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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 stimulates OCT-1-EGFP nuclear import. (A) InR1-G9 cells were transfected with Oct-1-EGFP, and treated with vehicle (control), or H2O2 (100 μM). After photo bleaching of the nuclear fluorescence, the time was recorded for the area to recover to the 50% intensity. (B) Comparison of recovery time and recovery rate between vehicle and H2O2 treated cells.
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Figure 3: H2O2 stimulates OCT-1-EGFP nuclear import. (A) InR1-G9 cells were transfected with Oct-1-EGFP, and treated with vehicle (control), or H2O2 (100 μM). After photo bleaching of the nuclear fluorescence, the time was recorded for the area to recover to the 50% intensity. (B) Comparison of recovery time and recovery rate between vehicle and H2O2 treated cells.

Mentions: Recent studies have indicated, with irradiation, DNA-PK activation could lead to enhanced phosphorylation of Oct-1 at its selected Ser/Thr residues [9]. This phosphorylation event may facilitate the binding of Oct-1 to its target gene promoters [9,14,15]. To investigate whether increased percentages of pattern I cells after H2O2 treatment is a result of increased Oct-1-EGFP nuclear import, we utilized Fluorescence Recovery After Photobleaching (FRAP). For FRAP analysis, the fluorescence molecule in the nuclei is bleached by a laser beam. By recording the time (normally in the ones to tens of seconds range) taken to reach 50% intensity when the fluorescence reaches a new plateau (or termed "50% recovery time") for the same bleached area, one can measure the capability of a molecule to move around over time. The "recovery rate" refers to as the percentage of the recovery observed from total cells bleached. For this study we transfected Oct-1-EGFP into the InR1-G9 cell line. Twenty-four h after the transfection, cells were treated with vehicle or H2O2. The treated cells were then subjected to the laser photobleaching. The 50% recovery time was recorded as an indicator of Oct-1-EGFP to move from cytoplasm to nuclear. The effect of H2O2 treatment on Oct-1-EGFP recovery time and the recovery rate by FRAP are shown in Figure. 3A and 3B. As summarized, for cells treated with vehicle, the 50% recovery time was about 10.12 seconds while the recovery rate is only about 33% (4 out of 12). However, for cells received H2O2 treatment, 50% recovery time was only 3.5 seconds and the recovery rate reached 79% (11 out of 14). These results clearly indicate that H2O2 treatment stimulates the import of Oct-1 from the cytoplasm into the nuclei, and further suggest that Oct-1 serve as a sensor for oxidative stress.


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 stimulates OCT-1-EGFP nuclear import. (A) InR1-G9 cells were transfected with Oct-1-EGFP, and treated with vehicle (control), or H2O2 (100 μM). After photo bleaching of the nuclear fluorescence, the time was recorded for the area to recover to the 50% intensity. (B) Comparison of recovery time and recovery rate between vehicle and H2O2 treated cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: H2O2 stimulates OCT-1-EGFP nuclear import. (A) InR1-G9 cells were transfected with Oct-1-EGFP, and treated with vehicle (control), or H2O2 (100 μM). After photo bleaching of the nuclear fluorescence, the time was recorded for the area to recover to the 50% intensity. (B) Comparison of recovery time and recovery rate between vehicle and H2O2 treated cells.
Mentions: Recent studies have indicated, with irradiation, DNA-PK activation could lead to enhanced phosphorylation of Oct-1 at its selected Ser/Thr residues [9]. This phosphorylation event may facilitate the binding of Oct-1 to its target gene promoters [9,14,15]. To investigate whether increased percentages of pattern I cells after H2O2 treatment is a result of increased Oct-1-EGFP nuclear import, we utilized Fluorescence Recovery After Photobleaching (FRAP). For FRAP analysis, the fluorescence molecule in the nuclei is bleached by a laser beam. By recording the time (normally in the ones to tens of seconds range) taken to reach 50% intensity when the fluorescence reaches a new plateau (or termed "50% recovery time") for the same bleached area, one can measure the capability of a molecule to move around over time. The "recovery rate" refers to as the percentage of the recovery observed from total cells bleached. For this study we transfected Oct-1-EGFP into the InR1-G9 cell line. Twenty-four h after the transfection, cells were treated with vehicle or H2O2. The treated cells were then subjected to the laser photobleaching. The 50% recovery time was recorded as an indicator of Oct-1-EGFP to move from cytoplasm to nuclear. The effect of H2O2 treatment on Oct-1-EGFP recovery time and the recovery rate by FRAP are shown in Figure. 3A and 3B. As summarized, for cells treated with vehicle, the 50% recovery time was about 10.12 seconds while the recovery rate is only about 33% (4 out of 12). However, for cells received H2O2 treatment, 50% recovery time was only 3.5 seconds and the recovery rate reached 79% (11 out of 14). These results clearly indicate that H2O2 treatment stimulates the import of Oct-1 from the cytoplasm into the nuclei, and further suggest that Oct-1 serve as a sensor for oxidative stress.

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