Document Detail


Definitive evidence using enucleated cytoplasts for a nongenomic basis for the cystic change in endoplasmic reticulum structure caused by STAT5a/b siRNAs.
MedLine Citation:
PMID:  23151802     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
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:
Jason E Lee; Yang-Ming Yang; Huijuan Yuan; Pravin B Sehgal
Publication Detail:
Type:  Journal Article; Research Support, N.I.H., Extramural     Date:  2012-11-14
Journal Detail:
Title:  American journal of physiology. Cell physiology     Volume:  304     ISSN:  1522-1563     ISO Abbreviation:  Am. J. Physiol., Cell Physiol.     Publication Date:  2013 Feb 
Date Detail:
Created Date:  2013-02-18     Completed Date:  2013-04-08     Revised Date:  2014-02-18    
Medline Journal Info:
Nlm Unique ID:  100901225     Medline TA:  Am J Physiol Cell Physiol     Country:  United States    
Other Details:
Languages:  eng     Pagination:  C312-23     Citation Subset:  IM    
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MeSH Terms
Descriptor/Qualifier:
Cells, Cultured
Cycloheximide / pharmacology
Dichlororibofuranosylbenzimidazole / pharmacology
Endoplasmic Reticulum / metabolism*,  ultrastructure
Endothelial Cells / metabolism,  ultrastructure
Gene Knockdown Techniques
Humans
Membrane Proteins / genetics,  metabolism
Nucleic Acid Synthesis Inhibitors / pharmacology
Protein Synthesis Inhibitors / pharmacology
Pulmonary Artery / cytology
RNA, Small Interfering / genetics*
STAT5 Transcription Factor / genetics*,  metabolism
Single-Cell Analysis
Tumor Suppressor Proteins / genetics*,  metabolism
Grant Support
ID/Acronym/Agency:
F31 HL-107013/HL/NHLBI NIH HHS; R01 HL-087176/HL/NHLBI NIH HHS; R03 HL114609/HL/NHLBI NIH HHS
Chemical
Reg. No./Substance:
0/CKAP4 protein, human; 0/Membrane Proteins; 0/Nucleic Acid Synthesis Inhibitors; 0/Protein Synthesis Inhibitors; 0/RNA, Small Interfering; 0/STAT5 Transcription Factor; 0/STAT5A protein, human; 0/STAT5B protein, human; 0/Tumor Suppressor Proteins; 53-85-0/Dichlororibofuranosylbenzimidazole; 98600C0908/Cycloheximide
Comments/Corrections

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