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Definitive evidence using enucleated cytoplasts for nongenomic effects of STAT5a/b siRNAs on ER structure
Author(s) -
Sehgal Pravin B,
Lee Jason E,
Yang YangMing,
Yuan Huijuan
Publication year - 2013
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.27.1_supplement.767.2
Subject(s) - microbiology and biotechnology , golgi apparatus , small interfering rna , gene knockdown , endoplasmic reticulum , calnexin , biology , cytoplast , gene silencing , chemistry , transfection , cytoplasm , gene , biochemistry , calreticulin
STAT5a/b species are well‐known as transcription factors that regulate nuclear gene expression. However, we observed that STAT5a associated with the Golgi apparatus in HPAECs and that siRNA‐mediated knockdown of STAT5a/b led to the rapid development of a dramatic cystic change in the endoplasmic reticulum (ER) characterized by deposition along cyst membranes and tubule‐to‐cyst boundaries of reticulon‐4 (RTN4/Nogo‐B), atlastin‐3 (ATL3) and Golgi fragmentation. Moreover, there was marked accumulation of the 63kDa cytoskeleton‐linking membrane protein and ER‐spacer CLIMP63/CKAP4 within the cysts. That the STAT5a/b‐siRNA‐induced cystic ER phenotype developed in the presence of a transcription inhibitor DRB had suggested that the mechanism was nongenomic. We have now definitively tested the requirement for the nucleus in eliciting the STAT5a/b‐siRNA‐induced cystic ER phenotype. Enucleated HPAEC cytoplasts were prepared using adherent 35 mm cultures using the cytochalasin B‐centrifugation method (typically yielding 65–75% enucleation). STAT5a/b siRNAs readily elicited the cystic ER phenotype including the marked luminal accumulation of CLIMP63 and Golgi fragmentation in the recovered HPAEC cytoplasts demonstrably lacking a nucleus. These studies provide unequivocal evidence for a nongenomic mechanism underlying the cystic change in ER structure elicited by STAT5a/b knockdown.