The role of the hypoxia-inducible factor 1 binding site in the induction of aquaporin-1 mRNA expression by hypoxia.

Aquaporin-1 (AQP1), a water channel protein, has been shown to play an important role in tumor growth and angiogenesis in mouse endothelial cells. We recently reported that the expression of AQP1 mRNA was induced in cultured human retinal vascular endothelial cells (HRVECs) under hypoxia. In the present study, HRVECs were cultured under normoxia or hypoxia (1% O(2)) to elucidate the mechanism of hypoxic induction of AQP1. AQP1 mRNA expression was increased 1.7 ± 0.24-fold under hypoxia compared with that under normoxia (p < 0.01). This increase was almost completely blocked by the transcriptional inhibitor actinomycin D (p < 0.01). The degradation of AQP1 mRNA showed no difference under normoxia or hypoxia. These data suggest that the hypoxia-induced expression of AQP1 results from RNA transcription. The sequence located from -1338 to -1334 bp is identical to the consensus sequence of the hypoxia-inducible factor 1 (HIF-1) binding site. The promoter activities of the two constructs including this putative HIF-1 binding site showed 2.0 ± 0.67-fold increase and 2.9 ± 1.9-fold increase under hypoxia when compared with those under normoxia. However, both deletion and mutation of the HIF-1 binding site abrogated this effect. These data suggest that this sequence mediates the transcriptional activation of AQP1 by hypoxia. The chromatin immunoprecipitation assay showed that HIF-1α bound to the putative HIF-1 binding site. In conclusion, hypoxia-induced expression of AQP1 requires transcriptional activation, and the HIF-1 binding site of the 5'-promoter is necessary for transcriptional activation in HRVECs.