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Endocrinology Vol. 145, No. 12 5629-5637
Copyright © 2004 by The Endocrine Society

Regulation of Steroidogenesis and Steroidogenic Acute Regulatory Protein in R2C Cells by DAX-1 (Dosage-Sensitive Sex Reversal, Adrenal Hypoplasia Congenita, Critical Region on the X Chromosome, Gene-1)

Youngah Jo and Douglas M. Stocco

Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, Lubbock, Texas 79430

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


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In the present study, steroidogenesis in two different Leydig tumor cell lines was compared. One, the MA-10 mouse tumor cell line, produces steroids and the steroidogenic acute regulatory (StAR) protein only when stimulated by trophic hormones and cAMP analogs. The other, the R2C rat tumor cell line, produces steroids and the StAR protein constitutively without stimulation. We observed that high levels of DAX-1 (dosage-sensitive sex reversal, adrenal hypoplasia congenita, critical region on the X chromosome, gene-1), a repressor of steroidogenesis and StAR gene expression, were present in MA-10 cells but not in R2C cells. Based upon this observation, we hypothesized that the absence of DAX-1 might result in constitutive steroidogenesis in R2C cells. To test this hypothesis, DAX-1 was overexpressed in the R2C cells using the Tet-on inducible gene expression system and resulted in a 45% decrease in steroid production, a 35% decrease in StAR protein, and a 39% decrease in cytochrome P450 side chain cleavage expression. Further, using retroviral infection with DAX-1, StAR expression and steroidogenesis were decreased 50–60% and 60% in R2C cells, respectively. These results corroborate previous findings that DAX-1 negatively regulates steroid synthesis through the inhibition of StAR expression and indicate that the absence of DAX-1 in R2C cells is, at least in part, responsible for the constitutive steroidogenesis and StAR expression observed.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE FIRST STEP in steroid biosynthesis, the conversion of cholesterol to pregnenolone by the cytochrome P450 cholesterol side-chain cleavage enzyme (P450scc), occurs in the mitochondria of steroidogenic cells present in the adrenals, ovaries, testes, and brain (1, 2, 3). However, it is the delivery of substrate cholesterol to the inner mitochondrial membrane site of the P450scc enzyme that constitutes the rate-limiting step in the steroidogenic cascade (4, 5). This step is dependent on a newly synthesized protein that was subsequently identified as the steroidogenic acute regulatory (StAR) protein (6, 7, 8), a protein required for the transfer of cholesterol to the inner mitochondrial membrane and the P450scc (9, 10, 11). The importance of the StAR protein in steroidogenesis was clearly demonstrated in the human disease, lipoid congenital adrenal hyperplasia, a potentially lethal condition caused by the failure of the newborn to synthesize steroid hormones. Individuals afflicted with this disease have normal steroidogenic enzyme activities (12, 13, 14), but the function of the StAR protein is impaired because of the presence of mutations in the StAR gene (15, 16, 17, 18, 19, 20, 21). The details involved in the discovery, characterization, and roles of the StAR protein in the regulation of acute steroidogenesis have been the subject of several reviews (22, 23, 24, 25).

MA-10 mouse Leydig tumor cells produce steroids and express StAR when stimulated with trophic hormones and cAMP analogs and as a result, have been used in many of the studies involving the role of StAR in steroidogenesis (26, 27, 28, 29, 30). In contrast, R2C rat Leydig tumor cells synthesize steroids and express StAR mRNA and protein constitutively in a cAMP-independent but cycloheximide-sensitive manner (31, 32). In addition, cAMP stimulation is not required for the internalization of plasma membrane cholesterol or the hydrolysis of cholesteryl esters in R2C cells (31, 32), observations consistent with a recent study in our laboratory (33). In that study, it was demonstrated that, in addition to elevated levels of StAR, R2C cells contained higher levels of the scavenger receptor type B class I (SR-BI), the high-density lipoprotein receptor that takes up cholesteryl esters from high-density lipoprotein and hormone-sensitive lipase (HSL), the enzyme that converts cholesterol esters to free cholesterol, when compared with MA-10 cells (33). Therefore, constitutive steroidogenesis in R2C cells may be attributed to the constitutive expression of proteins involved in the selective uptake of cholesteryl esters (SR-BI), their conversion to free cholesterol (HSL), and lastly, its transfer to the inner mitochondrial membrane (StAR). However, the mechanisms responsible for the elevated expression of these proteins in R2C cells remain unknown.

In the present study, we attempted to determine which factor(s) in the R2C cell line might be responsible for the greatly increased expression of StAR that supports the observed constitutive steroidogenesis. As a starting point, we cloned the promoter region of the R2C cell StAR gene and compared it with published rat promoter and mouse promoter sequences to determine whether this promoter contained any alterations in regulatory elements that might result in its constitutive expression. No significant differences in any of the sequences of these promoters were observed, nor were there any differences in promoter activity when tested in a reporter system. We then concentrated on the transcription factors known to activate the StAR promoter and screened them for their levels of expression in both cell lines. Finally, we also screened R2C cells for transcription factors known to negatively regulate StAR expression. We found that the transcription factor DAX-1, (dosage-sensitive sex reversal, adrenal hypoplasia congenita, critical region on the X chromosome, gene-1), a known repressor of the StAR gene, is absent in R2C cells. Because the absence of DAX-1 in R2C cells represented a potential cause for the observed results, we studied the effects of overexpressing DAX-1, using either the Tet-on inducible system or retroviral infection, on steroid production and StAR expression in this cell line.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Chemicals and plasmids
Doxycycline, puromycin, G418, and dibutyryl cAMP [(Bu)2cAMP] were purchased from Sigma (St. Louis, MO). The pTRE-myc vector was purchased from CLONTECH (Palo Alto, CA). The pMSCV-puro vector was a kind gift from Dr. Curt Pfarr (Texas Tech University Health Sciences Center).

Cell culture
MA-10 cells were grown in Waymouth’s MB752/1 medium (Sigma, St. Louis, MO) supplemented with 15% (vol/vol) heat inactivated horse serum (Invitrogen Life Technologies, Carlsbad, CA) in the presence of 0.4% (vol/vol) gentamicin (Invitrogen Life Technologies). R2C cells were grown in Waymouth’s MB752/1 medium supplemented with 15% horse serum and 2.5% (vol/vol) fetal bovine serum (FBS, Invitrogen Life Technologies) in the presence of 0.4% (vol/vol) gentamicin. PT67 cells were cultured in DMEM containing high glucose, 10% (vol/vol) FBS, and 100 U/ml of penicillin /streptomycin. All cells were incubated in 5% CO2 at 37 C.

Western blot analysis
Cells were plated as triplicate in six-well plates and treated with and without 1 mM (Bu)2cAMP for 6 h. After scraping, cells were transferred to microfuge tubes and centrifuged at 5000 rpm for 5 min. Media were discarded and pellets were washed with PBS minus calcium and magnesium PBS(–). For preparation of nuclear fractions, Buffer A [10 mM HEPES (pH 7.9), 1.5 mM MgCl2, 10 mM KCl, 0.1% Nonidet P-40, 0.5 mM dithiothreitol] was added to the pellets and placed on ice for 20 min to swell the plasma membranes (34). Cells were lysed by passage through a 26-gauge needle eight to 10 times. Lysates were centrifuged at 14,000 rpm for 20 sec. Supernatants were discarded and pellets were dissolved in Buffer C [20 mM HEPES (pH 7.9), 25% (vol/vol) glycerol, 0.42 M NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM phenylmethylsulfonyl fluoride, 0.5 mM dithiothreitol] for 30 min at 4 C to obtain the nuclear fraction (34). Nuclear factions were sonicated for 20 sec to shear genomic DNA. For preparation of total cell lysates, cells were washed twice with cold PBS(–) and lysed in lysis buffer [50 mM Tris (pH 7.4), 150 mM NaCl, 2 mM phenylmethylsulfonyl fluoride, 2 µg/ml of aprotinin, and 1% (vol/vol) Nonidet P-40]. Cells were collected and sonicated for 20 sec to shear genomic DNA. Protein concentration was measured by a colorimetric method using Coomassie Brilliant Blue G (35). Equal amounts of proteins were solubilized in 5x sample buffer [60 mM Tris-HCl (pH 6.8), 2% sodium dodecyl sulfate, 5% ß-mercaptoethanol, 1 mM EDTA, 25% glycerol, and 0.1% bromophenol blue], boiled for 10 min, and loaded onto a 12.5% acrylamide gel for subsequent SDS-PAGE (MiniProtein II System, Bio-Rad, Richmond, CA). Electrophoresis and transfer to polyvinylidene difluoride membranes were performed using the same methods previously described (36). Polyvinylidene difluoride membranes were incubated overnight at 4 C in blocking buffer (PBS, 0.25% Tween 20) containing 4% Carnation nonfat dry milk (Nestlé USA Inc., Solon, OH). The membranes were treated for 2 h with primary antibody against steroidogenic factor (SF)-1, Sp-1, GATA-4, ATF-1 (activating transcription factor-1), c-Jun, YY-1 (Yin and Yang-1), and chicken ovalbumin upstream promoter-transcription factor I (COUP-TFI) (purchased from Santa Cruz Biotechnology, Inc., Santa Cruz, CA), and CCAAT/enhancer binding protein-ß (C/EBPß), StAR, and DAX-1, kind gifts from Dr. Simon Williams (Texas Tech University Health Sciences Center), Dr. Walter Miller (University of California, San Francisco), and Dr. Paolo Sassone-Corsi (Université Louis Pasteur, Strasbourg, France), respectively. Membranes were then washed with PBS-T (PBS and Tween 20, 0.25%) three times for 20 min each. One-hour incubations were performed with the appropriate secondary antibody [antimouse (Promega, Madison, WI), -rabbit (Amersham, Arlington Heights, IL), -bovine (Santa Cruz), and -goat (Santa Cruz) IgG] conjugated with horseradish peroxidase. Membranes were washed three times with PBS-T for 20 min each time. Membranes were probed using enhanced chemiluminescence (ECL, PerkinElmer Life Sciences, Boston, MA) and exposed to x-ray film (Marsh Bio Products, Inc., Rochester, NY). Bands were quantified using the BioImage Visage 2000 (BioImage Corp., Ann Arbor, MI) and results were expressed as integrated OD (IOD).

Semiquantitative RT-PCR
Total RNA (2 µg) was used for reverse transcription reactions using the avian myeloblastosis virus reverse transcriptase (Promega) at 42 C, for 1 h. PCRs were performed using 2 µl of the reverse transcription reaction in the presence of deoxynucleotide triphosphate, Taq DNA polymerase (Promega), [{gamma}-32P] deoxy-CTP (2 µCi of 3000 Ci/mmol), and specific oligonucleotide primers for DAX-1 (forward: 5'-GCCGAGGGCCCCCTGGTGGGAC-3', reverse: 5'-TCCAGCATCATATCATCCATGCTGAC-3'). Ribosomal protein L19 (forward: 5'-GAAATCGCCAATGCCAACTC-3', reverse: 5'-TCTTAGACCTGCGAGCCTCA-3') was used as the loading control. Predicted sizes of DAX-1 and L19 PCR products were 550 bp, and 400 bp, respectively. Optimum conditions within the exponential phase of the amplification procedure were established.

RIA
Cells for measurement of progesterone synthesis were cultured in six-well plates as described above. To measure steroid production, MA-10 and R2C cells were incubated in serum-free Waymouth’s media in the absence and presence of (Bu)2cAMP for varying lengths of time and the media was recovered for RIA (36).

Selection of R2C clones expressing DAX-1 using the Tet-on inducible system
R2C cells were transfected with p172–1-Neo (Tet-on), a kind gift from Dr. Curt Pfarr (Texas Tech University Health Sciences Center), and screened using G418 (400 µg/ml). Cells exhibiting resistance to G418 were transiently transfected with a pTRE-Myc-Luc reporter vector (CLONTECH) for screening cells (R2C-Tet-on) containing low basal and high induction levels of luciferase activity when induced with doxycycline (data not shown). R2C-Tet-on cells were cotransfected with pTRE-DAX-1 and pTK-Puro and screened in the presence of puromycin (4 µg/ml). R2C-Tet-on clones expressing DAX-1 (R2C-Tet-on-DAX-1) were initially screened by selecting those clones demonstrating a decrease in steroid production and were confirmed by Western analysis of DAX-1 using a specific antibody.

Retroviral infection
DAX-1 expressing cells were produced using PT67 packaging cells transfected with the pMSCV-puro-DAX-1 using the Effectene reagent (QIAGEN, Valencia, CA). Two days later, puromycin (3 µg/ml) was added and stable PT67 clones that were able to survive in the presence of puromycin were screened by Western blot analysis to determine which clones were expressing DAX-1 (data not shown). Media from selected PT67 clones (Control, Clone 3, and Clone 6) was added to R2C cells. On each of the next 2 d, R2C cells were again infected with the viral particles in the same manner. On the fourth day of infection, R2C cells were washed with PBS(–) and incubated with serum-free Waymouth’s media for 6 h. Progesterone and StAR protein expression were determined by RIA and Western analysis, respectively.

Statistics
Experiments measuring steroid production were repeated at least three times using triplicate samples in each experiment. The data are expressed as the mean ± SE. Statistical analyses of data were performed using the Student’s t test (two-sample assuming unequal variances) and P < 0.05 was considered significantly different from controls.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Comparison of StAR expression and steroid production in MA-10 and R2C cells
The Leydig tumor cell lines, MA-10 and R2C, were compared to determine their levels of StAR protein and steroid production. Cells were incubated in serum-free Waymouth’s media for 6 h with or without (Bu)2cAMP stimulation. Figure 1AGo shows the expression levels of StAR protein. Unstimulated MA-10 cells show essentially no StAR expression and upon stimulation, produce high levels of StAR protein. R2C cells on the other hand have high levels of StAR protein both in the absence and presence of cAMP analog. The production of steroid in MA-10 and R2C cells was compared in Fig. 1BGo. MA-10 cells produce steroids only under stimulated condition, whereas R2C cells synthesize steroid in both the absence and presence of stimulation.



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  		FIG. 1. Comparison of StAR and steroid production in MA-10 and R2C cells with or without stimulation. Cells were stimulated in serum-free Waymouth’s media in the presence and absence of 1 mM (Bu)2cAMP for 6 h. A, Western blot analysis for StAR expression; B, progesterone production.

 
Screening for differential expression of transcription factors in MA-10 and R2C cells
Cloning of the StAR genomic DNA from R2C cells and comparison of its promoter sequence to those of the MA-10 and published rat sequences (35, 37) did not reveal any significant differences in their sequences and promoter activity assays were similar in the two cell lines (data not shown). These results led us to consider the levels of transcription factors known to positively and negatively regulate the StAR promoter in the two cell lines. We hypothesized that StAR gene transactivators may be expressed at higher levels in R2C cells than in MA-10 cells, or, that known StAR gene repressors may be lower in R2C than in unstimulated MA-10 cells. Therefore, a battery of transcription factors was screened in unstimulated and stimulated MA-10 and R2C cells to determine whether their expression levels were different in the two cell lines. Levels of known transactivators of the StAR gene, namely, ATF-1, SF-1, Sp-1, GATA-4, c-Jun, and C/EBPß (38, 39, 40, 41, 42, 43), were investigated in both cell lines. As shown in Fig. 2AGo, there were no significant changes in the levels of ATF-1 and SF-1, whereas the expressed levels of GATA-4, c-Jun, Sp-1, and C/EBPß were actually less in R2C cells when compared with MA-10 cells. The levels of YY-1, a known repressor of the StAR gene, appeared to be the same in both the MA-10 and R2C cells. The transcription repressor COUP-TFI levels were higher in MA-10 cells than in R2C cells, indicating that it might play a role in constitutive steroid production and StAR expression in R2C cells (44). However, the level of DAX-1, a well-known repressor of the expression of StAR and other steroidogenic enzymes in steroidogenic cells was very high in MA-10 cells but was undetectable in R2C cells. Based on previous observations (45, 46, 47, 48, 49), we decided to focus specifically on the role of DAX-1. To be certain that the human DAX-1 antibody would recognize the rat DAX-1 protein we tested it using rat adrenal protein from a 10-d-old animal, a source that is known to contain DAX-1 (44, 50). The antibody was able to detect the DAX-1 protein in rat adrenal samples, indicating that R2C cells do not express DAX-1 (data not shown). RT-PCR for DAX-1 was performed to determine the transcriptional level of DAX-1 mRNA in MA-10 and R2C cells. As shown in Fig. 2BGo, untreated MA-10 cells express high levels of DAX-1 mRNA, which decreased by 85% after stimulation when compared with controls. In contrast, R2C cells had no detectable DAX-1 mRNA under either unstimulated or stimulated conditions. Therefore, it was possible that constitutive StAR expression and steroidogenesis in R2C cells resulted from the absence of DAX-1, a repressor of StAR expression.



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  		FIG. 2. Screening for differential expression of transcription factors. A, Transcription factors in nuclear fractions of control and (Bu)2cAMP stimulated MA-10 and R2C cells were screened by Western blot analysis. B, mRNA levels for DAX-1 in MA-10 and R2C cells detected by RT-PCR. L19 mRNA was used as an internal control for RNA loading.

 
Expression of DAX-1 by cAMP analog in MA-10 cells
The decrease in DAX-1 after (Bu)2cAMP stimulation as illustrated in Fig. 2Go, A and B, was observed at 6 h. In the following experiments, MA-10 cells were stimulated and incubated for shorter and longer time periods and then analyzed by Western analysis for the levels of DAX-1, StAR, and the P450scc enzyme. In addition, the media were analyzed for progesterone production. The results demonstrated that the levels of DAX-1 began to decrease after 2 h of stimulation with 1 mM (Bu)2cAMP, and were maximally decreased at 8 h of stimulation (Fig. 3AGo). During these time periods, the protein levels of StAR and P450scc were increased. The lower panel in Fig. 3AGo shows the IOD of protein bands converted to a percentage of the maximal IOD. This graph shows a reverse correlation between DAX-1 and StAR or P450scc proteins. Progesterone production was maximal at 8 h of stimulation, a time that correlated with the highest level of StAR expression (Fig. 3BGo). StAR protein levels were decreased at 16 h and cumulative steroid levels were also decreased at this time point, an observation that will be discussed later. Collectively, after stimulation of MA-10 cells, the decreased level of DAX-1 and the increased levels of the P450scc and StAR paralleled the increase in progesterone synthesis.



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  		FIG. 3. Analysis of DAX-1, StAR, P450scc, and steroidogenesis in MA-10 cells for time courses. A, Western blot analysis of DAX-1, P450scc, and StAR proteins in MA-10 cells after stimulation with 1 mM (Bu)2cAMP. All bands of expressed protein were converted to the percentage of maximal IOD in a graph. The data are representative of an experiment performed twice with similar results. B, Steroid production in response to (Bu)2cAMP stimulation was measured using the same time courses as in (A).

 
DAX-1 expression in response to LH in rat Leydig cells
To determine whether the expression and hormonal regulation of DAX-1 in primary cultures of Leydig cells is similar to that occurring in MA-10 cells, 10-d-old rat Leydig cells (provided by Dr. Shafiq Khan, Texas Tech University Health Sciences Center) were stimulated with LH for 24 h and StAR protein was analyzed by Western blot analysis and testosterone production by RIA. Ten-day-old rat Leydig cells expressed high levels of DAX-1 (control) that was significantly decreased by LH stimulation (Fig. 4AGo). Conversely, StAR, P450scc, and testosterone production (Fig. 4BGo) were significantly increased after LH stimulation. Collectively, the inverse relationship between DAX-1 and StAR was observed in MA-10 cells, R2C cells, and primary cultures of rat Leydig cells.



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  		FIG. 4. Reverse correlation between DAX-1 and StAR, P450scc, and steroidogenesis following LH stimulation in primary cultures of 10-day old rat Leydig cells. A, Western analysis of DAX-1, P450scc, and StAR proteins in primary cultures of 10-d-old rat Leydig cells after 24 h of LH stimulation. The data are representative of three separate experiments that gave similar results. B, Testosterone production after LH stimulation was measured using the same treatment as in panel A.

 
DAX-1 overexpression in R2C cells by the Tet-on inducible expression system
Previous results suggest that DAX-1 is down-regulated by LH and (Bu)2cAMP stimulation, whereas StAR and steroid production are increased in both MA-10 cells and primary cultures of rat Leydig cells. Because of these results and the absence of DAX-1 in R2C cells, we tested the hypothesis that an overexpression of DAX-1 might result in decreases in StAR expression and steroid production. DAX-1 protein was overexpressed in R2C cells by using the Tet-on inducible protein expression system. First, the R2C-Tet-on clone was developed to express the reverse tetracycline-controlled transactivator that responds to tetracycline or doxycycline. The R2C-Tet-on clone was then cotransfected with the pTRE-DAX-1 vector to overexpress DAX-1 protein after doxycycline induction and with the pTK-Puro vector to screen the cotransfected cells in the presence of puromycin. The R2C-Tet-on-Puro clone was used as a negative control because it was resistant to puromycin but did not express DAX-1 protein (Fig. 5AGo). No changes in the synthesis of the P450scc enzyme, StAR protein or steroid were observed in this cell line (Fig. 5Go, A and B). In preliminary experiments, no adverse effects of doxycycline on steroidogenesis and StAR expression were observed in R2C cells (data not shown). The R2C-Tet-on-DAX-1 clone was resistant to puromycin and expressed DAX-1 when induced by doxycycline (Fig. 5AGo). In this clone, doxycycline treatment resulted in reductions in P450scc and StAR protein levels by approximately 39% and 35%, respectively, when compared with the noninduced group. Also, as expected, the R2C-Tet-on-DAX-1 clone demonstrated a 45% decrease in steroidogenesis following the induction of DAX-1 (Fig. 5BGo).



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  		FIG. 5. Overexpression of DAX-1 in R2C cells using the Tet-on inducible gene expression system. A, Western blot analysis was performed to determine the levels of StAR, P450scc, and DAX-1 in the Tet-on inducible DAX-1 expressing R2C clone (R2C-Tet-on-DAX-1) and puromycin-resistant R2C clone (R2C-Tet-on-puro) after induction for 18 h with doxycycline. After washing with PBS, the cells were incubated for 6 h in serum-free Waymouth’s media without/with doxycycline. The data shown in Fig. 5AGo are representative of an experiment performed four times with similar results. B, Steroid production was measured in R2C-Tet-on-puro and R2C-Tet-on-DAX-1 cells using the same treatment of the cells as in panel A. (*, P < 0.05, significantly different from noninduced group.)

 
Retroviral infection of DAX-1 into R2C cells
Problems encountered using the Tet-on inducible DAX-1 expression system were low transfection efficiency and a basal expression of DAX-1, even in the absence of doxycycline treatment as shown in Fig. 5AGo. In an attempt to overcome these difficulties and corroborate the observations made in the Tet-on inducible system, infection of R2C cells using retroviral particles was performed. PT67 cells were used to package the viral particles and transfection of these cells was performed using the DAX-1-containing pMSCV-puro-DAX-1 construct. Transfected PT67 cells were screened in the presence of puromycin. To obtain a control clone, we then screened clones that were transfected with pMSCV-puro-DAX-1, were resistant to puromycin but did not express DAX-1 (Fig. 6AGo, control). Additional clones were selected on the basis of elevated DAX-1 expression and Clones 3 and 6 were chosen for further studies. As shown in Fig. 6Go, infection of R2C cells with the media from Clones 3 and 6, resulted in the expression of DAX-1 protein and also in 50% and a 60% decreases, respectively, in StAR protein expression and corresponding approximately 60% decreases in steroid production. The results of these experiments indicated that the lack of DAX-1 expression in R2C cells is at least partially responsible for the observed constitutive steroidogenesis and StAR expression seen in these cells.



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  		FIG. 6. Overexpression of DAX-1 in R2C cells using the retroviral infection. The control is the clone that did not express DAX-1 but was resistant to the puromycin. Clones 3 and 6 were resistant to puromycin and expressed DAX-1. The media from the three clones were used to infect the R2C cells each day. After 72 h, the infected R2C cells were washed and incubated in serum-free Waymouth’s media for 6 h and analyzed for the expression of StAR and DAX-1 (A), and for the production of progesterone (B) (*, P < 0.0005, significantly different from control group). The data shown in panel A are representative of two separate experiments that gave similar results.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The constitutive synthesis of steroids and StAR expression in R2C cells has been known for more than a decade (Refs.8, 31, 32, 33 and 51). Recent findings demonstrated that R2C cells are well equipped for constitutive steroidogenesis, containing high levels of SR-BI, HSL, and StAR protein, all of which are required to support steroid production (33). However, the mechanism(s) involved in maintaining the constitutive steroidogenic phenotype of R2C cells has not been clearly defined. In the present study, we attempted to explore potential conditions within the R2C cells that might be responsible for the constitutive expression of the StAR protein and steroidogenesis. When we compared the organization of the genomic StAR genes, and the nucleotide sequence of the StAR promoter, there were no significant differences between MA-10 and R2C cells or between R2C cells and the published rat StAR sequence (data not shown). Thus, it appeared unlikely that differences in the promoter region in R2C cells would be responsible for constitutive StAR expression. This observation led us to hypothesize that it might be the complement of intracellular transcription factors in R2C cells that has the potential to activate the StAR gene and steroidogenesis.

An initial screening of several transcription factors known to activate the StAR gene resulted in the conclusion that none of these factors were likely to be responsible for the observed constitutive expression of StAR and steroid production. We then screened transcription factors known to be negative regulators of steroid production and StAR expression namely, YY-1, COUP-TFI, and DAX-1. We observed no differences in YY-1 in R2C and MA-10 cells, but both COUP-TFI and DAX-1 were significantly decreased in R2C cells when compared with MA-10 cells. In fact, DAX-1, the most thoroughly studied negative regulator of StAR expression, was completely absent at both the protein and mRNA levels in R2C cells indicating the possibility that its absence was responsible for the observed constitutive expression of StAR. This observation presented a compelling reason to focus further on DAX-1. Previous studies on the expression pattern of DAX-1 in mouse tissues demonstrated that it is expressed in thymus, lung, heart, hypothalamus, spleen, kidney, adrenal gland, ovary, and testes (50). A separate study demonstrated that it is highly expressed in rat Sertoli cells and that its expression was decreased in response to cAMP analog treatment (52). The results in the present study corroborate these earlier observations, as we found that both MA-10 cells and 10-d-old rat Leydig cells expressed DAX-1 endogenously, and further demonstrated that this expression was down-regulated by (Bu)2cAMP and LH, thus supporting our hypothesis that the absence of DAX-1 might be responsible for constitutive StAR expression and steroid production in R2C cells. In an earlier study (33), we observed that in addition to StAR, SR-BI, and HSL protein levels are constitutively higher in R2C cells. However, in the present study we did not determine whether overexpression of DAX-1 leads to a decrease in these proteins in R2C cells because the observed decrease in StAR protein levels would be sufficient to reduce steroid biosynthesis regardless of the effects of DAX-1 on either of these proteins.

DAX-1 is an unusual member of the nuclear-receptor superfamily of transcription factors in that it does not contain a typical zinc finger DNA binding domain. It is a well-characterized repressor of StAR gene transcription. Zazopoulos et al. demonstrated that the C terminus of DAX-1 was able to bind human StAR promoter sequences containing a hairpin-loop structure located upstream of a TATA-like sequence and block transcription of the StAR gene (45). The involvement of DAX-1 in inhibition of StAR and other steroidogenic genes was further studied using Y-1 mouse adrenocortical cells, which do not express endogenous DAX-1 (46). Overexpression of DAX-1 in these cells blocked the transcription of StAR, other steroidogenic genes (P450scc and 3ß-HSD), and the production of steroids. Those results were consistent with our present findings that R2C cells showed decreased synthesis of StAR and steroid production when DAX-1 was overexpressed using both the Tet-on inducible expression and retroviral infection systems.

Previous studies showed that the expression pattern of DAX-1 and the transcription factor SF-1 was colocalized during embryonic development (53, 54, 55) and that DAX-1 is involved in the control of transactivation of SF-1 for normal developmental differentiation. It was also reported that DAX-1 was able to inhibit the SF-1- or forskolin-induced transactivation of the StAR gene and steroid production (45, 46, 47, 48, 49). The protein kinase A (PKA)-dependent signaling transduction pathway plays a critical role in steroidogenesis, and SF-1 can be activated by phosphorylation through the PKA pathway resulting in the expression of steroidogenic genes such as StAR, P450scc, 3ß-HSD, aromatase, and the androgen and estrogen receptor genes (45, 46, 47, 48, 49, 53, 54, 55, 56, 57, 58, 59, 60). It would be of interest to determine whether the SF-1 present in R2C cells is constitutively phosphorylated and if the overexpression of DAX-1 can antagonize SF-1 action in these cells.

We also demonstrated that DAX-1 expression was decreased in (Bu)2cAMP-treated MA-10 cells and in LH-stimulated primary cultures of 10-d-old rat Leydig cells, resulting in increases in StAR and P450scc expression and steroid production. These results are analogous to previous data demonstrating that DAX-1 expression was decreased by stimulation with FSH, cAMP analogs, and angiotensin II (50, 52, 61, 62). Also, a recent study by Song et al. (63) using the K28 mouse testicular Leydig cell line demonstrated that DAX-1 mRNA was markedly decreased within 3 h by LH and forskolin stimulation. The levels of StAR protein and steroid production were maximal at 8 h and decreased at 16 h as shown in Fig. 3Go, A and B. Similar observations have been made in previous studies that used pregnant mare serum gonadotropin/human chorionic gonadotropin (hCG) (64, 65), cAMP analogs (27, 66) and T3 (67). The decrease in StAR protein level is most likely attributable to both the loss of LH receptors and the normal turnover of existing StAR protein. In MA-10 cells, it has previously been shown that internalization and degradation of bound hCG occurred within 12 h (68) and, as a result, the cells showed a time- and dose-dependent decrease in hCG binding capacity (69). Yet another study demonstrated that the mRNA levels of StAR and the LH receptor were maximal at 3 h after hCG or 8-bromo-cAMP stimulation and then gradually decreased between 6 and 24 h of stimulation (70). The authors suggested that over production of steroids might be toxic to the cells, thus resulting in a decrease of LH receptors and a concomitant decrease in steroid synthesis. Also, West et al. (71) demonstrated that in MA-10 and mLTC-1 mouse Leydig tumor cell lines, hCG and hCG receptor complexes were internalized and degraded within 2 h after incubation with (Bu)2cAMP, forskolin, and cholera toxin. In addition to the loss of LH receptors, the observed decrease in StAR protein is most likely a result of the normal turn over process for StAR that was calculated to have a half-life of approximately 5 h in rat granulosa cells (72). Therefore, the decrease in StAR at 16 h is probably caused by a combination of a decrease in LH receptors and the turn over of intramitochondrial StAR protein. The decrease in the cumulative level of progesterone at 16 h is a curious observation and is most likely because of the fact that progesterone in the medium can apparently be internalized by MA-10 cells and converted to a compound that does not react in the progesterone RIA (Stocco, D. M., personal observation). A similar observation was made by Rommerts et al. (73), who showed that MA-10 cells could metabolize 70–95% of added radiolabeled progesterone within 3 h. They found that these cells could convert progesterone to other steroids including 5{alpha}-pregnan-3ß-ol-20one, and thus the observed decrease in progesterone at 16 h seen in the present study would appear to be a result of this conversion.

Sandhoff and McLean (74) showed that prostaglandin F2{alpha} (PGF2{alpha}) induced the expression of DAX-1, and resulted in the repression of StAR mRNA levels in 10-d post-ovulated ovaries. We performed a similar experiment with PGF2{alpha} in MA-10 and R2C cells to attempt to induce the expression of DAX-1 endogenously and to observe the effect on the StAR expression and steroid synthesis. However, no expression of DAX-1 was detected following treatment with PGF2{alpha}. It is highly possible that the receptor for PGF2{alpha}, which has been identified in human (75), bovine (76), sheep (77), rat (78), and mouse (79), may not be present or functional in MA-10 and R2C cells. This may indicate that cellular signaling mechanisms in response to this compound function differently in the ovary and testes.

In R2C cells, a negative correlation was observed between DAX-1 and StAR. The extent of decrease in StAR and steroid production was approximately 40–60% after overexpression of DAX-1. This level of inhibition is somewhat less than that of the 80–90% decreases in StAR expression and steroid production observed in Y-1 cells after DAX-1 overexpression (48). The reasons for this difference are not clear, but it could be because of the length of time DAX-1 was expressed in those experiments vs. the time it was expressed in our studies. Although the data presented here indicate that it is likely that the absence of DAX-1 in R2C cells is partly responsible for the observed constitutive StAR expression and steroid production, it is also possible that other factors are involved. One possibility is the higher basal level of PKA activity in R2C cells compared with the basal PKA activity found in MA-10 cells (33). Because it was observed that treatment of R2C cells with H-89, a specific inhibitor of PKA activity, decreased StAR mRNA, SR-BI expression, and steroid production, the elevated PKA activity in R2C cells could be important in maintaining the constitutive steroidogenic phenotype. The PKA signaling pathway plays a critical role in the steroidogenic responses of cells after hormonal stimulation via the phosphorylation of transcription factors required to activate genes involved in steroid biosynthesis. Although we screened the total protein levels of transcription factors by Western blot analysis (shown in Fig. 2Go), we have not yet determined the phosphorylation status of these proteins. Therefore, it is possible that constitutive phosphorylation of one or more of these transcription factors could also be important in maintaining the constitutive steroidogenic phenotype of R2C cells.

In conclusion, we have attempted to determine one or more factors responsible for the constitutive steroidogenesis and StAR expression observed in R2C cells. Our results indicated that no significant differences were found in StAR promoter sequences or in StAR promoter activity between MA-10 and R2C cells. Our studies did, however, demonstrate that a repressor of StAR gene transcription, DAX-1, was present in high levels in MA-10 cells and was not expressed in R2C cells. Overexpression of DAX-1 in R2C cells decreased the expression of StAR and steroidogenesis by approximately 40–60%, indicating that its absence in R2C cells is at least partly responsible for the constitutive phenotype in these cells.


   Acknowledgments
 
The authors would like to thank Dr. Paolo Sassone-Corsi (Université Louis Pasteur, Strasbourg, France) for the monoclonal antibody (2F4) of DAX-1, and Dr. Curt Pfarr (Texas Tech University Health Sciences Center) for the vectors establishing the Tet-on inducible expression and retroviral infection systems.


   Footnotes
 
This investigation was supported by National Institutes of Health Grant HD-17481 and by the Robert A. Welch Foundation Grant B1-0028.

Abbreviations: ATF, Activating transcription factor; (Bu)2cAMP, dibutyryl cAMP; C/EBPß, CCAAT/enhancer binding protein-ß; COUP-TFI, chicken ovalbumin upstream promoter-transcription factor I; DAX-1, dosage-sensitive sex reversal, adrenal hypoplasia congenita, critical region on the X chromosome, gene-1; hCG, human chorionic gonadotropin; HSL, hormone-sensitive lipase; IOD, inegrated OD; P450scc, cytochrome P450 cholesterol side-chain cleavage enzyme; PGF2{alpha}, prostaglandin F2{alpha}; PKA, protein kinase A; SR-BI, scavenger receptor type B class I; StAR, steroidogenic acute regulatory; YY-1, Yin and Yang 1.

Received July 21, 2004.

Accepted for publication September 1, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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