Estrogen regulation of the insulin-like growth factor I gene transcription involves an AP-1 enhancer

As a step toward elucidating the physiological role of insulin-like growth factor-I (IGF-I) in mediating estrogen action, we sought to determine the molecular basis of the phenomenon. In HepG2 cells expressing exogenous estrogen receptors (ER), a reporter gene plasmid containing 600 base pairs of the chicken IGF-I promoter enhanced expression of luciferase 8.6-fold in response to 10(-6) M 17 beta-estradiol, indicating that the IGF-I promoter is a target of estrogen regulation. Although no conventional estrogen-responsive element was identified within the promoter fragment, the AP-1 motif located therein was shown to be essential; the estrogen-responsive enhancement of the Fos-Jun binding to the AP-1 motif, which takes place by means of post-translational modification, mediates the estrogen action. A direct or indirect interaction between the estrogen-ER complex and the Fos-Jun complex seems to facilitate the Fos-Jun binding to the target DNA. Although ER binding to the target DNA was not considered to be involved in the signaling pathway, the DNA binding domain-deficient ER did not mediate the phenomenon, providing support for the existence of a unique function of the DNA binding domain of ER in facilitating some protein-protein interaction. In conclusion, our present observations demonstrate that the chicken IGF-I gene promoter is controlled by estrogen through a unique pathway involving Fos, Jun, and the DNA binding domain of ER.     

Estradiol induction of retinoic acid receptors in human breast cancer cells

Retinoic acid inhibits proliferation and steroid receptor gene expression in human breast cancer cell lines. Retinoic acid receptors (RAR)alpha, -beta, and -gamma are expressed in these cells and the expression of RAR alpha is significantly greater in estrogen receptor (ER)-positive cells. This study was undertaken to determine whether the same relationship between RAR alpha and ER gene expression was present in human breast cancers and to explore the possibility that the higher level of RAR alpha in ER-positive cells was due to estrogen regulation of RAR alpha gene expression. RAR alpha and ER mRNA expression were determined by Northern blot analysis in 116 primary breast tumors; 94 (81%) tumors were ER-positive and of these 87 (93%) were also RAR alpha-positive. The coexpression of ER and RAR alpha was statistically significant (P = 0.0052 by chi 2 contingency analysis). There was also a positive correlation (by linear regression analysis) between the levels of expression of ER and RAR alpha mRNA (r2 = 0.251, P = 0.0001), which confirmed the relationship previously documented in breast cancer cell lines and suggested that RAR alpha expression may be modulated in breast cancer in vivo by estrogens acting via the ER. The ability of estradiol to regulate RAR alpha gene expression was examined in vitro using T-47D cells which had been rendered sensitive to estrogen by repeated passage in steroid-depleted medium. Estradiol increased RAR alpha gene expression, but not that of RAR beta or RAR gamma, in a concentration-dependent manner, with the effect being maximal at 10(-10) M and less marked at higher concentrations. The effect was rapid, being detectable 1 h after and maximal 6 h after treatment with 10(-10) M estradiol. Co-treatment of cells with estradiol and antiestrogens (tamoxifen or ICI 164384, 4 x 10(-7) M for 6 h) inhibited the estradiol induction of RAR alpha gene expression, demonstrating that the effect was ER mediated. The estradiol sensitivity of the effect was underscored by the demonstration that addition of untreated serum to cells growing under steroid-depleted conditions was sufficient to induce maximal RAR alpha gene expression. This effect was totally abolished by addition of ICI 164384. In summary, the demonstration that estradiol increased RAR alpha mRNA levels in breast cancer cells supports the hypothesis that the correlation between RAR alpha and ER gene expression in breast tumors and breast cancer cell lines is due to estradiol augmentation of RAR alpha gene expression.     

Characterization of heterologously expressed recombinant retinoic acid receptors with natural or synthetic retinoids

The first step in retinoid action is binding to their nuclear receptors. Therefore, characterization of binding characteristics of retinoids is of major importance. Human retinoic acid receptors alpha (hRAR alpha), hRAR beta, and mouse RAR gamma (mRAR gamma) were expressed heterologously in Escherichia coli as a recombinant glutathione S-transferase (GST) fusion protein. The expressed fusion proteins were functional and bound specifically to the all-trans-retinoic acid (RA). The dissociation constants (Kd) for RA were 1.4 nM for GST-hRAR alpha, 1.4 nM for GST-hRAR beta, and 3.3 nM for GST-mRAR gamma, respectively. The fusion proteins were further used for competitive displacement assays to determine the displacement constant (DC50) for other selected retinoids. All-trans-RA and 4-oxo-all-trans-RA have high affinity with all three receptors (DC50 = 0.8-55 nM). The 13-cis RA binds to hRAR alpha with low affinity, but not to other RARs evaluated here. All-trans-N-ethylretinamide, all-trans-retinylacetate, and an ethyl ester of tetrahydronaphthalene derivative had no affinity to any RARs. The hRAR alpha and mRAR gamma receptors did not bind a naphthalene carboxylic acid derivative of RA, but hRAR beta binds this chemical with high affinity. Results indicated that the three recombinant proteins were functional in binding various RA congeners. The affinity and binding data of these retinoids were compared to their observed teratogenic activity.     

Estrogen receptor does not directly regulate the murine Muc-1 promoter

Muc-1 is a heavily O-glycosylated, type 1 membrane glycoprotein present on the surface of polarized secretory uterine epithelial cells. Previous studies have shown that treatment of ovariectomized mice with 17-beta-estradiol (E2) strongly induces Muc-1 mRNA expression in an estrogen receptor (ER)-mediated fashion in the uterus. In this study, the 5.4 kb Muc-1 gene promoter has been isolated from a mouse genomic library and the proximal 1.85 kb region has been sequenced. Sequence analysis revealed the presence of one potential full estrogen response element (ERE) (GCTCGCGGTGACC) located at -748 to -735 bp in the Muc-1 promoter and several potential ERE half sites. Electrophoretic mobility shift assays (EMSA) showed that neither ERalpha nor ERbeta bind efficiently to this sequence. Transient cotransfection assays using constructs containing various deletion mutations of the 5' Muc-1 flanking sequences showed that E2 had no direct stimulation on promoter-driven reporter in NMuMG cells or primary mouse uterine epithelial cells, but did stimulate a consensus ERE CAT-reporter gene activity. In addition, E2-treatment of Weg-ER cells, a mouse uterine epithelial cell line stably expressing human ERalpha, did not restore endogenous Muc-1 expression or activate Muc-1 promoter-driven CAT activity. These results indicate that regions of the Muc-1 gene promoter within -1838 to +43 bp do not respond to E2 and ER stimulation and that ER alone is not sufficient to restore Muc1 gene expression. Deletion analyses also revealed that the sequence between -73 and +43 bp of the Muc-1 promoter is the minimal promoter region required for maximal Muc-1 promoter activity. Collectively, these results demonstrate that ER does not directly regulate the 1.85 kb murine Muc-1 gene promoter. Therefore, E2 control of uterine Muc-1 gene expression is likely to be indirect, i.e. mediated by stromal cell-derived factors.     

Identification and characterization of a putative bile acid-responsive element in cholesterol 7 alpha-hydroxylase gene promoter

Nucleotide sequences of a 7997-base pair SacI fragment spanning 3643 base pairs of the upstream promoter region to exon 4 of the rat cholesterol 7 alpha-hydroxylase gene (CYP7) have been determined. DNase I footprinting and electrophoretic mobility shift assay of the proximal promoter from nucleotides -346 to +36 revealed two protected regions which specifically shifted proteins in rat liver nuclear extracts. Footprint A (nucleotides -81 to -35) contained a cluster of overlapping sequence motifs of TGT3, steroid/thyroid hormone response elements (7 alpha TRE), hepatocyte nuclear factors 1 and 4, and CAAT/enhancer-binding protein alpha and has been shown to confer bile acid repression of the CYP7 gene promoter activity. Footprint B (nucleotides -148 to -129) contained a sequence motif HNF4. When footprint A (-101 to -49) or 7 alpha TRE (-73 to -55) sequence was linked upstream to a heterologous SV40 promoter/luciferase plasmid and transiently transfected into HepG2 cells, taurodeoxycholate suppressed the SV40 promoter activity. Electrophoretic mobility shift assays revealed that one or two bands shifted by the 7 alpha TRE or by a direct repeat sequence in 7 alpha TRE were absent when liver nuclear extracts of deoxycholic acid-treated rats were used. Similar gel shift patterns were also observed when human 7 alpha TRE or human liver nuclear extracts were used. The rat direct repeat sequence interacted with two polypeptides (M(r) = 57,000 and 116,000) in both rat and human liver nuclear extracts. These results suggest that hydrophobic bile acids may suppress the CYP7 gene expression by binding to a bile acid receptor which interacts with and prevents the binding of liver nuclear protein(s) to a bile acid-responsive element and that the core of bile acid-responsive element is a direct repeat.