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Molecular basis for vulnerability to mitochondrial and oxidative stress in a neuroendocrine CRI-G1 cell line.

Chandiramani N, Wang X, Margeta M - PLoS ONE (2011)

Bottom Line: Thus, differential vulnerability to mitochondrial toxins between these two cell types likely reflects differences in their ability to handle metabolically generated reactive oxygen species rather than differences in ATP production/utilization or in downstream apoptotic machinery.Genome-wide gene expression analysis and follow-up biochemical studies revealed that, in this experimental system, increased vulnerability to mitochondrial and oxidative stress was associated with (1) inhibition of ARE/Nrf2/Keap1 antioxidant pathway; (2) decreased expression of antioxidant and phase I/II conjugation enzymes, most of which are Nrf2 transcriptional targets; (3) increased expression of molecular chaperones, many of which are also considered Nrf2 transcriptional targets; (4) increased expression of β cell-specific genes and transcription factors that specify/maintain β cell fate; and (5) reconstitution of glucose-stimulated insulin secretion.In addition, the newly identified CRI-G1-RS cell line represents a new experimental model for investigating how endogenous antioxidants affect glucose sensing and insulin release by pancreatic β cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, University of California San Francisco, San Francisco, California, United States of America.

ABSTRACT

Background: Many age-associated disorders (including diabetes, cancer, and neurodegenerative diseases) are linked to mitochondrial dysfunction, which leads to impaired cellular bioenergetics and increased oxidative stress. However, it is not known what genetic and molecular pathways underlie differential vulnerability to mitochondrial dysfunction observed among different cell types.

Methodology/principal findings: Starting with an insulinoma cell line as a model for a neuronal/endocrine cell type, we isolated a novel subclonal line (named CRI-G1-RS) that was more susceptible to cell death induced by mitochondrial respiratory chain inhibitors than the parental CRI-G1 line (renamed CRI-G1-RR for clarity). Compared to parental RR cells, RS cells were also more vulnerable to direct oxidative stress, but equally vulnerable to mitochondrial uncoupling and less vulnerable to protein kinase inhibition-induced apoptosis. Thus, differential vulnerability to mitochondrial toxins between these two cell types likely reflects differences in their ability to handle metabolically generated reactive oxygen species rather than differences in ATP production/utilization or in downstream apoptotic machinery. Genome-wide gene expression analysis and follow-up biochemical studies revealed that, in this experimental system, increased vulnerability to mitochondrial and oxidative stress was associated with (1) inhibition of ARE/Nrf2/Keap1 antioxidant pathway; (2) decreased expression of antioxidant and phase I/II conjugation enzymes, most of which are Nrf2 transcriptional targets; (3) increased expression of molecular chaperones, many of which are also considered Nrf2 transcriptional targets; (4) increased expression of β cell-specific genes and transcription factors that specify/maintain β cell fate; and (5) reconstitution of glucose-stimulated insulin secretion.

Conclusions/significance: The molecular profile presented here will enable identification of individual genes or gene clusters that shape vulnerability to mitochondrial dysfunction and thus represent potential therapeutic targets for diabetes and neurodegenerative diseases. In addition, the newly identified CRI-G1-RS cell line represents a new experimental model for investigating how endogenous antioxidants affect glucose sensing and insulin release by pancreatic β cells.

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Related in: MedlinePlus

Differential expression of Nrf2, Keap1, and DJ-1.A. At baseline, Nrf2 protein level (apparent MW, 77 kDa) in                            the nuclear fraction is significantly lower in CRI-G1-RS than RR cells.                                B. At baseline, Keap1 protein level (apparent MW, 69                            kDa) in the whole cell lysate is significantly higher in CRI-G1-RS than                            RR cells. C. At baseline, DJ-1 protein level (apparent MW,                            21 kDa) in the whole cell lysate is significantly lower in CRI-G1-RS                            than RR cells. D and E. Treatment with 5 µM SF or 10                            µM tBHQ for 8 h (D) or 16 h (E)                            activated Nrf2 pathway to a similar extent in both CRI-G1-RR and RS                            cells. Apparent MW for lamin A was 68 kDa; for tubulin, 51 kDa. In                            panels A–C, statistical significance was determined by two-tailed                            Student's t-test; scatter plots of all data points are shown. In                            panels D and E, statistical significance was determined by two-way                            ANOVA; data are plotted as mean ± S.E.M.                            (n = 5). While there was no statistically                            significant difference between the two cell types overall, ad-hoc                            Bonferroni post-tests detected a statistically significant difference                            between DMSO-treated RR and RS samples (p<0.01), similar to the                            difference observed with untreated samples (panel A). *, p<0.05;                            ***, p<0.001.
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pone-0014485-g007: Differential expression of Nrf2, Keap1, and DJ-1.A. At baseline, Nrf2 protein level (apparent MW, 77 kDa) in the nuclear fraction is significantly lower in CRI-G1-RS than RR cells. B. At baseline, Keap1 protein level (apparent MW, 69 kDa) in the whole cell lysate is significantly higher in CRI-G1-RS than RR cells. C. At baseline, DJ-1 protein level (apparent MW, 21 kDa) in the whole cell lysate is significantly lower in CRI-G1-RS than RR cells. D and E. Treatment with 5 µM SF or 10 µM tBHQ for 8 h (D) or 16 h (E) activated Nrf2 pathway to a similar extent in both CRI-G1-RR and RS cells. Apparent MW for lamin A was 68 kDa; for tubulin, 51 kDa. In panels A–C, statistical significance was determined by two-tailed Student's t-test; scatter plots of all data points are shown. In panels D and E, statistical significance was determined by two-way ANOVA; data are plotted as mean ± S.E.M. (n = 5). While there was no statistically significant difference between the two cell types overall, ad-hoc Bonferroni post-tests detected a statistically significant difference between DMSO-treated RR and RS samples (p<0.01), similar to the difference observed with untreated samples (panel A). *, p<0.05; ***, p<0.001.

Mentions: To further delineate differences in Nrf2 pathway regulation between RR and RS cells, we determined the mRNA and protein expression levels of Nrf2 and its regulator Keap1 (Kelch-like ECH-associated protein 1). Under baseline conditions, Nrf2 is bound to actin cytoskeleton by Keap1, which prevents its translocation into the nucleus and targets it for ubiquitination and proteosomal degradation. In response to oxidative stress, kinase activation, or small molecule activators such as tert-butylhydroquinone (tBHQ) and sulforaphane (SF), Nrf2 is stabilized and translocates into the nucleus, where it binds the antioxidant response element (ARE) sequences in the promoter of its target genes and activates their transcription (reviewed in [24], [25]). We also determined mRNA and protein expression levels of DJ-1, which regulates Nrf2 stability in fibroblasts and the lung or lung-derived cell lines [26], [27], but not in neurons or astrocytes [28]. DJ-1 is a redox-sensitive protein [29], [30], mutations in which have been linked to an autosomal recessive form of Parkinson's disease [31]; it has recently been shown to protect β cells from various stress conditions [32]. The microarray data showed a modest but statistically significant decrease in expression of Nrf2 mRNA (Nfe2l2; log2fold change = −0.48, p-value = 0.0001); however, this finding was not confirmed by qRT-PCR (log2fold change = −0.28, p-value = 0.36; Table 1). Nonetheless, the expression of transcriptionally-active Nrf2 protein (assessed by immunoblotting of nuclear fractions with anti-Nrf2 antibody; Fig. 7A) was significantly lower in RS than in RR cells (53% or ∼2 fold decrease; n = 8, p<0.0001). In cytoplasmic fractions, Nrf2 protein level was below detection threshold in either cell type (data not shown). Significant differences in protein expression without major changes in mRNA expression suggest that regulation primarily occurs on the post-translational level. Consistent with this possibility, we found that RR and RS cells expressed different levels of Keap1 and DJ-1. Specifically, Keap1 was significantly upregulated in RS cells both on mRNA (microarray: log2fold change = 0.46, p-value = 2.46×10−7; qRT-PCR: log2fold change = 0.73, p-value = 0.0004; Table 1) and protein levels (35.6% or 1.36 fold increase; n = 10, p<0.05; Fig. 7B). In contrast, the expression of DJ-1/Park7 mRNA (microarray: log2fold change = −0.45, p-value = 5.87×10−7; qRT-PCR: log2fold change = 0.54, p-value = 0.005; Table 1) and protein (31.5% or 1.46 fold decrease; n = 7, p = 0.0002; Fig. 7C) was significantly decreased in RS cells. Given the reciprocal functions of Keap1 and DJ-1 in the regulation of Nrf2 pathway, both of these changes would be expected to result in destabilization of Nrf2 protein and subsequent downregulation of Nrf2 target genes in RS cells, as observed in the microarray and qRT-PCR datasets. Interestingly, treatment with 5 µM SF or 10 µM tBHQ for either 8 h (Fig. 7D) or 16 h (Fig. 7E) upregulated nuclear Nrf2 expression to the same extent in both RR and RS cells despite the baseline differences in Nrf2 pathway activity between the two cell types. (Visualizing low basal levels of nuclear Nrf2 protein in both cell types required long film exposures, raising a concern that much higher Nrf2 levels in SF- or tBHQ-treated samples were not accurately quantified. To address this possibility, band densitometry was also performed on short exposure films from the same experiments and showed essentially the same results [Supplemental Fig. S5].) Interestingly, tBHQ-induced activation of Nrf2 pathway was comparable to SF-induced activation following 8 h treatment (Figs. 7D and S5-A), but less strong following 16 h treatment (Figs. 7E and S5-B) in both cell types. Taken together, the data indicate that both the overall activity of Nrf2 pathway and expression level of many Nrf2-regulated antioxidant and detoxifying enzymes are decreased in CRI-G1-RS cells; these genetic changes are likely to mediate (or at least contribute to) increased vulnerability to mitochondrial and oxidative stress seen in these cells.


Molecular basis for vulnerability to mitochondrial and oxidative stress in a neuroendocrine CRI-G1 cell line.

Chandiramani N, Wang X, Margeta M - PLoS ONE (2011)

Differential expression of Nrf2, Keap1, and DJ-1.A. At baseline, Nrf2 protein level (apparent MW, 77 kDa) in                            the nuclear fraction is significantly lower in CRI-G1-RS than RR cells.                                B. At baseline, Keap1 protein level (apparent MW, 69                            kDa) in the whole cell lysate is significantly higher in CRI-G1-RS than                            RR cells. C. At baseline, DJ-1 protein level (apparent MW,                            21 kDa) in the whole cell lysate is significantly lower in CRI-G1-RS                            than RR cells. D and E. Treatment with 5 µM SF or 10                            µM tBHQ for 8 h (D) or 16 h (E)                            activated Nrf2 pathway to a similar extent in both CRI-G1-RR and RS                            cells. Apparent MW for lamin A was 68 kDa; for tubulin, 51 kDa. In                            panels A–C, statistical significance was determined by two-tailed                            Student's t-test; scatter plots of all data points are shown. In                            panels D and E, statistical significance was determined by two-way                            ANOVA; data are plotted as mean ± S.E.M.                            (n = 5). While there was no statistically                            significant difference between the two cell types overall, ad-hoc                            Bonferroni post-tests detected a statistically significant difference                            between DMSO-treated RR and RS samples (p<0.01), similar to the                            difference observed with untreated samples (panel A). *, p<0.05;                            ***, p<0.001.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3020905&req=5

pone-0014485-g007: Differential expression of Nrf2, Keap1, and DJ-1.A. At baseline, Nrf2 protein level (apparent MW, 77 kDa) in the nuclear fraction is significantly lower in CRI-G1-RS than RR cells. B. At baseline, Keap1 protein level (apparent MW, 69 kDa) in the whole cell lysate is significantly higher in CRI-G1-RS than RR cells. C. At baseline, DJ-1 protein level (apparent MW, 21 kDa) in the whole cell lysate is significantly lower in CRI-G1-RS than RR cells. D and E. Treatment with 5 µM SF or 10 µM tBHQ for 8 h (D) or 16 h (E) activated Nrf2 pathway to a similar extent in both CRI-G1-RR and RS cells. Apparent MW for lamin A was 68 kDa; for tubulin, 51 kDa. In panels A–C, statistical significance was determined by two-tailed Student's t-test; scatter plots of all data points are shown. In panels D and E, statistical significance was determined by two-way ANOVA; data are plotted as mean ± S.E.M. (n = 5). While there was no statistically significant difference between the two cell types overall, ad-hoc Bonferroni post-tests detected a statistically significant difference between DMSO-treated RR and RS samples (p<0.01), similar to the difference observed with untreated samples (panel A). *, p<0.05; ***, p<0.001.
Mentions: To further delineate differences in Nrf2 pathway regulation between RR and RS cells, we determined the mRNA and protein expression levels of Nrf2 and its regulator Keap1 (Kelch-like ECH-associated protein 1). Under baseline conditions, Nrf2 is bound to actin cytoskeleton by Keap1, which prevents its translocation into the nucleus and targets it for ubiquitination and proteosomal degradation. In response to oxidative stress, kinase activation, or small molecule activators such as tert-butylhydroquinone (tBHQ) and sulforaphane (SF), Nrf2 is stabilized and translocates into the nucleus, where it binds the antioxidant response element (ARE) sequences in the promoter of its target genes and activates their transcription (reviewed in [24], [25]). We also determined mRNA and protein expression levels of DJ-1, which regulates Nrf2 stability in fibroblasts and the lung or lung-derived cell lines [26], [27], but not in neurons or astrocytes [28]. DJ-1 is a redox-sensitive protein [29], [30], mutations in which have been linked to an autosomal recessive form of Parkinson's disease [31]; it has recently been shown to protect β cells from various stress conditions [32]. The microarray data showed a modest but statistically significant decrease in expression of Nrf2 mRNA (Nfe2l2; log2fold change = −0.48, p-value = 0.0001); however, this finding was not confirmed by qRT-PCR (log2fold change = −0.28, p-value = 0.36; Table 1). Nonetheless, the expression of transcriptionally-active Nrf2 protein (assessed by immunoblotting of nuclear fractions with anti-Nrf2 antibody; Fig. 7A) was significantly lower in RS than in RR cells (53% or ∼2 fold decrease; n = 8, p<0.0001). In cytoplasmic fractions, Nrf2 protein level was below detection threshold in either cell type (data not shown). Significant differences in protein expression without major changes in mRNA expression suggest that regulation primarily occurs on the post-translational level. Consistent with this possibility, we found that RR and RS cells expressed different levels of Keap1 and DJ-1. Specifically, Keap1 was significantly upregulated in RS cells both on mRNA (microarray: log2fold change = 0.46, p-value = 2.46×10−7; qRT-PCR: log2fold change = 0.73, p-value = 0.0004; Table 1) and protein levels (35.6% or 1.36 fold increase; n = 10, p<0.05; Fig. 7B). In contrast, the expression of DJ-1/Park7 mRNA (microarray: log2fold change = −0.45, p-value = 5.87×10−7; qRT-PCR: log2fold change = 0.54, p-value = 0.005; Table 1) and protein (31.5% or 1.46 fold decrease; n = 7, p = 0.0002; Fig. 7C) was significantly decreased in RS cells. Given the reciprocal functions of Keap1 and DJ-1 in the regulation of Nrf2 pathway, both of these changes would be expected to result in destabilization of Nrf2 protein and subsequent downregulation of Nrf2 target genes in RS cells, as observed in the microarray and qRT-PCR datasets. Interestingly, treatment with 5 µM SF or 10 µM tBHQ for either 8 h (Fig. 7D) or 16 h (Fig. 7E) upregulated nuclear Nrf2 expression to the same extent in both RR and RS cells despite the baseline differences in Nrf2 pathway activity between the two cell types. (Visualizing low basal levels of nuclear Nrf2 protein in both cell types required long film exposures, raising a concern that much higher Nrf2 levels in SF- or tBHQ-treated samples were not accurately quantified. To address this possibility, band densitometry was also performed on short exposure films from the same experiments and showed essentially the same results [Supplemental Fig. S5].) Interestingly, tBHQ-induced activation of Nrf2 pathway was comparable to SF-induced activation following 8 h treatment (Figs. 7D and S5-A), but less strong following 16 h treatment (Figs. 7E and S5-B) in both cell types. Taken together, the data indicate that both the overall activity of Nrf2 pathway and expression level of many Nrf2-regulated antioxidant and detoxifying enzymes are decreased in CRI-G1-RS cells; these genetic changes are likely to mediate (or at least contribute to) increased vulnerability to mitochondrial and oxidative stress seen in these cells.

Bottom Line: Thus, differential vulnerability to mitochondrial toxins between these two cell types likely reflects differences in their ability to handle metabolically generated reactive oxygen species rather than differences in ATP production/utilization or in downstream apoptotic machinery.Genome-wide gene expression analysis and follow-up biochemical studies revealed that, in this experimental system, increased vulnerability to mitochondrial and oxidative stress was associated with (1) inhibition of ARE/Nrf2/Keap1 antioxidant pathway; (2) decreased expression of antioxidant and phase I/II conjugation enzymes, most of which are Nrf2 transcriptional targets; (3) increased expression of molecular chaperones, many of which are also considered Nrf2 transcriptional targets; (4) increased expression of β cell-specific genes and transcription factors that specify/maintain β cell fate; and (5) reconstitution of glucose-stimulated insulin secretion.In addition, the newly identified CRI-G1-RS cell line represents a new experimental model for investigating how endogenous antioxidants affect glucose sensing and insulin release by pancreatic β cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, University of California San Francisco, San Francisco, California, United States of America.

ABSTRACT

Background: Many age-associated disorders (including diabetes, cancer, and neurodegenerative diseases) are linked to mitochondrial dysfunction, which leads to impaired cellular bioenergetics and increased oxidative stress. However, it is not known what genetic and molecular pathways underlie differential vulnerability to mitochondrial dysfunction observed among different cell types.

Methodology/principal findings: Starting with an insulinoma cell line as a model for a neuronal/endocrine cell type, we isolated a novel subclonal line (named CRI-G1-RS) that was more susceptible to cell death induced by mitochondrial respiratory chain inhibitors than the parental CRI-G1 line (renamed CRI-G1-RR for clarity). Compared to parental RR cells, RS cells were also more vulnerable to direct oxidative stress, but equally vulnerable to mitochondrial uncoupling and less vulnerable to protein kinase inhibition-induced apoptosis. Thus, differential vulnerability to mitochondrial toxins between these two cell types likely reflects differences in their ability to handle metabolically generated reactive oxygen species rather than differences in ATP production/utilization or in downstream apoptotic machinery. Genome-wide gene expression analysis and follow-up biochemical studies revealed that, in this experimental system, increased vulnerability to mitochondrial and oxidative stress was associated with (1) inhibition of ARE/Nrf2/Keap1 antioxidant pathway; (2) decreased expression of antioxidant and phase I/II conjugation enzymes, most of which are Nrf2 transcriptional targets; (3) increased expression of molecular chaperones, many of which are also considered Nrf2 transcriptional targets; (4) increased expression of β cell-specific genes and transcription factors that specify/maintain β cell fate; and (5) reconstitution of glucose-stimulated insulin secretion.

Conclusions/significance: The molecular profile presented here will enable identification of individual genes or gene clusters that shape vulnerability to mitochondrial dysfunction and thus represent potential therapeutic targets for diabetes and neurodegenerative diseases. In addition, the newly identified CRI-G1-RS cell line represents a new experimental model for investigating how endogenous antioxidants affect glucose sensing and insulin release by pancreatic β cells.

Show MeSH
Related in: MedlinePlus