Cyclin dependent kinase inhibitor p27(Kip1) is upregulated by hypoxia via an ARNT dependent pathway

Expression of cyclin dependent kinase (Cdk) inhibitor p27(Kip1), which blocks cell cycle progression from G(1) to S phase, can be regulated via multiple mechanisms including transcription, protein degradation, and translation. Recently, it was shown that p27(Kip1) plays an important role in the cellular response to hypoxia. However, the mechanisms involved in the hypoxia-induced regulation of p27(Kip1) expression are still not clear. In this study, we compare the expression of p27(Kip1) in two related murine hepatoma cell lines, Hepa-1 and c4. Hepa-1 produces functional aryl hydrocarbon receptor nuclear translocator (ARNT). c4 cells are derived from Hepa-1, but are ARNT deficient. Interestingly, we observed cell line-dependent effects of hypoxia on the expression of p27(Kip1). The level of p27(Kip1) protein in Hepa-1 cells is enhanced by hypoxia, but is reduced by hypoxia in c4 cells. Further investigation demonstrated that hypoxia-induced, ARNT-mediated, transactivation of the p27(Kip1) gene in Hepa-1 cells is responsible for the increase in p27(Kip1) protein. Once c4 cells were stably transfected with the wild type ARNT gene, a hypoxia-induced increase in p27(Kip1) mRNA was observed and reduction of p27(Kip1) protein caused by hypoxia was blocked. Hence, our data indicate that ARNT is involved in transcriptional upregulation of the p27(Kip1) gene under hypoxic conditions.