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Molecular Endocrinology, doi:10.1210/me.2005-0461
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Molecular Endocrinology 20 (11): 2711-2723
Copyright © 2006 by The Endocrine Society

Reciprocal Regulation of a Glucocorticoid Receptor-Steroidogenic Factor-1 Transcription Complex on the Dax-1 Promoter by Glucocorticoids and Adrenocorticotropic Hormone in the Adrenal Cortex

Brian M. Gummow, Joshua O. Scheys, Victoria R. Cancelli and Gary D. Hammer

University of Michigan Medical School, Department of Molecular and Integrative Physiology (B.M.G., G.D.H.), Program in Cellular and Molecular Biology (J.O.S.), Department of Internal Medicine, Division of Metabolism, Endocrinology and Diabetes (G.D.H.), Ann Arbor, Michigan 48109-2200

Address all correspondence and requests for reprints to: Gary D. Hammer, M.D., Ph.D., 109 Zina Pitcher Place, 1502 BSRB, Ann Arbor, Michigan 48109-2200. E-mail: ghammer{at}umich.edu.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Numerous genes required for adrenocortical steroidogenesis are activated by the nuclear hormone receptor steroidogenic factor 1 (SF-1) (NR5A1). Dax-1 (NR0B1), another nuclear hormone receptor, represses SF-1-dependent activation. Glucocorticoid products of the adrenal cortex provide negative feedback to the production of hypothalamic CRH and pituitary ACTH. We hypothesized that glucocorticoids stimulate an intraadrenal negative feedback loop via activation of Dax-1 expression. Reporter constructs show glucocorticoid-dependent synergy between SF-1 and glucocorticoid receptor (GR) in the activation of Dax-1, which is antagonized by ACTH signaling. We map the functional glucocorticoid response element between –718 and –704 bp, required for activation by GR and synergy with SF-1. Of three SF-1 response elements, only the –128-bp SF-1 response element is required for synergy with GR. Chromatin immunoprecipitation (ChIP) assays demonstrate that dexamethasone treatment increases GR and SF-1 binding to the endogenous murine Dax-1 promoter 10- and 3.5-fold over baseline. Serial ChIP assays reveal that that GR and SF-1 are part of the same complex on the Dax-1 promoter, whereas coimmunoprecipitation assay confirms the presence of a protein complex that contains both GR and SF-1. ACTH stimulation disrupts the formation of this complex by abrogating SF-1 binding to the Dax-1 promoter, while promoting SF-1 binding to the melanocortin-2 receptor (Mc2r) and steroidogenic acute regulatory protein (StAR) promoters. Finally, dexamethasone treatment increases endogenous Dax-1 expression and concordantly decreases StAR expression. ACTH signaling antagonizes the increase in Dax-1 yet strongly activates StAR transcription. These data indicate that GR provides feedback regulation of adrenocortical steroid production through synergistic activation of Dax-1 with SF-1, which is antagonized by ACTH activation of the adrenal cortex.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
NEGATIVE FEEDBACK REGULATION is a hallmark of endocrine systems, as exemplified by the hypothalamic-pituitary-adrenal (HPA) axis. In response to stress, CRH is released from the hypothalamus into the pituitary portal blood system (1, 2). CRH stimulates corticotrophs in the anterior pituitary to release ACTH, a cleavage product of the proopiomelanocortin gene, into the systemic blood system (3, 4, 5, 6). ACTH stimulates steroid production in the adrenal cortex through the activation of a variety of critical steroidogenic enzyme genes (7, 8, 9, 10, 11). Glucocorticoids, one class of hormones secreted by the adrenal cortex, have been shown to inhibit CRH production and secretion from the hypothalamus as well as proopiomelanocortin expression and ACTH secretion from the anterior pituitary in a classical negative feedback loop (12, 13, 14, 15, 16).

In the body, cholesterol is metabolized into two primary end-products: bile acids, synthesized in the liver, or steroid hormones, synthesized in the adrenal cortex, gonad, and brain. Hepatic cholesterol is converted into bile acids by a series of enzymatic reactions mediated by members of the cytochrome P450 class of enzymes (17). Expression of the enzymes regulating bile acid production is regulated in part by the monomeric nuclear hormone receptor liver receptor homolog-1 (LRH-1, officially designated NR5A2) (18, 19). In addition to promoting transcription of the genes involved bile acid synthesis, LRH-1 is required for expression of short heterodimer partner (SHP, officially designated NR0B2), an orphan nuclear receptor that has been shown to repress the transcriptional activity of other nuclear receptors (20, 21, 22). Recently, a novel mechanism of negative feedback regulation was described in the production of bile acids in the liver (23). Bile acid production in the liver is accompanied by an increase in SHP expression. It has been shown that the farnesoid X receptor (FXR), the nuclear receptor for bile acids, binds to the promoter for the SHP gene and synergizes with LRH-1 to activate SHP transcription. The increase in SHP levels results in SHP mediated repression of LRH-1-dependent transcription of CYP7A1, the gene that encodes the rate-limiting enzyme in bile acid synthesis (23).

There are several parallels between bile acid synthesis in the liver and steroid production in the adrenal cortex. The conversion of cholesterol into adrenal steroids is mediated by cytochrome P450 enzymes, and their expression is regulated by the monomeric nuclear receptor steroidogenic factor-1 (SF-1, officially designated NR5A1) (24, 25). Within the nuclear receptor superfamily, SF-1 is most homologous to LRH-1 (26). In addition to regulating the transcription of the genes encoding the adrenocortical steroidogenic enzymes, SF-1 is required for activation of the gene dose-sensitive sex reversal, adrenal hypoplasia congenita determining region on the X-chromosome-1 (Dax-1, officially designated NR0B1) (27, 28, 29, 30). Dax-1 encodes an orphan nuclear receptor that has been shown to bind to SF-1 and repress SF-1-dependent transcription by recruitment of corepressors such as Alien and nuclear corepressor (31, 32, 33, 34). Within the nuclear receptor superfamily, Dax-1 is most highly related to SHP because both factors lack the classical zinc finger DNA binding domain that characterizes most nuclear receptors (35). The metabolism of cholesterol in the adrenal cortex parallels the metabolism of cholesterol in the liver, both in the family of enzymes required for the conversion of cholesterol into its end products and the factors that regulate the expression of these enzymes. These parallels suggest that similar feedback mechanisms might regulate cholesterol metabolism in the adrenal cortex and the liver.

The regulation of Dax-1 transcription by SF-1 has been well characterized (27, 28, 29, 30). SF-1 has been shown to bind to three distinct cis-acting elements within the murine Dax-1 promoter (30). The most distal site was detected at –330 to –326 bp, and disruption of this site resulted in only a modest decrease in SF-1 responsiveness of a Dax-1 reporter (30). A second SF-1 binding site was found to be composed of two overlapping SF-1 response elements: one at –128 to –121 bp and another at –122 to –115 bp. SF-1 responsiveness was strongly reduced when this site was disrupted (30). The proximal SF-1 response element was localized to –80 to –72 bp and was also found to be responsible for significant portion of SF-1 responsiveness. Intriguingly, introduction of mutations into both the –128 to –115 composite site and the –80- to –72-bp site only decreased SF-1 mediated transcription by similar to disruption of the proximal response element alone (30).

Based on the similarities between bile acid synthesis in the liver and steroidogenesis in the adrenal cortex, we hypothesized that a negative feedback loop may regulate adrenocortical steroid production, whereby intraadrenal glucocorticoids inhibit adrenocortical steroidogenesis through a synergistic glucocorticoid receptor (GR)/SF-1-dependent activation of Dax-1 transcription in adrenocortical cells. The data presented below suggest that an intricate interplay of glucocorticoid and ACTH signaling tightly regulates steroid production within the adrenal cortex.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
GR Synergizes with SF-1 to Activate Dax-1 Expression
Using the FXR-mediated activation of SHP as a model of negative feedback regulation of bile acid synthesis in the liver, we hypothesized a similar method of negative feedback in adrenocortical steroid production. Cotransfection assays were performed to determine whether GR synergizes with SF-1 to activate Dax-1 expression. A reporter construct consisting of –2938 bp of the murine Dax-1 promoter driving luciferase was cotransfected with SF-1, GR or both into JEG3 placental carcinoma cells. The transfected cells were then treated with increasing doses of the GR agonist dexamethasone. As previously described, we observe an SF-1-dependent 11-fold activation of the Dax-1 reporter (Fig. 1AGo) that is not influenced by dexamethasone treatment. However, a statistically significant synergy between SF-1 and GR is observed after dexamethasone stimulation in a dose-dependent manner, showing a nearly 30-fold induction of the reporter construct at the maximal dose of dexamethasone of 10 µM. Such glucocorticoid concentrations are consistent with the 10- to 1000-fold increase in intraadrenal concentration of adrenocortical steroids (10–50 µM range) compared with serum levels (36).


Figure 1
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  		Fig. 1. GR Is Able to Activate the Dax-1 Promoter and Synergize with SF-1 in a Ligand-Dependent Manner

The functional GRE maps to between –800 and –700 bp of the Dax-1 promoter. A, In the presence of increasing concentrations of dexamethasone (10–9 to 10–5 M), GR activates a 2938-bp Dax-1 reporter construct in a dose-dependent manner with a maximum activation of 3-fold over baseline. Whereas dexamethasone has no effect on SF-1 mediated transcription in the absence of GR, dose-dependent synergy was observed in the presence of GR leading to 30-fold activation of the reporter. A total of 166 ng of pGL3B-mDX(2938), 50 ng of SF-1, 50 ng of GR, and 50 ng of myc-LacZ were transfected per condition in triplicate into JEG3 cells (n = 3). Raw data were normalized for transfection efficiency (LacZ), and values represent the mean fold activation over the reporter alone ± SEM. B, Serial deletions of the Dax-1 reporter were cotransfected with SF-1 and GR into JEG3 cells; cells transfected with GR were stimulated with 10–5 M dexamethasone 12 h before lysis. Although SF-1 mediated activation was maintained on all of the deletion constructs, GR responsiveness and synergy with SF-1 were lost on the –500-bp construct, indicating that the functional GRE resides between –1000 and –500 bp of the promoter. A total of 166 ng each of the reporter constructs, SF-1, and GR were transfected along with 50 ng of myc-LacZ per condition in triplicate into JEG3 cells (n = 3). Raw data were normalized for transfection efficiency (LacZ) and values represent the mean fold activation above basal reporter activity ± SEM. C, Serial deletions of the Dax-1 reporter were cotransfected with SF-1 and GR into JEG3 cells; cells transfected with GR were stimulated with 10–5 M dexamethasone 12 h before lysis. Whereas SF-1-mediated activation was maintained on all of the deletion constructs, GR responsiveness and synergy with SF-1 were lost on the –700 bp and shorter constructs, indicating that the functional GRE resides between –800 and –700 bp of the promoter. A total of 166 ng each of the reporter constructs, SF-1, and GR were transfected along with 50 ng of myc-LacZ per condition in triplicate into JEG3 cells (n = 3). Raw data were normalized for transfection efficiency (LacZ) and values represent the mean fold activation above basal reporter activity ± SEM. The asterisks represent statistically significant data as defined in Materials and Methods.

 
To localize the glucocorticoid response element (GRE) responsible for synergy with SF-1, we next developed 5' serial deletions of the Dax-1 promoter by PCR of –1500, –1000, and –500 bp of the promoter followed by cloning into the reporter plasmid pGL3 basic. Consistent with prior data indicating that the SF-1 response elements in the Dax-1 promoter are localized to the proximal –330 bp, SF-1 is able to activate all three deletion constructs similar to the full-length reporter construct (30) (Fig. 1BGo). Whereas both the –1500-bp construct and the –1000-bp construct are responsive to dexamethasone-induced GR mediated activation and statistically significant synergy with SF-1, GR mediated transcription and synergy with SF-1 are abrogated in the –500-bp reporter construct. These data demonstrate the GRE responsible for GR mediated synergy with SF-1 is located between –1000 and –500 bp of the Dax-1 promoter.

To further localize the region of the promoter containing glucocorticoid responsiveness, we next made serial deletions of –1000- to –500-bp region of the Dax-1 promoter to generate –900-, –800-, –700-, and –600-bp reporter constructs. As expected, SF-1 is able to activate all of the reporter constructs (Fig. 1CGo). The –900-bp reporter construct demonstrates significant glucocorticoid responsiveness that is similar to the –1000-bp construct. The –800-bp construct shows a 50% decrease in GR mediated activation, but a statistically significant synergy between SF-1 and GR is still observed on this promoter. The –700- and –600-bp constructs demonstrate a loss of GR-dependent activation and synergy with SF-1, similar to the –500-bp construct. These data suggest the cis-acting element required for GR activation of the Dax-1 promoter and synergy with SF-1 resides between –800 and –700, whereas another cis-acting element between –900 and –800 may further modulate GR-dependent transcription of Dax-1.

To determine which cis-acting elements are required for the glucocorticoid responsiveness of Dax-1, the sequence between –900 and –700 bp was analyzed using the Transcription Element Search System (http://www.cbil.upenn.edu/tess/) (37). Although no strong GREs are observed in the –900- to –800-bp region, a nearly consensus GRE is observed between –718 and –704 bp in the Dax-1 promoter (38). To determine whether the –718 site mediates GR activation of the Dax-1 promoter, we introduced point mutations into the 3' half-site of the element in the context of the –1000- and the –3000-bp reporter (39). Disruption of the –718 site has no effect on SF-1-mediated activation of the reporter construct (Fig. 2AGo). However, the reporter completely loses its glucocorticoid responsiveness, and GR is no longer capable of synergizing with SF-1 when the –718 site is mutated. The effect of this mutation is the same on both the –1000-bp construct and the full-length reporter, indicating that the –718 GRE is necessary for glucocorticoid-induced activation of Dax-1.


Figure 2
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  		Fig. 2. The –718 GRE Is Necessary for the Glucocorticoid Responsiveness of the Dax-1 Promoter and the –128 SF-1 Response Element Is Required for Synergy between SF-1 and GR

A, SF-1 and GR were cotransfected with Dax-1 reporter constructs containing point mutations in the –718 GRE into JEG3 cells. Cells that were transfected with GR were stimulated with 10–5 M dexamethasone 12 h before lysis. SF-1-mediated activation was maintained on all of the mutant constructs. GR-mediated activation and synergy with SF-1 were lost on the reporters containing mutations in the –718 GRE, demonstrating that the –718 GRE is necessary for GR-mediated activation of the Dax-1 promoter. A total of 166 ng each of the reporter constructs, SF-1, and GR were transfected along with 50 ng of myc-LacZ per condition in triplicate into JEG3 cells (n = 3). Raw data were normalized for transfection efficiency (LacZ) and values represent the mean fold activation above basal reporter activity ± SEM. B, Point mutations were generated in the –330 SF-1 response element, the –128 SF-1 response element, and the –80 SF-1 response element in all possible combinations. SF-1 and GR were cotransfected with reporter constructs into JEG3 cells; cells that were transfected with GR were also stimulated with 10–5 M dexamethasone 12 h before lysis. Synergy between SF-1 and GR was lost on all constructs where the –128 site was disrupted, whereas synergy was maintained on all constructs where the –128 site was intact. A total of 166 ng each of the reporter constructs, SF-1, and GR were transfected along with 50 ng of myc-LacZ per condition in triplicate into JEG3 cells (n = 3). Raw data were normalized for transfection efficiency (LacZ), and values represent the mean fold activation above basal reporter activity ± SEM. The asterisks represent statistically significant data as defined in Materials and Methods.

 
We also evaluated the roles of the three known SF-1 response elements on the synergy between SF-1 and GR. We introduced previously described mutations into the –330 to –326 (–330 site), the –128 to –115 composite site (–128 site), and the –80 to –72 (–80 site) SF-1 response elements (30). We also generated all combinations of double mutants and the triple mutant. Disruption of the –330 site results in a mild decrease in SF-1-mediated activation, but the significant synergy between SF-1 and GR is maintained on this construct (Fig. 2BGo). Mutation in the –128 site results in a profound decrease of SF-1 activation of Dax-1 and completely abrogates synergy between SF-1 and GR. A significant decrease in SF-1-dependent transcription is observed when the –80 response element is mutated but significant synergy between SF-1 and GR is retained. Although synergy between SF-1 and GR is also abolished in the –330/–128 and –128/–80 double mutants, SF-1 is able to synergize with GR on the –330/–80 double mutant, despite a strong decrease in SF-1-dependent activation. These data indicate that the –128 SF-1 response element is required for synergy with GR, whereas the –330 site and the –80 site are not required for this interaction. Finally, as expected, mutation in all three SF-1 response elements results in the loss of SF-1-dependent activation of the promoter and loss of synergy with GR.

The Activation of the Murine Dax-1 Promoter by SF-1 and GR Is Attenuated by ACTH in Adrenocortical Cells
To determine whether the observed GR and SF-1 synergism on the Dax-1 promoter functions in a relevant adrenocortical cell line, we performed cotransfection reporter assays in the mouse adrenocortical carcinoma cell line Y1. As expected, we observe a 3-fold increase in stimulation of the Dax-1 reporter construct due to SF-1 cotransfection (Fig. 3Go). Moreover, cotransfection of SF-1 and GR in the presence of dexamethasone results in a 7-fold induction of the promoter compared with the reporter alone. To examine how ACTH might influence glucocorticoid-mediated Dax-1 expression in the adrenal cortex, cells were also treated with 10–7 M ACTH. Consistent with published data that ACTH inhibits Dax-1 expression in adrenocortical cells (40), ACTH stimulation decreases the GR/SF-1 synergistic activation of Dax-1 transcription by 1.5-fold.


Figure 3
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  		Fig. 3. In the Presence of Dexamethasone, GR and SF-1 Can Synergistically Activate the Dax-1 Promoter in the Mouse Adrenocortical Cell Line Y1, and the Activation Can Be Inhibited by ACTH

SF-1 and GR were cotransfected with Dax-1 reporter into Y1 cells. In the presence of 10–7 M dexamethasone, GR increases SF-1-mediated transcription, and the increase can be abrogated by 10–7 M ACTH. Three hundred nanograms of pGL3B-mDax-1(2938), 166 ng of SF-1, GR, and 50 ng of myc-LacZ were transfected per condition in triplicate into Y1 cells (n = 3). Raw data were normalized for transfection efficiency (LacZ), and values represent the mean fold activation over the reporter alone ± SEM. The asterisks represent statistically significant data as defined in Materials and Methods.

 
SF-1 and GR Form a Complex on the Endogenous Murine Dax-1 Promoter
To determine whether GR binds to the –718 GRE of the endogenous murine Dax-1 promoter, chromatin immunoprecipitation (ChIP) assays were performed. Because Y1 adrenocortical cancer cells do not express Dax-1, we generated primary cell cultures of mouse adrenal glands for use in the ChIP assays (41). The primary cells were treated with dexamethasone, dexamethasone/ACTH, or vehicle (serum-free media) for 40 min before cross-linking. Sonicated cell lysates were then immunoprecipitated using either {alpha}-GR or {alpha}-hemagglutinin (HA) (nonspecific) rabbit polyclonal antibodies. The immunoprecipitates were reverse cross-linked and analyzed by quantitative PCR (qPCR) using both proximal primers (flanking the –718 GRE) and distal primers to demonstrate sufficient DNA shearing. The proximal Dax-1 promoter is immunoprecipitated by the {alpha}-GR antibodies, but not the {alpha}-HA antibodies, and dexamethasone treatment increases GR binding to the proximal promoter 14-fold over vehicle treatment (Fig. 4AGo). No increase in GR binding to the proximal promoter is observed when the cells are concomitantly stimulated with dexamethasone and ACTH, suggesting that ACTH signaling antagonizes dexamethasone-mediated recruitment of GR to the proximal Dax-1 promoter. The distal Dax-1 promoter is not immunoprecipitated, demonstrating that the DNA is sufficiently sheared, and the antibodies do not immunoprecipitate large chromosomal fragments that might interact with GR at additional/alternate contact points on the chromatin.


Figure 4
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  		Fig. 4. ChIP Analysis Reveals that Dexamethasone Promotes the Recruitment of GR and SF-1 to a Transcription Complex on the Dax-1 Promoter, Whereas ACTH Signaling Antagonizes Formation of this Complex

A, ChIP assays were performed on primary adrenal cells using {alpha}-GR antibodies after 40 min stimulation with 10–7 M ACTH and/or 10–5 M dexamethasone. Immunoprecipitates were analyzed by qPCR using primers designed against the proximal and distal Dax-1 promoter. The proximal promoter was immunoprecipitated by the {alpha}-GR antibodies, whereas the distal promoter was not. Rabbit polyclonal {alpha}-HA antibodies were used as a negative control. Experimental values were normalized to 1% input and values represent mean fold activation compared with untreated cells ± SEM (n = 3). B, ChIP assays were performed on primary adrenal cells using {alpha}-SF-1 antibodies after 40 min stimulation with 10–7 M ACTH and/or 10–5 M dexamethasone. Immunoprecipitates were analyzed by qPCR using primers designed against the proximal and distal Dax-1 promoter. The proximal promoter was immunoprecipitated by the {alpha}-SF-1 antibodies after dexamethasone stimulation but not ACTH stimulation, whereas the distal promoter was not. Rabbit polyclonal {alpha}-HA antibodies were used as a negative control. Experimental values were normalized to 1% input and values represent mean fold activation compared with untreated cells ± SEM (n = 3). C, Serial ChIP assays were performed on primary adrenal cells using {alpha}-GR antibodies followed by {alpha}-SF-1 antibodies after a 40-min stimulation with 10–7 M ACTH and 10–5 M dexamethasone. Immunoprecipitates were analyzed by qPCR using primers designed against the proximal and distal Dax-1 promoter. Dexamethasone stimulates a SF-1/GR complex on the proximal promoter, whereas ACTH signaling inhibits complex formation. Rabbit polyclonal {alpha}-HA antibodies were used as a negative control. Experimental values were normalized to 1% input and values represent mean fold activation compared with untreated cells ± SEM (n = 3). The asterisks represent statistically significant data as defined in Materials and Methods.

 
We next wanted to determine the effect of dexamethasone on SF-1 binding to the proximal Dax-1 promoter. After dexamethasone, concomitant dexamethasone and ACTH, or vehicle stimulation and cross-linking, lysates were immunoprecipitated with either {alpha}-SF-1 or {alpha}-HA (nonspecific) rabbit polyclonal antibodies. qPCR was performed using primers in the proximal Dax-1 promoter that flanked the –128 composite SF-1 response element, which was the only SF-1 response element necessary for synergy with GR, and primers in the distal Dax-1 promoter. As expected, the proximal Dax-1 promoter is immunoprecipitated by {alpha}-SF-1, but not {alpha}-HA, antibodies (Fig. 4BGo). Surprisingly, dexamethasone treatment increases SF-1 recruitment to the Dax-1 promoter 3.5-fold over baseline. Furthermore, concomitant treatment with dexamethasone and ACTH not only antagonizes dexamethasone mediated SF-1 recruitment to the promoter, but also abrogates basal SF-1 binding to the proximal promoter. Once again, the distal Dax-1 promoter is not immunoprecipitated, demonstrating sufficient chromatin shearing.

There are two possible explanations for dexamethasone-induced binding of SF-1 to the proximal Dax-1 promoter. First, GR binding and subsequent cofactor recruitment alter the local chromatin environment, making the promoter more readily available for SF-1 binding. Alternatively, SF-1 and GR may form a transcription complex on the proximal Dax-1 promoter, and increased binding of GR stabilizes SF-1 binding to the promoter. To determine whether SF-1 and GR form a transcription complex on the proximal Dax-1 promoter, we performed serial ChIP assays using antibodies against both SF-1 and GR. After dexamethasone stimulation and cross-linking, primary adrenal cell lysates were immunoprecipitated with {alpha}-GR antibodies. Immunoprecipitates were then washed, eluted, and subsequently immunoprecipitated with {alpha}-SF-1 or {alpha}-HA (nonspecific) rabbit polyclonal antibodies. Next, qPCR was performed using primers in the proximal Dax-1 promoter that flank the –128 composite SF-1 response element and primers in the distal Dax-1 promoter. The proximal, but not the distal, Dax-1 promoter is immunoprecipitated in this assay, demonstrating that GR and SF-1 form a complex on the proximal Dax-1 promoter (Fig. 4CGo). Furthermore, dexamethasone treatment results in a 3.5-fold increase in complex formation over baseline. Because this result mirrors the overall increase in SF-1 recruitment to the promoter after dexamethasone treatment, it is likely that GR stabilizes SF-1 binding to the promoter through the formation of a transcription complex and not by changing the chromatin environment of the promoter. Also, concomitant stimulation with dexamethasone and ACTH precludes formation of an SF-1/GR complex consistent with an ACTH-mediated antagonism of SF-1 recruitment to the proximal Dax-1 promoter.

SF-1 and GR Associate in a Protein Complex in Adrenocortical Cells
To further examine the association of GR and SF-1 in an adrenal cortical cell line, coimmunoprecipitation assays were performed in H295 cells. Western analysis demonstrates that levels of endogenous GR and SF-1 are consistent in vehicle, dexamethasone and dexamethasone plus ACTH treatment conditions (Fig. 5Go). Immunoprecipitation with {alpha}-SF-1 antibodies reveals that GR and SF-1 are present in a complex in adrenocortical cells (Fig. 5Go). Dexamethasone treatment appears to increase the amount of GR pulled down by SF-1, and ACTH appears to antagonize this increased pull-down. Although not quantitative, these coimmunoprecipitation data, together with the quantitative serial ChIP analysis, are consistent with dexamethasone increasing the association between GR and SF-1, and ACTH signaling disrupting the association.


Figure 5
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  		Fig. 5. GR and SF-1 Are Associated in Human Adrenocortical H295 Cells

H295 cells were treated with vehicle, 10–7 M dexamethasone (Dex), or 10–7 M Dex plus 10–7 M ACTH. Western analysis (upper panel) reveals that endogenous protein levels are consistent between the treatment groups. Coimmunoprecipitation was performed using rabbit polyclonal {alpha}-SF-1 antibodies. A complex between GR-1 and SF-1 is observed under each of these conditions (lower panel) (n = 3, representative data shown). IP, Immunoprecipitation; IB, immunoblot.

 
ACTH Induces Reciprocal Clearance of SF-1 from the Dax-1 Promoter and Recruitment of SF-1 to the StAR and Mc2r Promoters
In light of recent studies in our laboratory that show that ACTH signaling induces the recruitment of SF-1 to the promoters of adrenocortical steroidogenic genes (42), the observation that ACTH results in clearance from the Dax-1 promoter is intriguing. Therefore, to confirm whether ACTH can induce opposing effects on SF-1 binding to different adrenal-expressed promoters, we performed parallel ChIP assays of the Dax-1, Mc2r and StAR promoters on identical lysates after ACTH treatment. ChIP analysis in cultured primary adrenal cells using {alpha}-SF-1 antibodies reveals that, whereas ACTH signaling disrupts SF-1 binding to the Dax-1 promoter (Fig. 6Go), it induces a 5.5-fold increase in the SF-1 occupancy of the Mc2r promoter and an 8.9-fold increase in SF-1 recruitment to the StAR promoter.


Figure 6
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  		Fig. 6. ACTH Signaling Displaces SF-1 from the Dax-1 Promoter, While Recruiting SF-1 to the Mc2r and StAR Promoters

ChIP assays were performed on primary adrenal cells using {alpha}-SF-1 or {alpha}-HA antibodies after 40 min stimulation with 10–7 M ACTH. Immunoprecipitates were analyzed by qPCR using primers designed against the proximal and distal Dax-1, Mc2r, and StAR promoters. ACTH stimulation inhibited the SF-1 binding to the Dax-1 promoter observed at baseline. Conversely, ACTH treatment promoted the binding of SF-1 to the Mc2r and StAR promoters over baseline. Experimental values were normalized to 1% input and values represent mean fold activation compared with vehicle-treated cells ± SEM (n = 3). The asterisks represent statistically significant data as defined in Materials and Methods.

 
Dexamethasone Treatment and ACTH Signaling Conversely Regulate Endogenous Dax-1 and StAR Expression in Primary Adrenocortical Cells
To confirm our reporter assay and ChIP studies, we examined the ability of dexamethasone to stimulate Dax-1 expression in primary adrenocortical cells. Primary cells were treated with vehicle (serum-free media), dexamethasone, or dexamethasone plus ACTH for 18 h. RNA was collected, and qPCR studies were performed. In response to treatment with dexamethasone, there is a 4.5-fold increase in the expression of Dax-1 (Fig. 7Go). However, concomitant treatment with both dexamethasone and ACTH results in an attenuated response (2.8-fold increase in Dax-1 mRNA compared with basal expression). Conversely, there is a 4.7-fold decrease in StAR mRNA levels in response to dexamethasone treatment, whereas there is a 10.9-fold increase in response to both dexamethasone and ACTH.


Figure 7
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  		Fig. 7. Dexamethasone (Dex) Treatment Stimulates Endogenous Dax-1 Expression and Inhibits StAR Expression in Primary Adrenocortical Cells, Whereas the Reciprocal Effect Is Observed after ACTH Stimulation

Primary adrenocortical cells were stimulated with 10–5 M dexamethasone for 18 h. RNA was extracted from cells, reverse transcribed, and analyzed using qPCR. Dexamethasone treatment increases endogenous Dax-1 levels to a 4.5-fold induction over vehicle (serum-free medium)-treated cells. Furthermore, dexamethasone reduced StAR levels 4.7-fold compared with vehicle. After stimulation with both 10–5 M dexamethasone and 10–7 M ACTH, only a 2.8-fold increase in Dax-1 expression was observed, whereas StAR levels were increased 10.9-fold over vehicle. Experimental values were normalized to a glyceraldehyde-3-phosphate dehydrogenase internal standard. Data represent mean relative expression ± SEM (n = 3). The asterisks represent statistically significant data as defined in Materials and Methods.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Glucocorticoids are essential for life. Their actions modulate a variety of physiological processes to maintain homeostasis including glucose, protein, and fat metabolism along with antiinflammatory and immunosuppressive actions (43). Glucocorticoid levels need to be tightly regulated; glucocorticoid excess results in obesity and diabetes, as evident in patients with Cushing’s syndrome (44). Several homeostatic control mechanisms modulate glucocorticoid production in response to activation of the HPA axis. Increasing plasma glucocorticoid concentrations in the hypothalamus mediate a GR-dependent repression of CRH gene transcription, via a negative GRE within the CRH promoter (45). Likewise, GR binding to an imperfect GRE contributes to the inhibition of proopiomelanocortin gene expression in corticotrophs of the anterior pituitary in response to elevated glucocorticoids (46). Here we describe a negative feedback loop, whereby glucocorticoid production feeds back to inhibit adrenocortical transcription of the enzymes and cholesterol transporters required for glucocorticoid synthesis.

Molecular aspects of such feedback have been described previously. SF-1, the nuclear receptor responsible for activation of steroid hydroxylases and cholesterol transporters required for glucocorticoid synthesis, activates expression of Dax-1 (25, 30, 47). Dax-1 has been shown to inhibit SF-1-mediated transcription both in vitro and in vivo (31, 32, 33, 34, 35, 48). In the liver, the regulation of bile acid synthesis parallels the regulation of glucocorticoid production in the adrenal cortex. LRH, the liver homolog of SF-1, activates transcription of the enzymes required for bile acid synthesis and additionally activates expression of SHP, a negative regulator LRH transcription. It has been shown that bile acid synthesis induces the bile acid receptor, FXR, to synergize with LRH to activate SHP expression. As such, Dax-1 is an ideal candidate for intraadrenal negative feedback of glucocorticoid production.

We demonstrate that glucocorticoids stimulate GR mediated activation of Dax-1 in a dose-dependent manner. Additionally, in the presence of SF-1, glucocorticoids induce GR synergy with SF-1 to potentiate Dax-1 expression in a dose-dependent manner. The cis-acting element required for GR activation of Dax-1 maps between –800 and –700 bp with in the Dax-1 promoter, and we define the GRE responsible for GR-mediated transcription and synergy with SF-1 between –718 and –704 bp. Whereas three SF-1 response elements have been described within the Dax-1 promoter, we demonstrate that only the –128 composite SF-1 response element, not the 330 or the –80 response elements, is required for synergy with GR.

Other groups have examined the role of steroid regulation of Dax-1. It has been shown that androgens repress Dax-1 expression within the adrenal cortex. Furthermore, androgen receptor forms a transcription complex with SF-1, which inhibits the ability of SF-1 to transactivate the Dax-1 promoter (49). Although this suggests that negative feedback does not regulate androgen production, it should be noted that Dax-1 overexpression results in XY sex reversion in both mice and humans. Androgen receptor repression of Dax-1 may play a more developmental role than a role in the regulation of steroid production.

ChIP analysis reveals that glucocorticoid stimulation of primary adrenocortical cells resulted in a 14-fold recruitment of GR to the –718-bp region of the Dax-1 promoter. Because dexamethasone was unable to stimulate SF-1-dependent activation of our Dax-1 reporter construct (Fig. 1Go), we anticipated that SF-1 recruitment to the Dax-1 promoter would not be affected by glucocorticoid treatment. We were surprised to find that glucocorticoid treatment increased SF-1 recruitment to the proximal Dax-1 promoter 3.5-fold over baseline. Serial ChIP analysis and coimmunoprecipitation assays revealed that the increased SF-1 binding to the Dax-1 promoter was the result of increased complex formation between GR and SF-1 on the Dax-1 promoter. We further show that dexamethasone treatment induces increased levels of Dax-1 concomitant with decreases in the expression of the cholesterol transporter StAR. Because it is well established that Dax-1 antagonizes SF-1-mediated activation of genes involved in adrenocortical steroidogenesis (31, 32, 33, 34, 35), the decrease in StAR expression strongly suggests the presence of an intraadrenal negative feedback loop via Dax-1.

The similarity between the previously described molecular mechanism of feedback regulation in bile acid synthesis and the regulation of glucocorticoid synthesis suggests an evolutionarily conserved feedback loop that participates in cholesterol metabolism in both the liver and the adrenal cortex (Fig. 8Go). In response to elevated cholesterol, LXR{alpha} synergizes with LRH-1 to activate the enzymes required for bile acid synthesis (50, 51, 52). Accumulation of bile acids, in turn, shut down bile acid production by activation of SHP, which then represses transcription of the genes encoding the enzymes required for bile acid synthesis (23). However, adrenocortical steroid production is additionally regulated by a complex endocrine system of feed forward stimulation and negative feedback regulation, whereas the regulation of bile acid synthesis is much simpler. One can speculate that the presence of a similar negative feedback loop in the adrenal cortex evolutionarily predates the development of the endocrine control systems of the HPA axis.


Figure 8
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  		Fig. 8. The Intracellular Feedback Regulation of Adrenocortical Steroid Production Parallels the Regulation of Bile Acid Synthesis in the Liver

 
Interestingly, several groups have examined the effects of peptide hormones on the expression of Dax-1. Specifically, LH (53, 54), angiotensin II (55), and ACTH (40) have been shown to decrease Dax-1 levels in the gonads and adrenals. We confirm that ACTH signaling decreases endogenous levels of Dax-1 in the adrenal cortex, while promoting expression of the cholesterol transporter StAR. Moreover, the data presented suggest that the mechanism for the ACTH-induced repression of Dax-1 involves the disruption of a GR/SF-1 complex on the Dax-1 promoter, coincident with the recruitment of SF-1 to the StAR and Mc2r promoters. Because the decrease in SF-1 occupancy of the Dax-1 promoter and the increase in SF-1 occupancy of the StAR and Mc2r promoters are observed in the same immunoprecipitates, our data suggest that ACTH signaling differentially regulates SF-1 action in a promoter-specific manner.

Because Dax-1 is primarily expressed in the subcapsular region of the adrenal cortex coincident with the location of proliferating undifferentiated progenitor cells, we propose a model whereby the differentiated cortical cell generates glucocorticoids that provide an endocrine or paracrine (as opposed to autocrine) signal to the undifferentiated progenitor cells to activate Dax-1, which inhibits SF-1-mediated differentiation (steroidogenesis) until ACTH inactivates Dax-1 and initiates steroidogenesis as the cells migrate centripetally to populate the zonated cortex. Although an endocrine mechanism is more consistent with the well-characterized centripetal blood flow of the adrenal cortex (from outer to inner cortex), paracrine signaling through cellular diffusion remains a tenable alternative. Understanding the interplay between the glucocorticoid mediated feedback (via Dax-1) and the membrane-mediated signals (via ACTH) that regulate SF-1-dependent steroidogenesis within the adrenal cortex will help further elucidate the temporal and spatial regulation of differentiation in the adrenal cortex.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Cell Culture
JEG3 placental carcinoma cells were cultured in DMEM supplemented with 10% fetal calf serum. Transient transfection of JEG3 cells was performed using calcium phosphate coprecipitation (Promega, Madison, WI) according to the manufacturer’s directions. Y1 adrenocortical carcinoma cells were cultured in DMEM supplemented with 7.5% horse serum and 2.5% calf serum. Transient transfection of Y1 cells was performed using Fugene (Roche, Indianapolis, IN). H295 human adrenocortical carcinoma cells were cultured in RPMI supplemented with 2.5% calf serum and 1% insulin/transferrin/selenium (Invitrogen, Carlsbad, CA). Primary cells were derived from B6/SJL F2 mice. Adrenals were harvested, cleaned, and minced by hand. Single cell suspensions were generated by incubating minced adrenal glands in serum-free medium containing 2 mg/ml collagenase, 0.05 mg/ml deoxyribonuclease I, and 5 mg/ml BSA at 37 C for 45 min with mechanical disruption (pipetting up and down) every 15 min. Primary adrenal cells were plated on to fibronectin-coated plates in Knockout DMEM containing 15% Knockout serum replacement (Invitrogen) at a density of 16 adrenals per 6-cm culture plate. Primary cells were allowed to adhere to plates 48 h before treatment or use.

Plasmid Constructs
The following plasmids have been previously described: pGL3Basic-mDX(–2938), pci-neo-HA-SF-1, p6R GR and myc-LacZ (29, 56, 57). The plasmids pGL3Basic-mDX(–1500), pGL3Basic-mDX(–1000), pGL3Basic-mDX(–900), pGL3Basic-mDX (–800), pGL3Basic-mDX(–700), pGL3Basic-mDX (–600), and pGL3Basic-mDX(–500) were generated by PCR amplification from pGL3Basic-mDX(–2938) followed by cloning into the KpnI to NcoI fragment of pGL3Basic. See Table 1Go for PCR primer sequences and annealing temperatures. Point mutations in glucocorticoid response elements and SF-1 response elements were introduced using QuikChange Multi Site Directed Mutagenesis kit (Stratagene, La Jolla, CA) according to the manufacturer’s instructions. See Table 2Go for the oligonucleotide sequences used to generate these mutations.


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  		Table 1. PCR Primers Used to Generate Serial Deletions of the Dax-1 Promoter

 

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  		Table 2. Oligos Used to Generate Point Mutations within Response Elements of the Murine Dax-1 Promoter

 
Luciferase Assays
JEG3 cells were plated at a density of 5 x 104 cells per well into 24-well plates. Twenty-four hours after plating, cells were transiently transfected using calcium phosphate coprecipitation (Promega, Madison, WI). Thirty-six hours after transfection, cells were treated with either vehicle (serum-free medium) or dexamethasone. Cells were lysed 12 h after dexamethasone treatment, and a luciferase assay (Promega) was performed on a Veritas Microplate Luminometer (Turner BioSystems, Sunnyvale, CA). Luciferase assays were normalized for transfection efficiency by cotransfecting CMV-myc-LacZ and subsequent determination of ß-galactosidase activity using the galacton reagent (Applied Biosystems, Foster City, CA).

Y1 cells were plated at 4 x 104 cells per well in 24-well plates. Twenty-four hours after plating, cells were transiently transfected using Fugene (Roche, Indianapolis, IN). Thirty-six hours after transfection, cells were serum starved in DMEM + 0.05% BSA. Forty-eight hours after transfection, cells were treated with vehicle (serum-free medium), dexamethasone (100 nM) alone, or in combination with ACTH (100 nM) (Sigma, St. Louis, MO). Cells were lysed 14 h after treatment, and luciferase assays were performed and normalized for transfection efficiency by cotransfecting CMV-myc-LacZ and determination of ß-galactosidase activity using the galacton reagent (Applied Biosystems).

ChIP
ChIP assays were performed as previously described (42). Primary adrenocortical cells were treated with 10–5 M dexamethasone for 40 min, followed by cross-linking with 1% formaldehyde at 37 C for 10 min. Cells were washed twice with ice-cold PBS and lysed for 15 min in a buffer containing 1% SDS; 10 mM EDTA; and 50 mM Tris-HCl (pH 8.1) at 4 C. Lysates were sonicated at 30% power, 4 x 10 sec using a Sonic Dismembrator model 300 (Fisher Scientific, Pittsburgh, PA) to shear genomic DNA and centrifuged at 13,000 rpm for 10 min at 4 C. A total of 150 µl of supernatants were diluted into 1350 µl of a buffer containing 0.01% SDS; 1.1% Triton X-100; 1.2 mM EDTA; 16.7 mM Tris-HCl (pH 8.1); and 167 mM NaCl for a 1:10 dilution. The diluted lysates were immunocleared with 5µg sheared salmon sperm DNA (Invitrogen), 10 µl preimmune serum, and 60 µl protein A-Sepharose (50% slurry in 10 mM Tris-HCl (pH 8.1); 1 mM EDTA; 0.5 mg/ml BSA; 0.05% sodium azide; and 200 µg/ml sheared salmon sperm DNA) for 2 h at 4 C. Immunoprecipitation was performed overnight at 4 C using the following antibodies: polyclonal {alpha}-GR antibodies (clone P-20; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), polyclonal {alpha}-SF-1 antibodies (Upstate Biotechnologies, Lake Placid, NY), and polyclonal {alpha}-HA antibodies (clone Y-11, Santa Cruz). Immunoprecipitates were recovered with 40-µl protein A-Sepharose and 2 µg sheared salmon sperm DNA. Precipitates were washed for 5 min each with the following buffers: low salt buffer [0.1% SDS; 1% Triton X-100; 2 mM EDTA; 20 mM Tris-HCl (pH 8.1); and 150 mM NaCl], high-salt buffer (0.1% SDS; 1% Triton X-100; 2 mM EDTA; 20 mM Tris-HCl (pH 8.1); and 500 mM NaCl), and LiCl buffer (0.25 M LiCl; 1% Nonidet P-40; 1% deoxycholate; 1 mM EDTA; and 10 mM Tris-HCl, pH 8.1) followed by 2 x 5 min washes in Tris EDTA (pH 8.0). Immunoprecipitates were eluted from beads with 2 x 15 min in 125 µl elution buffer (1% SDS, 0.1 M NaHCO3). Ten microliters of 5 M NaCl were added to each of the 250-µl elutes, which were then reverse cross-linked at 65 C for 6 h. DNA fragments were purified using a PCR Purification Kit (QIAGEN, Valencia, CA) according to manufacturer’s instructions. Purified DNA fragments were analyzed by qPCR on a DNA Engine Opticon 2 (Bio-Rad, Hercules, CA) using Quantitect SYBR Green reagent (QIAGEN). Primer pairs used for qPCR are listed in Table 3Go.


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  		Table 3. PCR Primer Sequences Used in qPCR Assays

 
Serial ChIP
Serial ChIP assays were performed as previously described (58). Briefly, ChIP assays were performed as described above through the Tris EDTA (pH 8.0) washes. Complexes were eluted from primary immunoprecipitates by incubation in 10 mM DTT for 30 min at 37 C. Elutes were diluted 1:50 in re-ChIP buffer [1% Triton X-100; 2 mM EDTA; 150 mM NaCl; and 20 mM Tris-HCl (pH 8.1)] followed by immunoprecipitation with second antibodies (see above) at 4 C overnight. Immunocomplexes were recovered, washed, eluted, and analyzed as described above.

Coimmunoprecipitation
H295 cells grown in 10-cm plates were serum starved for 12 h in RPMI + 0.05% BSA and subsequently treated with vehicle (serum-free media), 10–7 M dexamethasone or 10–7 M dexamethasone plus 10–7 M ACTH. Cells were rinsed once with cold PBS and lysed in 600 µl of ice-cold lysis buffer [40 mM HEPES, 120 mM NaCl, 1 mM EDTA, 10 mM pyrophosphate, 10 mM glycerophosphate, 50 mM NaF, 0.5 mM orthovanadate, and EDTA-free protease inhibitors (Sigma)] containing 1% Triton X-100. Lysates were cleared by 10 min centrifugation at 13,000 rpm and nonspecific IgG binding precleared by incubation with Protein A agarose (Invitrogen) and subsequent washing. Immunoprecipitations were performed using polyclonal {alpha}-SF-1 antibodies (1 µg/immunoprecipitation; Upstate Biotechnology, Lake Placid, NY) and 40 µl protein A agarose followed by stringent washing with lysis buffer. Protein lysates were resolved on a 10% SDS-PAGE and transferred to nitrocellulose. Proteins were detected using polyclonal {alpha}-GR antibodies (Santa Cruz Biotechnology), followed by blotting with goat antirabbit horseradish peroxidase (Pierce, Rockford, IL). Detection was performed using Super Signal West Dura Extended Duration Substrate (Pierce).

RNA Isolation and Quantitative PCR
Primary adrenal cultures were prepared as described above and plated at a density of two adrenals per well in a 12-well plate treated with fibronectin. After allowing the cells to adhere for 48 h, they were stimulated with vehicle (serum-free medium), 10–5 M dexamethasone or 10–5 M dexamethasone plus 10–7 M ACTH for 18 h. RNA was isolated using Trizol (Invitrogen) according to the manufacturer’s instructions. One microgram of total RNA was reverse transcribed using the iScript Reverse Transcription Kit (BD Biosciences, San Diego, CA). Quantitative PCR was performed on a DNA Engine Opticon 2 (Bio-Rad) using Quantitect SYBR Green reagent (QIAGEN). Primer pairs used for qPCR are listed in Table 3Go.

Statistics
Statistical analysis was performed using a Student’s t test assuming equal variance. Statistical significance is defined as P ≤ 0.05.


   ACKNOWLEDGMENTS
 
We thank Jorge Iniguez (University of Michigan) for critical discussion of this manuscript, Alex Kim (University of Michigan) for technical assistance in the preparation of primary adrenocortical cells, and Tobias Else (University of Michigan) for crucial intellectual input to the preparation of this manuscript.


   FOOTNOTES
 
This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases/National Institutes of Health Grant RO1 DK62027 (to G.D.H.).

First Published Online July 20, 2006

Abbreviations: ChIP, Chromatin immunoprecipitation; Dax-1, dosage-sensitive sex reversal adrenal hypoplasia congenita critical region of the X-chromosome, gene 1; FXR, farnesoid X receptor; GR, glucocorticoid receptor; GRE, glucocorticoid response element; HA, hemagglutinin; HPA, hypothalamic-pituitary-adrenal; LRH-1, liver receptor homolog-1; Mc2r, melanocortin-2 receptor; qPCR, quantitative PCR; SF-1, steroidogenic factor-1; SHP, short heterodimer partner; StAR, steroidogenic acute regulatory protein.

Received for publication November 17, 2005. Accepted for publication July 10, 2006.


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 DISCUSSION
 MATERIALS AND METHODS
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NURSA Molecule Pages Link:

Nuclear Receptors:   DAX1  |  GR  |  SF-1
Ligands:   Dexamethasone



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