Tumor necrosis factor receptor I from patients with tumor necrosis factor receptor–associated periodic syndrome interacts with wild‐type tumor necrosis factor receptor I and induces ligand‐independent NF‐κB activation

Abstract

Objective. To investigate the molecular consequences of expressing mutated forms of tumor necrosis factor receptor I (TNFRI) as found in patients with TNFR-associated periodic syndrome (TRAPS).Methods. We cloned and expressed full-length wild-type (WT) and T50K and P46L variants of TNFRI using a new tightly regulated doxycycline-dependent expression system. This system enabled the study of molecular interactions between these receptors at both physiologic and pathophysiologic levels of expression.Results. We used chemical crosslinking on the cell surface to show that WT and mutant forms of TNFRI, derived from TRAPS patients, interact in the absence of TNF ligand. Doxycycline-controlled up-regulation of one TNFRI allele, either WT or mutant, caused downregulation of the other allele, indicating dynamic control of cell surface assembly. We also demonstrated that increased expression of mutant TNFRI (T50K) was associated with a parallel increase in NF-B p65 (RelA) subunit activation, which did not occur with increased expression of WT TNFRI.Conclusion. The T50K TRAPS-related variant is capable of sustaining inappropriate NF-B activation, resulting in persistent autoinflammation in target organs such as skin, synovial membrane, and the central nervous system. We conclude that some of the inflammatory processes seen in TRAPS do not involve direct interaction of TNF with its receptors, but that other proinflammatory mechanisms capable of up-regulating TNFRI expression may cause cellular activation through the NF-B signaling pathway.Tumor necrosis factor receptor-associated periodic syndrome (TRAPS; MIM no. 142680) is a dominantly inherited chronic inflammatory disorder caused by mutations in the extracellular domains of tumor necrosis factor receptor I (TNFRI), the gene for which is TNFRSF1A (1), and characterized by recurrent fevers and abdominal pain. The most common mutations involve cysteine residues, but several variants involving other residues have also been reported (1,2). More than 30 different pathogenic TNFRSF1A mutations have now been identified (see http://fmf.igh.cnrs.fr/infevers/ and ref.2), mostly located in either the first or second cysteine-rich N-terminal extracellular domain (CRD1 or CRD2) of TNFRSF1A (2,3). Impaired cleavage of the TNFRI (p55) ectodomain upon cellular activation has been demonstrated as a pathogenic mechanism in some but not all TNFRI variants (3-6) and did not relate to the severity of the phenotype (7,8).Biologic activities of TNF are mediated through 2 TNFRs, TNFRI and TNFRII (TNFR superfamily 1B

Altered lipid raft–associated proximal signaling and translocation of CD45 tyrosine phosphatase in B lymphocytes from patients with systemic lupus erythematosus

“…However, one explanation could be a negative feedback on CD45RB expression by the low molecular weight isoform detected in SLE B cells. In this regard, there is evidence that alternative isoforms exert negative feedback on the expression/function of other isoforms of the protein in question (44)(45)(46). Of relevance to our study, this observation could imply that the CD45 found in the LRs is an isoform different from CD45RB.…”

Protein misfolding and dysregulated protein homeostasis in autoinflammatory diseases and beyond

“…It has been postulated that TNF release stimulated by UPR activation may then signal through the wild-type TNFR1, generating an autocrine positive feedback loop enhancing TNF production [21,22]. It is also conceivable that mutant intracellular TNFR1 may still stimulate TNF production by activation of their intracellular death domains independent of receptorligand binding, particularly as this domain is rarely mutated in TRAPS.…”

Latency can be conferred to a variety of cytokines by fusion with latency-associated peptide from TGF-β

“…DNA sequences of all constructs were verified by sequencing at the Genome Centre, William Harvey Research Institute. Plasmid DNA was purified from E. coli DH5α using Qiagen maxiprep kits (Crawley, UK) and was transiently transfected into 293T cells using the calcium phosphate co-precipitation method as previously described [9]. For biological assays, 293T cells were incubated in 10% heat inactivated FBS in DMEM (Invitrogen) (with 100U/ml penicillin, 100µg/ml streptomycin and 2mM L-glutamine) and the supernatant collected 48 hours posttransfection.…”