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Hydrogen peroxide stimulates nuclear import of the POU homeodomain protein Oct-1 and its repressive effect on the expression of Cdx-2.

Wang P, Jin T - BMC Cell Biol. (2010)

Bottom Line: We found recently that in pancreatic and intestinal endocrine cells, Oct-1 also functions as a sensor for cyclic AMP (cAMP).Oct-1 binds to Cdx-2 promoter and represses its expression. cAMP elevation leads to increased nuclear exclusion of Oct-1, associated with reduced recruitment of nuclear co-repressors to the Cdx-2 promoter and increased Cdx-2 expression.Finally, increased Oct-1 nuclear content upon H2O2 treatment in a pancreatic alpha cell line was associated with reduced Cdx-2 and gcg mRNA expression.

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Affiliation: Div of Cell and Molecular Biology, Toronto General Research Institute, University Health Network, 10-354 Toronto Medical Discovery Tower, The MaRS Building, 101 College St, Toronto, Ontario M5G 1L7, Canada.

ABSTRACT

Background: The ubiquitously expressed POU homeodomain protein Oct-1 serves as a sensor for stress induced by irradiation. We found recently that in pancreatic and intestinal endocrine cells, Oct-1 also functions as a sensor for cyclic AMP (cAMP). The caudal homeobox gene Cdx-2 is a transactivator of proglucagon (gcg) and pro-insulin genes. Oct-1 binds to Cdx-2 promoter and represses its expression. cAMP elevation leads to increased nuclear exclusion of Oct-1, associated with reduced recruitment of nuclear co-repressors to the Cdx-2 promoter and increased Cdx-2 expression.

Results: We show in this study that inducing oxidative stress by hydrogen peroxide (H2O2) increased nuclear Oct-1 content in both pancreatic alpha and beta cell lines, as well as in a battery of other cells. This increase was then attributed to accelerated nuclear import of Oct-1, assessed by Fluorescence Recovery After Photobleaching (FRAP) using green fluorescence protein (EGFP) tagged Oct-1 molecule. H2O2 treatment was then shown to stimulate the activities of DNA-dependent protein kinase (DNA-PK) and c-jun N-terminal kinase (JNK). Finally, increased Oct-1 nuclear content upon H2O2 treatment in a pancreatic alpha cell line was associated with reduced Cdx-2 and gcg mRNA expression.

Conclusion: These observations suggest that Oct-1 functions as a sensor for both metabolic and stress/survival signaling pathways via altering its nuclear-cytoplasmic shuttling.

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H2O2 increased nuclear Oct-1 content, associated with reduced Cdx-2 and gcg expression. (A) InR1-G9 cells were treated with 100 μM H2O2 for 1 to 6 h, nuclear extract was prepared, followed by the analysis of Oct-1 and Cdx-2 expression by Western blotting. (B) The quantification results of Fig 5A. (C, D) InR1-G9 cells were treated with 100 μM H2O2 for 1 to 6 h. Cdx-2 (C) and gcg (D) mRNA expression was measured by real time RT-PCR.
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Figure 5: H2O2 increased nuclear Oct-1 content, associated with reduced Cdx-2 and gcg expression. (A) InR1-G9 cells were treated with 100 μM H2O2 for 1 to 6 h, nuclear extract was prepared, followed by the analysis of Oct-1 and Cdx-2 expression by Western blotting. (B) The quantification results of Fig 5A. (C, D) InR1-G9 cells were treated with 100 μM H2O2 for 1 to 6 h. Cdx-2 (C) and gcg (D) mRNA expression was measured by real time RT-PCR.

Mentions: Since Oct-1 serves as a transcriptional repressor of Cdx-2, expressed in pancreatic and intestinal endocrine cells, we wonder whether increased nuclear content of Oct-1 in response to oxidative stress reduces Cdx-2 expression in such an endocrine cell line. While H2O2 treatment increased nuclear Oct-1 content in the InR1-G9 cell line (Figure 5A, B), the inhibitory effect on Cdx-2 protein expression, however, was not notable until 6 h (Figure. 5A). This delay could be due to increased stability of Cdx-2 protein in response to a stress [23,24], We therefore directly assessed the effect of H2O2 treatment on Cdx-2 mRNA expression. As shown in Figure 5C, H2O2 treatment resulted in about 40% reduction in Cdx-2 mRNA level over the entire 6 h experimental period, confirming that oxidative stress represses Cdx-2 mRNA expression. Additionally, the expression of Gcg mRNA, which is a known downstream target of Cdx-2 in pancreatic α cells [25], is also reduced by H2O2 treatment, to approximately 50% level (Figure 5D).


Hydrogen peroxide stimulates nuclear import of the POU homeodomain protein Oct-1 and its repressive effect on the expression of Cdx-2.

Wang P, Jin T - BMC Cell Biol. (2010)

H2O2 increased nuclear Oct-1 content, associated with reduced Cdx-2 and gcg expression. (A) InR1-G9 cells were treated with 100 μM H2O2 for 1 to 6 h, nuclear extract was prepared, followed by the analysis of Oct-1 and Cdx-2 expression by Western blotting. (B) The quantification results of Fig 5A. (C, D) InR1-G9 cells were treated with 100 μM H2O2 for 1 to 6 h. Cdx-2 (C) and gcg (D) mRNA expression was measured by real time RT-PCR.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 5: H2O2 increased nuclear Oct-1 content, associated with reduced Cdx-2 and gcg expression. (A) InR1-G9 cells were treated with 100 μM H2O2 for 1 to 6 h, nuclear extract was prepared, followed by the analysis of Oct-1 and Cdx-2 expression by Western blotting. (B) The quantification results of Fig 5A. (C, D) InR1-G9 cells were treated with 100 μM H2O2 for 1 to 6 h. Cdx-2 (C) and gcg (D) mRNA expression was measured by real time RT-PCR.
Mentions: Since Oct-1 serves as a transcriptional repressor of Cdx-2, expressed in pancreatic and intestinal endocrine cells, we wonder whether increased nuclear content of Oct-1 in response to oxidative stress reduces Cdx-2 expression in such an endocrine cell line. While H2O2 treatment increased nuclear Oct-1 content in the InR1-G9 cell line (Figure 5A, B), the inhibitory effect on Cdx-2 protein expression, however, was not notable until 6 h (Figure. 5A). This delay could be due to increased stability of Cdx-2 protein in response to a stress [23,24], We therefore directly assessed the effect of H2O2 treatment on Cdx-2 mRNA expression. As shown in Figure 5C, H2O2 treatment resulted in about 40% reduction in Cdx-2 mRNA level over the entire 6 h experimental period, confirming that oxidative stress represses Cdx-2 mRNA expression. Additionally, the expression of Gcg mRNA, which is a known downstream target of Cdx-2 in pancreatic α cells [25], is also reduced by H2O2 treatment, to approximately 50% level (Figure 5D).

Bottom Line: We found recently that in pancreatic and intestinal endocrine cells, Oct-1 also functions as a sensor for cyclic AMP (cAMP).Oct-1 binds to Cdx-2 promoter and represses its expression. cAMP elevation leads to increased nuclear exclusion of Oct-1, associated with reduced recruitment of nuclear co-repressors to the Cdx-2 promoter and increased Cdx-2 expression.Finally, increased Oct-1 nuclear content upon H2O2 treatment in a pancreatic alpha cell line was associated with reduced Cdx-2 and gcg mRNA expression.

View Article: PubMed Central - HTML - PubMed

Affiliation: Div of Cell and Molecular Biology, Toronto General Research Institute, University Health Network, 10-354 Toronto Medical Discovery Tower, The MaRS Building, 101 College St, Toronto, Ontario M5G 1L7, Canada.

ABSTRACT

Background: The ubiquitously expressed POU homeodomain protein Oct-1 serves as a sensor for stress induced by irradiation. We found recently that in pancreatic and intestinal endocrine cells, Oct-1 also functions as a sensor for cyclic AMP (cAMP). The caudal homeobox gene Cdx-2 is a transactivator of proglucagon (gcg) and pro-insulin genes. Oct-1 binds to Cdx-2 promoter and represses its expression. cAMP elevation leads to increased nuclear exclusion of Oct-1, associated with reduced recruitment of nuclear co-repressors to the Cdx-2 promoter and increased Cdx-2 expression.

Results: We show in this study that inducing oxidative stress by hydrogen peroxide (H2O2) increased nuclear Oct-1 content in both pancreatic alpha and beta cell lines, as well as in a battery of other cells. This increase was then attributed to accelerated nuclear import of Oct-1, assessed by Fluorescence Recovery After Photobleaching (FRAP) using green fluorescence protein (EGFP) tagged Oct-1 molecule. H2O2 treatment was then shown to stimulate the activities of DNA-dependent protein kinase (DNA-PK) and c-jun N-terminal kinase (JNK). Finally, increased Oct-1 nuclear content upon H2O2 treatment in a pancreatic alpha cell line was associated with reduced Cdx-2 and gcg mRNA expression.

Conclusion: These observations suggest that Oct-1 functions as a sensor for both metabolic and stress/survival signaling pathways via altering its nuclear-cytoplasmic shuttling.

Show MeSH
Related in: MedlinePlus