ER-alpha Null HCC38 Cells Possess ER-alpha Variant ER36

Objective: Several studies have shown the presence of estrogen receptors (ER) in the cell membrane. We previously showed that the rapid activation of protein kinase C (PKC) in ER-alpha positive MCF-7 cells and ER-alpha negative HCC38 cells is membrane-mediated. Although HCC38 cells expressed mRNA for ER-beta, ER-beta protein was not detected, indicating that a non-traditional ER was responsible for PKC activation by estrogen. Recently, a novel 36,000 molecular weight variant of ER-alpha, ER36, was discovered. The present study was based on the hypothesis that HCC38 cells carry ER36 and this receptor is localizes in the cell membrane. Methods: Confluent cultures of MCF-7 and HCC38 breast cancer cells were treated with 17-beta estradiol for 9 and 90 minutes. Whole cell lysate and membrane fraction proteins were separate by SDS-PAGE and examined for ER36 by Western blot analysis. Additionally, MCF-7 and HCC38 cells were cultured on chamber slides and examined by laser confocal fluorescence microscopy to verify that ER36 was present in live cells. Results: Western blots of whole cell lysates and cell membranes showed the presence of ER36 and ER-beta in both MCF-7 and HCC38 cells at 9 and 90 min. Estrogen increased ER36 in the cell membrane in a time-dependent manner but had no effect on ER-beta. Confocal microscopy showed that ER36 was present throughout MCF-7 cells and HCC38 cells, whereas ER-alpha was localized to the peri-nuclear region as well as the nucleus of MCF-7 cells. Conclusion: Here we show that ER36 is present in ER-alpha null HCC38 cells, and to a lesser extent, ER-beta. This could explain the rapid estrogen-dependent increase in PKC in these cells; however more studies should be performed to understand the role of these receptors in the rapid membrane response. Suppported by Children's Healthcare of Atlanta.