Hueng-Sik Choi

An Orphan Nuclear Hormone Receptor That Lacks a DNA Binding Domain and Heterodimerizes with Other Receptors

SHP is an orphan member of the nuclear hormone receptor superfamily that contains the dimerization and ligand-binding domain found in other family members but lacks the conserved DNA binding domain. In the yeast two-hybrid system, SHP interacted with several conventional and orphan members of the receptor superfamily, including retinoid receptors, the thyroid hormone receptor, and the orphan receptor MB67. SHP also interacted directly with these receptors in vitro. In mammalian cells, SHP specifically inhibited transactivation by the superfamily members with which it interacted. These results suggest that SHP functions as a negative regulator of receptor-dependent signaling pathways.

A new orphan member of the nuclear hormone receptor superfamily that interacts with a subset of retinoic acid response elements.

We have identified and characterized a new orphan member of the nuclear hormone receptor superfamily, called MB67, which is predominantly expressed in liver. MB67 binds and transactivates the retinoic acid response elements that control expression of the retinoic acid receptor beta 2 and alcohol dehydrogenase 3 genes, both of which consist of a direct repeat hexamers related to the consensus AGGTCA, separated by 5 bp. MB67 binds these elements as a heterodimer with the 9-cis-retinoic acid receptor, RXR. However, MB67 does not bind or activate other retinoic acid response elements with alternative hexamer arrangements or any of several other wild-type and synthetic hormone response elements examined. The transactivation of retinoic acid response elements by MB67 is weaker than that conferred by the retinoic acid receptors but does not require the presence of all-trans retinoic acid, 9-cis-retinoic acid, or any exogenously added ligand. We propose that MB67 plays an important role in the complex network of proteins that govern response to retinoic acid and its metabolites.

Molecular Mechanism of Suppression of Testicular Steroidogenesis by Proinflammatory Cytokine Tumor Necrosis Factor Alpha

ABSTRACT Tumor necrosis factor alpha (TNF-α) has been demonstrated to inhibit steroidogenesis in Leydig cells at the transcriptional level of steroidogenic enzymes. However, the molecular mechanism of this observed gene repression is not well understood. We now demonstrate that nuclear factor κB (NF-κB) activated by TNF-α inhibits the transactivation of orphan nuclear receptors, which regulate the expression of steroidogenic-enzyme genes. TNF-α treatment suppressed the luteinizing-hormone-induced or Nur77/SF-1-stimulated promoter activity of steroidogenic-enzyme genes in Leydig cells. The TNF-α-mediated gene suppression was blocked by treatment with an inhibitor of NF-κB. In addition, overexpression of the p65 (RelA) subunit of NF-κB showed the same effect as TNF-α and inhibited Nur77 transactivation, suggesting the involvement of NF-κB activation in the observed gene repression. Physical association of Nur77 with p65 was revealed by mammalian two-hybrid, GST pull-down, and coimmunoprecipitation analyses. The NF-κB inhibition of Nur77 transactivation was likely due to the competition of p65 for Nur77 binding with coactivators. Finally, chromatin immunoprecipitation assays revealed that TNF-α treatment caused the recruitment of NF-κB to the promoter of the steroidogenic-enzyme P450c17 gene, supporting the hypothesis that the TNF-α-mediated gene repression involves NF-κB inhibition of the transcriptional activity of Nur77 and other orphan nuclear receptors. These findings provide a molecular mechanism underlying the inhibition of testicular steroidogenesis by proinflammatory cytokines.

Pharmacological Stimulation of NADH Oxidation Ameliorates Obesity and Related Phenotypes in Mice

OBJECTIVE Nicotinamide adenine dinucleotides (NAD+ and NADH) play a crucial role in cellular energy metabolism, and a dysregulated NAD+-to-NADH ratio is implicated in metabolic syndrome. However, it is still unknown whether a modulating intracellular NAD+-to-NADH ratio is beneficial in treating metabolic syndrome. We tried to determine whether pharmacological stimulation of NADH oxidation provides therapeutic effects in rodent models of metabolic syndrome. RESEARCH DESIGN AND METHODS We used β-lapachone (βL), a natural substrate of NADH:quinone oxidoreductase 1 (NQO1), to stimulate NADH oxidation. The βL-induced pharmacological effect on cellular energy metabolism was evaluated in cells derived from NQO1-deficient mice. In vivo therapeutic effects of βL on metabolic syndrome were examined in diet-induced obesity (DIO) and ob/ob mice. RESULTS NQO1-dependent NADH oxidation by βL strongly provoked mitochondrial fatty acid oxidation in vitro and in vivo. These effects were accompanied by activation of AMP-activated protein kinase and carnitine palmitoyltransferase and suppression of acetyl-coenzyme A (CoA) carboxylase activity. Consistently, systemic βL administration in rodent models of metabolic syndrome dramatically ameliorated their key symptoms such as increased adiposity, glucose intolerance, dyslipidemia, and fatty liver. The treated mice also showed higher expressions of the genes related to mitochondrial energy metabolism (PPARγ coactivator-1α, nuclear respiratory factor-1) and caloric restriction (Sirt1) consistent with the increased mitochondrial biogenesis and energy expenditure. CONCLUSIONS Pharmacological activation of NADH oxidation by NQO1 resolves obesity and related phenotypes in mice, opening the possibility that it may provide the basis for a new therapy for the treatment of metabolic syndrome.

BMP2 Protein Regulates Osteocalcin Expression via Runx2-mediated Atf6 Gene Transcription

Background: BMP2 activates UPR transducers during osteoblast differentiation. Results: BMP2 signaling increased ATF6 expression and cleavage, and activated ATF6 increased OC expression. Conclusion: BMP2 induced osteoblast differentiation through Runx2-dependent ATF6 expression, which directly regulates OC transcription. Significance: BMP2-induced mild ER stress positively regulates osteoblast differentiation via activation of UPR transducers, including ATF6. Bone morphogenetic protein 2 (BMP2) activates unfolded protein response (UPR) transducers, such as PERK and OASIS, in osteoblast cells. ATF6, a bZIP transcription factor, is also a UPR transducer. However, the involvement of ATF6 in BMP2-induced osteoblast differentiation has not yet been elucidated. In the present study, BMP2 treatment was shown to markedly induce the expression and activation of ATF6 with an increase in alkaline phosphatase (ALP) and OC expression in MC3T3E1 cells. In contrast, ATF6 activation by BMP2 was not observed in the Runx2−/− primary calvarial osteoblasts, and Runx2 overexpression recovered BMP2 action. BMP2 stimulated ATF6 transcription by enhancing the direct binding of Runx2 to the osteoblast-specific cis-acting element 2 (OSE2, ACCACA, −205 to −200 bp) motif of the Atf6 promoter region. In addition, the overexpression of ATF6 increased the Oc promoter activity by enhancing the direct binding to a putative ATF6 binding motif (TGACGT, −1126 to −1121 bp). The inhibition of ATF6 function with the dominant negative form of ATF6 (DN-ATF6) blocked BMP2- or Runx2-induced OC expression. Interestingly, OASIS, which is structurally similar to ATF6, did not induce Oc expression. ALP and Alizarin red staining results confirmed that BMP2-induced matrix mineralization was also dependent on ATF6 in vitro. Overall, these results suggest that BMP2 induces osteoblast differentiation through Runx2-dependent ATF6 expression, which directly regulates Oc transcription.

Regulation of Transforming Growth Factor-β Signaling and PDK1 Kinase Activity by Physical Interaction between PDK1 and Serine-Threonine Kinase Receptor-associated Protein

To gain more insights about the biological roles of PDK1, we have used the yeast two-hybrid system and in vivo binding assay to identify interacting molecules that associate with PDK1. As a result, serine-threonine kinase receptor-associated protein (STRAP), a transforming growth factor-β (TGF-β) receptor-interacting protein, was identified as an interacting partner of PDK1. STRAP was found to form in vivo complexes with PDK1 in intact cells. Mapping analysis revealed that this binding was only mediated by the catalytic domain of PDK1 and not by the pleckstrin homology domain. Insulin enhanced a physical association between PDK1 and STRAP in intact cells, but this insulin-induced association was prevented by wortmannin, a phosphatidylinositol 3-kinase inhibitor. In addition, the association between PDK1 and STRAP was decreased by TGF-β treatment. Analysis of the activities of the interacting proteins showed that PDK1 kinase activity was significantly increased by coexpression of STRAP, probably through the inhibition of the binding of 14-3-3, a negative regulator, to PDK1. Consistently, knockdown of the endogenous STRAP by the transfection of the small interfering RNA resulted in the decrease of PDK1 kinase activity. PDK1 also exhibited an inhibition of TGF-β signaling with STRAP by contributing to the stable association between TGF-β receptor and Smad7. Moreover, confocal microscopic study and immunostaining results demonstrated that PDK1 prevented the nuclear translocation of Smad3 in response to TGF-β. Knockdown of endogenous PDK1 with small interfering RNA has an opposite effect. Taken together, these results suggested that STRAP acts as an intermediate signaling molecule linking between the phosphatidylinositol 3-kinase/PDK1 and the TGF-β signaling pathways.

Hepatocyte Growth Factor Family Negatively Regulates Hepatic Gluconeogenesis via Induction of Orphan Nuclear Receptor Small Heterodimer Partner in Primary Hepatocytes

Hepatic gluconeogenesis is tightly balanced by opposing stimulatory (glucagon) and inhibitory (insulin) signaling pathways. Hepatocyte growth factor (HGF) is a pleiotropic growth factor that mediates diverse biological processes. In this study, we investigated the effect of HGF and its family member, macrophage-stimulating factor (MSP), on hepatic gluconeogenesis in primary hepatocytes. HGF and MSP significantly repressed expression of the key hepatic gluconeogenic enzyme genes, phosphoenolpyruvate carboxykinase (PEPCK), and glucose-6-phosphatase (Glc-6-Pase) and reduced glucose production. HGF and MSP activated small heterodimer partner (SHP) gene promoter and induced SHP mRNA and protein levels, and the effect of HGF and MSP on SHP gene expression was demonstrated to be mediated via activation of the AMP-activated protein kinase (AMPK) signaling pathway. We demonstrated that upstream stimulatory factor-1 (USF-1) specifically mediated HGF effect on SHP gene expression, and inhibition of USF-1 by dominant negative USF-1 significantly abrogated HGF-mediated activation of the SHP promoter. Elucidation of the mechanism showed that USF-1 bound to E-box-1 in the SHP promoter, and HGF increased USF-1 DNA binding on the SHP promoter via AMPK and DNA-dependent protein kinase-mediated pathways. Adenoviral overexpression of USF-1 significantly repressed PEPCK and Glc-6-Pase gene expression and reduced glucose production. Knockdown of endogenous SHP expression significantly reversed this effect. Finally, knockdown of SHP or inhibition of AMPK signaling reversed the ability of HGF to suppress hepatocyte nuclear factor 4α-mediated up-regulation of PEPCK and Glc-6-Pase gene expression along with the HGF- and MSP-mediated suppression of gluconeogenesis. Overall, our results suggest a novel signaling pathway through HGF/AMPK/USF-1/SHP to inhibit hepatic gluconeogenesis.

Glucotoxicity in the INS-1 rat insulinoma cell line is mediated by the orphan nuclear receptor small heterodimer partner.

Prolonged elevations of glucose concentration have deleterious effects on β-cell function. One of the hallmarks of such glucotoxicity is a reduction in insulin gene expression, resulting from decreased insulin promoter activity. Small heterodimer partner (SHP; NR0B2) is an atypical orphan nuclear receptor that inhibits nuclear receptor signaling in diverse metabolic pathways. In this study, we found that sustained culture of INS-1 cells at high glucose concentrations leads to an increase in SHP mRNA expression, followed by a decrease in insulin gene expression. Inhibition of endogenous SHP gene expression by small interfering RNA partially restored high-glucose–induced suppression of the insulin gene. Adenovirus-mediated overexpression of SHP in INS-1 cells impaired glucose-stimulated insulin secretion as well as insulin gene expression. SHP downregulates insulin gene expression via two mechanisms: by downregulating PDX-1 and MafA gene expression and by inhibiting p300-mediated pancreatic duodenal homeobox factor 1–and BETA2-dependent transcriptional activity from the insulin promoter. Finally, the pancreatic islets of diabetic OLETF rats express SHP mRNA at higher levels than the islets from LETO rats. These results collectively suggest that SHP plays an important role in the development of β-cell dysfunction induced by glucotoxicity.

Estrogen-related Receptor γ (ERRγ) Is a Novel Transcriptional Regulator of Phosphatidic Acid Phosphatase, LIPIN1, and Inhibits Hepatic Insulin Signaling

Background: The PAP function of LIPINs is involved in the regulation of intracellular lipid levels and hepatic insulin receptor signaling. Results: ERRγ-mediated induction of LIPIN1 results in the perturbation of hepatic insulin signaling through DAG-mediated activation of PKCϵ. Conclusion: ERRγ is a novel transcriptional regulator of LIPIN1. Significance: An ERRγ inverse agonist could ameliorate LIPIN1-mediated perturbation of hepatic insulin signaling. LIPINs have been reported to perform important roles in the regulation of intracellular lipid levels. Their mutations induce lipodystrophy, myoglobinuria, and inflammatory disorders. Recently, the phosphatidic acid phosphatase function of LIPINs has been associated with the perturbation of hepatic insulin receptor signaling via the diacylglycerol-mediated stimulation of PKCϵ activity. Here, we report that nuclear estrogen-related receptor (ERR) γ is a novel transcriptional regulator of LIPIN1. Overexpression of ERRγ significantly increased LIPIN1 expression in primary hepatocytes, whereas the abolition of ERRγ gene expression attenuated the expression of LIPIN1. Deletion and mutation analyses of the LIPIN1 promoter showed that ERRγ exerts its effect on the transcriptional regulation of LIPIN1 via ERRE1 of the LIPIN1 promoter, as confirmed by ChIP assay. We also determined that the gene transcription of LIPIN1 by ERRγ is controlled by the competition between PGC-1α and small heterodimer partner. Additionally, ERRγ leads to the induction of hepatic LIPIN1 expression and diacylglycerol production in vivo. Finally, an inverse agonist of ERRγ, GSK5182, restores the impaired insulin signaling induced by LIPIN1-mediated PKCϵ activation. Our findings indicate that the selective control of ERRγ transcriptional activity by its specific inverse agonist could provide a novel therapeutic approach to the amelioration of impaired hepatic insulin signaling induced by LIPIN1-mediated PKCϵ activation.

Small heterodimer partner interacts with NLRP3 and negatively regulates activation of the NLRP3 inflammasome

Excessive activation of the NLRP3 inflammasome results in damaging inflammation, yet the regulators of this process remain poorly defined. Herein, we show that the orphan nuclear receptor small heterodimer partner (SHP) is a negative regulator of NLRP3 inflammasome activation. NLRP3 inflammasome activation leads to an interaction between SHP and NLRP3, proteins that are both recruited to mitochondria. Overexpression of SHP competitively inhibits binding of NLRP3 to apoptosis-associated speck-like protein containing a CARD (ASC). SHP deficiency results in increased secretion of proinflammatory cytokines IL-1β and IL-18, and excessive pathologic responses typically observed in mouse models of kidney tubular necrosis and peritoneal gout. Notably, the loss of SHP results in accumulation of damaged mitochondria and a sustained interaction between NLRP3 and ASC in the endoplasmic reticulum. These data are suggestive of a role for SHP in controlling NLRP3 inflammasome activation through a mechanism involving interaction with NLRP3 and maintenance of mitochondrial homeostasis.