Abstract
Transcription factor tonicity-responsive enhancer-binding protein (TonEBP/NFAT5) is critical for osmo-adaptation and extracellular matrix homeostasis of nucleus pulposus (NP) cells in their hypertonic tissue niche. Recent studies implicate TonEBP signaling in inflammatory disease and rheumatoid arthritis pathogenesis. However, broader functions of TonEBP in the disc remain unknown. RNA sequencing was performed on NP cells with TonEBP knockdown under hypertonic conditions. 1140 TonEBP-dependent genes were identified and categorized using Ingenuity Pathway Analysis. Bioinformatic analysis showed enrichment of matrix homeostasis and cytokine/chemokine signaling pathways. C-C motif chemokine ligand 2 (CCL2), interleukin 6 (IL6), tumor necrosis factor (TNF), and nitric oxide synthase 2 (NOS2) were studied further. Knockdown experiments showed that TonEBP was necessary to maintain expression levels of these genes. Gain- and loss-of-function experiments and site-directed mutagenesis demonstrated that TonEBP binding to a specific site in the CCL2 promoter is required for hypertonic inducibility. Despite inhibition by dominant-negative TonEBP, IL6 and NOS2 promoters were not hypertonicity-inducible. Whole-disc response to hypertonicity was studied in an ex vivo organ culture model, using wild-type and haploinsufficient TonEBP mice. Pro-inflammatory targets were induced by hypertonicity in discs from wild-type but not TonEBP-haploinsufficient mice. Mechanistically, NF-κB activity increased with hypertonicity and was necessary for hypertonic induction of target genes IL6, TNF, and NOS2 but not CCL2 Although TonEBP maintains transcription of genes traditionally considered pro-inflammatory, it is important to note that some of these genes also serve anabolic and pro-survival roles. Therefore, in NP cells, this phenomenon may reflect a physiological adaptation to diurnal osmotic loading of the intervertebral disc.
Highlights
Transcription factor tonicity-responsive enhancer-binding protein (TonEBP/NFAT5) is critical for osmo-adaptation and extracellular matrix homeostasis of nucleus pulposus (NP) cells in their hypertonic tissue niche
In NP cells we have shown previously that TonEBP is important for osmoregulation and survival under hypertonic conditions [17]
TonEBP Regulates Pro-inflammatory Genes haploinsufficient mice, our results demonstrate that TonEBP and NF-B participate in activation of pro-inflammatory genes in response to hypertonic stimulus in NP cells
Summary
RNA Sequencing Reveals TonEBP as a Regulator of Pro-inflammatory Genes under Physiological Hypertonicity in NP— NP cells were transduced with either control (ShCTR) or TonEBP-specific (ShTonEBP) shRNA and cultured in hypertonic medium for 8 h to recapitulate the physiological state of the NP. Levels of IL6 and TNF␣ protein were unaffected by hypertonicity (Fig. 1, I and J), suggesting that acute transcript increases may be required for maintenance of baseline protein expression under hypertonicity To explore whether this regulation involved TonEBP, we analyzed the 2-kb promoter upstream of the transcription start site of these genes for potential TonEBP binding motifs (TonE). Expression of DN-TonEBP under isotonic conditions led to a slight reduction in IL6 promoter activity (Fig. 3E), but did not affect the NOS2 promoter (Fig. 3F). In control cells (ShCTR), we observed hypertonicity-dependent induction in mRNAs for TonEBP, TauT, CCL2, and TNF (Fig. 4, A–C and E) but not of IL6 or NOS2 (Fig. 4, D and F). The induction in SMIT mRNA was abolished in heterozygous animals, induction in TauT mRNA was less affected by TonEBP haploinsufficiency, indicating that some targets may be more sensitive to TonEBP modulation than others (Fig. 5, F and G)
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