| Definitive evidence using enucleated cytoplasts for a nongenomic basis for the cystic change in endoplasmic reticulum structure caused by STAT5a/b siRNAs. | |
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MedLine Citation:
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PMID: 23151802 Owner: NLM Status: MEDLINE |
Abstract/OtherAbstract:
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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. |
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Authors:
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Jason E Lee; Yang-Ming Yang; Huijuan Yuan; Pravin B Sehgal |
Publication Detail:
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Type: Journal Article; Research Support, N.I.H., Extramural Date: 2012-11-14 |
Journal Detail:
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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:
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Created Date: 2013-02-18 Completed Date: 2013-04-08 Revised Date: 2013-05-02 |
Medline Journal Info:
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Nlm Unique ID: 100901225 Medline TA: Am J Physiol Cell Physiol Country: United States |
Other Details:
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Languages: eng Pagination: C312-23 Citation Subset: IM |
Affiliation:
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Department of Cell Biology and Anatomy, New York Medical College, Valhalla, NY 10595, USA. |
Export Citation:
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| MeSH Terms | |
Descriptor/Qualifier:
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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:
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F31 HL-107013/HL/NHLBI NIH HHS; R01 HL-087176/HL/NHLBI NIH HHS; R03 HL114609/HL/NHLBI NIH HHS |
| Chemical | |
Reg. No./Substance:
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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; 66-81-9/Cycloheximide |
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine
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