Overexpression of cell membrane
aquaporins (AQPs) has recently been associated with
tumor formation, particularly with angiogenesis, cell migration and proliferation. Additionally, the
hypoxia inducible factor (HIF) family has been extensively implicated in
tumor growth and recent studies evidence interplay between AQP expression and HIF stability. Therefore, we decided to explore the effect that AQP overexpression has on the long-term stability of HIF-2α in PC12 cells exposed to chronic
hypoxia, characteristic of a growing
tumor. HIF-2α levels were analyzed in five PC12 clones with stable overexpression of different
proteins (AQP1, AQP3, AQP5, G6PD, and
GDNF), in PC12 transiently expressing G6PD or Kv4.2, and in wild-type PC12 cells. Overexpression of AQP1, 3 or 5 in PC12 cells (o-AQP-c) prevented the HIF-2α down-expression otherwise observed, after 16 h at 1% O2, in wt-PC12 and in PC12 overexpressing non-AQP
proteins. Longer HIF-2α stability was also observed in o-AQP-c exposed to
cobalt chloride or
dimethyloxallyl glycine. Normal
proteasome activity was confirmed in all clones analyzed. Levels of HIF target genes (PHD2 and 3,
VEGF, and PGK1) were 2-4 fold higher in hypoxic o-AQP-c than in wt-PC12 cells, and morphological changes in colony shape together with higher cell proliferation rates were observed in all o-AQP-c. Interestingly, analysis of PHD levels under normoxia revealed lower (50%) PHD3 expression in o-AQP-c than in controls. Our results indicate that AQP overexpression in PC12 cells prolongs HIF-2α stability during chronic
hypoxia, leading to higher level of induction of its target genes and likely conferring to these cells a more
tumor-like phenotype.