TNF-α promotes nuclear enrichment of the transcription factor TonEBP/NFAT5 to selectively control inflammatory but not osmoregulatory responses in nucleus pulposus cells

Abstract

Intervertebral disc degeneration (IDD) causes chronic back pain and is linked to production of proinflammatory molecules by nucleus pulposus (NP) and other disc cells. Activation of tonicity-responsive enhancer-binding protein (TonEBP)/NFAT5 by non-osmotic stimuli, including proinflammatory molecules, occurs in cells involved in immune response. However, whether inflammatory stimuli activate TonEBP in NP cells and whether TonEBP controls inflammation during IDD is unknown. We show that TNF-α, but not IL-1β or LPS, promoted nuclear enrichment of TonEBP protein. However, TNF-α-mediated activation of TonEBP did not cause induction of osmoregulatory genes. RNA sequencing showed that 8.5% of TNF-α transcriptional responses were TonEBP-dependent and identified genes regulated by both TNF-α and TonEBP. These genes were over-enriched in pathways and diseases related to inflammatory response and inhibition of matrix metalloproteases. Based on RNA-sequencing results, we further investigated regulation of novel TonEBP targets CXCL1, CXCL2, and CXCL3 TonEBP acted synergistically with TNF-α and LPS to induce CXCL1-proximal promoter activity. Interestingly, this regulation required a highly conserved NF-κB-binding site but not a predicted TonE, suggesting cross-talk between these two members of the Rel family. Finally, analysis of human NP tissue showed that TonEBP expression correlated with canonical osmoregulatory targets TauT/SLC6A6, SMIT/SLC5A3, and AR/AKR1B1, supporting in vitro findings that the inflammatory milieu during IDD does not interfere with TonEBP osmoregulation. In summary, whereas TonEBP participates in the proinflammatory response to TNF-α, therapeutic strategies targeting this transcription factor for treatment of disc disease must spare osmoprotective, prosurvival, and matrix homeostatic activities.

Keywords: NF-κB (NF-KB); NFAT transcription factor; NFAT5; TonEBP; chemokine; cytokine; inflammation; intervertebral disc; nucleus pulposus.

Figure 1.

TNF-α promotes nuclear localization of TonEBP without affecting transcript levels. A, TonEBP mRNA levels were unaffected by treatment with TNF-α, IL-1β, or LPS for 4–24 h; as expected, treatment with NaCl (110 mm) resulted in induction. B and C, levels of total cellular TonEBP did not change with TNF-α treatment for 4–24 h. D and E, nuclear and cytoplasmic fraction experiment clearly shows increased nuclear accumulation of TonEBP following TNF-α treatment for 24 h. No discernable depletion of TonEBP in cytoplasmic fraction was seen. Lamin and β-tubulin loading controls showed relatively high purity of fractions. F–I, treatment with IL-1β (F and G) and LPS (H and I) for 4–24 h did not affect the amount of nuclear TonEBP protein. J, immunofluorescent detection of TonEBP in NP cells following 24 h of treatment with NaCl or TNF-α. Inset shows high magnification image of cells. Scale bar, 100 μm. Quantitative measurements represent mean ± S.E. of ≥3 biological replicates. *, p ≤ 0.05; **, p ≤ 0.01. ns, not statistically significant. Neg. CTR, negative control.

Figure 2.

TonEBP activation under inflammatory conditions fails to induce transcription of its canonical osmoregulatory target genes. A, schematic depicting binary TonTAD-GAL4 system used to measure TonEBP-TAD activity. B, activity of TonEBP-TAD was unaffected by treatment with TNF-α, IL-1β, or LPS alone, but it was induced by NaCl. Co-treatment with TNF-α and NaCl dampened the TAD activation by NaCl alone. C, diagram showing taurine transporter (TauT) luciferase reporter, which contains an active, highly conserved TonEBP-binding site, TonE. D, although treatment with NaCl induced activity of the TauT promoter, treatment with TNF-α, IL-1β, or LPS had no effect. E–G, TauT (E), SMIT (F), and AR (G) mRNA levels did not significantly increase with TNF-α, IL-1β, or LPS treatment for 4–24 h, whereas 8 h of NaCl treatment resulted in induction. H, co-treatment of TNF-α along with NaCl did not affect NaCl-mediated induction of TauT, SMIT, or AR. Quantitative measurements represent mean ± S.E. of ≥3 biological replicates and, for transfection experiments, 3 technical replicates per biological replicate. *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; ****, p ≤ 0.0001; ns, not statistically significant.

Figure 3.

RNA sequencing reveals TonEBP as an important regulator of NP cell response to TNF-α. NP cells were transduced with either ShControl or ShTonEBP and cultured with or without TNF-α for 24 h, and RNA sequencing was performed. A, heat map depicting genes differentially expressed between shControl and shTonEBP under either basal or TNF-α-stimulated (24-h TNF-α) conditions. B, Venn diagram showing overlap between TonEBP-dependent genes in untreated versus TNF-α-treated cells. C, heat map depicting genes differentially expressed between control and TNF-α treatment groups. D, volcano plot depicting expression of transcripts that are differentially expressed between control and TNF-α treatment groups and controlled by TonEBP under TNF-α treatment. E, Venn diagrams showing overlap between genes positively or negatively regulated by TNF-α and also regulated by TonEBP. F, log2(−fold change) values for inflammation-related transcripts that were differentially expressed between control and TonEBP knockdown under TNF-α treatment; genes shown were statistically significant between the indicated experimental groups, as defined by adjusted p value <0.05. Quantitative measurements represent mean ± S.E. of ≥3 biological replicates.

Figure 4.

TonEBP regulates TNF-α-dependent expression of select genes linked to matrix catabolism. A, qRT-PCR confirmation of TonEBP knockdown. TonEBP and TauT mRNA levels were unaffected by TNF-α treatment in ShControl-transduced cells but decreased in cells transduced with ShTonEBP. B, TonEBP regulated transcript levels of matrix catabolic enzymes ADAMTS4, ADAMTS5, and MMP3 under TNF-α treatment. C, TNF-α treatment significantly decreases transcript levels of ACAN and COL2A1 in ShControl-transduced cells. Levels were unaffected by knockdown of TonEBP. Quantitative measurements represent mean ± S.E. of ≥3 biological replicates. *, p ≤ 0.05; **, p ≤ 0.01, ****, p ≤ 0.0001. ns, not statistically significant.

Figure 5.

TonEBP controls TNF-α-mediated induction of key cytokines, chemokines, and inflammatory molecules in NP cells. A, TNF-α treatment induced expression of TNF, NOS2, PHD3, and TLR2 mRNAs, which was significantly inhibited by TonEBP knockdown. B and C, expression level of CCL2 and IL-6 mRNA (B) and CCL2 protein (C) were induced by TNF-α treatment; this induction was blocked by TonEBP knockdown. D, IL-6 protein levels were decreased by TonEBP knockdown under TNF-α treatment condition. E and F, CXCL1/CINC1, CXCL2/CINC3, and CXCL3/CINC2 mRNA (E) and protein (F) levels were induced by TNF-α in cell transduced with ShControl and significantly decreased by TonEBP knockdown. Quantitative measurements represent mean ± S.E. of ≥3 biological replicates. *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; ****, p ≤ 0.0001. ns, not statistically significant.

Figure 6.

TonEBP controls the activity of the CXCL1 promoter via a highly conserved NF-κB-binding site. A, diagram showing predicted TonEBP- and NF-κB-binding sites in the rat CXCL1 proximal promoter spanning −1.5 kb upstream of the transcription start site. Predicted TonE is depicted by gray circle and NF-κB-binding sites by open squares. NF-κB-mutant reporter contains mutation in binding site 2 (star), which has high species conservation and activity in other cell types. B, under basal conditions, TonEBP increased activities of WT and TonE-mutant promoters, whereas NF-κB-mutant was unaffected. C, WT, TonE-, and NF-κB-mutant reporters were all induced by TNF-α treatment. During TNF-α treatment, addition of TonEBP further induced activity of WT and TonE-mutant reporters only. D, WT and TonE-mutant reporters, but not NF-κB-mutant, were induced by IL-1β treatment. During IL-1β stimulation, addition of TonEBP did not affect inducibility of any of the reporters, whereas DN-TonEBP blocked induction of WT and TonE-mutant reporters. E, none of the reporters responded to LPS treatment alone. Addition of TonEBP during LPS treatment induced activity in WT and TonE-mutant reporters only. F, addition of small amount (15 ng) of p65 plasmid resulted in a trend of increased activity in all reporters, which was further enhanced by addition of TonEBP. G, expression of TonEBP further increased TNF-α-dependent induction of NF-κB reporter activity. H, treatment with TNF-α, but not NaCl, induced mRNA expression of CXCL1. TNF-α-mediated induction was unaffected by co-treatment with NaCl. Quantitative measurements represent mean ± S.E. of ≥3 biological replicates and, for transfection experiments, 3 technical replicates per biological replicate. *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; ****, p ≤ 0.0001. ns, not statistically significant.

Figure 7.

TonEBP controls the CXCL2 promoter only under certain stimulatory conditions. A, diagram showing predicted TonEBP- and NF-κB-binding sites in the rat CXCL2 proximal promoter spanning −1.5 kb upstream of the transcription start site. TonEs are depicted by gray circles and NF-κB-binding sites by open squares. NF-κB-mutant reporter contains mutation in binding site 2 (star), which has high species conservation and activity in other cell types. B, WT and NF-κB-mutant (to a smaller extent) reporters were induced by TNF-α, whereas further addition of TonEBP or DN-TonEBP had no effect. C, WT reporter was induced by IL-1β, and further addition TonEBP or DN-TonEBP had no effect on reporter activity. D, LPS induced WT reporter activity, with further enhancement by addition of TonEBP. E, addition of small amount of p65 plasmid induced activity of both reporters. Addition of TonEBP along with p65 resulted in further induction of the NF-κB-mutant; there was a small trend of increase for the WT promoter. F, treatment with TNF-α, but not NaCl, increased CXCL2 mRNA expression. TNF-α-dependent induction was unaffected by co-treatment with NaCl. Quantitative measurements represent mean ± S.E. of ≥3 biological replicates and, for transfection experiments, 3 technical replicates per biological replicate. *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; ****, p ≤ 0.0001. ns, not statistically significant.

Figure 8.

TonEBP expression is correlated with levels of osmoprotective genes in human NP samples. A, immunohistochemical staining of TonEBP in non-degenerated (grades ≤4) and degenerated (grades >4) human NP samples. Isotype IgG was used as negative control for staining. B, percentage of cells immunopositive for TonEBP was not significantly different between non-degenerated and degenerated tissue samples. C–E, correlation between mRNA levels of TonEBP and osmoregulatory target genes TauT (p = 0.0044) (C), SMIT (p = 0.03) (D), and AR (p = 0.0554) (E) was seen in all tissue samples analyzed. F, schematic of proposed regulation of TonEBP by TNF-α and downstream transcriptional responses. Activation by either TNF-α or NaCl induces nuclear abundance of TonEBP, but only NaCl induces TonEBP transcript levels. Upon stimulation with TNF-α, TonEBP participates in cross-talk with NF-κB family members to promote transcription of proinflammatory genes. A, scale bar for ×40 images (left) is 100 μm, and scale bar for ×100 images (right) is 50 μm. ns, not statistically significant.