Cellular overexpression of Aquaporins slows down the natural HIF-2α degradation during prolonged hypoxia

Ana Galán-Cobo, Rocío Sánchez-Silva, Ana Serna, Irene Abreu-Rodríguez, Ana María Muñoz-Cabello, Miriam Echevarría

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.