Androgen Specificity of a Response Unit Upstream of the Human Secretory Component Gene Is Mediated by Differential Receptor Binding to an Essential Androgen Response Element

Guy Verrijdt, Erik Schoenmakers, Philippe Alen, Annemie Haelens, Ben Peeters, Wilfried Rombauts and Frank Claessens

Division of Biochemistry Faculty of Medicine Campus Gasthuisberg University of Leuven B-3000 Leuven, Belgium


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The expression of secretory component (SC), the epithelial receptor for poly-immunoglobulins, is regulated in a highly tissue-specific manner. In several tissues, e.g. lacrimal gland and prostate, SC synthesis is enhanced by androgens at the transcriptional level. In this study, we describe the presence of an androgen response unit, located 3.3 kb upstream of the sc transcription initiation site and containing several 5'-TGTTCT-3'-like motifs. Although each of these elements is implicated in the enhancer function, one element, the ARE1.2 motif, is found to be the main interaction site for the androgen receptor as demonstrated in in vitro binding assays as well as in transient transfection assays. A high-affinity binding site for nuclear factor I, adjacent to this ARE, is also involved in the correct functioning of the sc upstream enhancer. The ARE1.2 motif consists of an imperfect direct repeat of two core binding elements with a three-nucleotide spacer and therefore constitutes a nonconventional ARE. We demonstrate that this element displays selectivity for the androgen receptor as opposed to glucocorticoid receptor both in in vitro binding assays and in transfection experiments. Mutational analysis suggests that the direct nature of the half-site repeat is responsible for this selectivity. We have thus determined a complex and androgen-specific response unit in the far upstream region of the human SC gene, which we believe to be involved in its androgen responsiveness in epithelial cells of different organs such as prostate and lacrimal gland. We were also able to demonstrate that the primary sequence of a single nonconventional ARE motif within the enhancer is responsible for its androgen specificity.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Secretory component (SC), also known as polymeric immunoglobulin receptor, is a key molecule in the immune protection of epithelial tissues. Upon synthesis, the protein is targeted to the basolateral membrane of the epithelial cell (1), where it selectively binds dimeric IgA and pentameric IgM, released in the submucosal tissue by B lymphocytes. Upon binding, the complex is internalized by endocytosis and rapidly transported to the apical pole of the cell. The SC-IgA(M) complex is incorporated in the cell membrane and is subsequently proteolytically cleaved, releasing secretory IgA or IgM (S-IgA, S-IgM) in the external environment. Secretory Ig, therefore, consists of the extracellular globular domain of SC covalently bound to the Ig polymer (2).

SC is exclusively expressed in the epithelial cells of many different tissues [e.g. skin, lung, intestine, reproductive tract (3)]; its promoter is therefore highly specific for epithelial cells. The level of expression can be regulated by a wide variety of factors depending on the cell type of interest: the effect of the cytokines interferon-{gamma}, tumor necrosis factor-{alpha}, and interleukin-4 on sc expression in intestinal epithelial cells has been extensively studied (4, 5, 6, 7). Steroid hormones have been described to influence sc expression in epithelial tissues of the reproductive tract (8, 9, 10), the mammary gland (11), liver (12), and the lacrimal gland (13). In prostate epithelial cells, androgens enhance expression and secretion of SC (8). In the acinar epithelial cells of the lacrimal gland, androgen-stimulated sc expression is mediated, at least partly, by a rise in the sc mRNA content of the cell (14, 15). In primary cultures of lacrimal acinar cells, stimulation of sc expression by androgens is inhibited by actinomycin D as well as antiandrogens, clearly indicating a direct regulation of transcription (16).

Androgen stimulation of gene expression is mediated by androgen receptor (AR) binding to motifs resembling the 5'-TGTTCT-3' consensus binding sequence located within enhancer elements or promoters of androgen responsive genes. Together with the progesterone and mineralocorticoid receptor, the androgen and glucocorticoid receptors form the steroid receptor (SR) subfamily of nuclear receptors having similar DNA-binding domains (DBDs), hence having the same consensus recognition sequence (17). Imperfect palindromic repeats of this 5'-TGTTCT-3' motif in which the half-sites are separated by a three-nucleotide spacer are high-affinity binding sites for the members of this nuclear receptor subfamily (18). The mechanisms by which specificity of steroid hormone action through these response elements is regulated still remain, for the most part, unrevealed. Recent reports, however, discuss the possibility of specific recognition by the AR of sequences that are not recognized by the glucocorticoid receptor (GR) (19, 20).

In our earlier work, we cloned the 5'-region of the human sc gene (EMBL-Genbank accession numbers X95880 and X98765) and determined the major site of transcription initiation in prostate epithelial cells (21). Transcriptional activity of the proximal sc promoter was studied in the human HeLa and HepG2 cell lines. Brandtzaeg and co-workers (22) recently demonstrated the involvement of an E-box (from nucleotide (nt) -74 to -62) and an inverted repeat sequence (from nt -64 to -47) in the basal transcriptional activity of the proximal sc promoter. Piskurich et al. (4) postulated that IFN-{gamma} stimulation of sc transcription in human HT-29 colon carcinoma cells is mediated by an interferon stimulatory response element (ISRE) in the first exon of the gene. Work in our laboratory demonstrates activation of the ISRE by IRF-2, as well as the presence of a steroid hormone-regulatory element in the first exon of the SC gene (23).

In this report, we describe and analyze a genomic region, located 3.3 kb upstream of the transcription initiation site, that confers androgen responsiveness to the sc promoter as well as to a heterologous SV40 promoter. Furthermore, we demonstrate that this enhancer element shows a strong androgen selectivity in conferring steroid responsiveness to the homologous proximal promoter.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Delineation of a Functional Steroid Response Element Upstream of the Human SC Gene
In transient transfection experiments in T-47D cells, a reporter construct containing the sc genomic fragment from nt -3479 to nt +99 relative to the sc transcription initiation site (pSC3479Luc) is androgen responsive (induction factor: 3.5 ± 0.9 SEM), whereas a promoter fragment starting at nt -2507 (pSC2507Luc) is not (Fig. 1AGo). We have therefore inserted the region from nt -3479 to -2442 in front of either the homologous 537-bp sc proximal promoter (pSC537Luc) or the heterologous SV40 early promoter in the pGL3 promoter vector (pSV40Luc). Transcriptional activity of these constructs (pI-IVSC537Luc and pI-IVSV40Luc) is indeed responsive to androgens (the induction factors are 5.2 ± 0.9 and 11.8 ± 2.2 SEM, respectively (Fig. 1BGo)). Progressive deletion analysis of this fragment showed that the presence of a region between nt -3319 and -3141 (fragment II) is essential for androgen response in both promoter contexts. In these experiments, the activity of the androgen response unit (ARU) seems largely dependent on its environment, since induction factors mediated by the different fragments range from 5.2 (pI-IVSC537Luc) to 58 (pIISV40Luc).



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Figure 1. Delineation of the Androgen Response Unit in the Human sc Promoter Region

A, Transient transfection experiments in T-47D cells were performed with the reporter constructs indicated on the left. Induction factors upon stimulation with R1881 (1 nM) were calculated by dividing the luciferase values of the androgen-stimulated samples by the average luciferase value of the same sample, in triplicate, that was not stimulated. Horizontal bars represent the average induction factor of each reporter construct. As a positive control for androgen stimulation, a luciferase reporter vector driven by the MMTV-LTR was included in each experiment. B, A 1038-bp fragment (from nt -3479 to -2442) and sequential deletions of it from the 5'- to the 3'-end, were inserted in the pSC537Luc reporter vector (21 ). The same fragments, including sequential deletions of the 1038-bp fragment from the 3'- to the 5'-end, as well as the isolated fragments I to IV, were inserted in the pGL3 promoter vector. Androgen responsiveness of the resulting plasmids were evaluated in transient transfection experiments in T-47D cells. Luciferase reporter constructs are named as follows: p/identification of the upstream fragment inserted in front of the minimal promoter (see Materials and Methods)/the promoter (SV40 or the 537 bp sc proximal promoter)/Luc. Horizontal bars represent the average induction factor ± SEM, calculated as in panel A. For each promoter context, no significant differences were seen between the luciferase values of the nonstimulated samples. C, The core enhancer fragment (fragment II) was inserted in front of sc promoter fragments of different lengths driving the luciferase reporter gene. Induction factors are calculated as in panel A. Black bars represent the induction factors of reporter constructs containing only the sc promoter of the indicated length without fragment II. Open bars represent the induction factors of the reporter constructs containing the same sc promoter fragment of the indicated length, having fragment II inserted immediately in front of it. No difference in basal promoter activity was seen between sc promoter fragments of different lengths nor did the presence of fragment II have any effect on the basal activity of the different proximal promoters. D, Reporter constructs driven by the SV40 promoter and containing the sc upstream enhancer fragments as indicated at the left side of the figure are investigated in transient transfection experiments in T-47D cells. In pAR.NFSV40Luc, two copies of the AR.NF oligonucleotide are inserted in the pGL3 promoter vector and are oriented as depicted. Induction factors and SEM values are calculated as in panel A.

 
In the next step, we have inserted fragment II in front of sc promoter fragments of different lengths driving the luciferase reporter gene (Fig. 1CGo). sc Promoter fragments ranging from 3479 bp (pSC3479) to 1949 bp (pIISC1949Luc) show comparable responses to androgen stimulation. The 537-bp sc promoter, however, is remarkably inert, showing no significant androgen-stimulated transcription (pIISC537Luc). Androgen responsiveness is, however, restored in the 86-bp sc promoter construct (pIISC86Luc).

We have further delineated the minimal enhancer fragment by deletion analysis. Deletion of the 62 nt 5'-part of fragment II in the pIISV40Luc construct abolishes the androgen response in transient transfection assays in T-47D cells (Fig. 1DGo, p3'IISV40Luc), whereas deletion of the 113-bp downstream part of fragment II attenuates, but does not destroy, the androgen response (Fig. 1DGo, p5'IISV40Luc). Two copies of the 45-bp AR.NF oligonucleotide [nt -3319 to -3275], when inserted in front of the SV40 promoter, give rise to a strong androgen response of transcription (pAR.NFSV40Luc), indicating that this fragment contains the elements sufficient for conferring androgen responsiveness to a promoter.

Interaction of the AR- and GR-DBD with the sc Upstream Enhancer
In the sc upstream enhancer, four motifs resembling the 5'-TGTTCT-3' core SR consensus recognition sequence (called cores 1, 2, 3, and 4) are found (Fig. 2Go). Band shift assays were performed with DNA probes containing these core motifs: ARE1.2 (cores 1 and 2), ARE2.3 (cores 2 and 3), ARE3.0 (core 3), and NFsc (core 4). As a positive control, a probe containing the C3(1) ARE motif (24, 25) was used.



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Figure 2. Sequence of the 47-bp 5'-Part of the sc Upstream ARU Fragment II

The sequence from nt -3319 to -3273 is shown. The arrows indicate the positions and orientations of the four putative core SR binding motifs and are numbered accordingly. The monomer CTF/NF-I consensus site is boxed. Rectangles below the sequence represent the ARE1.2, 2.3, 3.0, NFsc, and AR.NF oligonucleotides that were used in band shift experiments. The nucleotides immediately below the sequence represent the mutations as they were introduced in the different motifs (lower strand) to destroy binding of the AR or CTF/NF-I. In the bottom line, below the ARE1.2 motif, the entire sequence of the ARE1.2 motif containing the T-to-A mutation at position 4 is depicted.

 
We demonstrate a specific binding of the AR-DBD to the ARE1.2 probe (Fig. 3Go). Furthermore, the AR-DBD binds to the ARE1.2 probe preferentially as a dimer, since the DNA/protein complex is localized at the same position in the gel as the DNA/AR-DBD complex in the lane containing the C3(1) ARE (lane 14). It has been shown previously that in the same experimental conditions a dimer of the AR- and GR-DBD binds to the C3(1) ARE (26). In contrast with exclusively dimeric AR-DBD binding to the ARE1.2 motif, only a low-affinity interaction of monomeric AR-DBD was seen with the ARE 2.3 and NFsc probes. Binding of dimeric AR-DBD was also observed with the ARE3.0 probe. In this case, however, a monomer band was always equally pronounced at the AR-DBD concentrations that were used.



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Figure 3. Band Shift Assays with Recombinant AR- and GR-DBD

Each oligonucleotide was incubated with 27 pmol of rat AR-DBD (lanes 2, 5, 8, 11, and 14) or 28 pmol of rat GR-DBD (lanes 3, 6, 9, 12, and 15) before submission to gel electrophoresis. The sequences and positions of the oligonucleotides are presented in Fig. 2Go. As a positive control, the C3(1 ) ARE (Refs. 24 and 25; lanes 13–15) was incubated with the same amounts of protein. The positions in the gel of the probes retarded by dimeric DBDs are indicated by arrowheads (open, GR-DBD, filled, AR-DBD). The arrows indicate the position of monomeric AR- (filled) and GR- (open) DBD.

 
The GR-DBD shows only a weak interaction with any of the tested probes (Fig. 3Go). Furthermore, the GR-DBD binds to these motifs only as a monomer, since the protein-DNA complexes migrate faster than does the AR-DBD bound to ARE1.2, whereas, if it were a dimer, it should migrate more slowly (cf. the C3(1)ARE control, compare lanes 14 and 15).

We have also performed comparative band shift assays with increasing amounts of both receptor DBDs using the ARE1.2, ARE2.3, ARE3.0, NFsc, AR.NF (which contains the cores 1, 2, 3, and 4) and, as a positive control, the C3(1) ARE probes. This revealed that the affinity of the AR-DBD for ARE1.2 or AR.NF is higher than for the ARE2.3, ARE3.0, and NFsc oligonucleotides. In this experiment, approximate values of the apparent dissociation constants (KS) for the AR-DBD interaction with the different probes were calculated: 40 ± 3 nM for the C3(1) ARE, 550 ± 30 nM for ARE1.2, and 600 ± 60 nM for the AR.NF probes. AR-DBD binding characteristics to ARE1.2 and AR.NF are identical. The KS value for binding of the AR-DBD to the ARE2.3, ARE3.0, and NFsc probes could not be determined with the amounts of AR-DBD used. Parallel experiments using the GR-DBD show very low affinity for any of the tested sc upstream enhancer sequences as compared with the C3(1) ARE. Again, due to the low affinities of the GR-DBD for these elements, the respective KS values could not be calculated with the amounts of GR-DBD used. A KS value of 77 ± 8 nM was calculated for interaction of the GR-DBD with the C3(1) ARE.

A NF-I Binding Site Flanks the sc ARE
To identify interactions of other transcription factors with the sc upstream enhancer, we have performed in vitro DNaseI footprinting experiments on fragment II with rat liver nuclear extracts as a source of ubiquitous transcription factors. A 24-bp region (nt -3295 to -3272) is protected from DNaseI digestion (Fig. 4AGo). In this window, two hypersensitive bands (the A at nt -3277 and the G at nt -3278) are present. Protection of the sequence from digestion is abolished by addition of an excess of cold competitor oligonucleotide containing the NF-I recognition sequence of the adenovirus origin of replication (27, lane 10) or a competitor oligonucleotide containing the footprinted sequence itself (NFsc, lane 6). Sequences both downstream and upstream of the two hypersensitive bands are affected in the same way by the competition; therefore, a single protein is probably responsible for the protection. High-affinity binding sites for other transcription factors [among others HNF-5 (28), PEA-3 (29), AP2 (30), AR/GR (24, 25), AP1 (31), and C/EBP (32)], were not able to compete for binding to the protected region.



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Figure 4. Identification of a NF-I Binding Site in the sc Upstream Enhancer

A, The 62-bp 5'-part of fragment II was used in DNase I footprinting experiments using rat liver nuclear extracts. Lanes 1 and 2 are the Maxam-Gilbert G and AG sequencing reactions. Bands in the digestion pattern depict the lower strand. In lane 3 the DNA was subjected to DNase I digestion in the absence of protein. Lane 4 shows the footprint pattern when DNA is digested after addition of 25 µg of rat liver nuclear extract. Lanes 5–11 show the footprint patterns in the presence of an approximate 1000-fold excess of cold competitor oligonucleotides containing binding sites for transcription factors [lane 5: HNF-5 (28 ), lane 7: AR/GR (24 25 ), lane 8 C/EBP (32 ), lane 9: PEA-3 (29 ), lane 10: NF-I (=NFAd (27 )), lane 11: AP1 (31 )]. In lane 6, the NFsc oligonucleotide containing the footprinted sequence is added. The sequence of the NF-I core recognition site is depicted at the left. B, In competition bandshift assays, the NFsc oligonucleotide (see Fig. 2Go) was incubated with approximately 12 µg of T-47D nuclear extract with or without an approximate 400-fold excess of the NFscmut (lane 3, cf. Figs 2Go and 5Go), NFAd (lane 4), or C3(1 )ARE (lane 5) oligonucleotide. C, The NFsc (lanes 1–4) and, as a positive control, the NFAd (lanes 5–8) oligonucleotides were incubated with T-47D nuclear extract in the presence of 1 µl of preimmune serum (PIS, undiluted in lane 3 and diluted 1:20 in lane 7) or 1 µl of serum containing anti-{alpha}-CTF/NF-I antibody (undiluted in lane 4 and diluted 1:20 in lane 8). The arrows indicate the positions of the supershifted bands.

 
As shown in Fig. 4BGo, band shift assays using nuclear extracts from T-47D cells show a high-affinity and specific binding of a protein to the NFsc oligonucleotide. Binding of this protein is fully competed for by addition of a 400-fold excess of cold competitor oligonucleotide containing the NF-I binding sequence of the adenovirus origin of replication (NFAd). This sequence consists essentially of a partial palindromic repeat of the 5'-TTGGC-3' NF-I core binding site separated by five nucleotides. The NFAdmut oligonucleotide, in which one of the two copies of the NF-I binding motif is mutated to 5'-TTTTA-3', is only moderately effective in competition experiments, since a more than 400-fold excess of this competitor oligonucleotide does not fully compete for binding of the factor (data not shown). Addition of a 400-fold excess of a nonspecific competitor does not influence the binding of the protein (Fig. 4BGo). Binding of CTF/NF-I to NFsc was confirmed by supershift experiments using a polyclonal antibody against the {alpha}-subunit of CTF/NF-I proteins (Fig. 4CGo). Inclusion of 1 µl of undiluted rabbit immune serum in the incubation mixture shifts the retarded band, whereas the same amount of undiluted preimmune serum does not. The same results were obtained when the NFAd motif was used as a radiolabeled probe.

Mutational Analysis of Putative Regulatory Elements in Fragment II
To asses the implication of each of the putative AR and NF-I binding sites, we have introduced point mutations destroying the binding of these proteins (see also Fig. 2Go) in them in the context of pIISV40Luc and investigated their effects on the androgen response (Fig. 5Go). Mutation of the core 1 sequence most dramatically decreases androgen stimulation of promoter activity (>90% reduction of induction), while mutations of cores 2, 3, or 4 have a less pronounced effect. Mutation of the NF-I binding site in fragment II (5'-TTGTGCAC-3' instead of 5'-TTTGGCAC-3', see also Fig. 2Go) results in a 70% decrease of androgen induction (pIImutNFSV40Luc). The effect of this mutation on NF-I binding was checked in band shift assays: a 400-fold excess of cold oligonucleotide containing the mutated sequence did not compete for NF-I binding to the wild-type NFsc oligonucleotide (Fig. 4BGo). Combined mutations of core 1 and the NF-I site (pIImut1+NFSV40Luc) or core 1 and core 2 combined with the NF-I site (pIImut1+2+NFLuc) completely abolish the androgen response.



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Figure 5. Mutational Analysis of Putative Functional Elements in Fragment II

The reporter constructs as depicted on the left of the figure were tested for androgen responsiveness in transient transfection experiments in T-47D cells. The point mutations are depicted in Fig. 2Go. The induction factors (calculated as in Fig. 1AGo) of each of the mutant constructs are represented as percentages of wild-type induction. In the schematic representation of the constructs, the mutated binding sites are omitted.

 
Functional Androgen Specificity of the sc Enhancer
Since in band shift assays, the GR-DBD does not bind the ARE1.2 motif, as opposed to strong dimeric AR-DBD binding to this element, we have tested the hypothesis that the sc upstream enhancer might function as an androgen-specific response unit. Therefore, we have transiently transfected T-47D cells with the pIISC86Luc and pSC3479 reporter vectors, together with an expression vector for the human AR or GR, and stimulated the cells for 24 h with increasing concentrations of androgens (from 10-11 to 10-7 M R1881) or glucocorticoids (from 10-10 to 10-6 M dexamethasone). As a positive control, we included in each experiment the pMMTVLuc reporter vector and stimulated these samples with 1 nM R1881 or dexamethasone. We indeed find that in T-47D cells the 86- bp sc promoter construct containing fragment II is strongly stimulated by androgens (up to 35 times with 10-7 M R1881) but is absolutely unresponsive to glucocorticoids in any of the concentrations that were used in the experiment (Fig. 6AGo). The pSC3479Luc reporter construct is also stimulated by androgens (up to 5-fold induction with 10-8 M R1881) and not by glucocorticoids. The pMMTVLuc reporter vector was induced 28- and 38-fold upon stimulation with 1 nM R1881 and dexamethasone, respectively.



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Figure 6. Evaluation of the Androgen Specificity of the sc Upstream Enhancer

A, T-47D cells were transiently transfected with the pIISC86Luc, pSC3479Luc, and pMMTVLuc reporter constructs and cotransfected with expression vectors for the human AR or GR. Cells were stimulated with increasing concentrations of either R1881 (lane 1, 0.01 nM; lane 2, 0.1 nM; lane 3, 1 nM; lane 4, 10 nM; lane 5, 100 nM, white bars) or dexamethasone (lane 1, 0.1 nM; lane 2, 1 nM; lane 3, 10 nM; lane 4, 100 nM; lane 5, 1 µM, black bars). Induction factors of the MMTV-LTR upon stimulation with 1 nM R1881 and dexamethasone are depicted in the third panel. Experiments were performed in duplicate at least twice independently. Induction factors and SEM values were calculated as in Fig. 1Go. B, The same experiment was performed in COS-7 cells. Cotransfections are now performed with AR and GR expression plasmids driven by the SV40 promoter to ensure equal and high levels of expression.

 
It is, however, known from literature that in transient transfections the mouse mammary tumor virus-long terminal repeat (MMTV-LTR) shows a 2- to 3- fold higher responsiveness to glucocorticoids as compared with androgens. Therefore, to exclude possible tissue-specific effects and to have a better control over the expression of the cotransfected AR or GR, we repeated the same experiments in COS-7 cells. AR and GR expression plasmids that were used in these experiments were both driven by the same SV40 promoter, ensuring a high and equal level of expression of both proteins. Binding assays using [H3]mibolerone and [H3]triamcinolone acetonide showed normal hormone binding characteristics and confirmed a high level of expression of both receptors (data not shown). The absolute induction factors of the sc promoter constructs are somewhat lower in COS-7 compared with T-47D cells. Nevertheless, the androgen specificity of their responses is clear over a broad range of hormone concentrations. The pIISC86Luc, however, seems somewhat responsive to dexamethasone. Dexamethasone induction of this reporter construct is, however, always at least 5-fold less compared with stimulation with the same amount of R1881. Stimulation of the pMMTVLuc construct in COS-7 cells with 1 nM R1881 resulted in an approximate 6-fold lower induction as compared with stimulation with 1 nM dexamethasone (a 28- and 183-fold induction, respectively).

Taken together, these functional data corroborate our in vitro finding that ARE1.2 is the predominant interaction site for the AR-DBD within the sc upstream ARU and that the same motif shows a strong preference for the AR compared with the GR.

The ARE1.2 Plays a Crucial Role in the Androgen Specificity of the sc Upstream Enhancer
It has been postulated previously (Refs. 19, 20 ; see also Discussion) that differential binding of the AR to direct repeats, rather than the classical inverted repeats, might account for androgen-specific transactivation . We have therefore increased the palindromic nature of the ARE1.2 motif by replacing the T at position 4 by an A (5'-GGCACTttcAGTTCT-3') and investigated its effect on AR- and GR-DBD binding (Fig. 7AGo). Whereas the GR-DBD is excluded from binding to the wild-type motif, it can specifically interact with the mutant motif. Furthermore, the GR-DBD binds exclusively as a dimer to this element, since no monomer band can be detected at any concentration of the GR-DBD that was used. The affinity of the AR-DBD for the mutated motif (KS = 486 nM) is slightly increased when compared with the wild-type element (KS = 580 nM, Fig. 7AGo).



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Figure 7. Mutational Analysis in Vitro and in Vivo of ARE1.2

A, Band shift assays compare binding of AR- and GR-DBD to the wild-type and mutated ARE1.2 motif. The wild-type ARE1.2 (ARE1.2 wt, left) and mutated ARE1.2 (ARE1.2 mut, right) are incubated with increasing amounts of AR-DBD: (in picomoles) lane 2, 0.7; lane 3, 1.7; lane 4, 3.4; lane 5, 7; lane 6, 10; lane 7, 14; lane 8, 27. In the bottom figure, the same probes are incubated with the same amounts of GR-DBD. B, The same mutation was introduced in the ARE1.2 motif in the context of pIISC86Luc. The wild-type or mutant pIISC86Luc was transfected in T-47D cells, cotransfected with expression plasmids for the human AR or GR, as appropriate. The cells were stimulated with either 1 nM R1881 (white bars) or 10 nM of dexamethasone (black bars). The horizontal bars represent the absolute induction factors of both reporter constructs, calculated as in Fig. 1Go. The sequences of the wild-type (top) and mutated (bottom) ARE1.2 motif are depicted on the left, the mutant nucleotide is indicated by an arrow.

 
The qualitative change in GR-DBD binding characteristics to the ARE1.2 motif due to the introduction of this point mutation is reflected by a clear change in the functionality of the sc upstream enhancer caused by the introducton of the same mutation in the ARE1.2 element in its original context within the ARU. Indeed, in transient transfection experiments in T-47D cells, the wild-type pIISC86 is stimulated a normal 12-fold upon stimulation with 1 nM R1881 and is insensitive to glucocorticoid stimulation, whereas the reporter construct carrying the mutated ARE1.2 motif is stimulated an approximate 10-fold upon stimulation with dexamethasone (Fig. 7BGo). The androgen responsiveness of the mutated construct is an approximate 3-fold higher compared with the wild-type construct, indicating that a relatively small increase in the affinity of the receptor’s DBD for the element in vitro can cause a marked increase in its functionality.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Delineation and Characterization of a Functional Androgen Response Unit 3.3 kb Upstream of the Human sc Promoter
The expression of SC, an essential factor in the proper functioning of our secretory immune system, is known to be under the control of androgens in prostate and lacrimal gland tissue (8, 13). In the lacrimal gland, this androgen responsiveness of SC production is correlated with a rise in sc mRNA content of the cell (14). In our previous work we have reported the cloning of the promoter region of the human SC gene as well as the identification of its transcription initiation site in human prostate epithelial cells and a delineation of the human sc minimal promoter (21). In this report, we search for the cis-acting elements that might be responsible for the observed androgen response of sc promoter activity. Since in T-47D cells the luciferase reporter construct driven by the 3479-bp sc promoter (pSC3479Luc) is clearly responsive to androgens, whereas a shorter 2507-bp sc promoter fragment is not, the enhancer element must reside in the sc upstream region from -2507 to -3479. This fragment is indeed found to confer androgen responsiveness to the heterologous SV40 promoter (Fig. 1BGo). By investigating the effects of progressive deletions of this region cloned in front of the SV40 promoter, we could delineate a 178-bp region (here called fragment II) from nt -3141 to -3319 that is not only essential for the mediation of androgen responsiveness of transcription, but also shows a very high stimulatory capacity (~60-fold stimulation of SV40 promoter activity; see Fig. 1BGo). The presence of regions flanking fragment II generally seems to have an inhibitory effect on its function, although clear functional regions surrounding the core fragment II could not be identified. Fragment I, however, located upstream of fragment II, seems to have a strong and reproducible inhibitory effect since its deletion causes a 2- to 5- fold increase in androgen responsiveness irrespective of the promoter context (compare in Fig. 1BGo pI-IVSC537Luc and pII-IVSC537Luc with pI-IVSV40Luc and pII-IVSV40Luc) or the presence of fragments III and/or IV (compare in the same figure pI-IVSV40Luc, pI-IIISV40Luc, and pI-IISV40Luc with pII-IVSV40Luc, pII-IIISV40Luc, and pIISV40Luc). Whether this inhibitory effect is due to actual transcription factor binding to cis-acting elements within fragment I, or is merely the consequence of a general change in the structural environment of the core enhancer fragment, remains to be elucidated. We attribute the ambiguous effects of the deletions of the fragments located downstream of fragment II to positional effects. It has been shown previously that the mere distance between a promoter and an isolated ARE or GRE can be a determining factor for the level of induction that can be conferred to the promoter by the enhancer (33, 34).

In the context of the homologous sc promoter, changing the distance between the core enhancer fragment II (from 3141 to 1949 nt from the transcriptional start point) and the proximal promoter has no severe effect on the stimulatory capacity of the ARU (Fig. 1CGo). The 537-bp sc proximal promoter, however, is remarkably insensitive to stimulation when the enhancer is inserted immediately upstream: promoter activity of the pIISC537Luc fragment is not stimulated to a significantly higher level than the pSC537Luc construct. The shorter 86-bp sc proximal promoter is, however, highly sensitive for androgen stimulation mediated by fragment II.

The 62-bp upstream part of fragment II was found to be necessary and sufficient for the mediation of androgen responsiveness of proximal promoter activity (Fig. 1DGo). Although the downstream 113-bp subfragment in itself is silent, its deletion causes an approximate 4-fold drop in androgen responsiveness. Again, whether the stimulatory effect of this fragment is due to protein binding to cis-acting elements within this region or is merely the consequence of environmental changes or positional effects, remains to be determined.

Of the 62-bp upstream part of fragment II, a 45-bp region contains sufficient elements to confer androgen responsiveness to a promoter (Fig. 1CGo, pAR.NFSV40Luc). This fragment contains four SR core binding elements as well as a consensus CTF/NF-I monomer binding motif (Fig. 2Go). The implication of each of these elements in the androgen responsiveness mediated by the enhancer was demonstrated by mutational analysis in transient transfection assays (Fig. 5Go). Strikingly, three of the SR monomer binding elements form a direct repeat in which each element is separated by a three-nucleotide spacer. The forth motif is located nine nucleotides downstream of the third. The CTF/NF-I binding element resides two nucleotides downstream of the third SR monomer binding motif and overlaps with the fourth. CTF/NF-I is known to be implicated in the functioning of several steroid response units described to date (35, 36, 37, 38). It recognizes a partial palindromic sequence (5'-TTGGCN5(T/G)CCA-3') although high-affinity binding has also been demonstrated for the single 5'-TTGGC-3' motif (39). In the sc upstream enhancer, only one half of the palindrome is present. The NF-I footprint in the sc upstream enhancer shows two hypersensitive bands downstream of the central motif (a G at position 18 and an A at position 19 in the footprint; Fig. 4AGo) similar to the NF-I footprint in the ARU in the first intron of the C3(1) gene of the rat prostatic binding protein (40). In the MMTV-LTR, the NF-I site is located at the border of a precisely positioned nucleosome and is not occupied by NF-I in the noninduced state (37, 41, 42). SR binding to the MMTV-LTR nucleosomal DNA in vivo is proposed to alter the nucleosome structure in such a way that NF-I can bind to its cognate sequence and activate transcription of the MMTV-LTR promoter (41). For the MMTV-LTR, it has been demonstrated that the orientation of the ARE/GRE elements on the nucleosomal surface is of great importance in the functionality of the element (42, 43).

All four of the SR core binding elements (each of them being the downstream binding element in the repeated motifs ARE1.2, ARE2.3, ARE3.0, and ARE4.0) are bound poorly by the GR-DBD in in vitro binding assays (Fig. 3Go). The GR-DBD binds to oligonucleotides containing these elements exclusively as a monomer, even at high concentrations (<=3 µM) of protein (data not shown). The affinities of the AR-DBD for the same elements are comparable to those of the GR-DBD except for the ARE1.2 motif containing cores 1 and 2 (Fig. 3Go). The affinity of the AR-DBD for this element is significantly higher compared with the other motifs. Furthermore, the AR-DBD binds to this element exclusively as a dimer even at lower concentrations of the protein (see also Fig. 7AGo). Since the AR-DBD shows very poor binding to the core 2 motif within the ARE2.3 motif, high-affinity and exclusively dimeric binding of the AR-DBD to the ARE1.2 motif strongly suggests a high degree of cooperativity in binding of the AR-DBD monomers. The overall affinity of the AR-DBD for this element is, however, still considerably lower when compared with the C3(1) ARE that was used as a positive control in the experiment. The discrepancy between AR- and GR-DBD binding to ARE1.2 is, however, clear and consistent.

ARE3.0 shows some degree of AR specificity in that, at higher concentrations of the AR-DBD, a dimer band appears whereas this is not the case with the GR-DBD. The presence of a monomeric band in the band shift assays, however, indicates that the dimer binding does not involve cooperativity between binding of the DBDs. Furthermore, the same KS values were calculated for AR-DBD binding to the AR.NF oligonucleotide (which contains cores 1, 2, 3, and 4) and the ARE1.2 motif (600 ± 60 nM and 550 ± 30 nM, respectively). Therefore, no cooperativity seems to exist between the ARE2.3, 3.0, or 4.0 elements and the ARE1.2 motif in the binding of the AR-DBD. In conclusion, the ARE1.2 is the strongest ARE within the sc upstream enhancer, although functional analysis of point mutations in the other elements clearly indicates their involvement in the functionality of the enhancer.

The sc Upstream Enhancer Is Androgen Specific
A good correlation exists between the mode of AR-DBD binding to a motif in our in vitro binding assays and its implication in the functionality of the sc upstream enhancer. Mutation of core 1, the downstream half-site within ARE1.2, the only motif that is specifically recognized by dimeric AR-DBD, has by far the most dramatic effect on the functioning of the enhancer (Fig. 5Go). Furthermore, the fact that the ARE1.2 element is not bound by the GR-DBD is correlated with the fact that the sc upstream enhancer does not confer glucocorticoid responsiveness to the homologous proximal sc promoter in transient transfection assays (Fig. 6Go).

The ARE1.2 motif (5'-GGCTCTttcAGTTCT-3') shows a striking resemblance to the PB-ARE-2 (5'-GGTTCTtggAGTACT-3'), another motif proposed to be specifically recognized by the AR (44). Both AREs can be considered direct repeats of the monomer binding elements with a three-nucleotide spacer, raising the possibility that the mechanism of AR specificity of binding to these motifs might be that the AR is able to bind a direct repeat, whereas the GR is not. Within the nuclear receptor core binding sites, the nucleotides at positions 2 and 5 (a guanine and a cytosine, respectively) are known to be essential for high-affinity binding of any member of the nuclear receptor superfamily (45, 46). The residues at positions 3 and 4 are known to be discriminative for binding of members of the two subfamilies of the nuclear receptors. High-affinity binding of a member of the SR family requires a thymidine at position 3; an adenine at this position turns the element into a binding element for a member of the RAR/RXR subfamily of nuclear receptors (45). Since one striking similarity between the sc ARE1.2 and the PB-ARE-2 is the thymidine residue at position 4 in the left half-site, it can be argued that this nucleotide is responsible for specific AR-DBD binding as opposed to GR-DBD. Position 4 is equivalent to position 3 of a half-site in the other orientation, which is therefore an A in the case of the sc ARE1.2 and the PB-ARE-2. We have provided further evidence confirming the validity of the aforementioned hypothesis by replacing the thymidine at position 4 of the ARE1.2 element with an adenine, increasing the palindromic nature of the repeat. Indeed, this mutation dramatically increases the affinity of the GR-DBD for the mutated element, whereas the affinity of the AR-DBD for the motif is hardly affected (Fig. 7AGo). Not only does the GR-DBD now bind to the element, it does so exclusively as a dimer, indicative for a highly cooperative binding of the GR-DBD to both half sites. Furthermore, the introduction of the same point mutation in ARE1.2 in the context of the sc upstream enhancer now allows the enhancer to confer glucocorticoid responsiveness to the sc proximal promoter in transient transfection assays (Fig. 7BGo). The androgen specificity of the sc upstream enhancer is therefore largely diminished essentially by converting the ARE1.2 element from an imperfect direct repeat into a partially palindromic repeat. We believe that these findings are a further confirmation of our hypothesis that transactivation by the AR can be mediated by AR binding to a direct repeat of its monomer binding motifs whereas the GR is not able to do so. Further investigation will be required, however, to establish whether or not this assumption will prove to be generally valid.

In conclusion, we have identified and functionally analyzed a complex and androgen-specific enhancer in the far upstream region of the human SC gene, which we believe is a likely candidate to be the key regulatory element in the steroid control of human sc expression. We have also demonstrated that the sc upstream ARU is androgen specific in vitro and in functional assays. From these findings, we believe that GR exclusion from binding to direct repeats of SR monomer binding elements, is an important mechanism that imposes androgen specificity on enhancer responsiveness.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
General Techniques
Restriction and modifying enzymes used in this study were obtained from Pharmacia Biotech (Uppsala, Sweden), Promega Corp. (Madison, WI), Boehringer Mannheim (Mannheim, Germany), or Life Technologies, Inc. (Gaithersburg, MD). Sequencing reactions were performed using the Pharmacia Autoread Sequencing Kit as described by Chen and Seeburg (47). The reaction products were separated on a polyacrylamide gel in the ALF sequencer, and data were analyzed using the ALF Manager software (Pharmacia Biotech). PCR reactions were performed on a Progene thermocycler (Techne, Cambridge, UK) using Taq DNA polymerase (Life Technologies, Inc.) or Takara Taq DNA polymerase (Takara Shuzo Co. Ltd., Shiga, Japan). The pGEM-T and pGEM-15Zf(-) cloning vectors and the pGL3 luciferase reporter vectors were purchased from Promega Corp.

Synthetic Oligonucleotides
Oligonucleotides used in this study were synthesized on a Biosearch Cyclone DNA synthesizer (Milligen Corp., Bedford, MA). Next to the T7 universal primer, the following primers were used in the generation, by PCR amplification, of sc upstream fragments (sc genomic sequences are in capitals):PCR1fwd (located from nt -3319 to -3296): 5'-actcgagctcTCCTAGAACTGAAAGAGCCTTTGG-3'; PCR1rev: 5'-catatgaattcCCAAAGGCTCTTTCAGTTCTAGGA-3'; PCR2fwd (from nt -3164 to -3141): 5'-actcgagctcGCTGAGTCCAGAGTCAGGAAAGTC-3'; PCR2rev: 5'-catatgaattcGACTTTCCTGACTCTGGACTCAGC-3'; PCR3fwd (nt -3006 to -2985): 5'-actcgagctcGGGCAATGGACTCTCTTGGCCT-3'; PCR3rev: 5'-catatgaattcAGGCCAAGAGAGTCCATTGCCC-3'; PCR4rev (nt -2467 to -2442): 5'-gagatgaattcAAGAAATAAGTTGTGTCCAGTTGTCC-3'. Figure 2Go depicts the AR.NF, ARE1.2, ARE2.3, ARE3.0, and NFsc oligonucleotides used in footprinting and/or band shift experiments. The upper strand oligonucleotides all have a 5'-CTAGC-3' extension at their 5'-ends as well as an additional A at their 3'-ends. The lower-strand oligonucleotides all have a 5'-GATCT-3' extension at their 5'-ends and an additional G at their 3'-ends. This generates NheI and BglII sticky ends at the 5'- and 3'-ends of the double- stranded oligonucleotides, respectively. The ARE1.2 mut and NFscmut oligonucleotides are identical to ARE1.2 and Nfsc, respectively, except for the mutation as depicted in Fig. 2Go. Next to these, the following oligonucleotides were used for competition or as radiolabeled probes in bandshift and or footprinting experiments: NFAd (27): 5'-ATTTTGGCTACAAGCCAATATGAT-3' and 5'-ATCATATTGGCTTGTAGCCAAAAT-3'; NFAdmut: 5'-ATTTTGGCTACAATAAAATATGAT-3' and 5'-ATCATATTTTATTGTAGCCAAAAT-3'. C3(1) ARE (24, 25): 5'-aagcttACATAGTACGTGATGTTCTCAAGg-3' and 5'-tcgacCTTGAGAACATCACGTACTATGTa-3'. The sequences of the PCR primers used for the introduction of point mutations in the putative core SR binding motifs in the sc upstream enhancer destroying AR or GR binding are identical to the wild-type sequence except for the G-to-T mutation at position 2 in the core motif. They all start at nt -3319 and all have a 5'-GGGGGA-3' extension at their 5'-end creating a BamHI restriction site in the amplified fragment. The oligonucleotides carrying the mutated cores 1, 2, 1+2, and the sc ARE1.2 mut oligonucleotide have their 3'-ends at nt -3290. The oligonucleotide for the introduction of the mutation in core 3 has its 3'-end at nt -3281; PCR oligonucleotides mutated in core 4 and the NF-I binding motif both have their 3' ends at nt -3272.

Luciferase Reporter Constructs
The reporter construct pSC3479Luc (Fig. 1AGo) was made by insertion of a NsiI fragment from a pGEM-15 construct containing a 4.1-kb XbaI fragment of the human SC gene (from nt -3479 to + 601) cloned downstream of the T7 RNA polymerase promoter, in the NsiI-digested 1257pGL construct described previously (21). The pSC2507Luc construct was made by digesting pSC3479Luc with SpeI and NheI, followed by self-ligation. The pSC1949Luc construct was made by digesting p3479Luc with StuI and XbaI followed by a fill-in of the overhanging ends and intramolecular ligation. Different sc upstream genomic fragments were generated by PCRs on the pGEM-15 construct containing the 4.1-kb XbaI fragment. PCR products originating from the primer combinations T7/PCR1rev (fragment I), T7/PCR3rev; T7/PCR4rev; PCR1fwd/PCR4rev, PCR2fwd/PCR4rev, and PCR3fwd/PCR4rev (fragment IV) were cloned in the pGEM-T cloning vector and inserted as SacI fragments from these plasmids in the correct orientation in the pGL3 promoter vector or in the pSC537Luc vector (as described in Ref. 21), as appropriate. PCR products from the primer combinations T7/PCR2rev; PCR1fwd/PCR2rev (fragment II); PCR1fwd/PCR3rev and PCR2fwd/PCR3rev (fragment III) were inserted into the pGEM-15 vector as EcoRI/SacI fragments. The T7/PCR2rev PCR product was cloned from the pGEM-15 construct as a XbaI fragment in the correct orientation in the NheI-digested pGL3 promoter vector. The PCR1fwd/PCR2rev, PCR1fwd/PCR3rev, and PCR2fwd/PCR3rev PCR products were inserted as XbaI/SacI fragments into the NheI/SacI-digested pGL3 promoter vector. Fragment II was cloned as a EcoRI/MluI fragment in the EcoRI/MluI-digested pSC2507Luc, pSC1949Luc, and pSC86Luc and as a XbaI/EcoRI fragment from a pGEM-15 subclone in pSC537Luc digested with NheI and EcoRI. The PCR1fwd/PCR2rev fragment in the pGL3 promoter vector was divided in a 62-bp upstream and a 113-bp downstream fragment (Fig. 1DGo) by digesting the plasmid with a combination of PstI and either KpnI (5' deletion) or EcoRI (3' deletion) followed by intramolecular ligation. Two copies of the AR.NF oligonucleotide were cloned in the SmaI site of pGEM-15 and subsequently as a XbaI/SacI fragment in the pGL3 promoter vector.

Point mutations of cores 1, 2, 3, and 4 putative SR binding sites and the NF-I site in the context of pIISV40Luc (as depicted in Fig. 2Go) were made by PCRs using combinations of the respective mutated forward primers (as described above) and the PCR2rev primer. PCR products were inserted as EcoRI/BamHI fragments into the pGEM-15 vector. From these constructs, the PCR fragments were subsequently inserted as XbaI/SacI fragments into the NheI/SacI-digested pGL3 promoter vector or as EcoRI/MluI fragments in the EcoRI/MluI-digested pSC86Luc (21).

Band Shift Assays and DNaseI Footprinting Reactions
Labeling of synthetic oligonucleotides or restriction fragments was performed by a fill-in reaction using the Klenow fragment of DNA polymerase I in the presence of [{alpha}-32P]dATP or [{alpha}-32P]dCTP (Amersham Pharmacia Biotech, Buckinghamshire, UK) to a specific activity of 5–10 x 103 cpm/fmol. Band shift assays were performed essentially according to De Vos et al. (48). Each binding mixture contained 50 ng/µl poly(dI/dC) as a nonspecific competitor. In competition experiments, a 400-fold excess of cold oligonucleotide was included in the binding mixture and incubated on ice for 10 min before the radiolabeled oligonucleotide was added. Samples were loaded on a nondenaturing 5% polyacrylamide/bis-acrylamide (29/1) gel and run at 120 V for 90 min at room temperature. The gel was dried and exposed to X-Omat-AR film (Eastman Kodak Co., Rochester, NY). For quantitative analysis, the dried gel was exposed to a PhosphoImager cassette and radioactivity was measured in a STORM 840 Phosphoimager (Molecular Dynamics, Inc., Sunnyvale, CA) using the Imagequant software provided by the manufacturer. For the calculation of the KS values, the Fig.P program (Fig.P Software Corp., Durham, NC) was used. The relative amount of radioactivity in the retarded bands was plotted as a function of the concentration of AR- or GR-DBD. Amounts of AR- and GR-DBD ranged from 17 nM to 2.7 µM. Binding curves were fitted to a function representing allosteric Hill kinetics.

In the supershift experiments, the antibody was added to the binding mixture and incubated at room temperature for 30 min before the radiolabeled probe was added. The mixture was then incubated an additional 30 min at room temperature.

The DNA fragment used in the footprinting experiments is an EcoRI/MluI restriction fragment from a pGEM-15 construct containing fragment II between the EcoRI and SacI sites. Footprinting reactions were performed essentially according to Lemaigre et al. (49). G and AG chemical cleavage reactions, according to Maxam and Gilbert (50), were performed on the same DNA fragment and were used as reference in each footprinting gel.

Preparation of Nuclear Extracts of the Rat AR- and GR-DBD
Rat liver nuclear extracts were prepared from the livers of 8-week-old male rats essentially as described by Wall et al. (51) and modified by Zhang et al. (52). Protein concentrations were determined by the Bradford method. Nuclear extracts from T-47D cells used in band shift assays were prepared according to Andrews and Faller (53). The DBD of the rat AR [Asp 533 to Asp 637 (54)] and GR [Ala 432 to Asn 533 (55)] were expressed in Escherichia coli as glutathione S-transferase (GST) fusion proteins and purified on a glutathione sepharose column to a concentration of 1 mg/ml of more than 95% pure protein, as was assessed by Coomassie-stained protein gels (56). The GST was removed by thrombin digestion.

Cell Culture and Transfection Experiments
T-47D human mammary gland carcinoma cells and COS-7 monkey kidney cells were obtained from the American Type Culture Collection (Manassas, VA). Cells were maintained in DMEM containing 1000 mg/liter glucose, supplemented with penicillin (100 IU/ml), streptomycin (100 µg/ml), and 10% FCS (Life Technologies, Inc.) at 37 C and 5% CO2. Transient transfections were performed by the calcium phosphate-DNA coprecipitation method mainly according to Claessens et al. (24). The first day, cells were plated in DMEM, supplemented with 5% charcoal-treated FCS, in 24-well tissue culture plates (Nunc, Roskilde, Denmark) at a density of 100,000 cells per well. The second day, cells were transfected with 1 µg of reporter construct per well. After 4 h, the cells were incubated for 1 min in 15% glycerol in 1 x PBS. The third day, medium was replaced with or without addition of the synthetic androgen methyltrienolone (R1881) or the synthetic glucocorticoid dexamethasone (Dex). Cells were incubated with hormone for 24 h. The fourth day, cells were harvested in 100 µl of Passive Lysis Buffer (Promega Corp.) according to the instructions of the manufacturer. Luciferase activity of 10–20 µl of cell lysate was measured in a Microlumat LB 96P luminometer (EG&G Berthold, Bad Wilstadt, Germany). In transfection experiments in T-47D cells, the reporter construct was cotransfected with either the pSV-AR0 human AR expression plasmid [as described by Brinkmann et al. (57)] or the pRSV-GR human GR expression plasmid (pRShGR{alpha}) described by Giguerre et al. (58), as appropriate (100 ng/well). In experiments in COS-7 cells, cotransfections were performed with the pSG5-hAR human AR expression plasmid [as described by Alen et al. (59)] or the pSG8-rGR rat GR expression plasmid (60). AR and GR expression levels in both cell lines were evaluated by a hormone binding assay using [H3]mibolerone (Amersham Pharmacia Biotech) or [H3]triamcinolone acetonide (Dupont New NEN, Boston, MA). Cells were transfected with 100 ng per ml medium of the appropriate SR expression plasmids, 100 ng per ml medium of CMV-ß-galactosidase expression plasmid, and 1.8 µg of carrier DNA. Retention of radioactivity at increasing concentrations of the radiolabeled hormones was compared between conditions with and without the addition of an excess of nonradiolabeled R1881 or dexamethasone. At saturating conditions, displaceable [H3]mibolerone binding of 0.85 (± 0.25) and 5.6 (± 1.8) fmol per µg protein was measured in T-47D and COS-7 cells, respectively. The capacity of displaceable [H3]triamcinolone acetonide binding of transfected T-47D and COS-7 cells at the same conditions were 0.33 (± 0.13) and 2.3 (± 0.17) fmol per µg protein, respectively. Transfection efficiencies were assessed by ß-galactosidase assays on parallel samples.

In the transfection assays using reporter plasmids, a luciferase reporter construct driven by the steroid-sensitive MMTV promoter (pMMTVLuc) was always included as a positive control (average induction: 89 ± 24 SEM). Luciferase values of the samples were normalized according to the protein concentration. Transfection experiments were performed in triplicate and repeated at least three times independently. In the generation of the dose- response curves upon stimulation with R1881 and dexamethasone in T-47D and COS-7 cells (Fig. 6Go), all samples were performed in duplicate and repeated at least twice independently. In the calculation of the SEM values, each independent experiment (in triplicate) is considered as one. In all transfection experiments, the activities of the reporter constructs driven by the different sc promoters constructs did not differ significantly. No influences of the length of the different sc promoters, of the presence of different upstream enhancer fragments, or the cotransfections with either GR or AR expression plasmids on the nonstimulated sc or SV40 promoter activities was seen in either T-47D or COS-7 cells. In T-47D cells, an average luciferase value of 1,600 light units per µg of cell lysate was measured for the nonstimulated MMTV-LTR; for the sc promoter or SV40 promoter-driven reporter constructs, average luciferase values were 14,000 and 9,000 light units per µg of cell lysate, respectively. In COS-7 cells average luciferase values are 6,000 and 23,000 for the nonstimulated MMTV-LTR and sc promoter-driven reporter constructs, respectively.


    ACKNOWLEDGMENTS
 
The anti-{alpha} CTF/NF-I polyclonal antibody was the kind gift of Dr. N. Tanese of the NYU Medical Center (New York, NY). The pSG8-rGR plasmid was a kind gift of Dr. Stunnenberg. The authors are grateful to H. Debruyn and R. Bollen for their excellent technical assistance and to V. Feytons for the expert synthesis of numerous oligonucleotides.


    FOOTNOTES
 
Address requests for reprints to: Dr. Frank Claessens, Division of Biochemistry, Faculty of Medicine, University of Leuven, Campus Gasthuisberg Herestraat 49, B-3000 Leuven, Belgium.

This work was supported by Grant 3.0048.94 of the Geconcerteerde Onderzoeksactie van de Vlaamse Gemeenschap, and by grants from the Inter Universitaire Attractie Pool, the Belgian Cancer Fund, the Fonds voor Geneeskundig en Wetenschappelijk Onderzoek, and the Vlaamse Wetenschappelijke Stichting. G.V. and P.A. were supported by a scholarship from the Vlaams Institut voor de Bevordering van het Wetenschappelijk-Technologisch Onderzoek in de Industrie. F.C. is a senior assistant of the Fonds Voor Wetenschappelijk Onderzoek.

Received for publication February 23, 1999. Revision received May 11, 1999. Accepted for publication June 3, 1999.


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 DISCUSSION
 MATERIALS AND METHODS
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