Chicken Ovalbumin Upstream Promoter-Transcription Factor Is a Negative Regulator of Steroidogenesis in Bovine Adrenal Glomerulosa Cells

Carine F. Buholzer, Jean-François Arrighi, Shahnaz Abraham, Vincent Piguet, Alessandro M. Capponi and Andérs J. Casal

Division of Endocrinology, Diabetology and Nutrition (C.B., A.M.C., A.J.C.) and Department of Dermatology and Venereology (J.-F.A., S.A., V.P.), University Hospital, CH-1211 Geneva 14, Switzerland

Address all correspondence and requests for reprints to: Professor Alessandro M. Capponi, Division of Endocrinology, Diabetology, and Nutrition, University Hospital, 24 rue Micheli-du-Crest, CH-1211 Geneva 14, Switzerland. E-mail: alessandro.capponi{at}medecine.unige.ch.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The octapeptide hormone, angiotensin II (AngII) and ACTH stimulate mineralocorticoid biosynthesis in the zona glomerulosa of the adrenal cortex in part by promoting the transcription of the gene coding for the steroidogenic acute regulatory (StAR) protein. We have examined whether chicken ovalbumin upstream promoter-transcription factor (COUP-TF), a member of the orphan nuclear receptor family of transcription factors, is involved in this transcriptional regulation. We analyzed COUP-TF and StAR mRNA and protein levels in bovine adrenal glomerulosa cells in primary culture. COUP-TF protein was readily detectable in nonstimulated cells, and AngII markedly reduced its expression in a time- and concentration-dependent manner (IC50 = 1 nM), to 46 ± 4.4% of control levels after 6 h, (n = 3; P < 0.01). This repression was paralleled by a marked decrease in COUP-TF mRNA levels, reaching 18 ± 8.8% of controls (n = 3, P < 0.01) after 6 h and by a 20-fold increase in aldosterone output. In bovine glomerulosa cells overexpressing COUP-TFI and -II, the induction of StAR mRNA and protein elicited by AngII was completely suppressed to control levels, and the aldosterone response was significantly reduced (from 4.8 ± 1.1-fold the basal value in mock-infected cells to 1.9 ± 0.5-fold and 2.2 ± 0.7-fold in COUP-TFI- and COUP-TFII-expressing cells, respectively; n = 3; P < 0.01 for both differences). Finally, by using electrophoretic mobility shift assays and chromatin immunoprecipitation, we have shown a direct interaction between COUP-TF and the proximal StAR promoter. These results suggest that COUP-TF exerts a tonic inhibition on steroidogenesis by repressing StAR protein expression and that activators of aldosterone biosynthesis lift this inhibition in part by repressing COUP-TF levels.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
THE MAIN MINERALOCORTICOID, aldosterone, is synthesized primarily in the zona glomerulosa of the adrenal cortex, under the control of three major physiological stimuli, the octapeptide hormone angiotensin II (AngII), ACTH, and extracellular potassium (K+) (1). Aldosterone is synthesized from cholesterol, the common precursor of all steroid hormones, which is stored within intracellular lipid droplets as cholesterol esters and mobilized to the mitochondrion by cholesterol ester hydrolase upon stimulation (2). The rate-limiting step in the activation of steroidogenesis is the delivery of cholesterol from the mitochondrial outer membrane to the inner membrane, where the cytochrome P450 side-chain cleavage enzyme is located (3). Within the mitochondria, cholesterol undergoes an enzymatic cascade leading eventually to the formation of aldosterone.

This crucial step of intramitochondrial cholesterol transfer is performed by the steroidogenic acute regulatory (StAR) protein (4, 5). The ultimate demonstration of the indispensable role of the StAR protein came from congenital lipoid adrenal hyperplasia, an inherited disease that leads to a dramatic deficiency in all steroid hormones and in which loss of function mutations have been discovered in the StAR gene (6).

Because the StAR protein mediates the rate-limiting step in steroidogenesis, its expression must be finely regulated. Indeed, in human NCI-H295R adrenocortical carcinoma cells, hormone-stimulated StAR protein synthesis and steroid production are prevented by inhibition of RNA synthesis (7). In bovine adrenal glomerulosa cells in primary culture, mimicking the calcium signal gener ated by AngII leads to increased expression of StAR mRNA and protein (8). Similarly, treatment of human NCI-H295R cells with AngII results in an increase in StAR mRNA levels within 30 min, with maximal levels being reached within 6 h (7, 9). These results clearly indicate a transcriptional regulation of AngII on StAR gene expression. Various consensus response elements have been identified in the StAR gene promoter (10). Although promoter mapping studies have provided extensive information on activators of the StAR gene, the role of transcription factors that repress StAR protein expression has received less attention. The best characterized repressor of the StAR gene is DAX-1 (dosage-sensitive sex reversal, adrenal hypoplasia congenita critical region on the X chromosome, gene 1), a member of the orphan nuclear receptor family (11, 12, 13). Our laboratory has recently demonstrated that AngII powerfully represses DAX-1 expression in bovine adrenal glomerulosa cells, thus leading to increased StAR expression and aldosterone production (14).

Another potential candidate for a negative regulation of the StAR gene is chicken ovalbumin upstream promoter-transcription factor (COUP-TF), which is usually present as two isoforms, COUP-TFI and COUP-TFII (15, 16). These orphan members of the steroid receptor superfamily play distinct and crucial roles in fetal development of the nervous (17) and cardiovascular (18) systems. In addition to these well-established developmental effects in fetal life, the following pieces of evidence suggest a direct or indirect interaction between COUP-TF and the promoter of the StAR gene and thus an involvement of COUP-TF in adult steroidogenic cell function. First, COUP-TF colocalizes in the nuclei of the adrenal cells with DAX-1 and steroidogenic factor-1 (SF-1), two transcription factors involved in steroidogenesis and in StAR regulation, and its expression is modulated in adrenocortical adenomas, COUP-TFI being lower in aldosterone-producing tumors than in normal tissue (19, 20) and COUP-TFII appearing to be increased in aldosteronoma cells (21). In normal adrenocortical tissue, COUP-TFI and -II expression levels appear to be qualitatively equivalent (20). Second, it has been recently shown that SF-1 and COUP-TF can activate and repress, respectively, bovine CYP17 gene transcription in Y-1 mouse adrenocortical cells (22, 23). Third, a similar competition between SF-1 and COUP-TF for a composite binding site has been demonstrated for other genes involved in steroidogenesis, including human aldosterone synthase, aromatase, and murine DAX-1 genes (23, 24, 25). Taken together, these data raise the question of a possible negative role of COUP-TF in the regulation of StAR gene expression.

In the present work, we have therefore examined whether COUP-TFs are involved in the regulation of basal and AngII- or ACTH-induced aldosterone biosynthesis in bovine adrenal glomerulosa cells in primary culture. Our results show clearly that COUP-TF is a negative regulator of mineralocorticoid biosynthesis.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
COUP-TF Protein Levels in Bovine Glomerulosa Cells and Modulation by AngII and Forskolin
We first examined whether bovine adrenal glomerulosa cells in primary culture express COUP-TF protein. Indeed, as shown in Fig. 1AGo, a robust COUP-TF signal was detected upon Western blot analysis of nuclear proteins from nonstimulated cells. When glomerulosa cells were challenged for 6 h with AngII, a concentration-dependent decrease in COUP-TF protein levels was observed, an inhibition to 46 ± 4.4% of control levels being reached with 10 nM AngII (n = 3; P < 0.01; Fig. 1BGo). Cultured bovine glomerulosa cells produced significant amounts of aldosterone into the medium (2.36 ± 0.2 fmol/µg protein/6 h), and AngII stimulation for 6 h increased aldosterone production 20-fold to 44.9 ± 5.2 fmol/µg/6 h (n = 3; P < 0.01; Fig. 1CGo). A similar activation of aldosterone production was obtained with forskolin, used as a mimicker of ACTH, to 42.2 ± 1.5 fmol/µg/6 h (n = 3; P < 0.01; Fig. 1CGo). To determine the kinetics of the effect of AngII on COUP-TF protein expression, we incubated glomerulosa cells with 10 nM AngII for various periods of time ranging from 0–15 h. As shown in Fig. 2Go, the inhibitory effect of AngII was time dependent: COUP-TF protein levels started to decrease after 30 min, and a complete abolition was reached within 15 h of treatment.



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Fig. 1. AngII Represses COUP-TF Protein Levels in a Concentration-Dependent Manner

Bovine adrenal glomerulosa cells were stimulated for 6 h with AngII at various concentrations. COUP-TF was determined in nuclear extracts and aldosterone was measured in the incubation medium as described in Materials and Methods. A, Representative Western blot of COUP-TF. B, Densitometric analysis of COUP-TF levels in AngII-stimulated glomerulosa cells. C, Aldosterone production in response to AngII (10 nM) or forskolin (Fk, 25 µM). Each value is the mean ± SEM of three experiments performed with separate cell preparations. **, P < 0.01 vs. unstimulated control (Ctrl).

 


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Fig. 2. Time-Dependent Repression of COUP-TF Protein by AngII

Bovine adrenal glomerulosa cells were treated with AngII (10 nM) for the indicated periods of time, after which COUP-TF protein was determined in nuclear extracts as described in Materials and Methods. A, Representative Western blot of COUP-TF. B, Densitometric analysis of COUP-TF levels in AngII-stimulated cells as a function of time, expressed as percentage of control values (Ctrl) at time zero. Each point is the mean value from two separate experiments.

 
AngII-Induced Repression of COUP-TF Is Independent of Protein Synthesis
AngII-induced inhibition of COUP-TF protein expression was paralleled by a decrease in COUP-TF mRNA levels, an inhibition to 18 ± 8.8% of controls (n = 3; P < 0.01) being reached with 10 nM AngII after 6 h, as shown in Fig. 3Go, A and B. To determine whether de novo protein synthesis is required for AngII-mediated COUP-TF repression, we treated bovine glomerulosa cells for 6 h with 10 nM AngII in the presence of 10 µg/ml cycloheximide, a protein synthesis inhibitor. Blockade of protein synthesis affected neither the inhibitory effect of AngII on COUP-TF mRNA expression (Fig. 3Go, A and B) nor its repression of COUP-TF protein levels in glomerulosa cells (Fig. 3Go, C and D). These observations suggest that de novo protein synthesis is not required for AngII-induced repression of COUP-TF. In this same series of experiments, forskolin was as potent as AngII in repressing COUP-TF mRNA (data not shown) and protein levels (Fig. 3Go, C and D).



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Fig. 3. Effect of AngII and Cycloheximide (CHX) Treatment on COUP-TF mRNA and Protein Levels

Bovine adrenal glomerulosa cells were treated for 6 h with AngII (10 nM) in the absence or in the presence of 10 µg/ml cycloheximide. COUP-TF mRNA and protein levels were determined by Northern blot and Western blot analysis, respectively, as described in Materials and Methods. A, Representative Northern blot of COUP-TF mRNA. B, Densitometric analysis of COUP-TF mRNA levels in glomerulosa cells. C, Representative Western blot of COUP-TF. D, Densitometric analysis of COUP-TF protein levels in glomerulosa cells. Values are means ± SEM from three separate experiments. **, P < 0.01 vs. control.

 
AngII Does Not Affect COUP-TF mRNA Stability
To determine whether AngII affects COUP-TF mRNA stability, we incubated control and AngII-stimulated adrenal glomerulosa cells in the presence of 1 µg/ml actinomycin D, a transcription inhibitor, for various periods of time and determined COUP-TF mRNA levels by Northern blot analysis. As shown in Fig. 4Go, in the presence of actinomycin, COUP-TFI mRNA decayed with a half-life of approximately 8 h, and AngII did not alter COUP-TFI mRNA stability over a 24-h period.



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Fig. 4. Effect of Actinomycin D on COUP-TFI mRNA Levels in AngII-Treated Bovine Adrenal Glomerulosa Cells

Bovine adrenal glomerulosa cells were treated with 2.5 µg/ml actinomycin D in the absence or in the presence of AngII (10 nM) for the indicated periods of time. COUP-TFI mRNA levels were determined by Northern blot analysis as described in Materials and Methods. A, Representative Northern blot of COUP-TFI. B, Densitometric analysis of COUP-TFI mRNA levels. Values are means ± SEM from three separate experiments.

 
Effect of COUP-TF Overexpression on StAR Expression and on Mineralocorticoid Production
Lentiviral vectors have proven to be highly useful infection tools. Indeed, HIV-derived lentiviral vectors efficiently integrate into nondividing cells (31, 32). Because bovine glomerulosa cells in primary culture are practically quiescent and in view of the relatively low transfection efficacy obtained in these cells with the usual transfection reagents, we used HIV-derived vectors to overexpress COUP-TFI and -II in glomerulosa cells. We first infected bovine glomerulosa cells with lentiviral vectors encoding green fluorescent protein (GFP) under control of the human phospholgycerate kinase (hPGK) promoter. The infected cells were analyzed for GFP fluorescence by flow-cytometric analysis 5 d after infection. Ninety-one percent of the cells infected with 500 ng p24 expressed GFP (data not shown). Bovine glomerulosa cells in primary culture were then infected with lentiviral vectors encoding for murine COUP-TFI or COUP-TFII under control of the hPGK promoter. Five days after infection, cells were incubated in the presence of 10 nM AngII for 6 h, and StAR, COUP-TFI, and COUP-TFII mRNA levels were determined by RT-PCR with bovine- and mouse-specific primers. As expected, no endogenous signal was detected in noninfected and mock-infected cells because we used murine probes (Fig. 5AGo). In contrast, the infected cells displayed robust expression of mouse COUP-TFI and -II mRNA, as determined by RT-PCR with mouse-specific primers. As shown in Fig. 5BGo, AngII treatment for 6 h induced an increase of StAR mRNA levels in noninfected and mock-infected cells [to 172 ± 14% and 165 ± 10% of controls, respectively (n = 3; P < 0.01; for both values)]. This increase was entirely blocked in cells overexpressing either COUP-TFI [104 ± 12% of controls; n = 3; not significantly different (NS; P > 0.05)] or COUP-TFII (82 ± 24% of controls; n = 3; NS) (Fig. 5Go, A and B).



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Fig. 5. Effect of COUP-TFI and II Overexpression on StAR mRNA Levels and Aldosterone Production in Bovine Adrenal Glomerulosa Cells

The COUP-TFI and -II isoforms were overexpressed in bovine adrenal glomerulosa cells using lentiviral vectors. StAR mRNA levels and aldosterone production were determined in control (Ctrl) and AngII-stimulated (10 nM, 6 h), noninfected (NI), mock-infected (Mock), and COUP-TFI- or COUP-TFII-infected cells as described in Materials and Methods. A, Representative ethidium bromide staining of murine COUP-TFI and -II mRNA amplified by RT-PCR in control glomerulosa cells and in cells infected with the pRRL-sin-ppt-hPGK viral vector, either mock or encoding for COUP-TFI or -II. B, Densitometric analysis of StAR mRNA in control cells and in cells challenged with AngII. C, Basal and AngII-stimulated aldosterone production in the medium of control and infected bovine glomerulosa cells. Values are means ± SEM, from three separate experiments. **, P < 0.01 vs. control.

 
Paralleling the effect on StAR mRNA under conditions of COUP-TF overexpression, we observed that the AngII-induced increase of StAR protein levels in nontransfected and mock-transfected cells (to 161 ± 5.8% and 125 ± 1.5% of controls, respectively; n = 3; P < 0.01 for both values) was entirely blocked in cells overexpressing COUP-TFI (87 ± 3.2% of controls; n = 3; NS) (Fig. 6Go).



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Fig. 6. Effect of COUP-TFI Overexpression on StAR Protein Levels in Bovine Adrenal Glomerulosa Cells

COUP-TFI was overexpressed in bovine adrenal glomerulosa cells using a classical transient transfection technique as described in Materials and Methods. StAR protein levels were determined by Western blot after 10 nM AngII treatment of the cells for 6 h. A, Representative Western blot. B, Densitometric analysis of StAR protein levels. Values are means ± SEM from three separate experiments. **, P < 0.01 vs. control. Coom., Staining of the gel with Coomassie blue to ensure that equal amounts of protein have been loaded.

 
Basal aldosterone production rate amounted to 3.5 ± 0.4 fmol/µg protein/6 h in both untreated noninfected and mock-infected cells and rose to 17.7 ± 3.6 fmol/µg protein/6 h in noninfected cells and to 16.7 ± 1.4 fmol/µg protein/6 h in mock-infected cells under AngII challenge (n = 3; P < 0.01; for both responses). In contrast, AngII only increased aldosterone production from 5.6 ± 0.2 to 10.7 ± 2.0 fmol/µg protein/6 h in cells overexpressing COUP-TFI and from 4.7 ± 1.2 to 10.7 ± 0.7 fmol/µg protein/6 h in cells overexpressing COUP-TFII (n = 3; P < 0.01 vs. AngII response in noninfected cells) (Fig. 5CGo).

Effect of COUP-TFI on StAR Promoter Activity
To gain further insight into the regulation of StAR gene expression by COUP-TFs, we performed a dual luciferase reporter assay in bovine adrenal glomerulosa cells in primary culture. The full-length 1.3-kb human StAR promoter region directing the expression of the luciferase reporter gene (Fig. 7AGo) was transfected into bovine glomerulosa cells. Treatment of the cells with AngII 10–8 M for 6 h significantly increased StAR promoter activity 2.2 ± 0.37-fold (n = 3; P < 0.01). In contrast, as shown in Fig. 7BGo, this AngII-induced effect was completely abolished in cells that had been cotransfected with either COUP-TFI (0.91 ± 0.07-fold) or COUP-TFII (1.01 ± 0.04-fold) cells (n = 3; NS vs. mock-transfected cells for both treatments).



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Fig. 7. COUP-TFI and -II Prevent StAR Promoter Activity by Directly Interacting with the StAR Promoter in Bovine Adrenal Glomerulosa Cells

A, COUP-TFI and -II were cotransfected with the StAR-luc reporter construct in bovine glomerulosa cells in primary culture as described in Materials and Methods. B, Densitometric analysis of the relative StAR promoter activity in mock-transfected and COUP-TFI- and COUP-TFII-overexpressing cells, in the presence and absence of 10 nM AngII. C, EMSA: nuclear extracts derived from control bovine adrenal glomerulosa cells or cells stimulated with 10 nM AngII were incubated with the labeled StAR promoter fragment probe (see Materials and Methods) in the absence or in the presence of a purified COUP-TFI antibody and submitted to PAGE followed by autoradiography. The EMSA is representative of three separate experiments yielding similar results. D, ChIP assay was performed on bovine adrenal glomerulosa cells using a purified COUP-TFI antibody for immunoprecipitation and specific primers corresponding to the StAR promoter for real-time PCR, as described in detail in Materials and Methods. Representative agarose gel of the amplification product. E, Densitometric analysis of the ChIP assay. Values are means ± SEM from three separate experiments. **, P < 0.01 vs. control.

 
Interaction of COUP-TFI with the StAR Promoter
To examine whether the transcription factor COUP-TFI interacts directly with the StAR promoter for its action, we performed EMSAs. Because direct competition between SF-1 and COUP-TF is known for binding to other gene promoters, we used a 23-bp DNA fragment containing the proximal SF-1 response element of the bovine StAR promoter as a probe. As shown in Fig. 7CGo, lanes 2 and 3, nuclear extracts of bovine glomerulosa cells shifted the labeled probe, generating at least two bands. When the nuclear extracts were preincubated with a purified antibody directed against COUP-TFI, the intensity of the shifted bands was dramatically reduced, thus suggesting that COUP-TFI binds to the proximal StAR promoter in vitro (lanes 4 and 5). No significant differences in the intensity of the shifted bands were observed with nuclear extracts from AngII-treated cells (lanes 3 and 5) in this qualitative assay.

Finally, this interaction was confirmed by in vivo analysis of the physical interaction between COUP-TFI and the StAR promoter, using the chromatin immunoprecipitation (ChIP) assay. As shown in Fig. 7Go, D and E, immunoprecipitation with a COUP-TFI antibody of nuclear extracts of glomerulosa cells, followed by real-time PCR of the StAR promoter, yielded a band of the expected size on agarose gel, indicating that the StAR promoter was immunoprecipitated with COUP-TFI and thus strongly suggesting an interaction between COUP-TFI and the proximal StAR promoter. Furthermore, when ChIP was performed on chromatin extracted from AngII-treated cells, a marked and significant decrease in the intensity of the StAR promoter band was observed (61.4 ± 2.8% of control; n = 3; P < 0.01).


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The StAR protein plays a rate-limiting, pivotal role in the activation of steroidogenesis (5). The present study was undertaken in an attempt to investigate the mechanism(s) of the known induction of StAR protein expression in adrenal glomerulosa cells in response to two physiological activators of aldosterone biosynthesis, the octapeptide hormone AngII and ACTH (9, 33). Specifically, we have examined a possible role of COUP-TF proteins in this process.

Four main conclusions can be drawn from the present work: 1) COUP-TF proteins are abundantly present in bovine adrenal glomerulosa cells in primary culture and are repressed by AngII and forskolin; 2) COUP-TFI and -II repress AngII-induced StAR mRNA and protein expression; 3) COUP-TFI and -II overexpression prevents AngII-induced mineralocorticoid biosynthesis; 4) COUP-TFI interacts directly with the proximal StAR promoter.

First, in agreement with previous immunohistochemical studies performed in mice (18), we detected high basal COUP-TF protein levels in resting bovine adrenal glomerulosa cells. In man, recent studies have shown that COUP-TF expression is inversely correlated with steroid production in adrenocortical tumors (20). These data suggested that this transcription factor could function as a repressor of steroidogenesis and that the mechanism of action of physiological activators of steroid production, such as AngII or ACTH (mimicked by forskolin), could operate, in part, through a reduction of COUP-TF protein levels. Indeed, as shown in the present study, both AngII and forskolin strongly diminished COUP-TF mRNA and protein expression, over a range of concentrations similar to those used to induce aldosterone production.

Second, we observed that COUP-TFI and -II repressed StAR mRNA and protein expression. Because the StAR protein controls the rate-limiting step of steroidogenesis, we hypothesized that COUP-TF might act at this level. COUP-TF is known to negatively regulate the transcriptional activity of SF-1 and to repress various actors in the steroidogenic cascade (23, 34), such as the gene encoding cytochrome P450 steroid 17{alpha}-hydroxylase, by competing with SF-1 for the binding to repeated cAMP-responsive sequences spaced by six nucleotides (repCRS2) (35). Furthermore, COUP-TF and SF-1 have also been shown to bind to overlapping binding sites involved in the regulation of the oxytocin gene in the bovine ovary (36). Although multiple SF-1 binding elements required for hormonal induction have been identified in the StAR protein gene 5'-flanking promoter region (10, 37, 38), COUP-TF has not been reported, so far, to bind to the StAR promoter or to affect its activity. Interestingly, our laboratory has demonstrated recently that the levels of another orphan nuclear receptor, DAX-1, which is known to repress StAR protein expression also by interacting negatively with SF-1 either directly or indirectly (12), are down-regulated by AngII and forskolin in bovine adrenal glomerulosa cells and that this effect is correlated inversely with StAR protein expression and aldosterone biosynthesis (14).

We therefore examined whether COUP-TF could repress StAR expression levels by overexpressing the transcription factor in bovine glomerulosa cells. To overcome the usually low rates of efficiency observed in these cells when utilizing classical transfection reagents, we have used an HIV-derived lentiviral system yielding high levels of gene delivery in primary cells (27, 28). Indeed, infected bovine glomerulosa cells expressed high amounts of exogenous, murine COUP-TFI and -II mRNA that could be distinguished from endogenous COUP-TF mRNAs by using species-specific primers for the RT-PCR amplification. Moreover, we show here that overexpression of either isoform of COUP-TF resulted in a complete suppression of AngII-induced StAR mRNA and protein induction, a finding strongly suggesting a direct or indirect repressor role for COUP-TF on StAR promoter activity. These results were indeed confirmed by luciferase reporter gene assay of the StAR promoter in cells overexpressing COUP-TF, which showed a dramatic reduction in AngII-induced StAR promoter activity. COUP-TFI and COUP-TFII were equally effective in repressing StAR mRNA expression, although only the latter has been shown to be expressed in the cortex of the adrenal gland during organogenesis in mice (18). However, both COUP-TFI and COUP-TFII have been shown to be present in normal and tumoral human adrenocortical tissue in immunohistological studies (19, 21), and we observed a similar expression pattern of the mRNA of both isoforms in bovine glomerulosa cells (data not shown).

Third, at the functional level, we observed an inverse relationship between mRNA COUP-TF levels and AngII-induced aldosterone output, a finding that corroborates our hypothesis that the hormone acts by lifting the repression exerted by COUP-TF on StAR gene expression. Interestingly, overexpression of COUP-TFs did not affect either basal StAR expression or basal aldosterone production. This result was actually expected, in view of the robust expression of endogenous COUP-TFs in bovine glomerulosa cells, which presumably already exert a maximal repressor effect on these parameters in the resting state. Only under conditions of stimulation with AngII, when endogenous COUP-TFs are repressed, is the effect of overexpressing exogenous COUP-TF visible, preventing the normal StAR induction and steroid production elicited by the hormone. In addition, COUP-TF is certainly not the sole regulator of StAR expression, and other factors, such as DAX-1 and SF-1 (14), most probably also contribute to basal StAR gene expression. This finding is also in agreement with clinical observations showing that COUP-TFI and -II protein levels are markedly reduced in steroid-producing human adrenal tumors, as assessed by Western blot analysis (19), but contrasts with immunohistological observations indicating that COUP-TFII is highly expressed in nuclei of aldosteronoma cells (21). One possible explanation for this discrepancy could be found in the different methodologies used in these studies, as immunoblotting yields more quantitative measures than immunocytochemistry.

Fourth, we have shown a direct physical interaction of COUP-TF with the proximal StAR promoter both in vitro and in vivo. In EMSAs performed with a probe corresponding to a region of the proximal StAR promoter known to be critical in the control by SF-1 of StAR gene expression, the intensity of two bands was dramatically reduced by preincubation of the nuclear extracts with the anti-COUP-TF antibody. This finding indicated that COUP-TF can interact either directly or indirectly with the StAR promoter. Whether the two shifted bands represent complexes of COUP-TF with other proteins remains to be elucidated.

Finally, ChIP assays allowed us to clearly demonstrate and quantify a direct binding of COUP-TF with the proximal StAR promoter. The decrease in StAR promoter yield in the ChIP assay observed after AngII treatment matches exactly the repression of COUP-TF that we have observed, thus confirming that AngII lifts a direct repression exerted by COUP-TF on the StAR promoter.

In summary, the data we have presented in this work indicates that the transcription factor COUP-TF, which is essentially known to play a developmental role in the adrenal cortex, is also implicated in modulating the differentiated function of adrenal glomerulosa cells, namely aldosterone biosynthesis. Under basal conditions, COUP-TF exerts a tonic inhibition on steroidogenesis by repressing StAR protein expression, thus preventing cholesterol substrate supply to the mitochondria. Challenge of glomerulosa cells with physiological activators of mineralocorticoid production lifts this inhibition by reducing COUP-TF protein levels and COUP-TF binding to the StAR promoter, thus allowing the StAR protein to be expressed in higher amounts, intramitochondrial cholesterol transfer to occur and, eventually, aldosterone synthesis to proceed.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Bovine Adrenal Zona Glomerulosa Cell Culture and Treatments
Bovine adrenal glands were obtained from a local slaughterhouse. Zona glomerulosa cells were prepared by enzymatic dispersion with dispase and purified on Percoll density gradient as reported previously (26). Primary cultures of purified glomerulosa cells were maintained in DMEM as described in detail elsewhere (26). The cells were grown in 10-cm petri dishes (1 x 107 cells per dish) and kept in serum-free medium for 24 h before experiments, which were performed on the third day of culture. Cells were then washed and incubated at 37 C in serum-free medium containing various agents, for varying periods of time as appropriate. At the end of the incubation period, the media were collected and cells were processed for protein or total RNA extraction as described below.

Generation of Lentiviral Vectors and Infection of Bovine Adrenal Glomerulosa Cells
Lentiviral vectors were generated as described previously (27, 28). COUP-TFI and -II 1.5-kb fragments were excised out of the original mouse plasmids pCDNA3-COUP-TFI and pCDNA3-COUP-TFII with BamHI and XhoI restriction enzymes. The digested fragments were separated on agarose and subsequently purified (JetQuick extraction kit, Genomed GmbH, Basel, Switzerland). COUP-TF cDNAs were then inserted into the pRRL-sin-ppt-hPGK vector digested with BamHI and SalI.

Lentiviral vectors were obtained by transient transfection of 293T human embryonic kidney cells. A total of 2 x 106 293T cells were seeded in 10-cm petri dishes 24 h before transfection, in DMEM containing 10% fetal calf serum and glutamine, in the presence of chloroquine (final concentration, 25 µM) and in a 5% CO2 atmosphere. Plasmid DNA (20 µg) was used for the transfection of one 10-cm dish using the calcium-phosphate precipitation method: 2.5 µg envelope-coding plasmid pMDG, 7.5 µg packaging plasmid pCMV{Delta}R8.91 (which expresses Gag, Pol, Tat, and Rev), and 10 µg of either COUP-TFI- or COUP-TFII-expressing pRRL-sin-ppt-hPGK plasmid. The cells were washed with PBS after 14–16 h and were incubated in serum-free DMEM in a 10% CO2 atmosphere. The conditioned medium was collected after another 24 h and filtered through 0.45-µm pore polyvinylidene difluoride filters. The vectors were concentrated 100-fold by one round of centrifugation at 50 000 x g for 90 min and resuspended for 20 min at room temperature in PBS. Titers of HIV vectors were determined by p24 antigen ELISA. Titers were found to range from 5 x 106 to 107 infectious units/ml. Stock of vectors was stored at –80 C until used for infection of the bovine glomerulosa cells. All infections were performed in 12-well plates, with 200,000 glomerulosa cells per well and 500 ng of p24 were used for each COUP-TF.

Determination of Aldosterone Production
Aldosterone content in incubation media was measured by direct RIA using a commercially available kit (Diagnostic Systems Laboratories, Webster, TX). Aldosterone production was normalized and expressed per microgram cellular protein.

Western Blot Analysis
For the determination of protein expression levels, bovine glomerulosa cells were washed twice in ice-cold PBS, and nuclear extracts were prepared according to the procedure of Schreiber et al. (29). Mitochondrial extracts were prepared as described elsewhere (30). Proteins were quantified using a protein microassay (Bio-Rad, Munich, Germany). Equal amounts of protein (15 µg) were resolved by 10% SDS-PAGE and transferred onto polyvinylidene difluoride membranes. Western blot analysis was carried out with rabbit antisera directed either against COUP-TF (kindly provided by Professor Ming-Jer Tsai, Houston, TX) or against StAR (kindly provided by Professor Douglas Stocco, Lubbock, TX). Immunoreactive proteins were visualized by the enhanced chemiluminescence method (Amersham Biosciences, Otelfingen, Switzerland).

Northern Blot Analysis
Glomerulosa cell total RNA was extracted using the RNAgents kit (Promega Corp., Zurich, Switzerland) according to the instructions of the manufacturer. For Northern blot analysis, 30 µg RNA were size fractionated on a 1% formaldehyde agarose gel, vacuum transferred onto Nytran membranes (Schleicher & Schuell, Dassel, Germany), and fixed by UV cross-linking. The integrity of the 18 S and 28 S RNA was checked by ethidium bromide staining of the gel. Hybridization was performed using the previously cloned 1.5-kb mouse COUP-TF cDNA. The cDNA was labeled with [{alpha}32P]dCTP using the Rediprime II random primer labeling kit from Amersham Biosciences. Northern blots were prehybridized in Rapid Hybridization Buffer (Amersham Biosciences) at 65 C for 30 min. The {alpha}32P-labeled probe (specific activity: 2 x 106 cpm/ng DNA) was then added, and the incubation was continued for 2.5 h at 65 C. Blots were washed for 5 min and 15 min successively at room temperature in 2x saline sodium citrate (SSC)-0.1% SDS, and then for 15 min in 1x SSC-0.1% SDS. The final wash was performed at 65 C for 15 min in 1x SSC-0.1% SDS. RNA-cDNA hybrids were visualized on Hyperfilms (Amersham Biosciences) after a 12- to 14-h exposure period. Blots were stripped and reprobed with mouse glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA (Ambion, Lugano, Switzerland) to assess RNA loading.

Semiquantitative RT-PCR
RT-PCR was used to evaluate StAR and COUP-TF mRNA abundance in response to various treatments. Total RNA (100 ng) was amplified by one-step RT-PCR Access system (Promega Corp., Madison, WI) according to the instructions of the manufacturer. The primers were: bovine StAR 5'-TGA AGA GCT TGT GGA GCG CA-3'(forward) and 5'-TGC GAG AGG ACC TGG TTG AT-3'(reverse), corresponding to positions +538–557 and +902–921, respectively (GenBank accession no. Y17259); mouse COUP-TFI 5'-ATG CAC TCA CAA ACG GGG AT-3' (forward) and 5'-ACT GTG CGA AGA GAG GGC AAT-3' (reverse), corresponding to positions +514–533 and +1121–1141, respectively (GenBank accession no. U07625); mouse COUP-TFII 5'-AAG CTG TAC AGA GAG GCA GGA-3' (forward) and 5'-AGA GCT TTC CGA ACC GTG TT-3' (reverse), corresponding to positions +475–495 and +1106–1125, respectively (GenBank accession no. X76653); GAPDH 5'-ATG GTG AAG GTC GGA GTG-3' (forward) and 5'-TGC AGA GAT GAT GAC CCT C-3' (reverse), corresponding to positions +82–99 and +426–444 (GenBank accession no. NM002046). Primers yielded products of the expected size corresponding to 362 bp for GAPDH, 383 bp for StAR, 627 bp for COUP-TFI, and 650 bp for COUP-TFII. PCR products (10 µl) were analyzed on 1.2% agarose gel and quantified by densitometry using a Computing Densitometer (Molecular Dynamics, Inc., Sunnyvale, CA). All mRNA levels were normalized to GAPDH mRNA.

Dual Luciferase Reporter Assay System
The StAR-luc reporter construct was kindly provided by Dr. Douglas Stocco. Briefly, the full-length 1.3-kb StAR promoter region was cloned into the plasmid vector pGL2 (Promega Corp.), which contains firefly luciferase as a reporter gene. Other plasmids used in these experiments included the pGL2 basic vector as a negative control, which contains no promoter sequence; pGL2 control used as a positive control, which places the luciferase gene under the control of the simian virus 40 (SV40) promoter; pRL-SV40 vector, which places the Renilla luciferase under the control of the SV40 promoter was used as an internal control; and pCR3.1 vector in which murine COUP-TFI or COUP-TFII cDNA were subcloned under the control of the cytomegalovirus promoter (kindly provided by Professor Ming-Jer Tsai, Houston, TX). Bovine glomerulosa cells were cotransfected with the various vectors according to the manufacturer’s protocol (Effectene Transfection Reagent kit, QIAGEN GmbH, Hilden, Germany). Cells were harvested 48 h after transfection, and extracts were made in Passive lysis buffer (Promega Corp.). Luciferase and Renilla activities were quantified using a Luminoskan Ascent (Labsystems, Catalys AG, Wallisellen, Switzerland). The luciferase assay results were normalized to Renilla activity to compensate for variations in transfection efficiency. Each treatment group contained at least triplicate cultures.

EMSA
Double-stranded DNA (5'-gatCACAGCCTTCAGCTGGAGGTATTT annealed with 5'-gatcAAATACCTCCAGCTGAAGGCTGTG) corresponding to nucleotides –49 to –26 of the bovine StAR promoter according to the sequence published by Rust et al. (39) was [{alpha}-32P]dATP labeled by Klenow (Promega Corp.) fill-in. Nuclear protein extracts (15 µg) were incubated for 20 min at room temperature with 30, 000 cpm/min of labeled probe and 2 µg of poly[d(IC)] in EMSA binding buffer. The reaction mixture was loaded onto on a nondenaturing 5% polyacrylamide gel for 2 h at 200 V in a cold room. Gels were dried and exposed to autoradiography films (Kodak Biomax MR, Sigma-Aldrich GmbH, Steinheim, Germany) for 72 h. In supershift experiments, 1 µg purified rabbit polyclonal anti-COUP-TFI (kindly provided by Dr. M. L. Dufau, Bethesda, MD) or anti-SF-1 (kindly provided by Professor K. Morohashi, Okasaki, Japan) antibody was added to the mixture 30 min before the labeled probe.

ChIP Assay
Chromatin cross-linking was performed by adding 1% formaldehyde to bovine glomerulosa cells at room temperature for 8 min. The reaction was stopped by adding glycine to a final concentration of 0.2 M. Cells were then washed twice with ice-cold PBS, collected in 5 ml PBS, and harvested by brief centrifugation. Cells were resuspended in ice-cold cell lysis buffer (10 mM Tris-HCl, pH 8.0; 1 mM EDTA; 0.5% Nonidet P-40; protease inhibitors), incubated on ice for 5 min, and briefly centrifuged. Cells were then resuspended in nuclear lysis buffer (10 mM Tris-HCl, pH 8.0; 1 mM EDTA; 1% Triton X-100; 0.5% NaDOC; 0.5% sarcosyl; 0.5 M NaCl; protease inhibitors), incubated at room temperature for 5 min while vortexing vigorously several times, and washed in PBS. Sonication of the cells was performed 30 times for 30 sec in TEN buffer (10 mM Tris-HCl, pH 8.0; 1 mM EDTA; 100 mM NaCl) with a Branson 250 sonifier, followed by centrifugation for 15 min at 14,000 rpm in an Eppendorf table centrifuge. Supernatant was collected and diluted 5-fold in dilution/incubation buffer (20 mM HEPES, pH 8.0; 0.2 M NaCl; 2 mM EDTA; 0.1% NaDOC; 1% Triton X-100; 100 µg/ml salmon sperm DNA; 1 mg/ml BSA; protease inhibitors) followed by immunoclearing with 20 µl protein A Sepharose CL-4B (Pharmacia Biotech AB, Uppsala, Sweden) for 30 min at room temperature, to which 5 µl of specific anti-COUP-TFI antibody were added for overnight incubation at 4 C. The mixture was then cleared from precipitated material by centrifugation at 10,000 rpm for 10 min. Supernatant was incubated with 12 µl Protein A Sepharose (Amersham Biosciences) for 1 h at room temperature. Precipitate was recovered by centrifugation at 3000 rpm for 1 min and washed seven times in RIPA buffer (50 mM HEPES, pH 7.6; 1 mM EDTA; 0.5 M LiCl; 0.7% NaDOC; 1% Nonidet P-40) and once in TE buffer (10 mM Tris-HCl; pH 8.0; 1 mM EDTA) and extracted twice with 100 µl elution buffer (100 mM Tris-HCl, pH 8.0; 1% SDS). Eluates were pooled, treated with proteinase K (100 µg/ml) for 2 h at 42 C, heated at 65 C overnight to reverse the formaldehyde cross-linking, and treated by phenol extraction and ethanol precipitation. The DNA pellet was dissolved in 15 µl of TE buffer. DNA (5 µl) was used for real-time PCR. The primers were located on the proximal part of the bovine StAR promoter: forward, 5'-AGA CTC CTG GTG AGG CAA TC-3'; and reverse, 5'-CTG CGG CCA GAT GAT GTG TT-3', according to the GenBank sequence (accession no. Y17260), and yielding an amplification product of 137 bp.

Analysis of Data
Results are expressed as means ± SEM. The mean values were compared by ANOVA using Fisher’s test. A value of P < 0.05 was considered as statistically significant. Quantification of immunoblots and autoradiograms was performed using a Molecular Dynamics Computing Densitometer.


    ACKNOWLEDGMENTS
 
We thank Dr. Ming-Jer Tsai (Baylor College of Medicine, Houston, TX) for his generous gift of COUP-TF cDNA and antibody; Dr. Maria Dufau (National Institutes of Health, Bethesda, MD) for the anti-COUP-TF antibody used in the ChIP assay; and Manuella Rey and Rachel Porcelli for their excellent technical assistance.


    FOOTNOTES
 
This work was supported by Swiss National Science Foundation Grant 3100A0-100797 (to A.M.C.) and by a grant from the Fondation Gustave Prévot (to A.M.C. and C.F.B.).

First Published Online September 16, 2004

Abbreviations: AngII, Angiotensin II; ChIP, chromatin immunoprecipitation; COUP-TF, chicken ovalbumin upstream promoter-transcription factor; DAX-1, dosage-sensitive sex reversal, adrenal hypoplasia congenita critical region on the X chromosome, gene 1; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GFP, green fluorescent protein; hPGK, human phospholgycerate kinase; SDS, sodium dodecyl sulfate; SF-1, steroidogenic factor 1; SSC, saline sodium citrate; StAR, steroidogenic acute regulatory; SV40, simian virus 40.

Received for publication February 10, 2004. Accepted for publication September 10, 2004.


    REFERENCES
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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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