Research Resource: Comparison of Gene Profiles From Wild-Type ERα and ERα Hinge Region Mutants

We showed previously that the hinge region of estrogen receptor (ER) alpha is involved in mediating its actions. The hinge 1 (H1) ER alpha mutant has disrupted nuclear localization and has lost interaction with c-JUN, but retains estrogen response element (ERE)-mediated functions. The hinge 2 + nuclear export sequence (H2NES) ER alpha mutant does not maintain nuclear translocation with hormone and no longer activates ERE target genes but does retain a nongenomic, nonnuclear, rapid-action response. Herein, we used the human endometrial cancer Ishikawa stable cell lines (Ishikawa/vector, Ishikawa/wild-type [WT] ER alpha, Ishikawa/H1 ER alpha, or Ishikawa/H2NES ER alpha) to characterize the biological activities of these 2 ER alpha hinge region mutants. We confirmed by confocal microscopy increased cytoplasmic ER alpha in the H1 ER alpha cell line and full cytoplasmic ER alpha localization in the H2NES ER alpha cell line. Luciferase assays using the 3xERE reporter showed activation of H1 ER alpha and H2NES ER alpha by estradiol (E-2) treatment, but using the endogenous pS2 reporter, luciferase activity was only seen with the H1 ER alpha cell line. Examining cell proliferation revealed that only the WTER alpha and H1 ER alpha cell lines increased proliferation after treatment. Using microarrays, we found that WT ER alpha and H1 ER alpha cluster together, whereas vector and H2NES ER alpha are most similar and cluster independently of E-2 treatment. These studies revealed that the nongenomic activities of ER alpha are unable to mediate proliferative changes or the transcriptional profile after treatment and demonstrate the importance of genomic action for ER alpha/E-2-mediated responses with the nongenomic actions of ER alpha being complementary to elicit the full biological actions of ER alpha.