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The novel S59P mutation in the TNFRSF1A gene identified in an adult onset TNF receptor associated periodic syndrome (TRAPS) constitutively activates NF-κB pathway.

Abstract

INTRODUCTION

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). 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.

METHODS

HEK-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.

RESULTS

TNF 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.

CONCLUSIONS

The 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.

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  • Authors+Show Affiliations

    ,

    University of Padova, Rheumatology Unit, Department of Medicine - DIMED, Via Giustiniani 2, 35128, Padova, Italy. eliana.greco@unipd.it. University of Padova, Laboratory Medicine, Department of Medicine - DIMED, Via Giustiniani 2, 35128, Padova, Italy. eliana.greco@unipd.it.

    ,

    University of Padova, Rheumatology Unit, Department of Medicine - DIMED, Via Giustiniani 2, 35128, Padova, Italy. ada.aita@studenti.unipd.it. University of Padova, Laboratory Medicine, Department of Medicine - DIMED, Via Giustiniani 2, 35128, Padova, Italy. ada.aita@studenti.unipd.it.

    ,

    University of Padova, Rheumatology Unit, Department of Medicine - DIMED, Via Giustiniani 2, 35128, Padova, Italy. paola.galozzi@unipd.it.

    ,

    University of Padova, Rheumatology Unit, Department of Medicine - DIMED, Via Giustiniani 2, 35128, Padova, Italy. alessandra.gava@gmail.com.

    ,

    University of Padova, Rheumatology Unit, Department of Medicine - DIMED, Via Giustiniani 2, 35128, Padova, Italy. paolo.sfriso@unipd.it.

    ,

    School of Life Sciences, The University of Nottingham, Queen's Medical Centre, Derby road, NG7 2UH, Nottingham, UK. ola.negm@nottingham.ac.uk. Medical Microbiology and Immunology Department, Faculty of Medicine, Mansoura University, Elgomhouria Street, 35516, Mansoura City, Egypt. ola.negm@nottingham.ac.uk.

    ,

    School of Life Sciences, The University of Nottingham, Queen's Medical Centre, Derby road, NG7 2UH, Nottingham, UK. paddy.tighe@nottingham.ac.uk.

    ,

    University of Padova, Rheumatology Unit, Department of Medicine - DIMED, Via Giustiniani 2, 35128, Padova, Italy. francescocaso@yahoo.it.

    ,

    University of Padova, Laboratory Medicine, Department of Medicine - DIMED, Via Giustiniani 2, 35128, Padova, Italy. filippo.navaglia@sanita.padova.it.

    ,

    Institute of Neuroscience of the National Research Council, Section of Padova, Corso Stati Uniti, 4, 3512, Padova, Italy. emanuela.dazzo@bio.unipd.it.

    ,

    Department of Biology, University of Padova, Via U. Bassi, 58/B, 35121, Padova, Italy. marzia.debortoli@unipd.it.

    ,

    Department of Biology, University of Padova, Via U. Bassi, 58/B, 35121, Padova, Italy. alessandra.rampazzo@unipd.it.

    ,

    Amyloidosis Research and Treatment Center, Biotechnology Research laboratories, Fondazione IRCSS Policlinico San Matteo and University of Pavia, Viale Camillo Golgi 19, 27100, Pavia, Italy. l.obici@smatteo.pv.it.

    ,

    Amyloidosis Research and Treatment Center, Biotechnology Research laboratories, Fondazione IRCSS Policlinico San Matteo and University of Pavia, Viale Camillo Golgi 19, 27100, Pavia, Italy. simona_donadei@yahoo.it.

    ,

    Amyloidosis Research and Treatment Center, Biotechnology Research laboratories, Fondazione IRCSS Policlinico San Matteo and University of Pavia, Viale Camillo Golgi 19, 27100, Pavia, Italy. gmerlini@unipv.it.

    ,

    University of Padova, Laboratory Medicine, Department of Medicine - DIMED, Via Giustiniani 2, 35128, Padova, Italy. mario.plebani@sanita.padova.it.

    ,

    School of Life Sciences, The University of Nottingham, Queen's Medical Centre, Derby road, NG7 2UH, Nottingham, UK. ian.todd@nottingham.ac.uk.

    ,

    University of Padova, Laboratory Medicine, Department of Medicine - DIMED, Via Giustiniani 2, 35128, Padova, Italy. daniela.basso@sanita.padova.it.

    University of Padova, Rheumatology Unit, Department of Medicine - DIMED, Via Giustiniani 2, 35128, Padova, Italy. leonardo.punzi@unipd.it.

    Source

    Arthritis research & therapy 17: 2015 Apr 03 pg 93

    MeSH

    Adult
    Age Factors
    Aged
    Apoptosis
    Case-Control Studies
    Cells, Cultured
    Familial Mediterranean Fever
    Genetic Predisposition to Disease
    HEK293 Cells
    Humans
    Immunoblotting
    Italy
    Male
    Mutation
    NF-kappa B
    Polymerase Chain Reaction
    Receptors, Tumor Necrosis Factor
    Receptors, Tumor Necrosis Factor, Type I
    Reference Values
    Risk Assessment
    Sampling Studies
    Signal Transduction

    Pub Type(s)

    Journal Article
    Research Support, Non-U.S. Gov't

    Language

    eng

    PubMed ID

    25888769

    Citation

    Greco, Eliana, et al. "The Novel S59P Mutation in the TNFRSF1A Gene Identified in an Adult Onset TNF Receptor Associated Periodic Syndrome (TRAPS) Constitutively Activates NF-κB Pathway." Arthritis Research & Therapy, vol. 17, 2015, p. 93.
    Greco E, Aita A, Galozzi P, et al. The novel S59P mutation in the TNFRSF1A gene identified in an adult onset TNF receptor associated periodic syndrome (TRAPS) constitutively activates NF-κB pathway. Arthritis Res Ther. 2015;17:93.
    Greco, E., Aita, A., Galozzi, P., Gava, A., Sfriso, P., Negm, O. H., ... Punzi, L. (2015). The novel S59P mutation in the TNFRSF1A gene identified in an adult onset TNF receptor associated periodic syndrome (TRAPS) constitutively activates NF-κB pathway. Arthritis Research & Therapy, 17, p. 93. doi:10.1186/s13075-015-0604-7.
    Greco E, et al. The Novel S59P Mutation in the TNFRSF1A Gene Identified in an Adult Onset TNF Receptor Associated Periodic Syndrome (TRAPS) Constitutively Activates NF-κB Pathway. Arthritis Res Ther. 2015 Apr 3;17:93. PubMed PMID: 25888769.
    * Article titles in AMA citation format should be in sentence-case
    TY - JOUR T1 - The novel S59P mutation in the TNFRSF1A gene identified in an adult onset TNF receptor associated periodic syndrome (TRAPS) constitutively activates NF-κB pathway. AU - Greco,Eliana, AU - Aita,Ada, AU - Galozzi,Paola, AU - Gava,Alessandra, AU - Sfriso,Paolo, AU - Negm,Ola H, AU - Tighe,Patrick, AU - Caso,Francesco, AU - Navaglia,Filippo, AU - Dazzo,Emanuela, AU - De Bortoli,Marzia, AU - Rampazzo,Alessandra, AU - Obici,Laura, AU - Donadei,Simona, AU - Merlini,Giampaolo, AU - Plebani,Mario, AU - Todd,Ian, AU - Basso,Daniela, AU - Punzi,Leonardo, Y1 - 2015/04/03/ PY - 2014/09/18/received PY - 2015/03/20/accepted PY - 2015/4/19/entrez PY - 2015/4/19/pubmed PY - 2016/1/20/medline SP - 93 EP - 93 JF - Arthritis research & therapy JO - Arthritis Res. Ther. VL - 17 N2 - INTRODUCTION: 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). 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. METHODS: HEK-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. RESULTS: TNF 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. CONCLUSIONS: The 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. SN - 1478-6362 UR - https://www.unboundmedicine.com/medline/citation/25888769/The_novel_S59P_mutation_in_the_TNFRSF1A_gene_identified_in_an_adult_onset_TNF_receptor_associated_periodic_syndrome__TRAPS__constitutively_activates_NF_κB_pathway_ L2 - https://arthritis-research.biomedcentral.com/articles/10.1186/s13075-015-0604-7 DB - PRIME DP - Unbound Medicine ER -