No abstract

Induction of cyclin D1 gene transcription by estrogen receptor ␣ (ER␣) plays an important role in estrogenmediated proliferation. There is no classical estrogen response element in the cyclin D1 promoter, and induction by ER␣ has been mapped to an alternative response element, a cyclic AMP-response element at ؊57, with possible participation of an activating protein-1 site at ؊954. The action of ER␤ at the cyclin D1 promoter is unknown, although evidence suggests that ER␤ may inhibit the proliferative action of ER␣. We examined the response of cyclin D1 promoter constructs by luciferase assay and the response of the endogenous protein by Western blot in HeLa cells transiently expressing ER␣, ER␣K206A (a derivative that is superactive at alternative response elements), or ER␤. In each case, ER activation at the cyclin D1 promoter is mediated by both the cyclic AMP-response element and the activating protein-1 site, which play partly redundant roles. The activation by ER␤ occurs only with antiestrogens. Estrogens, which activate cyclin D1 gene expression with ER␣, inhibit expression with ER␤. Strikingly, the presence of ER␤ completely inhibits cyclin D1 gene activation by estrogen and ER␣ or even by estrogen and the superactive ER␣K206A. The observation of the opposing action and dominance of ER␤ over ER␣ in activation of cyclin D1 gene expression has implications for the postulated role of ER␤ as a modulator of the proliferative effects of estrogen.

We find that tamoxifen is a potent activator of estrogen receptor (ER)- mediated induction of promoters regulated by AP-1 sites including the human collagenase gene promoter and constructs in which an AP-1 site is fused to the herpes thymidine kinase promoter. This contrasts with the inability of tamoxifen to activate otherwise identical promoters bearing classical estrogen response elements. Tamoxifen agonism at AP-1 sites is cell type specific, occurring in cell lines of uterine, but not of breast, origin. It thus parallels tamoxifen agonism in vivo. AP-1 proteins such as Jun or Jun/Fos are needed for tamoxifen stimulation, and tamoxifen increases the transcriptional efficiency of these proteins even when they are provided at optimal amounts. The DNA binding domain (DBD) of ER is required for tamoxifen activation at AP-1 sites. In contrast, estrogen activation is partially independent of this domain. This suggests the existence of two pathways of ER action at AP-1: an alpha (DBD-dependent) pathway activated by tamoxifen, and a beta (DBD-independent) pathway activated by estrogen. Fusing VP16 transcriptional activation functions to ER potentiates the beta, but not the alpha, pathway. We discuss models for the two pathways and the possibility that the AP-1 pathway is a major route by which ER affects target tissue growth and differentiation in vivo.

The activity of the AF-2 transcriptional activation function of nuclear receptors (NR) is mediated by the partially homologous transcriptional coactivators, glucocorticoid receptor interacting protein 1 (GRIP1)/transcriptional intermediary factor 2 (TIF2) and steroid receptor coactivator 1 (SRC-1). GRIP1 and SRC-1 bound nine different NRs and exhibited similar, but not identical, NR binding preferences. The most striking difference was seen with the androgen receptor, which bound well to GRIP1 but poorly to SRC-1. GRIP1 and SRC-1 contain three copies of the NR binding motif LXXLL (called an NR Box) in their central regions. Mutation of both NR Box II and NR Box III in GRIP1 almost completely eliminated functional and binding interactions with NRs, indicating that these two sites are crucial for most of GRIP1's NR binding activity. Interactions of GRIP1 with the estrogen receptor were more strongly affected by mutations in NR Box II, whereas interactions with the androgen receptor and glucocorticoid receptor were more strongly affected by NR Box III mutations. One isoform of SRC-1 has an additional NR Box (NR Box IV) at its extreme C terminus with an NR-binding preference somewhat different from that of the central NR-binding domain of SRC-1. GRIP1 has no NR Box in its C-terminal region and therefore no C-terminal NR-binding function. In summary, GRIP1 and SRC-1 have overlapping NR-binding preferences, but specific NRs display both coactivator and NR Box preferences that may contribute to the specificity of hormonal responses.

In the central nervous system, CRH regulates several affective states. Dysregulation of neuronal crh expression in the paraventricular nucleus of the hypothalamus correlates with some forms of depression, and amygdalar crh expression may modulate levels of anxiety. Because estrogens modulate these states, we sought to determine 17beta-estradiol (E2) effects on crh expression. CRH mRNA levels were measured in the AR-5 amygdaloid cell line by RT-PCR analysis. They increased by 1 min of E2 treatment, suggesting that crh behaves as an immediate-early gene. After peaking at 3 min, CRH mRNA returned to basal levels and then increased by 60 min. To dissect some of the molecular mechanisms underlying these events, we measured occupancy of the crh promoter by estrogen receptors (ERs) and coactivators, using chromatin immunoprecipitation. Because this promoter does not contain palindromic estrogen response elements, we targeted the region of a cAMP regulatory element (CRE), implicated in crh regulation. The temporal pattern of the mRNA response was mimicked by recruitment of ERalpha and -beta, phospho-CRE-binding protein, coactivators steroid receptor coactivator-1 and CRE-binding protein-binding protein (CBP), and an increase in histone 3 and 4 acetylation. Lastly, ERalpha and -beta loading were temporally dissociated, peaking at 1 and 3 min, respectively. The ER peaks were associated with coactivators and acetylation patterns. ERalpha associated with phospho-CRE-binding protein, CBP, steroid receptor coactivator-1, and increased acetylated histone 3. ERbeta associated with CBP and increased acetylated histone 4. The tight temporal correlation between E2-induced CRH mRNA levels and promoter occupancy by ERs strongly suggest that E2 regulates crh expression through an ERalpha- and/or ERbeta-CRE alternate pathway.