title: ER-α36, a novel variant of ER-α, mediates estrogen-stimulated proliferation of endometrial carcinoma cells via the PKCδ/ERK pathway.
abstract: BACKGROUND
  Recently, a variant of ER-α, ER-α36 was identified and cloned. ER-α36 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-α36 in growth regulation of endometrial Ishikawa cancer cells.
  
  
  METHODS
  The cellular localization of ER-α36 and ER-α66 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-α36 (Ishikawa/RNAiER36) and Ishikawa cells with shRNA knockdown of ER-α66 (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-α36 was expressed mainly on the plasma membrane and in the cytoplasm, while ER-α66 was predominantly localized in the cell nucleus. Both E2 and E2-BSA rapidly activated PKCδ not PKCα in Ishikawa cells, which could be abrogated by ER-α36 shRNA expression. E2-and E2-BSA-induced ERK phosphorylation required ER-α36 and PKCδ. However, only E2 was able to induce Camp-dependent protein kinase A (PKA) phosphorylation. Furthermore, E2 enhances cyclin D1/cdk4 expression via ER-α36.
  
  
  CONCLUSIONS
  E2 activates the PKCδ/ERK pathway and enhances cyclin D1/cdk4 expression via the membrane-initiated signaling pathways mediated by ER-α36, suggesting a possible involvement of ER-α36 in E2-dependent growth-promoting effects in endometrial cancer cells.
authors.affiliation: College of Life Sciences, Jilin University, Changchun, China.
authors.forename: Jing-Shan
authors.initials: JS
authors.lastname: Tong
authors.forename: Qing-Hua
authors.initials: QH
authors.lastname: Zhang
authors.forename: Zhen-Bo
authors.initials: ZB
authors.lastname: Wang
authors.forename: Sen
authors.initials: S
authors.lastname: Li
authors.forename: Cai-Rong
authors.initials: CR
authors.lastname: Yang
authors.forename: Xue-Qi
authors.initials: XQ
authors.lastname: Fu
authors.forename: Yi
authors.initials: Y
authors.lastname: Hou
authors.forename: Zhao-Yi
authors.initials: ZY
authors.lastname: Wang
authors.forename: Jun
authors.initials: J
authors.lastname: Sheng
authors.forename: Qing-Yuan
authors.initials: QY
authors.lastname: Sun
mesh_terms: D015153:Blotting, Western; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D019938:Cyclin D1 / Q000378:metabolism; D051358:Cyclin-Dependent Kinase 4 / Q000378:metabolism; D016889:Endometrial Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D004789:Enzyme Activation / Q000187:drug effects; D047628:Estrogen Receptor alpha / Q000235:genetics / Q000378:metabolism*; D004967:Estrogens / Q000494:pharmacology*; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism*; D005260:Female; D006801:Humans; D010766:Phosphorylation / Q000187:drug effects; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D051745:Protein Kinase C-delta / Q000378:metabolism*; D034622:RNA Interference; D012710:Serum Albumin, Bovine / Q000494:pharmacology; D015398:Signal Transduction / Q000187:drug effects
chemical_list: D047628:Estrogen Receptor alpha; D004967:Estrogens; D020033:Protein Isoforms; D019938:Cyclin D1; D012710:Serum Albumin, Bovine; D017868:Cyclic AMP-Dependent Protein Kinases; D051745:Protein Kinase C-delta; D051358:Cyclin-Dependent Kinase 4; D048049:Extracellular Signal-Regulated MAP Kinases