Assay variables.

The mechanism of signal transduction by the estrogen receptor (ER) is complex and not fully understood. In addition to the ER, a number of accessory proteins are apparently required to efficiently transduce the steroid hormone signal. In the absence of estradiol, the ER, like other steroid receptors, is complexed with Hsp90 and other molecular chaperone components, including an immunophilin, and p23. This Hsp90-based chaperone complex is thought to repress the ER's transcriptional regulatory activities while maintaining the receptor in a conformation that is competent for high-affinity steroid binding. However, a role for p23 in ER signal transduction has not been demonstrated. Using a mutant ER (G400V) with decreased hormone binding capacity as a substrate in a dosage suppression screen in yeast cells (Saccharomyces cerevisiae), we identified the yeast homologue of the human p23 protein (yhp23) as a positive regulator of ER function. Overexpression of yhp23 in yeast cells increases ER transcriptional activation by increasing estradiol binding in vivo. Importantly, the magnitude of the effect of yhp23 on ER transcriptional activation is inversely proportional to the concentration of both ER and estradiol in the cell. Under conditions of high ER expression, ER transcriptional activity is largely independent of yhp23, whereas at low levels of ER expression, ER transcriptional activation is primarily dependent on yhp23. The same relationship holds for estradiol levels. We further demonstrate that yhp23 colocalizes with the ER in vivo. Using a yhp23-green fluorescent protein fusion protein, we observed a redistribution of yhp23 from the cytoplasm to the nucleus upon coexpression with ER. This nuclear localization of yhp23 was reversed by the addition of estradiol, a finding consistent with yhp23's proposed role as part of the aporeceptor complex. Expression of human p23 in yeast partially complements the loss of yhp23 function with respect to ER signaling. Finally, ectopic expression of human p23 in MCF-7 breast cancer cells increases both hormone-dependent and hormone-independent transcriptional activation by the ER. Together, these results strongly suggest that p23 plays an important role in ER signal transduction.

Estradiol↗

PubMed1999

Transcriptional activation of insulin-like growth factor-binding protein-4 by 17beta-estradiol in MCF-7 cells: role of estrogen receptor-Sp1 complexes.

Insulin-like growth factor-binding protein-4 (IGFBP-4) is expressed in MCF-7 human breast cancer cells, and treatment of these cells with 17beta-estradiol (E2) resulted in induction of IGFBP-4 gene expression (>3-fold) and protein secretion (>6-fold). To identify genomic sequences associated with E2 responsiveness, the 5'-promoter region (-1214 to +18) of the IGFBP-4 gene was cloned into a vector upstream from the firefly luciferase reporter gene, and E2 induced a 10-fold increase in luciferase activity in MCF-7 cells transiently transfected with this construct. Deletion analysis of this region of the IGFBP-4 gene promoter identified two GC-rich sequences at -559 to -553 and -72 to -64 that were important for E2-induced trans-activation. Gel mobility shift assays using 32P-labeled -569 to -540 and -83 to -54 oligonucleotides from the IGFBP-4 gene promoter showed that Sp1 protein bound these oligonucleotides to form a retarded band, and the intensity of the band was competitively decreased after coincubation with unlabeled IGFBP-4-derived and consensus Sp1 oligonucleotides. Mutation of the GC-rich sites within these sequences resulted in loss of the retarded band formation. Wild-type human estrogen receptor did not bind directly to the IGFBP-4 oligonucleotides; however, human estrogen receptor enhanced Sp1-DNA binding in a concentration-dependent manner. The results of this study demonstrate that at least two GC-rich sequences at -559 to -553 and -72 to -64 are required for induction of IGFBP-4 gene expression by E2 in MCF-7 cells.

Estradiol↗