Molecular mechanisms of estrogen and antiestrogen action

Estrogen is a key regulatory hormone that affects numerous physiological processes. The estrogen receptors (ER), ER alpha and ER beta, play a central role in mediating the effects of both estrogens and antiestrogens. Estrogen signaling has proven to be multifaceted and the precise mechanism of action remains in many cases elusive. The work presented in this thesis provides further insight into the mechanisms that control the transcriptional regulation of estrogen and antiestrogen responsive genes.

The effect of tamoxifen and raloxifene on the sex hormone binding globulin (SHBG) gene was studied in human hepatocarcinoma cells (HepG2). Both tamoxifen and raloxifene functioned as estrogen agonists on the SHBG gene at doses higher than 1 mu M in a non-ER-dependent fashion. Both compounds displayed an additive effect to estrogen induced ER-dependent SHBG expression. Thus, we propose that the elevated SHBG levels observed in pre- menopausal women on adjuvant tamoxifen treatment may be explained by non-ER dependent tamixofen agonism.

The discovery of ER beta promises an opportunity to develop ligands. with improved tissue selectivity. Despite the great homology of the amino acids lining the ligand-binding cavities of ER alpha and ER beta, we showed selective effects of ER alpha and ER beta on an ERE-reporter in response to a set of ERligands. We showed that tamoxifen, 40H-tamoxifen and raloxifene displayed an ER alpha-selective partial agonism, whereas their effect via ER beta was antagonistic. In conclusion, our data indicated that it is possible to develop novel receptor subtype specific ligands that may have an improved tissue selectivity and side effect profile.

The pS2 gene is estrogen responsive in hepatocarcinoma cells (HepG2) in the presence of ER alpha (HepER3 cells). The estrogenic activity is mediated through an estrogen response element (ERE) in the 5'-flanking region of the pS2 gene, however, an AP1 response element located close to the ERE in the pS2 promoter was also essential for response to estrogen. The potentiation of pS2 promoter activity by the AP1 motif in response to estrogen was dependent on the ligand-binding domain of ER alpha. Furthermore, the presence of an intact AP1 element in the pS2 promoter sustained suppression of pS2 promoter activity by an LXXLL peptide. The phorbol ester PMA stimulated pS2 expression in both HepER3 and the parental, non-ER expressing, HepG2 cells although its activity was substantially less in HepG2 cells. The effect of PMA was mainly mediated through the AP1 element. In summary, the data suggest that the effect of estrogen is mediated through a crosstalk between the ERE and the AN response element and that ER alpha plays a crucial role in mediating the effect, not only of estrogen but also of PMA.

We show estrogen-induced synergistic activity by the p160 coactivator SRC- 1, mediated via the ERE and the AP1 response element in the pS2 promoter. In addition, we present data that support an interaction between the ERE and the AP1 motif via SRC-1. Also TIF-2, a related but distinct p160 coactivator, potentiated the estrogenic response of the pS2 gene, however, TIF-2 was less dependent on an intact AN response element in the pS2 promoter than SRC-1. Furthermore, the type of ERE in the pS2 promoter influenced the potentiation by SRC-1 ; in support of this, there was less dependence on the AP1 motif when the natural ERE was substituted for by a consensus ERE. These results highlight several mechanisms whereby fine-tuning of estrogen responsiveness of an individual gene may be achieved.

List of scientific papers

I. Barkhem T, Andersson-Ross C, Hoglund M, Nilsson S (1997). Characterization of the "estrogenicity" of tamoxifen and raloxifene in HepG2 cells: regulation of gene expression from an ERE controlled reporter vector versus regulation of the endogenous SHBG and PS2 genes. J Steroid Biochem Mol Biol. 62(1): 53-64.
https://pubmed.ncbi.nlm.nih.gov/9366498

II. Barkhem T, Carlsson B, Nilsson Y, Enmark E, Gustafsson J, Nilsson S (1998). Differential response of estrogen receptor alpha and estrogen receptor beta to partial estrogen agonists/antagonists. Mol Pharmacol. 54(1): 105-12.
https://pubmed.ncbi.nlm.nih.gov/9658195

III. Barkhem T, Haldosen LA, Gustafsson JA, Nilsson S (2002). pS2 Gene expression in HepG2 cells: complex regulation through crosstalk between the estrogen receptor alpha, an estrogen-responsive element, and the activator protein 1 response element. Mol Pharmacol. 61(6): 1273-83.
https://pubmed.ncbi.nlm.nih.gov/12021387

IV. Barkhem T, Haldosen LA, Gustafsson JA, Nilsson S (2002). Transcriptional synergism on the pS2 gene promoter between a p160 coactivator and estrogen receptor-alpha depends on the coactivator subtype, the type of estrogen response element, and the promoter context. Mol Endocrinol. 16(11): 2571-81.
https://pubmed.ncbi.nlm.nih.gov/12403846