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Endoplasmic reticulum stress response in an INS-1 pancreatic beta-cell line with inducible expression of a folding-deficient proinsulin.

Hartley T, Siva M, Lai E, Teodoro T, Zhang L, Volchuk A - BMC Cell Biol. (2010)

Bottom Line: To identify gene expression changes resulting from mutant insulin expression we performed microarray expression profiling and real time PCR experiments.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.ER-associated degradation is essential in maintaining cell survival in cells expressing mutant insulin.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Cellular and Molecular Biology, Toronto General Research Institute, University Health Network, 101 College Street, TMDT 10-706, Toronto M5G 1L7, Canada.

ABSTRACT

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.

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Expression of insulin 2(C96Y)-EGFP results in the induction of selected genes. Clone #4S2 cells were untreated (Control) or treated with 2 μg/ml doxycycline (+Dox) for the times indicated. Total RNA was isolated and the mRNA levels relative to cellular β-actin for Erdj4/Dnajb9 (A), Erdj5/Dnajc10 (B), Ero1β (C), Ero1α (D) were analyzed by real-time PCR as outlined in the methods. Shown are the mean ± SE of 3 independent experiments. *p < 0.05 relative to control.
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Figure 6: Expression of insulin 2(C96Y)-EGFP results in the induction of selected genes. Clone #4S2 cells were untreated (Control) or treated with 2 μg/ml doxycycline (+Dox) for the times indicated. Total RNA was isolated and the mRNA levels relative to cellular β-actin for Erdj4/Dnajb9 (A), Erdj5/Dnajc10 (B), Ero1β (C), Ero1α (D) were analyzed by real-time PCR as outlined in the methods. Shown are the mean ± SE of 3 independent experiments. *p < 0.05 relative to control.

Mentions: We performed additional real-time PCR analysis of other putative ER stress response genes to validate some of the microarray results (Figure 6). Consistent with the microarray data (Table 1) we observed an induction of Erdj4, but not the related gene Erdj5/Dnajc10, despite the fact that the latter has been shown to be induced by ER stress in other cell types [20,21]. In addition, although not detected by microarray, we observed induction of Ero1β, but not Ero1α (Figure 6C, D). The former, but not the latter, has been reported to be ER stress-inducible, which is consistent with our results [22]. It is important to note that doxycyline at the same concentration used in these studies added to a parental INS-1 cell line does not induce any changes in Grp78 or Chop mRNA or protein levels (results not shown), indicating that the changes observed are due to Ins2 (C96Y)-EGFP expression and not the small molecule.


Endoplasmic reticulum stress response in an INS-1 pancreatic beta-cell line with inducible expression of a folding-deficient proinsulin.

Hartley T, Siva M, Lai E, Teodoro T, Zhang L, Volchuk A - BMC Cell Biol. (2010)

Expression of insulin 2(C96Y)-EGFP results in the induction of selected genes. Clone #4S2 cells were untreated (Control) or treated with 2 μg/ml doxycycline (+Dox) for the times indicated. Total RNA was isolated and the mRNA levels relative to cellular β-actin for Erdj4/Dnajb9 (A), Erdj5/Dnajc10 (B), Ero1β (C), Ero1α (D) were analyzed by real-time PCR as outlined in the methods. Shown are the mean ± SE of 3 independent experiments. *p < 0.05 relative to control.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2921384&req=5

Figure 6: Expression of insulin 2(C96Y)-EGFP results in the induction of selected genes. Clone #4S2 cells were untreated (Control) or treated with 2 μg/ml doxycycline (+Dox) for the times indicated. Total RNA was isolated and the mRNA levels relative to cellular β-actin for Erdj4/Dnajb9 (A), Erdj5/Dnajc10 (B), Ero1β (C), Ero1α (D) were analyzed by real-time PCR as outlined in the methods. Shown are the mean ± SE of 3 independent experiments. *p < 0.05 relative to control.
Mentions: We performed additional real-time PCR analysis of other putative ER stress response genes to validate some of the microarray results (Figure 6). Consistent with the microarray data (Table 1) we observed an induction of Erdj4, but not the related gene Erdj5/Dnajc10, despite the fact that the latter has been shown to be induced by ER stress in other cell types [20,21]. In addition, although not detected by microarray, we observed induction of Ero1β, but not Ero1α (Figure 6C, D). The former, but not the latter, has been reported to be ER stress-inducible, which is consistent with our results [22]. It is important to note that doxycyline at the same concentration used in these studies added to a parental INS-1 cell line does not induce any changes in Grp78 or Chop mRNA or protein levels (results not shown), indicating that the changes observed are due to Ins2 (C96Y)-EGFP expression and not the small molecule.

Bottom Line: To identify gene expression changes resulting from mutant insulin expression we performed microarray expression profiling and real time PCR experiments.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.ER-associated degradation is essential in maintaining cell survival in cells expressing mutant insulin.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Cellular and Molecular Biology, Toronto General Research Institute, University Health Network, 101 College Street, TMDT 10-706, Toronto M5G 1L7, Canada.

ABSTRACT

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.

Show MeSH
Related in: MedlinePlus