Gene regulation in an MCF-7 cell line that naturally expresses an estrogen receptor unable to directly bind DNA.

In the described studies, we have developed a variant of the MCF-7 cell line, M-ERd3/g8, for analysis of 17-beta-estradiol (E2)-action without direct DNA interaction by E2-receptors. M-ERd3/g8 cells principally express the estrogen receptor alpha (ER) form ERDelta3 which, due to skipping of exon 3, lacks the second zinc finger of ER that is required for direct DNA interaction. This was achieved by introduction of siRNA targeting exon 3 to a Tamoxifen-selected MCF-7 variant, TMX 2-11, expressing approximately equal amount of full-length ER and ERDelta3 proteins. M-ERd3/g8 cells exhibited a normal nuclear ER localization, and ERDelta3 expression levels were similar to those for full-length ER protein in MCF-7 cells. Ser 118 phosphorylation of the ERDelta3 was triggered by E2 treatment. The expression of several well characterized E2-responsive markers was strongly modified in the M-ERd3/g8 cells. The E2-induction of progesterone receptor (PR) and HEM45 mRNAs was abolished. The effect on pS2 mRNA expression was complex: the pS2 mRNA levels fell approximately 50-fold in control M-ERd3/g8 cells. There was E2-induction of pS2-expression but with an altered temporal pattern. This was blocked by inhibitors of the p42/44 mitogen activated protein (MAP) kinase and inositol triphosphate (PI3) kinase pathways suggesting a role for cytoplasmic signaling pathways. Gene array analysis and real-time polymerase chain reaction (PCR) studies identified several genes whose expressions were induced in E2-treated M-ERd3/g8 cells. These included A-Myb, a homolog to the avian myoblastosis virus oncogene, carbonic anhydrase XII (CAXII), chemokine ligand 12 (CXCL-12), early growth response 3 (EGR 3), fibrinogen B beta (FibBbeta), along with serine protease 23 (SPUVE). The responses fell into several temporal patterns. A-Myb, CAXII, CXCL-12 and EGR 3 were E2-induced within 2 h. The expression of CXCL-12 and EGR 3 was persistent to 24 h, while that of A-Myb and CAXII was not persistent in M-ERd3/g8 cells. FibBbeta and SPUVE expression was not induced until times later than 6 h. Expression of none of the genes was elevated prior to 2 h, but the utilization of a 24 h time point for the gene array analysis may have eliminated the most transiently responsive genes. Immediate early 3 (IE3) was down-regulated by E2 in the M-ERd3/g8 cells but was transiently up-regulated during the 2-6 h period in MCF-7 cells. Basal levels of several of the genes were strongly reduced in M-ERd3/g8, compared to MCF-7. The studies suggest that M-ERd3/g8 cells provide a new model for studies of E2-action without direct ER binding to DNA and where E2-action must be via alternate pathways.