Identification, characterization and mechanism of the estrogen regulation of the gene encoding the sodium/hydrogen exchanger-regulatory factor (NHE-RF)

Estrogens are steroid hormones that influence growth, differentiation and functioning in multiple tissues of the body. Estrogens also play a role in the development and progression of breast cancer. Estrogens exert their influences on target tissues by diffusing through the cell membrane and binding to the estrogen receptor (ER), which acts as a ligand inducible transcription factor.; Thus far relatively few estrogen-regulated genes have been described. We have used differential display PCR to screen for primary estrogen-stimulated genes, and identified the sodium hydrogen exchanger regulatory factor (NHE-RF), as being rapidly and markedly increased by estrogen in breast cancer cells. Stimulation was selective for estrogenic ligands and was suppressed by antiestrogens. Induction was shown to occur via the ER pathway through several approaches, including the use of ER-negative cells containing a stably integrated ER. Among the tissues we examined we found that NHE-RF is widely expressed, with fairly high levels in mammary tissue.; We found that the NHE-RF gene is comprised of 6 exons, and 5 introns of variable size ranging from 231 by to >15 kb. The ribonuclease protection assay was used to define the transcription start site, and a reporter gene linked to progressive deletions was used to define the minimal promoter as well as locate two segments within the 5′-region responsible for estrogen inducibility of the gene. Both fragments lack a consensus ERE; however, the site most proximal to the translational start site contains one half-ERE, while the upstream fragment contains thirteen. Electrophoretic mobility gel shift and transfection assays demonstrated direct ER interaction with these half-ERE sites and a requirement for an intact DNA binding domain for ER regulation of NHE-RF.; Finally, studies were designed to help define the physiological role of estrogen-induction of the NHE-RF gene using two model systems. Immunocytochemistry was used to visualize NHE-RF in MCF-7 cells. Secondly, we created adenovirus that express the wild type or anti-sense NHE-RF RNA. By infecting cells with the adenovirus we may overexpress or functionally “knock out” the NHE-RF protein and monitor the resulting effects on cell signaling, fluid exchange and cell cytoarchitecture.