ER-α36, a Novel Variant of ER-α, Mediates Estrogen-Stimulated Proliferation of Endometrial Carcinoma Cells via the PKCδ/ERK Pathway

Background: Recently, a variant of ER-alpha, ER-alpha 36 was identified and cloned. ER-alpha 36 lacks intrinsic transcription activity and mainly mediates non-genomic estrogen signaling. The purpose of this study was to investigate the function and the underlying mechanisms of ER-alpha 36 in growth regulation of endometrial Ishikawa cancer cells. Methods: The cellular localization of ER-alpha 36 and ER-alpha 66 were determined by immunofluorescence in the Ishikawa cells. Ishikawa endometrial cancer control cells transfected with an empty expression vector, Ishikawa cells with shRNA knockdown of ER-alpha 36 (Ishikawa/RNAiER36) and Ishikawa cells with shRNA knockdown of ER-alpha 66 (Ishikawa/RNAiER66) were treated with E2 and E2-conjugated to bovine serum albumin (E2-BSA, membrane impermeable) in the absence and presence of different kinase inhibitors HBDDE, bisindolylmaleimide, rottlerin, H89 and U0126. The phosphorylation levels of signaling molecules and cyclin D1/cdk4 expression were examined with Western blot analysis and cell growth was monitored with the MTT assay. Results: Immunofluorescence staining of Ishikawa cells demonstrated that ER-alpha 36 was expressed mainly on the plasma membrane and in the cytoplasm, while ER-alpha 66 was predominantly localized in the cell nucleus. Both E2 and E2-BSA rapidly activated PKC delta not PKC alpha in Ishikawa cells, which could be abrogated by ER-alpha 36 shRNA expression. E2-and E2-BSA-induced ERK phosphorylation required ER-alpha 36 and PKCd. However, only E2 was able to induce Camp-dependent protein kinase A (PKA) phosphorylation. Furthermore, E2 enhances cyclin D1/cdk4 expression via ER-alpha 36. Conclusion: E2 activates the PKC delta/ERK pathway and enhances cyclin D1/cdk4 expression via the membrane-initiated signaling pathways mediated by ER-alpha 36, suggesting a possible involvement of ER-alpha 36 in E2-dependent growth-promoting effects in endometrial cancer cells.