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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.

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.

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H2O2 treatment leads to increased nuclear Oct-1 content. (A) Left panel, InR1-G9 cells were transfected with Oct-1-EGFP and treated with vehicle, or H2O2 (100 μM) for 1 h before confocal microscopy examination. The right panel shows the counting results. (B) InR1-G9 cells were transfected with myc-Oct-1 for 24 h, followed by vehicle or H2O2 (100 μM) treatment for 1 h. Immunofluorescence staining were conducted, Blue, nuclei, red, myc-Oct-1. Arrow indicates enhanced nuclear myc-Oct-1 or Oct-1-EGFP expression, while triangle indicates mixed expression in both nuclei and cytosol.
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Figure 2: H2O2 treatment leads to increased nuclear Oct-1 content. (A) Left panel, InR1-G9 cells were transfected with Oct-1-EGFP and treated with vehicle, or H2O2 (100 μM) for 1 h before confocal microscopy examination. The right panel shows the counting results. (B) InR1-G9 cells were transfected with myc-Oct-1 for 24 h, followed by vehicle or H2O2 (100 μM) treatment for 1 h. Immunofluorescence staining were conducted, Blue, nuclei, red, myc-Oct-1. Arrow indicates enhanced nuclear myc-Oct-1 or Oct-1-EGFP expression, while triangle indicates mixed expression in both nuclei and cytosol.

Mentions: We then examined whether H2O2 treatment affects sub-cellular distributions of Oct-1-EGFP. Oct-1-EGFP was transiently transfected into the InR1-G9 cell line for 24 h, followed by H2O2 treatment. We found that the treatment of InR1-G9 cells with 100 μM H2O2 for 0.5, 1, and 2 h led to increased percentages of pattern I cells. Figure 2A (left panel) shows our representative results after a 1 h H2O2 treatment, cells with nuclear Oct-1-EGFP expression (Pattern I) being sharply increased from 58% to 94% (right panel). We therefore suggest that H2O2 treatment significantly stimulates nuclear import of Oct-1, which is opposite to the effect induced by cAMP elevation. We then transfected InR1-G9 cells with myc-tagged Oct-1. 24 h after the transfection, cells were treated with vehicle or 100 μM H2O2 for 1 h before immunofluorescence staining, assessing the distribution of myc-tagged Oct-1. Representative images in Figure 2B indicate that H2O2 treatment increased nuclear myc-Oct immunofluorescence signal.


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 treatment leads to increased nuclear Oct-1 content. (A) Left panel, InR1-G9 cells were transfected with Oct-1-EGFP and treated with vehicle, or H2O2 (100 μM) for 1 h before confocal microscopy examination. The right panel shows the counting results. (B) InR1-G9 cells were transfected with myc-Oct-1 for 24 h, followed by vehicle or H2O2 (100 μM) treatment for 1 h. Immunofluorescence staining were conducted, Blue, nuclei, red, myc-Oct-1. Arrow indicates enhanced nuclear myc-Oct-1 or Oct-1-EGFP expression, while triangle indicates mixed expression in both nuclei and cytosol.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2913919&req=5

Figure 2: H2O2 treatment leads to increased nuclear Oct-1 content. (A) Left panel, InR1-G9 cells were transfected with Oct-1-EGFP and treated with vehicle, or H2O2 (100 μM) for 1 h before confocal microscopy examination. The right panel shows the counting results. (B) InR1-G9 cells were transfected with myc-Oct-1 for 24 h, followed by vehicle or H2O2 (100 μM) treatment for 1 h. Immunofluorescence staining were conducted, Blue, nuclei, red, myc-Oct-1. Arrow indicates enhanced nuclear myc-Oct-1 or Oct-1-EGFP expression, while triangle indicates mixed expression in both nuclei and cytosol.
Mentions: We then examined whether H2O2 treatment affects sub-cellular distributions of Oct-1-EGFP. Oct-1-EGFP was transiently transfected into the InR1-G9 cell line for 24 h, followed by H2O2 treatment. We found that the treatment of InR1-G9 cells with 100 μM H2O2 for 0.5, 1, and 2 h led to increased percentages of pattern I cells. Figure 2A (left panel) shows our representative results after a 1 h H2O2 treatment, cells with nuclear Oct-1-EGFP expression (Pattern I) being sharply increased from 58% to 94% (right panel). We therefore suggest that H2O2 treatment significantly stimulates nuclear import of Oct-1, which is opposite to the effect induced by cAMP elevation. We then transfected InR1-G9 cells with myc-tagged Oct-1. 24 h after the transfection, cells were treated with vehicle or 100 μM H2O2 for 1 h before immunofluorescence staining, assessing the distribution of myc-tagged Oct-1. Representative images in Figure 2B indicate that H2O2 treatment increased nuclear myc-Oct immunofluorescence signal.

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