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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 provoked DNA-PK and JNK activity.(A) InR1-G9 cells were treated with Zeocin (50, 100 or 200 μg/ml) for indicated time before harvested. Oct-1 expression in the nuclear extract (NE) was then assessed. (B) InR1-G9 cells were treated with vehicle, Zeocin (100 μg/ml) or H2O2 (00 μM) for 2 h before harvested for DNA-PK assay. The values are mean +/- S.E (n = 3). *, P < 0.05. (C, D) InR1-G9 cells were treated with forskolin/IBMX (C) or H2O2 (100 μM) (D) for indicated time. Whole cell lysates were utilized for assessing the expression of JNK and phosphorylated JNK, ERK and phosphorylated ERK.
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Figure 4: H2O2 provoked DNA-PK and JNK activity.(A) InR1-G9 cells were treated with Zeocin (50, 100 or 200 μg/ml) for indicated time before harvested. Oct-1 expression in the nuclear extract (NE) was then assessed. (B) InR1-G9 cells were treated with vehicle, Zeocin (100 μg/ml) or H2O2 (00 μM) for 2 h before harvested for DNA-PK assay. The values are mean +/- S.E (n = 3). *, P < 0.05. (C, D) InR1-G9 cells were treated with forskolin/IBMX (C) or H2O2 (100 μM) (D) for indicated time. Whole cell lysates were utilized for assessing the expression of JNK and phosphorylated JNK, ERK and phosphorylated ERK.

Mentions: Stress induced by irradiation was shown to cause DNA double strand break (DSB) and the activation of DNA-PK, followed by the recruitment of Ku protein complex to the DNA break ends. Zeocine, an antibiotic, was also shown to cause DSB. We then assessed whether oxidative tress induced by H2O2 treatment leads to increased DNA-PK activity. For this purpose, we have firstly verified that treating InR1-G9 cells with Zeocine, similar to H2O2 treatment, led to Oct-1 nuclear accumulation (Figure. 4A). This result allowed us to utilize Zeocine as a positive control to assess DNA-PK activity. As shown in Figure. 4B, DNA-PK activity in H2O2 treated InR1-G9 cells reached to a similar level as that in cells treated with Zeocine. We therefore conclude that in the pancreatic islet cell line InR1-G9, H2O2 treatment leads to a moderate but significant DNA-PK activation, associated with enhanced Oct-1 nuclear import.


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 provoked DNA-PK and JNK activity.(A) InR1-G9 cells were treated with Zeocin (50, 100 or 200 μg/ml) for indicated time before harvested. Oct-1 expression in the nuclear extract (NE) was then assessed. (B) InR1-G9 cells were treated with vehicle, Zeocin (100 μg/ml) or H2O2 (00 μM) for 2 h before harvested for DNA-PK assay. The values are mean +/- S.E (n = 3). *, P < 0.05. (C, D) InR1-G9 cells were treated with forskolin/IBMX (C) or H2O2 (100 μM) (D) for indicated time. Whole cell lysates were utilized for assessing the expression of JNK and phosphorylated JNK, ERK and phosphorylated ERK.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: H2O2 provoked DNA-PK and JNK activity.(A) InR1-G9 cells were treated with Zeocin (50, 100 or 200 μg/ml) for indicated time before harvested. Oct-1 expression in the nuclear extract (NE) was then assessed. (B) InR1-G9 cells were treated with vehicle, Zeocin (100 μg/ml) or H2O2 (00 μM) for 2 h before harvested for DNA-PK assay. The values are mean +/- S.E (n = 3). *, P < 0.05. (C, D) InR1-G9 cells were treated with forskolin/IBMX (C) or H2O2 (100 μM) (D) for indicated time. Whole cell lysates were utilized for assessing the expression of JNK and phosphorylated JNK, ERK and phosphorylated ERK.
Mentions: Stress induced by irradiation was shown to cause DNA double strand break (DSB) and the activation of DNA-PK, followed by the recruitment of Ku protein complex to the DNA break ends. Zeocine, an antibiotic, was also shown to cause DSB. We then assessed whether oxidative tress induced by H2O2 treatment leads to increased DNA-PK activity. For this purpose, we have firstly verified that treating InR1-G9 cells with Zeocine, similar to H2O2 treatment, led to Oct-1 nuclear accumulation (Figure. 4A). This result allowed us to utilize Zeocine as a positive control to assess DNA-PK activity. As shown in Figure. 4B, DNA-PK activity in H2O2 treated InR1-G9 cells reached to a similar level as that in cells treated with Zeocine. We therefore conclude that in the pancreatic islet cell line InR1-G9, H2O2 treatment leads to a moderate but significant DNA-PK activation, associated with enhanced Oct-1 nuclear import.

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