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(ER) Required for ER/Sp1-Mediated Activation of GC-Rich Promoters by Estrogens and Antiestrogens in Breast Cancer Cells
Department of Veterinary Physiology & Pharmacology (K.K., N.T., S.S.) and Department of Biochemistry & Biophysics (B.S.), Texas A&M University, College Station, Texas 77843-4466
Address all correspondence and requests for reprints to: Stephen H. Safe, Department of Veterinary Physiology & Pharmacology, Texas A&M University, 4466 TAMU, College Station, Texas 77843-4466. E-mail: ssafe{at}cvm.tamu.edu.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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(ER)/Sp1 activation of GC-rich gene promoters in breast cancer cells is dependent, in part, on activation function 1 (AF1) of ER, and this study investigates contributions of the DNA binding domain (C) and AF2 (DEF) regions of ER on activation of ER/Sp1. 17ß-Estradiol (E2) and the antiestrogens 4-hydroxytamoxifen and ICI 182,780 induced reporter gene activity in MCF-7 and MDA-MB-231 cells cotransfected with human or mouse ER (hER or MOR), but not ERß and GC-rich constructs containing three tandem Sp1 binding sites (pSp13) or other E2-responsive GC-rich promoters. Estrogen and antiestrogen activation of hER/Sp1 was dependent on overlapping and different regions of the C, D, E, and F domains of ER. Antiestrogen-induced activation of hER/Sp1 was lost using hER mutants deleted in zinc finger 1 [amino acids (aa) 185–205], zinc finger 2 (aa 218–245), and the hinge/helix 1 (aa 265–330) domains. In contrast with antiestrogens, E2-dependent activation of hER/Sp1 required the C-terminal F domain (aa 579–595), which contains a ß-strand structural motif. Moreover, in peptide competition experiments overexpression of a C-terminal (aa 575–595) F domain peptide specifically blocked E2-dependent activation of hER/Sp1, suggesting that F domain interactions with nuclear cofactors are required for ER/Sp1 action. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Ligand-activated estrogen receptor (ER) and ERß signaling pathways have been extensively investigated and involve interactions of ER homo- or heterodimers with estrogen responsive elements (EREs) in 17ß-estradiol (E2) responsive gene promoters (1, 2, 3, 4, 5, 6, 15, 16, 17, 18, 19). Several studies have also described ligand-activated ER action through activator protein 1 (AP1) elements in which transcriptional enhancement is associated with interactions between ER and the c-jun component of the AP1 complex (20, 21, 22, 23, 24, 25). Estrogens and antiestrogens activate ER/AP1; however, this response is ligand structure and cell context dependent, as antiestrogens exhibit minimal agonist activity in breast cancer cells. In contrast, ERß/AP1 is preferentially activated by antiestrogens but not E2 or diethylstilbestrol (20). Research in this laboratory has identified GC-rich motifs in promoters of several E2-responsive genes that are activated by ER/Sp1 and, like ER/AP1, this hormone-dependent response does not require direct binding of ER to promoter elements and is activated by HE11, a DNA-binding domain (DBD) deletion mutant of ER (26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36). Human ER (hER)/Sp1 is activated by estrogens and antiestrogens in MCF-7 and MDA-MB-231 breast cancer and LnCaP prostate cancer cells but not in HeLa cells. In contrast, hERß/Sp1 exhibits minimal activity in all cell lines examined (35). Studies with wild-type hER, various hERß chimeras, and mutants demonstrated that the activation function 1 (AF1) region of hER was required for hER/Sp1-mediated transactivation in MCF-7 and MDA-MB-231 breast cancer cells; however, this did not exclude a role for AF2 in this response (35).
This study further investigates estrogen and antiestrogen activation of hER/Sp1 action in MCF-7 and MDA-MB-231 breast cancer cells and AF1-independent domains of ER required for ligand-dependent transactivation. Estrogen activation of hER/Sp1 is observed in cells transfected with wild-type hER and deletions of one or both zinc fingers and point mutations in helix 12 (D538N, E542Q, and D545N) that block interactions with AF2-interacting coactivators. Activation of hER/Sp1 by E2 is not observed in hER mutants with deletion of the hinge region [amino acids (aa) 271–300] or helix 12 and the F domain (aa 538–595), whereas these latter mutants are activated by the antiestrogens 4-hydroxytamoxifen (4-OHT) and ICI 182,780. In contrast, antiestrogen activation of hER/Sp1 is not observed with zinc finger 1 or zinc finger 2 deletion mutants. Thus, hER/Sp1 activation by estrogens and antiestrogens is complex and requires multiple domains of hER.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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and ERß in Hormonal Activation of GC-Rich Promoters/Sp1 in breast cancer cells required the AF1 domain of hER (35), and activation by E2 was also observed in cells cotransfected with the DBD deletion mutant (aa 185–251) hER11 (26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36). The role of other domains of hER on estrogen and antiestrogen activation of hER/Sp1 has not been defined and is investigated in this study. Although hER11/Sp1 is activated by E2, deletion of the entire DBD resulted in loss of antiestrogen-induced transactivation (35, 37), and therefore initial studies determined the role of zinc fingers 1 and 2 deletion mutants on estrogen/antiestrogen activation of ER/Sp1.
Wild-type and zinc finger deletion mutants for hER ( and ß) and mouse ER (MOR) (Table 1
) were cloned into pcDNA3 and translated in vitro using [35S]methionine, and the radiolabeled proteins were separated by SDS-PAGE (Fig. 1A). The results show that in vitro translated proteins gave distinct bands with comparable intensities and the expected molecular weights, indicating that the 
ZF1 or ZF2 deletions did not cause unexpected frame shifts. The effects of ZF1 and ZF2 mutations on DNA binding were determined by gel EMSAs of the in vitro expressed proteins. The results showed that only wild-type hERs and MOR formed retarded bands after incubation with [32P]ERE (lanes 3, 7, and 10), whereas DNA-bound complexes were not observed with the zinc finger deletion mutants (Fig. 1B). Transcriptional activation assays in ER-negative MDA-MB-231 cells cotransfected with an ERE-dependent promoter (pERE3) and wild-type ER or ER deletion mutants showed that E2 induced activity only in cells transfected with wild-type hER ( or ß) or MOR (Fig. 1C). These results were consistent with the gel mobility shift assays showing that the zinc finger mutants do not bind EREs (Fig. 1B).
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or MOR (Fig. 2). In contrast to results obtained in cells transfected with pERE3, induction responses were observed for both zinc finger deletion mutants of hER and MOR. hERß and the DBD deletion mutants were only minimally active in MDA-MB-231 cells (
2-fold induction) and inactive in MCF-7 cells (data not shown). The fold-induction using wild-type and zinc finger deletion mutants of hER and MOR was lower in MDA-MB-231 than MCF-7 breast cancer cells due, in part, to higher basal activity in the former cell line. The highest induction responses using pSp13 and E2 were observed in MCF-7 cells transfected with ZF2 mutants; in MCF-7 cells, all the ER-DBD deletion mutants were more potent activators of pSp13 than wild-type hER or MOR. The overall pattern of induction responses was similar for wild-type hER and MOR and their zinc finger deletion mutants, and subsequent studies used only hER.
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/Sp1 interactions with GC-rich motifs have previously been identified (26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36), and these include retinoic acid receptor 1 and adenosine deaminase (29, 30). hER/Sp1 activation of constructs derived from GC-rich E2-responsive regions of the adenosine deaminase (pADA; -86 to -65) and retinoic receptor 1 (pRAR1; -79 to -49) gene promoters were investigated in MCF-7 and MDA-MB-231 cells transfected with wild-type or zinc finger deletion mutants of hER (Fig. 3). The pattern of induction by E2 was dependent on expression of wild-type or variant hER, promoter, and cell context. For example, hormone inducibility was higher in MDA-MB-231 cells transfected with either pADA or pRAR1 and wild-type hER compared with responses observed for the zinc finger deletion mutants. In MCF-7 cells, hormone inducibility was highest in cells transfected with pADA or pRAR1 and the ZF2 deletion mutant of hER.
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summarize the effects of E2 and the antiestrogens ICI 182,780 and 4-OHT on induction of luciferase activity in MCF-7 cells transfected with pSp13 and wild-type or zinc finger deletion mutants of hER. Both estrogens and antiestrogens induce transactivation in cells transfected with wild-type hER, whereas E2, but not the antiestrogens, were active in cells transfected with ZF1 or ZF2 deletion mutants of hER. These results suggest that antiestrogen-bound zinc finger mutants of hER are transcriptionally inactive and their recruitment of functional coactivators requires cooperative interactions that are directly or indirectly dependent on the zinc finger domains of hER. Although deletion of one or both zinc fingers resulted in loss of antiestrogen-dependent hER/Sp1 agonist activity, 4-OHT and ICI 182,780 inhibited E2-induced ER/Sp1 action using these DBD deletion constructs, suggesting that the antiestrogen-mediated responses are intact in cells expressing zinc finger mutants of hER. We also investigated the possibility that the failure to observe antiestrogen activation of zinc finger mutants of hER/Sp1 may be due to failure of the transfected constructs to accumulate in nuclei of breast cancer cells. However, results of immunofluorescent studies in MDA-MB-231 cells transfected with hER or the zinc finger mutants showed that wild-type and mutant constructs were primarily nuclear in cells treated with dimethylsulfoxide (DMSO) or E2/antiestrogens, although some perinuclear staining was observed with hERZF1 using ICI 182,780 (Fig. 4B).
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/Sp1 by E2, 4-OHT, and ICI 182,780 Does Not Require AF2-Helix 12-Coactivator Interactions/Sp1 by E2 was lost after deletion of aa 51–117; however, this did not exclude a role for AF2 alone or as a modifier of AF1-dependent hER/Sp1 action. hERTAF1 contains three aa mutations (D538N, E542Q, and D545N) that do not affect ligand binding but inactivate AF2 by selectively blocking interactions with AF2-dependent coactivators (38, 39, 40, 41). In MCF-7 cells transfected with pSp13 and hERTAF1, E2 significantly induced reporter gene activity, and ICI 182,780 and 4-OHT also slightly increased this response (Fig. 5A). Hormone-mediated transactivation was also observed in MCF-7 cells transfected with pSp13 and hERTAF1 containing deletions of zinc finger 1 or zinc finger 2 in the DBD; in contrast, the antiestrogens ICI 182,780 and 4-OHT did not activate luciferase activity using the hERTAF1 DBD mutants, and similar results were observed for hERZF1 and hERZF2 (Fig. 4A
). Increased hormone-induced transactivation was observed in MCF-7 cells transfected with pSp13 and hERZF1 or hERZF2 compared with wild-type hER (Figs. 2B and 4A). In contrast, deletion of zinc fingers 1 or 2 in hERTAF1 did not result in increased hormone responsiveness in MCF-7 cells transfected with the zinc finger deletion mutants compared with hERTAF1 (Fig. 5A). This suggests that helix 12 may contribute to E2-induced hERZF1/Sp1 and hERZF2/Sp1 action. hER19 and hERnull were also inactive, and this was consistent with previous studies showing the importance of AF1 for hER/Sp1 action (35). The results in Fig. 5B show that expression of the LXXLL peptide 2XF6 (38) significantly decreased hormone-induced transactivation in MDA-MB-231 cells transfected with pERE3, and inhibition was not observed in cells transfected with pSp13.
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with prototypical steroid receptor coactivators containing LXXLL motifs may not be critical for hER/Sp1 action. Coactivators of hER and other nuclear receptors have been extensively investigated, and these include AF2-dependent steroid receptor coactivators (SRCs) and AF1-dependent p68 RNA helicase (42, 43). However, many of these coactivators have not been investigated in breast cancer cells, and their coactivation of hER/Sp1 through LXXLL-dependent or -independent pathways has not been reported previously. Figure 5, C–E, summarizes the effects of selected coactivators on hER/Sp1 in breast cancer cells transfected with pSp13 and on hER/pERE3 in Chinese hamster ovary (CHO) cells, which have commonly been used for coactivation studies. MCF-7 cells were treated with 10 nM E2, transfected with pSp13 and hER, and different amounts (10, 50, and 100 ng) of expression plasmids for SRC-1, SRC-2 (glucocorticoid receptor interacting protein 1), SRC-3 (AIB1), and p68 RNA helicase, an AF1-dependent coactivator of ER (on an ERE promoter; Ref. 43). E2 induced activity (2-fold; Fig. 5C); however, cotransfection with SRC-1, SRC-2, SRC-3, or p68 did not enhance activity in this cell line, and similar results were observed for the RNA coactivator SRA or p300 and for higher amounts (500 ng) of transfected coactivators (data not shown). A parallel experiment was carried out in MDA-MB-231 cells (Fig. 5D), and E2 induced activity (2-fold), but cotransfection with SRC-1, SRC-2, SRC-3, and p68 did not enhance hER/Sp1 action. Moreover, many of these coactivators significantly inhibited the induction response in breast cancer cells. As a positive control for coactivation, we also investigated effects of p160 coactivators and p68 in CHO cells treated with 10 nM E2 and transfected with pERE3 and hER. This cell line has frequently been used to demonstrate coactivation of hER using ERE-dependent promoter-reporter constructs. E2 induced luciferase activity (8- to 15-fold) and SRC-1, SRC-2, and SRC-3 (but not p68) enhanced the induction response (Fig. 5E). Coactivation of hER/Sp1 by SRCs was not observed in ER-positive or negative breast cancer cell lines; this was consistent with the importance of AF1 for hER/Sp1-mediated transactivation (35). Surprisingly, we did not observe coactivation of hER or hER/Sp1 by the AF1-interacting coactivator p68 in ER-negative or -positive cell lines, suggesting that cell context modulates the effects of p68 as a coactivator.
Role of AF2/Hinge (DEF) Region for Activation of hER/Sp1 by Estrogen and Antiestrogens
We further investigated requirements for other regions within the DEF domains for activation of hER/Sp1 by estrogens and antiestrogens in MCF-7 cells. E2, 4-OHT, and ICI 182,780 did not induce luciferase activity in MCF-7 cells transfected with pSp13, whereas a 2.5- to 4.5-fold induction was observed by all three compounds in cells cotransfected with hER (Fig. 6A). E2 did not induce activity in MCF-7 cells transfected with hER(271–300) or hER(265–330) which contain deletions of the hinge (D) or hinge (D) plus helix 1 of the E domain. ICI 182,780 and 4-OHT were also inactive in cells transfected with hER(265–330), whereas induction by the antiestrogens was observed in cells transfected with hER(271–300). Estrogen/antiestrogen-dependent activation of hER/Sp1 was also investigated in MCF-7 cells transfected with a series of C-terminal deletion mutants, namely hER(538–595), hER(554–595), and hER(579–595). These mutants contain deletions of helix 12 (E) and the C-terminal F domain (538–595), the F domain (554–595) alone, and the ß-strand region of the F domain (579–595). In MCF-7 cells, both 4-OHT and ICI 182,780 induced luciferase activity in cells transfected with these hER deletion mutants, whereas E2 was inactive. The failure of E2 to induce transactivation in cells transfected with pSp13 and hER(579–595) suggests that the C-terminal aa 579–595, which contains a QKYYIT ß-strand motif (44), may be critical for transcriptional activation by E2 but not 4-OHT or ICI 182,780. We further confirmed the F domain requirement for hormonal activation of hER/Sp1 by examining a similar series of hER deletion mutants in MDA-MB-231 cells cotransfected with pSp13 or pERE3. E2 induced transactivation in cells cotransfected with pERE3 and hER(271–300) or hER(554–595), confirming results of previous studies in other cell lines showing that the hinge region and F domain are not necessary for hormonal activation of ER/pERE (42, 44, 45, 46, 47, 48). E2 did not induce transactivation in MCF-7 or MDA-MB-231 cells cotransfected with pSp13 and hER(554–595). Thus, hormonal activation of hER/Sp1 by E2 was dependent on the hinge (D) and F domains of hER, whereas these same regions of hER were not required for activation of pERE3.
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block hormone-induced transactivation of ERE-dependent promoters/genes (38, 39, 48). This has been extensively investigated with peptides containing LXXLL motifs that block coactivator interactions with ER (38, 39) and inhibit hormone-induced activation in cells transfected with pERE3 (Fig. 5B). Hormone-induced transactivation in MDA-MB-231 cells transfected with pERE3 was not significantly decreased after cotransfection with the F-ß strand peptide containing aa 575–595 from the F domain of hER fused to the DBD of the yeast GAL4 protein. In contrast, the F domain peptide blocked hormone-induced transactivation in MDA-MB-231 cells transfected with pSp13, whereas the 2XF6 peptide was inactive, and similar results were obtained with other peptides containing LXXLL sequences (data not shown). These results are consistent with the activity of wild-type and variant hER constructs and confirm that the F domain of ER is also essential for E2-dependent activation of hER/Sp1. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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, ERß, and other coregulatory proteins (7, 8, 9, 10, 11, 12, 13). The classical mechanism of ER activation involves ligand-dependent formation of ER dimers that bind consensus or nonconsensus EREs and recruit SRCs and other nuclear proteins that facilitate interactions with basal transcription factors (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13). In contrast, nonclassical pathways that involve ligand activation of ER/Sp1 and ER/AP1 do not require interactions of ER with promoter DNA but with other DNA-bound transcription factors, namely Sp1 and c-jun, respectively. Research in this laboratory has identified a number of E2-responsive genes regulated by ER/Sp1 (26) in breast cancer cells (27, 28, 29, 30, 31, 32, 33, 34, 35, 36), suggesting that the nonclassical pathways for activation of ER may play a significant role in cell context-dependent regulation of genes by E2 and SERMs.
Previous studies showed that both estrogens and antiestrogens activated a construct containing a GC-rich promoter (pSp1) in breast cancer cells and this response was AF1 dependent (35). Moreover, the DBD of hER was not required for activation by E2, whereas deletion of this region resulted in loss of transactivation by 4-OHT/ICI 182,780 (35, 54). We have further investigated the effects of selective mutations (deletions) of zinc fingers 1 and 2 on estrogen and antiestrogen activation of hER/Sp1 in breast cells. As expected, the zinc finger deletion mutants of hER, hERß, and MOR did not bind [32P]ERE in gel mobility shift assays or activate an ERE promoter (Fig. 1A). However, E2 activated pSp13, pADA, and pRAR in MCF-7 and MDA-MB-231 cells transfected with wild-type hER (and MOR) and both zinc finger mutants (Figs. 2 and 3), and similar results were obtained in MCF-7 cells transfected with wild-type and zinc finger mutants of hERTAF1 (Fig. 5A). In contrast, minimal responses were observed for hERß/Sp1 (data not shown) as previously reported (35). Zinc fingers 1 and 2 are important for DNA binding, and the D box region of zinc finger 2 plays a role in ER homodimerization (42, 55). The DBD of ER is also an important determinant for antiestrogen activation of ER/AP1 and ER/Sp1. Point mutations in zinc finger 1 either decreased (E207G/G208S), eliminated (K201A), or did not affect (E207A/G208A) ICI 182,780 activation of ER/AP1 in TSA cells, whereas an A227T mutation in zinc finger 2 resulted in loss of ICI 182,780 inducibility through an AP1 element (25). E2 decreased activation of ER/AP1 in MCF-7 and TSA cells, and this was also observed in all but one (K210a) of the DBD point mutants (25). We also investigated activation of hER/Sp1 by the zinc finger point mutants (25) in breast cancer cells, and minimal transactivation was observed after treatment with E2, 4-OHT, or ICI 182,780 (data not shown). In contrast, deletion of the DBD of hER did not affect activation of hER/Sp1 by E2 in breast cancer cells (35); however, our results show that both zinc fingers of hER and hERTAF1 are required for the activity of antiestrogens (Fig. 5). Thus, there are significant differences between hER/Sp1 and ER/AP1 and their requirements for regions within the DBD for activation by E2 and SERMs, suggesting that cell context-specific interactions of nuclear proteins with the DBD region of hER may be important for ligand-dependent activation of hER/Sp1. DBD-interacting proteins that coactivate hER and other hormone receptors have been reported (56, 57, 58), and current studies are investigating coactivation of hER/Sp1 by SNURF, a small RING finger protein initially identified as an androgen receptor DBD-interacting protein.
Previous studies with AF1 deletion mutants of hER showed that aa 51–117 were required for ER/Sp1-mediated responses (35); however, contributions of the DEF domains have not been determined. The AF2 domain of hER and other nuclear receptors is required for ligand-dependent activation of hER through classical DNA-dependent pathways, and this activation process involves recruitment of AF2-interacting coactivators (7, 8, 9, 10, 11, 12, 13). NR box (LXXLL) motifs in SRCs and other coactivators specifically interact with helix 12 of hER. D538N, E542Q, and D545N mutations in helix 12 give hERTAF1 abrogate interactions with most AF2-interacting coactivators and decrease transactivation from ERE promoters (38, 39, 40, 41). Maximal ER/AP1 activation by E2 requires intact activation surfaces of both AF1 and AF2, and AF2-dependent responses require helix 12 and the corresponding NR box interacting sites. Moreover, the AF1 domain of ER inhibits antiestrogen-induced ER/AP1 action (21). In contrast, both estrogens and antiestrogens activated hER and hERTAF1/Sp1, and overexpression of the NR box peptide 2XF6 (38) derived from SRC-2 did not affect activation of hER/Sp1 but inhibited ER on an ERE promoter (Fig. 5B). These results imply that regions of AF2 that interact with coactivators through their NR boxes are not necessary for hER/Sp1 action, and this is supported by studies showing that prototypical AF2-interacting SRCs did not enhance hER/Sp1-mediated transactivation in breast cancer cells transfected with pSp13 (Fig. 5). Interestingly, p68, an AF1-interacting protein, also did not enhance hER/Sp1 activation of a GC-rich promoter in breast cancer cells, indicating that AF1-dependent p68 coactivation of hER previously observed in COS-1 and HeLa cells transfected with an ERE promoter is also dependent on cell context (43). Results obtained with hERTAF1, the SRCs, and peptide competition experiments clearly define that some mechanistic differences between hormone-dependent activation of hER/Sp1 and hER are due, in part, to helix 12 of hER.
We further investigated other regions within the DEF domains required for ligand-dependent activation of hER/Sp1 (Fig. 6). The antiestrogens 4-OHT and ICI 182,780 activated hER/Sp1 in MCF-7 cells transfected with hER(271–300), a hinge region deletion mutant (Fig. 6A), whereas E2 was inactive. In contrast, E2 activated an ERE promoter in MDA-MB-231 cells transfected with hER(271–300) (Fig. 6B), and this was consistent with previous reports showing that the hinge region was not required for activation of ERE-dependent constructs (42). Deletion of the hinge region and helix 1 [i.e. hER(265–330)] resulted in loss of E2 and antiestrogen activation of hER and hER/Sp1 (Fig. 6, A and B), and the importance of helix 1 within the E domain for activation of ERE-dependent promoters has previously been reported (42). Pissios and co-workers (59) recently showed that E2, 4-OHT, and ICI 182,780 induced interactions of a helix 1-GAL4 chimeric protein with the ligand binding domain (LBD) or ER in a mammalian two-hybrid assay. Thus, helix 1 may stabilize ligand interactions with the LBD, and this process may be functional for both DNA-dependent and -independent mechanisms of ER action. However, the importance of helices 1 and 2 as interacting domains for other nuclear factors has not been determined.
Activation of hER/Sp1 by estrogens was also dependent on the C-terminal region of hER (aa 538–595), which encompassed part of helix 12 within the E domain (aa 538–553) and the F domain (aa 554–595), which potentially contains helix 13 and ß-strand motifs based on secondary structure calculations (42, 58). Helix 12 is required for E2-dependent activation of ER in cells transfected with an ERE promoter (42) (Fig. 6B), and similar results were observed for activation of hER/Sp1 by E2 (Fig. 6A). In contrast, both 4-OHT and ICI 182,780 activated hER(538–595)/Sp1, and this result coupled with antiestrogen activation of hERTAF1/Sp1 confirms that helix 12 is not required for this induction response by antiestrogens. The failure of E2 to activate hER(538–595)/Sp1 was not due to the requirement for helix 12 because activation was also not observed in cells transfected with hER(554–595) (i.e. F domain deletion) or hER(579–595) in which only the C-terminal ß-strand region of the F domain has been deleted. F domain deletions can modulate activation of ERE promoters by antiestrogens but have minimal effects on E2-mediated transactivation (45). However, the results in Fig. 6A clearly demonstrate that F domain aa 579–595 are required for activation of hER/Sp1 by E2. This was also confirmed in selective NR box (2XF6) and F domain (aa 575–595) peptide competition studies, which demonstrate preferential inhibition of hER/Sp1 action in cells transfected with the Fß expression plasmid (Fig. 6C). These results demonstrate that E2- and SERM-mediated activation of hER/Sp1 in breast cancer cells is complex and dependent on multiple overlapping and distinct regions of hER (Fig. 7). These domains of hER may impart unique structural features required for hER/Sp1 action and may also serve as binding sites for essential interacting nuclear coregulatory proteins. Decreased hER/Sp1-mediated transactivation in cells transfected with the F domain peptide (Fig. 6B) suggests that this region of hER may interact with other nuclear coregulatory proteins, and current studies are focused on identifying F domain-interacting factors and their function in breast cancer cells.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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-32P]ATP (3000 Ci/mmol) was purchased from NEN Life Science Products (Boston, MA). Polydeoxy-(inosinic-cytidylic)acid, and T4-polynucleotide kinase were purchased from Roche Molecular Biochemicals (Indianapolis, IN). All the restriction enzymes and modifying enzymes (T4 DNA ligase, calf intestinal alkaline phosphatase) used in this study were purchased from Promega Corp. (Madison, WI) or Roche Molecular Biochemicals. Plasmid preparation kits were purchased from QIAGEN (Valencia, CA), and 40% polyacrylamide was obtained from National Diagnostics (Atlanta, GA). All other chemicals were obtained from commercial sources at the highest quality available.
Cell Maintenance and Transient Transfection Assay
MCF-7, CHO, and MDA-MB-231 cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA). MCF-7, MDA-MB-231, and CHO cells were grown in DME/F12 (Sigma) supplemented with 2.2 g/liter sodium bicarbonate, 5% FBS, BSA (Sigma), and 10 ml/liter antibiotic/antimycotic solution (Sigma). Cells were cultured and maintained in 150-cm2 tissue culture dishes in a 37 C in 5% CO2-95% air. For transient transfection assays, cells were seeded onto six-well tissue culture plates in DME/F12 without phenol red supplemented with 2.2 g/liter sodium bicarbonate, 5% dextran-coated charcoal-stripped FBS, BSA, and 10 ml/liter antibiotic/antimycotic solution (Sigma). After 24 h, cells were transfected with the calcium phosphate method with 2 µg of luciferase reporter construct (pSp13, pERE3, pADA, and pRAR1), 250 ng pcDNA3/His/lacZ (Invitrogen, Carlsbad, CA) as a standard reference for transfection efficiency, and 1 µg or 100 ng (for cotransfection with pERE3) of the appropriate ER expression plasmid. In studies where variable amounts of coactivators were also used, the amount of DNA transfected was kept at a constant value by adding sufficient amount of empty vector. After 5–6 h, the media were removed and cells were shocked with 20% glycerol in PBS (pH 7.4) for 1 min. Cells were rinsed twice with 1 ml of PBS and treated with 5% charcoal-stripped DME/F12 either containing DMSO, E2 (10 nM), 4-OHT (1 µM), or ICI 182,780 (1 µM) for 36–40 h. After harvesting cells by scraping in 1x reporter lysis buffer (Promega Corp.), luciferase activity of aliquots of this extract were determined using the luciferase assay system (Promega Corp.). ß-Galactosidase activity was performed using Tropix Galacto-Light Plus assay system (Tropix, Bedford, MA). Light emission was detected on a lumicount micro-well plate reader (Packard Instruments, Meriden, CT), and luciferase reporter gene activity was corrected by normalizing against ß-galactosidase activity obtained from the same sample. Results are expressed as means ± SD with at least three determinations for each treatment group.
Oligonucleotides and Plasmids
hER expression plasmid was kindly provided by Dr. Ming-jer Tsai (Baylor College of Medicine, Houston, TX); ER-null and hERTAF1 and 2XF6 NR box peptide (fused to the yeast GAL4 DBD) were obtained from Dr. D. McDonnell (Duke University, Durham, NC). The hER deletion constructs hER11C, hER(265–330) (HE15), and hER(271–300) (HE12) were originally obtained from Dr. Pierre Chambon (Institut de Genetique et de Biologie Moleculaire et Cellulaire, Illkirch, France). MORs were generously provided by Dr. Malcom G. Parker (Imperial Cancer Research Fund, London, UK), and hERß was supplied by Dr. J. A. Gustafsson (Karolinska Institute, Huddinge, Sweden). Our experiments were carried out using a shorter variant form of ERß; however, in preliminary experiments using a longer form of ERß (provided by Dr. S. Mosselman, N.V. Organon, Oss, The Netherlands), minimal induction of ERß/Sp1 was also observed. SRC-1, SRC-2 (glucocorticoid receptor interacting protein 1), SRC-3 (AIB1), and p68 RNA helicase were graciously provided by Drs. B. O’Malley (Baylor College of Medicine), M. R. Stallcup (University of Southern California, Los Angeles, CA), P. Meltzer (National Cancer Institute, Bethesda, MD), and S. Kato (University of Tokyo, Tokyo, Japan), respectively. The hER cDNAs and the MOR cDNA were inserted into vectors pcDNA3 or pcDNA3.1/His C (Invitrogen, Carlsbad, CA) in this laboratory for in vitro translation and for expression in mammalian cells in transient transfection assays. For gel mobility shift assays, a consensus estrogen response element (ERE); 5'-GTC CAA AGT CAG GTC ACA GTG ACC TGA TCA AAG TT-3' (sense) was used and obtained from the Gene Technologies Laboratories, Texas A&M University (College Station, TX). The DNA oligonucleotides used for construction of plasmids were also synthesized by the Gene Technologies Laboratories (Texas A&M University). pXP1 luciferase reporter construct was obtained from ATCC, and the minimal TATA sequences were inserted into pXP1 in this laboratory. The following promoter sequences were cloned into HindIII and BamHI sites of pXP1 TATA-luciferase reporter construct: three consensus GC-rich Sp1 binding sites for pSp13 (5'-GCT TAT TCG ATC G)GG GCG GGG CGA GCA TTC GAT CGG GGC GGG GCG AGC ATT GAT CGG GGC GGG GGC GAG CG-3' (sense)]; and three consensus EREs for pERE3 (5'-AGC TTT CCG GAT CTA)GGT CAC TGT GAC CCG GGA TCC TAG GTC ACT GTG ACC CGG GAT CCT AGG TCA CTG TGA CCT GAT CAA AGT G-3' (sense)]. The GC-rich genomic promoter sequence from RAR1 gene (pRAR1; -79 to -49) (5'-AGC TTG ATT GGT CGG T)GG GCG GGC AGG GGC GGG CCT-3' (sense)] and the GC-rich genomic promoter sequence from the pADA gene (-86 to -65) (5'-AGC TTG GCG AGA G)GG CGG GCC CCG GGA-3' (sense)] were also cloned into HindIII and BamHI sites of pXP1 luciferase reporter construct. The GC-rich and ERE motifs are underlined.
Generation of ER Deletion Mutant Constructs
ER DBD deletion constructs were prepared by site-directed mutagenesis by overlap extension using the PCR as previously described (60). For example, hERZF1 in pcDNA3 was constructed by carrying out the following procedures. One set of primers (A1/B1) from the HindIII site (A1) in the multiple cloning site in pcDNA3 to the site before the first amino acid in the region to be deleted was amplified by PCR; the latter primer (B1) has an overlapping region of approximately 15 to 20 bp that starts at the next amino acid in the deletion construct. In addition, another set of primers beginning just after the last amino acid to be deleted to the HindIII site in hER cDNA sequence were also used and amplified by PCR. This second set of primers (A2/B2) contained a 15- to 20-bp overlapping region complementary to the last 15- to 20-bp DNA sequence in the first PCR product. Both PCR reaction products have their own regions of overlap, and these were coincubated to anneal the overlapping regions; this was followed by PCR amplification with the primer (A1) that starts at the multiple cloning site and the primer (B2) that starts at the hER cDNA sequence. The resulting PCR product containing the desired deletion and a unique restriction site at both ends (HindIII) was purified, digested with HindIII, and finally cloned into pcDNA3 construct to give the appropriate expression plasmid for electrophoretic mobility shift and transient transfection assays.
The primers used for the mutagenesis assays are summarized in Table 1. hERZF2 in pcDNA3 was also generated by using a unique HindIII restriction site for cloning into this vector. Generation of hERßZF1 and hERßZF2 used the unique NheI and EcoRI sites of previously modified hERß in pcDNA3.1 (35). MOR cDNA was inserted into EcoRI site of pMT2 mammalian expression vector that contains unique NotI and XbaI sites in MOR cDNA sequence suitable for cloning the PCR-amplified insert containing deletion of one zinc finger domain, into pMT2. The EcoRI fragment containing the desired deletion from pMT2 MOR vector was cloned into EcoRI site of pcDNA3.1 for in vitro translation. The hERTAF1 construct (in pcDNA3) has three point mutations (D538N, E542Q, and D545N) in AF2 (40, 41). hERTAF1ZF1 and hERTAF1ZF2 constructs were created by cloning the XbaI fragment (
0.7 kb) from hERTAF1 (in pcDNA3) into pcDNA3.1, and zinc finger mutants were prepared as described above. hER(538–595) was made by PCR amplification of wild-type hER using primer sets: (F1) 5'-TGC TAG CAT GAC CAT GAC CCT CCA CAC C-3' and (R1) 5'-GAC TCG AGT CAA GTG GGC GCA TGT AGG CGG TG-3'. hER(554–595) and hER(579–595) were also amplified with the same F1 primer above and (R2) 5'-GAC TCG AGT CAA GTG GGC GCA TGT AGG CGG TG-3' or (R3) 5'-TAC TCG AGT CAC AAG GAA TGC GAT GAA GTA GAG-3', respectively. The amplified fragments were digested and inserted into NheI and XhoI sites of pcDNA3.0 or 3.1 expression vector. GAL4-DBD fusion Fß peptide [21 amino acids from C-terminal end of hER (aa 575–595)] was made by using BamHI and HindIII sites within the multiple cloning region of pM vector (CLONTECH Laboratories, Inc., Palo Alto, CA) with (F) 5'-GAT CCG TTCA TCG CAT TCC TTG CAA AAG TAT TAC ATC ACG GGG GAG GCA GAG GGT TTC CCT GCC ACA GTC TGA A-3' and (R) 5'-AGC TTT CAG ACT GTG GCA GGA AAC CCT CTG CCT CCC CCG TGA TGT AAT ACT TTT GCA AGG AAT GCG ATG AAC G-3'. All constructs were mapped by restriction enzymes and sequenced to confirm that proper deletion or insertions were introduced into the target cDNA.
In Vitro Translation and Detection of the Translated Proteins
Wild-type ER and ER deletion mutants were synthesized in vitro using TNT T7 quick coupled transcription/translation System (Promega Corp.) in the presence or absence of [35S]methionine for EMSAs and separation by 10% SDS-PAGE.
EMSAs
The consensus [32P]ERE oligonucleotide was annealed and end labeled using T4-polynucleotide kinase and [-32P]ATP. To characterize DNA binding of wild-type ER and corresponding zinc finger deletion mutants, 0.5 µl of in vitro translated protein or 0.5 µl of unprogrammed lysate was incubated in 1x binding buffer (25% glycerol, 0.5 mM EDTA, 0.5 mM dithiothreitol, 50 mM potassium chloride, 10 mM HEPES at pH 8.0) for 5 min at 4 C. Radiolabeled consensus ERE oligonucleotide (60,000 cpm) was added to the reaction, and the reaction mixture was incubated at 25 C for 15 min. Samples were then applied to the gel and separated by polyacrylamide gel electrophoresis at 120 V in 0.9 mM Tris, 0.9 M borate, 2 mM EDTA (pH 8.0) for 2–3 h. Protein-DNA complexes were visualized by autoradiography using X-Omat film (Eastman Kodak Co., Rochester, NY).
Fluorescence Immunocytochemistry
MDA-MB-231 cells were subcultured in four-well Lab-Tek chambered slides (Nunc Inc., Naperville, IL) in DME/F12 medium without phenol red 5% FBS stripped with dextran-coated charcoal. After 24 h, cells were transiently transfected with 500 ng of hER or hER mutant expression plasmids. For transient transfection studies, cells were incubated with FuGENE Transfection Reagent (Roche) at 37 C for 5 h, followed by 24 h of recovery in DME/F12. Before fixation, slides were washed three times in Dulbecco’s PBS (DPBS) and then fixed for 10 min at -20 C at 100% methanol. For nuclear localization of ER, the rat monoclonal antibody raised against the N-terminal domain of the hER (H184; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) was diluted to a final concentration of 3 µg/ml in DPBS containing 0.5% BSA, 0.1% goat serum, and 0.3% Tween 20. Rat IgG at the same concentration was used as a control. Tween 20 (0.3%) was included in all antibody, blocking steps, and washes for nuclear localization of ER. Followed by incubation with H184 antibody for 16 h, cells were washed with DPBS (three times), then incubated for 1 h in a 1:200 dilution of fluorescein isothiocyanate-conjugated goat-antirat IgG (62–9511; Zymed Laboratories, Inc., South San Francisco, CA) in DPBS containing 0.1% goat serum. Cells were then washed (four times) over a period of 2 h and transferred to DPBS before coverslip mounting with ProLong Antifade mounting reagent (Molecular Probes, Inc., Eugene, OR). For each treatment, representative fluorescence images were recorded using an Axioplan microscope (Carl Zeiss, Thornwood, NY) equipped with a Hamamatsu chilled three charge-coupled device color camera (Hamamatsu, Japan) using Adobe Photoshop 5.0 (Adobe Systems, Seattle, WA) image capture software. Images from all treatment groups were captured at the same time using identical image capture parameters. Slides were subsequently washed three times in DPBS followed by a 1-h blocking step in 3% normal goat serum (G-9023; Sigma).
Statistics
For transient transfection studies, results are expressed as means ± SD for at least three separate experiments for each treatment group. Statistical differences (P < 0.05) between control (DMSO) and treatment groups or between E2-induced responses and treatment groups (coactivator experiments) were determined by ANOVA and Scheffe’s post hoc test.
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FOOTNOTES |
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Abbreviations: aa, Amino acids; ADA, adenosine deaminase; AF, activation function; AP1, activator protein 1; CHO, Chinese hamster ovary; DBD, DNA-binding domain; DMSO, dimethylsulfoxide; DPBS, Dulbecco’s PBS; E2, 17ß-estradiol; ER, estrogen receptor; ERE, estrogen responsive element; FBS, fetal bovine serum; hER, human ER; MOR, mouse ER; 4-OHT, 4-hydroxytamoxifen; RAR, retinoic acid receptor; SERM, selective ER modulator; SRC, steroid receptor coactivator.
Received for publication December 5, 2002. Accepted for publication January 27, 2003.
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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) of E2-induced activities are indicated; similar results were observed after transfecting higher amounts (500 ng) of each coactivator in both cell lines. Results are expressed as means ± SD for three separate experiments for each treatment group. E, Coactivation of hER
