A failure of TNFAIP3 negative regulation maintains sustained NF-κB activation in Sjögren’s syndrome

Abstract

Sjögren's syndrome (SS) is characterized by the features of systemic autoimmunity and exocrine gland dysfunction and inflammation. Deregulated cytokine production is known to contribute to the etiology of SS but the underlying molecular mechanism is still remains to be unclear. TNF-α-induced protein 3 or TNFAIP3 is involved in the negative feedback regulation of nuclear factor-κB (NF-κB) signaling in response to specific pro-inflammatory stimuli in different cell types. To define the contribution of TNFAIP3 to SS, the levels of TNFAIP3 expression in human salivary gland epithelial cells (SGEC) derived from active primary SS patients were analyzed. Histological analysis was performed on paraffin-embedded human Sjögren's samples and healthy tissues. In separate experiments, immunofluorescence staining, western blot analysis and quantitative real-time PCR for TNFAIP3 was conducted in SGEC from SS and healthy subjects. Our findings clearly demonstrate changes in levels of the protein and gene expression between healthy controls and SS patients, depicting a very weak positivity for TNFAIP3 in SS samples. TNFAIP3 was found down-regulated in SGECs derived from SS patients in comparison with controls, and the cells with down-regulated TNFAIP3 expression exhibited enhanced NF-κB activities. In addition, to investigate the role of TNFAIP3 in the activation of NF-κB, we depleted TNFAIP3 expression by siRNA in healthy SGEC after treatment with or without TNF-α. Intriguingly, the silencing of TNFAIP3 by its siRNA in healthy SGEC increased NF-κB activation that could explain the deregulated cytokines production observed in SS.

Sjögren’s syndrome pathological neovascularization is regulated by VEGF-A-stimulated TACE-dependent crosstalk between VEGFR2 and NF-κB

“…Our previous study demonstrated a higher transcriptional activity of NF-kB in SS than in normal subjects, suggesting that the NF-kB signal is important for modulating immune responses in human SGEC in this autoimmune condition. 30 Here we demonstrated that in anti-Ro/ SSA Abs-treated human SGEC increased VEGF-A expression was correlated with nuclear NF-kB translocation. The use of anti-VEGF-A mAb blocked the anti-Ro/SSA Abs-induced VEGF-A molecules and thus determined a remarkable decrease of NF-kB activation, whereas treatment with exogenous VEGF-A reversed the effect of VEGF-A neutralization.…”

Sjögren's syndrome autoantibodies provoke changes in gene expression profiles of inflammatory cytokines triggering a pathway involving TACE/NF-κB

“…NF-kB has been shown to be chronically active in many inflammatory autoimmune diseases such as inflammatory bowel disease, 34 rheumatoid arthritis 35 and systemic lupus erythematosus 36 as well as SS. 22 In fact, we previously demonstrated enhanced levels of NF-kB activation in SS as compared with normal subjects, suggesting that the NF-kB signal is important in human salivary gland cells for modulating immune responses. 22 In the present study we found that SS Abs are able to trigger the signal transduction pathways involved in NF-kB activation leading to enhanced gene expression of proinflammatory mediators.…”

“…22 In fact, we previously demonstrated enhanced levels of NF-kB activation in SS as compared with normal subjects, suggesting that the NF-kB signal is important in human salivary gland cells for modulating immune responses. 22 In the present study we found that SS Abs are able to trigger the signal transduction pathways involved in NF-kB activation leading to enhanced gene expression of proinflammatory mediators. As the induction of proinflammatory genes by anti-Ro/SSA Abs is complex and involves increased NF-kB-DNA binding, we tested the effect of TAPI-1 on NF-kB activation and NF-kB-mediated regulation of proinflammatory cytokines production.…”

“…Nuclear fractions were collected from cells using the nuclear extraction kit following the manufacturer's suggestions (Active Motif) and the technique was applied as described previously. 22 Transient Transfection with the Dominant Negative Version of IjB-a and Luciferase Assay To modulate NF-kB activity, healthy SGEC treated with antiRo/SSA Abs were transiently transfected with a dominant negative IkB-a vector (the plasmid with the dominantnegative version of IkB-a was a kind gift by Dr P Bauerle, University of Munich and Dr M Lienhard Schmitz, Deutsches Krebsforschungszentrum, Germany) or its control (empty vector, pCMV). This dominant negative super-repressor IkB-a contains mutations at residues 32 and 36 (the two inducible phosphorylation sites), which disrupt phosphorylation and subsequent degradation.…”

“…We previously reported that the transcription activity of NF-kB is higher in SS than in normal subjects and human SGECs have a prominent role in orchestrating the salivary gland inflammatory response, suggesting that the NF-kB signal is important in these cells for modulating immune responses. 22 NF-kB is a transcription factor family with multiple subunits, and the NF-kB subunits p50 and p65 are commonly associated with increased gene transcription. Here we found that anti-Ro/SSA Abs Anti-Ro/SSA trigger TACE/NF-jB axis S Lisi et al increased the NF-kB p65 nuclear immunofluorescence (Figures 2a and c), whereas ELISA used to detect NF-kB p65 subunits bound to the consensus DNA site of the identified region, demonstrated 2.65-fold more DNA binding by p65 in the nuclear extracts derived from anti-Ro/SSA Abs-treated SGEC as compared with untreated control SGEC (Figure 2b), suggesting that p65 transcription activity is higher after antiRo/SSA Abs treatment.…”