Enhancement of the SESN2-SHP cascade by melatonin ameliorates hepatic gluconeogenesis by inhibiting the CRBN-BTG2-CREBH signaling pathway.

Melatonin is involved in the regulation of various biological functions. Here, we explored a novel molecular mechanism by which the melatonin-induced sestrin2 (SESN2)-small heterodimer partner (SHP) signaling pathway protects against fasting- and diabetes-mediated hepatic glucose metabolism. Various key gene expression analyses were performed and multiple metabolic changes were assessed in liver specimens and primary hepatocytes of mice and human participants. The expression of the hepatic cereblon (CRBN) and b-cell translocation gene 2 (BTG2) genes was significantly increased in fasting mice, diabetic mice, and patients with diabetes. Overexpression of Crbn and Btg2 increased hepatic gluconeogenesis by enhancing cyclic adenosine monophosphate (cAMP)-responsive element-binding protein H (CREBH), whereas this phenomenon was prominently ablated in Crbn null mice and Btg2-silenced mice. Interestingly, melatonin-induced SESN2 and SHP markedly reduced hepatic glucose metabolism in diabetic mice and primary hepatocytes, and this protective effect of melatonin was strikingly reversed by silencing Sesn2 and Shp. Finally, the melatonin-induced SESN2-SHP signaling pathway inhibited CRBN- and BTG2-mediated hepatic gluconeogenic gene transcription via the competition of BTG2 and the interaction of CREBH. Mitigation of the CRBN-BTG2-CREBH axis by the melatonin-SESN2-SHP signaling network may provide a novel therapeutic strategy to treat metabolic dysfunction due to diabetes.

Growth hormone promotes hepatic gluconeogenesis by enhancing BTG2-YY1 signaling pathway.

Growth hormone (GH) is one of the critical factors in maintaining glucose metabolism. B-cell translocation gene 2 (BTG2) and yin yang 1 (YY1) are key regulators of diverse metabolic processes. In this study, we investigated the link between GH and BTG2-YY1 signaling pathway in glucose metabolism. GH treatment elevated the expression of hepatic Btg2 and Yy1 in primary mouse hepatocytes and mouse livers. Glucose production in primary mouse hepatocytes and serum blood glucose levels were increased during GH exposure. Overexpression of hepatic Btg2 and Yy1 induced key gluconeogenic enzymes phosphoenolpyruvate carboxykinase 1 (PCK1) and glucose-6 phosphatase (G6PC) as well as glucose production in primary mouse hepatocytes, whereas this phenomenon was markedly diminished by knockdown of Btg2 and Yy1. Here, we identified the YY1-binding site on the Pck1 and G6pc gene promoters using reporter assays and point mutation analysis. The regulation of hepatic gluconeogenic genes induced by GH treatment was clearly linked with YY1 recruitment on gluconeogenic gene promoters. Overall, this study demonstrates that BTG2 and YY1 are novel regulators of GH-dependent regulation of hepatic gluconeogenic genes and glucose production. BTG2 and YY1 may be crucial therapeutic targets to intervene in metabolic dysfunction in response to the GH-dependent signaling pathway.

Gluconeogenic signals regulate hepcidin gene expression via a CRBN-KLF15 axis.

Hepcidin (HAMP) is synthesized in the liver. It is a key ironregulatory hormone that controls systemic iron homeostasis. Cereblon (CRBN) and Kruppel-like factor 15 (KLF15) are known to regulate diverse physiological functions. In this study, we investigated the role of CRBN on hepatic hepcidin gene expression and production under gluconeogenic stimuli. Fasted mice as well as forskolin (FSK)- and glucagon (GLU)-treated mice had reduced serum iron levels but increased expression levels of hepatic Crbn and Klf15 and hepcidin secretion. MicroRNA (miRNA) expression analysis of fasted and Ad-Crbninfected mice revealed significant reduction of microRNA-639 (miR-639). Hepatic overexpression of Crbn elevated hepcidin expression and production along with Klf15 gene expression, whereas knockdown of Crbn and Klf15 markedly decreased FSK- and fasting-mediated induction of hepcidin gene expression and its biosynthesis in mouse livers and primary hepatocytes. Moreover, expression of KLF15 significantly increased the activity of hepcidin reporter gene. It was exclusively dependent on the KLF15-binding site identified within the hepcidin gene promoter. Overall, this study demonstrates that CRBN and KLF15 are novel mediators of gluconeogenic signal-induced hepcidin gene expression and production. Thus, CRBN and KLF15 might be novel potential therapeutic targets to intervene metabolic dysfunction. [BMB Reports 2021; 54(4): 221-226].

Monocytes enhance the inflammatory response to TLR2 stimulation in aortic valve interstitial cells through paracrine up-regulation of TLR2 level.

Background and Objectives: Chronic valvular inflammation associated with monocyte infiltration promotes calcific aortic valve disease (CAVD) progression. Further, innate immunity in aortic valve interstitial cells (AVICs), mediated by Toll-like receptors (TLRs), up-regulates cellular inflammatory, fibrogenic and osteogenic activities. Currently, the pro-inflammatory communication between monocytes and AVICs and the underlying mechanism are unclear. We hypothesized that monocytes up-regulate AVIC inflammatory activity. This study sought to characterize the interaction between monocytes and AVICs and to elucidate the mechanism underlying cell-to-cell communication. Methods and Results: AVICs, monocytes and co-cultures were exposed to a low concentration of TLR2 activator Pam3CSK4 (0.03 µg/ml). The TLR2 activator at this dose induced a marked increase in AVIC production of ICAM-1 and VCAM-1 only when co-cultured with monocytes. Adding conditioned medium from Pam3CSK4-treated monocytes (Pam3 CM, containing 0.1 µg/ml of Pam3CSK4) to AVIC culture (30% vol/vol; diluting Pam3CSK4 to 0.03 µg/ml) greatly increased the expression of adhesion molecules while adding conditioned medium from untreated monocytes (control CM) had no effect. Inhibition or knockdown of TLR2 in AVICs markedly reduced ICAM-1 and VCAM-1 expression induced by Pam3 CM. Further, Pam3 CM increased TLR2 levels in AVICs. Multiplex-ELISA analysis of Pam3 CM identified greater levels of TNF-α. Neutralization of TNF-α abolished the effect of Pam3 CM on AVIC TLR2 levels, resulting in marked attenuation of its potency in the induction of adhesion molecule expression. Conclusions: This study demonstrates that activated monocytes use paracrine signaling to sensitize AVICs for inflammatory responses to a low level of TLR2 activator. The mechanism of sensitization involves up-regulation of AVIC TLR2 levels by TNF-α from monocytes. Infiltrated monocytes in aortic valve tissue may exacerbate valvular inflammation by rendering AVICs hypersensitive to TLR2 activators.

Induction of SIRT1 by melatonin improves alcohol-mediated oxidative liver injury by disrupting the CRBN-YY1-CYP2E1 signaling pathway.

Alcoholic liver disease is the most prevalent chronic liver disease. Melatonin is known to control many vital processes. Here, we explored a novel molecular mechanism by which melatonin-induced SIRT1 signaling protects against alcohol-mediated oxidative stress and liver injury. Gene expression profiles and metabolic changes were measured in liver specimens of mice and human subjects. Expression levels of Cb1r, Crbn, Btg2, Yy1, pro-inflammatory cytokines, and Cyp2e1 were significantly enhanced in chronic alcohol-challenged mice and human subjects. Levels of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), hepatic CYP2E1 protein, and reactive oxygen species (ROS) were elevated in alcohol-fed WT mice but not in Cb1r antagonist-treated, Crbn null, or Yy1-silenced mice. Importantly, alcohol-induced Yy1 and Cyp2e1 expression, ROS amount, and liver injury were markedly diminished by melatonin treatment and the transduction of Sirt1 in mice, whereas this phenomenon was prominently ablated by silencing of Sirt1. Notably, SIRT1 physically interacted with YY1 and attenuated YY1 occupancy on the Cyp2e1 gene promoter. Melatonin-SIRT1 signaling ameliorates alcohol-induced oxidative liver injury by disrupting the CRBN-YY1-CYP2E1 signaling pathway. The manipulation of CRBN-YY1-CYP2E1 signaling network by the melatonin-SIRT1 pathway highlights a novel entry point for treating alcoholic liver disease.

Author Correction: B-cell translocation gene 2 enhances fibroblast growth factor 21 production by inducing Kruppel-like factor 15.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Differential neurodegenerative phenotypes are associated with heterogeneous voiding dysfunction in a coronavirus-induced model of multiple sclerosis.

Patients with multiple sclerosis (MS) develop a variety of lower urinary tract symptoms (LUTS). We previously characterized a murine model of neurogenic bladder dysfunction induced by a neurotropic strain of a coronavirus. In the present study, we further study the role of long-lasting neurodegeneration on the development of neurogenic bladder dysfunction in mice with corona-virus induced encephalitis (CIE). Long-term follow up study revealed three phenotypes of neurodegenerative symptom development: recovery (REC group), chronic progression (C-PRO group) and chronic disease with relapsing-remitting episodes (C-RELAP group). The levels of IL-1ß in REC group, IL-10 in C-RELAP group, and IL-1ß, IL-6, IL-10 and TNF-α in C-PRO group were diminished in the brain. The levels of TNF-α in REC group and INF-γ, IL-2, TGF-ß and TNF-α in the C-PRO group were also diminished in the urinary bladder. Mice in C-RELAP group showed a delayed recovery of voiding function. In vitro contractility studies determined a decreased basal detrusor tone and reduced amplitude of nerve-mediated contractions in C-RELAP group, whereas C-PRO group had elevated muscle-mediated contractions. In conclusion, mice with CIE developed three phenotypes of neurologic impairment mimicking different types of MS progression in humans and showed differential mechanisms driving neurogenic bladder dysfunction.

B-cell translocation gene 2 enhances fibroblast growth factor 21 production by inducing Kruppel-like factor 15.

Fibroblast growth factor 21 (FGF21) is a hormone that is vital for the regulation of metabolic homeostasis. In the present study, we report that Kruppel-like factor 15 (KLF15) is a novel mediator of b-cell translocation gene 2 (BTG2)-induced FGF21 biosynthesis. The expression levels of hepatic Fgf21, Btg2, and Klf15, and the production of serum FGF21 increased significantly in fasted and forskolin (FSK)-treated mice. The overexpression of Btg2 using an adenoviral delivery system elevated FGF21 production by upregulating Klf15 transcription. Interaction studies indicated that BTG2 was co-immunoprecipitated with KLF15 and recruited by the Fgf21 promoter. The disruption of hepatic Btg2 and Klf15 genes markedly attenuated the induction of Fgf21 expression and FGF21 biosynthesis in fasted mice. Similarly, the FSK-mediated induction of Fgf21 promoter activity was strikingly ablated by silencing of Btg2 and Klf15. Taken together, these findings suggest that KLF15 and BTG2 are mediators of fasting-induced hepatic FGF21 expression. Therefore, targeting BTG2 and KLF15 might be a therapeutically important strategy for combat metabolic dysfunction.

Association of genetic polymorphisms in the pore domains of mechano-gated TREK-1 channel with overactive lower urinary tract symptoms in humans.

AIMS: Mechanosensitivity of the urinary bladder is regulated by many factors including mechano-gated two-pore domain (K2 P, KCNK) potassium channels. TWIK-related K+ channel, TREK-1, is a predominantly expressed member of K2 P channel family in the human detrusor, and its expression and function are diminished in patients with overactive lower urinary tract symptoms (LUTS). The changes in channel activity may result from spontaneously occurring gene mutations. The aim of this study was to compare single nucleotide polymorphisms (SNPs) in TREK-1 channel between patients with LUTS and healthy donors. METHODS: Six SNPs (rs370266806, rs373919966, rs758937019, rs769301539, rs772497750, and rs775158737) in two pore domains of human TREK-1 gene were analyzed using TaqMan SNP genotyping assay with manufacturer-designed primers and allele-specific probes. The screening was done in control bladders and detrusor specimens from patients with overactive LUTS. Statistical analyses were performed using R, Fisher's exact test and Hardy-Weinberg Equilibrium. RESULTS: Six SNPs in two pore domains of the human TREK-1 gene were analyzed in human bladder specimens. The frequencies of rs758937019-CT genotype (P = 0.0016) and rs758937019-T allele (P = 0.0022) were significantly higher in the group with overactive LUTS. There was no significant association of rs775158737-GA genotype and rs775158737-A allele with the overactive LUTS, though they were present only in the overactive LUTS group. CONCLUSIONS: Our results provide evidence that altered expression and function of TREK-1 channel in patients with overactive LUTS could be due to genetic polymorphisms in the pore domains of TREK-1 channel (rs758937019).

Impact of Regular Cannabis Use on Biomarkers of Lower Urinary Tract Function.

OBJECTIVE: To evaluate the differences in the composition and quantities of urine peptides in regular cannabis users and nonusers by liquid chromatography tandem mass spectrometry analysis. MATERIALS AND METHODS: Urine specimens from healthy control subjects and cannabis users were utilized to identify the differences in the number and quantity of urine proteins by liquid chromatography tandem mass spectrometry analysis. Significantly altered proteins were determined by a permutation testing statistical method. Heat map, dendrogram, pathway, and network analyses were performed to assess the degree of expression and the potential relationships between proteins in both groups. RESULTS: A total of 1337 proteins were detected in both groups with 19 proteins being significantly altered in cannabis users. Innate immunity and carbohydrate metabolic pathways were highly linked with upregulated proteins in the cannabis group. Additionally, 91 proteins were present and 46 proteins were absent only in cannabis users in comparison with the control cohort. Our results suggest that regular use of cannabis is associated with significant alterations in a number of urinary peptides, with a large number of proteins present or absent only in cannabis users. Pathway analyses demonstrated an increased immune response in cannabis users compared with controls. CONCLUSION: Our observations potentially indicate activation (or inhibition) of specific signaling pathways in the lower urinary tract during chronic exposure to exogenous cannabinoids. Our study provides initial proteomic knowledge for future investigations on the potential role of exocannabinoids in the development of intravesical therapies to treat lower urinary tract disorders.

Altered expression and modulation of the two-pore-domain (K2P) mechanogated potassium channel TREK-1 in overactive human detrusor.

Detrusor overactivity (DO) is the abnormal response of the urinary bladder to physiological stretch during the filling phase of the micturition cycle. The mechanisms of bladder smooth muscle compliance upon the wall stretch are poorly understood. We previously reported that the function of normal detrusor is regulated by TREK-1, a member of the mechanogated subfamily of two-pore-domain potassium (K2P) channels. In the present study, we aimed to identify the changes in expression and function of TREK-1 channels under pathological conditions associated with DO, evaluate the potential relationship between TREK-1 channels and cytoskeletal proteins in the human bladder, and test the possibility of modulation of TREK-1 channel expression by small RNAs. Expression of TREK-1 channels in DO specimens was 2.7-fold decreased compared with control bladders and was associated with a significant reduction of the recorded TREK-1 currents. Isolated DO muscle strips failed to relax when exposed to a TREK-1 channel opener. Immunocytochemical labeling revealed close association of TREK-1 channels with cell cytoskeletal proteins and caveolins, with caveolae microdomains being severely disrupted in DO specimens. Small activating RNA (saRNA) tested in vitro provided evidence that expression of TREK-1 protein could be partially upregulated. Our data confirmed a significant downregulation of TREK-1 expression in human DO specimens and provided evidence of close association between the channel, cell cytoskeleton, and caveolins. Upregulation of TREK-1 expression by saRNA could be a future step for the development of in vivo pharmacological and genetic approaches to treat DO in humans.

Melatonin ameliorates alcohol-induced bile acid synthesis by enhancing miR-497 expression.

Alcoholic liver disease is a major cause of chronic liver disease worldwide, and cannabinoid receptor type 1 (CB1R) is involved in a diverse metabolic diseases. B-cell translocation gene 2 (BTG2) and yin yang 1 (YY1) are a potent regulator of biological conditions. Melatonin plays a crucial role in regulating diverse physiological functions and metabolic homeostasis. MicroRNAs are key regulators of various biological processes. Herein, we demonstrate that melatonin improves bile acid synthesis in the liver of alcohol-fed mice by controlling miR-497 expression. The level of bile acid and the expression of Cb1r, Btg2, Yy1, and bile acid synthetic enzymes were significantly elevated in the livers of Lieber-DeCarli alcohol-fed mice. The overexpression of Btg2 enhanced Yy1 gene expression and bile acid production, whereas disrupting the CB1R-BTG2-YY1 cascade protected against the bile acid synthesis caused by alcohol challenge. We identified an alcohol-mediated YY1 binding site on the cholesterol 7α-hydroxylase (Cyp7a1) gene promoter using promoter deletion analysis and chromatin immunoprecipitation assays. Notably, melatonin attenuated the alcohol-stimulated induction of Btg2, Yy1 mRNA levels and bile acid production by promoting miR-497. Overexpression of a miR-497 mimic dramatically diminished the increase of Btg2 and Yy1 gene expression as well as bile acid production by alcohol, whereas this phenomenon was reversed by miR-497 inhibitor. These results demonstrate that the upregulation of miR-497 by melatonin represses alcohol-induced bile acid synthesis by attenuating the BTG2-YY1 signaling pathway. The melatonin-miR497 signaling network may provide novel therapeutic targets for the treatment of hepatic metabolic dysfunction caused by the alcohol-dependent pathway.

Phosphoenolpyruvate carboxykinase and glucose-6-phosphatase are required for steroidogenesis in testicular Leydig cells.

Cyclic AMP (cAMP) induces steroidogenic enzyme gene expression and stimulates testosterone production in Leydig cells. Phosphoenolpyruvate carboxykinase (PEPCK) is expressed in Leydig cells, but its role has not been defined. In this study, we found that PEPCK and glucose-6-phosphatase (Glc-6-Pase) are increased significantly following cAMP treatment of mouse Leydig cells. Moreover, cAMP treatment increased recruitment of the cAMP-response element-binding transcription factor and decreased recruitment of the corepressor DAX-1 on the pepck promoter. Furthermore, cAMP induced an increase in ATP that correlated with a decrease in phospho-AMP-activated protein kinase (AMPK). In contrast, knockdown or inhibition of PEPCK decreased ATP and increased phospho-AMPK. Treatment with an AMPK activator or overexpression of the constitutively active form of AMPK inhibited cAMP-induced steroidogenic enzyme promoter activities and gene expression. Liver receptor homolog-1 (LRH-1) was involved in cAMP-induced steroidogenic enzyme gene expression but was inhibited by AMPK activation in Leydig cells. Additionally, inhibition or knockdown of PEPCK and Glc-6-Pase decreased cAMP-mediated induction of steroidogenic enzyme gene expression and steroidogenesis. Finally, pubertal mouse (8-week-old) testes and human chorionic gonadotropin-induced prepubertal mouse testes showed increased PEPCK and Glc-6-Pase gene expression. Taken together, these results suggest that induction of PEPCK and Glc-6-Pase by cAMP plays an important role in Leydig cell steroidogenesis.

Orphan nuclear receptor DAX-1 acts as a novel corepressor of liver X receptor alpha and inhibits hepatic lipogenesis.

DAX-1 (dosage-sensitive sex reversal adrenal hypoplasia congenital critical region on X chromosome, gene 1) is a member of the nuclear receptor superfamily that can repress diverse nuclear receptors and has a key role in adreno-gonadal development. Our previous report has demonstrated that DAX-1 can inhibit hepatocyte nuclear factor 4alpha transactivity and negatively regulate gluconeogenic gene expression (Nedumaran, B., Hong, S., Xie, Y. B., Kim, Y. H., Seo, W. Y., Lee, M. W., Lee, C. H., Koo, S. H., and Choi, H. S. (2009) J. Biol. Chem. 284, 27511-27523). Here, we further expand the role of DAX-1 in hepatic energy metabolism. Transfection assays have demonstrated that DAX-1 can inhibit the transcriptional activity of nuclear receptor liver X receptor alpha (LXRalpha). Physical interaction between DAX-1 and LXRalpha was confirmed Immunofluorescent staining in mouse liver shows that LXRalpha and DAX-1 are colocalized in the nucleus. Domain mapping analysis shows that the entire region of DAX-1 is involved in the interaction with the ligand binding domain region of LXRalpha. Competition analyses demonstrate that DAX-1 competes with the coactivator SRC-1 for repressing LXRalpha transactivity. Chromatin immunoprecipitation assay showed that endogenous DAX-1 recruitment on the SREBP-1c gene promoter was decreased in the presence of LXRalpha agonist. Overexpression of DAX-1 inhibits T7-induced LXRalpha target gene expression, whereas knockdown of endogenous DAX-1 significantly increases T7-induced LXRalpha target gene expression in HepG2 cells. Finally, overexpression of DAX-1 in mouse liver decreases T7-induced LXRalpha target gene expression, liver triglyceride level, and lipid accumulation. Overall, this study suggests that DAX-1, a novel corepressor of LXRalpha, functions as a negative regulator of lipogenic enzyme gene expression in liver.

DAX-1 acts as a novel corepressor of orphan nuclear receptor HNF4alpha and negatively regulates gluconeogenic enzyme gene expression.

DAX-1 (dosage-sensitive sex reversal adrenal hypoplasia congenital critical region on X chromosome, gene 1) is an atypical member of the nuclear receptor family and acts as a corepressor of a number of nuclear receptors. HNF4alpha (hepatocyte nuclear factor 4alpha) is a liver-enriched transcription factor that controls the expression of a variety of genes involved in cholesterol, fatty acid, and glucose metabolism. Here we show that DAX-1 inhibits transcriptional activity of HNF4alpha and modulates hepatic gluconeogenic gene expression. Hepatic DAX-1 expression is increased by insulin and SIK1 (salt-inducible kinase 1), whereas it is decreased in high fat diet-fed and diabetic mice. Coimmunoprecipitation assay from mouse liver samples depicts that endogenous DAX-1 interacts with HNF4alpha in vivo. In vivo chromatin immunoprecipitation assay affirms that the recruitment of DAX-1 on the phosphoenolpyruvate carboxykinase (PEPCK) gene promoter is inversely correlated with the recruitment of PGC-1alpha and HNF4alpha under fasting and refeeding, showing that DAX-1 could compete with the coactivator PGC-1alpha for binding to HNF4alpha. Adenovirus-mediated expression of DAX-1 decreased both HNF4alpha- and forskolin-mediated gluconeogenic gene expressions. In addition, knockdown of DAX-1 partially reverses the insulin-mediated inhibition of gluconeogenic gene expression in primary hepatocytes. Finally, DAX-1 inhibits PEPCK and glucose-6-phosphatase gene expression and significantly lowers fasting blood glucose level in high fat diet-fed mice, suggesting that DAX-1 can modulate hepatic gluconeogenesis in vivo. Overall, this study demonstrates that DAX-1 acts as a corepressor of HNF4alpha to negatively regulate hepatic gluconeogenic gene expression in liver.

Molecular characterization of SMILE as a novel corepressor of nuclear receptors.

SMILE (small heterodimer partner interacting leucine zipper protein) has been identified as a coregulator in ER signaling. In this study, we have examined the effects of SMILE on other NRs (nuclear receptors). SMILE inhibits GR, CAR and HNF4 alpha-mediated transactivation. Knockdown of SMILE gene expression increases the transactivation of the NRs. SMILE interacts with GR, CAR and HNF4 alpha in vitro and in vivo. SMILE and these NRs colocalize in the nucleus. SMILE binds to the ligand-binding domain or AF2 domain of the NRs. Competitions between SMILE and the coactivators GRIP1 or PGC-1 alpha have been demonstrated in vitro and in vivo. Furthermore, an intrinsic repressive activity of SMILE is observed in Gal4-fusion system, and the intrinsic repressive domain is mapped to the C-terminus of SMILE, spanning residues 203-354. Moreover, SMILE interacts with specific HDACs (histone deacetylases) and SMILE-mediated repression is released by HDAC inhibitor trichostatin A, in a NR-specific manner. Finally, ChIP (chromatin immunoprecipitation) assays reveal that SMILE associates with the NRs on the target gene promoters. Adenoviral overexpression of SMILE represses GR-, CAR- and HNF4 alpha-mediated target gene expression. Overall, these results suggest that SMILE functions as a novel corepressor of NRs via competition with coactivators and the recruitment of HDACs.

SMILE, a new orphan nuclear receptor SHP-interacting protein, regulates SHP-repressed estrogen receptor transactivation.

SHP (small heterodimer partner) is a well-known NR (nuclear receptor) co-regulator. In the present study, we have identified a new SHP-interacting protein, termed SMILE (SHP-interacting leucine zipper protein), which was previously designated as ZF (Zhangfei) via a yeast two-hybrid system. We have determined that the SMILE gene generates two isoforms [SMILE-L (long isoform of SMILE) and SMILE-S (short isoform of SMILE)]. Mutational analysis has demonstrated that the SMILE isoforms arise from the alternative usage of initiation codons. We have confirmed the in vivo interaction and co-localization of the SMILE isoforms and SHP. Domain-mapping analysis indicates that the entire N-terminus of SHP and the middle region of SMILE-L are involved in this interaction. Interestingly, the SMILE isoforms counteract the SHP repressive effect on the transactivation of ERs (estrogen receptors) in HEK-293T cells (human embryonic kidney cells expressing the large T-antigen of simian virus 40), but enhance the SHP-repressive effect in MCF-7, T47D and MDA-MB-435 cells. Knockdown of SMILE gene expression using siRNA (small interfering RNA) in MCF-7 cells increases ER-mediated transcriptional activity. Moreover, adenovirus-mediated overexpression of SMILE and SHP down-regulates estrogen-induced mRNA expression of the critical cell-cycle regulator E2F1. Collectively, these results indicate that SMILE isoforms regulate the inhibition of ER transactivation by SHP in a cell-type-specific manner and act as a novel transcriptional co-regulator in ER signalling.

Bisphenol A bis(2,3-dihydroxypropyl) ether (BADGE.2H2O) induces orphan nuclear receptor Nur77 gene expression and increases steroidogenesis in mouse testicular Leydig cells.

Bisphenol A bis (2,3-dihydroxypropyl) ether (BADGE.2H(2)O) is a component of commercial liquid epoxy resins commonly used in the food-packing industry and in dental sealants. There is evidence that it has significant estrogenic activity. Nur77 plays a crucial role in the regulation of certain genes involved in LH-mediated steroidogenesis in testicular Leydig cells. It was previously demonstrated that Bisphenol A (BPA) stimulates Nur77 gene induction and steroidogenesis. In this study, we investigated the effects of BADGE.2H(2)O on Nur77 gene expression and steroidogenesis. Northern blot analysis showed that it increased the expression of Nur77 mRNA and protein, and transient transfection assays demonstrated that it increased the promoter activity and transactivation of Nur77. It also increased the expression of certain steroidogenic genes, such as StAR and 3 beta-HSD. Finally, over-expression of a dominant negative Nur77 cDNA via adenoviral infection reduced BADGE.2H(2)O-mediated progesterone biosynthesis. These results indicate that BADGE.2H(2)O disrupts testicular steroidogenesis by increasing Nur77 gene expression.

The orphan nuclear receptor DAX-1 acts as a novel transcriptional corepressor of PPARgamma.

DAX-1 is an atypical nuclear receptor (NR) which functions primarily as a transcriptional corepressor of other NRs via heterodimerization. Peroxisome proliferator-activated receptor (PPAR) gamma is a ligand-dependent NR which performs a key function in adipogenesis. In this study, we evaluated a novel cross-talk mechanism between DAX-1 and PPARgamma. Transient transfection assays demonstrated that DAX-1 inhibits the transactivity of PPARgamma in a dose-dependent manner. DAX-1 directly competed with the PPARgamma coactivator (PGC)-1alpha for binding to PPARgamma. Endogenous levels of DAX-1 were significantly lower in differentiated 3T3-L1 adipocytes as compared to preadipocytes. Using a retroviral expression system, we demonstrated that DAX-1 overexpression downregulates the expression of PPARgamma target genes, resulting in an attenuation of adipogenesis in 3T3-L1 cells. Our results suggest that DAX-1 acts as a corepressor of PPARgamma and performs a potential function in the regulation of PPARgamma-mediated cellular differentiation.

Metformin inhibits hepatic gluconeogenesis through AMP-activated protein kinase-dependent regulation of the orphan nuclear receptor SHP.

OBJECTIVE: Metformin is an antidiabetic drug commonly used to treat type 2 diabetes. The aim of the study was to determine whether metformin regulates hepatic gluconeogenesis through the orphan nuclear receptor small heterodimer partner (SHP; NR0B2). RESEARCH DESIGN AND METHODS: We assessed the regulation of hepatic SHP gene expression by Northern blot analysis with metformin and adenovirus containing a constitutive active form of AMP-activated protein kinase (AMPK) (Ad-AMPK) and evaluated SHP, PEPCK, and G6Pase promoter activities via transient transfection assays in hepatocytes. Knockdown of SHP using siRNA SHP was conducted to characterize the metformin-induced inhibition of hepatic gluconeogenic gene expression in hepatocytes, and metformin-and adenovirus SHP (Ad-SHP)-mediated hepatic glucose production was measured in B6-Lep(ob/ob) mice. RESULTS: Hepatic SHP gene expression was induced by metformin, 5-aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside (AICAR), and Ad-AMPK. Metformin-induced SHP gene expression was abolished by adenovirus containing the dominant negative form of AMPK (Ad-DN-AMPK), as well as by compound C. Metformin inhibited hepatocyte nuclear factor-4alpha-or FoxA2-mediated promoter activity of PEPCK and G6Pase, and the inhibition was blocked with siRNA SHP. Additionally, SHP knockdown by adenovirus containing siRNA SHP inhibited metformin-mediated repression of cAMP/dexamethasone-induced hepatic gluconeogenic gene expression. Furthermore, oral administration of metformin increased SHP mRNA levels in B6-Lep(ob/ob) mice. Overexpression of SHP by Ad-SHP decreased blood glucose levels and hepatic gluconeogenic gene expression in B6-Lep(ob/ob) mice. CONCLUSIONS: We have concluded that metformin inhibits hepatic gluconeogenesis through AMPK-dependent regulation of SHP.