Emerging new role of NFAT5 in inducible nitric oxide synthase in response to hypoxia in mouse embryonic fibroblast cells

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Abstract
We previously described the protective role of the nuclear factor of activated T cells 5 (NFAT5) during hypoxia. Alternatively, inducible nitric oxide synthase (iNOS) is also induced by hypoxia. Some evidence indicates that NFAT5 is essential for the expression of iNOS in TLR-stimulated macrophages and that iNOS inhibition increase NFAT5 expression in renal ischemia/reperfusion. Here we studied potentials NFAT5-target genes stimulated by hypoxia in mouse embryonic fibroblasts (MEF) cells. We use three types of MEF cells associated with NFAT5 gene: NFAT5-Wild type (MEF-NFAT5+/+), NFAT5-knockout (MEF-NFAT5-/-), and NFAT5-dominant negative (MEF-NFAT5D/D) cells. MEF cells were exposed to 21% or 1%O2 in a time-course curve of 48h. We found that in MEF-NFAT5+/+ cells exposed to 1%O2, NFAT5 was upregulated, translocated into the nuclei and its transactivation domain (TAD) activity was induced, concomitant with iNOS, aquaporin 1 (AQP-1), and urea transporter 1 (UTA-1) upregulation. Interestingly, in MEF-NFAT5-/- or MEF-NFAT5D/D cells, the basal levels of iNOS and AQP-1 expression were strongly downregulated, but not for UTA-1. The upregulation of AQP-1, UTA-1, and iNOS by hypoxia was blocked in both NFAT5-mutated cells. The iNOS induction by hypoxia was recovered in MEF-NFAT5-/- MEF cells, when recombinant NFAT5 protein expression was reconstituted, but not in MEF-NFAT5D/D cells, confirming the dominant negative effect of MEF-NFAT5D/D cells. We did not see the rescue effect on AQP-1 expression. This work provides novel and relevant information, suggesting that iNOS expression by hypoxia-dependent partially of NFAT5, thus providing insight into the importance of the NFAT5 hypoxia response pathway in vivo
Funding Information
  • CONICYT-Fondecyt (1151157)

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