Reactive oxygen species regulate context-dependent inhibition of NFAT5 target genes

Abstract

The activation of nuclear factor of activated T cells 5 (NFAT5), a well-known osmoprotective factor, can be induced by isotonic stimuli, such as activated Toll-like receptors (TLRs). It is unclear, however, how NFAT5 discriminates between isotonic and hypertonic stimuli. In this study we identified a novel context-dependent suppression of NFAT5 target gene expression in RAW 264.7 macrophages stimulated with lipopolysaccharide (LPS) or a high salt (NaCl) concentration. Although LPS and NaCl both used NFAT5 as a core transcription factor, these stimuli mutually inhibited distinct sets of NFAT5 targets within the cells. Although reactive oxygen species (ROS) are essential for this inhibition, the source of ROS differed depending on the context: mitochondria for high salt and xanthine oxidase for TLRs. Specifically, the high salt-induced suppression of interleukin-6 (IL-6) production was mediated through the ROS-induced inhibition of NFAT5 binding to the IL-6 promoter. The context-dependent inhibition of NFAT5 target gene expression was also confirmed in mouse spleen and kidney tissues that were cotreated with LPS and high salt. Taken together, our data suggest that ROS function as molecular sensors to discriminate between TLR ligation and osmotic stimuli in RAW 264.7 macrophages, directing NFAT5 activity toward proinflammatory or hypertonic responses in a context-dependent manner.

RNA Sequencing Reveals a Role of TonEBP Transcription Factor in Regulation of Pro-inflammatory Genes in Response to Hyperosmolarity in Healthy Nucleus Pulposus Cells

“…Previous reports have demonstrated hypertonic induction of CCL2, IL6, and TNF (12,24), which was TonEBPdependent in some instances. On the other hand, hypertonicity suppressed LPS-mediated IL6 production in macrophages (16). These results suggest that the TonEBP-mediated response to hypertonicity is likely cell-type specific.…”

“…In addition, TonEBP controls transcription of several genes that are important for cell survival under hypertonic conditions independent of osmolyte accumulation (8 -11). Recently, studies have shown that TonEBP participates in hypertonicity, as well as LPS-mediated induction of certain pro-inflammatory genes in macrophages and other cell types (12)(13)(14)(15)(16). In NP cells we have shown previously that TonEBP is important for osmoregulation and survival under hypertonic conditions (17).…”

Inflammatory signals induce the expression of tonicity-responsive enhancer binding protein (TonEBP) in microglia

“…In peripheral macrophages, it has been shown that LPS induces the expression of TonEBP and that TonEBP, as a transcription factor, plays an essential part in the induction of multiple immunomodulatory genes, such as tnf, il6, and nos2 (iNOS) (Buxade et al, 2012). Importantly, such function of TonEBP has been suggested to be independent of its osmomodulatory role (Buxade et al, 2012;Kaltschmidt and Kaltschmidt, 2009;Kim et al, 2013). In fibroblast-like synoviocytes, proinflammatory cytokines, TNF-α and IL-1β could induce the expression and nuclear localization of TonEBP, processes which were also suggested to occur independent of changes in tonicity (Yoon et al, 2011).…”

Molecular Biology and Interactions in Intervertebral Disc Development, Homeostasis, and Degeneration, with Emphasis on Future Therapies: A Systematic Review

“…The second is the noncanonical pathway through TLR activation. While the latter pathways have not yet been verified in the IVD, Kim et al (2013b) demonstrated that the response of osmotic-response genes (AR, BGT1, SMIT) was lessened when macrophages were pretreated with LPS. Nevertheless, cells pretreated with hyperosmotic conditions showed a suppressed response to TLR stimuli (Johnson et al, 2014;Kim et al, 2013b).…”

“…While the latter pathways have not yet been verified in the IVD, Kim et al (2013b) demonstrated that the response of osmotic-response genes (AR, BGT1, SMIT) was lessened when macrophages were pretreated with LPS. Nevertheless, cells pretreated with hyperosmotic conditions showed a suppressed response to TLR stimuli (Johnson et al, 2014;Kim et al, 2013b). If these findings can be extended to the osmodependent TonEBP targets in the IVD such as aggrecan, Sox9, and GlcAT-I, their regulation could be negatively affected under inflammatory conditions such as disc degeneration.…”