Quantitative single-molecule imaging of TNFR1 reveals zafirlukast as antagonist of TNFR1 clustering and TNFα-induced NF-ĸB signaling.
Animals
Cell Line
Cytokines
/ biosynthesis
HeLa Cells
Humans
Indoles
Mice
NF-kappa B
/ metabolism
Phenylcarbamates
Protein Multimerization
/ drug effects
Receptors, Tumor Necrosis Factor, Type I
/ antagonists & inhibitors
Receptors, Tumor Necrosis Factor, Type II
/ metabolism
Signal Transduction
/ drug effects
Single Molecule Imaging
Sulfonamides
Tosyl Compounds
/ pharmacology
Transcription, Genetic
/ drug effects
Tumor Necrosis Factor-alpha
/ pharmacology
CysLTR1
Cysteine-Rich Domain (CRD)
Pre-Ligand Assembly Domain (PLAD)
Single-Molecule Localization Microscopy (SMLM)
Journal
Journal of leukocyte biology
ISSN: 1938-3673
Titre abrégé: J Leukoc Biol
Pays: England
ID NLM: 8405628
Informations de publication
Date de publication:
02 2021
02 2021
Historique:
received:
01
11
2019
revised:
22
04
2020
accepted:
23
04
2020
pubmed:
14
5
2020
medline:
24
3
2021
entrez:
14
5
2020
Statut:
ppublish
Résumé
TNFR1 is a crucial regulator of NF-ĸB-mediated proinflammatory cell survival responses and programmed cell death (PCD). Deregulation of TNFα- and TNFR1-controlled NF-ĸB signaling underlies major diseases, like cancer, inflammation, and autoimmune diseases. Therefore, although being routinely used, antagonists of TNFα might also affect TNFR2-mediated processes, so that alternative approaches to directly antagonize TNFR1 are beneficial. Here, we apply quantitative single-molecule localization microscopy (SMLM) of TNFR1 in physiologic cellular settings to validate and characterize TNFR1 inhibitory substances, exemplified by the recently described TNFR1 antagonist zafirlukast. Treatment of TNFR1-mEos2 reconstituted TNFR1/2 knockout mouse embryonic fibroblasts (MEFs) with zafirlukast inhibited both ligand-independent preligand assembly domain (PLAD)-mediated TNFR1 dimerization as well as TNFα-induced TNFR1 oligomerization. In addition, zafirlukast-mediated inhibition of TNFR1 clustering was accompanied by deregulation of acute and prolonged NF-ĸB signaling in reconstituted TNFR1-mEos2 MEFs and human cervical carcinoma cells. These findings reveal the necessity of PLAD-mediated, ligand-independent TNFR1 dimerization for NF-ĸB activation, highlight the PLAD as central regulator of TNFα-induced TNFR1 oligomerization, and demonstrate that TNFR1-mEos2 MEFs can be used to investigate TNFR1-antagonizing compounds employing single-molecule quantification and functional NF-ĸB assays at physiologic conditions.
Identifiants
pubmed: 32401398
doi: 10.1002/JLB.2AB0420-572RR
doi:
Substances chimiques
Cytokines
0
Indoles
0
NF-kappa B
0
Phenylcarbamates
0
Receptors, Tumor Necrosis Factor, Type I
0
Receptors, Tumor Necrosis Factor, Type II
0
Sulfonamides
0
Tosyl Compounds
0
Tumor Necrosis Factor-alpha
0
zafirlukast
XZ629S5L50
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
363-371Informations de copyright
© 2020 The Authors. Journal of Leukocyte Biology published by Wiley Periodicals, Inc. on behalf of Society for Leukocyte Biology.
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