An early study by our group indicated that Oct-1 co-transfection stimulated Cdx-2 promoter expression in the pancreatic and intestinal gcg producing cell lines . More recent investigations suggested that Oct-1 could function as a transcriptional repressor . We noticed that cAMP promoting agents, forskolin and IBMX, reduced nuclear Oct-1 content but stimulated Cdx-2 expression. Our further investigations revealed that Oct-1 is able to recruit nuclear co-repressors to Cdx-2 promoter and represses its transcription, while the activation of cAMP-Epac signaling increases Oct-1 nuclear-cytoplasmic shuttling . Thus, Oct-1 serves as a repressor of Cdx-2 and its downstream target Gcg in pancreatic and intestinal endocrine cells . As an abundantly expressed homeobox gene in gut, Cdx-2 plays a critical role in intestinal cell differentiation [23, 24, 27]. We have shown that it is expressed in intestinal endocrine L cells and stimulates Gcg transcription .
It has been shown that Oct1-/- cells are more hypersensitive to stress-inducing agents or treatment, such as ionizing radiation . In the present study, we have assessed the effect of H2O2 on cytoplasmic-nuclear shuttling of Oct-1. We show in the current study that H2O2 treatment led to increased Oct-1 nuclear localization by Western blotting, following sub-cellular fractionation; and confocal and fluorescence microcopy, following exogenous expression of EGFP or myc- tagged Oct-1. Increased nuclear shuttling was then directly assessed by FRAP. We found that H2O2 treatment stimulated the activity of DNA-PK; and that opposite to cAMP elevation, H2O2 stimulated JNK activity but not ERK activity. Finally, we show that increased nuclear Oct-1 content upon H2O2 treatment led to approximately 40-50% reduction of Cdx-2 and gcg mRNA expression, which was consistent with our notion that Oct-1 represses Cdx-2 expression. Since DNA-PK activity is responsible for Oct-1 phosphorylation upon irradiation , we speculate that increased Oct-1 nuclear shuttling in response to H2O2 treatment is a result of its phosphorylation by DNA-PK . This, nevertheless, needs to be further investigated.
Intensive investigations have shown that Oct-1 up- or down-regulates the expression of a large profile of target genes in different cell lineages and that this ubiquitously expressed transcription factor is involved in different categories of cellular and molecular activities, from transcriptional regulation to embryonic development [8, 28–30]. The role of Oct-1 in mediating metabolic as well as stress/survival signaling pathways, however, was recognized only recently [8, 14, 15, 31]. In addition to the repression of Cdx-2 expression, we have also shown that Oct-1 can repress the expression of the transcription factor carbohydrate responsive element binding protein (ChREBP) , which is important in facilitating liver lipogenesis . Insulin, however, stimulated ChREBP transcription. More importantly, the stimulatory effect of insulin was at least partially mediated by attenuating the repressive effect of Oct-1 . These observations collectively suggest that Oct-1 serves as a sensor for metabolic signaling pathways. It represses the expression of important master control genes, such as Cdx-2 in pancreatic islets and ChREBP in hepatocytes. Following the stimulation by a peptide hormone, such as insulin or those that utilize cAMP as the second messenger, Oct-1 is phosphorylated at certain Ser/Thr residues and excluded from the nucleus. This may represent a novel mechanism for peptide hormones in regulating gene expression.
Oct-1 is also known to act as a sensor for stress. Oct-1 deficiency in mice (Oct-1-/-) is embryonically lethal . Utilizing microarray expression profiling, Tantin et al. found that in Oct-1-/- fibroblasts a large profile of genes associated with cellular stress exhibited altered expression pattern . Furthermore, Tantin et al. and Schild-Poulter et al. have shown that in radiation induced stress, Oct-1 could be phosphorylated by DNA-PK at 13 potential Ser/Thr residues within the N terminus of the Oct-1 molecule. Consistent with this finding, Oct-1-/- fibroblasts are hypersensitive to γ irradiation, doxorubicin and H2O2 treatment, and contained elevated level of reactive oxygen species (ROS). Very recently, Kang et al. have demonstrated that Oct-1 is dynamically modulated by phosphorylation in vivo following the response to genotoxic and oxidative stress . The stress induced phosphorylated Oct-1 has a higher affinity for DNA binding. Additionally, the interaction between Oct-1 and a distinct group of target promoters is inducible by oxidative stress and these target promoters frequently contain conserved octamer binding sites . We present here that in a battery of cell lines and primary cells, nuclear Oct-1 content is elevated after H2O2 treatment. We suggest this is due to the result of Oct-1 shuttling from cytoplasm to nuclei, following the phosphorylation by DNA-PK.
Although the activation on DNA-PK by H2O2 is moderate, the stimulation was comparable with that of Zeocine treatment. Interestingly, Lebrun et al. have reported that DNA-PK could phosphorylate another pancreatic islet homeodomain protein PDX-1, and the phosphorylation accelerated PDX-1 proteasome degradation . Therefore, DNA-PK activation in response to oxidative stress may affect pancreatic islet hormone-gene expression through affecting both homeodomain protein expression and degradation. Furthermore, we observed increased JNK phosphorylation in the InR1-G9 cells in response to H2O2 treatment. Whether this activation is related to Oct-1 nuclear-cytoplamic shuttling deserves a further examination.
Our finding that cAMP elevation stimulates Oct-1 nuclear exclusion and that H2O2 treatment leads to increased nuclear Oct-1 content place Oct-1 in the centre of signaling cascades that are involved in response to both oxidative stress and hormones/neurotransmitters that utilize cAMP as the second messenger. For this matter, Oct-1 should not be simply considered as a repressor for a cluster of genes. Instead, it is a sensor for both metabolic and stress/survival signaling pathways. Indeed, a recent study shows that Oct-1 mediates the effect of oxidized LDL (oxLDL) in repressing the expression of vascular cytochrome P450 (CYP) monooxygenases . In the coronary arterial endothelial cells, knockdown of Oct-1 expression prevented oxLDL-mediated silencing of CYP expression . Therefore, Oct-1 activation in response to oxidative stress is among the pathological entity in metabolic dysfunction , and attenuating the function of Oct-1 improves the dysfunction.