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Molecular Endocrinology

Year: 2010  |  Volume: 24  |  Issue: 5  |  Page No.: 914 - 922

Identification of a Novel Estrogen Receptor-{alpha} Variant and Its Upstream Splicing Regulator

K Ohshiro, P Mudvari, Q. c Meng, S. K Rayala, A. A Sahin, S. A. W Fuqua and R. Kumar


Alternative splicing of precursor mRNA is a fundamental mechanism to generate multiple proteins from a single gene. Although constitutive and alternative mRNA splicing is temporally and spatially regulated, deregulation of mRNA splicing could cause development, progression, and metastasis of tumors. Through yeast two-hybrid screening of a human breast cDNA library using estrogen receptor- (ER) as bait, we identified a novel nuclear receptor box containing full-length protein, nuclear protein E3-3 (NPE3-3). Our results revealed that NPE3-3 associates with not only ER but also with splicing factors, serine/arginine-rich protein (SRp)-30c, SRp40, and splicing factor SC-35, suggesting that NPE3-3 is likely to be involved in regulation of mRNA splicing. Accordingly, transient expression of NPE3-3 in cells resulted in expected splicing of the CD44 control minigene. We also discovered that NPE3-3-overexpressing clones produced a novel, previously unrecognized, alternatively spliced variant of ER (termed ERV), which had a molecular size of 37 kDa composed of only exons 1, 2, 7, and 8. ERV was expressed and sequestered in the cytoplasm in MCF-7 cells stably overexpressing NPE3-3, suggesting its involvement in nongenomic hormone signaling. NPE3-3 clones exhibited up-regulation of ERK1/2 signaling, cyclin D1, and cathepsin D and enhanced tumor cell proliferation, migration, and tumorigenicity. Moreover, direct expression of the ERV in breast cancer cells stimulated ERK1/2 up-regulation and cyclin D1 expression. We found that ERV physically interacted with MAPK kinase (MEK)-1/2, and thus, an ERV and MEK1/2 complex could lead to the activation of the ERK1/2 pathway. Interestingly, NPE3-3 was up-regulated in human breast tumors. These findings revealed a role for NPE3-3 in alternative splicing and suggest that ER is a physiological target of NPE3-3, leading to a constitutive nongenomic signaling pathway in breast cancer cells.

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