Impaired Steroidogenic Factor 1 (NR5A1) Activity in Mutant Y1 Mouse Adrenocortical Tumor Cells

Claudia Frigeri, Jennivine Tsao, Waldemar Czerwinski and Bernard P. Schimmer

Banting and Best Department of Medical Research and Department of Pharmacology University of Toronto Toronto, Ontario, Canada M5G 1L6


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Mutants isolated from the Y1 mouse adrenocortical tumor cell line (clones 10r-9 and 10r-6) are resistant to ACTH because they fail to express the melanocortin-2 receptor (MC2R). In this study, we show that a luciferase reporter plasmid driven by 1800 bp of the proximal promoter region of the MC2R was expressed poorly in the mutant cells compared with parent Y1 cells. The differential expression of the MC2R in parent and mutant cells resulted from impaired activity of the orphan nuclear receptor NR5A1 (SF1) on the promoter as determined by 5'-deletion analysis. Furthermore, the activity of an SF1 expression plasmid on an SF1-dependent reporter plasmid was compromised in mutant clones. The site-specific DNA binding properties of SF1 from parent and mutant cells did not differ as determined in electrophoretic mobility shift assays, and the addition of the activation domain of VP16 to the amino terminus of SF1 restored the transcriptional activity of the protein. In addition, the levels of SF1 and other cofactors including WT1, CBP/p300, and steroid receptor coactivator 1 did not differ appreciably between parent and mutant cells. Taken together, these results suggest that ACTH resistance in the mutant clones resulted from a defect that affected the activation properties of SF1 rather than its DNA binding activity. Consistent with the observed impairment in SF1 function, other SF1-dependent genes, including Cyp11b1 and steroidogenic acute regulatory protein (StAR), were poorly expressed and global steroidogenesis, as evidenced by the metabolism of 22(R)-hydroxycholesterol to steroid products, was impaired. Interestingly, MC2R, Cyp11a, Cyp11b1, and StAR transcripts were not affected to the same degree, suggesting that each of these genes may have a different absolute requirement for SF1. These mutants thus provide an experimental paradigm to identify factors that influence SF1 function and to evaluate the relative importance of SF1 in the expression of genes essential for adrenal steroidogenesis.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The melanocortin-2 receptor (ACTH receptor, MC2R) is a member of the melanocortin receptor subfamily of G protein-coupled receptors (1) that is selectively expressed at high levels in cells of the adrenal cortex. The cell-selective expression of the mouse MC2R appears to be under the control of the proximal promoter region of the gene (bases -1808 to +104, relative to the transcription start site), as determined in transfection experiments using established cell lines. Distinct promoter regions that contribute to the adrenal cell-selective expression of the MC2R have been identified previously (2) and include: a regulatory element at -25 under control of the nuclear receptor NR5A1 (SF1),1 a negative control region from -1236 to -908, and an initiator-like core promoter that overlaps the transcription start site. The SF1 element enhances MC2R expression in those cell types where SF1 is expressed (3), whereas the negative control region seems to prevent MC2R expression in SF1-containing cells other than the adrenal cortex (2). SF1 elements also contribute to the cell-selective expression of the human MC2R gene (4).

To gain a better understanding of the factors that govern MC2R gene expression, we have investigated several mutants isolated from the Y1 mouse adrenocortical tumor cell line that fail to express the MC2R gene and consequently are ACTH resistant (5, 6, 7). In the present study, we demonstrate that the mutation leading to impaired MC2R expression also affects the transcriptional activity of the mouse MC2R promoter at the proximal SF1 site. Moreover, we demonstrate that the activation function of SF1, rather than its DNA binding activity, is compromised. The mutation leading to ACTH resistance adversely affected the expression of Cyp11b1 and the steroidogenic acute regulatory protein (StAR) and affected Cyp11a expression to a lesser degree. These mutants thus provide an experimental paradigm to identify factors that influence SF1 function and to evaluate the relative importance of SF1 in the expression of the MC2R and other genes essential for adrenal steroidogenesis.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
MC2R Promoter Activity in Parent and Mutant Clones
The 5'-flanking region and part of the 5'-untranslated sequence of the MC2R gene (bases -1808 to +104 relative to the transcription start site, Ref. 2) were placed ahead of a luciferase reporter gene and examined for activity in parental Y1 cells and in two MC2R-deficient mutants, clones 10r-9 and 10r-6. In parental Y1 cells, the MC2R promoter enhanced luciferase expression approximately 50-fold over levels obtained with the promoterless reporter plasmid (8). In contrast, the ACTH promoter was only 5% and 7% as effective in the MC2R-deficient mutants 10r-9 and 10r-6, respectively (Fig. 1Go; P < 0.05). The activity of the MC2R promoter also was markedly reduced in two other MC2R-deficient mutants (clones Y6 and OS3, data not shown). These observations indicate that the mutations affecting MC2R expression also affected the activity of the proximal promoter region of the MC2R gene.



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Figure 1. MC2R Promoter Activity in Parent Y1 and Mutant 10r-6 and 10r-9 Cells

Parent and mutant cells were transfected with luciferase reporter plasmids (1 µg) containing various lengths of the mouse MC2R promoter as indicated. Cells were harvested 48 h after transfection, and cell extracts were assayed for luciferase activity. Results were normalized for variations in transfection efficiency as described in Materials and Methods and expressed as percentages of the mean activity achieved with the -1808 to +104 MC2R promoter vector in Y1 cells ± SEM. Results obtained with the -1808 to +104 construct were from seven independent experiments; results obtained with the 5'-deletion constructs were averaged from six independent experiments.

 
To determine whether the differential expression of the MC2R in parent and mutant clones resulted from altered activity at a specific site within the promoter, we evaluated the effects of a limited set of 5'-deletions on reporter gene activity (Fig. 1Go). Deletion of the promoter to -106 resulted in a segment that retained the ability to support luciferase expression much more effectively in parent Y1 cells than in the MC2R-deficient mutants (P < 0.05) despite the removal of the negative regulatory region. This promoter construct had approximately 20% of the activity seen with the -1808 to +104 construct in Y1 cells, indicating the presence of another positive regulatory element in the upstream region of the promoter. Truncating the promoter to -13, so as to remove the SF1 site, resulted in a promoter with a lower activity that was equivalent in parent and mutant clones. These results raised the possibility that the compromised activity of the ACTH promoter in the mutants resulted from impaired activity at the SF1 site.

Transcriptional Activity of SF1 in Parent and Mutant Cells
To further evaluate SF1 function in parent and mutant clones, the expression of a reporter plasmid (p-25 Luc) containing two copies of the SF1 site from the MC2R promoter upstream of an SV40 core promoter and luciferase gene was examined in transient transfection assays. Adding SF1 sites before the core promoter did not enhance the basal expression of the reporter gene; however, it rendered the construct responsive to SF1. As shown in Fig. 2aGo, luciferase expression was increased approximately 10-fold (P < 0.05) when p-25 Luc was transfected into Y1 cells together with an SF1 expression plasmid; SF1 had no effect on luciferase expression from the reporter plasmid lacking SF1 sites (data not shown). In contrast, the SF1 expression plasmid did not enhance p-25 Luc expression in the ACTH-resistant mutants, 10r-9 and 10r-6 (Fig. 2aGo), supporting the hypothesis that SF1 function was compromised in the mutant clones.



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Figure 2. Transcriptional Activities of SF1 and VP16/SF1 at the Mouse MC2R SF1 Site

Parent and mutant cells were transfected with 1 µg of p-25 Luc in the absence (-) or presence (+) of 1 µg of expression plasmids encoding SF1 (panel a) or VP16/SF1 (panel b). Cells were harvested 48 h after transfection and assayed for luciferase activity as described in the legend to Fig. 1Go. Results were compiled from six experiments in panel a and from five experiments in panel b. In each case results were normalized to luciferase expression from a reporter plasmid under control of the SV40 promoter to correct for variations in transfection efficiencies among clones. Data are presented as means ± SEM.

 
SF1 function also was evaluated in parent and mutant clones using p-65 Luc, a reporter plasmid driven by multiple copies of the SF1 site at -65 in the Cyp21 gene (9). This SF1 site is distinct from most other SF1 sites in that it also binds the orphan nuclear receptor, NR4A1 (Nur77, Ref. 10) (see footnote 1), and thus permits a comparison of SF1 and Nur77 actions, each regulated by the cytomegalovirus (CMV) promoter and each acting on the same response element. In parent Y1 cells, the SF1 expression plasmid increased luciferase expression 56 ± 16-fold from p-65 Luc (n = 11). The SF1 expression plasmid was significantly less effective in the 10r-9 and 10r-6 mutants (P < 0.05), and displayed only 13% ± 2% (n = 10) and 34% ± 3% (n = 6) of the respective activities seen in Y1 cells (Fig. 3aGo). Optimally effective concentrations of the Nur77 expression plasmid increased luciferase expression from p-65 Luc approximately 275-fold in parent Y1 cells; in the mutants 10r-9 and 10r-6, the Nur77 vector was 1.5- to 2.0-fold more effective than in the parent cell line (Fig. 3bGo). These latter observations indicated that the mutations affecting MC2R expression affected SF1 function without affecting the activity of Nur77 or the transcriptional cofactors shared by these nuclear receptors.



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Figure 3. Transcriptional Activities of SF1, VP16/SF1, and Nur77 at the Cyp21 SF1 Site

Parent and mutant cells were transfected with 2.5 µg of p-65 Luc in the absence (-) or presence (+) of 0.5 µg (panel a) or 2.0 µg (panels b and c) of expression plasmids encoding SF1, VP16/SF1, or Nur77 as indicated. The activity of Nur77 on p-65 Luc (panel b) is to be compared with the activity of p-65 Luc alone as shown in panel a. Cells were harvested 48 h after transfection and assayed for luciferase activity as described in the legend to Fig. 1Go. Results were expressed as means ± SEM after normalization to luciferase expression from a reporter plasmid under control of the RSV promoter to correct for variations in transfection efficiencies among clones. The number of determinations (n) were: panel a,Y1 (n = 11), 10r-9 (n = 10), 10r-6 (n = 6); panel b, Y1 (n = 3), 10r-9 and 10r-6 (n = 4); panel c,Y1 + SF1 (n = 5), all others (n = 4).

 
Effects of VP16/SF1 in Parent and Mutant Cells
A VP16/SF1 chimeric protein also was evaluated for activity in parent and mutant cells. VP16/SF1 is a construct that retains the DNA binding specificity of SF1 and incorporates an activation domain from VP16 at the amino terminus (11). A VP16/SF1 expression plasmid was transfected into parent and mutant cells and evaluated for its ability to regulate gene expression from reporter plasmids under control of the SF1 sites of the MC2R promoter (Fig. 2bGo) or theCyp21 promoter (Fig. 3cGo). In parent Y1 cells, VP16/SF1 significantly enhanced expression from both reporter plasmids (P < 0.05) but did not affect the reporter plasmid lacking SF1 sites (data not shown). In the mutant clones, VP16/SF1 also significantly enhanced luciferase expression from both SF1-dependent reporter plasmids (P < 0.05) and gave levels of activity that approached those seen in parental Y1 cells (Figs. 2bGo and 3cGo). Thus, the addition of a strong activation domain to SF1 was sufficient to overcome the differential activity of SF1 on these reporter plasmids in parent and mutant clones. These results suggested that the mutations in 10r-9 and 10r-6 did not affect SF1 binding to DNA; rather, the mutation seemed to impair the activation function of SF1.

Levels of SF1, WT1, CBP (CREB-Binding Protein), p300, and Steroid Receptor Coactivator 1 in Parent and Mutant Clones
Despite the apparent loss of SF1 function in the mutants (e.g., Fig. 1Go), nuclear extracts from parent and mutant clones were not deficient in SF1 protein as determined on Western blots with an SF1-specific antiserum (Fig. 4Go). Inasmuch as SF1 interacts with a number of regulatory proteins, including WT1 (12), CBP/p300 (13), steroid receptor coactivator 1 (14, 15), Dax-1 (11, 16), and SOX9 (17), we also screened parent and mutant clones for the presence of several of these interacting proteins. As determined by Western blot analysis, mutant cells also were not deficient in WT1, p300, CBP, or steroid receptor coactivator 1 (Fig. 4Go).



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Figure 4. Levels of SF1, WT1, and SF1 Coactivators in Parent and Mutant Cells

Nuclear extracts (50 µg) from parent Y1 cells and from mutant 10r-6 and 10r-9 cells were electrophoresed on SDS-polyacrylamide gels (41 ) and electroblotted onto nitrocellulose membranes. The membranes were immunoblotted with a goat antibody against mouse steroid receptor coactivator 1 (SRC1), with rabbit antibodies against mouse WT1, CBP, and p300 (purchased from Santa Cruz Biotechnology, Inc., Santa Cruz, CA) or with a rabbit antibody against bovine SF1/AD4BP generously provided by K. Morohashi (National Institute of Basic Biology, Okazaki, Japan). Antigen-antibody interactions were detected using horseradish peroxidase-labeled secondary antibodies and the ECL detection system (Amersham Pharmacia Biotech).

 
DNA Binding Activity of SF1 in Parent and Mutant Clones
To assess the DNA binding activity of SF-1 more directly, electrophoretic mobility shift assays were conducted on nuclear extracts from parent and mutant clones (Fig. 5Go). Nuclear extracts from parent and mutant cells exhibited three shifted complexes when incubated with a labeled SF1 probe and then electrophoresed (lanes 1 and 7); one of the complexes represented SF1-specific DNA binding activity as indicated. The formation of this labeled complex was inhibited by an excess of oligonucleotides corresponding to the SF1 sites from the MC2R promoter (lanes 3 and 9) or the Cyp21 promoter (lanes 5 and 11). In contrast, the labeled complex persisted in the presence of an excess of an unrelated oligonucleotide (lanes 2 and 8) or in the presence of an excess of SF1-related oligonucleotides containing base substitutions that disrupted the SF1 binding sites (lanes 4, 6, 10, and 12; Refs. 2, 18). As shown in Fig. 6Go, similar concentrations of an unlabeled SF1 oligonucleotide were required to displace a labeled probe from SF1 complexes in parent and mutant extracts, indicating that the SF1 from parent and mutant cells had similar apparent affinities for their binding sites. Taken together, these results suggested that the failure of mutant clones to express the MC2R did not result from impaired binding of SF1 to DNA. Rather an inability of SF1 to activate transcription seemed to underlie the loss of MC2R expression.



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Figure 5. DNA Binding Activity of SF1 in Parent and Mutant Cells

Nuclear extracts from parent Y1 cells (lanes 1–6) and mutant 10r-6 cells (lanes 7–12) were incubated with a 32P-labeled SF1 probe (3.6 nM) in the absence (lanes 1 and 7) or presence of an 80-fold molar excess of unlabeled oligonucleotides as competitors. DNA binding activity was evaluated in an electrophoretic mobility shift assay. The competitors used corresponded to: the SF1 site from the MC2R promoter (lanes 3 and 9); the SF1 site from the Cyp21 promoter (lanes 5 and 11), mutated forms of the SF1 oligonucleotides (lanes 4 and 10 and lanes 6 and 12); an unrelated oligonucleotide corresponding to the sequence at -170 in the Cyp21 promoter (lanes 2 and 8). Shifted complexes were not observed in the absence of nuclear extract (not shown). The arrows identify the positions of the SF1 complex and free probe.

 


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Figure 6. Apparent Affinities of SF1 from Parent and Mutant Clones for Their DNA Sites

The DNA binding properties of SF1 from parent Y1 (filled circles) and mutant 10r-9 (open circles) and 10r-6 (filled squares) cells were assessed by electrophoretic mobility shift assays as described in Fig. 5Go. Each sample contained an 80-fold molar excess of the unrelated double-stranded oligonucleotide (-170) to minimize nonspecific interactions and varying concentrations of the MC2R SF1 oligonucleotide as indicated. The amount of labeled SF1 complex formed was determined by phosphorimage analysis using the Molecular Imager phosphorimager from Bio-Rad Laboratories, Inc. (Mississauga, Ontario, Canada) and normalized to the amount of shifted complex obtained with each extract in the absence of competitor. The results presented are averaged from two separate experiments.

 
Steroidogenic Activity in Parent and Mutant Cells
Since SF1 has also been implicated in the expression of other genes required for adrenal steroidogenesis (3), we examined the consequences of impaired SF1 function in our mutants on other components of the steroidogenic pathway. As shown in Fig. 7Go, the 10r-9 mutant had a markedly diminished capacity for steroidogenesis compared with parental Y1 cells. In Y1 cells, ACTH and other agents that raise intracellular levels of cAMP stimulated steroidogenesis approximately 7.5-fold. Not surprisingly, the MC2R-deficient 10r-9 mutant failed to respond to ACTH with increased steroidogenesis. The mutant, however, also did not respond to 8-Br-cAMP or forskolin and failed to convert 22(R)-hydroxycholesterol to steroid products, indicating a significant loss of steroidogenic capacity. The steroidogenic capacity of mutant 10r-6 cells was similarly affected (data not shown). As determined by Northern blot hybridization analysis, the loss of steroidogenic capacity was accompanied by diminished levels of transcripts encoding Cyp11b1 and StAR (Fig. 8Go)—two key proteins required for corticosteroid biosynthesis. Cyp11b1 transcripts in the mutants were reduced approximately 95% compared with parent Y1 cells and seemed to be more greatly affected than StAR transcripts, which were reduced approximately 75%. The levels of Cyp11a did not show as consistent a pattern of change as did the other transcripts. In the 10r-9 mutant, Cyp11a transcript levels were 45% of those seen in parental Y1 cells, whereas in the 10r-6 mutant, the levels of Cyp11a transcripts were reduced to approximately 20% (Fig. 8Go).



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Figure 7. Steroid Production from Endogenous Cholesterol and from Exogenous 22(R)-Hydroxycholesterol in Parent and Mutant Cells

Cultures of Y1 and 10r-9 cells were incubated for 6 h in the absence (basal) or presence of ACTH (5 mU/ml), forskolin (10 µM), 8-Br-cAMP (3 mM), or 22(R)-hydroxycholesterol (20 µM) as indicated. The medium from each sample was collected, extracted, and quantitated for fluorescent steroids as detailed in Materials and Methods. Results presented are means ± SEM (n = 3).

 


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Figure 8. Cyp11a, Cyp11b1, and StAR Transcripts in Parent and Mutant Cells

Total RNA (25 µg) from parent Y1 and mutant 10r-9 and 10r-6 clones was blotted onto nylon membranes and probed for Cyp11a1, Cyp11b1, StAR, and transketolase (Tkt) transcripts as described in Materials and Methods. A representative Northern blot is shown on the left. Results from three separate experiments, presented as percentages of the signals obtained in parent Y1 cells ± SEM, are shown on the right. Hybridization signals were quantitated by phosphorimage analysis using the Molecular Imager phosphorimager from Bio-Rad Laboratories, Inc. and normalized to the levels of transketolase transcripts to account for variations in mRNA recovery, loading, and transfer among samples.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
As demonstrated previously (7), ACTH resistance in mutant Y1 adrenocortical tumor cells results from a defect that markedly diminishes the transcription of the MC2R gene. As shown here, the loss of MC2R expression was correlated with impaired activity of SF1 at the MC2R promoter. In support of this hypothesis, we found that the proximal promoter region of the MC2R functioned poorly in the mutant clones (Fig. 1Go and Ref. 8); the differences in MC2R promoter activity in parent and mutant cells were abolished upon deletion of the SF1 site (Fig. 1Go); an SF1 expression plasmid regulated SF1-dependent reporter genes less effectively in mutant cells than in parent Y1 cells (Figs. 2bGo and 3aGo). The latter finding further indicated that the loss of SF1-dependent activity resulted from a secondary event affecting SF1 function rather than from a mutation in SF1 per se. Nevertheless, the loss of SF1 function seemed relatively specific since the activity of another orphan nuclear receptor, Nur77, was not compromised in the mutant clones (Fig. 3bGo).

The loss of SF1 function seemed to result from defects in SF1-dependent activation of gene expression rather than from DNA binding defects. These conclusions were supported by the findings that the DNA binding activities of SF1 from parent and mutant clones were indistinguishable (Figs. 5Go and 6Go). Furthermore, the addition of the VP16 activation domain to SF1 restored its ability to activate SF1-dependent reporter genes in mutant clones (Figs. 2Go and 3Go). Possible mechanisms underlying this loss of SF1 function include changes in posttranslational modification of the protein or changes in a coregulatory protein or activating ligand. Inspection of the SF1 sequence reveals consensus sites for phosphorylation by cAMP-dependent protein kinase, protein kinase C, mitogen-activated protein kinase, and casein-kinase II; only the mitogen-activated protein kinase site has been shown to be important for SF1 function, at least in the context of placental JEG cells (19). We have not yet examined SF1 phosphorylation in Y1 and mutant 10r-6 and 10r-9 cells; however, the mutant cells appear to have mitogen-activated protein kinase and cAMP-dependent protein kinase signaling pathways intact (B.P. Schimmer and T. Le, unpublished observations, and Ref. 6). Oxysterols can activate SF1 (20), provided that assays of SF1 function are carried out in appropriate cell types (e.g. CV1 cells) maintained in charcoal-absorbed delipidated serum. Since the assays of SF1 function described here were carried out in the presence of complete serum, it is unlikely that changes in oxysterol production contributed to the mutant phenotype, although contributions of other endogenous ligands cannot be excluded. Finally, we have shown that the levels of several regulatory proteins known to interact with SF1, including WT1 (12), CBP and p300 (13), and steroid receptor coactivator 1 (14, 15), are not altered in the mutants (Fig. 4Go). Nevertheless, it remains to be determined whether alterations that affect the activities of these or other transcription regulators contribute to the mutant phenotype in the Y1 mutants.

SF1 is also thought to be important for the cell-selective expression of other genes involved in adrenal steroid hormone biosynthesis, including the cytochrome P450 steroid hydroxylases, 3ß-hydroxysteroid dehydrogenase and StAR (reviewed in Ref. 3). The roles for SF1 in the expression of these genes, however, have been inferred from analyses of proximal promoter sequences of these genes in transient transfection assays. The finding that the 10r-9 and 10r-6 mutants had impaired SF1 activity raised the possibility that the mutants might provide a paradigm to examine the roles of SF1 in expression of other genes involved in steroidogenesis. As shown here, the mutants were unable to form fluorogenic steroid products from 22(R)-hydroxycholesterol (Fig. 7Go). 22(R)-Hydroxycholesterol is the first intermediate in the conversion of cholesterol to pregnenolone (21) and is efficiently metabolized to steroid products bypassing the hormonally controlled rate-limiting steps of cholesterol mobilization and metabolism (22). In parent Y1 cells, these steroid products are 20{alpha}-progesterone and 11ß,20{alpha}-dihydroxyprogesterone (23, 24). In association with this defect, transcripts encoding StAR, Cyp11a, and Cyp11b1 were also reduced in the mutant clones. Thus, the mutant clones appeared to have major defects in steroid biosynthesis, supporting the hypothesis that SF1 is important for the expression of genes required for steroidogenesis. Furthermore, these observations raise the possibility that transcripts encoding other steroidogenic enzymes also might be affected. Intriguingly, MC2R, Cyp11a, Cyp11b1, and StAR transcripts were not affected to the same degree. Whereas MC2R and Cyp11b1 transcripts were reduced to nearly undetectable levels, StAR transcripts were reduced by 75%, and Cyp11a transcripts exhibited greater variation (55% and 80% reduction) between the two clones (e.g., Fig. 8Go and Ref. 8). These observations suggest that each of these genes may have different absolute requirements for SF1. Interestingly, Cyp11a also is not dependent on SF1 for expression in placenta (25), spleen (26), primitive gut (27), and brain (28).

The mutant clones described here were isolated by virtue of their resistance to the growth-inhibiting effects of the diterpene, forskolin (6), and arose from single mutational events as determined by fluctuation analysis (29). As we have shown previously, the growth-inhibiting effects of forskolin in Y1 cells are cAMP mediated, and resistance results from a failure of forskolin to maximally activate adenylyl cyclase rather than from a defect in cAMP action (6). While the underlying mutations have yet to be identified, much of the phenotype, including loss of MC2R expression, seems to have resulted from impaired G protein ß/{gamma} activity. In functional reconstitution assays, G protein ß/{gamma} activity from mutant cells was impaired (30), and genes encoding wild–type ß/{gamma}, when transfected into the mutants, restored MC2R expression (8). These results thus raised the possibility that MC2R gene expression was impaired in the mutants because of an underlying defect in a Gß/{gamma}-dependent signaling process. The ß/{gamma}-dimer has been shown to regulate a number of signaling pathways that potentially impact on gene expression. These include both positive and negative effects on the cAMP-signaling cascade, activation of Ca2+ signaling pathways, and protein kinase C and activation of the mitogen-activated protein kinase cascade (31). The results presented here, demonstrating impaired SF1 function at the MC2R promoter, identify SF1 as a possible link between Gß/{gamma} and MC2R expression and offer a starting point to trace the signaling events that mediate ß/{gamma} effects on gene expression.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Oligonucleotides and Plasmids
The complementary oligonucleotides corresponding to SF1 sequence at -25 in the mouse MC2R promoter with BamHI and BglII overhanging ends were 5'-GATCCTTTAG TCAAGGTTAGGATAAA-3' and 5'-GATCTTTATCCTAACCTTGAGTAAAG-3'. The mutated MC2R SF1 oligonucleotides were blunt-end complementary sequences with the core sequence AAGGTT mutated to ATTGTT as described (2). The complementary oligonucleotides corresponding to the SF1 site at -140 in the Cyp21 promoter (5'-ACAGATTCTCCAAGGCTGAT-3'), the mutated SF1 site at -140 with the core sequence CAAGGCTG mutated to CTCGGCTG (18), and the SF1-independent site at -170 in Cyp21 (5'-TGATGGATGGTCCCATCTTTGATCC-3'; Ref. 32) were also described previously.

The plasmids used in this study included: a luciferase reporter plasmid under control of the mouse MC2R promoter (bases -1808 to +104; Ref. 2); a luciferase reporter plasmid under control of six copies of the element at -65 in the mouse Cyp21 gene (p65 Luc; Ref. 9); a luciferase reporter plasmid containing SV40 promoter and enhancer sequences (pGL3-control; obtained from Promega Corp., Madison, WI); a luciferase reporter plasmid under control of the Rous sarcoma virus promoter (from -142 to +36; obtained from Dr. H. Elsholtz, University of Toronto, Toronto, Ontario, Canada). Luciferase reporter plasmids containing 5'-deletions of the mouse MC2R promoter were prepared using the PCR with appropriate primers. The luciferase reporter plasmid, p-25 Luc, was prepared by ligating two copies of a double-stranded oligonucleotide corresponding the SF1 site at -25 in the mouse MC2R promoter into the BamHI site of the pGL3 promoter vector (a luciferase reporter plasmid containing the SV40 core promoter; Promega Corp.). The SF- 1 expression plasmid was prepared by subcloning mouse SF-1 cDNA (9) into the EcoRI site of pcDNA3.1/His (Invitrogen, Carlsbad, CA). This construct resulted in a cDNA encoding SF1 with (His)6 - and epitope-tags at the amino terminus under control of the CMV promoter. Nur77 (a 2.2-kb cDNA that includes the entire coding region) was isolated by K. L. Parker (University of Texas, Southwestern Medical Center, Dallas, TX) from a mouse adrenal cDNA library and subcloned into the CMV promoter-based expression plasmid pCMV5 (33).

Cells, Cell Culture, and Gene Transfer
The Y1 mouse adrenocortical tumor cell line used in this study is a stable subclone (34) of the population originally isolated by Yasumura et al. (35). ACTH receptor-deficient Y1 mutants, including clones 10r-9 and 10r-6, were isolated on the basis of their resistance to the growth-inhibiting effects of forskolin as described (6). Cells were cultured as monolayers at 36.5 C under a humidified atmosphere of 95% air-5% CO2 in nutrient mixture F10 supplemented with 15% heat-inactivated horse serum, 2.5% heat-inactivated FBS, and antibiotics. Tissue culture reagents were obtained from Canadian Life Technologies, Inc. (Burlington, Ontario, Canada).

For gene transfer experiments, cells (2 x 105) were replicate plated in 60-mm tissue culture dishes and grown for 3 days. Cells were transferred to {alpha}-minimal essential medium supplemented with serum and antibiotics and transfected with super-coiled plasmid DNA using a calcium phosphate precipitation technique described previously (36, 37). Cells were incubated with the DNA/calcium phosphate precipitates for 24 h, rinsed to remove the DNA, and incubated in fresh medium for an additional 48 h before being harvested for analysis. To correct for variations in transfection efficiencies, separate plates of cells were transfected with a luciferase expression vector under control of either the SV40 promoter/enhancer or the Rous sarcoma virus (RSV) promoter as indicated.

Electrophoretic Mobility Shift Assays
The DNA binding activity of nuclear extracts from parent and mutant cells was assayed in electrophoretic mobility shift assays essentially as described previously (38). Each reaction contained 5 µg nuclear protein, 3.6 nM labeled probe (50,000 cpm), 2 µg double-stranded poly(dI·dC) in a total of 20 µl of binding buffer (10 mM Tris·HCl, pH 7.5, 50 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol, and 5% glycerol). DNA-protein complexes were resolved by electrophoresis on 4% nondenaturing polyacrylamide gels and visualized by fluorography. The probe was prepared by annealing complementary oligonucleotides corresponding to the SF1 sequence at -25 in the MC2R promoter and filling in the 3'-BamHI/BglII ends using the Klenow fragment of DNA polymerase-I (Amersham Pharmacia Biotech, Baie d’Urfe, Quebec, Canada) in the presence of [{alpha}-32P]dGTP (3,000 Ci/mmol; Mandel Scientific Co., Guelph, Ontario, Canada).

Luciferase Activity
Cell extracts were prepared by scraping cells into a lysis buffer containing 50 mM Tris.2-[N-morpholino]ethanesulfonic acid, pH 7.8, 1% Triton X-100, 4 mM EDTA, and 1 mM dithiothreitol; extracts were clarified by centrifugation at 4 C. Cell supernatants (4 to 400 µg protein) were assayed for luciferase activity in a reaction cocktail (250 µl) containing 200 µl of cell extract, 45 mM Tris.2-[N-morpholino]ethanesulfonic acid, pH 7.8, 9 mM magnesium acetate, 3 mM disodium ATP, and 0.12 mM luciferin as described (39). Luminescence was measured using a Berthold Lumat LB Luminometer under conditions where signals were proportional to the amount of supernatant protein added.

Steroid Production
Cells (5 x 104) were plated in 60-mm tissue culture dishes, grown for 5–7 days, and then incubated for 6 h in 2 ml {alpha}-minimal essential medium containing serum and antibiotics. At the end of the incubation, steroids were extracted from the medium using methylene chloride and quantitated by fluorescence in 65% sulfuric acid-35% ethanol using corticosterone as a standard (23).


    ACKNOWLEDGMENTS
 
We thank Adrian Clark for the MC2R promoter-luciferase construct, Keith L. Parker for cDNAs encoding SF1 and Nur77, Jeff Milbrandt for the expression vector encoding the VP16/SF1 fusion protein and for the p-65 Luc reporter plasmid, Ken Morohashi for the SF1/AD4BP antiserum, and Harry Elsholtz for the RSV-Luc expression plasmid. Fanny Lee and Brian Wong provided expert technical assistance.


    FOOTNOTES
 
Address requests for reprints to: Bernard P. Schimmer, Ph.D., Banting and Best Department of Medical Research, University of Toronto, 112 College Street, Toronto, Ontario M5G 1L6 Canada.

This work was supported by research grants from the Medical Research Council of Canada. C.F. was supported by a studentship from the Medical Research Council of Canada and a University of Toronto Open Studentship Award.

1 Members of the nuclear receptor family are identified by official names as proposed under a unified nomenclature system (40 ). Back

Received for publication July 21, 1999. Revision received January 5, 2000. Accepted for publication January 10, 2000.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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