Emerging new role of NFAT5 in inducible nitric oxide synthase in response to hypoxia in mouse embryonic fibroblast cells
Journal
American Journal of Physiology-Cell Physiology
ISSN
0363-6143
1522-1563
Date Issued
2019
Author(s)
Yair Serman
Rodrigo A. Fuentealba
Consuelo Pasten
Jocelyn Rocco
Ben C. B. Ko
FLAVIO RIGOBERTO CARRION
Carlos E. Irarrázabal
Type
Resource Types::text::journal::journal article
URL Institutional Repository
Abstract
<jats:p> 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<jats:sup>+/+</jats:sup>), NFAT5 knockout (MEF-NFAT5<jats:sup>−/−</jats:sup>), and NFAT5 dominant-negative (MEF-NFAT5<jats:sup>Δ/Δ</jats:sup>) cells. MEF cells were exposed to 21% or 1% O<jats:sub>2</jats:sub> in a time course curve of 48 h. We found that, in MEF-NFAT5<jats:sup>+/+</jats:sup> cells exposed to 1% O<jats:sub>2</jats:sub>, 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<jats:sup>−/−</jats:sup> or MEF-NFAT5<jats:sup>Δ/Δ</jats:sup> 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<jats:sup>−/−</jats:sup> MEF cells, when recombinant NFAT5 protein expression was reconstituted, but not in MEF-NFAT5<jats:sup>Δ/Δ</jats:sup> cells, confirming the dominant-negative effect of MEF-NFAT5<jats:sup>Δ/Δ</jats:sup> 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. </jats:p>
Cite this document
Serman, Y., Fuentealba, R. A., Pasten, C., Rocco, J., Ko, B. C. B., Carrión, F., & Irarrázabal, C. E. (2019). Emerging new role of NFAT5 in inducible nitric oxide synthase in response to hypoxia in mouse embryonic fibroblast cells. American Journal of Physiology-Cell Physiology, 317(1), C31-C38. https://doi.org/10.1152/ajpcell.00054.2019
Subjects
aqp-1
;
hypoxia
;
inos
;
nfat5
;
uta-1
;
active transport, cell nucleus
;
animals
;
aquaporin 1
;
cell hypoxia
;
cells, cultured
;
fibroblasts
;
membrane transport proteins
;
mice
;
nitric oxide
;
nitric oxide synthase type ii
;
signal transduction
;
transcription factors
;
aquaporin 1
;
inducible nitric oxide synthase
;
transcription factor nfat
;
transcription factor nfat5
;
unclassified drug
;
aqp1 protein, mouse
;
aquaporin 1
;
carrier protein
;
inducible nitric oxide synthase
;
nfat5 protein, mouse
;
nitric oxide
;
nos2 protein, mouse
;
transcription factor
;
ut-a1 protein, mouse
;
membrane protein
;
toll like receptor
;
urea transporter 1
;
animal cell
;
article
;
cell hypoxia
;
controlled study
;
down regulation
;
embryo
;
fibroblast
;
gene knockout
;
mouse
;
nonhuman
;
priority journal
;
protein expression
;
signal transduction
;
upregulation
;
animal
;
cell culture
;
cell hypoxia
;
enzymology
;
fibroblast
;
genetics
;
metabolism
;
nucleocytoplasmic transport
;
enzyme activity
;
enzyme induction
;
gene
;
gene function
;
gene mutation
;
gene translocation
;
genetic transfection
;
luciferase assay
;
mef cell line
;
nfat5 gene
;
nuclear localization signal
;
plasmid
;
real time polymerase chain reaction
;
western blotting
;
wild type mouse