Short Heterodimer Partner Research Articles

Dietary cholesterol stimulates CYP7A1 in rats because farnesoid X receptor is not activated.

Cholesterol feeding upregulates CYP7A1 in rats but downregulates CYP7A1 in rabbits. To clarify the mechanism responsible for the upregulation of CYP7A1 in cholesterol-fed rats, the effects of dietary cholesterol (Ch) and cholic acid (CA) on the activation of the nuclear receptors, liver X-receptor (LXR-alpha) and farsenoid X-receptor (FXR), which positively and negatively regulate CYP7A1, were investigated in rats. Studies were carried out in four groups (n = 12/group) of male Sprague-Dawley rats fed regular chow (control), 2% Ch, 2% Ch + 1% CA, and 1% CA alone for 1 wk. Changes in mRNA expression of short heterodimer partner (SHP) and bile salt export pump (BSEP), target genes for FXR, were determined to indicate FXR activation, whereas the expression of ABCA1 and lipoprotein lipase (LPL), target genes for LXR-alpha, reflected activation. CYP7A1 mRNA and activity increased twofold and 70%, respectively, in rats fed Ch alone when the bile acid pool size was stable but decreased 43 and 49%, respectively, after CA was added to the Ch diet, which expanded the bile acid pool 3.4-fold. SHP and BSEP mRNA levels did not change after feeding Ch but increased 88 and 37% in rats fed Ch + CA. This indicated that FXR was activated by the expanded bile acid pool. When Ch or Ch + CA were fed, hepatic concentrations of oxysterols, ligands for LXR-alpha increased to activate LXR-alpha, as evidenced by increased mRNA levels of ABCA1 and LPL. Feeding CA alone enlarged the bile acid pool threefold and increased the expression of both SHP and BSEP. These results suggest that LXR-alpha was activated in rats fed both Ch or Ch + CA, whereas CYP7A1 mRNA and activity were induced only in Ch-fed rats where the bile acid pool was not enlarged such that FXR was not activated. In rats fed Ch + CA, the bile acid pool expanded, which activated FXR to offset the stimulatory effects of LXR-alpha on CYP7A1.

Inhibition of aromatase transcription via promoter II by short heterodimer partner in human preadipocytes.

Estrogen synthesis from C19 precursors is catalyzed by aromatase cytochrome P450. Overexpression of aromatase through atypical promoter usage (use of promoter II) in adipose tissue contributes to breast cancer development and progression. One tumor-derived factor that appears to contribute to this process is prostaglandin E2 (PGE2). A factor that regulates aromatase expression downstream of PGE2 is liver receptor homolog-1 (LRH-1). In a study of factors that inhibit LRH-1, we have examined the ability of short heterodimer partner (SHP) to inhibit aromatase transcription mediated by LRH-1 in preadipocytes. RT-PCR analysis indicated that both LRH-1 and SHP are expressed in human preadipocytes. To assess the effect of SHP on aromatase transcription, a transient transfection system was established using 3T3-L1 preadipocytes. Expression of SHP completely inhibited activity of an aromatase promoter II reporter gene induced by LRH-1. The combined treatment of forskolin and phorbol ester (which mimic PGE2) as well as LRH-1, which maximally induced reporter gene expression (140-fold), was also completely inhibited by SHP. This effect of SHP was mediated by inhibition of LRH-1 transcriptional activity, as measured by activity of GAL4-LRH-1 fusion constructs, and by inhibition of LRH-1 binding to promoter II. We conclude that SHP is a potent inhibitor of aromatase transcription in preadipocytes. Modulation of SHP expression and/or activity in adipose tissue may therefore have significant effects on aromatase expression and estrogen production in breast adipose tissue.

Feedback Regulation of Bile Acid Synthesis in Primary Human Hepatocytes: Evidence That Cdca Is the Strongest Inhibitor

Primary human hepatocytes were used to elucidate the effect of individual bile acids on bile acid formation in human liver. Hepatocytes were treated with free as well as glycine–conjugated bile acids. Bile acid formation and messenger RNA (mRNA) levels of key enzymes and the nuclear receptor short heterodimer partner (SHP) were measured after 24 hours. Glycochenodeoxycholic acid (GCDCA; 100 μmol/L) significantly decreased formation of cholic acid (CA) to 44% ± 4% of controls and glycodeoxycholic acid (GDCA) decreased formation of CA to 67% ± 11% of controls. Glycoursodeoxycholic acid (GUDCA; 100 μmol/L) had no effect. GDCA or glycocholic acid (GCA) had no significant effect on chenodeoxycholic acid (CDCA) synthesis. Free bile acids had a similar effect as glycine–conjugated bile acids. Addition of GCDCA, GDCA, and GCA (100 μmol/L) markedly decreased cholesterol 7α–hydroxylase (CYP7A1) mRNA levels to 2% ± 1%, 2% ± 1%, and 29% ± 11% of controls, respectively, whereas GUDCA had no effect. Addition of GDCA and GCDCA (100 μmol/L) significantly decreased sterol 12α–hydroxylase (CYP8B1) mRNA levels to 48% ± 5% and 61% ± 4% of controls, respectively, whereas GCA and GUDCA had no effect. Addition of GCDCA and GDCA (100 μmol/L) significantly decreased sterol 27–hydroxylase (CYP27A1) mRNA levels to 59% ± 3% and 60% ± 7% of controls, respectively, whereas GUDCA and GCA had no significant effect. Addition of both GCDCA and GDCA markedly increased the mRNA levels of SHP to 298% ± 43% and 273% ± 30% of controls, respectively. In conclusion, glycine–conjugated and free bile acids suppress bile acid synthesis and mRNA levels of CYP7A1 in the order CDCA > DCA > CA > UDCA. mRNA levels of CYP8B1 and CYP27A1 are suppressed to a much lower degree than CYP7A1. (Hepatology 2003;38:930–938).

Feedback regulation of bile acid synthesis in primary human hepatocytes: Evidence that CDCA is the strongest inhibitor

Primary human hepatocytes were used to elucidate the effect of individual bile acids on bile acid formation in human liver. Hepatocytes were treated with free as well as glycine-conjugated bile acids. Bile acid formation and messenger RNA (mRNA) levels of key enzymes and the nuclear receptor short heterodimer partner (SHP) were measured after 24 hours. Glycochenodeoxycholic acid (GCDCA; 100 μmol/L) significantly decreased formation of cholic acid (CA) to 44% ± 4% of controls and glycodeoxycholic acid (GDCA) decreased formation of CA to 67% ± 11% of controls. Glycoursodeoxycholic acid (GUDCA; 100 μmol/L) had no effect. GDCA or glycocholic acid (GCA) had no significant effect on chenodeoxycholic acid (CDCA) synthesis. Free bile acids had a similar effect as glycine-conjugated bile acids. Addition of GCDCA, GDCA, and GCA (100 μmol/L) markedly decreased cholesterol 7α-hydroxylase (CYP7A1) mRNA levels to 2% ± 1%, 2% ± 1%, and 29% ± 11% of controls, respectively, whereas GUDCA had no effect. Addition of GDCA and GCDCA (100 μmol/L) significantly decreased sterol 12α-hydroxylase (CYP8B1) mRNA levels to 48% ± 5% and 61% ± 4% of controls, respectively, whereas GCA and GUDCA had no effect. Addition of GCDCA and GDCA (100 μmol/L) significantly decreased sterol 27-hydroxylase (CYP27A1) mRNA levels to 59% ± 3% and 60% ± 7% of controls, respectively, whereas GUDCA and GCA had no significant effect. Addition of both GCDCA and GDCA markedly increased the mRNA levels of SHP to 298% ± 43% and 273% ± 30% of controls, respectively. In conclusion, glycine-conjugated and free bile acids suppress bile acid synthesis and mRNA levels of CYP7A1 in the order CDCA > DCA > CA > UDCA. mRNA levels of CYP8B1 and CYP27A1 are suppressed to a much lower degree than CYP7A1. (H epatology 2003;38:930-938.)

FXR-mediated down-regulation of CYP7A1 dominates LXRα in long-term cholesterol-fed NZW rabbits

We investigated how cholesterol feeding regulates cholesterol 7alpha-hydroxylase (CYP7A1) via the nuclear receptors farnesoid X receptor (FXR) and liver X receptor alpha (LXRalpha) in New Zealand white rabbits. After 1 day of 2% cholesterol feeding, when the bile acid pool size had not expanded, mRNA levels of the FXR target genes short-heterodimer partner (SHP) and sterol 12alpha-hydroxylase (CYP8B) were unchanged, indicating that FXR activation remained constant. In contrast, the mRNA levels of the LXRalpha target genes ATP binding cassette transporter A1 (ABCA1) and cholesteryl ester transfer protein (CETP) increased 5-fold and 2.3-fold, respectively, associated with significant increases in hepatic concentrations of oxysterols. Activity and mRNA levels of CYP7A1 increased 2.4 times and 2.2 times, respectively. After 10 days of cholesterol feeding, the bile acid pool size increased nearly 2-fold. SHP mRNA levels increased 4.1-fold while CYP8B declined 64%. ABCA1 mRNA rose 8-fold and CETP mRNA remained elevated. Activity and mRNA of CYP7A1 decreased 60% and 90%, respectively. Feeding cholesterol for 1 day did not enlarge the ligand pool size or change FXR activation, while LXRalpha was activated highly secondary to increased hepatic oxysterols. As a result, CYP7A1 was up-regulated. After 10 days of cholesterol feeding, the bile acid (FXR ligand) pool size increased, which activated FXR and inhibited CYP7A1 despite continued activation of LXRalpha. Thus, in rabbits, when FXR and LXRalpha are activated simultaneously, the inhibitory effect of FXR overrides the stimulatory effect of LXRalpha to suppress CYP7A1 mRNA expression.

Liver Receptor Homolog 1 Controls the Expression of Carboxyl Ester Lipase

The orphan nuclear receptor liver receptor homolog 1 (LRH-1) plays a central role in cholesterol homeostasis by regulating a number of hepatic and intestinal genes critical for reverse cholesterol transport and bile acid homeostasis. Herein, we describe the identification of carboxyl ester lipase (CEL) as a novel target of LRH-1 in pancreas, a tissue in which LRH-1 is abundantly expressed. In situ hybridization and gene expression studies demonstrate that both LRH-1 and CEL are co-expressed and confined to the exocrine pancreas. LRH-1 interacts with a consensus LRH-1 response element in the human CEL promoter, which is perfectly conserved in the rat gene, and induces CEL promoter activity in cotransfection assays. As reported for other LRH-1 target genes, the nuclear receptor short heterodimer partner represses LRH-1-induced CEL promoter activity. Chromatin immunoprecipitation demonstrates that binding of LRH-1 to the CEL promoter increases histone H4 acetylation corresponding with the activation of endogenous CEL gene transcription. Our data, identifying CEL as the first pancreatic LRH-1 target gene, indicate that LRH-1 is an important player in enterohepatic cholesterol homeostasis.

Liver Receptor Homologue-1 Mediates Species- and Cell Line-specific Bile Acid-dependent Negative Feedback Regulation of the Apical Sodium-dependent Bile Acid Transporter

Intestinal reclamation of bile salts is mediated in large part by the apical sodium-dependent bile acid transporter (ASBT). The bile acid responsiveness of ASBT is controversial. Bile acid feeding in mice results in decreased expression of ASBT protein and mRNA. Mouse but not rat ASBT promoter activity was repressed in Caco-2, but not IEC-6, cells by chenodeoxycholic acid. A potential liver receptor homologue-1 (LRH-1) cis-acting element was identified in the bile acid-responsive region of the mouse but not rat promoter. The mouse, but not rat, promoter was activated by LRH-1, and this correlated with nuclear protein binding to the mouse but not rat LRH-1 element. The short heterodimer partner diminished the activity of the mouse promoter and could partially offset its activation by LRH-1. Interconversion of the potential LRH-1 cis-elements between the mouse and rat ASBT promoters was associated with an interconversion of LRH-1 and bile acid responsiveness. LRH-1 protein was found in Caco-2 cells and mouse ileum, but not IEC-6 cells or rat ileum. Bile acid response was mediated by the farnesoid X receptor, as shown by the fact that overexpression of a dominant-negative farnesoid X-receptor eliminated the bile acid mediated down-regulation of ASBT. In addition, ASBT expression in farnesoid X receptor null mice was unresponsive to bile acid feeding. In summary cell line- and species-specific negative feedback regulation of ASBT by bile acids is mediated by farnesoid X receptor via small heterodimer partner-dependent repression of LRH-1 activation of the ASBT promoter.

Bile acid induced negative feedback regulation of the human ileal bile acid transporter is mediated by the farnesoid X-receptor (FXR), the short heterodimer partner (SHP), and the retinoid X receptor (RAR/RXR)

Bile acid induced negative feedback regulation of the human ileal bile acid transporter is mediated by the farnesoid X-receptor (FXR), the short heterodimer partner (SHP), and the retinoid X receptor (RAR/RXR)

Regulation of the Na+/taurocholate cotransporter Ntcp via short heterodimer partner (SHP)-dependent or SHP-independent mechanims depends on the type of cholestatic injury

Regulation of the Na+/taurocholate cotransporter Ntcp via short heterodimer partner (SHP)-dependent or SHP-independent mechanims depends on the type of cholestatic injury

Role of short heterodimer partner 9SHP) -dependent and -independent pathways for regulation of NA+/Taurocholate cotransporter (NTCP) expression in cholestatic mouse models

Role of short heterodimer partner 9SHP) -dependent and -independent pathways for regulation of NA+/Taurocholate cotransporter (NTCP) expression in cholestatic mouse models

Estrogen Receptor Inhibits IL-1 Induction of Gene Expression in the Mouse Liver

Estrogens have been suggested to modulate several inflammatory processes. Here, we show that IL-1 treatment induced the expression of approximately 75 genes in the liver of ovariectomized mice. 17-Ethinyl estradiol (EE) pretreatment reduced the IL-1 induction of approximately one third of these genes. Estrogen receptor (ER) was required for this inhibitory activity, because EE inhibition of IL-1-stimulated gene expression occurred in ER knockout mice, but not in ER knockout mice. EE treatment induced expression of 40 genes, including the transcriptional repressor short heterodimer partner and prostaglandin D synthase, known modulators of nuclear factor-B signaling. However, the ER agonists genistein and raloxifene both inhibited IL-1 gene induction without stimulating the expression of prostaglandin D synthase, short heterodimer partner, or other ERinducible genes, indicating that induction of gene expression was not required for ER inhibition of IL-1 signaling. Finally, the ability of EE to repress IL-1 gene induction varied among tissues. For example, EE inhibited IL-1 induction of lipopolysaccharide-induced c-x-c chemokine (LIX) in the liver, but not in the spleen or lung. The degree of EE repression did not correlate with ER expression. cAMP response element binding protein-binding protein (CBP)/p300 levels also varied between tissues. Together, these results are consistent with a model of in vivo ER interference with IL-1 signaling through a coactivatorbased mechanism. (Endocrinology 143: 2559–2570, 2002) EPSIS IS THE 11th leading cause of death in the United States and is a major cause of mortality in the intensive care unit, with mortality rates of 40 – 60% (1). The initial manifestation of sepsis is extensive inflammation, primarily due to increased TNF, IL-1, and IL-6 levels (2) resulting from bacterial products such as lipopolysaccharides (LPS). Animal models of sepsis have suggested that anticytokine therapy could provide benefit in sepsis (3). However, several randomized clinical trials of antiinflammatory therapies, including the use of naturally occurring antagonists or blocking antibodies directed against TNF or IL-1, have not shown any improvement in survival from sepsis (1), perhaps due to the redundant nature of cytokine signaling pathways. In humans, gender has recently been shown to be a significant predictor of survival from sepsis, with a hospital mortality rate of 70% for male patients, but only 26% for female patients in a prospective study (4). Surprisingly, although the women studied in this trial were postmenopausal, their plasma levels of 17-estradiol were in the high normal physiological range (250 pg/ml) and were significantly higher than those in the male patients, possibly due to

Estrogen receptor alpha inhibits IL-1beta induction of gene expression in the mouse liver.

Estrogens have been suggested to modulate several inflammatory processes. Here, we show that IL-1beta treatment induced the expression of approximately 75 genes in the liver of ovariectomized mice. 17alpha-Ethinyl estradiol (EE) pretreatment reduced the IL-1beta induction of approximately one third of these genes. Estrogen receptor alpha (ERalpha) was required for this inhibitory activity, because EE inhibition of IL-1beta-stimulated gene expression occurred in ERbeta knockout mice, but not in ERalpha knockout mice. EE treatment induced expression of 40 genes, including the transcriptional repressor short heterodimer partner and prostaglandin D synthase, known modulators of nuclear factor-kappaB signaling. However, the ER agonists genistein and raloxifene both inhibited IL-1beta gene induction without stimulating the expression of prostaglandin D synthase, short heterodimer partner, or other ER-inducible genes, indicating that induction of gene expression was not required for ER inhibition of IL-1beta signaling. Finally, the ability of EE to repress IL-1beta gene induction varied among tissues. For example, EE inhibited IL-1beta induction of lipopolysaccharide-induced c-x-c chemokine (LIX) in the liver, but not in the spleen or lung. The degree of EE repression did not correlate with ER expression. cAMP response element binding protein-binding protein (CBP)/p300 levels also varied between tissues. Together, these results are consistent with a model of in vivo ER interference with IL-1beta signaling through a coactivator-based mechanism.

A transcriptional inhibitor targeted by the atypical orphan nuclear receptor SHP.

SHP (short heterodimer partner, NROB2) is an atypical orphan member of the mammalian nuclear receptor family that consists only of a putative ligand-binding domain and thus cannot bind DNA. Instead, SHP acts as a transcriptional coregulator by inhibiting the activity of various nuclear receptors (downstream targets) via occupation of the coactivator-binding surface and active repression. However, repression mechanisms have remained elusive and may involve coinhibitory factors (upstream targets) distinct from known nuclear receptor corepressors. Here, we describe the isolation of mouse E1A-like inhibitor of differentiation 1 (EID1) as a candidate coinhibitor for SHP. We characterize the interactions between SHP and EID1 and identify two repression-defective SHP mutations that have lost the ability to bind EID1. We suggest histone acetyltransferases and histones as targets for EID1 action and propose that SHP inhibition of transcription involves EID1 antagonism of CBP/p300-dependent coactivator functions.

The agonist activity of tamoxifen is inhibited by the short heterodimer partner orphan nuclear receptor in human endometrial cancer cells.

Short heterodimer partner (SHP) is an orphan nuclear receptor that interacts with ER(alpha) and ERbeta and inhibits E2-induced transcription. We examined how SHP affects tamoxifen's estrogen agonist activity in endometrial cells. We report that SHP interacts with 4-hydroxytamoxifen (4-OHT) or E2-occupied ER(alpha) in a temperature-dependent manner in vitro. In transient transfection assays, SHP inhibited 4-OHT-stimulated reporter gene activity from an estrogen response element (ERE) in ER-positive RL95-2 but not in HEC-1A human endometrial carcinoma cells transfected with ER(alpha) or ERbeta. SHP inhibited E2-induced transcriptional activity in ER(alpha)- or ERbeta-transfected HEC-1A or Chinese hamster ovary-K1 cells. SHP inhibition of E2 activity was greater for ER(alpha) than ERbeta from the nonpalindromic ERE in the pS2 gene promoter in Chinese hamster ovary-K1 but not HEC-1A cells. Thus, ER subtype, cell type, and ERE sequence influence SHP repressor activity. An ER(alpha) mutant lacking activator function-1 showed reduced inhibition by SHP. In glutathione S-transferase pull-down experiments, SHP inhibited ER(alpha) dimerization, providing a possible mechanism to account for the inhibitory effect of SHP on ER activity. These results identify SHP as novel target for blocking 4-OHT agonist activity in endometrial cells.

The Agonist Activity of Tamoxifen Is Inhibited by the Short Heterodimer Partner Orphan Nuclear Receptor in Human Endometrial Cancer Cells

The Agonist Activity of Tamoxifen Is Inhibited by the Short Heterodimer Partner Orphan Nuclear Receptor in Human Endometrial Cancer Cells

Induction of short heterodimer partner 1 precedes downregulation of Ntcp in bile duct-ligated mice.

Cholestasis is associated with retention of bile acids and reduced expression of the Na(+)/taurocholate cotransporter (Ntcp), the major hepatocellular bile acid uptake system. This study aimed to determine whether downregulation of Ntcp in obstructive cholestasis 1) is a consequence of bile acid retention and 2) is mediated by induction of the transcriptional repressor short heterodimer partner 1 (SHP-1). To study the time course for the changes in serum bile acid levels as well as SHP-1 and Ntcp steady-state mRNA levels, mice were subjected to common bile duct ligation (CBDL) for 3, 6, 12, 24, 72, and 168 h and compared with sham-operated controls. Serum bile acid levels were determined by radioimmunoassay. SHP-1 and Ntcp steady-state mRNA expression were assessed by Northern blotting. In addition, Ntcp protein expression was studied by Western blotting and immunofluorescence microscopy. Increased SHP-1 mRNA expression paralleled elevations of serum bile acid levels and was followed by downregulation of Ntcp mRNA and protein expression in CBDL mice. Maximal SHP-1 mRNA expression reached a plateau phase after 6-h CBDL (12-fold; P < 0.001) and preceded the nadir of Ntcp mRNA levels (12%, P < 0.001) by 6 h. In conclusion, bile acid-induced expression of SHP-1 may, at least in part, mediate downregulation of Ntcp in CBDL mice. These findings support the concept that downregulation of Ntcp in cholestasis limits intracytoplasmatic accumulation of potentially toxic bile acids.

Characterization of the interaction between androgen receptor and a new transcriptional inhibitor, SHP.

SHP (short heterodimer partner) is an orphan nuclear receptor, first described for its interaction with nuclear receptors. This study explores a new way of inhibiting the androgen-signaling pathway. We demonstrated that SHP inhibited up to 97% of AR-induced activity. Characterization of AR/SHP interaction provided evidence of a clear ligand dependency. We also showed that the LXXI/LL motifs previously found on SHP mediated the interaction with the AR ligand-binding domain (AR-LBD), the motif responsible for the interaction being slightly different from that found with ER. The AR N-terminal domain (AR-NTD), in contrast to that of other nuclear receptors, accounts for most of the entire receptor transactivation potential. SHP also interacted with AR-NTD, thus stabilizing the interaction with AR. We demonstrated that SHP inhibited both AR-LBD and NTD-dependent transactivation, which evidenced for the first time a protein capable of inhibiting a steroid receptor amino-terminal-dependent transactivation. We further characterized the SHP mechanism of action by showing that SHP reversed AR coactivator-mediated activation. Conversely, FHL2 and TIF2 counteracted SHP-mediated inhibition of AR. SHP evidences a new way of inhibiting AR activity by competing with AR coactivators. This new type of inhibitor could dictate the activity of nuclear receptors, depending on the equilibrium between activators and inhibitors.

Suppression of sterol 12alpha-hydroxylase transcription by the short heterodimer partner: insights into the repression mechanism.

Cholesterol conversion to bile acids is subject to a feedback regulatory mechanism by which bile acids down-regulate their own synthesis. This regulation occurs at the level of transcription of several genes encoding enzymes in the bile acid biosynthetic pathway. One of these enzymes is sterol 12alpha-hydroxylase/CYP8B1 (12alpha-hydroxylase), the specific enzyme required for cholic acid synthesis. The levels of this enzyme determine the ratio of cholic acid to chenodeoxycholic acid and thus the hydrophobicity of the circulating bile acid pool. Previous studies from this laboratory showed that fetoprotein transcription factor (FTF) is required for 12alpha-hydroxylase promoter activity and bile acid-mediated regulation. Here, we report that the short heterodimer partner (SHP) suppresses 12alpha-hydroxylase promoter activity via an interaction with FTF. Hepatic nuclear factor-4 (HNF-4) binds and activates the 12alpha-hydroxylase promoter and is required for 12alpha-hydroxylase promoter activity. Although HNF-4 interacts with SHP, it is not involved in SHP-mediated suppression of 12alpha-hydroxylase promoter activity. FTF and not HNF-4 is the factor involved in regulation of 12alpha-hydroxylase promoter activity by bile acids through its interaction with SHP. Finally, interaction of SHP with FTF displaces FTF binding to its sites within the 12alpha-hydroxylase promoter. These results provide insights into the mechanism of action of bile acid-mediated regulation of sterol 12alpha-hydroxylase transcription.

TR4 Orphan Receptor Represses the Human Steroid 21-Hydroxylase Gene Expression through the Monomeric AGGTCA Motif

The human TR4 orphan receptor (TR4) is a member of the nuclear receptor superfamily. It functions as a transcriptional factor which regulates and controls many important physiological functions. It has been documented that TR4 may bind as a homodimer to a DNA response element containing two direct repeats of the AGGTCA consensus motif. Surprisingly, our data reveal that the expression of the human steroid 21-hydroxylase (21-OHase) gene could be repressed by TR4 via the monomeric AGGTCA motif (−228TR4RE) at its 5′ flanking region (nucleotide numbers 1431–1444, 5′-GGAAAAAGGTCAGG-3′). Electrophoretic mobility shift assay showed specific binding with a dissociation constant of 0.4 nM between TR4 and the monomeric −288TR4RE motif. However, TR4 does not form heterodimers with either retinoid X receptor alpha or SHP (short heterodimer partner) orphan receptor. Additionally, both dual-luciferase and chloramphenicol acetyltransferase assays demonstrated that TR4 can function as a repressor via the −228TR4RE of the 21-OHase gene. In conclusion, our data suggest that TR4 may bind to a monomeric DNA response element and play an important role in the suppression of the 21-OHase gene expression.

Short Heterodimer Partner (SHP) Orphan Nuclear Receptor Inhibits the Transcriptional Activity of Aryl Hydrocarbon Receptor (AHR)/AHR Nuclear Translocator (ARNT)

SHP (short heterodimer partner) is an orphan nuclear receptor lacking a DNA binding domain that interacts with nuclear receptors (NR) including thyroid receptor (TR), retinoic acid receptors (RAR and RXR), and estrogen receptors α and β (ERα and ERβ). SHP acts as a negative regulator of these receptors by inhibiting DNA binding and transcriptional activation. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) binds to arylhydrocarbon receptor (AHR), activating the AHR/AHR nuclear translocator (ARNT) heterodimer. We investigated the physical and functional interaction of SHP with AHR/ARNT. In RL95-2 human endometrial carcinoma cells, SHP inhibited TCDD-stimulated reporter activity from the AHR-responsive CYP1A1 and UGT1A6 gene promoters in a concentration-dependent manner. In GST pull-down assays, ARNT interacted directly with SHP in vitro, but AHR did not interact with GST-SHP. SHP inhibited AHR/ARNT–DNA binding in vitro. These results identify ARNT as a novel SHP target. We speculate a role for SHP in the suppression of agonist-activated AHR/ARNT activity.