Characterization of the Promoter Region of the Mouse Gene Encoding the Steroidogenic Acute Regulatory Protein

Kathleen M. Caron, Yayoi Ikeda, Shiu-Ching Soo, Douglas M. Stocco, Keith L. Parker and Barbara J. Clark

Departments of Medicine and Pharmacology Duke University Medical Center (K.M.C., Y.I., S.-C.S., K.L.P.) Durham, North Carolina 27710
Department of Cell Biology and Biochemistry Texas Tech University Health Science Center (D.M.S.) Lubbock, Texas 79430
Department of Biochemistry University of Louisville School of Medicine (B.J.C.) Louisville, Kentucky 40292


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Steroidogenic acute regulatory protein (StAR) delivers cholesterol to the inner mitochondrial membrane, where the cholesterol side-chain cleavage enzyme carries out the first committed step in steroid hormone biosynthesis. StAR expression is restricted to steroidogenic cells and is rapidly induced by treatment with trophic hormones or cAMP. We analyzed the 5'-flanking region of the mouse StAR gene to elucidate the mechanisms that regulate its cell-specific and hormone-induced expression. In transient transfection assays, a luciferase reporter gene driven by the StAR 5'-flanking region was preferentially expressed by steroidogenic Y1 adrenocortical and MA-10 Leydig cells in a cAMP-responsive manner. 5'-Deletion and site-directed mutagenesis studies identified a region between -254 and -113 that is essential for full levels of promoter activity. This region contains a binding site for the orphan nuclear receptor steroidogenic factor-1 (SF-1) that, although not required for hormone induction, is critical for basal promoter activity, thus implicating SF-1 in StAR expression. Analyses of knockout mice deficient in SF-1 further supported an important role for SF-1 in StAR gene expression. These studies provide novel insights into the mechanisms that regulate StAR gene expression and extend our understanding of SF-1’s global roles within steroidogenic cells. Molecular Endocrinology 11: 138–147, 1997)


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
A critical component of the regulated production of steroid hormones is the induction of their synthesis by pituitary trophic hormones. This hormonal induction is mediated predominantly by increases in intracellular cAMP and can be divided temporally into two phases: acute effects, which occur within seconds to minutes, and chronic effects, which require hours before they are manifest. The acute induction largely reflects increased mobilization and delivery of cholesterol, the precursor for all physiological steroid hormones, to the mitochondrial inner membrane, where it is metabolized to pregnenolone by the cytochrome P450 cholesterol side-chain cleavage enzyme (reviewed in Ref.1). In contrast, chronic effects of trophic hormones largely result from increased transcription of the genes that encode the steroidogenic enzymes, thereby maintaining optimal capacity for steroid production (reviewed in Ref.2).

Recent studies have implicated the steroidogenic acute regulatory protein (StAR) as an essential component of the acute response of steroidogenic cells to trophic hormone. StAR is a 30-kDa mitochondrial phosphoprotein whose expression is restricted to steroidogenic cells, where it is rapidly induced by trophic hormone in a manner that correlates with the acute stimulation of steroidogenesis (3, 4). In transient transfection experiments in both steroidogenic and nonsteroidogenic cells, StAR expression directly stimulated pregnenolone production (4, 5, 6). The onset of StAR expression during mouse embryonic development correlated closely with the onset of steroid hydroxylase expression and with the beginning of steroid hormone biosynthesis (5). Definitive proof for an essential role of StAR in regulated steroidogenesis came from studies of patients with lipoid congenital adrenal hyperplasia, a congenital disorder characterized by global defects in steroidogenesis (7). Analyses of patients with this disorder revealed mutations in the StAR gene that preclude the expression of functional StAR protein (8). These findings provided compelling biochemical and genetic evidence for the essential role of StAR in regulated steroidogenesis and strongly suggested that StAR mediates the acute regulation of steroid hormone biosynthesis.

To further our understanding of the mechanisms that regulate StAR gene expression, we recently isolated the mouse StAR gene and determined its structural organization and sequence (5). We further showed that StAR messenger RNA (mRNA) levels within steroidogenic cells were markedly increased by cAMP, suggesting that cAMP may induce StAR transcription. To extend these studies, we now characterize the mechanisms that regulate StAR expression in steroidogenic cells. Our results show that the 5'-flanking sequences of StAR can direct cell-specific and hormone-induced expression. They further implicate steroidogenic factor-1 (SF-1; also called adrenal-4-binding protein), an orphan nuclear receptor that plays key roles at multiple levels of the hypothalamic-pituitary-steroidogenic organ axis. Collectively, these results provide novel insights into the mechanisms that control the expression of this essential component of regulated steroidogenesis.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The 5'-Flanking Region of the StAR Gene Directs Cell-Selective and Hormone-Induced Expression
We previously reported the isolation and structural characterization of the mouse StAR gene (5). From these genomic clones, we isolated and sequenced ~3.6 kilobases of the StAR 5'-flanking region (Fig. 1Go). These sequences are numbered relative to the transcription initiation site, which was determined by primer extension analysis (Fig. 2Go). Unlike many tissue-specific and hormonally responsive genes, neither a canonical TATA box nor a consensus cAMP-responsive element (CRE) can be identified within this region. We previously noted a sequence at -135 (CCAAGGTGG, bottom strand) that resembles the consensus binding motif for the orphan nuclear receptor SF-1 (5). Further examination of the 5'-flanking sequence revealed two other potential binding sites for nuclear hormone receptors: the inverted repeat (AGGTCA GGACCT) located at -890 and a region at -42 (AGGCTG, bottom strand) that somewhat resembles the SF-1 consensus motif.



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Figure 1. Nucleotide Sequence of the 5'-Flanking Region of the Mouse StAR Gene

The sequence of 3646 bp of the mouse StAR 5'-flanking region is shown. The underlined sequence at -890 indicates an inverted repeat of the AGGTCA recognition motif for nuclear receptors. Two potential SF-1-binding sites at -135 and -42 are boxed. Dashed underlined sequences indicate repetitive motifs that resemble somewhat repetitive motifs in the human StAR promoter. The transcription initiation site, as determined by primer extension analysis, is designated +1 and is shown by the arrow.

 


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Figure 2. Primer Extension Analysis Maps the Mouse StAR Transcription Initiation Site

A primer complementary to positions 110–130 of the StAR cDNA was used in primer extension analysis with total RNA from mouse Y1 adrenocortical cells. As determined by comparison with end-labeled, HaeIII-digested {phi}X DNA markers, the size of the extended product was ~130 nucleotides.

 
To determine whether the StAR 5'-flanking region can direct cell-specific expression, the proximal 966 bp were placed upstream of the luciferase reporter gene, and the resulting plasmid (p-966StAR/luc) was transiently transfected into cultured mouse cell lines. As shown in Fig. 3Go, the StAR 5'-flanking region directed high levels of luciferase expression in two steroidogenic cell lines (mouse Y1 adrenocortical and MA-10 Leydig cells) and low levels of expression in two nonsteroidogenic cell lines (mouse L-TK- fibroblast and {alpha}T3 gonadotrope cells). The finding that StAR promoter activity is restricted to steroidogenic cells demonstrates that the 966 bp of the StAR 5'-flanking region are sufficient to direct appropriate cell-selective expression in transient transfection analyses.



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Figure 3. The StAR Promoter Is Preferentially Active in Steroidogenic Cell Lines

Y1 adrenocortical, MA-10 Leydig, {alpha}T3 gonadotrope, and L-TK- fibroblast cells were transiently transfected with 2.5 µg p-966StAR/luc as described in Materials and Methods. Where indicated, 8-Br-cAMP (1 mM) was added to the culture medium 36 h after transfection. Luciferase activities in cell lysates prepared 48 h after transfection were determined and normalized to those of the positive control plasmid pRSV2 luciferase, which contains the Rous sarcoma virus promoter/enhancer driving luciferase expression. Error bars indicate the SEMs.

 
As StAR expression is hormonally induced, we also analyzed the effect of cAMP treatment on StAR promoter activity in the same cell lines. As shown in Fig. 3Go (cross-hatched bars), treatment with cAMP significantly induced promoter activity in the steroidogenic Y1 adrenocortical and MA-10 Leydig cells. Significant increases were also observed in the very low levels of expression in L cells. These results demonstrate that the proximal 966 bp of the StAR promoter are sufficient for hormone-induced expression in steroidogenic and nonsteroidogenic cells. Studies with a reporter construct containing 3.6 kilobases of StAR 5'-flanking region showed that these additional sequences did not significantly increase basal or hormone-induced promoter activity over that seen with the proximal 966 bp (data not shown). We, therefore, concentrated our analyses of StAR gene regulation on the proximal 966 bp of the 5'-flanking region shown to be capable of directing both cell-selective and hormone-induced expression.

Elements Required for StAR Promoter Activity Are Located within 253 bp of the Transcription Initiation Site
We next performed 5'-deletion analyses to identify specific sequences within the StAR 5'-flanking region that regulate constitutive and hormone-induced promoter activity. Plasmids containing progressively decreasing amounts of StAR 5'-flanking region upstream of the human GH (hGH) reporter gene were transiently transfected into MA-10 Leydig and Y1 adrenocortical cells, with or without cAMP treatment, and promoter activity was measured by RIA for hGH (MA-10 cells) or by Northern blotting analyses of hGH transcripts (Y1 cells). As shown in Fig. 4AGo, progressive deletion of sequences from -966 to -426 and from -426 to -254 did not significantly impair basal or cAMP-induced promoter activity. In fact, there was a progressive increase in both basal and cAMP-induced activity, suggesting that a negative regulatory element may lie between -966 and -254. These results demonstrate that sequences within this region do not positively regulate StAR expression in MA-10 cells. Further deletion of the promoter from -254 to -113 considerably diminished basal StAR promoter activity to levels that did not differ significantly from the those of the promoterless negative control. Moreover, there was no response to cAMP treatment in cells transfected with the -113 construct. Similar results were obtained when the same plasmids were transiently transfected into Y1 adrenocortical cells (Fig. 4BGo). In addition, hGH transcripts expressed from the 5'-deletion plasmids were of the predicted size, strongly suggesting that they arose from the authentic initiation site. These results strongly suggest that the region between -254 and -113 contains an important positive regulator of StAR expression in both MA-10 Leydig cells and Y1 adrenocortical cells. Interestingly, this region contains the putative SF-1 binding site at -135.



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Figure 4. 5'-Deletion Analysis Reveals a Region Important for StAR Promoter Activity in Both MA-10 Leydig Cells and Y1 Adrenocortical Cells

A, MA-10 Leydig cells were transiently transfected in triplicate by the lipofectamine method with the indicated StAR 5'-deletion plasmids and pCMVß-gal. Twenty-four hours after transfection, the medium was replaced, and where indicated, the cells were treated with 1 mM Bt2cAMP. Levels of promoter activity were measured by RIA for hGH. Levels of hGH activities were normalized to ß-galactosidase activity and are expressed as a percentage of the basal activity of p-966StAR/hGH. Error bars depict the SEMs from three independent experiments. B, Y1 adrenocortical cells were transiently transfected in triplicate by the calcium phosphate precipitation method with 2.5 µg of the indicated StAR 5'-deletion plasmids. For reference, cells were also transfected with the positive control plasmid pXGH5, which contains the mouse metallothionine promoter driving hGH expression, and the promoterless plasmid pBSGH. Cells were treated with 1 mM 8-Br-cAMP 36 h after transfection, where indicated (+). Total RNA was harvested 48 h after transfection and used in Northern blot analysis with a hGH cDNA probe. A probe for {alpha}-tubulin was used to verify the amount and quality of the RNA.

 
Induction of StAR Transcripts by cAMP Is Blocked by Actinomycin D, but Does not Require de Novo Protein Synthesis
Previous analyses have implicated transcriptional induction as the predominant basis for trophic hormone induction of genes required for steroidogenesis (reviewed in Ref.2). However, the possible role of changes in mRNA stability and the requirement for de novo protein synthesis in hormone-induced gene expression appear to be gene dependent (9, 10, 11). Treatment of p-966StAR/luc with cAMP significantly increased StAR promoter activity (Fig. 3Go), suggesting that hormonal effects reflect increased StAR transcription. Consistent with this model, trophic hormone induction of StAR mRNA levels was abolished when cells were treated with actinomycin D to prevent the synthesis of new transcripts (Fig. 5Go). However, treatment of the cells with cycloheximide did not prevent the cAMP-related increases in StAR mRNA, indicating that cAMP induction of StAR mRNA is direct and does not require de novo protein synthesis.



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Figure 5. cAMP Induction of StAR mRNA Levels Requires Transcription, but not de Novo Protein Synthesis

MA-10 Leydig cells were treated for 2 h with 1 mM Bt2cAMP, Bt2cAMP plus 10 µg/ml actinomycin D (ACTD), or Bt2cAMP plus 7 µg/ml cycloheximide (CHX). Total RNA was isolated, and StAR and actin transcripts were quantitated by reverse transcription-PCR as described in Materials and Methods. Radiolabeled PCR products were quantitated using a PhosphoImager (Molecular Dynamics).

 
The Potential SF-1-Binding Site at -135 Interacts Specifically with SF-1
Our 5'-deletion studies implicated a region that includes a potential SF-1 site in basal and cAMP-induced StAR promoter activity. To determine whether SF-1 can bind this motif, we performed gel mobility shift assays with oligonucleotides containing this sequence and nuclear extracts from Y1 adrenocortical cells. As shown in Fig. 6Go, a probe containing the SF-1 motif located at -135 formed a prominent shifted complex that was competed in a dose-dependent manner by unlabeled oligonucleotides containing the homologous sequence (CCAAGGTGG), but not by unlabeled oligonucleotides containing a mutated SF-1-binding site (CCATATTAT). Moreover, preincubation of the nuclear extract with a polyclonal antiserum specific for the DNA-binding domain of SF-1 completely abolished complex formation (Fig. 6Go). Even though it was not identified as an important regulator of StAR expression in our 5'-deletion studies, the potential SF-1 motif at -42 also competed with the SF-1-related complex. Given that the region between -254 and -113 is important for StAR promoter activity, the finding that SF-1 interacts specifically with the -135 element is consistent with the model in which SF-1 regulates StAR gene expression.



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Figure 6. SF-1 Binds the Potential SF-1-Responsive Element at -135 in the StAR 5'-Flanking Region

An oligonucleotide corresponding to the potential SF-1-binding site at -135 of the mouse StAR 5'-flanking region (SF-1-1) was used in gel mobility shift assays with 10 µg nuclear extract from mouse Y1 adrenocortical cells. Where indicated, unlabeled oligonucleotides corresponding to the SF-1 site at -135 (SF-1-1), a mutated variant (mSF-1-1), or the proximal SF-1 binding site at -42 (SF-1-2) were included in the reactions at the indicated molar ratios to labeled probe. Where indicated, antiserum specific for SF-1 ({alpha}-SF-1) or preimmune serum were included in the binding reaction as described in Materials and Methods.

 
Site-Directed Mutagenesis Defines the Role of the -135 SF-1-Binding Site in StAR Promoter Activity
Although the 5'-deletion and gel shift assays suggested that SF-1 regulates StAR promoter activity, they did not directly address the roles of the SF-1 sites in StAR expression. We, therefore, used site-directed mutagenesis to mutate the SF-1 site at -135, either alone or in combination with the -42 site, within the context of p-966StAR/luc. Consistent with the 5'-deletion analyses shown in Fig. 4Go, mutation of the -135 SF-1-binding site decreased basal promoter activity in Y1 adrenocortical cells to ~50% of the level of the wild-type promoter (Fig. 7Go), revealing a role for this element in StAR promoter activity. No further impairment was seen when the -42 SF-1 site was also mutated. However, both mutated plasmids exhibited full levels of induction in response to cAMP, suggesting that hormone induction and regulation by SF-1 occur through different promoter elements. Similar qualitative results were obtained when mutated StAR promoter/hGH plasmids were transiently transfected in MA-10 Leydig cells (data not shown). Collectively, the 5'-deletion and site-directed mutagenesis studies thus define an important role for the SF-1 binding motif at -135 in StAR gene expression.



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Figure 7. Mutation of the SF-1 Binding Motif at -135 Impairs StAR Promoter Activity

Y1 adrenocortical cells were transiently transfected in triplicate with 0.5 µg of the indicated StAR promoter/luciferase plasmids. Thirty-six hours after transfection, the cells were treated with 1 mM 8-Br-cAMP (cross-hatched bars). Luciferase activities in cell lysates prepared 48 h after transfection were determined and normalized to those of the positive control plasmid pRSV2 luciferase. Error bars indicate the SEMs.

 
SF-1 Is Essential for StAR Gene Expression in Vivo
Our analyses of StAR expression in transfected cell lines strongly suggested that SF-1 positively regulates StAR expression. To address the role of SF-1 in StAR expression in vivo, we took advantage of knockout mice that are homozygously disrupted in the Ftz-F1 gene that encodes SF-1 (12). As a consequence of this gene knockout, the Ftz-F1-disrupted mice undergo degeneration of the developing gonads and adrenal primordia on approximately embryonic day 12 (E12). As StAR gene expression normally commences on E10.0 to E10.5, before the gonads have significantly degenerated, we were able to use these mice to examine the possible role of SF-1 in StAR expression in vivo. As shown in Fig. 8Go, StAR transcripts were readily detected in the genital ridges of wild-type embryos on both E10.5 and E11.5, but could not be detected in the urogenital ridges of Ftz-F1 knockout mice. To the extent that the developing gonads were still intact at these relatively early stages of gonadogenesis, these results implicate SF-1 as an essential regulator of StAR expression in vivo.



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Figure 8. StAR Transcripts Are not Detected in Knockout Mice Lacking SF-1

Mouse embryos were harvested and genotyped by Southern blotting analysis. Serial sagittal sections were prepared from wild-type (WT; left panels) and Ftz-F1-disrupted (right panels, -/-) embryos and analyzed by in situ hybridization with an antisense complementary RNA probe specific for StAR as described in Materials and Methods. Darkfield views from E10.5 (top panels) and E11.5 (middle panels) are shown above, with brightfield views of the E11.5 sections shown below. The arrow points to the developing gonad. G, Genital ridge.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
In this study, we explore the molecular mechanisms that regulate the expression of the mouse StAR gene. StAR has been implicated in the acute phase of regulated steroidogenesis, delivering cholesterol to the inner mitochondrial membrane where the cholesterol side-chain cleavage enzyme carries out the first steps in steroidogenesis. In keeping with its proposed function, StAR expression is restricted to the steroidogenic cells of the adrenal cortex and gonads (5). We now show that 966 bp of the StAR 5'-flanking region can direct cell-selective and hormone-induced expression in transient transfection experiments, suggesting that this region is important in both key components of StAR gene expression. We also demonstrate that the hormone-induced expression of StAR reflects increased transcription of StAR mRNA, but does not require de novo protein synthesis. Within the StAR 5'-flanking sequences, we identify a region from -254 to -113 that is essential for full levels of basal and hormone-induced promoter activity. This region contains a promoter element that is bound by the orphan nuclear receptor SF-1 and is essential for full levels of promoter activity, implicating SF-1 in the regulation of StAR expression. Finally, we show that StAR expression is markedly impaired in knockout mice deficient in SF-1, providing strong evidence that SF-1 positively regulates StAR gene expression in the intact mouse.

Our analyses of StAR promoter activity raise several interesting points. Perhaps most significantly, both transfection studies and analyses of knockout mice implicate the orphan nuclear receptor SF-1 in StAR gene regulation. Although cotransfection of Y1 cells with an SF-1 expression plasmid only slightly increased the promoter activity of the mouse StAR 5'-flanking sequences (data not shown), this modest induction probably reflects the fact that Y1 cells endogenously express high levels of SF-1. Consistent with this model, a similar role for SF-1 in the promoter activity of the human StAR gene has recently been proposed based on SF-1 induction of promoter activity of the human 5'-flanking sequences in transient transfection experiments in nonsteroidogenic cells (13).

Initially identified as an essential regulator of the cytochrome P450 steroid hydroxylases (14, 15), analyses of knockout mice established that SF-1 plays essential roles at multiple levels of the hypothalamic-pituitary-steroidogenic organ axis, establishing it as a key factor in reproduction (12, 16–19; reviewed in ref. 20). Although it is not entirely unexpected that SF-1 regulates StAR gene expression in view of its global roles in steroidogenesis and reproduction, the link between SF-1 and StAR identifies yet another target gene by which SF-1 determines steroidogenic competence.

Another important finding is that the StAR 5'-flanking region directs hormone-induced expression. Unlike many genes that are induced by cAMP, increases in StAR promoter activity apparently do not result from interactions between the transcriptional regulator cAMP-response element binding protein and its cognate CRE (21), as no sequence resembling the consensus CRE is found in the 5'-flanking region of the mouse (this report) or human (13) StAR genes. Nonetheless, our promoter analyses strongly suggest that StAR induction by cAMP at least partly involves transcriptional activation. In this respect, the StAR gene resembles those of several of the steroid hydroxylases, which also are induced by cAMP in the absence of a classical CRE (21). Although SF-1-binding sites have been implicated in the hormonal regulation of several steroid hydroxylases (reviewed in Ref.22), our site-directed mutagenesis studies demonstrate that the SF-1 sites in the StAR promoter are dispensable for hormone induction. Thus, an important goal for future studies will be to identify the precise region(s) of the StAR gene that underlies increased transcription in response to hormone induction.

Another key component of StAR expression is its tissue specificity, with expression in steroidogenic cells of the adrenal cortex and gonads, but not in those of the placenta. Our own studies indicate that the orphan nuclear receptor SF-1 is a key regulator of cell specificity. It is, nonetheless, clear that other mechanisms also must limit StAR expression to the steroidogenic cells of the adrenal cortex and gonads, as SF-1 is also expressed by pituitary gonadotropes and the ventromedial hypothalamic nucleus, regions that do not express StAR (5). Of interest in this regard, mutation of the SF-1 binding element does not impair StAR promoter activity as drastically as does deletion of the region from -254 to -113. It thus appears that other important regulatory elements reside within this part of the StAR 5'-flanking region. An important goal for future studies will be to identify and characterize these other regulatory elements, perhaps leading to a better understanding of the precise mechanisms that regulate this essential component of regulated steroidogenesis.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Materials
Radionucleotides were purchased from New England Nu-clear-DuPont (Boston, MA). Restriction and modification enzymes were purchased from Boehringer Mannheim (Indianapolis, IN). Reagents for sequencing were purchased from Amersham Life Science (Arlington Heights, IL). Reagents for cell culture, including lipofectamine reagent, were purchased from Life Technologies (Gaithersburg, MD) and Mediatech (Washington DC). 8-Bromo-cAMP (8-Br-cAMP) and (Bu)2cAMP were purchased from Sigma Chemical Co. (St. Louis, MO). PCR reagents were purchased from Perkin-Elmer (Norwalk, CT). The Double-Stranded Site Directed Mutagenesis Kit was obtained from Stratagene (La Jolla, CA). Reagents for luciferase assays and ß-galactosidase assays were purchased from Promega (Madison, WI). RIA kits for hGH were purchased from Nichols Institute (San Juan Capistrano, CA). Reagents for in situ hybridization were purchased from Novagen (Madison, WI).

Plasmids
Plasmids were made using previously isolated sequences of the StAR 5'-flanking region (5). Full-length and 5'-deletion plasmids with the indicated amounts of 5'-flanking sequences were cloned into a promoterless plasmid containing the hGH structural gene using PCR-based strategies and convenient restriction sites. Other plasmids used the same 5'-flanking sequences upstream of a luciferase reporter gene in the pGL2 basic plasmid (Promega). Mutated plasmids were generated by PCR-based strategies or double stranded site-directed mutagenesis. The SF-1 site at -135 was mutated from wild-type CCAAGGTGG (bottom strand) to TACGTAGTT (bottom strand). The SF-1 site at -42 was mutated from wild-type AGGCTG (bottom strand) to TACGTA (bottom strand).

Primer Extension Analysis
The transcription initiation site of StAR was mapped with the Drosophila Embryo Nuclear Extract In Vitro Transcription System (Promega) according to the manufacturer’s protocol. Total RNA was isolated from Y1 adrenocortical cells by the guanidinium isothiocyanate method using a standard protocol (23). A primer complementary to 110–130 bp of the complementary DNA (cDNA) was used to extend a ~130-nucleotide fragment, whose size was estimated relative to the migration of end-labeled, HaeIII-digested {phi}X DNA.

Cell Culture
Mouse Y1 adrenocortical, MA-10 Leydig, L-TK- fibroblast, and {alpha}T3 gonadotrope cells were cultured and transfected in triplicate by the CaPO4 precipitation technique (23) or by lipofectamine (4), using 0.5–2.5 µg of the reporter gene. Levels of hGH were assayed by RIA or Northern analysis 48 h after transfection. In experiments with cAMP stimulation, cells were treated with either 8-Br-cAMP for 12 h (Y1 studies) or (Bu)2cAMP for 2–24 h (MA-10 studies). Total RNA was isolated by the guanidinium isothiocyanate method using a standard protocol and subjected to Northern blot analyses using standard methods (23).

Reverse Transcription-PCR
MA-10 Leydig cells were either treated or untreated for 2 h with 1 mM (Bu)2cAMP alone plus 10 µg/ml actinomycin D or plus 7 µg/ml cycloheximide. Total RNA was extracted using the Trizol reagent (Life Technologies, Gaithersburg, MA) and semiquantitative reverse transcription-PCR was used to measure StAR mRNA levels. Random hexamer oligonucleotides (Pharmacia-LKB, Piscataway, NJ) were used for the RT reaction, which contained deoxy-NTPs, RNAsin, 2.5 mM MgCl2, 200 U Moloney murine leukemia virus-reverse transcriptase (Life Technologies) and 1 µg total RNA. For amplification of the cDNA, the reaction was "spiked" with [32P]deoxy-CTP to radiolabel the amplification products. After 25 cycles of amplification, the PCR products were separated on a 2% agarose gel, the gel was dried and exposed to a phosphoscreen, and the products were visualized and quantitated using a Molecular Dynamics PSF PhosphoImager (Sunnyvale, CA). StAR mRNA levels were normalized to ß-actin mRNA levels to determine the effect of treatment on StAR expression.

Gel Mobility Shift Assays
Complementary oligonucleotides corresponding to each of the putative SF-1 sequences were synthesized, annealed, and end labeled by Klenow fill-in reaction; these duplex oligonucleotides included SF-1-1 (5'-CCTCCCACCTTGGCCA-3', positions -142 to -126) and SF-1-2 (5'-TGCACAGCCTTCCACG-3', positions -49 to -34). Gel mobility shift assays were performed essentially as previously described (14). Briefly, labeled probes were incubated with 10 µg Y1 adrenocortical cell nuclear extract and 4 µg poly[d(I-C)]-poly-[d(I-C)] as nonspecific competitor. Where indicated, either unlabeled oligonucleotide competitors or antiserum specific for SF-1 were preincubated with the nuclear extract before probe addition. Samples were analyzed by electrophoresis on a 4% nondenaturing polyacrylamide gel. Gels were dried and exposed to x-ray film overnight.

In Situ Hybridization
Serial sagittal sections (6 µm) were deparaffinized and hybridized overnight at 50–55 C using an in situ hybridization kit according to recommended protocol. 35S-Labeled complementary RNA probes were prepared using T3 and T7 polymerases according to the protocol supplied with a kit purchased from Novagen (Madison, WI). After washes at high stringency, the slides were dipped in Kodak NTB-2 emulsion (Eastman Kodak, Rochester, NY) diluted 1:1. Exposures were carried out for 3–4 weeks. After exposure, slides were developed in Kodak D-19, fixed, and counterstained with methyl green. Both sense and antisense probes derived from the mouse StAR cDNA (5) were used, and all hybridizations included a control section of adult mouse adrenal gland.


    ACKNOWLEDGMENTS
 
We thank Dr. Mario Ascoli for the MA-10 cells; Dr. Pamela Mellon for the {alpha}T3 cells; Drs. Deepak Lala, Cameron Scarlett, Jerry Strauss, and Walter Miller for helpful discussions; and Jeana Meade, LeeAnn Baity, and Rebecca Combs for superb technical assistance.


    FOOTNOTES
 
Address requests for reprints to: Dr. Keith L. Parker, 323 CARL Building, Duke University Medical Center, Durham, North Carolina 27710.

This work was supported by the Howard Hughes Medical Institute; NIH Grants HL-48460 (to K.L.P.), HD-17481 (to D.M.S.), and DK-51656-01 (to B.J.C.); and NIEHS Grant ES06832-03 (to B.J.C.).

Received for publication February 19, 1996. Revision received October 17, 1996. Accepted for publication November 5, 1996.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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