Abstract
TonEBP/NFAT5 is a major regulator of the urinary concentrating process and is essential for the osmoadaptation of renal medullary cells. Focal adhesion kinase (FAK) is a mechanosensitive non-receptor protein tyrosine kinase expressed abundantly in the renal medulla. Since osmotic stress causes cell shrinkage, the present study investigated the contribution of FAK on TonEBP/NFAT5 activation. Osmotic stress induced time-dependent activation of FAK as evidenced by phosphorylation at Tyr-397, and furosemide reduces FAK Tyr-397 phosphorylation in the rat renal medulla. Both pharmacological inhibition of FAK and siRNA-mediated knockdown of FAK drastically reduced TonEBP/NFAT5 transcriptional activity and target gene expression in HEK293 cells. This effect was not mediated by impaired nuclear translocation or by reduced transactivating activity of TonEBP/NFAT5. However, TonEBP/NFAT5 abundance under hypertonic conditions was diminished by 50% by FAK inhibition or siRNA knockdown of FAK. FAK inhibition only marginally reduced transcription of the TonEBP/NFAT5 gene. Rather, TonEBP/NFAT5 mRNA stability was diminished significantly by FAK inhibition, which correlated with reduced reporter activity of the TonEBP/NFAT5 mRNA 3′ untranslated region (3′-UTR). In conclusion, FAK is a major regulator of TonEBP/NFAT5 activity by increasing its abundance via stabilization of the mRNA. This in turn, depends on the presence of the TonEBP/NFAT5 3′-UTR.
Highlights
Tonicity-responsive enhancer binding protein/nuclear factor of activated T cells 5 (TonEBP/NFAT5) is an osmosensitive transcription factor necessary for the urinary concentrating process and osmoadaptation of renal medullary cells (Miyakawa et al, 1999)
Since osmotic stress causes cell shrinkage, the present study investigated the contribution of Focal adhesion kinase (FAK) on TonEBP/NFAT5 activation
TonEBP/NFAT5 stimulates the expression of essential components of the urinary concentrating mechanism, including NKCC2, UT-A1, and ClC-K1 with its functional subunit Barttin, which are required for generating the corticomedullary osmotic gradient (Neuhofer and Beck, 2005; Fenton and Knepper, 2007; Küper et al, 2012b)
Summary
Tonicity-responsive enhancer binding protein/nuclear factor of activated T cells 5 (TonEBP/NFAT5) is an osmosensitive transcription factor necessary for the urinary concentrating process and osmoadaptation of renal medullary cells (Miyakawa et al, 1999). TonEBP/NFAT5 stimulates the expression of essential components of the urinary concentrating mechanism, including NKCC2, UT-A1, and ClC-K1 with its functional subunit Barttin, which are required for generating the corticomedullary osmotic gradient (Neuhofer and Beck, 2005; Fenton and Knepper, 2007; Küper et al, 2012b). The enhanced expression of these osmosensitive genes requires the interaction of TonEBP/NFAT5 with tonicity-responsive enhancer elements (TonE) in the promoter region of the respective target genes (Neuhofer and Beck, 2005; Jeon et al, 2006). Recent evidence suggests that TonEBP/NFAT5 is critically involved in the expression of various proinflammatory cytokines in cells exposed to local hypertonicity, as present under pathophysiological conditions (Machnik et al, 2009; Neuhofer, 2010; Küper et al, 2012a)
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