The primary aim of this study was to elucidate the role of the estrogen receptor (ER), a transcription factor involved in the nicotine- and 17β-estradiol (E2)-mediated up-regulation of α9-nAChR gene expression. A real-time polymerase chain reaction (PCR) assay was used to quantify the α9-nAChR mRNA expression levels of surgically isolated (n = 339) and laser-capture microdissected tissues (ER+ versus ER-, n = 6 per group). Chromatin immunoprecipitation (ChIP) and luciferase-promoter activity assays were used to investigate the ER-mediated transcriptional regulation of α9-nAChR gene expression. We observed that breast tumors with higher α9-nAChR mRNA expression levels (i.e., a mean fold ratio in the tumor/normal-paired samples of greater than tenfold) were associated with the lowest 5-year disease-specific survival rate (50%, dead/alive = 4/4, total = 8 patients, P = 0.006), in contrast to breast tumors with low levels (i.e., a mean fold ratio of less than onefold) of α9-nAChR expression (88%, dead/alive = 3/22, total = 25 patients). Furthermore, higher α9-nAChR mRNA expression levels were preferentially detected in ER+ tumor tissues in comparison to ER- tumor tissues (ER+ versus ER- patients: n = 160 vs. 72; mean fold ratios of α9-nAChR expression = 11 ± 3 vs. 6.7 ± 2.3 fold, respectively). In vitro promoter-binding assays demonstrated that the ER is a major transcription factor that mediates nicotine- and E2-induced up-regulation of α9-nAChR gene expression in MCF-7 cells. In conclusion, our data indicate that the ER plays a central role in mediating α9-nAChR gene up-regulation in response to either nicotine or E2 stimulation.
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