Analysis of mechanisms that determine dominant negative estrogen receptor effectiveness.

To analyze the mechanisms by which estrogen receptor (ER) activity is suppressed by dominant negative mutants, we examined the role of specific ER functions and domains in transcriptional repression. We previously described three transcriptionally inactive human ER mutants (the frameshift mutant S554fs, the point mutant L540Q, and the truncated receptor ER1-530), which act as effective dominant negative mutants, inhibiting the activity of wild type ER when they are coexpressed in mammalian cells. After additional mutational modifications, the ability of the ER mutants to suppress the activity of wild type ER was analyzed in cotransfection assays of the dominant negative mutants and wild type ER and an estrogen-responsive reporter gene (2ERE-TATA-CAT or 2ERE-pS2-CAT). Eliminating the ability of the three dominant negative mutants to bind to estrogen response element (ERE) DNA (by introducing three point mutations in their DNA binding domains) dramatically reduced, but did not completely abolish, the dominant negative activity of the ER mutants. The mutation G521R, which rendered the three mutants incapable of binding estradiol, also reduced, but did not abolish, their dominant negative activity. Immunoprecipitation with monoclonal or flag antibodies followed by Western blotting demonstrated that each of the original dominant negative ER mutants formed heterodimers with wild type ER. Rendering the dominant negative mutants dimerization deficient by the mutation L507R strongly reduced, but did not eliminate, their dominant negative activity. Deletion of the N-terminal A/B domain resulted in the nearly complete loss of inhibitory activity of the three dominant negative mutants. However, these double mutants retained their ability to heterodimerize with wild type ER, suggesting that dominant negative interference also occurs at an additional step beyond dimerization. Our data indicate that competition for ERE binding, formation of inactive heterodimers, and specific transcriptional silencing can all contribute to the dominant negative phenotype and that these receptors suppress the activity of wild type ER by acting at multiple steps in the ER-response pathway.

Repression of endogenous estrogen receptor activity in MCF-7 human breast cancer cells by dominant negative estrogen receptors.

We have investigated the ability of several transcriptionally inactive estrogen receptor (ER) mutants to block endogenous ER-mediated transcription in MCF-7 human breast cancer cells. In transient transfections of MCF-7 cells, two of the mutants, a frame-shifted ER (S554fs) and a point-mutated ER (L540Q), strongly inhibit the ability of endogenous wild-type ER to activate transcription of estrogen-regulated reporter plasmids. A third mutant, ER1-530, which is missing 65 residues from its carboxy-terminus, is a weaker repressor of estradiol-stimulated transcription. When an estrogen response element (ERE)-thymidine kinase-chloramphenicol acetyltransferase reporter gene is used, S554fs, L540Q, and ER1-530 suppress the transcriptional activity of endogenous MCF-7 ER by 87%, 97%, and 62%, respectively. The magnitude of dominant negative repression is promoter specific; when an ERE-pS2-chloramphenicol acetyltransferase reporter is employed, inhibition of endogenous ER activity by equivalent amounts of S554fs, L540Q, and ER1-530 ranges from 85-97%. Dose-response studies show the S554fs mutant to be the most potent of the three ER mutants as a repressor of estrogen action in these cells. In addition, elevated levels of intracellular cAMP, achieved by the addition of 3-isobutyl-1-methylxanthine plus cholera toxin to cells, fail to compromise the effectiveness of these mutants as dominant negative ERs despite the cAMP-enhanced transcriptional activity of ER. The mutants are also powerful repressors of the agonist activity of trans-hydroxytamoxifen-stimulated ER transcription. The dominant negative activity of the three mutants is lost when the A/B domain of these receptors is deleted, implying an important role for this N-terminal region of the ER in the ability of these mutants to inhibit endogenous wild-type ER activity. All in all, the data suggest that S554fs in particular is a reasonable candidate for studies designed to use a dominant negative ER to inhibit the estrogen- and tamoxifen-stimulated growth of human breast cancer cells.

Antiestrogens: mechanisms and actions in target cells.

Antiestrogens, acting via the estrogen receptor (ER) evoke conformational changes in the ER and inhibit the effects of estrogens as well as exerting anti-growth factor activities. Although the binding of estrogens and antiestrogens is mutually competitive, studies with ER mutants indicate that some of the contact sites of estrogens and antiestrogens are likely different. Some mutations in the hormone-binding domain of the ER and deletions of C-terminal regions result in ligand discrimination mutants, i.e. receptors that are differentially altered in their ability to bind and/or mediate the actions of estrogens vs antiestrogens. Studies in a variety of cell lines and with different promoters indicate marked cell context- and promoter-dependence in the actions of antiestrogens and variant ERs. In several cell systems, estrogens and protein kinase activators such as cAMP synergize to enhance the transcriptional activity of the ER in a promoter-specific manner. In addition, cAMP changes the agonist/antagonist balance of tamoxifen-like antiestrogens, increasing their agonistic activity and reducing their efficacy in reversing estrogen actions. Estrogens, and antiestrogens to a lesser extent, as well as protein kinase activators and growth factors increase phosphorylation of the ER and/or proteins involved in the ER-specific response pathway. These changes in phosphorylation alter the biological effectiveness of the ER. Multiple interactions among different cellular signal transduction systems are involved in the regulation of cell proliferation and gene expression by estrogens and antiestrogens.

Phosphorylation of the human estrogen receptor. Identification of hormone-regulated sites and examination of their influence on transcriptional activity.

We have used a transient transfection system with a cytomegalovirus-based vector expressing high levels of biologically active human estrogen receptor (ER) in COS-1 cells to study the phosphorylation of human ER and to identify major hormone-regulated phosphorylation sites. The features of phosphorylation of the wild-type ER were very similar to those previously observed for the endogenous ER in uterine cells: The ER exhibited a basal level of phosphorylation which was increased approximately 3-4-fold by estrogen (estradiol) and by antiestrogens (hydroxytamoxifen and ICI164,384), and phosphorylation was increased to an almost similar extent by activation of either protein kinase A or C signal transduction pathways with cholera toxin plus isobutyl methylxanthine (CT+IBMX) or phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), respectively. Phosphoamino acid analysis revealed that the phosphorylation occurred exclusively on serine residues in all cases. Tryptic phosphopeptide analysis of ER, using a two-dimensional peptide mapping procedure, revealed similar patterns for ER in cells treated with estradiol, antiestrogens or TPA; with CT+IBMX treatment, the same phosphopeptides were seen, but the relative phosphorylation of the different ER phosphotryptic peptides differed. In ER deleted of the NH2-terminal A and B (A/B) domains, estrogen and antiestrogen-stimulated phosphorylations were abolished, while the phosphorylation induced by CT+IBMX was maintained. This suggests that sites of phosphorylation enhanced by estradiol and antiestrogen, but not those induced by CT+IBMX, are located in the A/B domain. These results were further confirmed by comparing the tryptic phosphopeptide patterns of wild-type and A/B-deleted receptor upon estradiol and CT+IBMX treatments, and then by site-directed mutagenesis, by substituting alanines for the serine residues in the A/B domain (Ser104, Ser106, Ser118, Ser154, and Ser167) involved in known protein kinase consensus sequences. Comparison of the tryptic phosphopeptide patterns of wild-type ER and these mutant ERs allowed us to identify serine 104 and/or serine 106 and serine 118, all three being part of a serine-proline motif, the preferred substrate of proline-directed protein kinase, as major ER phosphorylation sites. When tested with two estrogen-responsive reporter gene constructs in several cell types, the mutant S104A, S106A, S118A showed a approximately 40% reduction in transactivation activity in response to E2, while the mutants S118A and S104A, S106A alone showed a approximately 15% decrease in transactivation. Our studies identify several serines in the NH2-terminal portion of the human ER as being major hormone-regulated phosphorylation sites.(ABSTRACT TRUNCATED AT 250 WORDS)