title: Thapsigargin down-regulates protein levels of GRP78/BiP in INS-1E cells.
abstract: Pancreatic β-cells have a well-developed endoplasmic reticulum (ER) and express large amounts of chaperones and protein disulfide isomerases (PDI) to meet the high demand for synthesis of proteins. We have observed an unexpected decrease in chaperone protein level in the β-cell model INS-1E after exposure to the ER stress inducing agent thapsigargin. As these cells are a commonly used model for primary β-cells and has been shown to be vulnerable to ER stress, we hypothesize these cells are incapable of mounting a chaperone defense upon activation of ER stress. To investigate the chaperone expression during an ER stress response, induced by thapsigargin in INS-1E cells, we used quantitative mass spectrometry based proteomics. The results displayed a decrease of GRP78/BiP, PDIA3 and PDIA6. Decrease of GRP78/BiP was verified by Western blot and occurred in parallel with enhanced levels of p-eIF2α and CHOP. In contrast to INS-1E cells, GRP78/BiP was not decreased in MIN6 cell or rat and mouse islets after thapsigargin exposure. Investigation of the decreased protein levels of GRP78/BiP indicates that this is not a consequence of reduced mRNA expression. Rather the reduction results from the combined effect of reduced protein synthesis and enhanced proteosomal degradation and possibly also degradation via autophagy. Induction of ER stress with thapsigargin leads to lower protein levels of GRP78/BiP, PDIA3 and PDIA6 in INS-1E cells which may contribute to the susceptibility of ER stress in this β-cell model.
authors.affiliation: Unit for Diabetes Research, Department of Clinical Science and Education, Södersjukhuset, Sjukhusbacken 10, Research Center 3rd Floor, Karolinska Institutet, 118 83 Stockholm, Sweden.
authors.forename: Victoria
authors.initials: V
authors.lastname: Rosengren
authors.forename: Henrik
authors.initials: H
authors.lastname: Johansson
authors.forename: Janne
authors.initials: J
authors.lastname: Lehtiö
authors.forename: Liselotte
authors.initials: L
authors.lastname: Fransson
authors.forename: Ake
authors.initials: A
authors.lastname: Sjöholm
authors.forename: Henrik
authors.initials: H
authors.lastname: Ortsäter
mesh_terms: D000818:Animals; D001343:Autophagy / Q000187:drug effects; D002460:Cell Line; D015536:Down-Regulation / Q000187:drug effects; D004721:Endoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism; D000091342:Endoplasmic Reticulum Chaperone BiP; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006360:Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism*; D066298:In Vitro Techniques; D050417:Insulin-Secreting Cells / Q000187:drug effects* / Q000378:metabolism*; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D040901:Proteomics; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases / Q000037:antagonists & inhibitors; D015398:Signal Transduction / Q000187:drug effects; D013312:Stress, Physiological / Q000187:drug effects; D019284:Thapsigargin / Q000494:pharmacology*; D051742:Transcription Factor CHOP / Q000235:genetics
chemical_list: C497251:Ddit3 protein, rat; D000091342:Endoplasmic Reticulum Chaperone BiP; D004791:Enzyme Inhibitors; C051684:GRP78 protein, rat; D006360:Heat-Shock Proteins; C000717248:Hspa5 protein, mouse; D012333:RNA, Messenger; D051742:Transcription Factor CHOP; D019284:Thapsigargin; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases