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

Greco E, Aita A, Galozzi P, Gava A, Sfriso P, Negm OH, Tighe P, Caso F, Navaglia F, Dazzo E, De Bortoli M, Rampazzo A, Obici L, Donadei S, Merlini G, Plebani M, Todd I, Basso D, Punzi L - Arthritis Res. Ther. (2015)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

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

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.

Show MeSH

Related in: MedlinePlus

NF-κB pathway in peripheral blood mononuclear cells. Control (wild-type (WT)) and TRAPS peripheral blood mononuclear cells unstimulated or stimulated for 10 minutes with TNF (6 ng/mL) or IL-1β (1 ng/mL). Western blot shows p-IκBα (Ser32) and p65 subunit of NF-κB (Ser536) and the corresponding β-actin, used as control. The histograms show semi-quantification of band intensities after normalization against the negative control (100%) (OD; Image J software, version 1.47 NIH, Bethesda, Maryland, USA). Columns indicate percent values.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC4416318&req=5

Fig5: NF-κB pathway in peripheral blood mononuclear cells. Control (wild-type (WT)) and TRAPS peripheral blood mononuclear cells unstimulated or stimulated for 10 minutes with TNF (6 ng/mL) or IL-1β (1 ng/mL). Western blot shows p-IκBα (Ser32) and p65 subunit of NF-κB (Ser536) and the corresponding β-actin, used as control. The histograms show semi-quantification of band intensities after normalization against the negative control (100%) (OD; Image J software, version 1.47 NIH, Bethesda, Maryland, USA). Columns indicate percent values.

Mentions: Figure 5 shows Western blot analysis of p-IκB-α (p-IκB-α Ser32) and of nuclear p65 component (p-NF-κB Ser536) in control and TRAPS PBMCs kept in culture for 24 hours unstimulated or stimulated with TNF and IL-1β. In basal conditions IκB-α phosphorylation was higher in TRAPS with respect to control PBMCs. In both control and TRAPS patients TNF reduced IκB-α phosphorylation. IL-1b reduced the phosphorylation of IkB-a in control PBMCs, while enhnacing it in TRAPS PBMCs. In the same experimental conditions, p65 activity in isolated nuclei was constitutively elevated only in S59P TRAPS PBMCs. Both TNF and IL-1β induced the p65 phosphorylation in controls and TRAPS PBMCs.Figure 5


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

Greco E, Aita A, Galozzi P, Gava A, Sfriso P, Negm OH, Tighe P, Caso F, Navaglia F, Dazzo E, De Bortoli M, Rampazzo A, Obici L, Donadei S, Merlini G, Plebani M, Todd I, Basso D, Punzi L - Arthritis Res. Ther. (2015)

NF-κB pathway in peripheral blood mononuclear cells. Control (wild-type (WT)) and TRAPS peripheral blood mononuclear cells unstimulated or stimulated for 10 minutes with TNF (6 ng/mL) or IL-1β (1 ng/mL). Western blot shows p-IκBα (Ser32) and p65 subunit of NF-κB (Ser536) and the corresponding β-actin, used as control. The histograms show semi-quantification of band intensities after normalization against the negative control (100%) (OD; Image J software, version 1.47 NIH, Bethesda, Maryland, USA). Columns indicate percent values.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4416318&req=5

Fig5: NF-κB pathway in peripheral blood mononuclear cells. Control (wild-type (WT)) and TRAPS peripheral blood mononuclear cells unstimulated or stimulated for 10 minutes with TNF (6 ng/mL) or IL-1β (1 ng/mL). Western blot shows p-IκBα (Ser32) and p65 subunit of NF-κB (Ser536) and the corresponding β-actin, used as control. The histograms show semi-quantification of band intensities after normalization against the negative control (100%) (OD; Image J software, version 1.47 NIH, Bethesda, Maryland, USA). Columns indicate percent values.
Mentions: Figure 5 shows Western blot analysis of p-IκB-α (p-IκB-α Ser32) and of nuclear p65 component (p-NF-κB Ser536) in control and TRAPS PBMCs kept in culture for 24 hours unstimulated or stimulated with TNF and IL-1β. In basal conditions IκB-α phosphorylation was higher in TRAPS with respect to control PBMCs. In both control and TRAPS patients TNF reduced IκB-α phosphorylation. IL-1b reduced the phosphorylation of IkB-a in control PBMCs, while enhnacing it in TRAPS PBMCs. In the same experimental conditions, p65 activity in isolated nuclei was constitutively elevated only in S59P TRAPS PBMCs. Both TNF and IL-1β induced the p65 phosphorylation in controls and TRAPS PBMCs.Figure 5

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

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

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.

Show MeSH
Related in: MedlinePlus