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Regular Article |
a Hormone Research Center, Chonnam National University, Gwangju 500-757, Republic of Korea
b Department of Life Science and Technology, Pohang University of Science and Technology, Pohang 790-784, Republic of Korea
c Department of Biology, Honam University, Gwangju 506-714, Republic of Korea
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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androgen receptor, Leydig cells, testis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The AR is a member of the nuclear-receptor superfamily that is a related group of ligand-inducible transcription factors [10, 11]. The overall structure of AR is similar to those of other nuclear receptors, with three separate functional domains: the variable N-terminal activation domain (AF1), the highly conserved central DNA-binding domain (DBD), and the C-terminal ligand-binding domain (LBD) [12]. A second, ligand-dependent activation function (AF2) has also been identified in the C-terminal part of the LBD [13, 14], although AF2 in AR is relatively weak compared with AF1 [15]. On ligand binding, AR undergoes conformational changes that facilitate the formation of AR dimers in complex with specific DNA sequences, called androgen response elements (AREs), to enhance transcription of target genes [16, 17]. Transcriptional activation of nuclear receptors appears to involve their interaction with general transcription factors and coactivators to increase the transcription rate of the target gene [18–20]. The number of coactivators reported to interact with AR is increasing, and they include TIF2, SNURF, ARA70, ARIP3, CBP/p300, Ubc9, PIAS1, ANPK, TRAM-1, and FHL2 [18, 21–30]. However, only a few testis-specific AR coactivators have been identified [27, 30].
Activating signal cointegrator 1 (ASC-1), originally cloned based on its interaction with thyroid receptor (TR) [31], has been shown to be a transcriptional coactivator of nuclear receptors for thyroid (TR), all-trans-retinoic acid, 9-cis-retinoic acid (RXR), and estrogen [32]. It promotes their transcriptional efficiencies, either alone or in cooperation with SRC-1 and CBP/p300. A putative zinc-finger motif in the autonomous transactivation domain of ASC-1 is involved in its association with nuclear receptors and other factors. Although ASC-1-binding sites involve the hinge domain of nuclear receptors, ASC-1 function appears to require the AF2-dependent factors, because it cannot coactivate a mutant receptor lacking the AF2 core domain. Cellular relocalization of ASC-1 under different cellular conditions has suggested that it may play a role in establishing distinct coactivator complexes on different cellular regulatory signals. However, to our knowledge, the physiological relevance and function of ASC-1 have not been studied.
The ASC-1 is expressed in human male reproductive organs such as testis and prostate [32]. In the present study, we first analyzed the expression pattern of ASC-1 in the mouse testis to examine its physiological role in the testis. Subsequently, we investigated the function of ASC-1 as an AR coregulator based on the first appearance of the testis-specific ASC-1 messages around the time of puberty as well as predominant expression of ASC-1 in the Leydig cells that express AR.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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All the LexA and B42 fusion vectors were constructed by cloning the indicated genes and their mutants in frame after LexA-DBD of p202PL [33] and B42-AD of pJG4-5 [33], respectively. Glutathione S-transferase (GST) fusion constructs were made using pGEX4T-1 (Amersham Pharmacia Biotech, Uppsala, Sweden), and plasmids for mammalian expression and for in vitro translation of AR and ASC-1 were constructed using the mammalian expression vector pcDNA3 (Invitrogen, San Diego, CA). All the plasmids containing full-length ASC-1 [32] and its deletion mutants, ASC-1A, ASC-1B, and ASC-1C, were provided by J.W.L. Mouse AR (a gift from Dr. D.J. Tindall at the Mayo Foundation, Rochester, MN) was cloned into the BamHI site of pcDNA3. A luciferase reporter plasmid containing two AREs of the C3 gene, pARE2-TATA-Luc [22], was a gift from Dr. J.J. Palvino (University of Helsinki, Finland).
Animals
BALB/C mice were purchased from a commercial supplier (Daehan Laboratories, Chung-buk, Korea). Animals were kept and bred in a cage with water and chow available and were maintained under controlled conditions (12L:12D, 50% humidity, 22°C). The day that litters were born was considered to be Day 1 of life. The ethical treatment of animals in this study was carried out according to National Institutes of Health standards.
Northern Blot Analysis
Total RNA was extracted from dissected tissues and cultured cells using Tri Reagent solution (Molecular Research Center, Inc., Cincinnati, OH). Twenty micrograms of total RNA were separated on a 1.2% (w/v) denaturing agarose gel, transferred onto Zeta-probe nylon membrane (Bio-Rad, Hercules, CA) in 10x SSC (standard saline citrate; 1x SSC: 0.15 M sodium chloride and 0.015 M sodium citrate) by the capillary transfer, and then immobilized under ultraviolet light. After overnight prehybridization, the membrane was hybridized at 42°C in a solution containing 50% (w/v) formamide, 10% dextran sulfate, 5x SSC, 1 mM EDTA, 100 µg/ml of denatured salmon sperm DNA, and a random-primed 32P-labeled mouse ASC-1 cDNA probe. After washing at 65°C for 20 min in 0.2x SSC and 0.1% (w/v) SDS as a final stringency, the membrane was exposed on Kodak x-ray film (Eastman Kodak, Rochester, NY) at -70°C. The membrane was then stripped and reprobed for glyceraldehyde phosphate dehydrogenase.
In Situ Hybridization
Testes were prefixed with 4% paraformaldehyde in potassium-buffered saline (PBS) by transcardiac perfusion, dissected out and further fixed at 4°C for 4–5 h in the same fixative, and then immersed in 20% sucrose in PBS overnight. The hybridization was performed as previously described [34] with minor modifications. In brief, after digestion with proteinase K and acetylation, cryostat sections (thickness, 20 µm) were hybridized at 60°C overnight with 35S-labeled mouse ASC-1 cRNA probe in 50% formamide, 10% dextran sulfate, 0.3 M NaCl, 10 mM Tris-HCl, 1 mM EDTA, 1x Denhardt solution, 500 µg/ml of carrier transfer RNA, and 10 mM dithiothreitol. Slides were washed under stringent conditions that included ribonuclease A (20 µg/ml) treatment at 37°C for 30 min and wash in final stringency of 0.1x SSC and then processed for liquid emulsion autoradiography (Kodak NBT-2). After exposure for 3 wk at 4°C, slides were developed, counterstained with hematoxylin and eosin, and taken for photomicrography. The sense cRNA probe was used as a control for nonspecific binding.
cDNA Library Screening
Approximately 7 x 104 pfu of mouse testis cDNA library packaged in 
gt 11 (Promega, Madison, WI) were transferred onto nitrocellulose filters (Amersham Pharmacia Biotech). Hybridization was carried out at 42°C overnight in a solution containing 50% formamide, 10% dextran sulfate, 5x SSC, 1 mM EDTA, 100 µg/ml of denatured salmon sperm DNA, and a random-primed 32P-labeled mouse ASC-1 cDNA probe. The positive cDNA clones were obtained by enzyme digestion and subcloned into pBluscript (Promega). Sequences of the cDNAs were analyzed using the ABI PRISM sequence analyzer (Perkin Elmer, Foster, CA).
Reverse Transcription-Polymerase Chain Reaction and Southern Blot Analysis
Total RNAs eluted from tissues were reverse transcribed and used for polymerase chain reaction (PCR) using the SuperScript One-Step Reverse Transcription (RT)-PCR system (Life Technologies, Gaithersburg, MD) according to the supplier's instructions. A 1746-base pair (bp) product spanning the whole open reading frame (ORF) region was amplified using forward (5'-ATG GCG GTG GCT GGG GCG G-3') and reverse (5'-TCA GAC AGC TTT ATT CTG CTT CAT T-3') primers corresponding to the mouse ASC-1. The RT-PCR products were run on 1% agarose gel and transferred onto Zeta-probe nylon membrane (Bio-Rad). After prehybridization for 3 h, the blot was hybridized with a random-primed 32P-labeled mouse ASC-1 cDNA probe overnight at 68°C in a buffer containing 6x SSC, 5x Denhardt, 0.5% SDS, and 100 µg/ml of denatured salmon sperm DNA. The membrane was washed at room temperature in 2x SSC and 0.1 % SDS, followed by 0.2x SSC and 0.1% SDS at 68°C for 90 min as a final stringency, and exposed on Kodak x-ray film at -70°C.
Yeast Two-Hybrid Assay
Plasmids encoding LexA fusions and B42 fusions were cotransformed into Saccaromyces cerevisiae EGY48 containing the lacZ reporter plasmid, SH/1834 [33]. The transformants grown on a plate of selective medium were then incubated in the same liquid medium, but also containing 2% galactose, at 30°C for 3 h. An equal amount of cells were harvested, resuspended in a buffer (60 mM Na2HPO4, 40 mM NaH2PO4, 10 mM KCl, 1 mM MgSO4, and 50 mM 2-mercaptoethanol, pH 7.0), and lysed with 0.1% of SDS and 10% chloroform at 30°C for 15 min. The liquid ß-galactosidase assays were carried out as described previously [35].
GST Pull-Down Assay
Escherichia coli BL21 cells transformed with pGEX-4T1 only and pGEX expressing different regions of ASC-1 or AR were grown at 37°C, and the synthesis of GST fusion proteins was induced for 3 h by addition of 0.1 mM isopropylthio-ß-D-galactoside as a final concentration. The GST fusion proteins were isolated with glutathione-Sepharose-4B beads (Pharmacia, Biotech AB, Uppsala, Sweden), washed twice with PBS, and then incubated with 35S-labeled methionine proteins produced by in vitro translation using the TNT-coupled transcription-translation system (Promega) under the conditions recommended by the manufacturer. Specifically bound proteins were eluted from the beads with 40 mM reduced glutathione in 50 mM Tris (pH 8.0) and analyzed by 10% SDS-PAGE and autoradiography.
Transient Transfection Assays
CV-1, HeLa, and 15P-1 cells were maintained in Dulbecco modified Eagle medium (Life Technologies) in the presence of 10% fetal bovine serum. Twenty-four hours before transfection, cells were plated in 24-well plates (3 x 104 cells/well) and transfected with the indicated amount of expression plasmids, the reporter plasmid pARE2-TATA-Luc, and control lacZ expression plasmid pCMVß using Superfect reagent (Qiagen, Hilden, Germany) according to the manufacturer's instructions. Total amounts of expression vectors were kept constant by adding appropriate amounts of pcDNA3. Twenty-four hours after transfection, the medium was replaced with fresh medium containing 10% charcoal-stripped serum and either 10 nM testosterone or vehicle (ethanol). Cells were harvested 24 h after hormone treatment, and luciferase and ß-galactosidase activities were assayed as described previously [36]. The levels of luciferase activity were normalized to the lacZ expression.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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To examine the function of ASC-1 in the testis, we investigated its expression pattern in the mouse testis. Northern blot analysis revealed that ASC-1 mRNA is expressed in multiple forms in adult mouse testis, with testis-specific stronger bands of approximately 2.4 and 2.6 kilobases (kb) and a weaker, 3.4-kb mRNA that was also detected at a relatively low level in all mouse tissues examined (Fig. 1A). In the mouse testis, the expression of testis-specific ASC-1 mRNAs was developmentally regulated, whereas the expression of the ubiquitously expressed form was relatively constant during testis development (Fig. 1B). The testis-specific ASC-1 mRNAs were undetectable at 14 days, were present at a low level at 24 days, and were increased dramatically in intensity from 33 to 56 days. It is intriguing that the onset of their expression coincided with puberty.
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Cloning of a Mouse Testis-Specific Variant of ASC-1 mRNA
To identify the testis-specific transcripts, we screened a mouse testis cDNA library and cloned another form of ASC-1 cDNA (2629 bp). Sequence analysis revealed that the mouse testis-specific cDNA has exactly the same coding region with the previously reported mouse ASC-1 cDNA (1797 bp, cloned from a liver cDNA library, GenBank accession no. AF197574) but with different nucleotide sequences in the 5'-untranslated region (5'-UTR). To confirm that the cDNA clone is a testis-specific form of ASC-1, RT-PCR was carried out with three different primer sets (Fig. 2B). The results showed that with primer set 1 (primers 1–4) and 2 (primers 2–4), the expected PCR products were produced with RNA from only testis, not from liver and kidney (Fig. 2B, upper and middle), whereas with primer set 3 (primers 3 and 4), which spans the ORF of ASC-1, the expected PCR product was produced with RNA from all tissues (Fig. 2B, bottom). These RT-PCR analyses demonstrated that the testis-specific ASC-1 cDNA is not an artifact generated during the construction of the cDNA library and that mouse testis expresses bona fide testis-specific transcripts of ASC-1. For more definitive evidence, Northern blot analyses were also carried out with three probes spanning different regions of the testis-specific ASC-1 cDNA. The result showed that the probes (nucleotides 1–195 and 628–825) spanning the 5'-UTR detect only testis-specific transcripts, whereas the probe (nucleotides 1040–1957, corresponding to nucleotides 216–1133 of the mouse liver ASC-1 cDNA) spanning the ORF detects all ASC-1 transcripts (Fig. 2C). These results also suggest that all ASC-1 mRNAs likely share the same coding region and are alternative splicing products. This postulation is further supported by several lines of experimental results. First, Southern blot analysis showed that the mouse has a single copy of the ASC-1 gene (Fig. 2D), indicating transcription of different forms of ASC-1 mRNA from the same gene. Second, RT-PCR of total testis RNA with 11 ASC-1 primer sets (combinations from six sense and three antisense primers located in different exons of the coding region; unpublished data) gave only a single expected size of PCR product (data not shown). Finally, with four different probes covering the whole ORF region in the mouse ASC-1 cDNA, the same results as those of Northern blot analyses were obtained, detecting all the ASC-1 mRNAs as detailed in Figure 1 (data not shown).
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ASC-1 Is Highly Expressed in Leydig Cells of the Testis
To identify the testicular cells expressing ASC-1 mRNAs, in situ hybridization analysis was performed with cryostat sections of the mouse adult testis using antisense mouse ASC-1 cRNA probe (nucleotides 391–707, corresponding to testis-specific ASC-1 nucleotides 1214–1780). Although some signals were observed in the cells at the periphery of the seminiferous tubules, peritubular cells, and, perhaps, spermatogonia, ASC-1 mRNAs were strongly detected within the interstitial compartment, especially in the Leydig cells (Fig. 3, C and E), which express AR. Negative control with sense probe showed only background level of signal (Fig. 3D). The ASC-1 signals were hardly detected in the testis of 14-day-old mice (data not shown). Along with the relative abundance of testis-specific ASC-1 transcripts in the adult testis (Fig. 1), this result suggests that the strong ASC-1 signals in interstitial cells represent at least testis-specific ASC-1 transcripts.
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The expression of ASC-1 in testicular somatic cells was further supported by RT-PCR with total RNA from adult testis with hypogonadism (hpg) that consists mostly of somatic cells because of the arrest of spermatogenesis by the diplotene stage. The expected 1.75-kb PCR product was produced from hpg testis as well as the controls, wild-type (wt) testis and liver (Fig. 3F). By Southern blot analysis of the same gel with mouse ASC-1 cDNA probe, the 1.75-kb PCR product was confirmed to be amplified from ASC-1 mRNAs (Fig. 3F). These results suggest that the message for ASC-1 is expressed in the hpg testis, probably in the somatic cells. However, whether the hpg testis expresses the ubiquitous and/or testis-specific forms of ASC-1 mRNA is unclear from these experiments.
ASC-1 Interaction with AR in Yeast
Because ASC-1 mRNAs were increased around the onset of spermatogenesis and highly expressed in the interstitial cells, which reflects AR expression, the interaction of ASC-1 with AR was investigated. Yeast liquid ß-galactosidase assay was performed using AR fused to LexA DNA-binding domain and ASC-1 cloned in-frame to B42 activation domain. As shown in Figure 4, LexA-AR fusion protein itself showed weak androgen-dependent autonomous transactivation activity, whereas B42-ASC-1 alone showed negligible activity. The presence of both partners, however, induced strong activation of the ß-galactosidase reporter. This interaction of ASC-1 with AR was androgen (testosterone and dihydrotestosterone)-dependent (Fig. 4 and results not shown). Further analyses of the ligand specificity in yeast with estradiol, progesterone, dexamethasone, and retinoic acid revealed that none of these hormones induced a detectable AR interaction with ASC-1 at the concentration of 10 nM (data not shown), indicating the stringent androgen dependence of the AR interaction with ASC-1.
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To determine which region of the ASC-1 is able to interact with AR, yeast liquid ß-galactosidase assay was performed using B42 fusion proteins of ASC-1 fragments: full-length ASC-1 (residues 1–581), the N-terminal (ASC-1A, residues 1–124), the middle portion containing the zinc-finger domain (ASC-1B, residues 125–280), and the C-terminal part (ASC-1C, residues 266–581) (Fig. 5A). The results showed that testosterone at 100 nM promotes the interaction of AR with ASC-1B as much as with the full-length ASC-1 (Fig. 5B), indicating that ASC-1B containing the zinc-finger domain is sufficient to interact with AR.
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To investigate which individual domain of AR is responsible for its interaction with ASC-1, we coexpressed LexA fusion proteins of AR deletion mutants (Fig. 5C) together with B42-ASC-1 in the yeast liquid ß-galactosidase assay (Fig. 5D). The AR-hLBD was able to interact with ASC-1 in a ligand-dependent manner as strongly as the full-length AR, whereas it did not interact with B42 only. In contrast to the full-length AR, however, AR-hLBD interaction with ASC-1 showed more stringent androgen dependence, producing undetectable ß-galactosidase activity in the absence of testosterone, which may be caused by the lack of the AF1 domain of AR. Coexpression of LexA-AR-AF1DBDh with B42-ASC-1 strongly increased ß-galactosidase activity irrespective of the presence of testosterone. This likely results from the constitutive transactivation activity of the AF1 domain of AR in the absence of the LBD [37, 38], because coexpression of LexA-AR-AF1DBDh with B42 empty vector also showed strong ß-galactosidase activity in the absence and presence of testosterone. Thus, it can hardly be judged from this experiment whether AR-AF1DBDh interacts with ASC-1. Taken together, the results suggest that the hLBD of AR is the major contributor to the androgen-dependent interaction with ASC-1.
Direct Interaction of ASC-1 with AR In Vitro
Based on the results that ASC-1 interacts with AR in yeast cells, physical interactions between ASC-1 and AR and the region of each protein responsible for their interaction were assessed by GST pull-down experiments. [35S]methionine-labeled AR produced by in vitro translation was allowed to bind the GST fusion proteins of the full-length ASC-1, ASC-1A, ASC-1B, and ASC-1C (Fig. 6A). Whereas AR could interact with the full-length ASC-1, a stronger interaction was observed with ASC-1B containing a zinc-finger motif. Given the results that ASC-1 interacts with the hLBD of AR in yeast and that ASC-1 generally binds to the hinge domain of nuclear receptors, involvement of the AR-hinge region in the direct interaction with ASC-1 was tested using 35S-labeled methionine ASC-1 and GST fusion protein of AR-hLBD or AR-LBD (Fig. 6B). A specific retention of ASC-1 protein was observed for the samples with GST-AR-hLBD, but little retention was observed with GST-AR-LBD. The binding between AR and ASC-1 was not significantly affected by androgen in the GST pull-down experiments. Similar observations have been reported for other nuclear-receptor coregulators [39–43]. These may be caused by improper protein folding of the GST fusions or an as-yet-unknown factor in cells that prevents the interaction of ASC-1 and AR in the absence of androgen. The results from the GST pull-down experiments are consistent with those from the yeast two-hybrid experiments, and they suggest that the ASC-1 region containing the zinc-finger motif and the hinge of AR mainly contribute to the interaction between AR and ASC-1.
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ASC-1 Activates AR Transactivation
Because ASC-1 has been shown to interact with AR (Figs. 4–6), we next attempted to determine whether ASC-1 could influence androgen-induced AR transactivation. The AR and ASC-1 expression plasmids, along with a minimal reporter gene construct regulated by two AREs in front of E1b TATA sequence (pARE2-TATA-Luc [16]), were transiently transfected into CV-1 cells, and the effects on luciferase activity were measured. As shown in Figure 7A, AR activated the expression of the reporter gene by approximately 7-fold in the presence of testosterone. Coexpressed ASC-1 further enhanced this androgen-dependent AR transactivation up to approximately 2.8-fold, whereas ASC-1 only had no effect on the expression of the reporter gene. Similar results were obtained with HeLa cells (data not shown).
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To estimate the modulation capacity of ASC-1 on AR transactivation in comparison with other AR coregulators, coactivators (ARA70 and p300) [23, 25], a corepressor (Smad3) [44], and ASC-1 were cotransfected with AR into 15P-1 Sertoli cells, and their effects on the reporter expression were compared. As shown in Figure 7B, ASC-1 is able to enhance the transactivation of AR to an extent comparable with those of other coactivators.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The ASC-1 was highly expressed in Leydig cells in the interstitial compartment of mouse adult testis (Fig. 3E), which are the same cells that express AR [6, 7]. Considering the relative abundance of the testis forms of ASC-1 message in the adult testis (Fig. 1), the strong intensity of the signals in the in situ hybridization analysis suggests that the Leydig cells express at least testis-specific ASC-1 mRNAs, for which the onset of expression coincides with puberty (Fig. 1B). Coincidence of the expression of AR and ASC-1 in testicular somatic cells and their increased expression during sexual maturation implicate ASC-1 as a coregulator for AR in vivo.
Expression of testis-specific ASC-1 mRNAs was not detected in Leydig cell lines, MA10 and TM3 (results not shown). Along with their expression from puberty, this suggests that expression of testis-specific ASC-1 mRNAs may require developing germ cells and is possibly regulated by communication among testicular cells (germ cells, Sertoli cells, myoid cells, and Leydig cells) involving paracrine and autocrine factors. Indeed, there have been reports that expression of some genes, such as serotonin receptor, sertolin, testicular haploid expressed gene, neural cell adhesion molecule, and glycosaminoglycan, are regulated by such testicular cell-cell communications [45–49].
The interaction between ASC-1 and AR is readily detectable in a yeast two-hybrid system and GST pull-down assay (Figs. 5D and 6B). Like TR and, probably, RXR, which interact with ASC-1 through their hinge domain [32], AR also interacts with ASC-1 through its hinge region. The ASC-1 has a putative zinc-finger motif that provides binding sites for nuclear receptors and coactivators [32]. The ASC-1B, containing the zinc-finger domain (123 amino acids) and an extra 43 amino acids, also appears to be sufficient for binding to AR, as demonstrated by the yeast two-hybrid assay and the GST pull-down analysis (Figs. 5B and 6A). These results raise the possibility that ASC-1 may function in a similar mode for different nuclear receptors, functioning in conjugation with other coregulators.
In a spermatogenic cycle, different stages of spermatogenesis have different sensitivity to androgen [50–52]. This could be achieved by regulating the expression of AR [7] and/or by controlling AR transactivation in a constant state of ligand activation. During the last several years, many studies have shown AR coregulators that are expressed in a ubiquitous or testis-specific manner and that function in an AR-specific or -nonspecific way. They may act in concert to control the fine-tuning of AR activity under different cellular conditions in the testis. The ASC-1, which is coexpressed and associates with AR in testicular somatic cells, may also have such a role in spermatogenesis.
In conclusion, our data demonstrated that ASC-1, associated with AR in a ligand-dependent manner, modulates the AR-dependent transactivation. Its concomitant expression with AR in the testis enhances the possibility that ASC-1 functions as an AR coregulator in vivo. Further studies of ASC-1, for example, with null mutants of testis-specific forms by the knockout mouse approach may provide strong insight regarding the physiological function(s) of ASC-1 in the testis.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence. FAX: 82 62 530 0500; klee{at}chonnam.ac.kr
3 The first two authors contributed equally to this work
Received: 1 April 2002.
First decision: 26 April 2002.
Accepted: 20 June 2002.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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