SF-1 (Steroidogenic Factor-1), C/EBPß (CCAAT/Enhancer Binding Protein), and Ubiquitous Transcription Factors NF1 (Nuclear Factor 1) and Sp1 (Selective Promoter Factor 1) Are Required for Regulation of the Mouse Aldose Reductase-Like Gene (AKR1B7) Expression in Adrenocortical Cells

Christelle Aigueperse, Pierre Val, Corinne Pacot, Christian Darne, Enzo Lalli, Paolo Sassone-Corsi, Georges Veyssiere, Claude Jean and Antoine Martinez

UMR Centre National de la Recherche Scientifique 6547 Physiologie Comparée et Endocrinologie Moléculaire (C.A., P.V., C.P., C.D., G.V., C.J., A.M.) Université Blaise Pascal Clermont II, Complexe Universitaire des Cézeaux 63177 Aubière cedex, France
Institut de Génétique et de Biologie Moléculaire et Cellulaire (E.L., P.S.C.), Centre National de la Recherche Scientifique, INSERM Université Louis Pasteur, BP 163 67404 Illkirch, Strasbourg, France


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The MVDP (mouse vas deferens protein) gene encodes an aldose reductase-like protein (AKR1B7) that is responsible for detoxifying isocaproaldehyde generated by steroidogenesis. In adrenocortical cell cultures, hormonal regulation of MVDP gene occurs through the cAMP pathway. We show that in adrenals, the pituitary hormone ACTH regulates MVDP gene expression in a coordinate fashion with steroidogenic genes. Cell transfection and DNA-binding studies were used to investigate the molecular mechanisms underlying MVDP gene regulation in Y1 adrenocortical cells. Progressive deletions of upstream regulatory regions identified a -121/+41 fragment that was sufficient for basal and cAMP-mediated transcriptional activities. Gel shift assays showed that CTF1/nuclear factor 1 (NF1), CCAAT enhancer binding protein-ß (C/EBPß), and selective promoter factor 1 (Sp1) factors bound to cis-acting elements at positions -76, -61, and -52, respectively. We report that the cell-specific steroidogenic factor-1 (SF-1) interacts specifically with a novel regulatory element located in the downstream half-site of the proximal androgen response element (AREp) at position -102. Functional analysis of SF-1 and NF1 sites in the -121/+41 promoter showed that mutation of one of them decreases both constitutive and forskolin-stimulated promoter activity without affecting the fold induction (forskolin stimulated/basal). Individual mutations of C/EBP and Sp1 sites resulted in a loss of more than 50% of the cAMP-dependent induction. When both sites were mutated simultaneously, cAMP responsiveness was nearly abolished. Thus, in adrenocortical cells, both SF-1 and NF1 are required for high expression of the MVDP promoter while Sp1 and C/EBPß functionally interact in an additive manner to mediate cAMP-dependent regulation. Furthermore, we report that MVDP gene regulation is impaired in stably transfected Y1 clones expressing DAX-1. Taken together, our findings suggest that detoxifying enzymes of the aldose reductase family may constitute new potential targets for regulators of adrenal and gonadal differentiation and function, e.g. SF-1 and DAX-1. (Molecular Endocrinology 15: 93–111, 2001)


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The adult adrenal cortex is divided into three morphologically and functionally distinguishable zones characterized by different patterns of steroidogenic enzyme gene expression (1). The zona glomerulosa produces the mineralocorticoid aldosterone, whereas the zona fasciculata synthesizes glucocorticoids: cortisol in the human and bovine species, corticosterone in rodents. The zona reticularis cells produce cortisol and, in primates, the adrenal androgen dehydroepiandrosterone (1). It is also well established that adrenocortical cell functions are regulated by the circulating hormones ACTH and angiotensin II acting on the inner adrenocortical zones and the outer zona glomerulosa, respectively (2, 3). cAMP acts as a second intracellular messenger to transduce the effect of ACTH both acutely and in a long-term manner (2). The acute effect, which occurs within seconds to minutes, increases biosynthesis by making cholesterol available to the steroidogenic enzymes (4). Biochemical and genetic evidence have implicated the steroidogenic acute regulatory (StAR) protein, which accelerates cholesterol transport into the inner mitochondrial membrane, as an essential component of the acute response (4, 5). The long-term effects of ACTH, achieved after many hours, result from increased transcription of several key cAMP-responsive genes that encode steroidogenic enzymes (2). Previous studies have shown that a common factor in this pathway is the orphan nuclear receptor steroidogenic factor 1 (SF-1) (6). SF-1 has been shown to regulate the transcription of many genes involved in steroidogenesis, including steroid hydroxylase genes (7, 8, 9) and the StAR protein gene (10). SF-1 also regulates the expression of genes involved in the steroidogenesis regulation such as the LHß subunit (11), the ACTH receptor (12), and the GnRH receptor (13). Whether SF-1 is hormonally regulated is still unclear. It has been shown that DAX-1 (dosage-sensitive sex reversal-adrenal hypoplasia congenita critical region on the X-chromosome, gene 1) gene expression is up-regulated by SF-1 in vitro as in vivo (14, 15) and that DAX-1 inhibits SF-1-mediated transactivation (16). Finally, DAX-1 has been proposed to be involved in the regulation of steroidogenesis by repressing StAR, P450scc, and 3ß-hydroxysteroid dehydrogenase (3ß-HSD) expression (17). Thus, it seems that regulation of SF-1 target genes is ensured by antagonistic pathways that are necessary to establish proper development and functions of steroidogenic organs.

The first step of steroidogenesis is the cleavage of the cholesterol side chain by P450scc, resulting in the formation of pregnenolone and isocaproaldehyde (4-methyl pentanal), which is further metabolized to isocaproic acid and isocapryl alcohol (18). While detailed mechanisms of enzymatic catalysis and gene regulation have been extensively studied for steroidogenic enzymes, little is known about those involved in steroid metabolism. Recent studies have suggested that members of the aldo-keto reductase (AKR) family could be related to steroid metabolism. First, it has been shown that degradation of steroids by reduction of their aldehyde and ketone groups is not primarily due to specific dehydrogenases but results from the broad specificity of enzymes of the AKR family (19, 20). Second, members of this family may be responsible for detoxifying aldehydes generated by steroidogenesis or derived from lipid peroxidation in mammalian adrenals (21). The AKR family consists of at least 42 members that catalyze the reduction of a broad range of substrates including aldoses, aliphatic and aromatic aldehydes and ketones, prostaglandins, and xenobiotics such as chemotherapeutic drugs (see Ref. 22 for review and nomenclature). Among the AKR family, aldose reductase has been implicated in the etiology of diabetic complications (23). A subgroup of the AKR family, including the mouse vas deferens protein (MVDP: AKR1B7) (24), the mouse fibroblast growth factor 1-regulated protein (FR-1: AKR1B8) (25), and the Chinese hamster ovary cell-derived CHO reductase (AKR1B9) (26), is closely related to aldose reductase. The presence of human homologs of this subgroup has recently been reported (27, 28). Although MVDP represents a major secretory component of the vas deferens, this expression profile is restricted to the mouse species (29). More recently, MVDP was shown to be highly expressed in the adrenals from various rodents under the control of the hypothalamo-pituitary-adrenal axis (30, 31). This evolutionary conserved expression of MVDP is restricted to the zona fasciculata and, in human and murine adrenocortical cells, the MVDP gene is induced by cAMP at both mRNA and protein levels (31). The involvement of MVDP in steroidogenic functions was recently demonstrated by our group (32). In adrenocortical cells and in adrenal glands, MVDP plays an essential role in detoxifying isocaproaldehyde generated by the conversion of cholesterol to pregnenolone, which is the rate-limiting step in steroidogenesis.

In light of these data, our objective was to examine the MVDP upstream regulatory regions for potential binding sites for transcription factors that may be involved in the hormonal regulation of MVDP gene transcription in adrenocortical cells. Deletion and site- directed mutagenesis allowed us to identify a proximal promoter region spanning positions -121 to +41 that confers basal and cAMP-induced regulation through the combinatorial action of SF-1, nuclear factor 1 (NF1), C/EBPß, and selective promoter factor (Sp1) factors. Furthermore, we demonstrate that SF-1 interacts with a novel regulatory element lying within a previously characterized androgen responsive element. Our reports also suggest that MVDP, a gene encoding a detoxifying enzyme, may be considered as a novel target gene for SF-1/DAX-1 antagonistic actions.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
MVDP Expression in Adrenals Is Regulated by ACTH
As previously shown, the MVDP gene is highly expressed in adrenal glands from adult mouse (Fig. 1Go). To determine whether or not the accumulation of MVDP mRNA in adrenals was under the control of pituitary ACTH, as has been shown for steroidogenic genes, hormonal treatments were performed. In adult mice treated for 5 days with dexamethasone to inhibit ACTH secretion, MVDP mRNA levels were dramatically decreased by more than 4-fold when compared with control animals (vehicle treated) (Fig. 1Go). A 2-day ACTH administration to dexamethasone-treated animals was sufficient to induce a 9-fold increase in MVDP mRNA levels. As shown in Fig. 1Go, the responsiveness of MVDP gene to variations of ACTH concentrations was similar to that of steroidogenic genes such as P450scc and StAR the mRNA levels of which were decreased by 3-fold and induced over 3-fold in the same experimental conditions.



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Figure 1. Hormonal Regulation of MVDP in Adrenal Glands

mRNA levels of MVDP, P450scc, and StAR were determined by Northern blotting using pooled adrenals from six adult mice treated with vehicle alone (5 days), dexamethasone (5 days, DEX), and dexamethasone (5 days) plus ACTH for the last 2 days. Analyses were performed using 25 µg of total RNAs. Blots were sequentially probed with the indicated 32P-labeled cDNA probes.

 
MVDP Transcription Start Site in Adrenals
In vas deferens, a major transcription start site has been located on an adenine residue 46 nucleotides upstream from the ATG initiation codon (33). RNAse protection experiments were performed to map the transcription start site in adrenal glands (Fig. 2Go). Total RNAs from adrenals and vas deferens were hybridized to a radiolabeled riboprobe encompassing the previously identified transcription start site. Nonhybridized RNAs were digested by RNAse, and resistant complexes were resolved on a sequencing gel. As shown by the similar profiles in the two organs, the transcription start site is the same in both adrenals and vas deferens and maps to the previously identified start site (33). This result was also confirmed by 5'-RACE (rapid amplification of cDNA ends) experiments (data not shown).



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Figure 2. Identification of MVDP Transcription Start Site in Adrenals

Total RNA from mouse adrenals (10 µg) and vas deferens (2 µg) was subjected to a RNase protection reaction with a probe encompassing the previously identified transcription start site (33 ). Digestion-resistant hybrids were resolved in a 6% polyacrylamide denaturing gel next to a dideoxy sequencing reaction of the probe. Note that RNA migration is slightly retarded compared with DNA in such denaturing gels. Arrowhead indicates a major transcription start site. Asterisks indicate minor start sites.

 
A Proximal Region (-121 to +41) of the MVDP Gene Is Sufficient for Both Constitutive and cAMP-Induced Expression
Our previous results showed that a 3-h treatment of Y1 adrenocortical cells with forskolin is sufficient to reach maximal levels of MVDP mRNA, suggesting that cAMP may regulate MVDP gene expression at transcriptional levels (31). To determine DNA regions that confer basal and cAMP-induced transcription, a series of 5'-deletion mutants of the 1.8-kb upstream regulatory region of the MVDP gene were fused to the chloramphenicol acetyltransferase (CAT) reporter gene and then transiently transfected into Y1 cells. All MVDP promoter constructs had significant transcriptional activity. Compared with the p0.16 CAT plasmid activity (referred to as 100%), basal CAT activity varied slightly after 5'-deletions from 35% with the p1.8 CAT plasmid to 115% with the p0.5 CAT construct (Fig. 3Go). In contrast, a drastic decrease (83%) of CAT activity was observed when the sequence from -121 to -40 was deleted (pTATA CAT plasmid). These results suggest that the region between -121 to-40 contains important elements for constitutive expression of the MVDP promoter in Y1 adrenal cells.



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Figure 3. Identification of Regulatory Regions Responsible for Constitutive and cAMP-Induced Expression of MVDP Promoter in Y1 Cells

Restriction fragments from MVDP gene 5'-flanking region were cloned upstream of the CAT reporter gene. Nucleotide numbering is according to the transcription start site, which is indicated by an arrow. CAT activity was determined in protein extracts of Y1 cells transfected with 1.5 µg of each MVDP-CAT construct and incubated in the absence or presence of 10-5 M forskolin (24 h). Results were expressed as percentages of the mean value achieved with p0.16 CAT construct. Fold activation represents the forskolin-stimulated reporter activity divided by the unstimulated level. Values are means ± SEM of at least five independent transfections.

 
After transfection, cells were incubated for 24 h in the absence or presence of forskolin. All constructs showed significant increases in CAT activity (from 3.3 to 46.3-fold) in the presence of forskolin. This effect was specifically mediated by regulatory regions of the MVDP gene since the activity of the SV-40 enhancer/promoter sequence (control pSV2-CAT vector) was unaffected by this treatment (fold induction, 1.1). Thus, it appears that cAMP stimulation of the MVDP gene was achieved, at least in part, at the transcriptional level. The minimal promoter construct limited to the TATA sequence itself (-40/+41) was able to ensure a low level of forskolin-dependent transcription (3.3-fold). On the other hand, a 13.4-fold induction was achieved by the p0.16 CAT plasmid, indicating that cAMP responsiveness of the MVDP minimal promoter was markedly potentiated by the -121/-40 region. Thus, cis elements located between positions -121 to -40 were required to direct strong cAMP-induced transcription. However, the region between -1,285 to +41 gave a higher induction of CAT activity, suggesting that the sequence spanning positions -1,285 to -121 contains important element(s) needed for full cAMP inducibility. Next, the -121 to -40 region sequence was examined to identify putative protein-binding sites that might mediate cAMP-stimulated expression of MVDP in Y1 cells. Surprisingly, sequence analysis of the 162 bp did not reveal any regulatory motifs that resemble the canonical CRE (cAMP response element). This region contains several putative binding sites for known transcription factors including the selective promoter factor 1 (Sp1, -52 to -44), C/EBP (-61 to -54), nuclear factor 1 (NF1, -76 to -63), and androgen receptor (AR, -111 to -97). Gel mobility shift assays were then performed to further examine the nuclear proteins that bind to various regions of the proximal promoter.

Nuclear Proteins from Y1 Cells Bind Specifically to the Sp1, C/EBP, and NF1 Binding Sites of the MVDP Promoter
As shown in Fig. 4AGo, lane 1, addition of nuclear extracts from Y1 cells to end-labeled oligonucleotide containing the -52 Sp1 site (Table 1Go) resulted in the formation of three retarded bands. Complex 1 was the major protein/DNA interaction detected. All three complexes were specific as judged by the ability of an excess of unlabeled -52 Sp1 or canonical Sp1 oligonucleotides to abolish the presence of the shifted bands (Fig. 4AGo, lanes 3 and 4). This pattern was not affected by an excess of oligonucleotides containing a mutated Sp1 site (-52 Sp1m, Table 1Go) (Fig. 4AGo, lane 7). The addition of anti-Sp1 antibodies in electrophoretic mobility shift assay (EMSA) experiments consistently abrogated the formation of complexes 1 and 2 (Fig. 4AGo, lane 10). Sp1 is not the only protein binding to GC boxes. Additional factors such as early growth response protein-1 (Egr-1) and the Wilms’ tumor suppressor (WT1) may also bind to these GC-rich sequences or related motifs (Table 1Go). Thus, oligonucleotides containing high-affinity binding sites for WT1 (WTE) or for Egr-1 were designed and used as competitors. As shown in Fig. 4AGo, lane 5, complex 3 could be competed by an excess of the WTE oligonucleotide, whereas complexes 1 and 2 were unaffected. However, none of the three complexes were affected by monoclonal or polyclonal antibodies against WT1 (not shown). In addition, none of the three complexes could be displaced by either an excess of unlabeled Egr-1 oligonucleotide (Fig. 4AGo lane 6) or by addition of antibodies directed against Egr-1 (not shown). Although the nature of the minor complex 3 remains unknown, these results show that in Y1 cells, proteins of the Sp1 family generate the major binding complexes to the -52/-44 element of the MVDP gene. Because cAMP induced the MVDP proximal promoter activity, we examined whether the relative amount of factors bound to the -52 Sp1 probe was changed in nuclear extracts from forskolin-treated cells. There was no apparent effect of forskolin treatment on the binding activity of the Sp1 protein family (Fig. 4AGo, compare lanes 1 and 2).



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Figure 4. Binding of Sp1, C/EBPß, and NF1, Present in Y1 Nuclear Extracts, to the -121/+41 Promoter of the MVDP Gene

A, Identification of proteins binding to the -52 Sp1 binding site. Y1 nuclear proteins (5 µg), with or without 24 h stimulation with forskolin, were incubated with a 32P-labeled probe corresponding to the -52 Sp1 binding site in the absence or presence of a 50-fold molar excess of unlabeled competitor oligonucleotides. The competitors were either the unlabeled wild-type Sp1 site (-52 Sp1), canonical Sp1 site (Sp1c), WT1 (WTE) and Egr-1 binding sites (Table 1Go), mutant version of the MVDP Sp1 site (-52 Sp1m), or an unrelated site (-76 NF-1). Arrows indicate the three specific DNA-protein complexes. Complexes 1 and 2 were abrogated upon addition of Sp1-specific antibody. B, Identification of proteins binding to the -61 C/EBP binding site. Five micrograms of nuclear proteins prepared from unstimulated or forskolin-stimulated Y1 cells or 3 µl of in vitro translated C/EBPß were incubated with 32P-labeled probe corresponding to the -61 C/EBP binding site. The competitors were either the wild-type C/EBP binding site (-61 C/EBP), canonical C/EBP site (C/EBPc), mutant version of the MVDP C/EBP site (-61 C/EBPm), or -76 NF1 binding site. Arrow indicates the DNA-protein complex. Specific complexes were supershifted upon addition of C/EBPß-specific antibody. C, Identification of proteins binding to the -76 NF1 binding site. Y1 nuclear proteins (5 µg), with or without 24 h stimulation with forskolin, were incubated with a 32P-labeled probe corresponding to the -76 NF1 binding site in the absence or presence of a 50-fold molar excess of unlabeled competitor oligonucleotides. The competitors were either the wild-type NF1 site (-76 NF1), canonical NF-1 site (NF1c), mutant version of the MVDP NF-1 site (-76 NF1m), or -52 Sp1 binding site. Arrow indicates the prominent DNA/protein complex. The major part of this complex was shifted upon addition of anti-CTF1/NF1 antibody. NS, Nonspecific complexes. Asterisks indicate supershifted complexes.

 

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Table 1. Sequences of the Oligonucleotides Used in EMSAs

 
As shown in Fig. 4BGo, lane 1, incubation of nuclear extracts from Y1 cells with the -61 C/EBP-labeled probe resulted in the formation of a retarded complex that probably contains several proteins. The specificity of the retarded complex was checked by competition with an excess of unlabeled oligonucleotides: either homologous (-61 C/EBP, lane 5), canonical (C/EBPc, lane 6), mutated (-61 C/EBPm, lane 7), or unrelated (Egr-1, lane 8). As shown in Table 1Go, the -61 C/EBP binding site was more related to the enhancer core motif than to the GCAAT motif. The intensity of the specific complex increased when nuclear extracts from forskolin-treated Y1 cells were used (Fig. 4BGo, compare lanes 1 and 2). The addition of an antibody directed against C/EBPß reduced the mobility but did not alter the whole DNA-protein complexes, indicating that other interactions may occur (Fig. 4BGo, lanes 3 and 4). The resulting supershifted complexes resolved into two distinct bands, and only the intensity of the upper band was increased by forskolin treatment (Fig. 4BGo, compare lanes 3 and 4). Note that the intensity of the remaining nonsupershifted complex was also increased upon forskolin stimulation. Antibodies against members of the ATF/CREB family, such as ATF1, ATF2, ATF3, and ATF4, did not affect the profile of the proteins bound to the -61 C/EBP oligonucleotide, suggesting that the main binding activity of this MVDP promoter region is due to C/EBP proteins (not shown). To further characterize this binding site, EMSA were performed using in vitro translated C/EBPß protein produced in rabbit reticulocyte lysate. A specific DNA-protein complex was formed when the -61 C/EBP probe was incubated with in vitro translated C/EBPß that was absent in unprogrammed lysate (Fig. 4BGo, compare lane 9 and 13). This complex was completely supershifted by anti-C/EBPß antibody and was specifically competed by an excess of unlabeled -61 C/EBP oligonucleotide or by a canonical C/EBPc site. Note that the supershifted complex comigrates with the upper band formed with nuclear extracts (Fig. 4BGo, compare lanes 3, 4, and 10). Taken together, these data demonstrate that forskolin treatment increases expression, availability, or DNA-binding activity of C/EBPß and other yet unidentified protein bands to the -61 C/EBP element of MVDP promoter.

When a labeled oligonucleotide containing the putative -76 NF1 binding site was incubated with Y1 cells nuclear extracts, one prominent DNA-protein complex was observed (Fig. 4CGo, lane 1). This interaction was competed by a 50-fold molar excess of the unlabeled homologous (-76 NF1) or canonical NF1 (NF1c) oligonucleotides whereas no competition was observed with either the unrelated Sp1 or mutated NF1 oligonucleotides (Fig. 4CGo, lanes 2–6). The prominent complex was markedly reduced and supershifted by anti-CTF1/NF1 antibody (Fig. 4CGo lane 8), indicating that CTF1 is a major component of the binding activity. As described above for the -52 Sp1 probe, the intensity of the -76 NF1 complex was unaffected by forskolin stimulation of Y1 cells (Fig. 4CGo, compare lanes 1 and 2).

SF-1 Binds to the Androgen Response Element Sequence
When a 33-bp labeled oligonucleotide harboring the proximal MVDP androgen response element (AREp) sequence (Table 1Go) was incubated with nuclear extracts from Y1 cells, a major DNA protein complex was observed (Fig. 5AGo, lane 1). This could not be due to the AR because Y1 cells lack such a receptor (31). Addition of increasing levels of the unlabeled AREp oligonucleotide gradually diminished the DNA-protein complex but failed to completely eliminate it (Fig. 5AGo, lanes 2–4), suggesting a specific but rather low-affinity interaction. As SF-1 has been shown to be an important regulator of several genes involved in adrenal development and function (Ref. 6 for review), we used a known SF-1 binding site as DNA competitor and an anti-SF-1 antibody (directed against SF-1 DNA-binding domain) in mobility shift assays. As shown (Fig. 5AGo, lane 5; Fig. 5BGo, lane 2), the major DNA-protein complex was efficiently competed by a 50-fold molar excess of the unlabeled probe containing a high-affinity SF-1 binding site from the mouse 21-hydroxylase (21-OH) gene promoter (Table 1Go) and was eliminated by addition of an antibody to SF-1 (Fig. 5AGo, lane 7). As expected, no competitive effect was observed when the unrelated -76 NF1 oligonucleotide was used (Fig. 5BGo, lane 3). To further characterize this binding site, EMSAs were carried out using SF-1 protein produced in rabbit reticulocyte lysate. We showed that the in vitro translated SF-1 protein generated an identical gel shift binding pattern to that observed with Y1 nuclear extracts (Fig. 5BGo, compare lanes 1 and 4). Furthermore, the complex was either partially or totally abolished by a 50-fold molar excess of unlabeled oligonucleotides corresponding to the MVDP AREp and SF-1 binding site from the 21-OH gene, respectively (Fig. 5BGo, lanes 5 and 6). Unprogrammed control lysate failed to form any specific complex with MVDP AREp probe (Fig. 5BGo, lane 7). Collectively, these data show that the transcriptional activator SF-1 is able to interact in a specific manner to the MVDP AREp probe.



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Figure 5. SF-1 Binds to an Unusual Site Contained within the Proximal ARE (AREp)

Identification of proteins binding to the AREp. Y1 nuclear extracts (5 µg) (A) and in vitro translated SF-1 protein produced in rabbit reticulocyte lysate (2 µl) (B) were incubated with a 32P-labeled probe corresponding to the MVDP AREp sequence in the absence or presence of an excess of unlabeled competitor oligonucleotides. The competitors were either the wild-type AREp, a known SF-1 binding site from the mouse 21-OH gene, or the -76 NF1 binding site. Arrow indicates the DNA-protein complex. The specific DNA-protein complex was eliminated upon addition of the anti-SF-1 antibody. L, Unprogrammed lysate. Asterisk indicates binding activity of endogenous reticulocyte lysate proteins.

 
As shown in Table 1Go, the region extending from -117 to -93 lacks a consensus SF-1 binding site (PyCAAGGPyPy or PuPuAGGTCA). A second potential ARE (AREd) has been described in the 5'-flanking region of the MVDP gene at position -1,186 (35). To check whether AREd was a potential binding site for SF-1, this site was assayed for its ability to bind Y1 cell nuclear proteins in EMSA. The DNA-protein complex formed was not affected by a 50-fold excess of unlabeled oligonucleotide containing a high-affinity SF-1 site of the 21-OH gene (Fig. 6AGo, lanes 1 and 2), indicating that the protein interacting with the probe is not SF-1. These data confirm that the binding of SF-1 to AREp is sequence specific and suggest that nucleotides that differ between AREp and AREd sequences may be important for SF-1 binding. The AR binds a motif consisting of an imperfect palindromic repeat of the core sequence 5'-TGTTCT-3', separated by three nonconserved nucleotides (Table 1Go and Fig. 6DGo). To characterize this noncanonical SF-1 binding site more precisely, oligonucleotides containing point mutations within or adjacent to the core ARE sequence were used. The first mutant used in these studies had the downstream half-site and 3'-flanking nucleotides of AREp changed to that of AREd (Mut 1). As shown in Fig. 6AGo, lane 4, this replacement appeared to disrupt SF-1 binding. When compared with the MVDP AREp core sequence TGTTCT, AREd contains a C instead of a T at position 3 (TGCTCT). This substitution introduced in AREp (Mut 2) also prevented SF-1 binding (Fig. 6AGo, lane 5). The G-to-T substitution in the downstream half-site of AREp (Mut 3) abolished SF-1 protein binding (Fig. 6AGo, lane 6), suggesting that this nucleotide is also involved in SF-1/DNA interactions. Mutations generated in the three nonconserved spacer base pairs (Mut 4) or in the upstream half-site of the palindrome (Mut 5, 6, 7) did not affect SF-1 binding significantly (Fig. 6AGo, lanes 7–10). To confirm the data obtained with Y1 nuclear extracts, in vitro translated SF-1 was incubated with either wild-type or mutated AREp probes, and SF-1-containing complexes were checked by competition with an excess of the specific SF1 oligonucleotide from the 21-OH gene. As shown in Fig. 6BGo, in vitro translated SF-1 bound to AREp probe while no SF-1-containing complexes were formed using Mut1, -2, or -3 probes (lanes 5–10). The asterisk indicates an upper migrating band competed by 21-OH oligonucleotide that was unrelated to SF-1 since it was also detected with unprogrammed lysate. Finally, experiments using unlabeled DNA as competitor (Fig. 6CGo) showed that the complex bound to AREp probe (lane 1) was unaffected by a 200-fold excess of AREd or Mut 3 oligonucleotides (lanes 2 and 3) whereas a 50-fold excess of a high-affinity binding site for SF-1 (from the 21-OH promoter, Fig. 6CGo, lane 4) was sufficient to abolish it. Thus, it appears that SF-1 binding activity is retained by sequences beginning at position -102 and spanning the downstream palindromic hexanucleotide of AREp.



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Figure 6. Characterization of the SF-1 Binding Site within AREp Oligonucleotide

A, Nuclear extracts (5 µg) from Y1 cells were incubated with 32P-labeled probes corresponding to the distal androgen response element of the MVDP promoter (AREd, see also Table 1Go), AREp, or mutant versions of AREp (Mut 1 to Mut 7). B, In vitro translated SF-1 protein (2 µl) was incubated with the indicated 32P-labeled probes in the absence or presence of an excess of the unlabeled high-affinity SF-1 binding oligonucleotide from 21-OH gene. L, Unprogrammed lysate. C, Nuclear extracts (5 µg) from Y1 cells were incubated with a 32P-labeled probe corresponding to AREp in the absence or presence of the indicated molar excess of unlabeled competitor oligonucleotides. D, The sequences of ARE probes are shown. The two half-sites of ARE motifs are shown in bold, and the nucleotide substitutions are underlined. The bases important for SF-1 binding are boxed.

 
SF-1 and NF1 Binding Sites Are Required for Constitutive Expression of the MVDP Gene
EMSA studies demonstrated the authenticity of the Sp1, C/EBP, NF1, and SF-1 binding sites found in the proximal upstream region of the MVDP gene. To determine whether these factors could affect either constitutive or cAMP-induced expression of MVDP promoter, additional reporter constructs were generated in which the different binding sites were mutated in the context of the proximal promoter. CAT reporter constructs used for these experiments were driven by the wild-type -121/+41 proximal promoter or by mutant forms carrying substitutions within one or two of these sites. All the mutations disrupted the specific DNA-protein complexes observed in gel shifts (see above results). As shown in Fig. 7Go, mutation in the -102 SF-1 site, which abolished SF-1 binding (0.16 SF-1m), resulted in a 77% reduction in basal CAT activity. Mutation in the -76 NF1 binding site had similar effects: basal CAT activity was reduced by 81%. When expressed as fold-increase over basal level, the constructions lacking NF1 or SF-1 binding sites were able to mediate a significant stimulation of CAT activity in response to forskolin, and the fold-inductions of the two reporters were relatively unchanged compared with the wild-type promoter. However, forskolin- induced CAT activity, expressed as percentage conversion, was significantly reduced by 82% and 86% after mutation of SF-1 or NF1 binding sites, respectively. These data indicate that both the -102 SF-1 and -76 NF1 binding sites are required to ensure full transactivation of the MVDP proximal promoter.



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Figure 7. Functional Activity of SF-1, NF1, C/EBPß, and Sp1 Binding Sites in Both Constitutive and cAMP-Induced Expression of the MVDP Proximal Promoter

Y1 cells were transfected with 1.5 µg of the wild-type or mutated versions of the p0.16 CAT construct, and reporter activity was measured from forskolin-stimulated or unstimulated cells. Results were expressed as percentages of the mean value achieved with the p0.16 CAT construct. Fold activation represents the forskolin-stimulated reporter activity divided by the unstimulated level. Values are means ± SEM of at least four independent transfections. *, Significantly different (P < 0.01) from basal activity observed with control (p0.16 CAT).

 
To determine whether the differential expression of wild-type and -102 SF-1 mutated MVDP promoters in Y1 cells resulted from the presence of SF-1 factor, we compared their activities in steroidogenic cells that are devoid of SF-1, the placental JEG-3 cell line (Fig. 8AGo). Disruption of the -102 SF-1 site within the p.016 promoter resulted in a 77% decrease in reporter activity in Y1 cells whereas this mutation had virtually no effect in JEG-3 cells. We next examined the ability of exogenous SF-1 to transactivate MVDP promoter through the -102 site in SF-1-deficient nonsteroidogenic CV1 cells. A SF-1 expression vector was cotransfected with the wild-type and mutant promoter construct. MVDP p0.16Luc proximal promoter was stimulated approximately 3.1-fold by SF-1, and this effect was lost when the -102 site was mutated (Fig. 8BGo). Taken together, these data indicate that differences between wild-type and -102 site mutant promoters activities were abolished in steroidogenic SF-1-deficient cells (JEG-3) and that transactivation of the MVDP proximal promoter by exogenous SF-1 requires intact -102 site.



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Figure 8. Transcriptional Activity of SF-1 at the AREp Motif of MVDP Proximal Promoter

A, Y1 and SF-1-deficient JEG-3 cells were transfected with 1.5 and 3 µg of CAT reporter plasmids, respectively, driven either by the wild-type (p0.16) or -102 site mutant (p0.16 SF-1 m) promoters. Results were expressed as percentages of the mean activity achieved with the p0.16 CAT construct. B, CV1 cells were cotransfected with 3 µg of the wild-type (p0.16 Luc) or mutated version at the -102 site (p0.16 SF-1 m Luc) and 5 ng of SF-1 expression vector or empty vector. Results were expressed as percentages of the mean activity achieved with the p0.16 Luc plasmid cotransfected with the empty vector. Values are means ± SEM of at least four independent transfections.

 
Cooperativity between Sp1 and C/EBP in cAMP-Induced Activation of the MVDP Promoter
As shown in Fig. 7Go, the mutation of Sp1 binding site decreased both basal and forskolin-induced CAT activities by 80% and 95%, respectively. The construct retained cAMP responsiveness, but the fold-increase (6.9-fold) was lower than that determined with the wild-type promoter (13.4-fold). The mutant promoter for C/EBP binding was more effective in driving basal expression of the CAT reporter gene (+119%) than the wild-type promoter (p0.16 CAT). Expressed as fold-increase over basal values, the ability of forskolin to elicit cAMP responsiveness was reduced by at least 50% after mutation of the C/EBP binding site. To determine whether the Sp1 and C/EBP binding sites could account for full cAMP stimulation of MVDP proximal promoter, plasmids containing double mutations were generated. As observed in Fig. 7Go, the double mutation of both -52 Sp1 and -61 C/EBP binding sequences reduced the forskolin responsiveness of the p0.16 CAT promoter to the residual induction level observed with pTATA construct. When the SF-1 binding site was mutated in the context of C/EBP or Sp1 mutant promoters, fold-activation by forskolin treatment was either not affected or reduced to values observed with Sp1 mutant promoter, respectively. These results indicate that a cooperative or additive functional interaction between Sp1 and C/EBPß seems critical for cAMP activation of MVDP proximal promoter in Y1 cells.

To better understand the respective roles of C/EBPß and Sp factors in MVDP gene transcription, we performed transactivation assays in a nonsteroidogenic cell line, CV1 cells, that lacks C/EBPß expression (Fig. 9Go). Increasing amounts of C/EBP expression vector were cotransfected with the wild-type promoter or mutant promoters for C/EBP and/or Sp1 sites. The p0.16 CAT promoter was stimulated in a dose-dependent manner, and this effect was diminished by approximately 67% and 77% when either the -61 C/EBP or -52 Sp1 sites were mutated, respectively (Fig. 9Go, 25 ng). Mutation of both sites nearly abolished the responsiveness of MVDP promoter to C/EBPß. These data indicate that full C/EBPß-dependent activation of the MVDP promoter requires the integrity of both -61 C/EBP and -52 Sp1 sites.



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Figure 9. Requirement of -61 C/EBP and -52 Sp1 Sites for Transactivation of MVDP Proximal Promoter by C/EBPß

CV1 cells were cotransfected with 8 µg of wild-type or mutated versions of the p0.16 CAT construct and increasing amounts (25 and 100 ng) of C/EBPß expression vector or empty vector. Results were expressed as percentages of the mean value achieved with p0.16 CAT plasmid cotransfected with the empty vector. Values are means ± SEM of at least four independent transfections. *, Significantly different (P < 0.01) between activity of wild-type and C/EBP mutant version plasmid with 25 ng C/EBPß expression vector. °, Significantly different (P < 0.05) between activity of wild-type and C/EBP mutant version plasmid with 100 ng C/EBPß expression vector.

 
Forskolin-Induced MVDP Expression in Y1 cells Is Inhibited by DAX-1
It has been shown that DAX-1 can affect steroidogenesis in Y1 cells by inhibiting the expression of genes involved in critical steps of this process (17). To test whether DAX-1 can affect MVDP expression, we used stably transfected Y1 clones expressing human DAX-1 (Y1/hDAX-1) and control cells containing the empty plasmid expressing the neomycin-resistance gene (Y1/neo) (38). Constitutive MVDP mRNA levels are very low in Y1/neo cells as well as in Y1/hDAX-1 cells (Fig. 10AGo). Forskolin treatment led to the expected increase of MVDP mRNA levels in Y1/neo cells, the induction being maximal 3 h after addition of the activator. Conversely, MVDP transcripts stayed at very low levels in Y1/hDAX-1 cells after forskolin treatment. The same results have been obtained by Western blot analysis of cell extracts: MVDP concentrations were up-regulated by forskolin in control cells but not in Y1 clones expressing hDAX-1 (Fig. 10BGo). These results demonstrate that the expression of DAX-1 in Y1 cells significantly impaired cAMP-stimulated expression of the MVDP gene at least by controlling mRNA levels.



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Figure 10. MVDP Forskolin-Induced Expression Is Inhibited by hDAX1 Expression in Y1 Cells

Stably transfected Y1 cells expressing hDAX-1 (Y1/hDAX-1) and control cells only expressing the neomycin-resistance gene (Y1/neo) were cultured in the presence of 10-5 M forskolin (Fsk) for the indicated times. A, Northern blot analysis. Twenty five micrograms of total RNAs were loaded per well and hybridized with either 32P-labeled MVDP cDNA or ß-actin cDNA probes. B, Western blot analysis of 15 µg proteins. After SDS-PAGE the proteins were transferred to nitrocellulose and incubated with anti-MVDP polyclonal antibody (dilution 1:3,000).

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The level and pattern of expression of an eukaryotic gene are mainly governed by the combinatorial action of multiple transcriptional activators and repressors binding to distinct promoter and enhancer elements (39). Using Y1 murine adrenocortical cells, our studies demonstrate that basal and cAMP-induced activities of the MVDP proximal promoter involve both cell-specific (SF-1), more restricted (C/EBPß), and ubiquitous (NF1, Sp1) transcriptional activators that act in combination.

Several lines of evidence indicate that the transcriptional activator SF-1 interacts with a cis-acting regulatory element positioned at -102 within the proximal ARE (AREp) of the MVDP gene. First, a known pure SF-1 binding site [from murine 21-steroid hydroxylase gene promoter, position -140 (37)] competed efficiently for the formation of the DNA/protein complex in gel mobility shift assays. Second, immunoreactivity in gel mobility shift assay using an anti-SF-1 antibody provides evidence that the protein in Y1 nuclear extracts interacting with the cis-acting sequence is closely related to SF-1. Third, a specific DNA-protein complex was formed when the AREp probe was incubated with in vitro translated SF-1 that was absent in unprogrammed lysate. Finally, expression of exogenous SF-1 transactivates MVDP promoter depending on the integrity of the AREp site. SF-1 is an orphan member of the nuclear receptor superfamily expressed in all major steroidogenic tissues, binding as a monomer to consensus response elements PyCAAGGPyPy or PuPuAGGTCA in the upstream region of a number of genes (40, 41). Surprisingly, the AREp probe we used in our experiments does not contain a SF-1 consensus sequence. Two recent examples of noncanonical SF-1 site usage are the murine DAX-1 and the Inhibin {alpha}-promoters that are both stimulated via cryptic SF-1 sites located at proximal positions -126 and -137, respectively (42, 43). These two sites share a common sequence TCAG/TGGCCA but seem unrelated to AREp. Although the precise sequence involved in SF-1 binding to AREp remains to be determined, mutation analyses demonstrate that this binding site resides in the downstream half-site of the AREp sequence (5'-TGTTCT-3'). Within this binding motif, the GT dinucleotide (underlined) is critical for SF-1 binding since mutation of either G or T abolished the interaction with SF-1. However, the importance of these nucleotides is dependent upon the sequences flanking the AREp half-site since an identical TGTTCT motif lying within the distal ARE (AREd), but in the opposite strand (AGAACA), is unable to bind SF-1. Interestingly, the GT dinucleotide was highly conserved in functional AREs identified in a number of androgen-regulated genes, and it has been shown that in vitro, as well as in vivo, a G nucleotide at position 2 of the downstream half-site is essential for AR/DNA interaction (35, 44, 45). This SF-1 binding region has been previously shown to be a functional ARE in human mammary carcinoma cells (35). This suggests that in different tissues the same DNA motif may respond differently to the binding of several nuclear receptors. Mutation within this SF-1 binding site nearly abolishes basal CAT activity, suggesting that constitutive expression of the gene in adrenocortical Y1 cells requires the presence of promoter sequence containing an SF-1 recognition element. SF-1 is known to regulate a large number of genes involved in steroidogenesis and reproduction (reviewed in Ref. 6). Within the murine adrenocortical cells, it has been shown that SF-1 stimulated expression of the cytochrome P450 steroid hydroxylases (6), StAR (10, 46), high density lipoprotein (HDL) receptor (47), and ACTH receptor (12). Our data regarding the MVDP (AKR1B7) gene expression are the first to demonstrate that SF-1 regulates a gene involved in the detoxification of products of the steroidogenesis. Taken together, these results suggest that SF-1 plays a critical role in the coordinated expression of ACTH receptor, proteins involved in delivery of cholesterol, steroidogenic enzymes responsible for the biosynthesis of steroid hormones, and nonsteroidogenic enzymes involved in the reduction of reactive aldehydes generated by steroidogenesis (32).

Mutational analysis also identified a second element that is required for basal expression of the MVDP promoter. The NF1 binding site appears to be equally important for MVDP gene transcription as its mutation nearly abolishes basal promoter activity in Y1 cells. Thus, both SF-1 and NF1 binding sites must be intact for constitutive transactivation of the MVDP promoter in adrenocortical cells. In addition to the vas deferens expression that is characteristic of the mouse species, MVDP is essentially expressed in steroidogenic cells (Ref. 31 and our unpublished results), and the NF1/DNA interaction cannot explain the restricted pattern of MVDP. Members of the CTF/NF1 family are ubiquitous factors known to function as positive regulators of gene transcription (48). Our results rather suggest that SF-1 participates in the adrenocortical expression of the MVDP gene.

In common with many cAMP-regulated steroidogenic genes, the MVDP proximal promoter does not contain any consensus CRE, suggesting the existence of yet uncharacterized cAMP-dependent regulatory factors. Surprisingly, each steroidogenic gene appears to be regulated by a different mechanism, even though the transcription of these genes is temporally coordinated (49). Recent studies have shown that whatever the mechanism involved, SF-1 is a common factor in this pathway that mediates basal and/or hormone-induced expression of the steroid hydroxylase genes (reviewed in Ref. 6). After mutation within SF-1 or NF1 binding sites, the absolute cAMP-induced CAT activities of the two reporters were strongly reduced. However, cAMP fold-induction values were preserved, indicating that the SF-1 and NF1 sites are important for full transcriptional responsiveness but dispensable for the cAMP-dependent regulation of the MVDP gene in Y1 cells. Thus, SF-1 may represent a tissue-specific factor rather than a mediator of cAMP induction.

All experimental features, including EMSAs and functional analyses using mutated constructs, showed that maximal responsiveness to cAMP required two cis-acting components: the Sp1 and C/EBP binding sites. Inactivation of any one component decreased the cAMP responsiveness measured as fold-induction, suggesting that cAMP stimulation of MVDP promoter activity is mediated by both actions of the transcription factors bound to the -52 Sp1 and -61 C/EBP elements. Although originally associated with constitutive transcription, it is now evident that Sp1 also regulates several inducible genes. The promoter region of the genes encoding the steroid hydroxylase usually contains atypical cAMP-responsive sequences unrelated to the classical CRE/CREB system (reviewed in Ref. 49). It has recently been shown that Sp1 is important for the regulation of the CYP11A gene through binding to an element required for cAMP responsiveness (50, 51, 52). The mechanism by which Sp1 confers cAMP responsiveness to the MVDP promoter has not yet been determined. Possible mechanisms include regulation of the level of Sp1 protein, Sp1 activation by posttranslational modification, affinity for DNA, and interaction with other factors. In adrenocortical cells the levels of Sp1 protein are not affected by forskolin treatment (52). Recent data have demonstrated that PKA (protein kinase A) phosphorylates Sp1 and up-regulates its DNA-binding and trans-activating properties (53). On the other hand, it has been recently reported that both Sp1 and SF-1 are required to regulate the cAMP-dependent expression of bovine CYP11A gene (54) and that the cooperation between these two factors is mediated by a direct protein-protein interaction and/or by the common activator CBP (CREB binding protein) (55). Whether or not the -52 Sp1 element of the MVDP promoter is involved in the same way in transmitting a cAMP response remains to be determined. We have previously shown that exposure of T47D cells to (Bu)2cAMP had no effect on the basal expression of CAT driven by the MVDP proximal promoter (56). Our site-directed mutagenesis showed that the SF-1 binding site is required for high MVDP promoter activity in the absence as well as in the presence of forskolin. We do not know yet how cAMP responsiveness is restricted to Y1 cells, but one explanation could be that Y1-enriched specific factors such as SF-1 may interact with Sp1 bound to the MVDP promoter to confer cAMP induction.

Immunoreactivity and in vitro translated protein in gel shift assays as well as transactivation experiments demonstrated that C/EBPß-related proteins are essential components of the binding activity at position -61. The C/EBP family has been associated with differentiation, lipid biosynthesis, and metabolism (Ref. 57 for review). C/EBPß has been implicated in the immune response and recently in female reproduction (57, 58). Mutation of the -61 C/EBP binding site increases basal promoter activity to 119% of wild-type levels, suggesting that C/EBPß acts as a repressor of the MVDP gene. A few cases of repressor effects have been described in relation to the C/EBP family. Two isoforms of C/EBPß are generated from a single mRNA: the full-length protein (also called LAP: liver enriched activator protein) and a truncated protein (LIP: liver enriched inhibitor protein) that is a transcriptional repressor (59). Thus, the ability of C/EBPß to activate or repress a promoter is probably determined, at least in part, by the LAP/LIP ratio. Our results also define C/EBPß as a possible cAMP-regulated activator of MVDP gene transcription in Y1 cells. C/EBPß expressed in other steroidogenic cells, such as Leydig (60) and granulosa cells (58), is believed to play an important role in regulating the expression of genes involved in steroid hormone synthesis. In support of a role of C/EBPß in mediating the cAMP-dependent regulation of gene expression in steroidogenic cells, recent data have revealed that this factor is involved in transcriptional activation of the murine and human StAR gene (61, 62). However, when the C/EBP binding sites identified in the StAR gene were mutated, a pronounced decrease in basal promoter activity occurred in Leydig or granulosa cells, but the fold-stimulation by cAMP was unaffected (61, 62). As shown by mutational analysis, the -61 C/EBP site participates in cAMP responsiveness of the MVDP gene promoter expressed as fold-induction. Thus, it seems that participation of C/EBP factors in cAMP responsiveness could be achieved by a different mechanism in Y1 cells. In agreement with this possibility, we found that forskolin treatment increased the intensity of the C/EBPß complexes bound to the -61 C/EBP site in nuclear extracts of Y1 cells. In some cell types, increases in intracellular cAMP levels have been shown to up-regulate C/EBPß via different mechanisms including the level of expression and the subcellular localization of the protein (63, 64). Moreover, the activity of C/EBPß may also be controlled at the posttranslational level, presumably by the PKA pathway (65). Since the stimulation of MVDP gene expression by forskolin does not require continuous protein synthesis (31), it seems likely that cAMP exerts its action through subcellular localization or posttranslational modifications of C/EBPß proteins. An alternative explanation is that forskolin treatment increases the ratio of LAP to LIP as reported in Sertoli cells (64).

Mutational analyses identified two critical regulatory elements for cAMP responsiveness of the MVDP promoter. The two adjacent -52 Sp1 and -61 C/EBP binding sites appear to be important for cAMP- induced transactivation as their mutation similarly decreased the fold-activation. cAMP inducibility was nearly abolished when both Sp1 and C/EBP sites were mutated. Consistent with these observations, in CV1 cells, C/EBP fails to transactivate efficiently the p0.16 CAT MVDP promoter when either the -52 Sp1 or -61 C/EBP sites are mutated, implying that a cooperative functional interaction between Sp1 and C/EBPß is required for cAMP activation of the MVDP promoter in Y1 cells. In support of this model, it has been reported that C/EBPß, but not C/EBP{alpha}, is able to cooperate with Sp1 at the level of both transactivation and DNA binding to their adjacent sites, to regulate the expression of the rat CYP2D5 gene (66, 67). However, gel shift assays using a probe containing both -61 C/EBP and -52 Sp1 sites (-69 to -37, Table 1Go) did not reveal any higher order Sp1-C/EBP-DNA complex formation, suggesting that mechanisms other than cooperative binding might be involved in MVDP promoter cAMP activation (data not shown). cAMP responsiveness has been shown to be fulfilled by SF-1 in the case of CYP17 gene (9), SF-1 plus Sp1 in the CYP11A gene (54), SF-1 plus C/EBP in the StAR gene (61, 62), and Sp1 plus C/EBP in the MVDP gene (present results).

Inappropriate DAX-1 expression causes defect in adrenal and gonadal development and function (Ref. 68 for review). DAX-1 is supposed to act in these processes at least by suppressing SF-1-mediated transcription through different mechanisms, including interaction with SF-1 (16, 69) and/or binding to DNA stem-loop structures formed in the promoters of target genes (17, 38). Interestingly, we found that DAX-1 is also able to repress cAMP-induced MVDP gene expression in Y1 cells but the mechanism involved will need further investigation. Indeed, in addition to the -102 SF-1 site, analysis of more upstream DNA sequence reveals the presence of four putative SF-1 binding sites at positions -279, -458, -503, and -578 that could constitute additional potential targets for SF-1/DAX1 actions. These data are the first report pointing out detoxifying enzymes of the aldose reductase family as new potential targets of SF-1/DAX-1 functional interactions in steroidogenic organs.

In conclusion, we show that a complex array of nuclear proteins bind to the MVDP promoter. At least four functional interactions have been demonstrated within the -121/-40 bp fragment to be sufficient for basal and hormonal-regulated expression. The SF-1 protein, which binds to an unusual cis-acting element located in the proximal ARE, regulates the basal level of MVDP gene expression and boosts the overall response to forskolin. In addition, the adjacent NF1 binding site contributes equally to these two processes. Our study also establishes the role of Sp1 and C/EBPß as important transducers of the cAMP-positive signal and points out DAX-1 as a negative actor in this pathway. Further studies will be required to determine whether the interactions between the regulatory proteins could be direct or through the recruitment of coactivators/corepressors to the MVDP promoter.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Animals
Mice of the Swiss strain (CD-1; Charles River Laboratories, Inc. Saint Aubin Les Elbeuf, France) were used. To investigate the role of pituitary ACTH, adult males (8 weeks old) were injected sc with dexamethasone acetate for 5 days (75 µg dissolved in sesame oil twice daily, Sigma, St. Louis, MO) or with dexamethasone acetate (5 days) plus ACTH im (1–2 daily, Synacthene, Novartis Pharma S.A., Rueil-Malmaison, France) for the last 2 days. Control animals received injection of vehicle only. Animals were killed 6 h after the last injection. Adrenals were removed, frozen in liquid nitrogen, and stored at -70 C until Northern blot analysis.

RNase Protection Assay
A 32P-labeled antisense riboprobe complementary to MVDP sequences from -185 to +94, encompassing the previously identified transcription start site (33), was synthesized using RiboProbe Combination System-T7/SP6 kit from Promega Corp. (Madison, WI); 2.5 105 dpm of the probe were coprecipitated with each total RNA sample (adrenals, 10 µg; vas deferens, 2 µg). The pellet was then resuspended in 30 µl of hybridization buffer [80% formamide, 40 mM piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES), pH 6.4, 0.4 M sodium acetate, 1 mM EDTA] and incubated at 85 C for 5 min. After an overnight incubation at 45 C, nonhybridized RNAs were digested by RNAse ONE ribonuclease (adrenals, 7 U; vas deferens, 5 U; Promega Corp.) for 45 min at 37 C in 300 µl of digestion buffer (10 mM Tris, pH 7.5, 5 mM EDTA, 200 mM sodium acetate). Reaction was stopped by addition of 20 µl 10% SDS and 5 µl 10 mg/ml proteinase K, followed by incubation at 37 C for 15 min. Proteins were then removed by phenol-chloroform-isoamyl alcohol extraction. Digestion- resistant RNA-RNA hybrids were precipitated, resuspended in 5 µl loading dye (80% formamide, 10 mM EDTA, 0.1% bromophenol blue, 0.1% xylene cyanol, 0.1% SDS) and run on a 6% polyacrylamide denaturing sequencing gel. A molecular size marker was provided by a dideoxy sequence of the DNA fragment used as the probe template, run next to the RNase mapping reactions.

5'-RACE-PCR
Five'-RACE-PCR has been performed as described in Ref. 70 with the following exceptions. Ten micrograms total RNA from adrenals and vas deferens were used as template. cDNA were purified using QiaQuick PCR purification kit (QIAGEN, Hilden, Germany). Oligonucelotidic single-stranded anchor: PO3-ACTATCGATTCTGGAACCTTCAGAGG-NH2. Anchor PCR primer: 5'-CCACCTCTGAAGGTTCCAGAATCGATAG-3'. MVDP specific primers were 5'-GTGATACACATAGGCACAGTCAAT-3' (external primer, complementary to MVDP cDNA sequence from position 127 to 150, where A of ATG translation start codon is 1) for the first 35 cycles and 5'-AATGGCCGCCTTCACGGCTTCCTT-3' (internal primer, complementary to MVDP cDNA sequence from positions 85 to 108) for the next 35 cycles. DNA was denatured for 5 min at 94 C, and amplification was carried out using Takara rTaq (Boehringer Ingelheim Bioproducts, Heidelberg, Germany), for 35 cycles with the following parameters: denaturation, 94 C for 45 sec; annealing 60 C (first 35 cycles) or 63 C (next 35 cycles) for 45 sec; then synthesis, 72 C for 2 min. A 10-min synthesis period was finally applied to samples. One microliter of a fifth dilution of the reaction was used to set up the second 35-cycle amplification round, using internal primer. An aliquot of the PCR reactions was run in a 2% agarose gel in Tris-acetate-EDTA electrophoresis buffer, blotted to a nylon membrane (Hybond N+, Amersham Pharmacia Biotech France SA, Les Ulis, France) and hybridized with a 5'-32P-labeled oligonucleotide probe specific to the first exon (5'-GCATCTTGGCTTTGGTACT-3', position 22 to 40) overnight at 37 C. Sequencing was carried out using a T7 sequencing KIT (Pharmacia Biotech, Piscataway, NJ) after cloning into pGEM-T Easy (Promega Corp.).

Northern Blot Analysis
Total RNAs were extracted from Y1/hDAX-1, Y1/neo cells, or adrenals with RNAzol (Bioprobe Systems, Montreuil-sous-Bois, France), according to the manufacturer’s instructions. Total RNA (25 µg) was electrophoresed in a 1% (wt/vol) agarose, 2.2 M formaldehyde gel and transferred to nylon filters. A 5-min UV exposure covalently bound RNAs to the filters. Probes used in these experiments include: MVDP, a 1.2-kb EcoRI-BamHI fragment of MVDP cDNA; StAR, a RT-PCR-generated fragment from murine StAR cDNA; and P450scc, a generous gift from Dr. M. Begeot (INSERM-INRA U418, Lyon, France). The cDNAs were labeled with 32P-dCTP using the Megaprime DNA Labeling System (Amersham Pharmacia Biotech, Les Ulis, France). Prehybridization was carried for 1.5 h at 65 C in a solution containing 3 x SSC (standard sodium citrate buffer), polyvinylpyrrolidone (0.2%), Ficoll (0.2%), polyethyleneglycol (5%), glycine (1%), SDS salt (1%), and 100 µg/ml denatured salmon sperm DNA. Hybridization was performed overnight at 65 C in the same solution containing 1 x 106 cpm/ml of 32P-labeled probes. Filters were washed at 65 C. To normalize the quantities of MVDP mRNAs, Northern blots were stripped and rehybridized in the buffer described above, at 65 C with a 32P-labeled ß-actin cDNA probe. Autoradiography was carried out by exposing the blots to x-ray films (Cronex, Sterling Diagnostic, Newark, DE) for 48 h with an intensifying screen at -80 C.

Western Blot Analysis
A fraction of cell samples was collected before RNA extraction (RNA Plus, Quantum Bioprobe, Montreuil-sous-Bois, France). The concentration of soluble proteins was determined by the Bradford method (Bio-Rad Laboratories, Inc. München, Germany). Soluble cellular extracts (15 µg proteins) were subjected to SDS-PAGE on 10% acrylamide gels and transferred to nitrocellulose membranes according to the manufacturer’s instructions (Bio-Rad Laboratories, Inc.). Nonspecific protein binding sites were blocked by incubation of nitrocellulose for 1 h at room temperature in TBST [50 mM Tris-HCl (pH 8), 150 mM NaCl, 0.1% (vol/vol) Tween 20] containing 5% (wt/vol) nonfat dry milk, and incubation with the polyclonal anti-MVDP antibody (diluted 1:10,000) was carried out for 1 h at room temperature as previously described (71). After three washes in TBST, the sheets were incubated for 1 h at room temperature with the second antibody (peroxidase-conjugated antirabbit IgG) diluted 1:15,000. The blots were washed again, and peroxidase activity was detected by autoradiography with the ECL enhanced chemiluminescence system (Amersham Pharmacia Biotech).

Cell Culture and Transfections
Murine Y1 adrenocortical tumor cells (72) and human placental JEG-3 were grown in DMEM-Ham’s F-12 medium (1:1) supplemented with 10% FCS, 2 mM glutamine, penicillin (100 U/ml), and streptomycin (100 µg/ml) (Life Technologies Ltd, Paisley, UK).

DAX-1-expressing Y1 cell lines were a generous gift from E. Lalli (38). Stably transfected Y1 clones were obtained using the expression plasmids pSG.DAX-1 (73) and pD383, which carry the neomycin-resistance gene under the control of the phosphoglycerate kinase gene promoter. Y1/hDAX-1 and Y1/neo cells were grown in DMEM-Ham’s F-12 medium (1:1) supplemented with 15% horse serum, 2.5% FCS, 500 µg/ml geneticin (G418) (Roche Molecular Biochemicals, Meylan, France), 2 mM glutamine, penicillin (100 U/ml), and streptomycin (100 µg/ml).

One day before transfection, Y1 or JEG-3 cells were plated at a density of 1 x 106 cells/60-mm dish. MVDP-CAT reporter plasmids (1.5 or 3 µg) were transfected into cultured cells with FuGENE6 transfection reagent according to the manufacturer’s instructions (Roche Molecular Biochemicals). Twenty-four hours after transfection, the medium was replaced, and the cells were treated or not with forskolin [10-5 M, dissolved in dimethylsulfoxide (DMSO)] for 24 h. Cells were harvested 48 h after lipofection and subjected to CAT assays.

CV1 cells were transfected by the calcium phosphate method as previously described (74). Twelve hours before transfection, cells were seeded at 0.5 106 cells per 60-mm dish or at 0.25 x 106 cells per well in six-well culture plates for CAT assays or luciferase assays, respectively. CV1 cells were transferred to DMEM supplemented with 5% FCS and transfected with various amounts of effector and reporter plasmids along with carrier DNA to equalize the total amount of DNA to 10 µg for CAT assays (8 µg CAT reporter construct) and 5 µg for luciferase assays (3 µg luciferase reporter construct). Cells were incubated with the DNA/calcium phosphate precipitates for 12 h, rinsed to remove DNA, and incubated in fresh medium for an additional 36 h before being harvested for analysis. All transfection experiments were performed at least four times. To correct the variations in transfection efficiencies, separate plates of cells were transfected with a CAT reporter plasmid under the control of the SV40 promoter/enhancer (pSV2-CAT) as indicated.

CAT and Luciferase Assays
For CAT assays, cells were lysed by four rounds of freezing and thawing. The cell lysates were heat treated 10 min at 65 C and clarified by centrifugation. Under conditions where signals were proportional to the amount of supernatant added, 3–40 µl of supernatant were combined with 2 µl of [14C]chloramphenicol (1.850 Mbq/ml, NEN Life Science Products, Boston, MA), 2.5 µl of 8 mM acetylcoenzyme A, and 250 mM Tris-HCl, pH 7.8, to a final volume of 100 µl. The mixtures were incubated at 37 C for 3 h, and the reactions were stopped by extraction with 550 µl ethyl acetate. The products were separated by TLC, acetylated forms of [14C] chloramphenicol were excised individually, and radioactivity was counted in a scintillation counter.

For luciferase assays, cell extracts were prepared by scraping using 150 µl luciferase assay system reporter lysis buffer (Promega Corp., Lyon, France); extracts were then clarified by centrifugation at 13,000 x g at 4 C. Supernatants (40 µl) were assayed for luciferase activity using a luciferase assay kit (Genofax A, Prodemat, Anduse, France) exactly as described by the protocol provided with the kit. Luminescence was measured using a Berthold LB 96V Microplate Luminometer (Perkin-Elmer, Courta Boeuf, France) under conditions in which signals were proportional to the amount of supernatant added.

Plasmid Constructs
All plasmid constructs were prepared using standard methods (75). MVDP-CAT reporter plasmids contain -1,804 to +41 bp (and deletions thereof) of the murine MVDP promoter sequence linked to the coding region of bacterial CAT gene in the pBlCAT3 vector as previously described (35). pTATA CAT was constructed as follows: a DNA fragment encompassing regions from -40 to +41 of the MVDP gene was amplified by a standard PCR reaction using p0.16 CAT plasmid as a template. PCR products were cloned into a PstI restriction site of the pBlCAT3 vector. Resulting plasmid was completely sequenced to confirm the presence and orientation of the expected insert. Mutations of the AREp (-117/-93) and NF1 (-84/-59) binding site contained in the p0.16 CAT construct have been described previously (76). The Gene Editor kit (Promega Corp., Lyon, France) was used to introduce the other mutations into p0.16 CAT. The oligonucleotides used to generate the mutations are shown in Table 1Go (-52 Sp1m, -61 C/EBPm, -76 NF1m) and Fig. 6DGo (Mut 3). Resulting plasmids were partially sequenced to confirm that the sites had been mutated as expected. Luciferase reporter plasmids, p0.16 Luc and p0.16 SF1m Luc, were constructed by introducing into the pGL3 plasmid (Promega Corp., Lyon, France), the -121/+41 promoter fragment either intact or mutated (Mut 3) within the AREp sequence, respectively.

In vitro translation experiments were performed using pGEM-SF-1 plasmid that was constructed as follows: a 2-kb EcoRI insert containing the murine SF-1 cDNA was excised from the pCMV5 plasmid (kindly provided by Dr. K.L. Parker, University of Texas Southwestern Medical Center, Dallas, TX) and introduced into the EcoRI site of pGEM-T Easy plasmid (Promega Corp., Lyon, France). The pCDNA1-LAP plasmid expressing C/EBPß under the control of a cytomegalovirus (CMV) promoter was kindly provided by Dr. U. Schibler (University of Geneva, Geneva, Switzerland) and used for both transfections and in vitro translation experiments.

EMSA
Nuclear extracts were prepared from confluent Y1 adrenocortical cell cultures. Briefly, cell monolayers were rinsed once with PBS and scraped in 500 µl of 10% glycerol PBS. The cells were pelleted by centrifugation at 500 x g for 5 min, washed in 500 µl buffer A (20 mM HEPES, 50 mM KCl, 1 mM EDTA, 0.25 mM EGTA, 0.15 mM spermine, 0.5 mM spermidine, 0.5 mM sucrose, 1 mM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol, 1% aprotinin) and pelleted. Pellets were resuspended in 500 µl buffer A with 0.5% Nonidet P-40 and vortexed. The cells were expanded for 15 min on ice. The homogenates were centrifuged 10 min at 2,500 x g and rinsed with buffer A without NP-40. After a centrifugation at 2,500 x g for 10 min to pellet the nuclei, 25–50 µ l buffer C (20 mM HEPES, 0.45 M NaCl, 1 mM EDTA, 0.25 mM EGTA, 0.15 mM spermine, 0.5 mM spermidine, 0.5 mM sucrose, 1 mM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol, 1% aprotinin) were added, and the samples were expanded for 10 min on ice. The nuclear lysate was then centrifuged for 20 min at 13,000 x g at 4 C, and the supernatant was placed into a fresh microfuge tube and stored at -80 C.

To produce in vitro translated SF-1 or CEBPß, pGEM-SF-1 or pCDNA1-LAP plasmids were transcribed using SP6 or T7 polymerase, respectively, and translated using a TNT kit (Promega Corp., Lyon, France).

Oligonucleotides used in this study are listed in Table 1Go and Fig. 6DGo. Ten picomoles of the double-stranded oligonucleotides were labeled with T4 polynucleotide kinase and [{gamma}-32P dATP] (110 TBq/mmol, Amersham Pharmacia Biotech, Les Ulis, France). All probes were purified by precipitation. Binding reactions were performed by mixing 5 µg Y1 nuclear extract or 1–3 µl in vitro translated protein with 0.05 pmol of probe, in the presence or absence of 50- to 200-fold molar excess of unlabeled competitor in a 1x binding buffer (20 mM HEPES, 0.2 mM EDTA, 100 mM NaCl, 100 mM KCl, 10 mM MgCl2, 8 mM spermidine, 4 mM dithiothreitol, 200 µg/ml albumin, 8% Ficoll) and 1 µg poly(deoxyinosinic-deoxycytidylic)acid. Where indicated, antiserum specific for SF-1, Sp1, C/EBPß, ATF family (ATF 1–4) (Santa Cruz Biotechnology, Inc. Santa Cruz, CA) or CTF1/NF1 (kindly provided by Dr. Naoko Tanese, NYU Medical Center, New York, NY) were incubated 10 min after probe addition. Complexes were resolved by electrophoresis through 6% nondenaturing polyacrylamide gels in 0.5 x Tris-borate-EDTA (TBE) electrophoresis buffer. The gels were dried and autoradiographed.


    ACKNOWLEDGMENTS
 
The authors would like to thank Anne-Marie Lefrançois, Michèle Manin, and Jean-Marc Lobaccaro for critical reading of the manuscript. We wish to thank Alain Halère for his excellent technical assistance.


    FOOTNOTES
 
Address requests for reprints to: Dr. Antoine Martinez, UMR CNRS 6547, Physiologie Comparée et Endocrinologie Moléculaire, Université Blaise Pascal, Clermont II, Complexe Universitaire des Cézeaux, 24 avenue des Landais, 63177 Aubière cedex, France. E-mail: antoine.martinez{at}geem.univ-bpclermont.fr

Received for publication January 18, 2000. Revision received September 14, 2000. Accepted for publication September 20, 2000.


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 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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