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

• Published in: American Journal of Physiology: Cell Physiology 2019-07, Vol.317 (1), p.C31-C38
• Main Authors: Serman, Yair, Fuentealba, Rodrigo A, Pasten, Consuelo, Rocco, Jocelyn, Ko, Ben C B, Carrión, Flavio, Irarrázabal, Carlos E
• Format: Article
• Language: English
• Subjects: Active Transport, Cell Nucleus; Animals; Aquaporin 1; Aquaporin 1 - genetics; Aquaporin 1 - metabolism; Cell Hypoxia; Cells, Cultured; Embryo fibroblasts; Fibroblasts; Fibroblasts - enzymology; Hypoxia; Ischemia; Macrophages; Mammalian cells; Membrane Transport Proteins - genetics; Membrane Transport Proteins - metabolism; Mice; NF-AT protein; Nitric oxide; Nitric Oxide - metabolism; Nitric Oxide Synthase Type II - genetics; Nitric Oxide Synthase Type II - metabolism; Nitric-oxide synthase; Oxygen depletion; Reperfusion; Rodents; Signal Transduction; Toll-like receptors; Transcription factors; Transcription Factors - genetics; Transcription Factors - metabolism; Urea; Urea Transporters
• ISSN: 0363-6143; 1522-1563
• DOI: 10.1152/ajpcell.00054.2019

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 Toll-like receptor-stimulated macrophages and that iNOS inhibition increases NFAT5 expression in renal ischemia-reperfusion. Here we studied potential NFAT5 target genes stimulated by hypoxia in mouse embryonic fibroblast (MEF) cells. We used three types of MEF cells associated with NFAT5 gene: NFAT5 wild type (MEF-NFAT5 ), NFAT5 knockout (MEF-NFAT5 ), and NFAT5 dominant-negative (MEF-NFAT5 ) cells. MEF cells were exposed to 21% or 1% O in a time course curve of 48 h. We found that, in MEF-NFAT5 cells exposed to 1% O , NFAT5 was upregulated and translocated into the nuclei, and its transactivation domain activity was induced, concomitant with iNOS, aquaporin 1 (AQP-1), and urea transporter 1 (UTA-1) upregulation. Interestingly, in MEF-NFAT5 or MEF-NFAT5 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-NFAT5 cells, confirming the dominant-negative effect of MEF-NFAT5 cells. We did not see the rescue effect on AQP-1 expression. This work provides novel and relevant information about the signaling pathway of NFAT5 during responses to oxygen depletion in mammalian cells and suggests that the expression of iNOS induced by hypoxia is dependent on NFAT5.