The regulation of M1/M2 polarization in liver macrophages is closely associated with the progression of nonalcoholic steatohepatitis (NASH); however, the mechanism involved in this process remains unclear. Here, we describe the orphan nuclear receptor retinoic-acid-related orphan receptor α (RORα) as a key regulator of M1/M2 polarization in hepatic residential Kupffer cells (KCs) and infiltrated monocyte-derived macrophages. RORα enhanced M2 polarization in KCs by inducing the kruppel-like factor 4. M2 polarization was defective in KCs and bone-marrow-derived macrophages of the myeloid-specific RORα null mice, and these mice were susceptible to HFD-induced NASH. We found that IL-10 played an important role in connecting the function of M2 KCs to lipid accumulation and apoptosis in hepatocytes. Importantly, M2 polarization was controlled by a RORα activator, JC1-40, which improved symptoms of NASH. Our results suggest that the M2-promoting effects of RORα in liver macrophages may provide better therapeutic strategies against NASH.

There is increasing evidence that the retinoic acid receptor-related orphan receptor a (RORa) plays an important role in the regulation of metabolic pathways, particularly of fatty acid and cholesterol metabolism; however, the role of RORa in the regulation of hepatic lipogenesis has not been studied. Here, we report that RORa attenuates hepatic steatosis, probably via activation of the adenosine monophosphate (AMP)-activated protein kinase (AMPK) and repression of the liver X receptor a (LXRa). First, RORa and its activator, cholesterol sulfate (CS), induced phosphorylation of AMPK, which was accompanied by the activation of serine-threonine kinase liver kinase B1 (LKB1). Second, the activation of RORa, either by transient transfection or CS treatment, decreased the TO901317-induced transcriptional expression of LXRa and its downstream target genes, such as the sterol regulatory element binding protein-1 (SREBP-1) and fatty acid synthase. RORa interacted physically with LXRa and inhibited the LXRa response element in the promoter of LXRa, indicating that RORa interrupts the autoregulatory activation loop of LXRa. Third, infection with adenovirus encoding RORa suppressed the lipid accumulation that had been induced by a free-fatty-acid mixture in cultured cells. Furthermore, we observed that the level of expression of the RORa protein was decreased in the liver of mice that were fed a high-fat diet. Restoration of RORa via tail-vein injection of adenovirus (Ad)-RORa decreased the high-fat-diet-induced hepatic steatosis. Finally, we synthesized thiourea derivatives that activated RORa, thereby inducing activation of AMPK and repression of LXRa. These compounds decreased hepatic triglyceride levels and lipid droplets in the high-fat-diet-fed mice. Conclusion: We found that RORa induced activation of AMPK and inhibition of the lipogenic function of LXRa, which may be key phenomena that provide the beneficial effects of RORa against hepatic steatosis. (HEPATOLOGY 2012;55:1379-1388 A n increasing number of populations in the world suffer from fatty liver, which is a disease defined as hepatic fat accumulation greater than 5% of the liver wet weight. The major causes of fatty liver are obesity, diabetes, hyperlipidemia, drugs, and metabolic disorders.1 Although this relativelyAbbreviations: ACC, acetyl-CoA carboxylase; Ad-RORa, adenovirus-RORa; AICAR, aminoimidazole carboxamide ribonucleotide; AKT2, v-akt murine thymoma viral oncogene homolog 1; AMPK, adenosine monophosphate (AMP)-activated protein kinase; ATP, adenosine triphosphate; BODIPY, borondipyrromethene; CA-AMPK, constitutively active AMPK; ChIP, chromatin immunoprecipitation; CS, cholesterol sulfate; Cyp7b1, oxysterol 7a-hydroxylase; DBD, DNA binding domain; FA, fatty acid; FAS, fatty acid synthase; FFA, free fatty acid; HFD, high-fat diet; LBD, ligand binding domain; LKB1, serine-threonine kinase liver kinase B1; LXRa, liver X receptor a; LXRE, LXR response element; NADH, reduced nicotinamide adenine dinucleotide; p, phosphorylated; RORa, retinoic acid rec...

Under low oxygen tension, cells increase the transcription of specific genes involved in angiogenesis, erythropoiesis, and glycolysis. Hypoxia-induced gene expression depends primarily on stabilization of the ␣ subunit of hypoxia-inducible factor-1 (HIF-1␣), which acts as a heterodimeric trans-activator with the nuclear protein known as the aryl hydrocarbon receptor nuclear translocator (Arnt). The resulting heterodimer (HIF-1␣/Arnt) interacts specifically with the hypoxia-responsive element (HRE), thereby increasing transcription of the genes under HRE control. Our results indicate that the 90-kDa heat-shock protein (Hsp90) inhibitor radicicol reduces the hypoxia-induced expression of both endogenous vascular endothelial growth factor (VEGF) and HRE-driven reporter plasmids. Radicicol treatment (0.5 g/ml) does not significantly change the stability of the HIF-1␣ protein and does not inhibit the nuclear localization of HIF-1␣. However, this dose of radicicol significantly reduces HRE binding by the HIF-1␣/Arnt heterodimer. Our results, the first to show that radicicol specifically inhibits the interaction between the HIF-1␣/Arnt heterodimer and HRE, suggest that Hsp90 modulates the conformation of the HIF-1␣/Arnt heterodimer, making it suitable for interaction with HRE. Furthermore, we demonstrate that radicicol reduces hypoxia-induced VEGF expression to decrease hypoxia-induced angiogenesis.Cells adapt to hypoxia by up-regulating the transcription of specific genes involved in angiogenesis, erythropoiesis, and glycolysis. Pathologically, tumor hypoxia contributes directly to enhanced glucose metabolism and angiogenesis, which are major features of malignant progression. The genes up-regulated during hypoxia include vascular endothelial growth factor (VEGF), erythropoietin, and several glycolytic enzymes. These diverse, targeted genes are induced by a common trans-activator, hypoxia-inducible factor 1 (HIF-1) (Iyer et al., 1998;Bruick and McKnight, 2001b;Semenza, 2002).HIF-1 was first identified as a heterodimeric trans-activator composed of two subunits, HIF-1␣ and -␤, both of which belong to the growing family of basic-helix-loop-helix-PAS (bHLH-PAS) proteins, including period (Per), Arnt, and single-minded (Sim). The bHLH-PAS proteins share common characteristics: first, a bHLH-PAS protein dimerizes with a specific partner protein through the HLH-PAS domain. Second, a partner such as the aryl hydrocarbon receptor (AhR) or HIF-1␣ is activated by specific stimuli (i.e., xenobiotics or low oxygen tension, respectively) before translocating to the nucleus, where it heterodimerizes with a partner protein. Alternatively, Arnt, another bHLH-PAS protein, is constitutively located in the nucleus and interacts with several

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Multiple cellular signaling pathways that control metabolism and survival are activated when cell are incubated under hypoxic conditions. Activation of the hypoxia inducible factor (HIF)-1 promotes expression of genes that increase the capacity to cope with the stress imposed by a reduced oxygen environment. Here we show that the orphan nuclear receptor estrogen related receptor γ (ERRγ) plays a critical role in hypoxia–mediated activation of pyruvate dehydrogenase kinase 4 (PDK4) gene expression. ERRγ mRNA and protein levels were increased by hypoxia or desferrioxamine (DFO) treatment in hepatoma cell lines. Co-expression of HIF-1α and β increased ERRγ promoter activity as well as mRNA expression, while knockdown of endogenous HIF-1α reduced the hypoxia-mediated induction of ERRγ. In addition, hypoxia also increased the promoter activity and mRNA level of PDK4 in HepG2 cells. Adenovirus mediated-overexpression of ERRγ specifically increased PDK4 gene expression, while ablation of endogenous ERRγ significantly decreased hypoxia-mediated induction of PDK4 gene expression. Finally, GSK5182, an inverse agonist of ERRγ, strongly inhibited the hypoxia-mediated induction of PDK4 protein and promoter activity. Regulation of the transcriptional activity of ERRγ may provide a therapeutic approach for the regulation of PDK4 gene expression under hypoxia.