PKC-α contributes to high NaCl-induced activation of NFAT5 (TonEBP/OREBP) through MAPK ERK1/2.
High NaCl in the renal medullary interstitial fluid powers the concentration of urine but can damage cells. The transcription factor nuclear factor of activated T cells 5 (NFAT5) activates the expression of osmoprotective genes. We studied whether PKC-α contributes to the activation of NFAT5. PKC-α protein abundance was greater in the renal medulla than in the cortex. Knockout of PKC-α reduced NFAT5 protein abundance and expression of its target genes in the inner medulla. In human embryonic kidney (HEK)-293 cells, high NaCl increased PKC-α activity, and small interfering RNA-mediated knockdown of PKC-α attenuated high NaCl-induced NFAT5 transcriptional activity. Expression of ERK1/2 protein and phosphorylation of ERK1/2 were higher in the renal inner medulla than in the cortex. Knockout of PKC-α decreased ERK1/2 phosphorylation in the inner medulla, as did knockdown of PKC-α in HEK-293 cells. Also, knockdown of ERK2 reduced high NaCl-dependent NFAT5 transcriptional activity in HEK-293 cells. Combined knockdown of PKC-α and ERK2 had no greater effect than knockdown of either alone. Knockdown of either PKC-α or ERK2 reduced the high NaCl-induced increase of NFAT5 transactivating activity. We have previously found that the high NaCl-induced increase of phosphorylation of Ser(591) on Src homology 2 domain-containing phosphatase 1 (SHP-1-S591-P) contributes to the activation of NFAT5 in cell culture, and here we found high levels of SHP-1-S591-P in the inner medulla. PKC-α has been previously shown to increase SHP-1-S591-P, which raised the possibility that PKC-α might be acting through SHP-1. However, we did not find that knockout of PKC-α in the renal medulla or knockdown in HEK-293 cells affected SHP-1-S591-P. We conclude that PKC-α contributes to high NaCl-dependent activation of NFAT5 through ERK1/2 but not through SHP-1-S591.
Department of Medicine, Uniformed Services University of the Health Sciences, Bethesda, Maryland;
Hong
H
Wang
Systems Biology Center, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland; and.
Joan D
JD
Ferraris
Renal Division, School of Medicine, Emory University, Atlanta, Georgia.
Janet D
JD
Klein
Renal Division, School of Medicine, Emory University, Atlanta, Georgia.
Jeff M
JM
Sands
Systems Biology Center, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland; and.
Maurice B
MB
Burg
Department of Medicine, Uniformed Services University of the Health Sciences, Bethesda, Maryland; xiaoming.zhou@usuhs.edu.
Xiaoming
X
Zhou
D000818:Animals; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D007668:Kidney / Q000201:enzymology*; D020935:MAP Kinase Signaling System; D008810:Mice, Inbred C57BL; D050778:NFATC Transcription Factors / Q000378:metabolism*; D010766:Phosphorylation; D051571:Protein Kinase C-alpha / Q000378:metabolism*; D012965:Sodium Chloride / Q000378:metabolism
D050778:NFATC Transcription Factors; D012965:Sodium Chloride; D051571:Protein Kinase C-alpha; D048049:Extracellular Signal-Regulated MAP Kinases
Src homology 2 domain-containing phosphatase 1; hypertonicity; inner medulla; nuclear factor of activated T cells 5; osmotic response element-binding protein; serine-591-phosphorylated Src homology 2 domain-containing phosphatase 1; tonicity-responsive enhancer-binding protein; urinary concentration