Regulation of Steroidogenesis and the Steroidogenic Acute Regulatory Protein by a Member of the cAMP Response-Element Binding Protein Family

Pulak R. Manna, Matthew T. Dyson, Darrell W. Eubank, Barbara J. Clark, Enzo Lalli, Paolo Sassone-Corsi, Anthony J. Zeleznik and Douglas M. Stocco

Department of Cell Biology and Biochemistry (P.R.M., M.T.D., D.W.E., D.M.S.), Texas Tech University Health Sciences Center, Lubbock, Texas 79430; Department of Biochemistry (B.J.C.), University of Louisville School of Medicine, Louisville, Kentucky 40292; Institute de Génétique et de Biologie et Moléculaire et cellulaire (E.L., P.S.-C.), CNRS-INSERM, 67085 Strasbourg, France; and Department of Cell Biology and Physiology (A.J.Z.), University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261

Address all correspondence and requests for reprints to: Douglas M. Stocco, Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, Lubbock, Texas 79430. E-mail: doug.stocco{at}ttmc.ttuhsc.edu


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The mitochondrial phosphoprotein, the steroidogenic acute regulatory (StAR) protein, is an essential component in the regulation of steroid biosynthesis in adrenal and gonadal cells through cAMP-dependent pathways. In many cases transcriptional induction by cAMP is mediated through the interaction of a cAMP response-element binding protein (CREB) family member with a consensus cAMP response element (CRE; 5'-TGACGTCA-3') found in the promoter of target genes. The present investigation was carried out to determine whether a CRE-binding protein (CREB) family member [CREB/CRE modulator (CREM) family] was involved in the regulation of steroidogenesis and StAR protein expression. Transient expression of wild- type CREB in MA-10 mouse Leydig tumor cells further increased the levels of (Bu)2cAMP-induced progesterone synthesis, StAR promoter activity, StAR mRNA, and StAR protein. These responses were significantly inhibited by transfection with a dominant-negative CREB (A-CREB), or with a CREB mutant that cannot be phosphorylated (CREB-M1), the latter observation indicating the importance of phosphorylation of a CREB/CREM family member in steroidogenesis and StAR expression. The CREB/CREM-responsive region in the mouse StAR gene was located between -110 and -67 bp upstream of the transcriptional start site. An oligonucleotide probe (-96/-67 bp) containing three putative half-sites for 5'-canonical CRE sequences (TGAC) demonstrated the formation of protein-DNA complexes in EMSAs with recombinant CREB protein as well as with nuclear extracts from MA-10 or Y-1 mouse adrenal tumor cells. The predominant binding factor observed with EMSA was found to be the CREM protein as demonstrated using specific antibodies and RT-PCR analyses. The CRE elements identified within the -96/-67 bp region were tested for cAMP responsiveness by generating mutations in each of the CRE half-sites either alone or in combination. Although each of the CRE sites contribute in part to the CREM response, the CRE2 appears to be the most important site as determined by EMSA and by reporter gene analyses. Binding specificity was further assessed using specific antibodies to CREB/CREM family members, cold competitors, and mutations in the target sites that resulted in either supershift and/or inhibition of these complexes. We also demonstrate that the inducible cAMP early repressor markedly diminished the endogenous effects of CREM on cAMP-induced StAR promoter activity and on StAR mRNA expression. These are the first observations to provide evidence for the functional involvement of a CREB/CREM family member in the acute regulation of trophic hormone-stimulated steroidogenesis and StAR gene expression.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
STEROID BIOSYNTHESIS IN response to trophic hormone stimulation is a de novo protein synthesis requiring a process that is regulated by the delivery of cholesterol from the outer to the inner mitochondrial membrane where it is cleaved by the cholesterol side chain cleavage cytochrome P450 enzyme (1, 2). Among the candidate regulatory proteins involved in this transfer, the steroidogenic acute regulatory protein (StAR) has essentially all of the required characteristics (2, 3). The strongest evidence for the critical role of StAR in regulating steroidogenesis has been demonstrated in patients suffering from congenital lipoid adrenal hyperplasia, in which both adrenal and gonadal steroid biosyntheses are markedly impaired due to mutations in the StAR gene (4). An essentially identical phenotype is observed in StAR null mice (5, 6).

Transduction of signals by a number of hormones and neurotransmitters is known to occur via the activation of adenylate cyclase, which increases intracellular cAMP levels and activates PKA. The expression of the StAR protein in the adrenals and gonads is stimulated through the cAMP signaling pathway and is closely correlated with the acute steroidogenic response of these cells to tropic hormone stimulation (7, 8). Several transcription factors and processes are involved in regulating both StAR expression and steroidogenesis, but none so far can fully account for their rapid stimulation in response to increases in cAMP.

Response to cAMP at the gene level is typically mediated through a palindromic conserved sequence (5'-TGACGTCA-3') referred to as the cAMP response element (CRE) (9, 10, 11). However, the StAR gene lacks a consensus CRE, a situation also found in several steroid hydroxylase genes that are regulated by cAMP (12). CRE sequences investigated to date demonstrate that the 5'-TGACG is highly conserved in comparison to the 3'-TCA (13). A large family of basic-leucine zipper (bZip) CRE-binding factors, including CRE-binding protein (CREB), CRE modulator protein (CREM), and activating transcription factor (ATF-1), have been shown to interact with this sequence. Both activators and repressors of transcription can be found within the CRE-binding factor family, which can homo- and heterodimerize using a specific interaction code (14, 15, 16). Three genes, CREB, CREM, and ATF-1, share extensive homology, constitute the CREB/CREM subfamily (CREB/CREM), and mediate transcriptional activation (17).

CREB/CREM plays a major role in the growth and developmental processes of many organs, is required for survival, and mediates a variety of biological functions (10, 17, 18). CREB/CREM is activated after phosphorylation by PKA as well as other kinases, and its transcriptional activities are regulated by multiple signaling pathways (9, 14, 16). Transcriptional increases in response to cAMP are observed after the phosphorylation of CREB at serine-133 (19, 20) or CREM at serine-117 (21), and its binding to the nuclear protein CBP (CREB binding protein). Recent studies have demonstrated that neither a mutant form of CREB, unable to be phosphorylated by PKA (CREB-M1), nor a dominant-negative form of CREB (A-CREB) is capable of activating transcription (22, 23, 24). Also, transcriptional activation by CREB/CREM is greatly affected by expression of CREM isoforms that include DNA-binding and dimerization domains but lack transactivation domains. In particular, the CREM inducible cAMP early repressor (ICER) acts as a powerful repressor of cAMP-induced transcription (25, 26). Transcriptional repression by ICER has been shown to occur via its binding to the CRE sites of target gene promoters or by the formation of inactive heterodimers with CREB or other associated transactivators (13, 25, 26).

Considerable progress has been made regarding the regulation of hormone-stimulated steroidogenesis and StAR expression. However, the mechanism for the acute regulation of StAR transcription by cAMP is still not completely understood. Based on these considerations, the present investigation was undertaken to evaluate the potential involvement of CREB/CREM family members in the regulation of steroidogenesis and StAR expression. Our findings provide evidence for the first time that a CREB/CREM family member, most probably CREM, is involved in increases in the levels of steroid synthesis, StAR promoter activity, StAR mRNA, and StAR protein expression in steroidogenic cells.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Involvement of CREB/CREM in the Regulation of Steroidogenesis
The potential role of CREB/CREM in the regulation of (Bu)2cAMP-induced progesterone (P) production was determined. These findings demonstrated that nonstimulated MA-10 cells transiently expressing WT-CREB significantly increased (P < 0.05) basal P production, while (Bu)2cAMP-stimulated P levels observed were increased by 30–40% when compared with mock-transfected cells (expression vector not containing CREB) (Table 1Go). Expression of either A-CREB or CREB-M1 resulted in an approximately 30% decrease in (Bu)2cAMP-stimulated P levels when compared with that seen in control cells. However, neither A-CREB nor CREB-M1 significantly altered basal P levels in the absence of cAMP stimulation. These results demonstrate that a CREB/CREM family member is involved in the regulation of steroid hormone biosynthesis in mouse Leydig cells.


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Table 1. Effect of WT-CREB, A-CREB, and CREB-M1 on Basal and (Bu)2cAMP-Induced P Production in MA-10 Cells

 
The Role of CREB/CREM in StAR Expression
To determine whether StAR was involved in the observed stimulation of P synthesis in response to CREB/CREM expression, StAR mRNA and protein levels were analyzed in CREB transfected cells. As illustrated in Fig. 1AGo, MA-10 cells expressing wild-type CREB (WT-CREB) modestly, but consistently, increased basal StAR mRNA expression (P < 0.05), an observation in keeping with the observed increase in basal P production. (Bu)2cAMP-induced StAR mRNA expression in WT-CREB transfected cells was significantly elevated to approximately 3-fold of that seen in mock-transfected cells. Similar to the observation with P synthesis, when MA-10 cells were transfected with A-CREB or CREB-M1, (Bu)2cAMP-stimulated levels of StAR mRNA were decreased to a level approximately 30–40% below that seen in control cells. Using the same experimental paradigms it was also determined that the mitochondrial 30-kDa StAR protein content was slightly but consistently increased in nonstimulated MA-10 cells after transfection with WT-CREB and was increased approximately 2-fold by expression of WT-CREB in (Bu)2cAMP-stimulated cells. After expression of A-CREB or CREB-M1 there was a 40% decrease in StAR protein levels in (Bu)2cAMP-stimulated cells when compared with controls (Fig. 1BGo). These results demonstrate the potential involvement of a CREB/CREM family member in StAR expression and demonstrate a direct relationship between StAR expression and steroidogenesis.



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Figure 1. Effect of Expression of WT-CREB, A-CREB, and CREB-M1 on (Bu)2cAMP-Stimulated StAR mRNA and StAR Protein Expression in MA-10 Cells

Cells were transiently transfected with different CREB expression plasmids (2 µg each) using Fugene-6 transfection reagent as described in Materials and Methods. After 36 h of transfection, cells were stimulated for 6 h in the absence (Basal) or presence of 500 µmol/liter of (Bu)2cAMP (cAMP). Total RNA and mitochondrial protein were isolated from each group separately and subjected to RT-PCR and Western blotting to determine StAR expression. Shown is a representative autoradiogram of cAMP-induced StAR mRNA (panel A) and a representative Western blot of 30-kDa StAR protein (panel B) expression in mock (MT)-, WT-CREB-, A-CREB-, and CREB-M1-transfected cells. The integrated optical density (IOD) of StAR expression was quantitated in both cases and normalized in the case of RT-PCR to L19 expression. The IOD values represent the mean ± SEM of three to five independent experiments.

 
Time-Dependent Increase in cAMP-Stimulated StAR mRNA and P Levels in Relation to CREB/CREM
The temporal relationship between StAR expression and steroidogenesis in response to CREB/CREM was investigated. Figure 2Go shows the time-dependent increases in the steady-state levels of StAR mRNA in control and CREB-expressing cells in response to (Bu)2cAMP stimulation. Expression of WT-CREB marginally increased (P < 0.05) basal StAR mRNA but significantly elevated (Bu)2cAMP-induced StAR mRNA levels, reaching a maximum between 6 and 8 h and being 3 times higher than mock-transfected cells. Thereafter, the levels of StAR mRNA decreased. The magnitude of response was evident within 1 h of cAMP induction displaying a 70% increase in StAR mRNA levels in CREB-transfected cells compared with mock-transfected cells.



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Figure 2. Temporal Pattern of (Bu)2cAMP-Induced StAR mRNA Expression in Mock- and WT-CREB-Transfected MA-10 Cells

Cells were transfected with either empty vector (mock; MT) or WT-CREB (2 µg each) as described in Fig. 1Go. After 36 h of transfection, cells were washed and stimulated for 0–12 h in the presence of 500 µmol/liter (Bu)2cAMP. Total RNA was extracted at the indicated times and subjected to RT-PCR analysis of StAR mRNA expression as described in the legend of Fig. 1Go and Materials and Methods. Panel A illustrates a representative autoradiogram of cAMP-induced StAR mRNA expression at different times. Panel B depicts IOD values of StAR mRNA expression from three independent experiments (± SEM) after normalization to the L19 bands.

 
The accumulation of P in the media at each time point was also determined in mock- and WT-CREB-transfected cells after (Bu)2cAMP stimulation and paralleled the increases in StAR mRNA expression (Fig. 3Go). The levels of P significantly increased (P < 0.01) after 1 h of (Bu)2cAMP stimulation, while WT-CREB increased P levels by approximately 35% over mock-transfected cells by 6 and 8 h. The P production began to decrease beyond 8 h in both cases as did that of StAR mRNA, further demonstrating a close correlation between cAMP-stimulated StAR expression and steroidogenesis.



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Figure 3. Temporal Pattern of (Bu)2cAMP-Induced P Levels in Mock- and WT-CREB-Transfected MA-10 Cells

Cells were transiently transfected with empty vector (MT) or WT-CREB (2 µg each) as described in Fig. 2Go. Thirty-six hours after transfection, cells were washed and stimulated with 500 µmol/liter (Bu)2cAMP for 0–12 h. P levels in the media at each time point were assayed by RIA. The results are the mean ± SEM of five independent experiments.

 
Determination of CREB/CREM-Responsive Region(s) in the 5'-Flanking Region of the Mouse StAR Gene
To identify the functional region involved in the CREB/CREM increase in StAR mRNA levels, different mouse StAR promoter/luciferase constructs (-966, -254, -110, and -68 StAR/luc) were generated and assessed for (Bu)2cAMP-induced luciferase activity in MA-10 cells. As shown in Fig. 4Go, using the -966 bp fragment, (Bu)2cAMP-stimulated luciferase activity was increased approximately 4-fold in the WT-CREB-transfected cells when compared with the mock-transfected control group. Further deletion of the 5'-flanking region to -254 bp yielded a similar 4-fold (Bu)2cAMP-induced luciferase response with WT-CREB transfection, indicating that the regulatory elements remained present within this region. Transfection with the -110 bp fragment caused both a decreased basal (not illustrated) and (Bu)2cAMP-induced promoter activity in mock-transfected cells; however, the fold stimulation in WT-CREB-transfected cells was similar to that of longer StAR reporters. Conversely, even though there was an increase in WT-CREB-mediated cAMP-induced luciferase activity when the -68 bp fragment was used, its activity was markedly decreased to about 15% of that seen with the longer constructs, demonstrating the presence of element(s) responsive to CREB/CREM family members in the -110 to -68 bp region of the mouse StAR promoter.



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Figure 4. 5'-Flanking Deletion of the Mouse StAR Gene for Determination of the CREB-Responsive Region(s)

MA-10 cells were transiently transfected with one of the 5'-deleted StAR promoter/luciferase constructs (-966 StAR/luc, -254 StAR/luc, -110 StAR/luc, and -68 StAR/luc) either alone (StAR/luc+cAMP) or in combination with WT-CREB (StAR/luc+CREB+cAMP) in the presence of pRL-SV40 (renilla luciferase for determining transfection efficiency) as described in Materials and Methods. After 36 h of transfection, cells were incubated for a further 6 h without or with (Bu)2cAMP (500 µmol/liter). Luciferase activity in the cell lysates was determined and expressed as relative light units (RLU, fold activation) where fold increases after (Bu)2cAMP stimulation over respective controls are presented. The data represent the mean ± SEM of five independent experiments.

 
Role of CREB/CREM in cAMP-Dependent StAR Promoter Activity in MA-10, Y-1, and COS-1 Cells
The potential involvement of CREB/CREM in StAR promoter/luciferase activity was further studied in MA-10, Y-1, and COS-1 cells using the -254 StAR/luc segment of the StAR promoter. The -254 bp fragment was chosen since, unlike the -110 StAR/luc fragment, there was no decrease in the basal StAR promoter/luciferase response (Fig. 4Go). The data presented in Fig. 5AGo show that (Bu)2cAMP treatment of mock-transfected MA-10 cells resulted in a 6.6-fold stimulation of StAR promoter activity over untreated cells. Transient expression of WT-CREB in these cells resulted in a 2-fold increase in basal promoter activity but a highly significant 4-fold increase in the cAMP-stimulated luciferase response as compared with the mock-transfected group. Transfection of Y-1 cells with WT-CREB resulted in a modest increase in basal StAR promoter activity but in an approximately 2.3-fold increase in (Bu)2cAMP-stimulated promoter activity compared with mock transfected cells (Fig. 5BGo). Cotransfection of the cells with A-CREB modestly decreased basal but strongly impaired cAMP-induced luciferase activity in both MA-10 and Y-1 cells. Similarly, CREB-M1 essentially abolished the cAMP-stimulated StAR promoter/luciferase activity in both cell types. Nonsteroidogenic COS-1 cells transfected with different CREB expression plasmids did not show any significant cAMP-induced activation of the StAR promoter/luciferase response. It was noted, however, that WT-CREB markedly increased basal StAR promoter activity, suggesting that a CREB/CREM family member modulates StAR promoter in COS-1 cells in a cAMP-independent manner (Fig. 5CGo).



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Figure 5. Expression of WT-CREB, A-CREB, and CREB-M1 on cAMP-Induced StAR Promoter Activity

MA-10 (panel A), Y-1 (panel B), and COS-1 (panel C) cells were transiently transfected for 36 h with -254 StAR/luc in the presence or absence of different CREB constructs as described in Materials and Methods. To control the variation in transfection efficiency, pRL-SV40 was included in each assay. Cells were then stimulated for 6 h in the absence (Basal) or presence (cAMP) of 500 µmol/liter (Bu)2cAMP. Luciferase activity in the cell lysates was determined and expressed as RLU (luciferase/renilla). The data represent the mean ± SEM of two to five independent experiments.

 
Identification of CREB/CREM-Responsive Element(s) Within the -110/-67 bp Region of the StAR Promoter
The -110/-67 bp region is reasonably conserved among the StAR promoters of different species (Fig. 6AGo), including mouse (27), rat (28), ovine (GenBank accession no. AF086814), human (29), monkey (GenBank accession no. AY007224), and pig (30) and revealed three 5'-canonical CRE half-site sequences that could be potential binding sites for CREB/CREM.



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Figure 6. Identification of CREB/CREM Responsive Element(s) Between -110 and -67 bp of the Mouse StAR Gene

A, Sequence alignment of mouse, rat, ovine, human, monkey, and pig StAR promoters depicting three putative CRE half-sites CRE1, CRE2, and CRE3 (bold), and transcription factor-binding sites for SF-1 and AP-1 (highlighted in gray) and C/EBP (boxed). The asterisks represent the conserved bases in the StAR promoters of different species. B, Representative phosphorimage of an EMSA using -96 to -67 bp of the mouse StAR promoter as a radiolabeled oligonucleotide probe as described in Materials and Methods. The reactions include: rec CREB protein (lanes 1 and 2), rec CREB plus ATF-1 Ab (lane 2), NE from nonstimulated MA-10 cells (lane 3) or stimulated with 0.5 mmol/liter (Bu)2cAMP (cAMP; lane 4), 0.5 mmol/liter cAMP plus ATF-1 Ab (lane 5); transfected with WT-CREB plus cAMP (lane 6); transfected with WT-CREB plus cAMP plus ATF-1 Ab (lane 7); and in Y-1 NE (lanes 8–11). Formation of specific protein-DNA complexes with rec CREB protein (a) or with NE is marked as I, II, and III, respectively. Arrows indicate the supershifted position (SS) of protein-DNA complexes. The migration of free probe is shown. The experiments were repeated three to five times.

 
The CRE half-sites in the -110 and -67 bp region were tested for CREB/CREM protein-DNA binding using EMSA. As illustrated in Fig. 6BGo, a radioactively labeled oligonucleotide probe (-96 to -67 bp) containing the three CRE half-sites demonstrated the presence of a specific protein-DNA complex with recombinant CREB (rec CREB) (a, lane 1). Furthermore, nuclear extracts (NE) prepared from MA-10 (lanes 3–7) and Y-1 (lanes 8–11) cells also showed formation of three protein-DNA complexes (I, II, and III) with the -96 to -67 bp region. As demonstrated previously (31), treatment of MA-10 cells with (Bu)2cAMP resulted in increased formation of protein-DNA complex I (lane 4) while only modestly increasing complexes II and III. Complex I apparently corresponded to that observed with rec CREB protein (a, lane 1) and WT-CREB-transfected, cAMP-stimulated NE from MA-10 (lane 6) or Y-1 (lane 10) cells. ATF-1, an antibody (Ab) that recognizes CREB/CREM-1/ATF-1 in immunoblots supershifted the rec CREB protein-DNA complex (lane 2), as well as complexes formed with NE from WT-CREB-transfected, cAMP-stimulated cells (lanes 7 and 11). However, ATF-1 Ab did not supershift these complexes when cAMP-stimulated NE from untransfected MA-10 cells was used; rather, a modest inhibition was observed (compare lanes 4 and 5). These data demonstrate that rec CREB can bind to the StAR promoter, presumably at one or more of the canonical 5'-CRE half-site(s), and also suggest that a CREB/CREM family member present in NE from MA-10 and Y-1 cells binds to the -96/-67 bp region of the mouse StAR promoter.

Identification of the CREB/CREM Family Member Interacting with the StAR Promoter
To determine the identity of the endogenous protein(s) that bind to the -96/-67 bp region of the mouse StAR promoter, (Bu)2cAMP-stimulated MA-10 NE binding was assessed in EMSA using antisera specific to the CREB/CREM/ATF-1 family members (Fig. 7AGo). A specific antiserum to CREM family members (CREM1) markedly decreased protein-DNA complexes (lane 3) when compared with its preimmune serum (lane 2) or a nonimmune IgG (lane 10). In contrast, different antibodies to CREB/CREM family members CREB and ATF-1 had either modest or no effects on protein-DNA complexes (lanes 4–8). Also, CCAAT/enhancer binding protein ß Ab neither supershifted nor showed significant inhibition of protein-DNA complexes (lane 9). Therefore, these data suggest that the MA-10 NE species that bind to the -96/-67 region are predominantly CREM proteins.



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Figure 7. Determination of the Binding and Relative Expression Levels of Endogenous CREB/CREM Family Involved in StAR Gene Expression

A, EMSA reactions with (Bu)2cAMP-stimulated MA-10 NE (lanes 1–9) with the 32P-labeled oligonucleotide probe (-96 to-67) in the absence or presence of Abs to different CREB family members. Protein-DNA complexes in MA-10 NE are marked as I, II, and III. B, Formation of protein-DNA complexes with rec CREB protein (a, lanes 1–2) or MA-10 NE (I, II, and III, lanes 3–6) in the absence or presence of Abs to different CREB family members using -83/-67 bp as the probe. The arrow indicates supershift (SS) of the retarded complexes. These experiments were repeated three to five times, and a representative phosphorimage from each group is illustrated. C, RT-PCR analysis of CREM and CREB mRNAs in MA-10, Y-1, and COS-1 cells. Total RNA was extracted from these cells and subjected to RT-PCR analysis of CREM and CREB gene expression as described in Materials and Methods. A representative experiment from four different experiments is shown.

 
The involvement of CREM protein in protein-DNA binding as shown with EMSA was further assessed using a shorter oligonucleotide probe (-83 to -67) containing the CRE2 and CRE3 sites (Fig. 7BGo). The results obtained with this shorter probe were similar to those observed with -96/-67 bp probe and demonstrated qualitatively similar protein-DNA binding with either rec CREB protein (lanes 1 and 2) or NE prepared from (Bu)2cAMP-stimulated MA-10 cells (lanes 3–6). However, the binding observed at complex I was decreased using this probe (-83/-67 bp), suggesting that CRE1 may play a role in the formation of protein-DNA complexes. Importantly, CREM1-specific Ab abolished protein-DNA complexes (lane 5) while CREB-1 Ab had no effect on them (lane 6). These data further confirm the presence of CREM proteins in MA-10 NE that bind to the CRE half-sites.

Since CREM proteins appeared to be the major species binding to the CRE sites, the relative abundance of CREB and CREM mRNAs in MA-10, Y-1, and COS-1 cells were determined using RT-PCR analysis (Fig. 7CGo). Two conclusions can be drawn from these results. 1) Both MA-10 and Y-1 cells contain several different sized CREM transcripts while there appears to be only one CREB transcript in these cells. 2) There is a significant increase in the total amount of CREM transcripts when compared with the CREB transcript. Similar results were also obtained in the nonsteroidogenic COS-1 cell line (Fig. 7CGo). These results further support our EMSA studies and are in agreement with previous data showing CREM to be the predominant transcript in the testis (32, 33).

Relative Importance of CRE Half-Sites
The importance of the three CRE half-sites was further assessed by generating mutations in each CRE half-site either alone or in combination, followed by competition analysis using EMSA. The data presented in Fig. 8Go demonstrate that protein-DNA complexes observed with the -96/-67 bp oligonucleotide probe using (Bu)2cAMP-stimulated MA-10 NE (lanes 1–15) were effectively inhibited by 100-fold molar excess addition of its unlabeled sequences (-96/-67; lane 2). CREM protein-DNA binding specificity was also assessed by competition with a consensus CRE sequence (lane 3). The protein-DNA complexes were competed with 100-fold molar excess oligonucleotides bearing mutations in CRE1 (TGACCC to Tccgga; lane 4), CRE1 affecting steroidogenic factor 1 (SF-1) binding (CRE1/SF-1) (TGACCC to gaAttC; lane 5), and CRE3 (lane 7), indicating these sites did not affect CREM binding. However, mutations in CRE1&3 sites (lane 11) demonstrated relatively weaker competition, suggesting that both sites had a modest effect on these complexes. On the other hand, mutation of the CRE2 site either alone (lane 6) or in combination with other CRE sites, i.e. CRE1&2 (lane 10), CRE2&3 (lanes 12 and 13), and CRE1,2,3 (lanes 14 and 15), were not able to compete with these complexes, suggesting the greater importance of the CRE2 site in CREM protein-DNA binding. In fact, mutations in the CRE2 and CRE3, or the CRE1,2,3 sites comprised of different mutated bases (see Materials and Methods) demonstrated qualitatively similar results (lanes 12 and 14). In addition, radioactively labeled -96/-67 StAR probes containing mutations in CRE1 or CRE3 alone showed formation of protein-DNA complexes with MA-10 NE similar to -96/-67, while the formation of these complexes was markedly affected in a probe containing the CRE2 mutation (data not shown). It is noteworthy that the CRE2 site is analogous to an AP-1 binding motif (TGACTGA) previously identified (31). Hence, a consensus AP-1 sequence was used in competition experiments (lane 9) and showed strong competition with the protein-DNA complexes. Since CREM family members can also bind to AP-1 sites (34, 35), a point mutation was introduced in the AP-1 site (TGACgGA; lane 8), and the resulting sequence demonstrated similar competition with the protein-DNA complexes. These data are consistent with the hypothesis that complex formation on the CRE2/AP-1 binding site was predominantly CREM proteins. Furthermore, we have observed that the Ap-1 family members, cfos and Fra-2 markedly decrease CREM protein-DNA binding with the -97/-67 StAR probe while jun members have little or no effects on these complexes (data not shown).



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Figure 8. The Importance of the CRE Half-Sites Within -96 to -67 bp Region of the StAR Gene

(Bu)2cAMP (0.5 mmol/liter) induced MA-10 NE was used in EMSA as described in Materials and Methods. The following unlabeled oligonucleotide competitors were used at 100-fold molar excess: self (-96/-67 StAR, lane 2), consensus CRE (Cons CRE, lane 3), and consensus AP-1 (Cons AP-1, lane 9). Also the following unlabeled probes consisting of mutations in CRE1 (lane 4), CRE1 and SF-1 (CRE1/SF-1; lane 5), CRE2 (lane 6), CRE3 (lane 7), AP-1 point mutation (AP-1 pt Mut; lane 8), or double and triple mutations in CRE sites as indicated (lanes 10–15) were used. MA-10 protein-DNA complexes are marked as I, II, and III. These experiments were repeated three times, and the data from one representative experiment are shown. The migration of free probe is shown in each lane.

 
To obtain even more insight into these mechanisms, specific mutations were generated in each of the CRE half-sites, either alone or in combination, in the -151/-1 bp fragment, and StAR promoter/luciferase activity was determined. This construct was used since the -110 bp StAR fragment demonstrated decreased basal promoter activity. MA-10 cells transfected with -151 StAR/luc demonstrated an approximately 4-fold increase in the (Bu)2cAMP-stimulated luciferase response over nonstimulated cells (Fig. 9Go). Mutation of the CRE1 site (TGACCC to Tccgga) did not significantly affect cAMP-stimulated reporter promoter activity, although there was an approximately 35% decrease in basal activity when compared with the wild-type StAR promoter. On the other hand, mutation of the CRE2 site resulted in an approximately 50% decrease in both basal and cAMP-induced promoter activity as compared with controls. Mutation of the CRE3 site had essentially no effect on basal or cAMP-stimulated StAR promoter/luciferase responses. Interestingly, mutation in the bases of CRE1 involved in SF-1 binding (CRE1/SF-1) caused approximately 80% decreases in basal and cAMP-stimulated activities. Attenuation of basal and cAMP-stimulated promoter responses was consistently observed when mutations generated in the CRE1/SF-1 site were assessed in combination with mutations in CRE2 and/or CRE3, with the CRE1/SF-1 and CRE2 double mutation and the triple mutation having an equally significant effect (Fig. 9Go). Consistent with these observations, transfection of WT-CREB with the -151 CRE2 mutant significantly diminished the CREM-dependent cAMP induction of StAR promoter activity while there was little or no effect of mutating the CRE1 or CRE3 sites, respectively (data not shown). These data further confirm the importance of CRE2 in CREM-induced StAR promoter activity and demonstrate the site specificity of these CRE elements in cAMP responsiveness.



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Figure 9. Functional Assessment of the CRE Half-Sites in StAR Promoter/Luciferase Activity

MA-10 cells were transiently transfected with different -151 StAR constructs containing mutations in one or more CRE sites (as indicated), in the presence of pRL-SV40 as described in Materials and Methods. pGL3-Basic (pGL3) was used as a control. After 36 h of transfection, cells were incubated for a further 6 h without or with (Bu)2cAMP (500 µmol/liter). Luciferase activity in the cell lysates was determined and expressed as relative light units (RLU, luciferase/renilla). These experiments were repeated three to five times, and data (±SEM) from one representative experiment in triplicate are presented.

 
Effect of ICER on Endogenous CREM-Mediated cAMP-Stimulated StAR Promoter Activity and StAR mRNA Expression
The functional involvement of CREM on StAR promoter activity and StAR expression was further assessed in MA-10 cells after cotransfection with ICER. As before, expression of WT-CREB with the -254 StAR/luc promoter significantly (P < 0.01) increased (Bu)2cAMP-induced StAR promoter activity (Fig. 10AGo) and StAR gene expression (Fig. 10BGo) as compared with mock-transfected cells. Cells transiently transfected with ICER alone or in combination with WT-CREB marginally affected basal responses. However, the cAMP-stimulated level of StAR promoter/luciferase activity was inhibited by approximately 90% (Fig. 10AGo), and StAR mRNA expression by 80% (Fig. 10BGo), in WT-CREB+ICER-transfected cells. P levels in the media of the corresponding incubations demonstrated qualitatively similar results (data not shown). These data are again consistent with the involvement of endogenous CREM in cAMP-induced StAR expression and steroid biosynthesis in MA-10 cells.



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Figure 10. Effects of WT-CREB, ICER, and Their Combination on cAMP-Induced StAR Promoter Activity and StAR mRNA Expression in MA-10 Cells

Cells were transiently transfected with -254 StAR/luc and either empty expression vector (MT), ICER, WT-CREB (CREB) or both CREB+ICER expression plasmids. Panel A illustrates the StAR promoter/luciferase activity in different incubations. The variation in transfection efficiency was monitored by a pRL-SV40 plasmid cotransfected in each assay as described in Materials and Methods. After 36 h of transfection, cells were incubated for 6 h without (Basal) and with (Bu)2cAMP (500 µmol/liter), and luciferase activity in the cell lysates was determined and expressed as RLU (luciferase/renilla). Data represent the mean ± SEM of four experiments. To determine StAR mRNA expression, MA-10 cells were transfected either with WT-CREB, ICER alone, or in combination as above. After 36 h, cells were stimulated without (Basal) or with 500 µmol/liter (Bu)2cAMP. Panel B, Total RNA was extracted from different treatment groups and subjected to RT-PCR analysis for StAR mRNA expression as described in the legend of Fig. 1Go and in Materials and Methods. A representative autoradiogram from three independent experiments with similar results is shown. The IOD values represent the mean ± SEM of three experiments.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The StAR protein plays a critical role in steroidogenesis by mediating the intramitochondrial transport of cholesterol to the cholesterol side chain cleavage cytochrome P450 enzyme (1, 2, 36). Rapid expression of StAR in response to trophic hormone is required to initiate and maintain ongoing steroid hormone biosynthesis, and this expression is regulated via the cAMP second messenger signaling pathway. Given the importance of StAR expression and the purported role of cAMP in this process, we explored the possible involvement of one or more of the members of the CREB/CREM family of transcription factors in steroidogenesis and StAR expression. The CREB/CREM family is distinguished by their DNA-binding bZip domains and mediate part of the transcriptional response to cAMP signaling (11, 37, 38, 39). To more fully understand the role of CREB/CREM in this regulation, the present investigation used three different inhibitors of CREB/CREM function: a dominant-negative A-CREB that blocks the DNA binding activity in a dimerization-dependent fashion (23), CREB-M1 (Ser 133Ala substitution) that is unable to be phosphorylated by PKA (20, 40), and the transcription repressor, ICER, which forms inactive heterodimers (13, 25, 26).

Our results demonstrate that cells expressing WT-CREB significantly increased cAMP-dependent steroid production, StAR promoter activity, StAR mRNA, and StAR protein, thus showing a potential role for a CREB/CREM family member. In addition, 5'-deletion analysis of the mouse StAR gene identified the presence of CREB/CREM responsive element(s) between -110 and -68 bp of the transcription start site, a region containing three half-sites for 5'-canonical CRE sequences. An oligonucleotide probe (-96/-67 bp) containing these CRE half-sites showed protein-DNA binding with rec CREB protein or NE prepared from MA-10 cells. Utilizing a series of antibodies for various members of the CREB/CREM family, it was determined that the CREB/CREM family member, CREM, was the predominant form binding to the CRE sites. The quantities of CREM and CREB transcripts were established by RT-PCR, and it was found that CREM transcripts were present in greater abundance than was the CREB transcript in both MA-10 and Y-1 cells. The role of CREM was further strengthened by the observation that ICER inhibited the ability of endogenous CREM proteins to induce steroid hormone biosynthesis and StAR mRNA expression in response to cAMP. These results clearly indicate an important role of CREB/CREM family members, most notably CREM, in the cAMP-mediated regulation of steroidogenesis and StAR expression and thus provide a potential new mechanism for the rapid stimulation of StAR by cAMP.

The observations obtained with CREB-M1 indicates the importance of phosphorylation and provides a mechanism for the rapid increase in StAR expression by PKA activation (19, 20). Our findings are consistent with previous data demonstrating that CREB-M1 inhibited somatostatin gene transcription and adversely affected survival in transfected F9 teratocarcinoma, rat granulosa, and Sertoli cells, respectively (19, 22, 41). Similarly, our observation that endogenous CREM-dependent, cAMP-induced steroid production and StAR expression were markedly decreased by ICER is consistent with previous studies demonstrating that ICER down-regulated the CRE-mediated transcription of the CREB gene in Sertoli cells (42) and suppressed both basal and cAMP-induced expression of the inhibin {alpha}-subunit in granulosa cells (43).

As discussed above, while StAR expression is regulated through a PKA-dependent pathway, the StAR promoter lacks a consensus CRE. In such cases it is possible that alternate elements are involved in CREB/CREM function, a phenomenon that has previously been observed for cAMP-regulated genes in which nonconsensus CREs mediate hormonal induction (12). Our data on the 5'-flanking region of the mouse StAR gene indicates that the majority of the acute cAMP responsive region was present between -110 and -68 bp upstream of the transcriptional start site. This is the same region required for CREB/CREM responsiveness and supports the observation made in a previous study (31). We identified three 5'-canonical CRE half-sites within this region that are well conserved among different species. Indeed, our data clearly show that an oligonucleotide probe (-96/-67 bp) containing these CRE sites resulted in the formation of specific protein-DNA complexes with rec CREB or with NE from MA-10 and Y-1 cells. Specific mutations in the CRE half-sites within the -96/-67 bp region demonstrated the predominant involvement of the CRE2 site in CREM binding and in cAMP responsiveness. However, CREB activation of a promoter can require the binding of multiple CRE binding factors to several CREs rather than the binding of a single dimer (44, 45, 46). Thus, it is possible that the CRE1 and CRE3 sites may also play roles, albeit lesser ones, in the expression of the StAR gene.

The CRE2 site maps to the CREs identified in two previous studies and referred to as C/EBPß-3 (47) or the C/EBPß/nonconsensus activating protein (AP-1)/nuclear receptor half-site (CAN) (31). In those studies, a mutation of the AP-1 site (the same mutation as the CRE2 mutation in the current studies), or a different mutation in the C/EBPß-3 site both abolished protein-DNA binding and reduced basal and cAMP/FSH-responsive StAR promoter activity by approximately 50% (31, 47). Members of the AP-1, CREB/CREM/ATF-1, and C/EBP families are bZip proteins that recognize similar DNA sequences, and certain ATF/CREB factors can heterodimerize with AP-1 (34, 35) and C/EBP (48) family members depending on the binding site. Further studies must be performed to determine whether bZip family heterodimers can form in a tissue-specific fashion on the CRE2 site and whether these heterodimers are important for the cAMP induction of StAR expression.

Previous EMSA studies demonstrated C/EBPß binding to -87 to -70 of the StAR promoter in ovarian extracts (47); however, in subsequent studies, Abs to C/EBPß and AP-1 family members did not affect complex formation in MA-10 NE bound to a similar region (-87/-64, see Ref. 31). Moreover, as there was a cAMP-dependent increase in MA-10 NE binding (our data and Ref. 31) and since we found rec CREB bound to -96/-67 of the StAR promoter, Ab supershift experiments coupled with the use of specific Abs were performed to determine whether this region bound endogenous CREB/CREM protein(s) present in MA-10 extracts. Indeed, the protein-DNA complexes were essentially abolished in the presence of CREM1-specific Ab, while antisera to CREB and ATF-1 family members demonstrated only a modest or no inhibition of these complexes. We also corroborated previous results (31) indicating that C/EBPß does not appear to bind to this region.

While a role for AP-1 or other bZip family members cannot be eliminated, the present observations suggest that CREM proteins are the predominant binding species in MA-10 cells. Consistent with our EMSA data, RT-PCR analyses demonstrated that the total amount of CREM mRNA is much higher than CREB mRNA in MA-10 and Y-1 cells. These findings are in agreement with previous studies using Northern and in situ analyses which demonstrated that the number of CREM transcripts in the testis are markedly higher than those of CREB (32, 33). In the adult testis CREM transcripts exclusively encode for the activator form of CREM, CREM{tau} (17). Thus, it would appear that CREM is the predominant endogenous protein in MA-10 NE binding to the CRE-2 site. Moreover, our EMSA experiments using MA-10 cells transfected with WT-CREB suggest that the protein-DNA complex changes (from complex I, II, and III) to resemble that of rec CREB (complex I only) are presumably due to titration effects. In contrast, there was no apparent change in complex formation in Y-1 cells either in response to cAMP or when transfected with WT-CREB. Hence, our data corroborate previous work (31, 47) suggesting that there are cell type-specific binding differences in -96/-67 of the StAR promoter.

Several studies have identified three elements that bind the transcription factors SF-1 (our work and Ref. 31), C/EBP (47, 49, 50), and GATA-4 (31, 47) within the -110 to -64 bp region of the StAR promoter, and each site influenced basal and/or cAMP induction. However, interaction of these factors with CREB/CREM family members needs further exploration. Most likely, in the absence of CREB/CREM binding, the cAMP-dependent response remains partially intact due to coactivator recruitment by other transcription factors bound to this specified region of the StAR promoter. We propose that the SF-1, CRE2/AP-1/C/EBPß-3, and GATA sites might function as a complex cAMP response unit. In support of this, studies on the phosphoenol-pyruvate carboxykinase, aromatase, and inhibin-{alpha} genes demonstrated that CREB synergizes with other transcription factors to mediate cAMP responsiveness (40, 46, 51, 52). In addition, there is extensive documentation of the cooperation between SF-1 and C/EBPß, GATA-4, or Sp1 in the regulation of StAR gene transcription and steroidogenesis (31, 49, 53, 54). The interaction of CREB/CREM with other transcription factor(s) and/or coactivators that may bind within this region of the StAR promoter and be required for full promoter activity will be the subject of future investigations. Moreover, as aromatase and other steroidogenic enzymes previously shown to be regulated by cAMP contain elements for SF-1 and/or GATA, these genes may also be regulated by a complex cAMP response unit containing a nonconsensus CRE site similar to the StAR promoter.

In conclusion, we present evidence the CREB/CREM family member, CREM, is functionally important in MA-10 and Y-1 cells for maximal cAMP-dependent steroidogenesis and StAR gene expression. The mechanism was found to be predominantly mediated through the binding of CREM to the CRE2 site; however, all three CRE elements identified and characterized within the -96/-67 bp region of the mouse StAR gene mediate at least part of the response. We also propose that trophic hormone-stimulated steroid biosynthesis in other steroidogenic cells may be mediated by similar mechanisms.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Construction of StAR Promoters and CRE Mutants
Various truncations in the 5'-flanking region of the mouse StAR gene (-966 bp, -254 bp, -110 bp, and -68 bp) were cloned upstream of a luciferase reporter gene into the pGL2 basic vector (Promega Corp., Madison, WI), as described previously (27).

The -151/-1 bp region of the StAR promoter was synthesized using a PCR-based cloning strategy using -254-StAR-pGL2 as the template. The 5' primer 5'-TAGCTCGAGTCTGCTCCCTCCCACCTTGGCCAGC-3' and the 3'-primer 5'-CTAAAGCTTGGCGCAGATCAAGTGCGCTGCCT-3', contain a XhoI and HindIII site (underlined) at the 5' end, respectively. The amplicon is then subcloned into PCR2.1-TOPO vector following the TOPO-TA cloning method of the manufacturer (CLONTECH Laboratories, Inc., Palo Alto, CA). The XhoI and HindIII fragments were cleaved from the StAR-PCR2.1 vector, purified, and subcloned into the XhoI and HindIII cloning sites of the pGL3 basic vector (Promega Corp.), which contains firefly luciferase as a reporter gene. The identity of the inserted XhoI and HindIII fragments was confirmed by sequencing using the pGL3 clockwise 5'-CTAGCAAAATAGGCTGTCCC-3' and counterclockwise 5'-TGGAAGACGCCAAAAACATAAAG-3' primers, corresponding to a region 3' of the multiple cloning site and the beginning of the region encoding luciferase.

Plasmids containing a single mutation in the CRE half-sites were generated using the Quikchange site-directed mutagenesis kit (Stratagene, La Jolla, CA) using -151/-1 StAR-pGL3 as the template. Double or triple mutations in the CRE sites were generated using appropriate CRE single/double mutation vectors as the PCR template. The sense strand of the oligonucleotide sequences used were [mutated (Mut) bases in boldface lowercase letters]: CRE1 Mut, 5'-GGCAATCATTCCATCCTTccggaTCTGCACAATGAC-3'; CRE1/SF1 Mut, 5'-GCAATCATTCCATCCTgaAttCTCTGCACAATGAC-3'; CRE2 Mut 5'-CCTTGACCCTCTGCACAATagaTcttGACTTTTTTATCTC-3'; CRE3 Mut 5'-CCCTCTGCACAATGACTGAgatCTTTTTTATCTC-3'; CRE2&3 Mut 5'-CCTTGACCCTCTGCACAAgcggccgcGACTTTTTTATCTCAAG-3'.

Specific mutations were tested by restriction endonuclease digestion using Sau 3A I (CRE1 Mut), EcoRI (CRE1/SF-1 Mut), BglII (CRE2 Mut and CRE3 Mut), or NotI (CRE2&3 Mut). Finally, a XhoI and HindIII fragment containing the mutations was religated into the pGL3 vector. Assessment of these mutations was confirmed by sequencing on a PE Biosystems 310 Genetic Analyzer (Perkin Elmer) at the Texas Tech University Biotechnology Core Facility.

RNA Extraction and Quantitative RT-PCR
Total RNA was extracted from different treatment groups using Trizol reagent (Life Technologies, Inc., Gaithersburg, MD). The isolation and amplification of MA-10 StAR cDNA were carried out utilizing the mouse StAR cDNA sequence (3), as previously described (55). Briefly, the following primer pairs were used for StAR amplification: the forward primer, 5'-GACCTTGAAAGGCTCAGGAAGAAC-3', and the reverse primer, 5'-TAGCTGAAGATGGACAGACTTGC-3', spanning bases -51 to -27 and 931–908, respectively. The variation in RT-PCR efficiency was corrected for using an L19 ribosomal protein gene, which was amplified using the following primer pairs: the forward 5'-GAAATCGCCAATGCCAACTC-3' and the reverse 5'-TCTTAGACCTGCGAGCCTCA-3' (56). RT and PCR of StAR and L19 were run sequentially in the same assay tube using 2 µg of total RNA, and the parameters including the number of cycles used were optimized to be in the exponential phase as previously described (55, 57).

CREB and CREM cDNAs were generated with 2 µg of total RNA using the specific primer pairs: CREB forward, 5'-GCAGTGACGGAGGAGCTTGTAC-3' (bases 101–122) and CREB reverse, 5'-TCTGATTTGTGGCAGTAAAG-3' (bases 1,138–1,157); CREM forward, 5'-ACTGGGCAAATTTCAATCCCTGC-3' (bases 88–110), and CREM reverse, 5'-CAAACTTCCGGGCGATGCAGCCATC-3' (bases 765–789), respectively (58, 59, 60). cDNAs were amplified by PCR for 32 cycles (94 C for 75 min, 58 C for 2 min, and 72 C for 3 min). The identity of all RT-PCR products was confirmed by automated sequencing (as above) as described previously (55), and the sequences obtained corresponded to previously published findings (3, 56, 58, 59, 60).

The molecular sizes of the PCR products (StAR, CREB, CREM, and L19) were determined in 1.2% agarose gels. The gels were stained with ethidium bromide (CREB and CREM). For StAR and L19, the gels were vacuum dried and exposed to Hyperfilm (Amersham Pharmacia Biotech, Little Chalfont, Buckinghamshire, UK) at 4 C between 1 and 3 h, and relative levels of different signals were quantitated by densitometry.

Preparation of Mitochondria and Immunodetection of StAR Protein
Isolation of mitochondria was carried out as follows. In brief, cells were homogenized in TSE buffer (10 mmol/liter Tris, 250 mmol/liter sucrose, 100 mmol/liter EDTA, pH 7.4) containing protease inhibitors (1 mmol/liter dithiothreitol, 1 mmol/liter phenylmethylsulfonyl fluoride, 2 mg/liter leupeptin, 2 mg/liter aprotinin) at 1,200 rpm for 30 passes with a Potter-Elvehjem homogenizer. Mitochondrial isolation was carried out by differential centrifugation, as previously described (3, 57).

Mitochondrial proteins (15–20 µg) were solubilized in sample buffer (25 mmol/liter Tris-Cl, pH 6.8, 1% SDS, 5% ß-mercaptoethanol, 10% glycerol, and 0.01% bromophenol blue) and loaded onto 12% SDS-polyacrylamide gels (Mini Protean II System, Bio-Rad Laboratories, Inc., Hercules, CA), as described by Laemmli (61). Electrophoresis was performed at 200 V for 1 h, and the proteins were electrophoretically transferred onto Immuno-blot polyvinylidene difluoride membranes (Bio-Rad Laboratories, Inc.). The membranes were incubated in a blocking buffer (Tris-buffered saline, containing 0.1% Tween-20 and 5% nonfat dry milk) for 2 h at room temperature, followed by incubation with anti-StAR peptide antibodies (3, 57). Immunodetection of StAR protein was performed using the ECL Western blotting detection kit (Amersham Pharmacia Biotech, Piscataway, NJ) and after exposure of the membranes to Hyperfilm (Amersham Pharmacia Biotech) for the appropriate times, bands were quantitated using computer- assisted image analysis (Visage 2000, BioImage, Ann Arbor, MI).

Preparation of NE and EMSAs
The NE from different treatment groups were prepared as described previously (62, 63), slightly modified to achieve higher purity. Briefly, cells were washed and collected with 0.01 mol/liter PBS and centrifuged at 1,000 x g for 5 min. The pellet was resuspended in buffer A containing protease inhibitors (10 mmol/liter HEPES, 1.5 mmol/liter MgCl2, 10 mmol/liter KCl, 0.5 mmol/liter dithiothreitol, 0.1% Nonidet P-40, 1 mmol/liter phenylmethylsulfonyl fluoride, 2 mg/liter leupeptin, 2 mg/liter aprotinin, pH 7.9). After centrifugation, the crude nuclear pellet was resuspended and allowed to swell for 20–30 min in a rotator at 4 C in buffer C (buffer A containing 10 mmol/liter HEPES, 0.42 mmol/liter NaCl, 0.2 mmol/liter EDTA, 25% glycerol, pH 7.9). After removing the debris by centrifugation at 12,000 x g for 3 min, the NE was assayed directly or stored at -80 C.

The different mouse StAR oligonucleotide probes were annealed by heating sense and antisense primers to 65 C for 5 min in annealing buffer (10 mmol/liter Tris-Cl, 100 mmol/liter NaCl, 1 mmol/liter EDTA, pH 7.5) and then slowly cooled over 2 h to room temperature. The following sense primers of the oligonucleotides and mutated (Mut) sequences (lowercase bold) were used:

-96/-67 StAR 5'-GGTGACCCTCTGCACAATGACTGATGACTTTT-3'

-96/-67 StAR CRE1 Mut 5'-GGTccggaTCTGCACAATGACTGATGACTTTT-3'

-96/-67 StAR CRE1/SF-1 Mut 5'-GGgaAttCTCTGCACAATGACTGATGACTTTT-3'

-96/-67 StAR CRE2/AP-1 Mut 5'-GGTGACCCTCTGCACAATagaTctTGACTTTT-3'

-96/-67 StAR CRE3 Mut 5'-GGTGACCCTCTGCACAATGACTGAgatCTTTT-3'

-96/-67 StAR AP-1 point (pt) Mut 5'-GGTGACCCTCTGCACAATGACgGATGACTTTT-3'

-96/-67 StAR CRE1&2 Mut: 5'-GGTccggaTCTGCACAATagaTctTGACTTTT-3'

-96/-67 StAR CRE1&3 Mut: 5'-GGTccggaTCTGCACAATGACTGAgatCTTTT-3'

-96/-67 StAR CRE2&3 Mut: 5'-GGTGACCCTCTGCACAATagaTctgatCTTTT-3'

-96/-67 StAR CRE1,2,3 Mut: 5'-GGTccggaTCTGCACAATagaTctgatCTTTT-3'

-96/-67 StAR CRE2&3 Mut no. 2: 5'-GGTGACCCTCTGCACAAgcggccgcGACTTTT-3'

-96/-67 StAR CRE1,2,3 Mut no. 2: 5'-GGgaAttCTCTGCACAAgcggccgcGACTTTT-3'

-83/-67 StAR: 5'-GGAATGACTGATGACTTTT-3'

Consensus AP-1 (Ref. 34): 5'-GGCGCTTGATGAGTCAGCCGGAA-3'

Consensus CRE (Ref. 9): 5'-GGAGAGATTGCCTGACGTCAGAGAGCTAG-3'

The 5'-GG overhangs present in the doubled-stranded oligonucleotides (4 pmol/liter each) were end-labeled with [{alpha}32P]-dCTP (DuPont Easytides, 6000 Ci/mmol, Amersham Pharmacia Biotech) using Klenow (Promega Corp.) at 25 C for 15 min. The probes were purified using Probe Quant spin columns (Amersham Pharmacia Biotech). DNA-protein binding assays were carried out under optimized conditions as described previously (49, 63, 64). Briefly, 10 µg NE or 1.5 µg rec CREB protein were incubated in 1x reaction buffer (10 mM HEPES, 1 mmol/liter EDTA, 4% Ficoll, 10 mmol/liter dithiothreitol, 1 µg poly dI·dC, 40 ng/µl BSA, pH 7.9) for 15 min at room temperature before the addition of 32P-labeled double-stranded DNA probe (0.5 pmol/liter, ~100,000 cpm). When antiserum was used, binding reactions were incubated an additional 45 min on ice before the addition of the labeled DNA. The following Abs were used in EMSA reactions: monoclonal mouse anti-human ATF-1 Ab (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), which maps to the DNA binding and dimerization domain and is reactive with ATF-1 p35, CREB-1 p43, and CREM1; CREM1 Ab, which is specific for CREM and cross-reacts with CREB only at very high protein concentrations (32, 65); CREB-1 Ab, which is specific for CREB (65), and C/EBPß Ab, which is specific for C/EBPß (49, 66). In addition, polyclonal rabbit antihuman ATF-2 (specific for ATF-2), polyclonal rabbit antihuman ATF-3 (specific for ATF-3), polyclonal goat antihuman CREB-2 (specific for CREB-2 and designated ATF-4), and mouse IgG were purchased from Santa Cruz Biotechnology, Inc. When competitors were used, 50 pmol/liter (100-fold molar excess) of unlabeled oligonucleotide competitors were added at the same time as the radiolabeled probe and incubated for 15 min. The entire reaction was then subjected to electrophoresis at 200 V for about 1.5 h through a 5% polyacrylamide gel in 0.5x TBE buffer (90 mmol/liter Tris-borate, 2 mmol/liter EDTA, pH 8.0). The gels were dried and then exposed to phosphor screens. Detection and quantitation of the protein-DNA complexes were performed using a phosphorimaging device (Molecular Dynamics, Inc., Sunnyvale, CA).

Cell Culture, Transfections, and Luciferase Assays
The MA-10 mouse Leydig tumor cells (67) were a generous gift from Dr. Mario Ascoli (Department of Pharmacology, University of Iowa College of Medicine, Iowa City, IA). These cells were maintained in HEPES-buffered Weymouth’s MB/752 medium supplemented with 15% heat-inactivated horse serum (HS; Life Technologies, Inc., Gaithersburg, MD) containing 40 mg/liter of gentamicin sulfate, in a humidified atmosphere of 95% air and 5% CO2. The Y-1 (mouse adrenocortical tumor) and COS-1 (African green monkey kidney) cells were obtained from American Type Culture Collection (Manassas, VA) and grown, respectively, in Y-12K medium supplemented with 15% HS and 2.5% FBS and DMEM with high glucose plus 10% (FBS), containing 50,000 U/liter penicillin and 50 mg/liter of streptomycin (Life Technologies, Inc.

Transfections were carried out when the cells were 65–75% confluent using FuGENE 6-transfection reagent (Roche Diagnostics Corp., Indianapolis, IN) according to the manufacturer’s instructions under optimized conditions (55). Briefly, MA-10 cells were transfected with 2 µg of a particular CREB expression construct or WT-CREB (CREB341) plus ICER II {gamma} (ICER) expression constructs (2 µg each) where indicated (see Ref. 25) for studying both StAR expression and steroidogenesis. The levels of P in the media were measured by RIA as described previously (68).

For promoter studies, cells were transfected with CREB expression plasmids together with equal amounts (1:1) of various truncations in the 5'-flanking region of the StAR gene (-966 StAR/luc, -254 StAR/luc, -110 StAR/luc, and -68 StAR/luc) as specified elsewhere, in the presence of 10 ng pRL-SV40 vector (a plasmid that constitutively expresses renilla luciferase) to normalize for transfection efficiency. Reporter assays were also carried out in combination with WT-CREB and ICER. The effects of mutating the three CRE half-sites either alone or in combination on StAR promoter/luciferase activity were performed in MA-10 cells transfected with 1.0 µg of different -151 StAR-pGL3 plasmids.

Luciferase activity in the cell lysates was determined by the Dual-luciferase reporter assay system (Promega Corp.). Briefly, after washing, 300 µl of the reporter lysis buffer were added to the cells. The cellular debris was pelleted by centrifugation at 14,000 x g at 4 C, and the supernatant was measured for relative light units in a Monolight 2010 luminometer (Analytical Luminescence Laboratory, San Diego, CA) following the manufacturer’s instructions.

Statistical Analysis
The data presented are the mean ± SEM. Statistical analysis was performed by ANOVA using Statview (Abacus Concepts Inc., Berkeley, CA) followed by Fisher’s protected least significant differences test (Fisher’s PLSD). P < 0.05 was considered statistically significant.


    ACKNOWLEDGMENTS
 
The authors would like to thank Dr. M. Montminy (Department of Cell Biology, Harvard Medical School, Boston, MA) and Dr. R. H. Goodman (Oregon Health Sciences University, Portland, OR) for the generous gifts of CREB-M1 and WT-CREB, respectively. We thank Dr. S. Williams (Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, Lubbock, TX) for providing us with C/EBPß antiserum, and Dr. M. R. Waterman (Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, TN) for the CREB-1 antiserum for this study. The -151/-1 pGL3-StAR plasmid was a generous gift from Dr. X. J. Wang (Texas Tech Health Sciences Center). The technical assistance of Deborah Alberts is gratefully acknowledged.


    FOOTNOTES
 
This work was supported in part by NIH Grant HD-17481 and by the Robert A. Welch Foundation.

Abbreviations: Ab, Antibody; ATF-1, activating transcription factor 1; bZip, basic-leucine zipper; CRE, cAMP response element; CREB, CRE binding protein; CREM, CRE modulator; ICER, inducible cAMP early repressor; Mut, mutant; NE, nuclear extracts; P, progesterone; rec CREB, recombinant CREB; SF-1, steroidogenic factor 1; StAR, steroidogenic acute regulatory protein; WT-CREB, wild-type CREB.

Received for publication January 19, 2001. Accepted for publication September 25, 2001.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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