Analysis of redox system regulating the expression of HIF-lα in squamous cell carcinomas

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We analyzed the mechanism how endogenous ROS induced HIF-lα expression on both mRNA and protein levels under normoxic and hypoxic conditions by using oral squamous cell carcinoma cell lines transfected with manganese-superoxide dismutase siRNA (SI cells) and scrambled control siRNA (mock cells). The results are as follows.1. The interaction between HIF-lα and pVHL proteins and the following ubiquitination of HIF-lα protein were suppressed in SI cells as compared with mock cells. However, significant differences on both mRNA and protein levels of PHD 1-3 expression were not observed.2. The phosphorylation of ERK and Akt was induced in SI cells under both normoxic and hypoxic conditions and these effects were suppressed by treatment with ERK inhibitor (PD98059) and Akt inhibitor (LY294002).3. The phosphorylation levels of p70S6K, elF-4E and 4E-BP1, which are located downstream of ERK and Akt and regulate the translation of HIF-la, were also more increased in SI cells as compared with mock cells. These effects were strongly suppressed by treatment with PD98059 and LY294002.4. Although mRNA level and promoter activity of HIF-lα was not influenced by both inhibitors in mock cells, the obvious suppressive effect was observed in SI cells, especially under normoxic conditions.5. Although the expression of HIF-lα protein was completely suppressed by ERK and Akt inhibitors under normoxic conditions, only PD98059 suppressed the HIF-lα expression under hypoxic conditions.These results suggest that endogenous ROS regulate the expression of HIF-lα through the two mechanisms; (1) the inhibition of the interaction between HIF-lα and pVHL, the following ubiquitination and degradation of HIF-lα protein and (2) the promotion of the transcription and translation of HIF-lα by the activation of ERK and Akt signal pathways.