Aspartate 351 of estrogen receptor alpha is not crucial for the antagonist activity of antiestrogens.

The antagonist activity of antiestrogens is due to the presence of a long carbon side chain at positions 7alpha or 11beta or equivalent on their steroid or steroid-like skeletons. These side chains establish hydrophobic interactions with amino acids of the estrogen receptor alpha (ERalpha) ligand binding domain. In addition, a hydrogen bond formed between amino acid Asp-351 and the tertiary amine present at the end of the side chain of partial antiestrogens is considered to be crucial for their antiestrogenicity. Here, we have investigated the role of Asp-351 in antiestrogen action in transiently transfected HeLa and MDA-MB-231 cells. Our results indicate that disruption of the negative charge at position 351 does not increase the agonist activity of partial antiestrogens and thus that the hydrogen bond with the antiestrogen side chain is not determinant in positioning the side chain in an antagonist position. The negative charge at position 351 was not required for transcriptional activity in the presence of hormone, but its presence was necessary for basal activity of the wild-type receptor and constitutive activities of mutants L536P and Y537A, suggesting a role of Asp-351 in stabilizing the active conformation of ERalpha. This stabilizing role of Asp-351 could be due to interaction of Asp-351 with the amide group of the peptide bond between Leu-539 and Leu-540 in helix 12 observed in the active conformation of the ERalpha ligand binding domain.

Different classes of coactivators recognize distinct but overlapping binding sites on the estrogen receptor ligand binding domain.

We have analyzed interaction of coactivators with the wild-type estrogen receptor alpha (ER), HEG0, and a mutant, L536P-HEG0, which is constitutively active in several transiently transfected cells and a HeLa line that stably propagates an estrogen-sensitive reporter gene. Different classes of coactivators do not recognize the ER ligand binding domain (LBD) in the same manner. Steroid receptor coactivator-1 (SRC-1), amplified in breast cancer-1 (AIB-1), transcriptional intermediary factor-1 (TIF-1), transcriptional intermediary factor-2 (TIF-2), and receptor interacting protein 140 (RIP140) interacted with HEG0 and L536P-HEG0 in the presence of estradiol, but generally not in the presence of anti-estrogens. However, ICI164,384 stimulated some interaction of RIP140 with LBDs. SRC-1, AIB-1, and RIP140 interacted constitutively with the L536P ER, whereas TIF-1 and TIF-2 interacted only weakly in the absence of hormone. Reciprocal competition for binding to the ER LBD was observed between different classes of coactivators. Moreover, coexpression of RIP140 blocked enhanced transactivation by HEG0 observed in the presence of TIF-2, suggesting that RIP140 may play a negative role in ER signaling. We conclude that constitutive activity of L536P-HEG0 is manifested to similar degrees in different cell types and likely arises from constitutive coactivator binding; different classes of coactivators recognize distinct but overlapping binding sites on the ER LBD. Finally, the observation that L536P-HEG0 interacted constitutively with AIB-1, a coactivator that has been implicated in ER signaling in breast and ovarian cancer, suggests that similar mutations in the ER may contribute to hormone-independent proliferation of breast and ovarian cells.

Estrogen response elements can mediate agonist activity of anti-estrogens in human endometrial Ishikawa cells.

Anti-estrogens like hydroxytamoxifen (OHT) have mixed agonist/antagonist activities, leading to tissue-specific stimulation of cellular proliferation. Partial agonist activity of OHT can be observed in vitro in endometrial carcinoma cells like Ishikawa. Here, we have compared several anti-estrogens (including extensively characterized OHT and pure anti-estrogens such as ICI164, 384 and RU58,668, which are devoid of uterotrophic activity) for their capacity to stimulate promoters containing estrogen response elements (EREs) or AP1-binding sites (12-O-tetradecanoylphorbol-13-acetate response elements, TREs), the two types of DNA motifs known to mediate transcriptional stimulation by estrogen receptors. Assays were performed in Ishikawa cells either by transient transfection or by using cell lines with stably propagated reporter vectors. In transient transfection experiments, none of the anti-estrogens displayed agonist activity on the promoters tested. In contrast, significant transcriptional stimulation was observed with low concentrations of OHT and RU39,411 in Ishikawa cells stably propagating reporter constructs containing a minimal ERE3-TATA promoter. In addition, micromolar concentrations of OHT, but not of RU39,411, stimulated stably propagated AP1-responsive reporter constructs. No transcriptional stimulation of ERE- or TRE-containing promoters was observed with the pure anti-estrogens ICI164,384 and RU58,668. These results indicate that the presence of estrogen response elements in promoters is sufficient to mediate cell-specific agonism of anti-estrogens at the transcriptional level, and that stimulation of AP1 activity may be restricted to a subset of anti-estrogens possessing agonist activity on EREs. In addition, our results suggest that transient transfections do not fully recapitulate in vivo conditions required to observe agonist activity of anti-estrogens.