Publications by authors named "Yoon-Kwang Lee"

28 Publications

  • Page 1 of 1

The Mitochondrial mitoNEET Ligand NL-1 Is Protective in a Murine Model of Transient Cerebral Ischemic Stroke.

Pharm Res 2021 May 12;38(5):803-817. Epub 2021 May 12.

Department of Pharmaceutical Sciences, School of Pharmacy, West Virginia University, 1 Medical Center Drive, Morgantown, West Virginia, 26506, USA.

Purpose: Therapeutic strategies to treat ischemic stroke are limited due to the heterogeneity of cerebral ischemic injury and the mechanisms that contribute to the cell death. Since oxidative stress is one of the primary mechanisms that cause brain injury post-stroke, we hypothesized that therapeutic targets that modulate mitochondrial function could protect against reperfusion-injury after cerebral ischemia, with the focus here on a mitochondrial protein, mitoNEET, that modulates cellular bioenergetics.

Method: In this study, we evaluated the pharmacology of the mitoNEET ligand NL-1 in an in vivo therapeutic role for NL-1 in a C57Bl/6 murine model of ischemic stroke.

Results: NL-1 decreased hydrogen peroxide production with an IC of 5.95 μM in neuronal cells (N2A). The in vivo activity of NL-1 was evaluated in a murine 1 h transient middle cerebral artery occlusion (t-MCAO) model of ischemic stroke. We found that mice treated with NL-1 (10 mg/kg, i.p.) at time of reperfusion and allowed to recover for 24 h showed a 43% reduction in infarct volume and 68% reduction in edema compared to sham-injured mice. Additionally, we found that when NL-1 was administered 15 min post-t-MCAO, the ischemia volume was reduced by 41%, and stroke-associated edema by 63%.

Conclusion: As support of our hypothesis, as expected, NL-1 failed to reduce stroke infarct in a permanent photothrombotic occlusion model of stroke. This report demonstrates the potential therapeutic benefits of using mitoNEET ligands like NL-1 as novel mitoceuticals for treating reperfusion-injury with cerebral stroke.
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http://dx.doi.org/10.1007/s11095-021-03046-4DOI Listing
May 2021

Hepatic lipid homeostasis by peroxisome proliferator-activated receptor gamma 2.

Liver Res 2018 Dec 20;2(4):209-215. Epub 2018 Dec 20.

Department of Integrative Medical Sciences, College of Medicine, Northeast Ohio Medical University, Rootstown, OH, USA.

Peroxisome proliferator-activated receptor gamma (PPARγ or PPARG) is a ligand-activated transcription factor belonging to the nuclear hormone receptor superfamily. It plays a master role in the differentiation and proliferation of adipose tissues. It has two major isoforms, PPARγ1 and PPARγ2, encoded from a single gene using two separate promoters and alternative splicing. Among them, PPARγ2 is most abundantly expressed in adipocytes and plays major adipogenic and lipogenic roles in the tissue. Furthermore, it has been shown that PPARγ2 is also expressed in the liver, specifically in hepatocytes, and its expression level positively correlates with fat accumulation induced by pathological conditions such as obesity and diabetes. Knockout of the hepatic gene ameliorates hepatic steatosis induced by diet or genetic manipulations. Transcriptional activation of in the liver induces the adipogenic program to store fatty acids in lipid droplets as observed in adipocytes. Understanding how the hepatic gene expression is regulated will help develop preventative and therapeutic treatments for non-alcoholic fatty liver disease (NAFLD). Due to the potential adverse effect of hepatic gene deletion on peripheral tissue functions, therapeutic interventions that target PPARγ for fatty liver diseases require fine-tuning of this gene's expression and transcriptional activity.
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http://dx.doi.org/10.1016/j.livres.2018.12.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6594548PMC
December 2018

Hairy and enhancer of split 6 prevents hepatic lipid accumulation through inhibition of expression.

Hepatol Commun 2017 12 8;1(10):1085-1098. Epub 2017 Nov 8.

Department of Integrative Medical Sciences Northeast Ohio Medical University Rootstown Ohio.

Peroxisome proliferator-activated receptor gamma (PPARγ) is a master regulator for white adipocyte differentiation and lipid storage. The increased level of hepatic PPARγ2 isoform reprograms liver for lipid storage and causes abnormal fat accumulation in certain pathophysiologic conditions. The current study aimed to investigate a role of transcriptional repressor hairy and enhancer of split 6 (HES6) in the regulation of expression and hepatic steatosis induced by diet. Liver-specific overexpression of using adenovirus reduced messenger RNA levels by 90% and hepatic triglyceride accumulation by 22% compared to the levels in mice injected with an adenoviral empty vector with Western diet feeding. In sharp contrast, silencing gene expression using short hairpin RNA increased hepatic lipid accumulation and messenger RNA levels by 70% and 4-fold, respectively. To locate hepatocyte nuclear factor 4 alpha (HNF4α) binding site(s), through which repressional activity of HES6 is mediated, a 2.5-kb promoter-driven luciferase reporter was constructed for transient transfection assays. Subsequently, chromatin immunoprecipitation and electrophoretic mobility shift assays were performed. An HNF4α binding consensus sequence was identified at 903 base pairs upstream from the transcription start site of . Deletion or point mutation of the sequence in a luciferase reporter containing the promoter abolished HNF4α-mediated activation in HeLa cells. Chromatin immunoprecipitation and electrophoretic mobility shift assays further confirmed direct recruitment and binding of HNF4α to the site. Gene expression analysis with liver samples from subjects with nonalcoholic steatohepatitis suggested that the axis of the Hes6-Hnf4a-Pparg2 transcriptional cascade is also responsible for hepatic fat accumulation in humans. : HES6 represses gene expression, thereby preventing hepatic lipid accumulation induced by chronic Western diet feeding or pathophysiologic conditions. ( 2017;1:1085-1098).
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http://dx.doi.org/10.1002/hep4.1120DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5721401PMC
December 2017

Reversal of metabolic disorders by pharmacological activation of bile acid receptors TGR5 and FXR.

Mol Metab 2018 03 11;9:131-140. Epub 2018 Jan 11.

Department of Integrative Medical Sciences, Northeast Ohio Medical University, Rootstown, OH, 44272, USA. Electronic address:

Objectives: Activation of the bile acid (BA) receptors farnesoid X receptor (FXR) or G protein-coupled bile acid receptor (GPBAR1; TGR5) improves metabolic homeostasis. In this study, we aim to determine the impact of pharmacological activation of bile acid receptors by INT-767 on reversal of diet-induced metabolic disorders, and the relative contribution of FXR vs. TGR5 to INT-767's effects on metabolic parameters.

Methods: Wild-type (WT), Tgr5, Fxr, Apoe and Shp mice were used to investigate whether and how BA receptor activation by INT-767, a semisynthetic agonist for both FXR and TGR5, could reverse diet-induced metabolic disorders.

Results: INT-767 reversed HFD-induced obesity dependent on activation of both TGR5 and FXR and also reversed the development of atherosclerosis and non-alcoholic fatty liver disease (NAFLD). Mechanistically, INT-767 improved hypercholesterolemia by activation of FXR and induced thermogenic genes via activation of TGR5 and/or FXR. Furthermore, INT-767 inhibited several lipogenic genes and de novo lipogenesis in the liver via activation of FXR. We identified peroxisome proliferation-activated receptor γ (PPARγ) and CCAAT/enhancer-binding protein α (CEBPα) as novel FXR-regulated genes. FXR inhibited PPARγ expression by inducing small heterodimer partner (SHP) whereas the inhibition of CEBPα by FXR was SHP-independent.

Conclusions: BA receptor activation can reverse obesity, NAFLD, and atherosclerosis by specific activation of FXR or TGR5. Our data suggest that, compared to activation of FXR or TGR5 only, dual activation of both FXR and TGR5 is a more attractive strategy for treatment of common metabolic disorders.
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http://dx.doi.org/10.1016/j.molmet.2018.01.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5870099PMC
March 2018

Small heterodimer partner deletion prevents hepatic steatosis and when combined with farnesoid X receptor loss protects against type 2 diabetes in mice.

Hepatology 2017 12 30;66(6):1854-1865. Epub 2017 Oct 30.

Department of Molecular and Integrative Physiology, University of Illinois, Urbana-Champaign, Urbana, IL.

Nuclear receptors farnesoid X receptor (FXR) and small heterodimer partner (SHP) are important regulators of bile acid, lipid, and glucose homeostasis. Here, we show that global Fxr Shp double knockout (DKO) mice are refractory to weight gain, glucose intolerance, and hepatic steatosis when challenged with high-fat diet. DKO mice display an inherently increased capacity to burn fat and suppress de novo hepatic lipid synthesis. Moreover, DKO mice were also very active and that correlated well with the observed increase in phosphoenolpyruvate carboxykinase expression, type IA fibers, and mitochondrial function in skeletal muscle. Mechanistically, we demonstrate that liver-specific Shp deletion protects against fatty liver development by suppressing expression of peroxisome proliferator-activated receptor gamma 2 and lipid-droplet protein fat-specific protein 27 beta.

Conclusion: These data suggest that Fxr and Shp inactivation may be beneficial to combat diet-induced obesity and uncover that hepatic SHP is necessary to promote fatty liver disease. (Hepatology 2017;66:1854-1865).
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http://dx.doi.org/10.1002/hep.29305DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5696047PMC
December 2017

Structure-activity and in vivo evaluation of a novel lipoprotein lipase (LPL) activator.

Bioorg Med Chem Lett 2017 01 21;27(2):303-308. Epub 2016 Nov 21.

Department of Pharmaceutical Sciences, Northeast Ohio Medical University, Rootstown, OH 44272, United States.

Elevated triglycerides (TG) contribute towards increased risk for cardiovascular disease. Lipoprotein lipase (LPL) is an enzyme that is responsible for the metabolism of core triglycerides of very-low density lipoproteins (VLDL) and chylomicrons in the vasculature. In this study, we explored the structure-activity relationships of our lead compound (C10d) that we have previously identified as an LPL agonist. We found that the cyclopropyl moiety of C10d is not absolutely necessary for LPL activity. Several substitutions were found to result in loss of LPL activity. The compound C10d was also tested in vivo for its lipid lowering activity. Mice were fed a high-fat diet (HFD) for four months, and treated for one week at 10mg/kg. At this dose, C10d exhibited in vivo biological activity as indicated by lower TG and cholesterol levels as well as reduced body fat content as determined by ECHO-MRI. Furthermore, C10d also reduced the HFD induced fat accumulation in the liver. Our study has provided insights into the structural and functional characteristics of this novel LPL activator.
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http://dx.doi.org/10.1016/j.bmcl.2016.11.053DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5366985PMC
January 2017

Farnesoid X receptor activation increases reverse cholesterol transport by modulating bile acid composition and cholesterol absorption in mice.

Hepatology 2016 10 30;64(4):1072-85. Epub 2016 Jul 30.

Department of Integrative Medical Sciences, Northeast Ohio Medical University, Rootstown, OH.

Unlabelled: Activation of farnesoid X receptor (FXR) markedly attenuates development of atherosclerosis in animal models. However, the underlying mechanism is not well elucidated. Here, we show that the FXR agonist, obeticholic acid (OCA), increases fecal cholesterol excretion and macrophage reverse cholesterol transport (RCT) dependent on activation of hepatic FXR. OCA does not increase biliary cholesterol secretion, but inhibits intestinal cholesterol absorption. OCA markedly inhibits hepatic cholesterol 7α-hydroxylase (Cyp7a1) and sterol 12α-hydroxylase (Cyp8b1) partly through inducing small heterodimer partner, leading to reduced bile acid pool size and altered bile acid composition, with the α/β-muricholic acid proportion in bile increased by 2.6-fold and taurocholic acid (TCA) level reduced by 71%. Overexpression of Cyp8b1 or concurrent overexpression of Cyp7a1 and Cyp8b1 normalizes TCA level, bile acid composition, and intestinal cholesterol absorption.

Conclusion: Activation of FXR inhibits intestinal cholesterol absorption by modulation of bile acid pool size and composition, thus leading to increased RCT. Targeting hepatic FXR and/or bile acids may be useful for boosting RCT and preventing the development of atherosclerosis. (Hepatology 2016;64:1072-1085).
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http://dx.doi.org/10.1002/hep.28712DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5033696PMC
October 2016

Cholesteryl ester transfer protein alters liver and plasma triglyceride metabolism through two liver networks in female mice.

J Lipid Res 2016 08 27;57(8):1541-51. Epub 2016 Jun 27.

Tennessee Valley Healthcare System, Department of Veterans Affairs, Nashville, TN Department of Molecular Physiology and Biophysics Vanderbilt University Medical Center, Nashville, TN Division of Diabetes, Endocrinology and Metabolism, Department of Medicine, Vanderbilt University Medical Center, Nashville, TN

Elevated plasma TGs increase risk of cardiovascular disease in women. Estrogen treatment raises plasma TGs in women, but molecular mechanisms remain poorly understood. Here we explore the role of cholesteryl ester transfer protein (CETP) in the regulation of TG metabolism in female mice, which naturally lack CETP. In transgenic CETP females, acute estrogen treatment raised plasma TGs 50%, increased TG production, and increased expression of genes involved in VLDL synthesis, but not in nontransgenic littermate females. In CETP females, estrogen enhanced expression of small heterodimer partner (SHP), a nuclear receptor regulating VLDL production. Deletion of liver SHP prevented increases in TG production and expression of genes involved in VLDL synthesis in CETP mice with estrogen treatment. We also examined whether CETP expression had effects on TG metabolism independent of estrogen treatment. CETP increased liver β-oxidation and reduced liver TG content by 60%. Liver estrogen receptor α (ERα) was required for CETP expression to enhance β-oxidation and reduce liver TG content. Thus, CETP alters at least two networks governing TG metabolism, one involving SHP to increase VLDL-TG production in response to estrogen, and another involving ERα to enhance β-oxidation and lower liver TG content. These findings demonstrate a novel role for CETP in estrogen-mediated increases in TG production and a broader role for CETP in TG metabolism.
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http://dx.doi.org/10.1194/jlr.M069013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4959869PMC
August 2016

Enhanced ethanol catabolism in orphan nuclear receptor SHP-null mice.

Am J Physiol Gastrointest Liver Physiol 2016 05 11;310(10):G799-807. Epub 2016 Mar 11.

Department of Integrative Medical Sciences, Northeast Ohio Medical University, Rootstown, Ohio

Deficiency of the orphan nuclear hormone receptor small heterodimer partner (SHP, NR0B2) protects mice from diet-induced hepatic steatosis, in part, via repression of peroxisome proliferator-activated receptor (PPAR)-γ2 (Pparg2) gene expression. Alcoholic fatty liver diseases (AFLD) share many common pathophysiological features with non-AFLD. To study the role of SHP and PPARγ2 in AFLD, we used a strategy of chronic ethanol feeding plus a single binge ethanol feeding to challenge wild-type (WT) and SHP-null (SHP(-/-)) mice with ethanol. The ethanol feeding induced liver fat accumulation and mRNA expression of hepatic Pparg2 in WT mice, which suggests that a high level of PPARγ2 is a common driving force for fat accumulation induced by ethanol or a high-fat diet. Interestingly, ethanol-fed SHP(-/-) mice displayed hepatic fat accumulation similar to that of ethanol-fed WT mice, even though their Pparg2 expression level remained lower. Mortality of SHP(-/-) mice after ethanol binge feeding was significantly reduced and their acetaldehyde dehydrogenase (Aldh2) mRNA level was higher than that of their WT counterparts. After an intoxicating dose of ethanol, SHP(-/-) mice exhibited faster blood ethanol clearance and earlier wake-up time than WT mice. Higher blood acetate, the end product of ethanol metabolism, and lower acetaldehyde levels were evident in the ethanol-challenged SHP(-/-) than WT mice. Ethanol-induced inflammatory responses and lipid peroxidation were also lower in SHP(-/-) mice. The current data show faster ethanol catabolism and extra fat storage through conversion of acetate to acetyl-CoA before its release into the circulation in this ethanol-feeding model in SHP(-/-) mice.
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http://dx.doi.org/10.1152/ajpgi.00343.2015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4895871PMC
May 2016

Thyroid Hormone Regulates the mRNA Expression of Small Heterodimer Partner through Liver Receptor Homolog-1.

Endocrinol Metab (Seoul) 2015 Dec 20;30(4):584-92. Epub 2015 Oct 20.

Department of Internal Medicine, Seoul National University College of Medicine, Seoul, Korea.

Background: Expression of hepatic cholesterol 7α-hydroxylase (CYP7A1) is negatively regulated by orphan nuclear receptor small heterodimer partner (SHP). In this study, we aimed to find whether thyroid hormone regulates SHP expression by modulating the transcriptional activities of liver receptor homolog-1 (LRH-1).

Methods: We injected thyroid hormone (triiodothyronine, T3) to C57BL/6J wild type. RNA was isolated from mouse liver and used for microarray analysis and quantitative real-time polymerase chain reaction (PCR). Human hepatoma cell and primary hepatocytes from mouse liver were used to confirm the effect of T3 in vitro. Promoter assay and electrophoretic mobility-shift assay (EMSA) were also performed using human hepatoma cell line.

Results: Initial microarray results indicated that SHP expression is markedly decreased in livers of T3 treated mice. We confirmed that T3 repressed SHP expression in the liver of mice as well as in mouse primary hepatocytes and human hepatoma cells by real-time PCR analysis. LRH-1 increased the promoter activity of SHP; however, this increased activity was markedly decreased after thyroid hormone receptor β/retinoid X receptor α/T3 administration. EMSA revealed that T3 inhibits specific LRH-1 DNA binding.

Conclusion: We found that thyroid hormone regulates the expression of SHP mRNA through interference with the transcription factor, LRH-1.
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http://dx.doi.org/10.3803/EnM.2015.30.4.584DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4722415PMC
December 2015

Signal Transduction Mechanisms of Alcoholic Fatty Liver Disease: Emer ging Role of Lipin-1.

Curr Mol Pharmacol 2017 ;10(3):226-236

Department of Pharmaceutical Sciences. 0.

Lipin-1, a mammalian phosphatidic acid phosphatase (PAP), is a bi-functional molecule involved in various signaling pathways via its function as a PAP enzyme in the triglyceride synthesis pathway and in the nucleus as a transcriptional co-regulator. In the liver, lipin-1 is known to play a vital role in controlling the lipid metabolism and inflammation process at multiple regulatory levels. Alcoholic fatty liver disease (AFLD) is one of the earliest forms of liver injury and approximately 8-20% of patients with simple steatosis can develop into more severe forms of liver injury, including steatohepatitis, fibrosis/ cirrhosis, and eventually hepatocellular carcinoma (HCC). The signal transduction mechanisms for alcohol-induced detrimental effects in liver involves alteration of complex and multiple signaling pathways largely governed by a central and upstream signaling system, namely, sirtuin 1 (SIRT1)-AMP activated kinase (AMPK) axis. Emerging evidence suggests a pivotal role of lipin-1 as a crucial downstream regulator of SIRT1-AMPK signaling system that is likely to be ultimately responsible for development and progression of AFLD. Several lines of evidence demonstrate that ethanol exposure significantly induces lipin-1 gene and protein expression levels in cultured hepatocytes and in the livers of rodents, induces lipin-1-PAP activity, impairs the functional activity of nuclear lipin-1, disrupts lipin-1 mRNA alternative splicing and induces lipin-1 nucleocytoplasmic shuttling. Such impairment in response to ethanol leads to derangement of hepatic lipid metabolism, and excessive production of inflammatory cytokines in the livers of the rodents and human alcoholics. This review summarizes current knowledge about the role of lipin-1 in the pathogenesis of AFLD and its potential signal transduction mechanisms.
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http://dx.doi.org/10.2174/1874467208666150817112109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4879108PMC
February 2019

The orphan nuclear receptor small heterodimer partner is required for thiazolidinedione effects in leptin-deficient mice.

J Biomed Sci 2015 May 8;22:30. Epub 2015 May 8.

Department of Molecular and Cellular Biology, Baylor College of Medicine, One Baylor Plaza, Houston, Texas, USA.

Background: Small heterodimer partner (SHP, NR0B2) is involved in diverse metabolic pathways, including hepatic bile acid, lipid and glucose homeostasis, and has been implicated in effects on the peroxisome proliferator-activated receptor γ (PPARγ), a master regulator of adipogenesis and the receptor for antidiabetic drugs thiazolidinediones (TZDs). In this study, we aim to investigate the role of SHP in TZD response by comparing TZD-treated leptin-deficient (ob/ob) and leptin-, SHP-deficient (ob/ob;Shp(-/-)) double mutant mice.

Results: Both ob/ob and double mutant ob/ob;Shp(-/-) mice developed hyperglycemia, insulin resistance, and hyperlipidemia, but hepatic fat accumulation was decreased in the double mutant ob/ob;Shp(-/-) mice. PPARγ2 mRNA levels were markedly lower in ob/ob;Shp(-/-) liver and decreased to a lesser extent in adipose tissue. The TZD troglitazone did not reduce glucose or circulating triglyceride levels in ob/ob;Shp(-/-) mice. Expression of the adipocytokines, such as adiponectin and resistin, was not stimulated by troglitazone treatment. Expression of hepatic lipogenic genes was also reduced in ob/ob;Shp(-/-) mice. Moreover, overexpression of SHP by adenovirus infection increased PPARγ2 mRNA levels in mouse primary hepatocytes.

Conclusions: Our results suggest that SHP is required for both antidiabetic and hypolipidemic effects of TZDs in ob/ob mice through regulation of PPARγ expression.
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http://dx.doi.org/10.1186/s12929-015-0133-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4489392PMC
May 2015

Farnesoid X Receptor Agonist Represses Cytochrome P450 2D6 Expression by Upregulating Small Heterodimer Partner.

Drug Metab Dispos 2015 Jul 29;43(7):1002-7. Epub 2015 Apr 29.

Departments of Pharmacy Practice (H.J.) and Biopharmaceutical Sciences (X.P., H.J.), College of Pharmacy, University of Illinois at Chicago, Chicago, Illinois; and Department of Integrative Medical Sciences, Northeast Ohio Medical University, Rootstown, Ohio (Y.K.L.)

Cytochrome P450 2D6 (CYP2D6) is a major drug-metabolizing enzyme responsible for eliminating approximately 20% of marketed drugs. Studies have shown that differential transcriptional regulation of CYP2D6 may contribute to large interindividual variability in CYP2D6-mediated drug metabolism. However, the factors governing CYP2D6 transcription are largely unknown. We previously demonstrated small heterodimer partner (SHP) as a novel transcriptional repressor of CYP2D6 expression. SHP is a representative target gene of the farnesoid X receptor (FXR). The objective of this study is to investigate whether an agonist of FXR, 3-(2,6-dichlorophenyl)-4-(3'-carboxy-2-chlorostilben-4-yl)oxymethyl-5-isopropylisoxazole (GW4064), alters CYP2D6 expression and activity. In CYP2D6-humanized transgenic mice, GW4064 decreased hepatic CYP2D6 expression and activity (by 2-fold) while increasing SHP expression (by 2-fold) and SHP recruitment to the CYP2D6 promoter. CYP2D6 repression by GW4064 was abrogated in Shp(-/-);CYP2D6 mice, indicating a critical role of SHP in CYP2D6 regulation by GW4064. Also, GW4064 decreased CYP2D6 expression (by 2-fold) in primary human hepatocytes, suggesting that the results obtained in CYP2D6-humanized transgenic mice can be translated to humans. This proof of concept study provides evidence for CYP2D6 regulation by an inducer of SHP expression, namely, the FXR agonist GW4064.
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http://dx.doi.org/10.1124/dmd.115.064758DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4468439PMC
July 2015

All-trans-retinoic acid ameliorates hepatic steatosis in mice by a novel transcriptional cascade.

Hepatology 2014 May 26;59(5):1750-60. Epub 2014 Mar 26.

Department of Integrative Medical Sciences, Northeast Ohio Medical University, Rootstown, OH.

Unlabelled: Mice deficient in small heterodimer partner (SHP) are protected from diet-induced hepatic steatosis resulting from increased fatty acid oxidation and decreased lipogenesis. The decreased lipogenesis appears to be a direct consequence of very low expression of peroxisome proliferator-activated receptor gamma 2 (PPAR-γ2), a potent lipogenic transcription factor, in the SHP(-/-) liver. The current study focused on the identification of a SHP-dependent regulatory cascade that controls PPAR-γ2 gene expression, thereby regulating hepatic fat accumulation. Illumina BeadChip array (Illumina, Inc., San Diego, CA) and real-time polymerase chain reaction were used to identify genes responsible for the linkage between SHP and PPAR-γ2 using hepatic RNAs isolated from SHP(-/-) and SHP-overexpressing mice. The initial efforts identify that hairy and enhancer of split 6 (Hes6), a novel transcriptional repressor, is an important mediator of the regulation of PPAR-γ2 transcription by SHP. The Hes6 promoter is specifically activated by the retinoic acid receptor (RAR) in response to its natural agonist ligand, all-trans retinoic acid (atRA), and is repressed by SHP. Hes6 subsequently represses hepatocyte nuclear factor 4 alpha (HNF-4α)-activated PPAR-γ2 gene expression by direct inhibition of HNF-4α transcriptional activity. Furthermore, we provide evidences that atRA treatment or adenovirus-mediated RAR-α overexpression significantly reduced hepatic fat accumulation in obese mouse models, as observed in earlier studies, and the beneficial effect is achieved by the proposed transcriptional cascade.

Conclusions: Our study describes a novel transcriptional regulatory cascade controlling hepatic lipid metabolism that identifies retinoic acid signaling as a new therapeutic approach to nonalcoholic fatty liver diseases.
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http://dx.doi.org/10.1002/hep.26699DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4008145PMC
May 2014

Eicosanoids in metabolic syndrome.

Adv Pharmacol 2013 ;66:157-266

Biochemistry and Molecular Pathology, Department of Integrative Medical Sciences, Northeast Ohio Medical University, Rootstown, Ohio, USA.

Chronic persistent inflammation plays a significant role in disease pathology of cancer, cardiovascular disease, and metabolic syndrome (MetS). MetS is a constellation of diseases that include obesity, diabetes, hypertension, dyslipidemia, hypertriglyceridemia, and hypercholesterolemia. Nonalcoholic fatty liver disease (NAFLD) is associated with many of the MetS diseases. These metabolic derangements trigger a persistent inflammatory cascade, which includes production of lipid autacoids (eicosanoids) that recruit immune cells to the site of injury and subsequent expression of cytokines and chemokines that amplify the inflammatory response. In acute inflammation, the transcellular synthesis of antiinflammatory eicosanoids resolve inflammation, while persistent activation of the autacoid-cytokine-chemokine cascade in metabolic disease leads to chronic inflammation and accompanying tissue pathology. Many drugs targeting the eicosanoid pathways have been shown to be effective in the treatment of MetS, suggesting a common linkage between inflammation, MetS and drug metabolism. The cross-talk between inflammation and MetS seems apparent because of the growing evidence linking immune cell activation and metabolic disorders such as insulin resistance, dyslipidemia, and hypertriglyceridemia. Thus modulation of lipid metabolism through either dietary adjustment or selective drugs may become a new paradigm in the treatment of metabolic disorders. This review focuses on the mechanisms linking eicosanoid metabolism to persistent inflammation and altered lipid and carbohydrate metabolism in MetS.
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http://dx.doi.org/10.1016/B978-0-12-404717-4.00005-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3675900PMC
August 2013

Opposing regulation of cytochrome P450 expression by CAR and PXR in hypothyroid mice.

Toxicol Appl Pharmacol 2012 Sep 3;263(2):131-7. Epub 2012 Apr 3.

Department of Internal Medicine, Seoul National University College of Medicine, Republic of Korea.

Clinical hypothyroidism affects various metabolic processes including drug metabolism. CYP2B and CYP3A are important cytochrome P450 drug metabolizing enzymes that are regulated by the xenobiotic receptors constitutive androstane receptor (CAR, NR1I3) and pregnane X receptor (PXR, NR1I2). We evaluated the regulation of the hepatic expression of CYPs by CAR and PXR in the hypothyroid state induced by a low-iodine diet containing 0.15% propylthiouracil. Expression of Cyp3a11 was suppressed in hypothyroid C57BL/6 wild type (WT) mice and a further decrement was observed in hypothyroid CAR-/- mice, but not in hypothyroid PXR-/- mice. In contrast, expression of Cyp2b10 was induced in both WT and PXR-/- hypothyroid mice, and this induction was abolished in CAR-/- mice and in and CAR-/- PXR-/- double knockouts. CAR mRNA expression was increased by hypothyroidism, while PXR expression remained unchanged. Carbamazepine (CBZ) is a commonly used antiepileptic that is metabolized by CYP3A isoforms. After CBZ treatment of normal chow fed mice, serum CBZ levels were highest in CAR-/- mice and lowest in WT and PXR-/- mice. Hypothyroid WT or PXR-/- mice survived chronic CBZ treatment, but all hypothyroid CAR-/- and CAR-/- PXR-/- mice died, with CAR-/-PXR-/- mice surviving longer than CAR-/- mice (12.3±3.3 days vs. 6.3±2.1 days, p=0.04). All these findings suggest that hypothyroid status affects xenobiotic metabolism, with opposing responses of CAR and PXR and their CYP targets that can cancel each other out, decreasing serious metabolic derangement in response to a xenobiotic challenge.
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http://dx.doi.org/10.1016/j.taap.2012.03.017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3399920PMC
September 2012

Dissociation of diabetes and obesity in mice lacking orphan nuclear receptor small heterodimer partner.

J Lipid Res 2011 Dec 23;52(12):2234-2244. Epub 2011 Sep 23.

Department of Integrative Medical Sciences, Northeast Ohio Medical University, Rootstown, OH; Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX and. Electronic address:

Mixed background SHP(-/-) mice are resistant to diet-induced obesity due to increased energy expenditure caused by enhanced PGC-1α expression in brown adipocytes. However, congenic SHP(-/-) mice on the C57BL/6 background showed normal expression of PGC-1α and other genes involved in brown adipose tissue thermogenesis. Thus, we reinvestigated the impact of small heterodimer partner (SHP) deletion on diet-induced obesity and insulin resistance using congenic SHP(-/-) mice. Compared with their C57BL/6 wild-type counterparts, SHP(-/-) mice subjected to a 6 month challenge with a Western diet (WestD) were leaner but more glucose intolerant, showed hepatic insulin resistance despite decreased triglyceride accumulation and increased β-oxidation, exhibited alterations in peripheral tissue uptake of dietary lipids, maintained a higher respiratory quotient, which did not decrease even after WestD feeding, and displayed islet dysfunction. Hepatic mRNA expression analysis revealed that many genes expressed higher in SHP(-/-) mice fed WestD were direct peroxisome proliferator-activated receptor alpha (PPARα) targets. Indeed, transient transfection and chromatin immunoprecipitation verified that SHP strongly repressed PPARα-mediated transactivation. SHP is a pivotal metabolic sensor controlling lipid homeostasis in response to an energy-laden diet through regulating PPARα-mediated transactivation. The resultant hepatic fatty acid oxidation enhancement and dietary fat redistribution protect the mice from diet-induced obesity and hepatic steatosis but accelerate development of type 2 diabetes.
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http://dx.doi.org/10.1194/jlr.M016048DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3220290PMC
December 2011

A nuclear-receptor-dependent phosphatidylcholine pathway with antidiabetic effects.

Nature 2011 May 25;474(7352):506-10. Epub 2011 May 25.

Program in Developmental Biology, Baylor College of Medicine, Houston, Texas 77030, USA.

Nuclear hormone receptors regulate diverse metabolic pathways and the orphan nuclear receptor LRH-1 (also known as NR5A2) regulates bile acid biosynthesis. Structural studies have identified phospholipids as potential LRH-1 ligands, but their functional relevance is unclear. Here we show that an unusual phosphatidylcholine species with two saturated 12 carbon fatty acid acyl side chains (dilauroyl phosphatidylcholine (DLPC)) is an LRH-1 agonist ligand in vitro. DLPC treatment induces bile acid biosynthetic enzymes in mouse liver, increases bile acid levels, and lowers hepatic triglycerides and serum glucose. DLPC treatment also decreases hepatic steatosis and improves glucose homeostasis in two mouse models of insulin resistance. Both the antidiabetic and lipotropic effects are lost in liver-specific Lrh-1 knockouts. These findings identify an LRH-1 dependent phosphatidylcholine signalling pathway that regulates bile acid metabolism and glucose homeostasis.
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http://dx.doi.org/10.1038/nature10111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3150801PMC
May 2011

Forkhead box transcription factor O1 inhibits cholesterol 7alpha-hydroxylase in human hepatocytes and in high fat diet-fed mice.

Biochim Biophys Acta 2009 Oct 20;1791(10):991-6. Epub 2009 May 20.

Department of Integrative Medical Sciences, Northeastern Ohio Universities Colleges of Medicine and Pharmacy, 4209 State Route 44, Rootstown, OH 44272, USA.

The conversion of cholesterol to bile acids is the major pathway for cholesterol catabolism. Bile acids are metabolic regulators of triglycerides and glucose metabolism in the liver. This study investigated the roles of FoxO1 in the regulation of cholesterol 7alpha-hydroxylase (CYP7A1) gene expression in primary human hepatocytes. Adenovirus-mediated expression of a phosphorylation defective and constitutively active form of FoxO1 (FoxO1-ADA) inhibited CYP7A1 mRNA expression and bile acid synthesis, while siRNA knockdown of FoxO1 resulted in a approximately 6-fold induction of CYP7A1 mRNA in human hepatocytes. Insulin caused rapid exclusion of FoxO1 from the nucleus and resulted in the induction of CYP7A1 mRNA expression, which was blocked by FoxO1-ADA. In high fat diet-fed mice, CYP7A1 mRNA expression was repressed and inversely correlated to increase hepatic FoxO1 mRNA expression and FoxO1 nuclear retention. In conclusion, our current study provides direct evidence that FoxO1 is a strong repressor of CYP7A1 gene expression and bile acid synthesis. Impaired regulation of FoxO1 may cause down-regulation of CYP7A1 gene expression and contribute to dyslipidemia in insulin resistance.
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http://dx.doi.org/10.1016/j.bbalip.2009.05.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2743791PMC
October 2009

Aberrant regulation and modification of heat shock factor 1 in senescent human diploid fibroblasts.

J Cell Biochem 2009 Feb;106(2):267-78

Department of Cell Biology and Neuroscience, Rutgers State University of New Jersey 604 Allison Road, Piscataway, New Jersey 08854, USA.

Induction of the heat shock response (HSR), determined by hsp70-luciferase reporter and HSP70 protein expression, is attenuated as a function of age of the IMR-90 human diploid fibroblasts. To better understand the underlying mechanism, we evaluated changes in the regulation and function of the HSF1 transcription factor. We show that the activation of HSF1 both in vivo and in vitro was decreased as a function of age, and this was attributable to a change in the regulation of HSF1 as the abundance of HSF1 protein and mRNA was unaffected. HSF1 was primarily cytosolic in young cells maintained at 37 degrees C, and heat shock promoted its quantitative nuclear translocation and trimerization. In old cells, some HSF1 was nuclear sequestered at 37 degrees C, and heat shock failed to promote the quantitative trimerization of HSF1. These changes in HSF1 could be reproduced by treating young cells with H2O2 to stunt them into premature senescence. Flow cytometry measurement of peroxide content showed higher levels in old cells and H2O2-induced premature senescent cells as compared to young cells. Experiments using isoelectric focusing and Western blot showed age-dependent changes in the mobility of HSF1 in a pattern consistent with its S-glutathiolation and S-nitrosylation; these changes could be mimicked by treating young cells with H2O2. Our results demonstrated dynamic age-dependent changes in the regulation but not the amount of HSF1. These changes are likely mediated by oxidative events that promote reversible and irreversible modification of HSF1 including S-glutathiolation and S-nitrosylation.
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http://dx.doi.org/10.1002/jcb.21997DOI Listing
February 2009

Liver receptor homolog-1, an emerging metabolic modulator.

Front Biosci 2008 May 1;13:5950-8. Epub 2008 May 1.

Department of Molecular and Cellular Biology, Baylor College of Medicine, 1 Baylor Plaza, Houston, TX 77030, USA.

The liver receptor homolog-1 (LRH-1; NR5A2) belongs to nuclear hormone receptor superfamily, and is expressed mainly in the liver, intestine, exocrine pancreas, and ovary. It binds DNA as a monomer, and is best known as a regulator of hepatic expression of the key bile acid biosynthetic enzyme cholesterol 7alpha hydroxylase (Cyp7A1). It is also expressed in embryonic stem cells and the initial stages of embryonic development, and the very early lethality of LRH-1 knockout mice highlights its essential developmental role. Recent crystal structures of LRH-1 and its closest relative steroidogenic factor-1 (SF-1; NR5A1) identified phospholipids as potential ligands. This intriguing discovery raises the possibility of an unexpected new class of nuclear receptor signaling molecules, but the broader functional roles of LRH-1 and these new ligands remain to be established.
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http://dx.doi.org/10.2741/3128DOI Listing
May 2008

Loss of orphan receptor small heterodimer partner sensitizes mice to liver injury from obstructive cholestasis.

Hepatology 2008 May;47(5):1578-86

Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX 77030, USA.

Unlabelled: The orphan nuclear hormone receptor small heterodimer partner (SHP) regulates the expression of several genes involved in bile acid homeostasis in the liver. Because bile acid toxicity is a major source of liver injury in cholestatic disease, we explored the role of SHP in liver damage induced by common bile duct ligation (BDL). Shp(-/-) mice show increased sensitivity in this model of acute obstructive cholestasis, with greater numbers of bile infarcts and higher mortality than wild-type C57BL/6 mice. This increased sensitivity could not be accounted for by differences in expression of bile acid homeostatic genes 2 or 5 days after BDL. Instead, higher basal expression of such genes, including the key biosynthetic enzyme cholesterol 7alpha hydroxylase (Cyp7A1) and the bile salt export pump, is associated with both an increase in bile flow prior to BDL and an increase in acute liver damage at only 1.5 hours after BDL in Shp(-/-) mice, as shown by bile infarcts. At 3 hours, Cyp7A1 expression still remained elevated in Shp(-/-) with respect to wild-type mice, and the hepatic and serum bile acid levels and total hepatobiliary bile acid pool were significantly increased. The increased sensitivity of mice lacking SHP contrasts with the decreased sensitivity of mice lacking the farnesoid X receptor (FXR; nuclear receptor subfamily 1, group H, member 4) to BDL, which has been associated with decreased intraductal pressure and fewer bile infarcts.

Conclusion: We propose that differences in acute responses to BDL, particularly the early formation of bile infarcts, are a primary determinant of the differences in longer term sensitivity of the Fxr(-/-) and Shp(-/-) mice to acute obstructive cholestasis.
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http://dx.doi.org/10.1002/hep.22196DOI Listing
May 2008

Phosphorylation of the hinge domain of the nuclear hormone receptor LRH-1 stimulates transactivation.

J Biol Chem 2006 Mar 25;281(12):7850-5. Epub 2006 Jan 25.

Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas 77030, USA.

The nuclear receptor LRH-1 (NR5A2) functions to regulate expression of a number of genes associated with bile acid homeostasis and other liver functions, but mechanisms that modulate its activity remain unclear. We have found that mitogenic stimuli, including treatment with phorbol myristate (PMA), increase LRH-1 transactivation. This response maps to the hinge and ligand binding domains of LRH-1 and is blocked by the mitogen-activated protein kinase ERK1/2 inhibitor U0126. LRH-1 is a phosphoprotein and hinge domain serine residues at 238 and 243 are required for effective phosphorylation, both in vitro and in cells. Preventing phosphorylation of these residues by mutating both to alanine decreases PMA-dependent LRH-1 transactivation and mimicking phosphorylation by mutation to positively charged aspartate residues increases basal transactivation. Although serine phosphorylation of the hinge of SF-1 (NR5A1), the closest relative of LRH-1, confers a similar response, the specific targets differ in the two closely related orphan receptors. These results define a novel pathway for the modulation of LRH-1 transactivation and identify specific LRH-1 residues as downstream targets of mitogenic stimuli. This pathway may contribute to recently described proliferative functions of LRH-1.
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http://dx.doi.org/10.1074/jbc.M509115200DOI Listing
March 2006

The orphan receptor SHP and the three-hybrid interference assay.

Methods Enzymol 2003 ;364:152-9

Department of Molecular and Cellular Biology, Baylor College of Medicine, One Baylor Plaza, Houston, Texas 77030, USA.

The three-hybrid interference method described here is similar to previous interference assays based on the two-hybrid system. In these approaches the presence of a third protein disrupts the interaction between the two different hybrid proteins. The three-hybrid interference assay circumvents a potential problem that arises when the third protein is itself a direct transcriptional repressor by preventing the decrease in transcriptional readout that could occur if the third protein is recruited indirectly to the two-hybrid protein complex. This three-hybrid method should be applicable to any third protein that is a transcriptional repressor. To use this method to explore the effects of such repressors on protein-protein interactions, however, it is important to confirm that the VP16-repressor fusion does not retain active repressor function. It is also appropriate to compare the effects of the VP16-repressor fusion to those with the repressor alone. Finally, it is essential to confirm any conclusions from this or any other single method to study protein-protein interactions with alternative independent approaches.
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http://dx.doi.org/10.1016/s0076-6879(03)64009-7DOI Listing
December 2003

Resistance of SHP-null mice to bile acid-induced liver damage.

J Biol Chem 2003 Nov 21;278(45):44475-81. Epub 2003 Aug 21.

Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas 77030, USA.

The orphan nuclear hormone receptor SHP (gene designation NROB2) is an important component of a negative regulatory cascade by which high levels of bile acids repress bile acid biosynthesis. Short term studies in SHP null animals confirm this function and also reveal the existence of additional pathways for bile acid negative feedback regulation. We have used long term dietary treatments to test the role of SHP in response to chronic elevation of bile acids, cholesterol, or both. In contrast to the increased sensitivity predicted from the loss of negative feedback regulation, the SHP null mice were relatively resistant to the hepatotoxicity associated with a diet containing 0.5% cholic acid and the much more severe effects of a diet containing both 0.5% cholic acid and 2% cholesterol. This was associated with decreased hepatic accumulation of cholesterol and triglycerides in the SHP null mice. There were also alterations in the expression of a number of genes involved in cholesterol and bile acid homeostasis, notably cholesterol 12alpha-hydroxylase (CYP8B1), which was strongly reexpressed in the SHP null mice, but not the wild type mice fed either bile acid containing diet. This contrasts with the strong repression of CYP8B1 observed with short term bile acid feeding, as well as the effects of long term feeding on other bile acid biosynthetic enzymes such as cholesterol 7alpha-hydroxylase (CYP7A1). CYP8B1 expression could contribute to the decreased toxicity of the chronic bile acid treatment by increasing the hydrophilicity of the bile acid pool. These results identify an unexpected role for SHP in hepatotoxicity and suggest new approaches to modulating effects of chronically elevated bile acids in cholestasis.
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http://dx.doi.org/10.1074/jbc.M305258200DOI Listing
November 2003

Redundant pathways for negative feedback regulation of bile acid production.

Dev Cell 2002 Jun;2(6):721-31

Department of Molecular and Cellular Biology, Houston, TX 77030, USA.

The orphan nuclear hormone receptor SHP has been proposed to have a key role in the negative feedback regulation of bile acid production. Consistent with this, mice lacking the SHP gene exhibit mild defects in bile acid homeostasis and fail to repress cholesterol 7-alpha-hydroxylase expression in response to a specific agonist for the bile acid receptor FXR. However, this repression is retained in SHP null mice fed bile acids, demonstrating the existence of compensatory repression pathways of bile acid signaling. We provide evidence for two such pathways, based on activation of the xenobiotic receptor PXR or the c-Jun N-terminal kinase JNK. We conclude that redundant mechanisms regulate this critical aspect of cholesterol homeostasis.
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http://dx.doi.org/10.1016/s1534-5807(02)00187-9DOI Listing
June 2002

Differential regulation of the orphan nuclear receptor small heterodimer partner (SHP) gene promoter by orphan nuclear receptor ERR isoforms.

J Biol Chem 2002 Jan 8;277(3):1739-48. Epub 2001 Nov 8.

Department of Biology, Hormone Research Center, Chonnam National University, Kwangju, 500-757, Republic of Korea.

The orphan nuclear receptor small heterodimer partner (SHP; NR0B2) interacts with a wide array of nuclear receptors and represses their transcriptional activity. SHP expression is regulated by several other members of the nuclear receptor superfamily, including the orphan receptors SF-1 and LRH-1, and the bile acid receptor FXR. We have found that the SHP promoter is also activated by the estrogen receptor-related receptor gamma (ERRgamma) but not the related ERRalpha and ERRbeta isoforms. SHP and ERRgamma mRNAs are coexpressed in several tissues, including pancreas, kidney, and heart, confirming the potential relevance of this transactivation. ERRgamma transactivation is dependent on only one of five previously characterized DNA-binding sites for SF-1, and this element differs from previously reported ERR response elements. However, treatment with the histone deacetylase inhibitor trichostatin A significantly increased ERRalpha and ERRbeta activity on this element indicating that the lack of activity of ERRalpha and -beta may depend on their association with co-repressor in vivo. Furthermore, using protease sensitivity assays on DNA bound receptors it was demonstrated that DNA sequence of different response elements may cause allosteric modulation of ERR proteins, which in turn may be responsible for the differential activities of these receptors on different response elements. SHP inhibits ERRgamma transactivation and physically interacts with all three members of ERR subfamily, as demonstrated by both yeast two-hybrid and biochemical assays. As with other SHP targets, this interaction is dependent on the AF-2 coactivator-binding site of ERRgamma and the previously described N-terminal receptor interaction domain of SHP. Several recently described SHP mutations associated with moderate obesity in humans block the inhibition of ERRgamma activity. Overall, these results identify a new autoregulatory loop controlling SHP gene expression and significantly extend the potential functional roles of the three ERRs.
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http://dx.doi.org/10.1074/jbc.M106140200DOI Listing
January 2002

Dual mechanisms for repression of the monomeric orphan receptor liver receptor homologous protein-1 by the orphan small heterodimer partner.

J Biol Chem 2002 Jan 19;277(4):2463-7. Epub 2001 Oct 19.

Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas 77030, USA.

The orphan nuclear hormone receptor liver receptor homologous protein-1 (LRH-1; NR5A2, also known as FTF), an unusual receptor that binds DNA as a monomer, is an essential regulator of expression of a rate-limiting enzyme in bile acid formation, cholesterol 7-alpha-hydroxylase. In a classic negative feedback loop that is a crucial component of the complex regulation of cholesterol metabolism, cholesterol 7-alpha-hydroxylase expression is decreased when bile acid levels are high. This repression is thought to be based on the bile acid-dependent induction of expression of the orphan receptor small heterodimer partner (SHP) NR0B2, which inhibits the activity of LRH-1. We have explored the molecular basis for this important regulatory effect by characterizing the mechanisms by which mouse and human SHP inhibit LRH-1-mediated transactivation. Both SHP proteins specifically interact with the AF-2 transactivation domain of LRH-1 both in vivo and in vitro. This domain is a common target for coactivator interaction, and the SHP proteins can compete with p160 coactivators for binding to LRH-1. In addition to the N-terminal receptor interaction domain, SHP includes a C-terminal domain with autonomous repression function. Neither a deletion nor a point mutation specifically affecting this domain blocked the ability to interact with LRH-1 to compete for coactivator binding or to repress LRH-1 transactivation. However, the relative ability of these mutants to inhibit LRH-1-mediated transactivation was markedly decreased. We conclude that the proposed central role of SHP in cholesterol metabolism is based on a two-step mechanism that is dependent on both coactivator competition and direct transcriptional repression.
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http://dx.doi.org/10.1074/jbc.M105161200DOI Listing
January 2002