Upregulation of HIF-1 alpha in Endometrial Carcinoma Cells
Upregulation of HIF-1 alpha in Endometrial Carcinoma Cells
Evidence has been provided that HIF-1α expression is regulated not only by post-translational modification but also involved transcriptional control by NF-κB/p65, from the following findings. First, short-term treatment of cells with CoCl2 and H2O2 resulted in a rapid and transient increase in HIF-1α mRNA expression, along with stabilization of nuclear p65. At the same time, pretreatment with the transcriptional inhibitor actinomycin D resulted in suppression of the mRNA and protein expression induced by CoCl2 treatment. Second, cells transiently overexpressing p65 exhibited upregulation of HIF-1α mRNA and protein expression through activation of the promoter, in line with the data showing significantly higher pp65 LI values in HIF-1α-positive as compared with HIF-1α-negative components in Em Cas. Third, p65 was capable of binding directly to the three specific regulatory sequences, including nucleotides −377/−368, +149/+158, and +244/+253, within the promoter, as evidenced by inhibition of promoter activity by mutations in the three sequences. In contrast, previous studies demonstrated that activated NF-κB was bound to the putative responsive site at −197/−188 of the promoter in human pulmonary artery smooth muscle cells. Other cell type-specific transcriptional factors may also contribute to the promoter-specific mechanism of action.
In contrast to transcriptional upregulation of HIF-1α by NF-κB, overexpression of HIF-1α also caused an increase in NF-κB-mediated transcription, indicating the existence of a positive feedback loop system. This conclusion is supported by our results of high levels of pp65 expression in HIF-1α-positive components of Em Cas. The significantly lower cell proliferation in HIF-1α-positive carcinomatous lesions may be due to the relatively low-oxygenated status, as hypoxia caused cell cycle arrest through a mechanism that was either dependent (p53, p21, and bcl-2) or independent (GADD153) of HIF-1α. In fact, long-term treatment of Ishikawa cells with CoCl2 resulted in a significant decrease in growth, leading to upregulation of p27.
A rapidly growing body of evidence indicates that hypoxia triggers a functional switch in β-catenin signaling, as well as PI3K/Akt. In this study, CoCl2-meditated hypoxia caused considerable accumulation of nuclear β-catenin, probably due to inactivation of the Akt/GSK-3β-mediated degradation system. This is consistent with the results of significantly higher levels of nuclear β-catenin in HIF-1α-positive lesions of Em Cas. However, the association appeared to be indirect, on the basis of our findings, in one hybrid assay. Given the evidence of a direct association between p300 and either HIF-1α or β-catenin, it is likely that the co-activator may be essential for cross-talk between the two signaling components in Em Ca cells.
Another finding of interest in this study was the transient upregulation of HIF-1α expression that was observed in normal endometrial glandular but not stromal components in early secretory stages, despite relatively well-oxygenated tissues. A similar finding was also observed in a case of Sox7, which may be important for changes in cell kinetics from proliferative to secretory stages in the normal endometrium. Interestingly, HIF-1α protein has been shown to be upregulated under normoxic condition in response to growth factors, hormones, coagulation factors, cytokines, and vasoactive peptide, allowing us to speculate that other factors, in particular estrogen, may serve as positive regulators for HIF-1α expression during the menstrual cycle, independent of O2 tension. In contrast, the relatively higher HIF-1α expression in both glandular and stromal components in menstrual stages may be simply due to hypoxic effects.
Our observations suggest a model for the regulation and function of HIF-1α in Em Ca cells (Figure 7). Activation of the NF-κB pathway due to a variety of stimuli, such as hypoxic condition and several cytokines, causes transactivation of the HIF-1α gene, while HIF-1α can enhance NF-κB-mediated transcriptional activity, through a positive feedback loop system. Overexpression of HIF-1α is also associated indirectly with excess β-catenin, probably due to the gene mutations and hypoxic exposure, by interaction with p300, and this in turn leads to transactivation of their target genes, resulting in tumor adaptation to microenvironmental hypoxia in Em Ca cells.
(Enlarge Image)
Figure 7.
Schematic representation of associations among hypoxia-inducible factor (HIF)-1α, nuclear factor-κB (NF-κB), and β-catenin in response to microenvironment factors including hypoxia in endometrial carcinoma (Em Ca) cells.
In conclusion, our results suggest that associations with HIF-1α and the NF-κB pathway, as well as β-catenin/p300 complexes, may participate in modulating changes in tumor kinetics in response to hypoxic stress in Em Cas.
Discussion
Evidence has been provided that HIF-1α expression is regulated not only by post-translational modification but also involved transcriptional control by NF-κB/p65, from the following findings. First, short-term treatment of cells with CoCl2 and H2O2 resulted in a rapid and transient increase in HIF-1α mRNA expression, along with stabilization of nuclear p65. At the same time, pretreatment with the transcriptional inhibitor actinomycin D resulted in suppression of the mRNA and protein expression induced by CoCl2 treatment. Second, cells transiently overexpressing p65 exhibited upregulation of HIF-1α mRNA and protein expression through activation of the promoter, in line with the data showing significantly higher pp65 LI values in HIF-1α-positive as compared with HIF-1α-negative components in Em Cas. Third, p65 was capable of binding directly to the three specific regulatory sequences, including nucleotides −377/−368, +149/+158, and +244/+253, within the promoter, as evidenced by inhibition of promoter activity by mutations in the three sequences. In contrast, previous studies demonstrated that activated NF-κB was bound to the putative responsive site at −197/−188 of the promoter in human pulmonary artery smooth muscle cells. Other cell type-specific transcriptional factors may also contribute to the promoter-specific mechanism of action.
In contrast to transcriptional upregulation of HIF-1α by NF-κB, overexpression of HIF-1α also caused an increase in NF-κB-mediated transcription, indicating the existence of a positive feedback loop system. This conclusion is supported by our results of high levels of pp65 expression in HIF-1α-positive components of Em Cas. The significantly lower cell proliferation in HIF-1α-positive carcinomatous lesions may be due to the relatively low-oxygenated status, as hypoxia caused cell cycle arrest through a mechanism that was either dependent (p53, p21, and bcl-2) or independent (GADD153) of HIF-1α. In fact, long-term treatment of Ishikawa cells with CoCl2 resulted in a significant decrease in growth, leading to upregulation of p27.
A rapidly growing body of evidence indicates that hypoxia triggers a functional switch in β-catenin signaling, as well as PI3K/Akt. In this study, CoCl2-meditated hypoxia caused considerable accumulation of nuclear β-catenin, probably due to inactivation of the Akt/GSK-3β-mediated degradation system. This is consistent with the results of significantly higher levels of nuclear β-catenin in HIF-1α-positive lesions of Em Cas. However, the association appeared to be indirect, on the basis of our findings, in one hybrid assay. Given the evidence of a direct association between p300 and either HIF-1α or β-catenin, it is likely that the co-activator may be essential for cross-talk between the two signaling components in Em Ca cells.
Another finding of interest in this study was the transient upregulation of HIF-1α expression that was observed in normal endometrial glandular but not stromal components in early secretory stages, despite relatively well-oxygenated tissues. A similar finding was also observed in a case of Sox7, which may be important for changes in cell kinetics from proliferative to secretory stages in the normal endometrium. Interestingly, HIF-1α protein has been shown to be upregulated under normoxic condition in response to growth factors, hormones, coagulation factors, cytokines, and vasoactive peptide, allowing us to speculate that other factors, in particular estrogen, may serve as positive regulators for HIF-1α expression during the menstrual cycle, independent of O2 tension. In contrast, the relatively higher HIF-1α expression in both glandular and stromal components in menstrual stages may be simply due to hypoxic effects.
Our observations suggest a model for the regulation and function of HIF-1α in Em Ca cells (Figure 7). Activation of the NF-κB pathway due to a variety of stimuli, such as hypoxic condition and several cytokines, causes transactivation of the HIF-1α gene, while HIF-1α can enhance NF-κB-mediated transcriptional activity, through a positive feedback loop system. Overexpression of HIF-1α is also associated indirectly with excess β-catenin, probably due to the gene mutations and hypoxic exposure, by interaction with p300, and this in turn leads to transactivation of their target genes, resulting in tumor adaptation to microenvironmental hypoxia in Em Ca cells.
(Enlarge Image)
Figure 7.
Schematic representation of associations among hypoxia-inducible factor (HIF)-1α, nuclear factor-κB (NF-κB), and β-catenin in response to microenvironment factors including hypoxia in endometrial carcinoma (Em Ca) cells.
In conclusion, our results suggest that associations with HIF-1α and the NF-κB pathway, as well as β-catenin/p300 complexes, may participate in modulating changes in tumor kinetics in response to hypoxic stress in Em Cas.
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