Abstract
IntroductionMutations in the TNFRSF1A gene, encoding tumor necrosis factor receptor 1 (TNF-R1), are associated with the autosomal dominant autoinflammatory disorder, called TNF receptor associated periodic syndrome (TRAPS). TRAPS is clinically characterized by recurrent episodes of long-lasting fever and systemic inflammation. A novel mutation (c.262 T > C; S59P) in the TNFRSF1A gene at residue 88 of the mature protein was recently identified in our laboratory in an adult TRAPS patient. The aim of this study was to functionally characterize this novel TNFRSF1A mutation evaluating its effects on the TNF-R1-associated signaling pathways, firstly NF-κB, under particular conditions and comparing the results with suitable control mutations.MethodsHEK-293 cell line was transfected with pCMV6-AC construct expressing wild-type (WT) or c.262 T > C (S59P), c.362G > A (R92Q), c.236C > T (T50M) TNFRSF1A mutants. Peripheral blood mononuclear cells (PBMCs) were instead isolated from two TRAPS patients carrying S59P and R92Q mutations and from five healthy subjects. Both transfected HEK-293 and PBMCs were stimulated with tumor necrosis factor (TNF) or interleukin 1β (IL-1β) to evaluate the expression of TNF-R1, the activation of TNF-R1-associated downstream pathways and the pro-inflammatory cytokines by means of immunofluorescent assay, array-based technique, immunoblotting and immunometric assay, respectively.ResultsTNF induced cytoplasmic accumulation of TNF-R1 in all mutant cells. Furthermore, all mutants presented a particular set of active TNF-R1 downstream pathways. S59P constitutively activated IL-1β, MAPK and SRC/JAK/STAT3 pathways and inhibited apoptosis. Also, NF-κB pathway involvement was demonstrated in vitro by the enhancement of p-IκB-α and p65 nuclear subunit of NF-κB expression in all mutants in the presence of TNF or IL-1β stimulation. These in vitro results correlated with patients’ data from PBMCs. Concerning the pro-inflammatory cytokines secretion, mainly IL-1β induced a significant and persistent enhancement of IL-6 and IL-8 in PBMCs carrying the S59P mutation.ConclusionsThe novel S59P mutation leads to defective cellular trafficking and to constitutive activation of TNF-R1. This mutation also determines constitutive activation of the IL-1R pathway, inhibition of apoptosis and enhanced and persistent NF-κB activation and cytokine secretion in response to IL-1β stimulation.Electronic supplementary materialThe online version of this article (doi:10.1186/s13075-015-0604-7) contains supplementary material, which is available to authorized users.
Highlights
Mutations in the TNFRSF1A gene, encoding tumor necrosis factor receptor 1 (TNF-R1), are associated with the autosomal dominant autoinflammatory disorder, called TNF receptor associated periodic syndrome (TRAPS)
The TNF-R1 presents an extracellular domain involved in ligand binding, a transmembrane domain involved in receptor solubilization and an intracellular region where death domains (DD) are involved in signal transduction [5,6]
T > C), located in exon 3, results in a proline for serine amino acid substitution (S59P) at residue 88 of the mature protein. We studied another TRAPS patient who carried in heterozygosis the common single-base mutation (c.362G > A) in exon 4 (Figure 1, panel B), resulting in an arginine for glutamine amino acid substitution (R92Q) at residue 121 of the mature protein
Summary
Mutations in the TNFRSF1A gene, encoding tumor necrosis factor receptor 1 (TNF-R1), are associated with the autosomal dominant autoinflammatory disorder, called TNF receptor associated periodic syndrome (TRAPS). Tumor necrosis factor receptor-associated periodic syndrome (TRAPS; OMIM 142680) is the second most common inherited autosomal dominant autoinflammatory disease [1]. It is caused by mutations in the TNFRSF1A gene located on chromosome 12p13 and encodes the 55 kDa receptor for tumor necrosis factor receptor 1 (TNF-R1). The binding of TNF to TNF-R1 causes recruitment of several intracellular adaptor proteins leading to downstream signaling events that activate the inflammatory (complex I) and apoptotic (complex II) processes. Complex I activation prevails and leads to NF-κB activation and transcription of anti-apoptotic and pro-inflammatory genes [7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27]
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