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REPRODUCTION-DEVELOPMENT |
Department of Physiology (P.K., J.L., P.S., I.H., M.P.) and Turku Graduate School of Biomedical Sciences (P.K., P.S.), University of Turku, 20520 Turku, Finland
Address all correspondence and requests for reprints to: Matti Poutanen, Ph.D., Department of Physiology, University of Turku, Kiinamyllynkatu 10, FIN-20520 Turku, Finland. E-mail: . matti.poutanen{at}utu.fi
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Introduction |
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Zimmermann et al. (11) demonstrated that steroidogenic factor 1 (SF-1), also known as adrenal 4-binding protein, is a one of the key regulators of RLF promoter activity. SF-1 is a member of the nuclear orphan receptor superfamily, sharing a common structural organization with this group. A characteristic zinc finger in the DNA-binding domain of its N-terminal region, a ligand-binding domain, and an activation function 2 sequence in the C terminus are well conserved among members of this family (12). SF-1 binds as a monomer to its recognition elements, PyCAAGGPyPyPu and PuPuAGGTCA motifs. These DNA motifs are recognized by the two zinc fingers of SF-1, leading to high affinity binding.
SF-1 was first observed to regulate the expression of genes for steroidogenic enzymes in the adrenal gland and gonads (13). Subsequently, several investigators have shown that this orphan nuclear receptor plays an important role as a transcriptional regulator of a variety of target genes, including those for aromatase (14), LHß (15), FSH (16), steroidogenic acute regulatory protein (17), cholesterol side-chain cleavage enzyme (18), DAX-1 (dosage-sensitive sex reversal-adrenal hypoplasia congenita critical region on the X chromosome) (19), and anti-Mullerian hormone (20).
In accordance with its documented functions, SF-1 expression has been detected in steroid hormone-producing cells, including testicular Leydig and Sertoli cells, ovarian thecal and granulosa cells, and adrenocortical cells (21, 22). Some other endocrine cells, such as those of the ventro-medial nucleus of the hypothalamus, as well as pituitary gonadotrope cells, also express SF-1 (23, 24). SF-1 plays an essential role in the embryonic development of these tissues, as shown in studies on targeted disruption of the SF-1 gene, with resulting pleiotropic impairment of function of the hypothalamic-pituitary-gonadal and hypothalamic-pituitary-adrenal axes (25, 26). Collectively, these data support the role of SF-1 as a key regulator of endocrine development and function.
Interestingly, DAX-1, another orphan nuclear receptor, has been shown to repress the transcriptional activity of SF-1. The human DAX-1 gene is located on the X chromosome at region p21, which, when duplicated, gives rise to XY females, a condition referred to as dosage-sensitive sex reversal. DAX-1 mutations are associated with the pathogenesis of adrenal hypoplasia congenita and hypogonadotropic hypogonadism (27, 28). The remarkable similarities between SF-1-null mice and humans with DAX-1 mutations in cases of adrenal hypoplasia congenita raise the possibility that SF-1 and DAX-1 work together as essential regulators of the hypothalamic-pituitary-steroidogenic axis. Indeed, there is full overlap between the sites of expression of the SF-1 and DAX-1 genes (29). Several investigators have shown that SF-1-mediated trans-activation is inhibited by DAX-1 via direct interaction with SF-1 (30, 31, 32) or through binding to single-stranded hairpin DNA structures (33).
In this study we characterized in detail the SF-1-dependent regulation of the murine RLF promoter and investigated the influence of DAX-1 on RLF promoter activity in vitro.
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Materials and Methods |
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). These 5'-deletion fragments were subcloned in front of the luciferase reporter gene (pBL-Luc) to obtain RLF-luciferase reporter constructs.
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), dNTPs (each 25 mmol/liter), and 50 µl 1x reaction buffer in the presence of 1 µl Pfu DNA polymerase. The PCR conditions included 16 cycles of denaturing at 95 C for 30 sec, annealing at 55 C for 1 min, and extension at 68 C for 13 min. The parental DNA template was digested by adding 1 µl DpnI restriction endonuclease for 1 h at 37 C. Five microliters of the PCR reaction were used to transform XL-1 Blue Supercompetent cells (Stratagene). The mutations were verified by restriction mapping using EcoRI and by sequencing both strands using a fluorescent dye terminator reaction (Prism Ready Reaction Dye Termination Cycle Sequencing Kit) and an automated sequencer (Perkin-Elmer Corp., Foster City, CA).
Cell culture and transfections
Mouse tumor Leydig cells (mLTC-1) (35) were cultured in HEPES-buffered Waymouth’s medium supplemented with 9% heat-inactivated horse serum (Life Technologies, Inc., Paisley, UK) and 4.5% FCS (Bioclear, Wilts, UK) and containing 50 mg gentamicin/liter (Biological Industries, Haemek, Israel). The KK-1 cells were derived from a granulosa cell tumor of a transgenic mouse expressing the simian virus 40 T antigen under the murine inhibin 
-subunit promoter (36). KK-1 and human embryonic kidney (HEK-293) cells were cultured in DMEM-Ham’s F-12 (1:1; Sigma, St. Louis, MO) supplemented with 10% heat-inactivated FCS containing 50 IU penicillin/liter and 0.5 µg streptomycin/ml (Sigma). The cells were cultured at 37 C in a humidified atmosphere containing 5% CO2.
Transfections were carried out at 70–80% confluence of the cells on six-well plates. Fugene 6 transfection reagent (Roche Molecular Biochemicals, Mannheim, Germany) was used for transfecting the mLTC-1 and HEK-293 cells, and Lipofectamine (Life Technologies, Inc.) was used for KK-1 cells, according to the instructions of the manufacturers. For cotransfection experiments, various amounts of expression plasmids for human SF-1 (pCMV119+-SF-1) and human DAX-1 (pBKCMV-hDAX-1) were included in the transfection reaction, and the total amount of DNA was kept constant by adding carrier DNA (empty expression vector). Transfected cells were incubated for 24 h before harvesting and functional analysis. For measuring luciferase activity, medium was replaced with 100 µl lysis buffer [12.5 mM Tris-HCl (pH 7.8), 10 mM NaCl, 0.4 mM EDTA, 0.2 mM MgSO4, 1 mM dithiothreitol, and 0.2% Triton X-100], followed by incubation for 5 min at room temperature. Cell suspension was collected and briefly centrifuged, and 20 µl cell lysate were pipetted into a well of an opaque 96-well plate (Wallac, Inc., Turku, Finland). One hundred microliters of assay buffer [40 mM Tris-HCl (pH 7.8), 0.5 mM ATP, 10 mM MgSO4, 0.5 mM EDTA, 10 mM dithiothreitol, 0.5 mM coenzyme A, and 0.5 mM luciferin] were added, and luciferase activity was analyzed by using a Victor Multicounter (Wallac, Inc.). The luciferase values were normalized for transfection efficiency by relating them to values obtained for a cotransfected pCMV-ß-galactosidase construct and/or to the amount of total protein. ß-Galactosidase activity was measured in the same lysates, according to a protocol recommended by Invitrogen (Carlsbad, CA), and protein concentrations were determined by the Bradford method (37).
RNA analysis
Total RNA was isolated from different cell lines and mLTC-1 cells transfected with DAX-1 expression vector, using the acid guanidinium thiocyanate method (38). Twenty micrograms of denatured total RNA were resolved on 1.2% denaturing agarose gel and transferred onto nylon membranes (Hybond XL, Amersham Pharmacia Biotech, Uppsala, Sweden) by employing the capillary transfer method. The membranes were prehybridized for 4 h at 42 C in a solution containing 3x SSC (1x SSC is 0.15 M NaCl and 0.015 M sodium citrate, pH 7.0), 5x Denhardt’s solution, 1% SDS, and 0.1 mg heat-denatured calf thymus DNA/liter. Hybridization was carried out at the same temperature overnight in the same solution after the addition of the [-32P]dCTP-labeled probe. For preparing the SF-1 cDNA probe, an EcoRI/PstI-digested fragment of SF-1 cDNA was labeled with [-32P]dCTP, using the Prime-A-Gene labeling kit (Promega Corp., Madison, WI). The labeled probe was purified by using a Sephadex G-50 nick column (Pharmacia Biotech). After hybridization, the membrane was washed twice with 2x SSC and 0.1% SDS at room temperature for 30 min and twice at 42 C with 0.1x SSC and 0.1% SDS. The membranes were exposed for 4–10 h to a phosphorimager plate (BAS-5000, Fujifilm II, Tokyo, Japan).
RT-PCR was used to measure differences in RLF expression between different cell lines. RT and amplification were performed in the same tube so that 2 µg deoxyribonuclease I (Life Technologies, Inc.)-treated total RNA were mixed with 25 U AMV RT (Promega Corp.), 40 U of the ribonuclease inhibitor RNasin (Promega Corp.), 400 nM dNTP, and 5 U Dynazyme-DNA polymerase in 1x PCR buffer (Finnzymes, Espoo, Finland). Reaction mixture was divided into two tubes containing 30 pmol RLF-specific or L19 ribosomal-specific primers (Table 1). L19 ribosomal primers were chosen to coamplify ribosomal protein for monitoring equal PCR amplification efficiency. Samples were heat denatured and reverse transcribed by incubation at 48 C for 30 min and were amplified with following cycles: 94 C for 1 min (2 min for the first cycle), 60 C for 50 sec, and 72 C for 40 sec (5 min for last cycle). Ten to 35 cycles of PCR were tested to find the exponential phase for both RLF and L19 amplification. Twenty and 25 cycles were chosen for L19 and RLF, respectively (data not shown). After RT-PCR reaction, samples were loaded onto a 1.3% agarose gel to obtain a 219-bp fragment of the RLF gene and a 408-bp fragment of the L19 ribosomal protein gene.
EMSA
Nuclear extracts were prepared from control and DAX-1-transfected mLTC-1 cells as previously described (39). Complementary oligonucleotides corresponding to the three SF-1-binding sites (Table 1) identified within the RLF promoter were annealed. The annealed oligomers with 5'-GG overhangs were labeled with [-32P]dCTP (3000 Ci/mmol; Amersham Pharmacia Biotech) using the Klenow fragment of DNA polymerase I. In the binding reaction, 10 µg nuclear extract were incubated for 30 min on ice with 2 µg poly(dI:dC) and 20 fmol labeled probe in binding buffer [12 mM HEPES (pH 7.8), 60 mM KCl, 12% glycerol, 4 mM Tris-Cl, 1 mM EDTA, and 1 mM dithiothreitol]. For competition experiments, the protein extract was first incubated for 30 min on ice with a 50- or 200-fold molar excess of unlabeled competitor DNA or rabbit anti-SF-1 polyclonal antiserum (1:10 or 1:100; obtained from Dr. K. Morohashi, Kyushu University, Fukuoka, Japan) before addition of [-32P]dCTP-labeled DNA. After the binding reaction, the products were resolved by electrophoresis through a 5% nondenaturing polyacrylamide gel. After electrophoresis in 0.25x Tris borate-EDTA buffer, the gel was dried and subjected to autoradiography.
Immunoblotting
Nuclear extracts from various cell lines were resolved by 7.5% SDS-PAGE; transferred to nitrocellulose membrane (Hybond ECL, Amersham Pharmacia Biotech) by electroblotting; preincubated for 2 h in Tris-buffered saline, 1% Tween 20, and 5% nonfat dried milk; and washed three times for 10 min each time in the same buffer without milk. Incubation with rabbit anti-SF-1 antiserum (1:5000) was performed overnight at 4 C. The filter was washed and incubated for 1 h with antirabbit antiserum (1:1000) linked with horseradish peroxidase. After three washes, the immunocomplexes were visualized using an ECL Western blotting detection kit (Amersham Pharmacia Biotech) and exposing the membranes for 2–10 min to Kodak x-ray films (Eastman Kodak Co., Rochester, NY). The immunospecific bands were quantified using Tina software (Raytest, Stranbenhardt, Germany).
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Results |
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). These include a consensus TATA box sequence and one consensus binding site for Sp1 (GGGCGG), at position -53 to -48 (in relation to the translation initiation codon). In addition, one putative recognition motif (TACAAAG) for the sex-determining region Y chromosome (SRY) transcription factor was identified, at position -218 to -212. Four potential binding sites for transcription factor SF-1 were located at positions -64 to -56 (SF-1/A), -115 to -107 (SF-1/B), -144 to -136 (SF-1/C), and -479 to -471 (SF-1/D).
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, the promoter fragments, of different lengths, displayed their highest activity in the mLTC-1 Leydig and KK-1 granulosa luteal cells, and the relative promoter activities of the deletion fragments were similar in both cell types. However, promoter activities were, on the average, more than 3-fold higher in mLTC-1 cells than in KK-1 cells, and the 188-bp fragment demonstrated a 29-fold increase over the promoterless control plasmid (pBL-0Luc) in mLTC-1 cells. Compared with the 188-bp promoter, a dramatic decrease in luciferase activity was observed with the 104- and 55-bp deletion fragments (70% and 90% lower, respectively). An identical phenomenon was observed in KK-1 cells, with a 60% decrease in promoter activity for the shortest fragments. It should be pointed out that the 188-bp promoter fragment contained three of the four putative binding sites for SF-1, whereas the 104-bp fragment contained one, and the 55-bp fragment contained none. The promoter region between bp -700 to -188 bp did not show any marked effect on promoter activity, as similar luciferase gene activities were detected with the reporter genes 700-luc and 188-luc. However, the longest fragment (4.3 kb) showed 65–66% lower activity in mLTC-1 and KK-1 cells compared with the 188-bp fragment. The minimal length of the promoter able to drive luciferase expression in mLTC-1 and KK-1 cells derived from steroidogenic cells resided in the 188-bp fragment. In MSC-1 and HEK 293 cells, the RLF promoter showed significantly less activity, and the activity of the 188-bp fragment was only 2-fold higher than that of the promoterless plasmid. Other promoter fragments also showed similar low basal activity in these cells.
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) was highest in mLTC-1 cells, followed by KK-1 cells. A very low level of SF-1 expression was detected in MSC-1 cells, whereas no expression was seen in HEK-293 cells. Western blot analysis of the 53-kDa SF-1 protein showed an identical result, except that the difference in SF-1 expression between mLTC-1 and KK-1 cells was even greater at the protein level. Endogenous RLF expression was also detected by RT-PCR in both mLTC-1 and KK-1 cells, but not in MSC-1 cells, and the level of expression was clearly higher in mLTC-1 cells compared with KK-1cells (Fig. 3C).
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). However, mutating the SF-1/B site (MUT B) repressed the activity most effectively, down to 19% that of the wild-type promoter, whereas MUT A and MUT C constructs showed clear, but less profound, inhibition (67% and 53%, respectively). Combinations of SF-1-binding site mutations did not result in a further decrease in promoter activity compared with MUT B. The 55-bp promoter fragment, lacking all SF-1-binding sites, showed only a low basal activity (10% that of the 188-bp promoter). Furthermore, the 104-bp fragment, containing only the proximal SF-1-binding site, showed activity (28% the activity of the 188-bp promoter) similar to that of MUT BC (22%), where two distal SF-1 sites were mutated.
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). Interestingly, the promoter activity in KK-1 cells is similar to that in mLTC-1 cells when all putative SF-1 binding motifs were mutated; the activity was 28% of the maximal promoter activity found in mLTC-1 cells. Transfecting exogenous SF-1 into KK-1 cells, furthermore, markedly increased the promoter activity in KK-1 cells (data not shown). Hence, the data indicate that in KK-1 cells the promoter activity is limited by the amount of SF-1 present in the cells. However, RLF promoter is not solely dependent on SF-1 in steroidogenic cells in vitro. In mLTC-1 and KK-1 cells the promoter activities of the 188-bp fragment with all the SF-1 motifs mutated were 6- and 8-fold higher, respectively, than the luciferase activity obtained with the promoterless plasmid. The promoter activities in KK-1 cells with the various deletion fragments (Fig. 2) further indicate that the elements involved in SF-1-independent transcription are colocalized within the 188-bp fragment containing the three SF-1 binding motifs.
To further characterize the relative importance of SF-1-binding sites in regulation of the RLF promoter, we conducted trans-activation experiments in nonsteroidogenic HEK-293 (Fig. 4C). In these cells the 188-bp wild-type construct was almost nonfunctional, but responded markedly to the cotransfected SF-1 expression vector with a 40-fold increase in luciferase activity. All SF-1-binding site mutations abolished SF-1 enhancement drastically. Identically to that found in mLTC-1 cells, the strongest effect was always detected by mutating the SF-1/B motif, and SF-1-induced expression was almost completely inhibited by the mutation. As expected, the shortest (55-bp) promoter fragment, which does not have any SF-1-binding sites, did not respond to cotransfected SF-1. The activity of the 104-bp fragment, with only the proximal SF-1-binding site, was similar to that evoked by MUT BC, having the same functional SF-1 site.
SF-1 is able to bind to the RLF promoter
Using EMSA, we determined whether SF-1 is able to bind to the putative SF-1 recognition motifs located in the 188-bp proximal promoter fragment. Nuclear extracts were purified from mLTC-1, KK-1, MSC-1, and HEK 293 cells and incubated with specific radiolabeled double-stranded (ds) oligonucleotides (SF-1/A, SF-1/B, and SF-1/C) containing the putative SF-1 recognition sequences. All three SF-1 sites formed a single protein/DNA complex with mLTC-1 cell nuclear extract, and its intensity could be reduced or removed completely by 50- or 200-fold molar excesses of the corresponding wild-type ds oligonucleotides used as competitors (Fig. 5). Mutated SF-1 ds oligonucleotides were unable to compete for the protein/DNA complexes. To ensure that the protein identified by EMSA was SF-1, we added a rabbit polyclonal antibody directed against full-length bovine SF-1 protein (40) to the binding reaction. The SF-1 antiserum added at a dilution of 1:100 reduced the intensity, whereas a higher concentration totally abolished protein/DNA complex formation. Nonimmunized rabbit serum was not able to displace the complex (data not shown). In nuclear extracts from KK-1 cells we were able to detect a weak protein/DNA complex, using the same oligonucleotides as those described above, whereas no binding was seen when nuclear extracts from MSC-1 or HEK 293 cells were used (data not shown). The EMSA analyses further indicated that the radiolabeled oligonucleotides corresponding the SF-1/A and SF-1/B motifs were efficiently competed with the SF-1/C for the SF-1 binding, whereas the opposite was not found to the same extent. This suggests that the SF-1/C motif has the highest binding affinity toward SF-1.
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). We also assessed SF-1 mRNA levels in these cells exposed to DAX-1. The data showed that SF-1 mRNA was not affected by increasing amounts of transfected DAX-1 (Fig. 6B).
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). In the presence of DAX-1, the activity of the 188-bp wild-type RLF promoter construct was only 6% of that found with SF-1 only. EMSA was then used to determine whether DAX-1 has an effect on SF-1 binding to the RLF promoter. The data indicated that in the presence of DAX-1, SF-1 binding was reduced, especially to the SF-1/A and SF-1/B motifs, whereas the effect was less clear on the SF-1/C motif (Fig. 8). This was in accordance with the trans-activation studies. The activity of a MUT AB fragment was inhibited 32% by DAX-1, whereas the MUT AC and MUT BC were inhibited 64% and 51%, respectively (Fig. 7).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the present study we demonstrated that the RLF promoter is highly active in mLTC-1 Leydig cells and moderately active in KK-1 granulosa cells, whereas no activity for the promoter was seen in nonsteroidogenic MSC-1 Sertoli cells or HEK 293 cells. This was in accordance with the presence of endogenous RLF mRNA expression in mLTC-1 and KK-1 cells. As in a recent report by Zimmermann et al. (11), we found that activation of the RLF promoter in cells originating from steroidogenic tissues was associated with the immediate 188-bp 5'-flanking region. The promoter sequence between bp 700 and 188 showed no marked effect in promoter activity, suggesting that the most distal SF-1-binding site may not be crucial for RLF promoter activity. When a 4.3-kb promoter fragment was used, transcriptional activity was reduced by about half, compared with the 700- to 188-bp promoters, suggesting that repressor elements exist in this region.
The 188-bp 5'-flanking region, with full transcriptional activity, contains three functional binding sites for SF-1. The data indicate that the maximal RLF promoter activation in vitro in various cell lines is related to the level of SF-1 protein, suggesting an important role for SF-1 in regulating RLF gene expression. Mutating any of the SF-1-binding sites inhibited RLF promoter activity significantly in mLTC-1 cells, and even more markedly in nonsteroidogenic HEK 293 cells. In KK-1 cells a 3-fold lower promoter activity was found compared with mLTC-1 cells. Accordingly, an approximately 5-fold lower mRNA level for SF-1 was found compared with the mLTC-1 cells, and the difference was even greater at the protein level. The results of cotransfection studies, using a cell line devoid of endogenous SF-1 expression (HEK-293), further confirmed a direct trans-activating role of SF-1. In HEK-293 cells the promoter activity was solely dependent on transfected SF-1. However, in mLTC-1 and KK-1 cells, a clear SF-1 independent trans-activation was observed that was 6- to 8-fold higher than that found with the promoterless plasmid. This indicates that in addition to SF-1 other cell-specific transcription factors involved in the activation of RLF are present in steroidogenic mLTC-1 and KK-1 cells. This was further indicated by the fact that by mutating the SF-1-binding sites, a stronger suppression of the promoter activity was seen in HEK-293 cells compared with mLTC-1 cells.
The relative importance of the three SF-1-binding sites was analyzed by mutation analysis of the 188-bp promoter. Identically to that shown previously (11), our data clearly indicated that all SF-1-binding sites contributed to RLF induction, and this binding could be reversed by SF-1 antiserum. This type of competition, termed gel-shift abrogation, has been described previously, and it relies on the ability of the anti-SF-1 antibody to effectively block accessibility of the DNA to the SF-1 DNA-binding domain (11, 42). However, studies in both mLTC-1 Leydig cells and nonsteroidogenic 293 cells indicated that mutating the SF-1/B-binding site results in maximal down-regulation of activity of the promoter. Hence, in contrast to what has been previously suggested (11), our data indicate that the SF-1-binding site located at position -115 to -107 relative to the translation initiation codon has the strongest trans-activation function. Furthermore, we demonstrated that all three SF-1-binding sites were able to specifically bind SF-1. Surprisingly, from the three SF-1-binding sites analyzed, the most distal SF-1 binding motif (SF-1/C) was found to have the highest affinity toward SF-1, but did not result in the highest transcriptional activation. This indicates that the location of the SF-1 binding motif is critical to properly recruit other factors for transcriptional initiation.
However, the cell-specific expression of RLF cannot be determined by the expression of SF-1 only. In addition to Leydig cells, SF-1 is also expressed, for example, in Sertoli cells, granulosa cells, adrenal cortical cells, and pituitary gonadotropes (21, 22, 23, 24), whereas RLF is expressed mainly in Leydig cells. However, RLF is also expressed at low levels in ovary, especially in luteal cells and postnatal thecal cells (4, 5). The expression of the endogenous RLF together with the promoter activity in KK-1 cells are in line with the luteal phenotype of this granulosa cell line (36). It has been shown that SF-1 can interact with several other coregulators, such as DAX-1 (32), N-CoR (31), Alien (43), and ER and ERß (44), which therefore may also play central roles in the regulation SF-1 target genes. Moreover, ER and ER-related receptors and ß can bind to SF-1 binding sites (45, 46) and regulate transcription. ER-related receptors can even modulate promoter activity without ligand. Inhibitory effects of DAX-1 have been demonstrated with several genes regulated by SF-1, such as steroidogenic acute regulatory protein (33) and LHß (47). In this study we showed that DAX-1 suppresses RLF promoter activity in a dose-dependent fashion, and DAX-1 could abolish the SF-1-mediated induction of RLF promoter activity almost completely. Several studies have been carried out to clarify the mechanisms behind the repression. The results suggest that DAX-1 is able to act in various ways: by direct protein-protein interaction (30), by binding to DNA hairpin loops close to an SF-1-binding site (33), by direct down-regulation of SF-1 mRNA (48, 49), by recruiting corepressors (31), and/or by blocking SF-1 synergy with its coactivators (32, 47). Our studies did not reveal the precise mechanism behind the DAX-1 inhibition of RLF promoter activity, but we can exclude some of the proposed alternatives. We saw no SF-1 mRNA down-regulation in DAX-1-transfected mLTC-1 Leydig cells, an observation similar to that noted by Oba et al. (50) with adrenal Y1 cells. In this study we show that DAX-1 interferes with SF-1 binding to its recognition sequences directly or by acting through other factors involved in SF-1-dependent RLF promoter activity. The possible interactions between these proteins are probably weak, as we detected no shifted protein/DNA complexes in EMSA when nuclear extracts were obtained from DAX-1-transfected mLTC-1 cells. This finding is in agreement with other reports showing direct interaction between SF-1 and other transcription factors, which, however, were not detected by gel mobility shifts (30, 32, 51). There is a coordinated expression of SF-1 and DAX-1 during gonadal development (29, 52). Hence, we conclude that the interplay between SF-1 and DAX-1 could well be one of the mechanisms regulating mouse RLF gene expression during Leydig cell development. It is likely, however, that SF-1 and DAX-1 also interact with other corepressors and coactivators, forming complex transcription machinery that sustains the timing and cell specificity of RLF gene expression in endocrine tissues, especially in the developing testis. Interestingly, the elements involved in the SF-1-independent activity of the mouse RLF promoter, observed in steroidogenic KK-1 and mLTC-1 cells, were colocalized in the promoter fragment including the three SF-1-binding sites. Indeed, maternal exposure to estrogens in vivo down-regulates RLF expression in rodents without affecting SF-1 expression in fetal Leydig cells (53, 54). Hence, suppression of RLF expression by estrogens is thought to be one of the mechanisms leading to cryptorchidism in perinatally estrogen-exposed rodents. Further studies are required, however, to clarify the roles of estrogenic substances in regulation of the RLF promoter.
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Acknowledgments |
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Footnotes |
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Abbreviations: CMV, Cytomegalovirus; ds, double-stranded; mRLF, mouse relaxin-like factor; RLF, relaxin-like factor; SF-1, steroidogenic factor 1.
Received August 27, 2001.
Accepted for publication November 5, 2001.
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References |
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-subunit promoter/simian virus T-antigen fusion gene: characterization of ovarian tumors and establishment of gonadotropin-responsive granulosa cell lines. Mol Endocrinol 9:616–627 (estrogen receptor-related receptor-). Mol Endocrinol 13:764–773, but not by ERß. EMBO J 18:4270–4279[CrossRef][Medline]
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