title: Definitive evidence using enucleated cytoplasts for a nongenomic basis for the cystic change in endoplasmic reticulum structure caused by STAT5a/b siRNAs.
abstract: STAT5a/b species are well known as transcription factors that regulate nuclear gene expression. In a novel line of research in human pulmonary arterial endothelial cells (HPAECs), we previously observed that STAT5a associated with the Golgi apparatus 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 the proteins reticulon-4 (RTN4; also called Nogo-B) and the ER-resident GTPase atlastin-3 (ATL3) and Golgi fragmentation. We now report that STAT5a can be observed in ER sheets in digitonin-permeabilized HPAECs and that anti-STAT5a cross- immunopanned ATL3 but not RTN4. Moreover, there was marked accumulation of the 63-kDa cytoskeleton-linking membrane protein and ER-spacer CLIMP63 (also called cytoskeleton-associated protein 4, CKAP4) and KDEL-mCherry within the cysts. That the STAT5a/b-siRNA-induced cystic ER phenotype developed in the presence of the transcription inhibitor 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole (DRB) had suggested that the mechanism was independent of the transcription factor functions of STAT5a/b, i.e., 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 using enucleated cytoplasts for a nongenomic mechanism(s) underlying the cystic change in ER structure elicited by STAT5a/b knockdown.
authors.affiliation: Department of Cell Biology and Anatomy, New York Medical College, Valhalla, NY 10595, USA.
authors.forename: Jason E
authors.initials: JE
authors.lastname: Lee
authors.forename: Yang-Ming
authors.initials: YM
authors.lastname: Yang
authors.forename: Huijuan
authors.initials: H
authors.lastname: Yuan
authors.forename: Pravin B
authors.initials: PB
authors.lastname: Sehgal
mesh_terms: D002478:Cells, Cultured; D003513:Cycloheximide / Q000494:pharmacology; D004004:Dichlororibofuranosylbenzimidazole / Q000494:pharmacology; D004721:Endoplasmic Reticulum / Q000378:metabolism* / Q000648:ultrastructure; D042783:Endothelial Cells / Q000378:metabolism / Q000648:ultrastructure; D055785:Gene Knockdown Techniques; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D019384:Nucleic Acid Synthesis Inhibitors / Q000494:pharmacology; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D011651:Pulmonary Artery / Q000166:cytology; D034741:RNA, Small Interfering / Q000235:genetics*; D050799:STAT5 Transcription Factor / Q000235:genetics* / Q000378:metabolism; D059010:Single-Cell Analysis; D025521:Tumor Suppressor Proteins / Q000235:genetics* / Q000378:metabolism
chemical_list: C081876:CKAP4 protein, human; D008565:Membrane Proteins; D019384:Nucleic Acid Synthesis Inhibitors; D011500:Protein Synthesis Inhibitors; D034741:RNA, Small Interfering; D050799:STAT5 Transcription Factor; C494092:STAT5A protein, human; C494095:STAT5B protein, human; D025521:Tumor Suppressor Proteins; D004004:Dichlororibofuranosylbenzimidazole; D003513:Cycloheximide