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

Yair Serman, Rodrigo A. Fuentealba, Consuelo Pasten, Jocelyn Rocco, Ben C.B. Ko, Flavio Carrión, Carlos Ernesto Irarrazabal Muñoz

Research output: Contribution to journalArticlepeer-review

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 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% O2 in a time course curve of 48 h. We found that, in MEF-NFAT5+/+ cells exposed to 1% O2, 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 recombi-nant 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.

Original languageEnglish
Pages (from-to)C31-C38
JournalAmerican Journal of Physiology - Cell Physiology
Volume317
Issue number1
DOIs
StatePublished - 2019

Bibliographical note

Funding Information:
This work was supported by Fondo Nacional de Desarrollo Científico y Tecnológico (FONDECYT) Grant 1151157.

Publisher Copyright:
© 2019 the American Physiological Society.

Keywords

  • AQP-1
  • Hypoxia
  • INOS
  • NFAT5
  • UTA-1

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