Endoplasmic reticulum stress response in an INS-1 pancreatic beta-cell line with inducible expression of a folding-deficient proinsulin.
BACKGROUND
Cells respond to endoplasmic reticulum stress (ER) stress by activating the unfolded protein response. To study the ER stress response in pancreatic beta-cells we developed a model system that allows for pathophysiological ER stress based on the Akita mouse. This mouse strain expresses a mutant insulin 2 gene (C96Y), which prevents normal proinsulin folding causing ER stress and eventual beta-cell apoptosis. A double-stable pancreatic beta-cell line (pTet-ON INS-1) with inducible expression of insulin 2 (C96Y) fused to EGFP was generated to study the ER stress response.


RESULTS
Expression of Ins 2 (C96Y)-EGFP resulted in activation of the ER stress pathways (PERK, IRE1 and ATF6) and caused dilation of the ER. To identify gene expression changes resulting from mutant insulin expression we performed microarray expression profiling and real time PCR experiments. We observed an induction of various ER chaperone, co-chaperone and ER-associated degradation genes after 24 h and an increase in pro-apoptotic genes (Chop and Trib3) following 48 h of mutant insulin expression. The latter changes occurred at a time when general apoptosis was detected in the cell population, although the relative amount of cell death was low. Inhibiting the proteasome or depleting Herp protein expression increased mutant insulin levels and enhanced cell apoptosis, indicating that ER-associated degradation is maintaining cell survival.


CONCLUSIONS
The inducible mutant insulin expressing cell model has allowed for the identification of the ER stress response in beta-cells and the repertoire of genes/proteins induced is unique to this cell type. ER-associated degradation is essential in maintaining cell survival in cells expressing mutant insulin. This cell model will be useful for the molecular characterization of ER stress-induced genes.
Hartley
Taila
T
Division of Cellular and Molecular Biology, Toronto General Research Institute, University Health Network, 101 College Street, TMDT 10-706, Toronto M5G 1L7, Canada.
Siva
Madura
M
Lai
Elida
E
Teodoro
Tracy
T
Zhang
Liling
L
Volchuk
Allen
A
D000818:Animals; D017209:Apoptosis / Q000235:genetics; D002460:Cell Line; D004721:Endoplasmic Reticulum / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D020869:Gene Expression Profiling; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D007328:Insulin / Q000235:genetics / Q000378:metabolism*; D050417:Insulin-Secreting Cells / Q000378:metabolism* / Q000473:pathology; D051379:Mice; D008817:Mice, Mutant Strains; D046228:Microarray Analysis; D011384:Proinsulin / Q000235:genetics / Q000378:metabolism*; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D015202:Protein Engineering; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D019076:Transgenes / Q000235:genetics; D056811:Unfolded Protein Response / Q000235:genetics
C514817:Ins1 protein, mouse; D007328:Insulin; D011993:Recombinant Fusion Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D011384:Proinsulin; D046988:Proteasome Endopeptidase Complex