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Seung Won Ahn,
Gil-Tae Gang,
Surendar Tadi, Balachandar Nedumaran,
Yong Deuk Kim,
Ji Hoon Park,
Gi Ryang Kweon,
Seung-Hoi Koo,
Keesook Lee,
Ryun-Sup Ahn,
Yong-Hyeon Yim,
Chul-Ho Lee,
Robert A Harris,
Hueng-Sik Choi
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ABSTRACT: 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 (G6Pase) are increased significantly following cAMP treatment of mouse Leydig cells. Moreover, cAMP treatment increased recruitment of the cAMP response element-binding transcription factor (CREB) and decreased recruitment of the corepressor DAX-1 on the pepck promoter. Furthermore, cAMP induced an increase in ATP which correlated with a decrease in phospho-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 (CA-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 G6Pase decreased cAMP-mediated induction of steroidogenic enzyme gene expression and steroidogenesis. Finally, pubertal mice (8-week-old) testis and hCG-induced prepubertal mouse testis showed increased PEPCK and G6Pase gene expression. Taken together, these results suggest that induction of PEPCK and G6Pase by cAMP plays an important role in Leydig cell steroidogenesis.
Journal of Biological Chemistry 10/2012; · 4.77 Impact Factor
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ABSTRACT: 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.
Journal of Biological Chemistry 03/2010; 285(12):9221-32. · 4.77 Impact Factor
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ABSTRACT: 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 4α 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 α (LXRα). Physical interaction between
DAX-1 and LXRα was confirmed Immunofluorescent staining in mouse liver shows that LXRα 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 LXRα. Competition analyses demonstrate that DAX-1 competes with the coactivator SRC-1 for repressing LXRα transactivity.
Chromatin immunoprecipitation assay showed that endogenous DAX-1 recruitment on the SREBP-1c gene promoter was decreased in the presence of LXRα agonist. Overexpression of DAX-1 inhibits T7-induced LXRα target gene
expression, whereas knockdown of endogenous DAX-1 significantly increases T7-induced LXRα target gene expression in HepG2 cells. Finally, overexpression of DAX-1 in mouse liver decreases T7-induced LXRα target gene expression, liver triglyceride level, and lipid accumulation. Overall,
this study suggests that DAX-1, a novel corepressor of LXRα, functions as a negative regulator of lipogenic enzyme gene expression
in liver.
Journal of Biological Chemistry 03/2010; 285(12):9221-9232. · 4.77 Impact Factor
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ABSTRACT: 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. HNF4α (hepatocyte
nuclear factor 4α) 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 HNF4α 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 HNF4α 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-1α and HNF4α under fasting and refeeding, showing that DAX-1
could compete with the coactivator PGC-1α for binding to HNF4α. Adenovirus-mediated expression of DAX-1 decreased both HNF4α-
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 HNF4α to negatively regulate hepatic gluconeogenic
gene expression in liver.
Journal of Biological Chemistry 10/2009; 284(40):27511-27523. · 4.77 Impact Factor
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ABSTRACT: 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.
Journal of Biological Chemistry 09/2009; 284(40):27511-23. · 4.77 Impact Factor
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ABSTRACT: 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.
Nucleic Acids Research 06/2009; 37(12):4100-15. · 8.03 Impact Factor
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ABSTRACT: 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.
Biochemical Journal 12/2008; 416(3):463-73. · 4.90 Impact Factor
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ABSTRACT: 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.
Molecules and Cells 07/2008; 26(1):74-80. · 2.18 Impact Factor
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ABSTRACT: 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.
Biochemical and Biophysical Research Communications 06/2008; 370(2):264-8. · 2.48 Impact Factor
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Yong Deuk Kim,
Keun-Gyu Park,
Yong-Soo Lee,
Yun-Yong Park,
Don-Kyu Kim, Balachandar Nedumaran,
Won Gu Jang,
Won-Jea Cho,
Joohun Ha,
In-Kyu Lee,
Chul-Ho Lee,
Hueng-Sik Choi
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ABSTRACT: 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).
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.
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.
We have concluded that metformin inhibits hepatic gluconeogenesis through AMPK-dependent regulation of SHP.
Diabetes 03/2008; 57(2):306-14. · 8.29 Impact Factor
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ABSTRACT: 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) γ 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 PPARγ. Transient transfection assays demonstrated that DAX-1 inhibits the transactivity of PPARγ in a dose-dependent manner. DAX-1 directly competed with the PPARγ coactivator (PGC)-1α for binding to PPARγ. 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 PPARγ target genes, resulting in an attenuation of adipogenesis in 3T3-L1 cells. Our results suggest that DAX-1 acts as a corepressor of PPARγ and performs a potential function in the regulation of PPARγ-mediated cellular differentiation.
Biochemical and Biophysical Research Communications.
