Estrogen-Induced Retinoic Acid Receptor {alpha}1 Gene Expression: Role of Estrogen Receptor-Sp1 Complex

Gulan Sun, Weston Porter and Stephen Safe

Department of Veterinary Physiology and Pharmacology Texas A & M University College Station, Texas 77843-4466


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Retinoic acid receptor {alpha}1 (RAR{alpha}1) gene expression is induced by 17ß-estradiol (E2) in estrogen receptor (ER)-positive breast cancer cells, and the -100 to -49 region of the RAR{alpha}1 gene promoter was previously shown to be required for E2-responsiveness. This region of the RAR{alpha}1 promoter was further analyzed using the following oligonucleotides: -100 to -49 (RAR4); -79 to -56 (RAR3); -79 to -49 (RAR2); -100 to -58 (RAR1); and their derived promoter reporter constructs (pRAR4, pRAR3, pRAR2, and pRAR1). In transient transfection studies in MCF-7 human breast cancer cells, pRAR2 and pRAR1 were E2-responsive; both of the RAR{alpha}1 gene promoter inserts contained two GC-rich sites and bound Sp1 protein in gel mobility shift assays. Using wild-type [32P]RAR2 and oligonucleotides mutated in one or both GC-rich sites, it was shown that ER enhanced Sp1 binding to both sites, but a ternary ER-Sp1-DNA complex was not observed in gel mobility shift assays. In transient transfection assays, each of the GC-rich motifs were sufficient for E2-induced transactivation. In ER-negative MDA-MB-231 cells transiently transfected with pRAR2, E2 responsiveness was observed only in cells cotransfected with wild-type ER or 11C-ER containing a deletion of the DNA-binding domain but not with ER variants that express activation function-1 (AF-1) or AF-2. Using a similar approach, it was shown that the GC-rich sites in RAR1 were also sufficient for ER activation. These results demonstrate that interaction of a transcriptionally active ER/Sp1 complex with GC-rich motifs is required for hormone inducibility of the downstream region of the RAR{alpha}1 gene promoter.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Retinoic acid receptors (RARs) are ligand-activated members of the nuclear receptor superfamily, which includes steroid hormone, vitamin D, retinoid, thyroid hormone, and a large number of orphan receptors (1, 2, 3, 4). This superfamily of receptors plays an important role in the physiology of target organs/cells, particularly with respect to cell differentiation and development (5, 6, 7). The expression and functional activity of nuclear receptors as transcription factors are highly regulated and dependent on multiple factors including ligand structure, dimeric partners, cis-acting genomic binding sites, coactivators, corepressors, and histone modification (acetylation/deacetylation) (1, 2, 8).

The three known subtypes of the RAR ({alpha}, ß, and {gamma}) bind all-trans and 9-cis-retinoic acid (5, 6, 7, 9, 10, 11); ligand-activated RARs are differentially expressed throughout development and exhibit discrete and overlapping functions (5, 6, 7, 12, 13, 14, 15). Retinoids have been extensively used as antineoplastic agents for treatment of epithelial- and mesenchymal-derived tumors (16, 17, 18, 19). For example, retinoids inhibit basal and 17ß-estradiol (E2)-induced proliferation and gene expression in human breast cancer cell lines and carcinogen-induced mammary tumor development and growth in female Sprague-Dawley rats (20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38). RAR{alpha}1 is highly expressed in estrogen receptor (ER)-positive breast cancer cell lines and is required for retinoid-induced inhibition of human breast cancer cell proliferation and E2-induced gene expression. A recent report also showed that retinoids inhibit growth of some ER-negative breast cancer cell lines that expressed RAR{alpha}1 (26, 28).

Several studies have shown that E2 induces RAR{alpha}1 gene expression and reporter gene activity in human breast cancer cells transiently transfected with constructs containing RAR{alpha}1 gene promoter inserts linked to reporter genes (20, 21, 24, 25, 27). Rishi and co-workers (25) identified an estrogen responsive element (ERE) half-site(N)10Sp1 motif [GGTGA(N)10-GGCGGG] at -82 to -62 in the RAR{alpha} gene promoter that was responsible for E2-induced transactivation in breast cancer cells. In contrast, Elgort and co-workers (27) identified two E2-responsive regions in the -491 to +36 sequence of the RAR{alpha} gene promoter using HepG2 cells cotransfected with the ER. The upstream sequence at -491 to -455 bp contained two GGTCA half-sites and a GC-rich downstream region (-79 to -49) which bound Sp1 protein. Neither of these promoter regions directly bound to the ER in gel mobility shift assays.

This study reinvestigates E2-responsiveness of the more proximal region in the RAR{alpha}1 gene promoter. The RAR4 oligonucleotide (-100 to -49) contained both the upstream Sp1(N)10ERE half-site and downstream GC-rich motifs that were previously identified as promoter sequences required for E2-induced transactivation (25, 27). Deletion analysis of the -79 to -49 region of the RAR{alpha}1 gene promoter showed that the GC-rich Sp1 protein-binding sites at -68 to -62 and -59 to -52 were required for E2-responsiveness. Analysis of the upstream sequence (-100 to -58, RAR1) showed that ER did not bind to this region of the promoter, and ER activation was associated with interaction of ER/Sp1 with GC-rich motifs. These results complement recent studies using a consensus Sp1 oligonucleotide that first demonstrated that ER and Sp1 proteins physically interact to form a functional transcription factor complex (39).


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
In MCF-7 cells treated with 1 nM E2 and transiently transfected with pRAR4, which contained a -100 to -49 RAR{alpha}1 gene promoter insert, there was a 3.3-fold increase in chloramphenicol acetyltransferase (CAT) activity compared with cells treated with solvent [dimethylsulfoxide (DMSO)] (Fig. 1Go). Similar results were obtained using pRAR2, which contained the -79 to -49 RAR{alpha}1 gene promoter insert, whereas pRAR3 (-79 to -56 insert) was not E2-responsive. In MCF-7 cells transiently transfected with pRAR1, treatment with E2 also significantly induced (3.4-fold) CAT activity. E2 responsiveness of pRAR2 was compared with constructs containing mutations in the GC-rich sites at -59 to -52 (pRAR2•m1) and -68 to -62 (pRAR2•m2) (Fig. 2Go). One nanomolar E2 did not induce CAT activity in cells transiently transfected with both mutant plasmids (Fig. 2Go); however, 10 nM E2 caused a 3.4- and 5.2-fold induction response using pRAR2•m1 and pRAR2•m2, respectively. Thus, each of the GC-rich sites was sufficient for E2-responsiveness. RAR1 contains GC-rich sites at -68 to -62 and -94 to -88 and an ERE half-site motif at -82 to -78 (25). Previous studies reported that E2-induced transactivation of pRAR1 required an intact ERE half-site (25); however, pRAR1•m1, which is mutated in the ERE half-site, was also E2-responsive (Fig. 2Go), suggesting that only the GC-rich motifs are required for hormone inducibility in MCF-7 cells.



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Figure 1. Effect of E2 on CAT Activity in MCF-7 Cells Transfected with pRAR1, pRAR2, pRAR3, and pRAR4

Cells were transiently cotransfected with 5 µg hER and 10 µg pBL/TATA CAT (vector), pRAR1, pRAR2, pRAR3, and pRAR4 constructs. Cells were treated with DMSO or 1 nM E2. The transient transfection and CAT assays were performed as described in Materials and Methods. E2 induced a significant (P < 0.05) 3.4-, 2.5- and 3.3-fold increase in CAT activity in cells transfected with pRAR1, pRAR2, and pRAR4, respectively. Results are means ± SD for three replicate determinations for each treatment group.

 


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Figure 2. E2 Responsiveness of Wild-Type and Mutant Constructs Containing RAR{alpha} Gene Promoter Inserts

Cells were transiently cotransfected with 5 µg hER and 10 µg pRAR2, pRAR2·m1, pRAR2·m2, and pRAR1·m1 constructs, respectively. The transient transfection and CAT assays were performed as described in Materials and Methods. Cells were treated with DMSO and 1 or 10 nM E2. The results are means ± SD for at least three separate determinations. Significant induction by 1 nM E2 was observed in cells transfected with pRAR2; 10 nM E2 significantly induced CAT activity with all constructs.

 
The results in Fig. 3Go compare the binding of Sp1 protein (0, 5, 10, or 20 ng) to a consensus [32P]Sp1 oligonucleotide with Sp1 binding to [32P]RAR2, [32P]RAR2•m1, and [32P]RAR2•m2. Formation of a retarded band (bound DNA) was observed using all the oligonucleotides, indicating that the GC-rich sites identified in the -79 to -49 region of the RAR{alpha}1 gene promoter bound Sp1 protein. The results also showed that near-saturation of Sp1 binding to consensus [32P]Sp1 was observed at the lowest amount of Sp1 protein, whereas only minimal or nondetectable binding to the other oligonucleotides (lanes 6, 10, and 14) was observed using the same amount of Sp1 protein. Relative intensities of the retarded band using the highest amount (20 ng) of Sp1 (protein) and consensus [32P]Sp1, [32P]RAR2, [32P]RAR2•m1, and [32P]RAR2•m2 were 100, 20, 27, and 15, respectively. Thus, the GC-rich sites within RAR2 bound Sp1 protein with lower affinity than the consensus Sp1 oligonucleotide. Mobilities of retarded bands using [32P]RAR2, [32P]RAR2·m1, or [32P]RAR2·m2 gave a single retarded band with comparable mobilities. These data suggest that of the GC-rich sites in RAR2 initially bound only one Sp1 molecule/DNA under the conditions of this assay, which was limiting in Sp1 protein. Incubation of [32P]RAR2 with a large excess of Sp1 protein gave a second band with decreased mobility (data not shown), suggesting that binding can occur at both GC-rich sites.



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Figure 3. Binding of Sp1 Protein to 32P-Labeled RAR2, RAR2·m1, RAR2·m2, and Consensus Sp1 Oligonucleotides

Sp1 protein (0, 5, 10, or 20 ng) was incubated with [32P]Sp1 (lanes 1–4), [32P]RAR2 (lanes 5–8), [32P]RAR•m1 (lanes 9–12), and [32P]RAR2·m2 (lanes 13–16), respectively. Gel mobility shift analysis was performed as described in Materials and Methods. Sp1 bands (see arrow) were visualized by autoradiography. Sp1 protein formed a retarded band with all of the oligonucleotides; in contrast, a retarded band was not observed using [32P]RAR2·m3, which is mutated in both Sp1 sites (data not shown).

 
Results of competitive binding studies (Fig. 4Go) showed that wild-type Sp1 protein (5 ng) bound consensus [32P]Sp1 oligonucleotide to form a retarded band (lane 2, bound DNA), and the intensity of this band was not decreased by competition with 400-fold excess of unlabeled mutant Sp1 oligonucleotide (lane 4). The intensity of the Sp1-[32P]Sp1 (oligo) retarded band was decreased by coincubation with 0.5–5.0 pmol unlabeled RAR2 (lanes 5–7), RAR2•m1 (lanes 8–10), and RAR2•m2 (lanes 11–13) oligonucleotides. In contrast, competition with RAR2•m3, which contains mutations in both GC-rich sites, did not significantly decrease intensity of the retarded band (data not shown). Thus, results in Figs. 3Go and 4Go demonstrate in both direct and indirect binding gel mobility shift assays that GC-rich sites within RAR2 interact with Sp1 protein.



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Figure 4. Competition of Sp1-[32P]Sp1 Complex Formation with Unlabeled RAR2, RAR2·m1, and RAR2·m2 Oligonucleotides

The [32P]Sp1 oligonucleotide was incubated with 5 ng Sp1 protein (lanes 2–13); 5 pmol unlabeled wild-type and mutant consensus Sp1 oligonucleotides (lanes 3 and 4), 0.5, 1.5, and 5 pmol of RAR2 (lanes 5–7), RAR2·m1 (lanes 8–10), and RAR2·m2 (lanes 11–13) oligonucleotides were used for competition. Gel mobility shift analysis was performed as described in Materials and Methods. The retarded bands (see arrow) were visualized by autoradiography. Wild-type consensus Sp1 and RAR2, RAR2·m1, and RAR2·m2 oligonucleotides all significantly decreased intensity of the Sp1-[32P]Sp1-retarded band. Mutant Sp1 (lane 4) and RAR2·m3 (data not shown) oligonucleotides did not decrease retarded band intensity.

 
Recent studies have shown that consensus Sp1 oligonucleotide and GC-rich sites from heat shock protein 27 are E2-responsive in transient transfection studies (39). Hormone inducibility was associated with formation of an ER/Sp1 complex. ER did not directly bind DNA; however, in gel mobility shift assays, ER enhanced Sp1-DNA binding, but a ternary complex (ER/Sp1-DNA) was not detected using this technique. The results in Fig. 5Go show that [32P]RAR2 binds Sp1 protein (lane 3) but not ER protein (lane 2); incubation of [32P]RAR2 and Sp1 protein plus 200, 400, or 800 fmol ER (lanes 4–6) resulted in a concentration-dependent increase in formation of the Sp1-[32P]RAR2-retarded band (bound DNA, upper). Intensity of the retarded band was increased by 2.8-fold at the highest concentration of ER (lane 6). Intensity of the ER-enhanced retarded band (lane 6) was competitively decreased by coincubation with 400-fold excess unlabeled consensus Sp1 and RAR2 oligonucleotides (lanes 7 and 8) but not by mutant consensus Sp1 oligonucleotide (lane 9). Incubation of [32P]ERE and ER gave a retarded ER-ERE band (bound DNA, lower) (lane 11), which was more mobile than the Sp1-DNA complexes.



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Figure 5. ER Enhances Binding of Sp1 Protein to [32P]RAR2

Different amounts of hER (0, 200, 400, and 800 fmol) (lanes 3–6) were incubated with 3 ng Sp1 protein and [32P]RAR2. Gel mobility shift analysis was performed as described in Materials and Methods. The retarded band intensity values relative to that of Sp1 binding alone (lane 3, 100 ± 14) were 136 ± 13, 242 ± 20, and 279 ± 23 (lanes 4–6, respectively) (means ± SD for three determinations). hER (lanes 5 and 6) significantly enhanced the intensity of the Sp1-[32P]RAR2-retarded band (P < 0.05), whereas binding of hER (800 fmol) to [32P]RAR2 was not observed (lane 2) compared with [32P]ERE (lanes 11). However, the intensity of the bound-DNA complex band can be significantly decreased by competition with consensus Sp1 or RAR2 oligonucleotides (lanes 7 and 8) but not by unlabeled mutant Sp1 oligonucleotide (lane 9).

 
The results summarized in Fig. 6Go show that wild-type [32P]RAR2 bound Sp1 protein to form a retarded band (lane 2), and coincubation with ER (200–800 fmol) enhanced intensity of the retarded band (lanes 3–5). Incubation of Sp1 protein alone with [32P]RAR2•m1 (lane 7) or [32P]RAR2•m2 (lane 12) resulted in retarded band formation, and coincubation with ER (200–800 fmol) resulted in a concentration-dependent increase in retarded band intensity (lanes 8–10 and 13–15, respectively).



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Figure 6. ER Enhances Binding of Sp1 Protein to [32P]RAR2, [32P]RAR2·m1, and [32P]RAR2·m2 in Gel Mobility Shift Assays

Different amounts of hER (0, 200, 400, and 800 fmol) were incubated with 3 ng Sp1 protein and [32P]RAR2 (lanes 2–5), [32P]RAR2·m1 (lanes 7–10), and [32P]RAR2·m2 (lanes 12–15). Gel mobility shift analysis was performed as described in Materials and Methods. The retarded band intensity values relative to that of Sp1 binding alone (lane 2, 100 ± 14; lane 7, 100 ± 16; and lane 12, 100 ± 12) were 136 ± 13, 242 ± 20, 279 ± 23 (lanes 3–5), 216 ± 26, 300 ± 22, and 371 ± 11 (lanes 8–10); 352 ± 24, 517 ± 30, and 629 ± 15 (lanes 13–15), respectively (means ± SE from three determinations). Compared with band intensities observed after incubation of Sp1 protein alone with the [32P]oligonucleotides, ER significantly enhanced (P < 0.05) retarded band intensity (lanes 4, 5, 10, 14, and 15).

 
It was previously reported that in gel mobility shift assays RAR1 binds ER and Sp1 protein using nuclear extracts and in vitro translated ER (25). The interaction of ER, Sp1, and ER/Sp1 with [32P]RAR1 has been reinvestigated in gel mobility shift assays (Fig. 7Go). [32P]RAR1 binds Sp1 protein (lane 3) but not ER protein (lane 2); however, coincubation of [32P]RAR1, Sp1, and ER resulted in enhanced (1.7-fold) intensity of the [32P]RAR1-Sp1 retarded band (lanes 4–6, bound DNA). These results show that ER did not directly bind [32P]RAR1 but ER enhanced binding of [32P]RAR1 to the Sp1 protein.



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Figure 7. ER Enhances Binding of Sp1 to [32P]RAR1

Sp1 protein (3 ng) alone (lane 3) and different amounts of ER (200, 400, and 800 fmol) (lanes 4–6) were incubated with [32P]RAR1. Gel mobility shift analysis was performed as described in Materials and Methods. Retarded band intensity values relative to that of Sp1 binding [32P]RAR1 alone (lane 6, 100) were 172 ± 7.7, 174 ± 24, and 173 ± 7.5 (lanes 4–6, respectively) (means ± SE from three determinations). ER protein alone did not bind [32P]RAR1 (lane 2).

 
Previous studies in ER-negative MDA-MB-231 cells with constructs containing Sp1 oligonucleotide inserts showed that E2 induced reporter gene activity in cells cotransfected with WT-ER or 11C-ER (DNA binding domain-deficient) expression plasmids (39). Comparable studies using pRAR2 gave similar results (Fig. 8Go) and also showed that transfection with 19C-ER and 15C-ER, which contain only activation function-2 (AF-2) and AF-1, respectively, did not result in E2-induced transactivation. These results show that the GC-rich sites at -68 to -62 and -59 to -52 in the RAR{alpha}1 gene proximal promoter region are primarily responsible for E2-responsiveness via interactions with a transcriptionally active ER/Sp1 complex.



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Figure 8. Effects of Wild-Type or Variant ER on CAT Activity Induced by E2 in MDA-MB-231 Cells Cotransfected with pRAR2

MDA-MB-231 cells were cotransfected with pRAR2 plus hER, H11C-ER, 15C-ER, 19C-ER, or pCDNA3 (as control) (total amount of DNA was kept constant). The transient transfection and CAT assays were performed as described in Materials and Methods. Cells were treated with DMSO (C, light bars) or 10 nM E2 (E2, dark bars). Significant induction of CAT activity was observed only in cells cotransfected with wild-type ER or 11C-ER (P < 0.05).

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Several reports (20, 21, 24, 25, 27) have demonstrated that E2 induces RAR{alpha}1 gene expression, and at least two regions of the RAR{alpha}1 gene promoter at -491 to -455 and -100 to -49 were sensitive to E2-induced transactivation (27). Contributions of the two E2-responsive regions of the RAR{alpha}1 gene promoter may also be dependent on cellular context. In transient transfection studies in HepG2 cells, the upstream region (-491 to -455) was the more important contributor to E2 responsiveness (27), whereas the downstream sequence (-100 to -49) played the major role in E2-mediated transactivation in breast cancer cells (25). Analysis of the downstream region of the promoter by transient transfection and gel mobility shift assays gave conflicting results. Rishi and co-workers (25) showed that an ERE(1/2)(N)10Sp1 (-100 to -58) motif bound the ER and Sp1 proteins in gel mobility shift assays and was E2-responsive in transient transfection studies. These data were consistent with other reports showing that an ERE(1/2)(N)xSp1 sequence also played a role in ER-mediated transactivation of c-myc, creatine kinase B, cathepsin D, and heat shock protein 27 gene expression (40, 41, 42, 43, 44, 45). In contrast, Elgort and co-workers (27) showed that E2-responsiveness of the downstream RAR{alpha}1 gene promoter sequence was localized in the -79 to -49 region, which contained two GC-rich sequences but not the ERE(1/2).

We have reinvestigated the E2-responsiveness of the proximal promoter region of the RAR{alpha}1 gene in transient transfection studies utilizing constructs containing the -100 to -49 (pRAR4), -100 to -58 (pRAR1), -79 to -49 (pRAR2), and -79 to -56 (RAR3) RAR{alpha}1 gene promoter inserts. The insert in pRAR4 encompasses both the ERE(1/2)(N)10Sp1 (RAR1) and GC-rich (RAR2) promoter sequences and, not surprisingly, was E2-responsive (Fig. 1Go). Both E2-responsive RAR1 (-100 to -58) and RAR2 (-79 to -49) regions of the RAR1{alpha} gene promoter contain two GC-rich sites. Recent studies in this laboratory have demonstrated that Sp1-binding sites are potentially hormone-responsive via formation of ER/Sp1 protein-GC rich (DNA) complexes (39). Therefore, this study reexamined the role of GC-rich elements in the RAR1{alpha} gene promoter as mediators of ER activation. Rishi and co-workers (25) previously reported that [32P]RAR1 bound ER in a gel mobility shift assay and that the ERE half-site was required for this binding and for E2-induced transactivation of pRAR1. E2-responsiveness of pRAR1 was also observed in our study (Fig. 1Go); however, the results also showed that [32P]RAR1 did not directly bind the ER in a gel mobility shift assay (Fig. 7Go) and pRAR1·m1, which is mutated in the ERE half-site, was E2-responsive (Fig. 2Go). Thus, only the GC-sites in RAR1 were required for ER activation, and results of gel mobility shift assays showed that [32P]RAR1 bound Sp1 protein, and unlabeled RAR1 and Sp1 oligonucleotides competitively decreased binding of Sp1 protein to [32P]RAR1 (Fig. 7Go). Moreover, ER enhanced Sp1 binding to [32P]RAR1 (Fig. 7Go), indicating that the GC-rich Sp1-binding sites are critical elements for ER activation of RAR1.

Elgort and co-workers (27) previously reported that RAR2 region of the promoter (-79 to -49) contained two GC-rich sites that bound Sp1 but not ER protein and was E2-responsive in transient assays. Therefore, we investigated the role of one or both of the GC-rich sites within RAR2 in mediating E2-responsiveness. pRAR2·m3 is mutated in both downstream GC-rich sites and was not active in transient transfection studies (data not shown); however, as previously reported (27), hormone responsiveness was observed in transient transfection studies using pRAR2 (-79 to -49) (Figs. 1Go and 2Go) suggesting that the Sp1 sites are required for inducibility. E2 induced CAT activity in transient transfection studies with constructs containing mutations in the -68 to -62 (pRAR2·m2) or -59 to -52 (pRAR2·m1) sites (Fig. 2Go), suggesting that either of the two GC-rich sites are sufficient for ER-mediated transactivation of pRAR2.

Gel mobility shift assays clearly showed that RAR2, RAR2·m1, RAR2·m2, and RAR1 bound Sp1 protein to form a retarded band or competitively decreased Sp1-[32P]Sp1 in competition assays (Figs. 3Go, 4Go, and 7Go). Although ER and Sp1 physically interact (39), coincubation of both proteins with a consensus [32P]Sp1 oligonucleotide resulted only in formation of an Sp1-[32P]Sp1 complex in which ER enhanced the rate of complex formation and retarded band intensity (39). Similar results were obtained in this study using [32P]RAR2, [32P]RAR2•m1, [32P]RAR2·m2, and [32P]RAR1 ( Figs. 5–7GoGoGo). The failure of ER to supershift the Sp1-DNA complex but to enhance Sp1-DNA binding has previously been observed in other studies showing that HTLV-1 Tax, SREPB, and cyclin D1 enhanced binding of bZip, Sp1, and ER to their respective enhancer elements, respectively (46, 47, 48). The results with the RAR2 (-79 to -49) region of the RAR{alpha}1 gene promoter demonstrate that both GC-rich sites bind Sp1 protein in gel mobility shift assays, and intensities of both retarded bands were enhanced by coincubation with ER, which is consistent with results of transactivation assays (Fig. 2Go).

It has also been reported that both wild-type ER and 11C-ER (but not 19C-ER or 15C-ER) enhance Sp1 binding to GC-rich elements in gel mobility shift assays, confirming that the effect of ER does not require DNA binding (39). In ER-negative MDA-MB-231 cells, E2 induced CAT activity in cells cotransfected with pRAR2 and wild-type ER or 11C-ER, which does not contain the DNA-binding domain of the ER (Fig. 8Go). In contrast, no induction response was observed in cells cotransfected with 15C-ER or 19C-ER, which express AF-1 or AF-2 domains of the ER, respectively. These results were comparable to previous studies using gel mobility shift assays and constructs containing a consensus Sp1 oligonucleotide insert and further support hormone-induced transactivation via ER/Sp1 interactions with GC-rich Sp1-binding sites (39).

Results of this study demonstrate that the GC-rich motifs in the proximal region of the RAR{alpha}1 gene promoter are functional E2-responsive enhancer sequences in which ER-mediated transactivation is independent of ER-DNA interactions. Recent studies in the laboratory have identified Sp1-binding sites in the c-fos protooncogene promoter that are also functional enhancer elements for ER/Sp1-mediated gene expression in MCF-7 cells (49). These hormone-induced responses do not require interaction of the ER with DNA and are similar to ER-AP1 interactions (50). Sp1-binding sites are common motifs in promoters of diverse cellular and viral genes, most of which are hormone-independent. The reasons for differential sensitivity of Sp1-binding sites in gene promoters are unknown but could be due to specific interactions with other nuclear proteins including coactivators. Current studies in this laboratory are focused on identifying other E2-responsive GC-rich motifs within gene promoter sequences and determining their cell-, promoter region-, and ligand-dependent functionality.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Chemicals, Cells, and Oligonucleotides
MCF-7 and MDA-MB-231 cells were obtained from the American Type Culture Collection (Rockville, MD). Cells were maintained in MEM with phenol red and supplemented with 10% FCS plus 0.2 x antibiotic/antimycotic solution, 0.035% sodium bicarbonate, 0.011% sodium pyruvate, 0.1% glucose, 0.24% HEPES, and 6 x 10-7% insulin. Cells were incubated in an air-carbon dioxide (95:5) atmosphere at 37 C and passaged every 3–5 days without becoming confluent. DME/F12 without phenol red, PBS, acetyl coenzyme A, E2, and 100 x antibiotic/antimycotic solution were purchased from Sigma Chemical Co. (St. Louis, MO). FCS was obtained from Intergen (Purchase, NY). MEM was purchased from Life Technologies (Grand Island, NY). [{gamma}-32P]ATP (3000 Ci/mmol) and [14C]chloramphenicol (53 mCi/mmol) were purchased from NEN Research Products (Boston, MA). Poly deoxy-(inosinic-cytidylic) acid [poly d(I-C)], restriction enzymes (HindIII and BamHI), and T4-polynucleotide kinase were purchased from Boehringer Mannheim (Indianapolis, IN). The human estrogen receptor (hER) expression plasmid was kindly provided by Dr. Ming-jer Tsai (Baylor College of Medicine, Houston, TX). The hER deletion mutants 11C-ER, 15C-ER, and 19C-ER were kindly provided by Dr. Pierre Chambon (Strasbourg, France). Sp1 protein and bacculovirus-expressed hER proteins were purchased from Promega (Madison, WI), and Panvera (Madison, WI), respectively. Plasmid preparation kit was purchased from Qiagen (Santa Clarita, CA); 40% polyacrylamide was obtained from National Diagnostics (Atlanta, GA). All other chemicals and biochemicals were the highest quality available from commercial sources. DNA oligonucleotides (Table 1Go) were synthesized by the Gene Technologies Laboratory, Texas A & M University (College Station, TX).


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Table 1. List of DNA Oligonucleotides

 
Cloning
The pBL/TATA-CAT plasmid was made by digesting the pBLCAT2 vector with BamHI and XhoI to remove the thymidine kinase promoter; the double-stranded E1B-TATA oligonucleotide containing complementary 5'-overhangs was then inserted into the corresponding sites (39). The oligonucleotides from the human RAR{alpha}1 promoter listed above were cloned into the pBL/TATA-CAT at the HindIII and BamHI sites to give pRAR1, pRAR2, pRAR3, pRAR4, pRAR1·m1 pRAR2·m1, pRAR2·m2, and pRAR2·m3 plasmids, respectively. All ligation products were transformed into DH5{alpha}-competent Escherichia coli cells, plasmids were isolated, and correct clonings were confirmed by restriction enzyme mapping and DNA sequencing using the Sequitherm cycle sequencing kit from Epicentre Technologies (Madison, WI). Plasmid preparation for transfection was carried out by alkaline lysis followed by cesium chloride gradient centrifugation (2x) or by using a Qiagen Plasmid Mega Kit.

Transient Transfection and CAT Centrifugations
Cultured MCF-7 and MDA-MB-231 cells were seeded in 5% charcoal-stripped DME/F12 medium in 100-mm plates for 16 h and then transiently transfected by the calcium phosphate method with 10 µg reporter plasmid and 5 µg of wild-type hER or variant ER expression plasmids in pCDNA3-Neo (InVitrogen, Inc., Carlsbad, CA). The empty vector pcDNA3.1 (InVitrogen) was used to bring the total DNA content to 15 µg. E2 responsiveness in MCF-7 cells was observed only after cotransfection with WT-ER (or 11C-ER), and this has been observed in other studies using E2-responsive constructs due to overexpression of the plasmids (39, 40, 41, 44). After incubation for 14–16 h, media was changed and cells were treated with the appropriate chemicals in DMSO for 44 h. Cells were then washed with PBS and harvested by scraping, and then lysed in 200 µl of 0.25 M Tris-Cl (pH 7.6) by three cycles of freeze (1.5 min)-thaw (1.5 min) sonication (3 min). Cell debris was pelleted and protein concentration was determined by the method of Bradford using BSA as standard. An aliquot of cell lysate was brought to 120 µl with 0.25 M Tris-Cl (pH 7.6) and incubated with 1 µl [14C]chloramphenicol (53 mCi/mmol) and 42 µl of 4 mM acetyl coenzyme A for an appropriate time at 37 C. The reaction was stopped by vortexing with 300 µl ethyl acetate. After vortexing for 30 sec and centrifuging at 16,000 x g for 1 min at 20 C, a 250-µl aliquot of ethyl acetate was evaporated in vacuo, resuspended in 20 µl ethyl acetate, spotted on a TLC plate (Whatman Ltd., Maidstone, England), and developed using a 95:5 chloroform-methanol solvent mixture. The percent protein conversion into acetylated chloramphenicol was quantitated using the counts/min obtained from the Betagen Betascope 603 blot analyzer (Tritech, Annapolis, MD). CAT activity was calculated as the percentage of that observed in cells treated with DMSO (arbitrarily set at 100). TLC plates were subjected to autoradiography using a Kodak X-Omat film (Eastman Kodak, Rochester, NY) for 20 h.

Electrophoretic Mobility Shift Assays
Oligonucleotides were annealed and labeled at the 5'-end using T4-polynucleotide kinase and [{gamma}-32P]ATP. Gel electrophoretic mobility shift assays were performed by incubating 0–20 ng pure Sp1 protein (Promega, Madison, WI) in 25 µl of 1 x binding buffer (6% glycerol, 1 mM MgCl2, 0.5 mM EDTA, 0.5 mM dithiothreitol, 50 mM NaCl, 10 mM Tris-HCl, pH 8.0), 0.1 mg/ml of BSA. After incubation for 10 min at 4 C, 32P-labeled oligonucleotides (50,000 cpm) were added to the reaction mixture in the presence of 0.5 µg poly d(I-C) and incubated for an additional 15 min at 25 C. Excess unlabeled DNA for competition studies was added before the addition of 32P-labeled oligonucleotides. The following procedure was used for ER-enhanced Sp1 binding studies: 1) 200–800 fmol pure hER protein in 1 x binding buffer containing 40 mM E2 and BSA was incubated for 15 min at 4 C; 2) 1–5 ng Sp1 protein was added to the mixture and incubated on ice for 5 min; 3) 32P-labeled oligonucleotides (50,000 cpm) were added to the reaction mixture in the presence of 0.5 µg poly d(I-C), and the mixture was incubated for an additional 15 min at 25 C. Samples were loaded onto a 5% polyacrylamide gel (acrylamide-bisacrylamide ratio, 30:0.8) and run in 1 x TBE buffer (0.09 M Tris, 0.09 boric acid, and 2 mM EDTA, pH 8.3) at 110 V. Protein-DNA binding was visualized by autoradiography and quantitated by densitometry using the Zero-D software package (Molecular Dynamics, Sunnyvale, CA) and a Sharp JX-330 scanner (Sharp Corp., Mahwah, NJ) and subjected to autoradiography using a Kodak X-Omat film for the appropriate time at -80 C.

Statistical Analysis
Statistical significance was determined by ANOVA and Scheffe’s test, and the levels of probability are noted. Results are expressed as means ± SD for at least three separate experiments.


    FOOTNOTES
 
Address requests for reprints to: Stephen Safe, Department of Veterinary Physiology and Pharmacology, Texas A & M University, College Station, Texas 77843-4466.

This work was supported by NIH Grant CA-76636, the Robert A. Welch Foundation, and the Texas Agricultural Experiment Station. S.S. is a Sid Kyle Professor of Toxicology.

Received for publication October 17, 1997. Revision received February 11, 1998. Accepted for publication February 25, 1998.


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 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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