NF-κB signalling pathway, whose expression was found to be transcriptionally upregulated by RORα1 via a ROR response element in the IκBα promoter. Taken together, these data identify RORα1 as a potential target in the treatment of chronic inflammatory diseases, including atherosclerosis and rheumatoid arthritis
Hierarchical clustered heat map of mean relative expression values for undifferentiated and differentiated CD14<sup>+</sup> human peripheral blood mononuclear cells (PBMC, M(GC) and M(GC)LPS/IFNγ) and monocyte-like cell lines (MCLCs ± PMA) including THP-1, U-937 and HL-60s.
<p>PBMCs were differentiated using 10 ng/mL granulocyte-macrophage colony stimulating factor (GM-CSF) for 6 days to give M(GC) and activated using 100 ng/mL LPS and 20 μg/mL IFNγ for 24 h to generate M(GC)LPS/IFNγ. MCLCs were differentiated using 16 ng/mL phorbol-12-myristate-13-acetate (PMA) for 48 h. Data represent n = 3–10; raw data, including the relative expression ± SEM used to generate this heat map, are shown in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0197177#pone.0197177.s002" target="_blank">S2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0197177#pone.0197177.s003" target="_blank">S3</a> Tables</b>. All qPCR data were normalized against both housekeeper genes <i>GAPDH</i> and <i>ACTB2</i> and the values calculated as described in the Methods. Relative expression values were then calculated versus the relevant non-differentiated cell type. Where no expression was detected a value of 0 was used. Thirty-five genes that encode for inflammatory chemokines, cytokines, adipokines and their relevant receptors were selected as they are associated with inflammation or have been implicated in the development and/or progression of obesity-induced insulin resistance. In addition, small subsets of genes encoding for regulatory factors and enzymatic processes that have been implicated in the pathogenesis of T2DM were profiled. Differential gene expression data were analysed using R version 3.4.1 and hierarchical clustering was performed using complete linkage method with Euclidean distance measure.</p