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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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Inhibiting Herp expression increases insulin 2 (C96Y)-EGFP levels and enhances apoptosis. A. Clone #4S2 cells were untreated or treated with 2 μg/ml doxycycline for the times indicated and cell lysates were prepared and immunoblotted. B. Clone #4S2 cells were transfected with control siRNA or an siRNA directed against Herp and treated or not with doxycycline for 48 h. Cells were lysed and immunoblotted as indicated. A low and high exposure of the anti-GFP western blot is shown. Results from two of four independent experiments are shown. C. Immunoblots were quantified and the mean relative expression of Herp from 4 independent experiments is presented. D. Clone #4S2 cells were transfected with control or Herp siRNA and 48 h later apoptosis was measured by immunobloting for cleaved caspase 3. Results from two experiments are shown. E. Clone #4S2 cells were transfected as in (D) and 24 h later doxycycline was added or not for 48 h and apoptosis was measured by immunobloting for cleaved caspase 3. INS-1 cells treated or not with 0.3 mM straurosporine (St.) was included as a positive control for cleaved caspase 3.
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Figure 9: Inhibiting Herp expression increases insulin 2 (C96Y)-EGFP levels and enhances apoptosis. A. Clone #4S2 cells were untreated or treated with 2 μg/ml doxycycline for the times indicated and cell lysates were prepared and immunoblotted. B. Clone #4S2 cells were transfected with control siRNA or an siRNA directed against Herp and treated or not with doxycycline for 48 h. Cells were lysed and immunoblotted as indicated. A low and high exposure of the anti-GFP western blot is shown. Results from two of four independent experiments are shown. C. Immunoblots were quantified and the mean relative expression of Herp from 4 independent experiments is presented. D. Clone #4S2 cells were transfected with control or Herp siRNA and 48 h later apoptosis was measured by immunobloting for cleaved caspase 3. Results from two experiments are shown. E. Clone #4S2 cells were transfected as in (D) and 24 h later doxycycline was added or not for 48 h and apoptosis was measured by immunobloting for cleaved caspase 3. INS-1 cells treated or not with 0.3 mM straurosporine (St.) was included as a positive control for cleaved caspase 3.

Mentions: As an alternative approach for inhibiting ERAD we targeted the Herp protein which was found to be induced early by mutant insulin expression (Table 1). Herp has been implicated in ERAD of certain misfolded proteins [23], although whether it is required for misfolded insulin degradation has not been examined. We first confirmed that the Herp protein is induced in response to mutant insulin expression (Figure 9A). To perturb ERAD function, we blunted the induction of Herp expression using siRNA (Figure 9B, C). Reducing Herp expression by about 40-50% resulted in an increase in the steady-state levels of the mutant insulin protein, even in the absence of doxycycline induction (Figure 9B). This also increased apoptosis levels in the population relative to control siRNA transfected cells (Figure 9D). With doxycyline induction for 48 h the levels of Ins2 (C96Y)-EGFP in Herp siRNA-treated cells was increased relative to control siRNA transfected cells, but the effect was small (Figure 9B). In some experiments we also observed a reduction in the degradation fragments in Herp siRNA-treated cells (see Figure 9B, Expt.#2, low exposure), but this was not a consistent finding (Figure 9B, Expt.#1). The inconsistent results may be due to the inefficient knock-down of the Herp protein achieved in these studies. Regardless, the levels of apoptosis in the population depleted of Herp expression was significantly higher compared to control siRNA-transfected cells as measured by cleaved caspase 3 levels (Figure 9E). Altogether, these results suggest that the mutant insulin is an ERAD target and that Herp function is required for its degradation and that reducing Herp levels increases mutant insulin levels and sensitizes the cells to apoptosis.


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)

Inhibiting Herp expression increases insulin 2 (C96Y)-EGFP levels and enhances apoptosis. A. Clone #4S2 cells were untreated or treated with 2 μg/ml doxycycline for the times indicated and cell lysates were prepared and immunoblotted. B. Clone #4S2 cells were transfected with control siRNA or an siRNA directed against Herp and treated or not with doxycycline for 48 h. Cells were lysed and immunoblotted as indicated. A low and high exposure of the anti-GFP western blot is shown. Results from two of four independent experiments are shown. C. Immunoblots were quantified and the mean relative expression of Herp from 4 independent experiments is presented. D. Clone #4S2 cells were transfected with control or Herp siRNA and 48 h later apoptosis was measured by immunobloting for cleaved caspase 3. Results from two experiments are shown. E. Clone #4S2 cells were transfected as in (D) and 24 h later doxycycline was added or not for 48 h and apoptosis was measured by immunobloting for cleaved caspase 3. INS-1 cells treated or not with 0.3 mM straurosporine (St.) was included as a positive control for cleaved caspase 3.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 9: Inhibiting Herp expression increases insulin 2 (C96Y)-EGFP levels and enhances apoptosis. A. Clone #4S2 cells were untreated or treated with 2 μg/ml doxycycline for the times indicated and cell lysates were prepared and immunoblotted. B. Clone #4S2 cells were transfected with control siRNA or an siRNA directed against Herp and treated or not with doxycycline for 48 h. Cells were lysed and immunoblotted as indicated. A low and high exposure of the anti-GFP western blot is shown. Results from two of four independent experiments are shown. C. Immunoblots were quantified and the mean relative expression of Herp from 4 independent experiments is presented. D. Clone #4S2 cells were transfected with control or Herp siRNA and 48 h later apoptosis was measured by immunobloting for cleaved caspase 3. Results from two experiments are shown. E. Clone #4S2 cells were transfected as in (D) and 24 h later doxycycline was added or not for 48 h and apoptosis was measured by immunobloting for cleaved caspase 3. INS-1 cells treated or not with 0.3 mM straurosporine (St.) was included as a positive control for cleaved caspase 3.
Mentions: As an alternative approach for inhibiting ERAD we targeted the Herp protein which was found to be induced early by mutant insulin expression (Table 1). Herp has been implicated in ERAD of certain misfolded proteins [23], although whether it is required for misfolded insulin degradation has not been examined. We first confirmed that the Herp protein is induced in response to mutant insulin expression (Figure 9A). To perturb ERAD function, we blunted the induction of Herp expression using siRNA (Figure 9B, C). Reducing Herp expression by about 40-50% resulted in an increase in the steady-state levels of the mutant insulin protein, even in the absence of doxycycline induction (Figure 9B). This also increased apoptosis levels in the population relative to control siRNA transfected cells (Figure 9D). With doxycyline induction for 48 h the levels of Ins2 (C96Y)-EGFP in Herp siRNA-treated cells was increased relative to control siRNA transfected cells, but the effect was small (Figure 9B). In some experiments we also observed a reduction in the degradation fragments in Herp siRNA-treated cells (see Figure 9B, Expt.#2, low exposure), but this was not a consistent finding (Figure 9B, Expt.#1). The inconsistent results may be due to the inefficient knock-down of the Herp protein achieved in these studies. Regardless, the levels of apoptosis in the population depleted of Herp expression was significantly higher compared to control siRNA-transfected cells as measured by cleaved caspase 3 levels (Figure 9E). Altogether, these results suggest that the mutant insulin is an ERAD target and that Herp function is required for its degradation and that reducing Herp levels increases mutant insulin levels and sensitizes the cells to apoptosis.

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