ER-alpha 36, a novel variant of ER-alpha, mediates estrogen stimulated proliferation of endometrial carcinoma cells via the PKC delta/ERK pathway

Jing-Shan Tong, Qinghua Zhang, Zhen-Bo Wang, Sen Li, Cai-Rong Yang, Xue-Qi Fu, Yi Hou, Zhao-Yi Wang, Jun Sheng, Qing-Yuan Sun

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Background: Recently, a variant of ER-a, ER-a36 was identified and cloned. ER-a36 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-a36 in growth regulation of endometrial Ishikawa cancer cells. Methods: The cellular localization of ER-a36 and ER-a66 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-a36 (Ishikawa/RNAiER36) and Ishikawa cells with shRNA knockdown of ER-a66 (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-a36 was expressed mainly on the plasma membrane and in the cytoplasm, while ER-a66 was predominantly localized in the cell nucleus. Both E2 and E2-BSA rapidly activated PKCd not PKCa in Ishikawa cells, which could be abrogated by ER-a36 shRNA expression. E2-and E2-BSA-induced ERK phosphorylation required ER-a36 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-a36. Conclusion: E2 activates the PKCd/ERK pathway and enhances cyclin D1/cdk4 expression via the membrane-initiated signaling pathways mediated by ER-a36, suggesting a possible involvement of ER-a36 in E2-dependent growth-promoting effects in endometrial cancer cells
Original languageEnglish
Article numbere15408
Number of pages11
JournalPLoS ONE
Issue number11
Publication statusPublished - 2010
Externally publishedYes

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