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: Jason E Lee, Yang-Ming Yang, Huijuan Yuan, Pravin B Sehgal
• Journal: American journal of physiology. Cell physiology (Am J Physiol Cell Physiol) Vol. 304 Issue 4 Pg. C312-23 (Feb 15 2013) ISSN: 1522-1563 [Electronic] United States
• PMID: 23151802 (Publication Type: Journal Article, Research Support, N.I.H., Extramural)

Chemical References

• CKAP4 protein, human
• Membrane Proteins
• Nucleic Acid Synthesis Inhibitors
• Protein Synthesis Inhibitors
• RNA, Small Interfering
• STAT5 Transcription Factor
• STAT5A protein, human
• STAT5B protein, human
• Tumor Suppressor Proteins
• Dichlororibofuranosylbenzimidazole
• Cycloheximide

Topics

• 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)