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Endocrinology, doi:10.1210/en.2003-0987
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Endocrinology Vol. 145, No. 2 781-789
Copyright © 2004 by The Endocrine Society

Nuclear Factor-{kappa}B Activates Transcription of the Androgen Receptor Gene in Sertoli Cells Isolated from Testes of Adult Rats

Liying Zhang, Martin Charron, William W. Wright, Bandana Chatterjee, Chung S. Song, Arun K. Roy and Terry R. Brown

Division of Reproductive Biology (L.Z., M.C., W.W.W., T.R.B.), Department of Biochemistry and Molecular Biology, Johns Hopkins Bloomberg School of Public Health, Baltimore, Maryland 21205; and Department of Molecular Medicine (B.C., C.S.S., A.K.R.), Institute of Biotechnology, University of Texas Health Science Center at San Antonio, San Antonio, Texas 78245

Address all correspondence and requests for reprints to: Terry R. Brown, Ph.D., Department of Biochemistry and Molecular Biology, Johns Hopkins Bloomberg School of Public Health, Room W3606, 615 North Wolfe Street, Baltimore, Maryland 21205. E-mail: tbrown{at}jhsph.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The androgen receptor (AR) in Sertoli cells mediates the actions of testosterone on spermatogenesis. However, the transcription factors responsible for AR gene regulation in Sertoli cells remain unknown. In this study, we determined that nuclear factor-{kappa}B (NF-{kappa}B) regulates transcription of AR in primary cultures of Sertoli cells isolated from testes of adult rats. Electrophoretic mobility shift and antibody supershift assays with nuclear extracts prepared from Sertoli cells identified two binding sites, termed {kappa}B1 at -491/-482 bp and {kappa}B2 at -574/-565 bp, upstream of the transcription start site of the AR gene that bind the NF-{kappa}B subunits, p50 and p65. DNAse I footprint analyses showed that binding of the p50 NF-{kappa}B subunit protected the same regions on the rat AR promoter. Analyses of AR promoter-luciferase reporter gene activity after transfection of primary cultures of Sertoli cells demonstrated that mutation of the {kappa}B2 site or combined mutation of the {kappa}B1 and {kappa}B2 sites reduced activity by 40%. Preferential binding of the transcriptionally active p65/p50 heterodimer to the {kappa}B2 site rather than to the {kappa}B1 site supported these observations. Overexpression of the NF-{kappa}B p65 and p50 subunits in Sertoli cells increased activity from the wild-type AR promoter and the promoter with mutation of the {kappa}B1 site, but not the {kappa}B2 site. Activity was further stimulated by CBP (CREB binding protein), a coactivator of p65 transcriptional activity. Taken together, our data show that NF-{kappa}B is an activator of AR gene transcription in Sertoli cells and may be an important determinant of androgen activity during spermatogenesis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
SPERMATOGENESIS IS DEPENDENT upon a number of endocrine, paracrine, and autocrine regulators, with androgens playing an essential role in the maintenance of spermatogenesis in rats and humans. Studies in the rat have established that maintenance, as well as restoration of spermatogenesis after experimental induction of oligospermia due to hormonal insufficiency, are androgen dependent (1, 2). Sertoli cells that line the seminiferous tubules are considered to be the target cells for androgen action required for maturation of the adjacent germ cells. The effects of androgens, primarily testosterone within the testis, are mediated via the androgen receptor (AR) that functions as a ligand-activated transcription factor to mediate the effects of testosterone on spermatogenesis (3). Expression of a functional AR in Sertoli cells is required for fertility (4, 5). Interestingly, testicular concentrations of testosterone, specifically within the seminiferous tubules, exceed the levels in peripheral blood by 40- to 50-fold (6). These findings suggest that testosterone levels within the testis are not a limiting factor for androgen action under normal physiological conditions. Rather, changes in AR expression, such as the well-documented changes in AR level that occur during the different stages of the spermatogenic cycle in testes of sexually mature rats (7, 8, 9), are likely to represent an important regulatory mechanism by which the effects of testosterone on spermatogenesis can be modulated.

Previous studies have used immature Sertoli cells from prepubertal rats to examine the effects of FSH, testosterone, thyroid hormone, and other factors on the regulation of AR gene expression in Sertoli cells (10, 11, 12, 13, 14). The isolation of purified preparations of Sertoli cells from testes of sexually immature rats is facilitated by the relative absence of germ cells at early developmental stages. However, the relevance of studies based upon Sertoli cells from immature rats are marked by the incomplete process of germ cell maturation that precedes the pubertal increase in androgen biosynthesis. Sertoli cells undergo major structural and functional changes in vivo during sexual maturation (15, 16, 17). These changes are accompanied by the establishment of specific associations between populations of maturing germ cells and the adjacent Sertoli cells that define the 14 morphological stages of the cycle of the seminiferous epithelium in testes of fertile, adult rats (18). Moreover, the level of AR expression in Sertoli cells varies according to the different stages defined within the spermatogenic cycle (7, 8, 9). However, the mechanisms responsible for the transcriptional control of the AR gene in Sertoli cells of the sexually mature, adult rat testis remain unknown. Therefore, the goal of this study was to begin to shed light on the transcription factors involved in regulation of the AR gene in Sertoli cells of the adult rat testis.

Recently, the expression of two members of the nuclear factor-{kappa}B (NF-{kappa}B) transcription factor family, p65 (Rel A) and p50/p105, were described in nuclear extracts of rat Sertoli cells (18A ). The p50 subunit is able to bind to DNA, but it must complex with the p65 subunit that contains an activation domain necessary for formation of the transcriptionally active NF-{kappa}B heterodimer (19). In Sertoli cells from testes of sexually immature rats, NF-{kappa}B activates transcription of the cAMP-response element binding (CREB) protein gene (18, 20) and was recently shown to stimulate activity of the AR promoter (21). By contrast, increased expression of NF-{kappa}B in the liver of aging rats was shown to repress AR gene expression (22). In this study, we investigated the role of NF-{kappa}B in the regulation of AR gene transcription in Sertoli cells from the testes of adult rats.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Primary culture of rat Sertoli cells
Sertoli cells were isolated from seminiferous tubules of sexually mature (60–70 d) Sprague Dawley rats (Charles River Laboratory, Wilmington, MA) as described in detail (23). Tissue culture medium used in these studies was a 1:1 mixture of DMEM-Nutrient Mixture F-12 (DMEM/F-12) containing 15 mM HEPES buffer, L-glutamine, and pyridoxine hydrochloride. Testes were decapsulated and digested with enzyme solutions. Highly purified clumps of Sertoli cells from the dispersed cell mixture were obtained by filtration and by a series of sedimentations at unit gravity. Sertoli cell/germ cell aggregates were cultured on 30-mm diameter filter inserts (Millipore, Bedford, MA) coated with matrigel (Collaborative Research, Waltham, MA) in DMEM/F-12 medium supplemented with hormones and antibiotic (1 µg/ml vitamin E, 0.4 µM retinoic acid, 10 µg/ml insulin, 5 µg/ml transferrin, 1 µM testosterone, 1 ng/ml epidermal growth factor, 40 µg/ml vitamin C, 10 ng/ml FSH, and 10 µg/ml gentamicin). On the following day, the culture medium was removed, and residual germ cells were removed by osmotic shock induced by brief incubation of cells for 2 min in 50 mM Tris-HCl (pH 7.4), as described by Galdieri et al. (24). Fresh DMEM/F-12 medium supplemented with hormones and antibiotic was added to the Sertoli cells. Sertoli cell cultures were judged to be of 95% or greater purity as assessed by microscopic examination. The use of animals for the experiments described in these studies was approved by the Institutional Animal Care and Use Committee of the Johns Hopkins University.

Preparation of nuclear extracts
Sertoli cells were isolated as described above from sexually mature Sprague Dawley rats and cultured overnight on a dried film of matrigel (1:55 dilution). Cells were recovered by the addition of trypsin, and nuclear extracts were prepared in accordance with the methods of Roy et al. (25) and Dignam et al. (26). The extracts were stored as aliquots in a liquid nitrogen freezer. Protein concentration was determined by the method of Bradford using the assay reagents from Bio-Rad (Hercules, CA).

EMSA
32P-radiolabeled DNA probes were generated by labeling of annealed complementary oligonucleotides containing the AR promoter-specific NF-{kappa}B binding sites plus flanking sequences. The nucleotide sequences of the wild-type and mutant oligonucleotides are provided in Table 1Go. The overhangs were filled by a labeling reaction containing 10 mM Tris-HCl (pH 7.5), 5 mM MgCl2, 7.5 mM dithiothreitol, 10 pmol annealed double-stranded oligonucleotide, 7.5 U Klenow DNA polymerase (New England Biolabs, Beverly, MA), 0.02 mM each of deoxy ATP, GTP, and TTP, and 0.33 µM {alpha}-32P deoxy CTP (specific activity = 3000 Ci/mmol). The reaction was incubated at 25 C for 30 min. The unincorporated nucleotides were removed by separation using poly-prep chromatography columns (Bio-Rad) filled with Sephadex G-50 Fine (Amersham Biosciences, Piscataway, NJ). Binding reactions were performed by incubating 32P-labeled {kappa}B probes (20,000 cpm) with Sertoli cell nuclear extracts at room temperature for 20 min in the presence of 0.02 mg/ml polydeoxyinosinic deoxycytidylic acid, 1.6% Ficoll, 10 mM HEPES (pH 7.9), 1 mM EDTA, 0.75 mM dithiothreitol, 5 mM MgCl2, and 50 mM KCl. The protein-DNA complexes were resolved via 5% PAGE under nondenaturing conditions. The gel was then dried and exposed to autoradiographic film.


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  		TABLE 1. The sequences of the NF-{kappa}B oligonucleotides used for EMSA

 
For competition experiments, a 50-fold molar excess of double-stranded unlabeled competitor oligonucleotides containing the wild-type or mutant NF-{kappa}B binding sites were co-incubated with radiolabeled {kappa}B probes and nuclear extracts. For supershift assays, specific antibodies (1 µg) against p65, p50, c-Rel, Rel-B, and p52 (Santa Cruz Biotechnology, Santa Cruz, CA) were preincubated with the Sertoli cell nuclear extracts for 15 min at room temperature before the addition of 20,000 cpm of 32P-labeled {kappa}B probes. To generate the titration curve, the dried gel was exposed to phosphorimager plates. The image was scanned using a Typhoon 9200 Imaging System (Amersham Biosciences), and the intensity of each DNA-protein complex was quantified using the ImageQuant Software that was provided with the instrument.

DNAse I footprinting assay
The radiolabeled DNA probe corresponding to the coding strand was generated by PCR amplification of the AR promoter region using the 32P-labeled sense primer directed at -600/-580 and unlabeled antisense primer directed at -300/-321 positions. The footprinting assay was performed using conditions described previously (27). Briefly, recombinant p50 (Promega, Madison, WI) mixed with DNA binding buffer was incubated with 2 µg polydeoxyinosinic deoxycytidylic acid at room temperature for 5 min. The gel-purified labeled probe (10 fmol and 50,000 cpm) was subsequently added to the reaction mixture and incubated for an additional 30 min. BSA was used in the control experiment to produce the unprotected ladder pattern from DNAse I-digested fragments. The reaction mixture was brought to 2 mM CaCl2 and 10 mM MgCl2, and the DNA was digested with 40 ng DNAse I (Roche Molecular Biochemicals, Basel, Switzerland) for 30 sec on ice. The digested DNA fragments were subjected to organic extraction before analysis on a sequencing gel.

Construction of rat AR gene reporter plasmid and site-directed mutagenesis
The -717/+560 AR promoter plasmid was generated by PCR of the wild-type rat AR promoter (rARp)/5'UTR (-1040/+560)-luciferase pGL2 plasmid template (27), in the presence of an AR promoter specific primer and a vector-specific primer. The DNA fragments were digested with restriction enzymes and purified by gel electrophoresis. DNA fragments were then inserted into the pGL2 basic vector (Promega) and verified by DNA sequencing. Point mutations were introduced into the {kappa}B1 and {kappa}B2 binding sites on the rARp using the Altered Sites II in vitro Mutagenesis kit (Promega) according to the manufacturer’s instructions. The newly created mutant rARp sequences were checked with the Transcription Element Search System (TESS; http://www.cbil.upenn.edu/tess/) to ensure that no new binding sites for other transcription factors were created by mutagenesis. The introduction of mutations was verified by sequencing. Three mutant plasmids were generated with {kappa}B1mut containing GGGA->TTCT mutations, {kappa}B2mut containing GGGA->ATCT mutations, and {kappa}B1/{kappa}B2mut containing the mutations at both sites. For expression plasmids, the p50 and p65 subunits of NF-{kappa}B were expressed from a cytomegalovirus (CMV)-driven promoter (28). The mouse CREB-binding protein (CBP) expression vector was constructed in the pRC/RSV vector (29).

Transient transfections and luciferase assays
Lipofectamine (Invitrogen, Carlsbad, CA) was used for transient transfection of Sertoli cells according to the manufacturer’s instructions. Mature Sertoli cells were plated in 6-well tissue culture plates coated with matrigel at 1.2 x 106 cells per well. The rARp-Luc reporter construct contained -717 to +560 bp of the rat AR gene and the firefly luciferase reporter gene in the pGL2 plasmid. pRL-CMV (Promega), containing the Renilla luciferase reporter gene driven by the CMV promoter, was used as an internal control for transfection efficiency. Mature Sertoli cells were grown for 48 h before the addition of the DNA-Lipofectamine transfection mixture containing rARp-Luc plasmid (2.5 µg) and pRL-CMV (25 ng). After 5 h, the transfection mixture was removed, and cells were incubated for another 16–20 h before harvesting and assay of luciferase activity. In all cases, the total amount of DNA in each transfection was kept constant by the addition of the corresponding empty expression vector. The firefly luciferase and Renilla luciferase activities were assayed using the Dual Luciferase Assay kit (Promega) and a TD-20/20 luminometer (Turner Designs, Sunnyvale, CA) following the manufacturer’s instruction. All assays were conducted in triplicate, and each experiment was repeated three to five times. The data were expressed as the relative luciferase activity based on the ratio of firefly luciferase light units to Renilla luciferase light units for each data point. For the purpose of comparison, the relative luciferase activity of the wild-type promoter was set to a value of 1.0, and the relative luciferase activity for each of the other experimental groups was normalized to that for the wild-type control.

Statistical analyses
All data indicated as significant were analyzed using ANOVA at the 0.05 confidence level.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Two NF-{kappa}B motifs located upstream of the transcription start site of the rat AR gene bind nuclear proteins from Sertoli cells of adult testes
A map of the putative regulatory elements present within an approximately 1-kb genomic DNA fragment upstream of the transcription start site of the rat AR gene (GenBank accession no. L15617) was established using TESS. Three putative NF-{kappa}B cis-acting elements (termed {kappa}B1, {kappa}B2, and {kappa}B3) are present within this region. The nucleotide sequences of these three sites are presented in Table 1Go. EMSA was performed to test whether nuclear proteins prepared from primary cultures of adult rat Sertoli cells could bind to each of the three putative NF-{kappa}B regulatory elements contained within double-stranded DNA fragments formed by annealing synthetic oligonucleotides based upon the AR genomic nucleotide sequence. The {kappa}B1 and {kappa}B2 probes each formed two DNA-protein complexes (designated as complex I and II) when incubated with nuclear protein extracts from Sertoli cells obtained from adult rat testes, whereas the binding of nuclear proteins to the {kappa}B3 site was undetectable (Fig. 1AGo). The formation of complexes I and II at the {kappa}B1 and {kappa}B2 sites differed; the {kappa}B2 probe preferentially formed the slower migrating complex I, whereas the {kappa}B1 probe formed more of the faster migrating complex II (Fig. 1Go). Competition assays were conducted to determine the specificity of protein binding to the {kappa}B1 and {kappa}B2 motifs. Formation of complexes I and II was abolished when the nuclear protein extracts were incubated with 50-fold molar excess of unlabeled wild-type {kappa}B1 or {kappa}B2 probe, but a 50-fold molar excess of the respective unlabeled mutant probes (Table 1Go) had no effect on complex formation (Fig. 1BGo). We did not perform competition assays for the {kappa}B3 motif because protein binding was not detected in the initial EMSA experiment. As expected, when the mutant probes for {kappa}B1 and {kappa}B2 were radiolabeled and incubated with nuclear protein extracts, no binding was detected by EMSA (data not shown). These results show that Sertoli cell nuclear proteins can specifically bind to the {kappa}B1 and {kappa}B2 motifs located upstream of the transcription start site of the AR gene.



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  		FIG. 1. Binding of nuclear proteins extracted from primary cultures of Sertoli cells isolated from testes of adult rats to radiolabeled oligonucleotides containing the putative NF-{kappa}B binding sites within the rat AR gene promoter. A, 32P-labeled oligonucleotides containing the putative NF-{kappa}B binding motifs, {kappa}B1, {kappa}B2, and {kappa}B3, plus flanking sequences were incubated with nuclear protein extracts (20 µg) prepared from cultured Sertoli cells. The putative NF-{kappa}B binding motifs, {kappa}B1, {kappa}B2, and {kappa}B3, and their location relative to the transcription start site of the rat AR gene are shown in Table 1Go. B, Specificity of binding of Sertoli cell nuclear proteins to the {kappa}B1 and {kappa}B2 motifs. Nuclear protein extracts (20 µg) from cultured Sertoli cells were incubated with 32P-labeled NF-{kappa}B probes, {kappa}B1 and {kappa}B2, in the absence (-) or the presence of a 50-fold molar excess of either an unlabeled wild-type NF-{kappa}B probe (wt) or an unlabeled mutant NF-{kappa}B probe (M) as competitor. The nucleotide sequences of the mutant probes are shown in Table 1Go. Protein-DNA complexes were resolved by electrophoresis on 5% PAGE and visualized by autoradiography. The formation of specific protein-DNA complexes I and II are indicated by the arrows. Data are representative of at least three independent experiments.

 
DNAse I footprinting confirms binding of the p50 DNA-binding subunit of NF-{kappa}B to two sites on the rARp
To confirm the binding of the p50 DNA-binding subunit of the NF-{kappa}B family to the rARp, we incubated the -600/-300-bp radiolabeled fragment of the AR promoter with recombinant p50 protein before DNAse I digestion of the DNA (Fig. 2Go). The binding of p50 protein protected two regions at -476/-500 and -560/-583 bp of the rARp from digestion with DNAse I. These regions correspond to the {kappa}B1 (-491/-482) and {kappa}B2 (-574/-565) sites for NF-{kappa}B binding predicted from the TESS database analysis and our previous EMSA experiment. The specificity of p50 binding and nuclease protection was confirmed using BSA as a negative control.



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  		FIG. 2. DNAse I footprinting of the rARp by the p50 subunit of NF-{kappa}B. The vertical lines at the left mark the protected regions within the radiolabeled rat AR probe (-600/-300 bp), and their locations relative to the transcription start site of the rat AR gene are indicated numerically. Lanes 1 and 4, Incubation of the DNA probe with BSA. Lanes 2 and 3, The DNA probe was incubated with 1 µl or 2 µl of purified recombinant p50 (1 gel shift unit/µl; Promega) before DNAse I digestion.

 
The NF-{kappa}B subunits, p65 and p50, bind to the {kappa}B1 and {kappa}B2 motifs
To identify the specific Sertoli cell nuclear proteins that bind to the {kappa}B1 and {kappa}B2 motifs, antibodies directed against the known NF-{kappa}B subunits, p65, p50, c-Rel, RelB, and p52, were used in EMSA supershift assays (Fig. 3Go). Previous studies have reported that NF-{kappa}B p65 and p50 subunits are expressed in rat Sertoli cells (18), but the expression of the other subunits remained unknown. Using Sertoli cell nuclear protein extracts from the testes of sexually mature rats and the oligonucleotide probes for {kappa}B1 and {kappa}B2, the p50 subunit-specific antibody diminished the intensity of both DNA-protein complexes I and II and gave rise to a distinctly larger complex that exhibited a supershift (Fig. 3Go, A and B, lane 2). The p65 subunit-specific antibody diminished the intensity of complex I, but not complex II, for both the {kappa}B1 and {kappa}B2 probes. A larger complex as evidence of a supershift by the p65 subunit-specific antibody was observed with the {kappa}B2 probe (Fig. 3Go, A and B, lane 3). The use of commercial antibodies against c-Rel, RelB, and p52 did not affect the intensity or position of complexes I and II formed with the {kappa}B1 and {kappa}B2 probes, thus suggesting that these NF-{kappa}B subunits are not expressed in rat Sertoli cells or do not bind in appreciable amounts to the selected {kappa}B motifs (Fig. 3Go, A and B, lanes 4–6). However, as a cautionary note, we did not determine positive controls for these antibodies with the specific NF-{kappa}B proteins derived from rat. The preimmune IgG was used as a negative control (Fig. 3Go, A and B, lane 7). These results indicate that complex I contains the p65/p50 heterodimer because it was affected by antisera against both the p65 and p50 subunits, whereas complex II was affected only by the p50 antisera and likely contains the p50/p50 homodimer.



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  		FIG. 3. NF-{kappa}B p50 and p65 subunits in Sertoli cell nuclear extracts bind to the oligonucleotides containing the {kappa}B1 and {kappa}B2 sites within the rARp. Nuclear protein extracts (20 µg) from cultured Sertoli cells isolated from the testes of adult rats were incubated with radiolabeled oligonucleotides containing the {kappa}B1 (A) and {kappa}B2 (B) binding sites plus flanking sequences. Specific antibodies against p50, p65, c-Rel, RelB, and p52 subunits or preimmune serum (IgG; lanes 2–7 in A and B) were preincubated with the Sertoli cell nuclear proteins before the addition of 32P-labeled NF-{kappa}B probes. Protein-DNA complexes were resolved by electrophoresis on 5% PAGE and visualized by autoradiography. The DNA-protein complexes, I and II, are indicated by arrows, and the supershifted slower migrating complexes in the presence of the antibody to the p50 subunit (lane 2 in A and B) and the antibody to the p65 subunit (lane 3 in A and B) are indicated by additional arrows. Data are representative of at least three independent experiments.

 
{kappa}B1 and {kappa}B2 motifs bind NF-{kappa}B proteins with different affinities
Results from EMSA indicated that both p65/p50 heterodimers and p50/p50 homodimers are able to bind to the {kappa}B1 and {kappa}B2 motifs to form complex I and complex II, respectively. However, as seen in Fig. 1Go, the relative binding affinities at each site appeared to differ based upon the autoradiographic intensities associated with the respective DNA-protein complexes. The p65/p50 heterodimers bound preferentially at the {kappa}B2 site, whereas the p50/p50 homodimers bound more avidly to the {kappa}B1 site. To better understand the function of these two NF-{kappa}B binding sites in regulating the AR promoter activity, we tested the relative affinity of the {kappa}B1 and {kappa}B2 sites for NF-{kappa}B proteins. As shown in Fig. 4Go, A and B, the addition of increasing amounts of Sertoli cell nuclear protein resulted in an increase in the autoradiographic intensity of the DNA-protein complexes formed on EMSA. The relative intensity of each DNA-protein complex formed in Fig. 4Go, A and B, was quantified and is shown graphically in Fig. 4CGo. The intensity of complex I formed by the binding of the transcriptionally active p65/p50 heterodimer to the {kappa}B2 site was much greater than the intensity of p65/p50 binding at the {kappa}B1 site. The intensity of complex II formed by the binding of the transcriptionally inactive p50/p50 homodimers for the {kappa}B1 and {kappa}B2 sites was intermediate between the intensities for formation of complex I. Moreover, the formation of complex I was predominant at the {kappa}B2 site, whereas formation of complex II was greater at the {kappa}B1 site. Protein binding at the {kappa}B1 and {kappa}B2 sites reached saturation after the addition of approximately 40 µg of total nuclear protein. Most importantly, the formation of the transcriptionally active p65/p50 complex at the {kappa}B2 site was about 10-fold higher than at the {kappa}B1 site as shown at the plateau of the individual protein titration curves (Fig. 4CGo).



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  		FIG. 4. Sertoli cell nuclear protein binding to the {kappa}B1 and {kappa}B2 sites form complexes I and II with different affinities. 32P-labeled oligonucleotides containing the {kappa}B1 (A) or {kappa}B2 (B) NF-{kappa}B binding motifs plus flanking sequences were incubated with increasing amounts of Sertoli cell nuclear protein (0–80 µg). Protein-DNA complexes were resolved by electrophoresis on 5% PAGE and visualized by autoradiography. The formation of specific protein-DNA complexes I and II are indicated by the arrows. C, Titration curve for the binding of nuclear protein to the {kappa}B1 and {kappa}B2 binding sites. To generate the titration curve, the dried gel was exposed to phosphorimager plates. The images were scanned, and the relative intensity of each DNA-protein complex, I or II, formed at the {kappa}B1 and {kappa}B2 binding site was quantified using ImageQuant Software and graphed as a function of the amount of nuclear protein.

 
The {kappa}B2 motif regulates the rARp activity in Sertoli cells
Primary cultures of Sertoli cells isolated from the testes of adult rats were transiently transfected with various AR promoter-luciferase reporter constructs to determine whether NF-{kappa}B can regulate AR promoter activity. The wild-type construct, rARp (-717/+560)-Luc, containing the putative NF-{kappa}B binding sites was mutated at the {kappa}B1 and/or {kappa}B2 sites. In the construct, {kappa}B1mut, the GGGA sequence in the {kappa}B1 motif was mutated to TTCT. In the {kappa}B2mut construct, the sequence GGGA was mutated to ATCT. In the construct {kappa}B1/{kappa}B2mut, both the {kappa}B1 and {kappa}B2 sites were mutated as described previously. The mutations introduced in the {kappa}B1 and {kappa}B2 motifs were the same as those in the mutant oligonucleotides (Table 1Go) tested in the EMSA and shown to be specific for DNA-protein binding in Fig. 1Go. Therefore, we tested whether the introduction of these mutations into the AR promoter affected its activity. The DNA constructs {kappa}B1mut, {kappa}B2mut, and {kappa}B1/{kappa}B2mut were transfected into primary cultures of Sertoli cells from adult rat testes. As shown in Fig. 5Go, reporter gene activity was reduced by 40% in cells transfected with the construct {kappa}B2mut or {kappa}B1/{kappa}B2mut compared with that of cells transfected with the wild-type AR promoter construct. By contrast, mutation of the {kappa}B1 site ({kappa}B1mut) had no effect. Therefore, the binding of NF-{kappa}B to the {kappa}B2 site was necessary for normal AR promoter activity in Sertoli cells.



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  		FIG. 5. Effect of mutation of the NF-{kappa}B binding sites on activity of the rARp in primary cultures of Sertoli cells prepared from the testes of adult rats. Sertoli cells were transfected with plasmids containing the wild-type (wt) and mutant rARp (-717/+560)-Luc reporter (2.5 µg) and the internal control DNA plasmid pRL-CMV (25 ng). The mutant promoter-reporter constructs ({kappa}B1mut and {kappa}B2mut) were created by substitution of the first four consecutive bases within the 10-bp NF-{kappa}B consensus binding sequence (see Table 1Go). The promoter activity was expressed as the ratio of firefly luciferase reporter to Renilla luciferase internal control. For the purpose of comparison, the relative luciferase activity of the wild-type promoter was set to a value of 1.0, and the relative luciferase activity of the mutant promoters was calculated relative to the wild-type control. Two independent preparations of each plasmid were used in transfection experiments. Data represent the mean ± SE of triplicate assays based upon five independent experiments. *, Different from wild-type control, P < 0.05.

 
NF-{kappa}B stimulated the AR promoter activity in Sertoli cells from adult rats
The role of NF-{kappa}B in the regulation of the rARp was further studied in transient transfection assays in which the p50 and p65 subunits were overexpressed in primary cultures of Sertoli cells from adult rat testes (Fig. 6Go). Overexpression of p65 alone and p65/p50 resulted in a 2.5- to 3-fold induction of promoter activity in Sertoli cells transfected with the wild-type and {kappa}B1mut AR promoter constructs. NF-{kappa}B-mediated induction of the AR promoter was abolished in cells transfected with the {kappa}B2mut and {kappa}B1/{kappa}B2mut constructs. When p50 alone was overexpressed in Sertoli cells, the activities of the wild-type and {kappa}B1mut constructs were reduced by 40%, but there was no effect on the activities of the {kappa}B2 and the {kappa}B1/{kappa}B2mut constructs (Fig. 6Go).



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  		FIG. 6. Effects of p65 and p50 subunit expression on rARp activity in primary cultures of Sertoli cells prepared from the testes of adult rats. Sertoli cells were transfected with rARp (-717/+560)-Luc reporter plasmids (2.5 µg) containing either the wild-type rARp (wt), an rARp with mutant {kappa}B1 motif ({kappa}B1mut), an rARp with mutant {kappa}B2 motif ({kappa}B2mut), or an rARp with mutant {kappa}B1 and {kappa}B2 motifs ({kappa}B1mut/{kappa}B2mut), as shown in Table 1Go. Cells were cotransfected with additional plasmid DNA (2 µg total DNA) consisting of the empty expression vector (pCMV5; 2 µg) or with cDNA expression vectors for the p65 (2 µg) or p50 (2 µg) subunits alone and their combination (1 µg each plasmid). pRL-CMV (25 ng) was included as an internal control. The promoter activity was expressed as the ratio of firefly luciferase reporter to Renilla luciferase internal control. For the purpose of comparison, the relative luciferase activity of the wild-type promoter was set to a value of 1.0, and the relative luciferase activity of the mutant promoters was calculated relative to the wild-type control. Two independent preparations of each plasmid were used in transfection experiments. Data represent the mean ± SE of triplicate assays within each group based upon five independent experiments.

 
CBP enhances NF-{kappa}B-mediated activation of the AR promoter in Sertoli cells from adult rats
NF-{kappa}B-dependent gene expression involves a growing number of transcriptional coactivators that function by facilitating or bridging the sequence-specific activators to the basal transcriptional machinery and altering chromatin structure (30). CBP/p300 interacts with the p65 subunit of NF-{kappa}B to enhance its ability to activate transcription (31, 32). Recent studies by Chaudhary et al. (33) demonstrated that both CBP and p300 proteins are expressed in rat Sertoli cells and CBP/p300 appears to be involved in regulating FSH-mediated activation of the transferrin gene promoter. On the basis of this information, we tested whether CBP was able to further enhance the NF-{kappa}B-mediated activation of the AR promoter in Sertoli cells. As shown in Fig. 7Go, overexpression of p65 alone increased AR promoter activity in cells transfected with the wild-type and {kappa}B1mut constructs but not in cells transfected with the {kappa}B2mut or {kappa}B1/{kappa}B2mut constructs. Overexpression of CBP alone caused a 3-fold induction of AR promoter activity with the wild-type and the mutated AR promoter constructs. When both CBP and p65 were overexpressed, a further increase in AR promoter activity was observed with the wild-type and {kappa}B1mut constructs, but not with the {kappa}B2mut and {kappa}B1/{kappa}B2mut constructs. In comparing the results for fold induction shown in Figs. 6Go and 7Go, it should be noted that different amounts and combinations of the plasmid constructs were used in these experiments to address the effects of p65/p50 (Fig. 6Go) or p65/CBP (Fig. 7Go). Taken together, these data suggest that the {kappa}B2 site is required for NF-{kappa}B-mediated induction of AR promoter activity in Sertoli cells from adult rat testes and that CBP is able to further enhance the activity of NF-{kappa}B through its interaction with p65 at the {kappa}B2 binding site.



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  		FIG. 7. Effects of p65 and CBP on AR promoter activity in primary cultures of Sertoli cells prepared from the testes of adult rats. Sertoli cells were transfected with rARp (-717/+560)-Luc reporter plasmids (2.5 µg) containing either the wild-type rARp (wt), an rARp with mutant {kappa}B1 motif ({kappa}B1mut), an rARp with mutant {kappa}B2 motif ({kappa}B2mut), or an rARp with mutant {kappa}B1 and {kappa}B2 motifs ({kappa}B1mut/{kappa}B2mut) as shown in Table 1Go. Cells were cotransfected with additional plasmid DNA consisting of the empty expression vectors, pRC/RSV (4 µg) and pCMV5 (0.4 µg), or with cDNA expression vectors for p65 (0.4 µg) alone, CBP (4 µg) alone, or their combination. pRL-CMV (25 ng) was included as an internal control. The promoter activity is expressed as the ratio of firefly luciferase reporter to Renilla luciferase internal control. For the purpose of comparison, the relative luciferase activity of the wild-type promoter alone was set to a value of 1.0. The relative luciferase activities for the wild-type or mutant promoters with or without the addition of expression plasmids for p65 and/or CBP were calculated relative to the wild-type promoter as control. Data represent the mean ± SE of triplicate assays within each group based upon five independent experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Testosterone is required for spermatogenesis, and its primary actions are mediated via the AR in Sertoli cells (34). The local concentration of testosterone within the testis is 40–50 times higher than in peripheral blood that affects other androgen target tissues (6). AR localization within Sertoli cell nuclei increases progressively from stage II through VII of the spermatogenic cycle and subsequently declines during stage VIII to become barely detectable in stages IX to XIII (7, 8). These findings establish the paradigm that androgen-dependent gene transcription during spermatogenesis is determined by the level of AR expression in Sertoli cells and is stage specific. Our studies were initiated to identify and characterize the function of nuclear transcription factors that regulate the stage-specific pattern of AR expression in Sertoli cells.

For the current study, three putative NF-{kappa}B binding sites ({kappa}B1, {kappa}B2, and {kappa}B3) were identified within the -667/-482 bp region of the rARp sequence using the TESS database of known transcription factor DNA binding sites. The prototypical, inducible NF-{kappa}B subunits, p65 and p50, are expressed in Sertoli cells at all stages of the spermatogenic cycle, and their nuclear levels are highest during stages XIV to VII (18). NF-{kappa}B was shown to regulate CREB gene expression in immature Sertoli cells (20). NF-{kappa}B increases in Sertoli cells at stages in which round spermatids, that secrete TNF-{alpha}, are present within the seminiferous tubules (35). TNF-{alpha} receptors are expressed by Sertoli cells (35, 36), and this cytokine induces NF-{kappa}B DNA binding activity (18).

NF-{kappa}B proteins function as dimeric transcription factors to control genes regulating a broad range of biological processes (37, 38). In the canonical pathway, NF-{kappa}B proteins are inactive and bound by inhibitory {kappa}B (I{kappa}B-{alpha}, -ß, or -{gamma}) proteins. Proinflammatory cytokines, growth factors, and antigen receptors activate I{kappa} kinase (I{kappa}K) complexes ({alpha}, ß, and NF-{kappa}B essential modulator) to phosphorylate I{kappa}B proteins. Phosphorylation of I{kappa}B leads to its ubiquitination and proteasomal degradation, freeing the NF-{kappa}B protein complexes. Active NF-{kappa}B (e.g. p65/p50) complexes are subsequently activated by phosphorylation and translocated to the nucleus where, either alone or in combination with other transcription factors, they alter target gene expression.

Interestingly, Delfino et al. (21) also identified three putative NF-{kappa}B binding sites within the rARp sequence using the TFSEARCH database. The sites we designated as {kappa}B1 and {kappa}B2 were also predicted by TFSEARCH, but a third {kappa}B site was located at -951/-942. We demonstrated that NF-{kappa}B proteins from nuclei of primary cultures of Sertoli cells isolated from testes of adult rats specifically bind only to the {kappa}B1 and {kappa}B2 motifs within the AR promoter region. Coincidentally, these same two sites, {kappa}B1 and {kappa}B2, preferentially bound NF-{kappa}B in the report by Delfino et al. (21). In general, NF-{kappa}B proteins bind to a DNA motif with the consensus nucleotide sequence, GGGRNNYYCC, as deduced from the naturally occurring NF-{kappa}B binding sites within the regulatory regions of different genes (39). The {kappa}B1 and {kappa}B2 sites were identical to the consensus-binding motif, whereas the consensus third G nucleotide is replaced by C within the {kappa}B3 sequence (Table 1Go). Among a series of gene-specific NF-{kappa}B binding sites surveyed by Hirano et al. (40), the sequence of three consecutive G nucleotides was invariant, and substitution of the second G by C resulted in the loss of NF-{kappa}B binding (41). This may explain why we, as well as Delfino et al. (21) who used extracts from Sertoli cells of immature rats, only detected NF-{kappa}B binding at the {kappa}B1 and {kappa}B2 sites.

Supershift assays with NF-{kappa}B subunit specific antisera identified p65/p50 subunit heterodimers and p50/p50 subunit homodimers in the slower and faster migrating DNA-protein complexes, respectively, bound to {kappa}B1 and {kappa}B2 probes. Whereas the NF-{kappa}B p50 subunit is able to bind to DNA, it lacks a transactivation domain (42). By comparison, the NF-{kappa}B p65 subunit contains a transactivation domain and, thereby, is the transcriptionally active subunit when it dimerizes with p50. Thus, the preferential binding of the transcriptionally active p65/p50 heterodimer to the {kappa}B2 site provides an explanation for why the {kappa}B2 site enhanced AR promoter activity in cultured Sertoli cells. However, p50/p50 homodimers are not inactive but rather are able to repress (43) or activate gene transcription (44, 45), depending upon interaction with accessory nuclear proteins. Zhong et al. (43) demonstrated that p50/p50-histone deacetylase-1 complexes bind to DNA and suppress NF-{kappa}B-dependent gene expression. p50/p50 homodimers can interact with other proteins, such as Bcl-3, which recruits proteins containing histone acetylase activity to induce expression of genes, such as P-selectin (44). In our experiments, overexpression of p50 led to inhibition of rARp activity. This inhibitory effect may be mediated via recruitment of histone deacetylase-1 or by direct competition with the transcriptionally active p65/p50 heterodimer for binding to the NF-{kappa}B regulatory sites within the AR gene promoter (40, 46).

The EMSA data indicated the preferential binding of the transcriptionally active p65/p50 heterodimer at the {kappa}B2 site. The titration curve (Fig. 4CGo) demonstrated that the intensity of complex I, comprised of the p65/p50 heterodimer, was about 10-fold higher for the {kappa}B2 site than for the {kappa}B1 site at saturating amounts of nuclear protein. The binding affinities of the p65/p50 and p50/p50 dimers for the NF-{kappa}B binding sites, as well as the relative levels of p65 and p50 expression in Sertoli cells, may directly influence the formation of these two complexes. Therefore, even though the titration experiment was not quantitative, it provided an estimation of the relative affinity for each DNA-protein complex at the two sites. The higher binding affinity for the transcriptionally active p65/p50 heterodimer with the {kappa}B2 site explained our functional analyses of AR promoter activity. Mutation of the {kappa}B2 site caused a 40% decrease in basal AR promoter activity and the loss of induction when p65 and p50 were overexpressed in Sertoli cells. Several possibilities exist for the differing functions of the {kappa}B1 and {kappa}B2 sites. One is that the nucleotide sequences (underlined) of these two NF-{kappa}B binding sites differ for {kappa}B1 (GGGACTCTCC) and {kappa}B2 (GGGAATTCCC). In vitro studies have shown that specific {kappa}B binding sites exhibit a preference for certain NF-{kappa}B dimers and, in turn, that each dimer binds some sites with higher affinity than others (47, 48, 49). Second, the position and context of the {kappa}B1 and {kappa}B2 sites within the promoter may also affect their function relative to other transcription factors interacting with cis-acting elements located on either side of these {kappa}B sites. It is noteworthy that the same {kappa}B2 binding site that is stimulatory in Sertoli cells in the present study was previously reported to inhibit AR promoter activity in the liver of aging rats (22).

Recent studies have shown that the coactivator CBP and its homolog p300, both of which contain histone acetyltransferase activity domains, interact with and enhance the transcriptional activity of p65 (31, 32). CBP was found to associate with p65 via two interaction sites, one of them depending on a phosphorylated Ser residue at position 276 (50). In our study, CBP further enhanced NF-{kappa}B-mediated stimulation of rARp activity in Sertoli cells, an effect specific to the {kappa}B2 site. Coactivation by CBP was observed when CBP alone was overexpressed in Sertoli cells or when it was coexpressed with p65. The effect of CBP alone suggests its possible interaction with other transcription factors to enhance AR promoter activity.

Our data and the recent report of Delfino et al. (21) support a stimulatory role for NF-{kappa}B in AR gene transcription in Sertoli cells. By contrast, Supakar et al. (22) previously reported that NF-{kappa}B functions as a negative regulator of AR gene transcription in rat liver by binding to the {kappa}B2 site in the promoter region. Therefore, NF-{kappa}B plays opposite roles in the regulation of the same gene in the context of different cell environments (51). This may be due to the relative abundance of p50 and p65 subunits or the differential expression of coactivators and/or corepressors, or their combined effects, in these tissues. The EMSA data presented by Supukar et al. (22) show that the {kappa}B2 site preferentially bound the p50/p50 homodimer present in rat liver nuclear extracts. Based on our observations when p50 was overexpressed in Sertoli cells, binding of the p50/p50 homodimer repressed activity of the AR promoter. Further investigation of the relative expression levels of NF-{kappa}B subunits and other regulatory molecules within the NF-{kappa}B pathway, such as I{kappa}B and I{kappa}K proteins, in Sertoli cells are needed to clarify the actions of NF-{kappa}B and its effects on AR gene transcription. The expression of intratesticular cytokines or growth factors that affect the NF-{kappa}B pathway in Sertoli cells should also be characterized.

In summary, our findings support a role for NF-{kappa}B in the stimulation of AR gene expression in Sertoli cells. The ability of NF-{kappa}B to induce AR gene promoter activity in primary cultures of adult rat Sertoli cells suggests that NF-{kappa}B may be an important factor in the androgen regulation of spermatogenesis. Considering the dramatic changes that occur in AR expression during the various stages of the spermatogenic cycle (7, 8, 9), it will be important to determine whether other transcription factors and coactivators/corepressors in Sertoli cells also contribute to the activation and repression of AR gene transcription.


   Acknowledgments
 
This work is dedicated to the memory of our friend, colleague, and co-author, Dr. Arun K. Roy, whose untimely death occurred while this article was under review. We thank Albert S. Baldwin, Jr. (University of North Carolina, Chapel Hill) for the p65 and p50 expression vectors and Richard H. Goodman (Oregon Health Science University) for the pRC/RSV and mouse CBP expression vectors. Recombinant FSH was provided by Dr. Albert F. Parlow (National Hormone and Peptide Program). DNA oligonucleotides were synthesized in the DNA synthesis core facility supported by the National Institute of Environmental Health Sciences Center Program (P30, ES 03819).


   Footnotes
 
This work was supported by NIH/National Institute of Child Health and Human Development through cooperative agreement U54 HD36209 (to T.R.B.) as part of the Specialized Cooperative Centers Program in Reproduction Research and by R37 AG10486 (to A.K.R. and B.C.).

This work was presented in part at the 83rd Annual Meeting of The Endocrine Society, Denver, CO, June 20–23, 2001, and was submitted as partial fulfillment of the doctoral degree requirements (L.Z.) at Johns Hopkins University, January 2003.

Abbreviations: AR, Androgen receptor; CBP, CREB binding protein; CMV, cytomegalovirus; CREB, cAMP-response element binding protein; I{kappa}B, inhibitory {kappa}B; NF-{kappa}B, nuclear factor-{kappa}B; rARp, rat androgen receptor promoter; TESS, Transcription Element Search System.

Received August 4, 2003.

Accepted for publication October 16, 2003.


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 Results
 Discussion
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Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals