Nuclear Receptor SHP Research Articles

Depicting a novel regulatory Bcl2/SHP/lncRNA H19 network in bile acid homeostasis (605.12)

Objective: The aim of this study is to investigate a novel function of the anti‐apoptotic protein Bcl2 in bile acid (BA) homeostasis. Methods: Bcl2 was overexpressed using adenoviruses in male C57BL6 livers. After two weeks, BA homeostasis, liver morphology, fibrosis, and inflammation were analyzed. RNA‐seq and GC/MS were used to identify changes in gene expression and metabolites, respectively. Results: Hepatic overexpression of Bcl2 largely induced serum BA and FGF15 levels and decreased total BA pool size and fecal BA output. Severe hepatocyte necrosis, fibrosis, and Kupffer cell activation were observed, which was accompanied by a marked induction of long non‐coding RNA H19. RNA‐seq identified significant downregulation of genes in bile acid synthesis and transport, but upregulation of genes in collagen formation and inflammatory response by Bcl2. The increases in TCA cycle and lipid metabolites were the most drastic metabolomics changes. Using a series of cell based biochemistry and molecular biology approaches, we found that Bcl2 induced rapid nuclear receptor SHP protein degradation via activation of the caspase 8‐caspase 3 pathway. Furthermore, bile acid directly induced H19 expression in cultured primary hepatocytes that was independent of Bcl2 activation by bile acid. Importantly, Bcl2 and H19 were both dramatically increased in human cirrhotic livers and in mouse cholestatic liver fibrosis models. The dysregulation of the Bcl2/SHP/H19 network resulted in the disruption of bile acid homeostasis which contributed to the development of cholestatic liver fibrosis. Conclusions: Our results uncovered a unique metabolic regulatory network that integrated Bcl2 and SHP with lncRNA H19 to control BA homeostasis.Grant Funding Source: Supported by DK080440 (L.W).

Circadian clock mediates ER stress signaling in alcoholic fatty liver (1116.12)

[Purpose] This is the first study to investigate how alcohol regulates ER stress and fatty liver through nuclear receptor SHP mediated liver circadian clock machinery.[Methods] The NIAAA EtOH binge model was established using 8 weeks old, male C57BL/6 (WT) and SHP‐/‐ (SKO) mice (Nat Protoc 2013; 8:627‐637). In brief, the mice were acclimatized to a control liquid diet for 5 days, then fed with 5% Lieber‐DeCarli diet or pair‐fed for 10 days. By day 11, the mice were gavaged maltose (control) or EtOH solutions at zeitgeber time (ZT) 3 (9 am) and sacrificed at ZT12, 18, 0, and 6.[Results] Metabolomics by GC/MS revealed a global alteration by alcohol in the oscillation of serum intermediate metabolites in pathways of glucose, lipid and amino acid metabolism in SKO mice. The rhythmic expression of core clock genes (Clock, Bmal1, Npas2) and nuclear receptors (Rorα, Rorγ, Rev‐erbα) were disturbed by alcohol. Hepatic lipid metabolic genes and ER stress markers exhibited strong oscillations which were perturbed by alcohol. Chop was a clock controlled gene through the Rorα/Rorγ/Rev‐erbα/SHP regulatory cascade. Liver SREBP‐1c protein cleavage and TG content were drastically induced in control SKO livers, which was markedly decreased by alcohol in both WT and SKO mice.[Conclusions] SHP mediated clock machinery is significantly disrupted by alcohol, which in turn perturbs ER stress to control AFL.Grant Funding Source: Supported by AHA and NIDDK

Correction for Zhi et al., Structural insights into gene repression by the orphan nuclear receptor SHP

Correction for Zhi et al., Structural insights into gene repression by the orphan nuclear receptor SHP

MicroRNA-433 Inhibits Liver Cancer Cell Migration by Repressing the Protein Expression and Function of cAMP Response Element-binding Protein

We show for the first time that potent microRNA-433 (miR-433) inhibition of expression of the cAMP response element-binding protein CREB1 represses hepatocellular carcinoma (HCC) cell migration. We identified a miR-433 seed match region in human and mouse CREB1 3'-UTRs. Overexpression of miR-433 markedly decreased human CREB1 3'-UTR reporter activity, and the inhibitory effect of miR-433 was alleviated upon mutation of its binding site. Ectopic expression of miR-433 reduced CREB1 protein levels in a variety of human and mouse cancer cells, including HeLa, Hepa1, Huh7, and HepG2. Human CREB1 protein levels in highly invasive MHCC97H cells were diminished by expression of miR-433 but were induced by miR-433 antagomir (anti-miR-433). The expression of mouse CREB1 protein negatively correlated with miR-433 levels in nuclear receptor Shp(-/-) liver tissues and liver tumors compared with wild-type mice. miR-433 exhibited a significant repression of MHCC97H cell migration, which was reversed by anti-miR-433. Overexpressing miR-433 inhibited focus formation dramatically, demonstrating that miR-433 may exert a tumor suppressor function. Knockdown of CREB1 by siRNAs impeded MHCC97H cell migration and invasion and antagonized the effect of anti-miR-433. Interestingly, CREB1 siRNA decreased MHCC97H cell proliferation, which was not influenced by anti-miR-433. Overexpressing CREB1 decreased the inhibitory activity of miR-433. The CpG islands surrounding miR-433 were hypermethylated, and the DNA methylation agent 5'-aza-2'-deoxycytidine, but not the histone deacetylase inhibitor trichostatin A, drastically stimulated the expression of miR-433 and miR-127 in HCC cells. The latter is clustered with miR-433. The results reveal a critical role of miR-433 in mediating HCC cell migration via CREB1.

Abstract 077: Small Heterodimer Partner, An Orphan Nuclear Receptor, Inhibits Cardiac Hypertrophy

Background: Small heterodimer partner (SHP; NR0B2) is an atypical orphan nuclear receptor lacking DNA binding domain. SHP directly modulates the activities of other nuclear receptors and regulates a variety of cellular events such as cell differentiation, proliferation, and metabolism in various tissues. However, the role of SHP in heart has not yet been elucidated. Thus, in this study, we tried to investigate the functional roles of SHP in heart physiology and in the development of cardiac hypertrophy. Methods and Results: We observed that SHP knock-out mice elicited cardiac hypertrophic features determined by heart weight to body weight or to tibia length ratios. Fetal genes, such as atrial natriuretic factor (ANF) or beta myosin heavy chain (βMHC) were significantly up-regulated in SHP knockout mice heart. In neonatal rat ventricular cardiomyocytes (NRVCs), phenylephrine (PE) reduced promoter activation of SHP and decreased protein level of SHP. Adenovirus-mediated over-expression of SHP (Adeno-SHP) significantly reduced hypertrophic responses induced by PE as assayed by [3H]-leucine incorporation, Nppa promoter activity, and cell size measurement. Adeno-SHP significantly reduced hypertrophy-associated proteins. In contrast, knock-down of SHP by small hairpin RNA, decreased both Myh7 and Nppa promoter activities, whereas it up-regulated ANF or α-tubulin expressions. Metfomin (N,N-Dimethylimidodicarbonimidic diamide), an anti-diabetic agent, up-regulated SHP in dose-response fashion. PE-induced activation of Nppa promoter and [3H]-leucine incorporation were completely blocked by metformin. PE-induced down-regulation of SHP was blunted by simultaneous treatment of metformin. Metformin-mediated antihypertrophic action was not observed when the SHP was down-regulated by small interfering RNA against to SHP. Conclusions: These results suggest that atypical orphan nuclear receptor SHP prevents cardiac hypertrophy and it mediates metformin-mediated antihypertrophic responses, implicating that theses signal cascades may serve as a novel therapeutic target of treatment of hypertrophic cardiomyopathy patients.

Characterization of the Mitochondrial Localization of the Nuclear Receptor SHP and Regulation of Its Subcellular Distribution by Interaction with Bcl2 and HNF4α

The nuclear receptor small heterodimer partner SHP was shown recently to translocate to the mitochondria, interact with Bcl2, and induce apoptosis in liver cancer cells. However, the exact mitochondrial localization of SHP remains to be determined. In addition, the detailed interaction domains between SHP and Bcl2 have not been characterized. Using biochemistry and molecular biology approaches, we demonstrate that SHP is localized to the mitochondrial outer membrane. Interestingly, compared with the full-length SHP, the N-terminal deleted protein exhibits increased expression in the mitochondria and decreased expression in the nucleus. GST pull-down assays demonstrate that the interaction domain of SHP shows the strongest interaction with Bcl2. Furthermore, the interaction of Bcl2 with SHP is completely abolished by deletion of the Bcl2 transmembrane domain (TM), whereas deletion of the Bcl2 BH1 domain enhances the interaction. As expected, AHPN, a synthetic SHP ligand, markedly augments the direct protein-protein interaction between Bcl2 and SHP. Ectopic expression of hepatocyte nuclear factor 4 alpha (HNF4α) results in exclusive nuclear translocation of SHP proteins that contain either the full-length or the N-terminal domain, but has a minimal effect on the subcellular distribution of SHP protein containing only the interaction domain or repression domain. These results indicate that the N-terminal domain of SHP is important for itsnuclear translocation via HNF4α. Overall, this study provides novel insights into the domains of SHP that are critical for its shutting between different subcellular compartments.

Genome-wide transcriptome analysis identifies novel gene signatures implicated in human chronic liver disease

The molecular mechanisms behind human liver disease progression to cirrhosis remain elusive. Nuclear receptor small heterodimer partner (SHP/Nr0b2) is a hepatic tumor suppressor and a critical regulator of liver function. SHP expression is diminished in human cirrhotic livers, suggesting a regulatory role in human liver diseases. The goal of this study was to identify novel SHP-regulated genes that are involved in the development and progression of chronic liver disease. To achieve this, we conducted the first comprehensive RNA sequencing (RNA-seq) analysis of Shp(-/-) mice, compared the results with human hepatitis C cirrhosis RNA-seq and nonalcoholic steatohepatitis (NASH) microarray datasets, and verified novel results in human liver biospecimens. This approach revealed new gene signatures associated with chronic liver disease and regulated by SHP. Several genes were selected for validation of physiological relevance based on their marked upregulation, novelty with regard to liver function, and involvement in gene pathways related to liver disease. These genes include peptidoglycan recognition protein 2, dual specific phosphatase-4, tetraspanin 4, thrombospondin 1, and SPARC-related modular calcium binding protein-2, which were validated by qPCR analysis of 126 human liver specimens, including steatosis, fibrosis, and NASH, alcohol and hepatitis C cirrhosis, and in mouse models of liver inflammation and injury. This RNA-seq analysis identifies new genes that are regulated by the nuclear receptor SHP and implicated in the molecular pathogenesis of human chronic liver diseases. The results provide valuable transcriptome information for characterizing mechanisms of these diseases.

Nuclear Receptors HNF4α and LRH-1 Cooperate in Regulating Cyp7a1 in Vivo

Fibroblast growth factor 19 (FGF19) is a postprandial enterokine induced by the nuclear bile acid receptor, FXR, in ileum. FGF19 inhibits bile acid synthesis in liver through transcriptional repression of cholesterol 7α-hydroxylase (CYP7A1) via a mechanism involving the nuclear receptor SHP. Here, in a series of loss-of-function studies, we show that the nuclear receptors HNF4α and LRH-1 have dual roles in regulating Cyp7a1 in vivo. First, they cooperate in maintaining basal Cyp7a1 expression. Second, they enable SHP binding to the Cyp7a1 promoter and facilitate FGF19-mediated repression of bile acid synthesis. HNF4α and LRH-1 promote active transcription histone marks on the Cyp7a1 promoter that are reversed by FGF19 in a SHP-dependent manner. These findings demonstrate that both HNF4α and LRH-1 are important regulators of Cyp7a1 transcription in vivo.

Cross-Regulation of Protein Stability by p53 and Nuclear Receptor SHP

We report here a novel interplay between tumor suppressor p53 and nuclear receptor SHP that controls p53 and SHP stability. Overexpression of p53 causes rapid SHP protein degradation, which does not require the presence of Mdm2 and is mediated by the proteosome pathway. Overexpressing SHP alone does not affect p53 stability. However, SHP destabilizes p53 by augmentation of Mdm2 ubiquitin ligase activity toward p53. The single amino acid substitution in the SHP protein SHPK170R increases SHP binding to p53 relative to SHP wild-type, whereas SHPG171A variant shows a diminished p53 binding. As a result of the cross-regulation, the tumor suppressor function of p53 and SHP in inhibition of colon cancer growth is compromised. Our findings reveal a unique scenario for a cross-inhibition between two tumor suppressors to keep their expression and function in check.

E2F1 is a potential novel regulator of liver fibrosis by targeting Egr‐1 through nuclear receptor SHP

ObjectiveEgr‐1 is a critical regulator of liver fibrosis. However, the transcriptional network that governs Egr‐1 expression remains largely unknown. This study aimed at identifying novel transcriptional factors controlling Egr‐1 expression and liver fibrosis.MethodsLuciferase reporter and ChIP assays were used to elucidate the mechanisms. Mouse models of liver fibrosis induced by bile‐duct ligation (BDL), CCl4, or 3,5‐diethoxycarbonyl‐1,4‐dihydrocollidine were used as in vivo models.ResultsThe SHP‐/‐ mice showed increased fibrosis in response to BDL, which was reversed in hepatocyte SHP‐overexpressed mice. The Egr‐1 promoter was activated by E2F1, and the activation was abrogated by co‐expression of SHP and EID1 in both human hepatoma Huh7 and stellate cells LX2. ChIP assays confirmed the association of E2F1 and SHP with the Egr‐1 promoter, and GST pull‐down confirmed a direct interaction between E2F1 and SHP. Interestingly, the loss of E2F1 had no effect in CCl4‐intoxicated mice, but significantly decreased liver fibrosis of the biliary type. Importantly, E2F1 and Egr‐1 expression were dramatically increased in human cirrhotic liver, which was inversely correlated with diminished SHP expression.ConclusionsEgr‐1 expression is regulated by a transcription network involving E2F1 and SHP. E2F1 may function as a novel fibrogenic gene. Supported by T32CA092347 (Y.Z) and DK080440 (L.W).

An autoregulatory feedback loop between nuclear receptor SHP and Mdm2 that controls Mdm2, SHP and p53 protein stability

MethodsImmunoprecipitation, Western blots, in vitro ubiquitination assays, luciferase reporter assays, qPCR, ChIP assays, and apoptosis assays etc.ResultsMdm2 attenuates SHP expression by promoting SHP protein ubiquitination and downregulating SHP mRNA, and SHP in turn stabilizes Mdm2 protein by abrogating Mdm2 selfubiquitination. p53 reduces SHP protein through proteasome, but not phosphorylation and acetylation, mediated SHP degradation by Mdm2, and inhibits SHP mRNA by transcriptional repression. SHP destabilizes p53 by augmentation of Mdm2 ubiquitin ligase activity toward p53. Such cross‐regulation critically depends on the physical interaction of SHP with Mdm2 through the SHPK170 residue. Consequently, SHP antagonizes p53 dependent induction of apoptosis and inhibition of tumor growth by AHPN and 3‐Cl‐AHPC in colon cancer cells.ConclusionsThe Mdm2‐SHP‐p53 interplay represents a novel component of Mdm2/p53 crosstalk that dictates p53 activity and function.This work was supported by the National Institutes of Health (DK080440) to L.W.

Zinc-induced Dnmt1 expression involves antagonism between MTF-1 and nuclear receptor SHP.

Dnmt1 is frequently overexpressed in cancers, which contributes significantly to cancer-associated epigenetic silencing of tumor suppressor genes. However, the mechanism of Dnmt1 overexpression remains elusive. Herein, we elucidate a pathway through which nuclear receptor SHP inhibits zinc-dependent induction of Dnmt1 by antagonizing metal-responsive transcription factor-1 (MTF-1). Zinc treatment induces Dnmt1 transcription by increasing the occupancy of MTF-1 on the Dnmt1 promoter while decreasing SHP expression. SHP in turn represses MTF-1 expression and abolishes zinc-mediated changes in the chromatin configuration of the Dnmt1 promoter. Dnmt1 expression is increased in SHP-knockout (sko) mice but decreased in SHP-transgenic (stg) mice. In human hepatocellular carcinoma (HCC), increased DNMT1 expression is negatively correlated with SHP levels. Our study provides a molecular explanation for increased Dnmt1 expression in HCC and highlights SHP as a potential therapeutic target.

Mdm2 is a novel activator of ApoCIII promoter which is antagonized by p53 and SHP inhibition

We examined the effect of Mdm2 on regulation of the ApoCIII promoter and its cross-talk with p53 and nuclear receptor SHP. Overexpression of Mdm2 markedly enhanced ApoCIII promoter activity by HNF4α. A direct association of Mdm2 protein with the HNF4α protein was observed by co-immunoprecipitation. Ectopic expression of p53 decreased HNF4α activation of the ApoCIII promoter and antagonized the effect of Mdm2. Co-expression of SHP further strengthened p53 inhibition and abolished Mdm2 activation of the ApoCIII promoter. Mdm2 inhibited p53-mediated enrichment of HNF4α to the ApoCIII promoter while simultaneously reducing p53 binding and increasing recruitment of SHP to the ApoCIII promoter. The results from this study implicate a potentially important function of Mdm2 in regulation of lipoprotein metabolism.

NUCLEAR RECEPTORS

NUCLEAR RECEPTORS

SHP works a double shift to control TLR signaling

Innate immune responses need to be tightly controlled to avoid autoimmune and inflammatory diseases. The atypical orphan nuclear receptor SHP has now been identified as a negative regulator of Toll-like receptor–induced activation of the transcription factor NF-κB.

Hypoxia induces expression of COX-2 through the homeodomain transcription factor CDX1 and orphan nuclear receptor SHP in human endometrial cells

Endometriosis, the presence of ectopic endometrial tissue outside the uterine cavity, is a common disease affecting women during their reproductive years. The aim of this study was to identify the molecular mechanism of transcriptional regulation of inflammatory cyclooxygenase-2 (COX-2) gene during endometriosis by hypoxia. Hypoxia induced COX-2 expression in endometrial cells together with the induction of the orphan nuclear receptor SHP and intestinal-specific transcription factor Caudal-related transcription factor 1 (CDX1). Hypoxia-inducible factor (HIF)-1α was responsible for SHP induction mediated by a hypoxia. In addition, we observed that ectopic expression of CDX1 enhanced COX-2 gene expression in hypoxia-dependent fashion. Additionally, we evaluated that induction of CDX1 by hypoxia was mediated by SHP. Expression of COX-2, CDX1, SHP and HIF-1α mRNA in hypoxia-treated human endometrial cells were significantly higher than normal control cells. These results suggest that the SHP and CDX1 expression increased by hypoxia play an active role in inducing inflammatory COX-2 expression in the pathogenesis of endometriosis.

Nuclear receptor SHP inhibition of Dnmt1 expression via ERRγ

We describe a transcriptional mechanism regulating the expression of Dnmt1 by nuclear receptors. We show that ERRγ functions as a transcriptional activator of mouse and human Dnmt1 expression by direct binding to its response elements (ERE1/ERE2) in the dnmt1/DNMT1 promoters. The induction of Dnmt1 by ERRγ is repressed by SHP through SHP inhibition of ERRγ transactivity, diminishing ERRγ recruitment to the Dnmt1 promoter, and altering the conformation of local chromatin from an active mode by ERRγ to an inactive mode. Our study provides the first evidence for nuclear receptor mediated regulation of Dnmt1 expression through ERRγ and SHP crosstalk. Structured summary of protein interactions ERR gamma physically interacts with SHP by anti tag coimmunoprecipitation ( View interaction).

Nuclear Receptor SHP as a Potential Therapeutic Target for Liver Cancer

Small heterodimer partner (SHP, NR0B2) is a nuclear receptor that exerts influence over the expression of several nuclear receptors and transcription factors. We have previously shown that SHP is involved in several metabolic and cellular processes. Of particular interest, SHP appears to play a crucial role as a tumor suppressor. We have recently published work demonstrating that SHP knockout (SHP-/-) mice spontaneously develop hepatocellular carcinoma. We have discovered that SHP is a potent inhibitor of cellular proliferation, and that activation of SHP induces apoptosis and inhibits tumor growth. In collateral studies, we have also found that SHP is a key regulator of microRNA gene transcription and through this process can activate apoptosis. Furthermore, we have also shown that SHP expression can be influenced by the degree of methylation. The ability to modulate key cellular processes, particularly cellular proliferation and apoptosis, has heightened interest in the ability of this protein to function as a therapeutic target in hepatomas, as well as other neoplasms. Future research will also examine the sensitivity and specificity of SHP to function as a biomarker for hepatocellular carcinoma. Keywords: Nuclear receptor, gene transcription, cell cycle, apoptosis, liver cancer.

Novel Polymorphisms of Nuclear Receptor SHP Associated with Functional and Structural Changes

We identified three heterozygous nonsynonymous single nucleotide polymorphisms in the small heterodimer partner (SHP, NROB2) gene in normal subjects and CADASIL (cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy)-like patients, including two novel missense mutations (p.R38H, p.K170N) and one of the previously reported polymorphism (p.G171A). Four novel heterozygous mutations were also identified in the intron ((Intron)1265T-->A), 3'-untranslated region ((3'-UTR)101C-->G, (3'-UTR)186T-->C), and promoter ((Pro)-423C-->T) of the SHP gene. The exonic R38H and K170N mutants exhibited impaired nuclear translocation. K170N made SHP more susceptible to ubiquitination mediated degradation and blocked SHP acetylation, which displayed lost repressive activity on its interacting partners ERRgamma and HNF4alpha but not LRH-1. In contrast, G171A increased SHP mRNA and protein expression and maintained normal function. In general, the interaction of SHP mutants with LRH-1 and EID1 was enhanced. K170N also markedly impaired the recruitment of SHP, HNF4alpha, HDAC1, and HDAC3 to the apoCIII promoter. Molecular dynamics simulations of SHP showed that G171A stabilized the nuclear receptor boxes, whereas K170N promoted the conformational destabilization of all the structural elements of the receptor. This study suggests that genetic variations in SHP are common among human subjects and the Lys-170 residue plays a key role in controlling SHP ubiquitination and acetylation associated with SHP protein stability and repressive function.

Targeting orphan nuclear receptor SHP in the treatment of metabolic diseases

Importance of the field: The orphan nuclear receptor small heterodimer partner (SHP; NR0B2) is an atypical nuclear receptor that contains a ligand-binding domain, but lacks the conserved DNA-binding domain. Since its discovery, SHP has been identified as a key transcriptional regulatory factor of genes involved in diverse metabolic pathways.Areas covered in this review: We performed a Medline/Pubmed search to find published studies on the role of SHP in the regulation of metabolism in the liver, pancreatic islets, blood vessel, and kidney and on the feasibility of using SHP as a therapeutic target in metabolic disease.What the reader will gain: In this review, we discuss the function of SHP as regulator of cholesterol, lipid and glucose metabolism, and the role of SHP in metabolic and fibrotic diseases.Take home message: The reviewed studies suggested that SHP could be a major target for therapeutic intervention in metabolic and fibrotic diseases, including metabolic syndrome and its complications. However, for SHP-targeted therapy, there are some outstanding issues, including the small number of known inducers of SHP and the lack of sufficient data in humans.