Simple and highly sensitive assay system for TNFR2-mediated soluble- and transmembrane-TNF activity.
Drugs that target tumor necrosis factor-alpha (TNF) are particularly important in the treatment of severe inflammatory progression in rheumatoid arthritis, Crohn's disease and psoriasis. Despite the central role of the TNF/TNF receptor (TNFR) in various disease states, there is a paucity of information concerning TNFR2 signaling. In this study, we have developed a simple and highly sensitive cell-death based assay system for analyzing TNFR2-mediated bioactivity that can be used to screen for TNFR2-selective drugs. Using a lentiviral vector, a chimeric receptor was engineered from the extracellular and transmembrane domain of human TNFR2 and the intracellular domain of mouse Fas and the recombinant protein was then expressed in TNFR1(-/-)R2(-/-) mouse preadipocytes. Our results demonstrate that this chimeric receptor is capable of inducing apoptosis by transmembrane- as well as soluble-TNF stimuli. Moreover, we found that our bioassay based on cell death phenotype had an approximately 80-fold higher sensitivity over existing bioassays. We believe our assay system will be an invaluable research tool for studying TNFR2 and for screening TNFR2-targeted drugs.
Abe
Yasuhiro
Y
Laboratory of Pharmaceutical Proteomics, National Institute of Biomedical Innovation, Ibaraki, Osaka, Japan.
Yoshikawa
Tomoaki
T
Kamada
Haruhiko
H
Shibata
Hiroko
H
Nomura
Tetsuya
T
Minowa
Kyoko
K
Kayamuro
Hiroyuki
H
Katayama
Kazufumi
K
Miyoshi
Hiroyuki
H
Mukai
Yohei
Y
Yoshioka
Yasuo
Y
Nakagawa
Shinsaku
S
Tsunoda
Shin-Ichi
S
Tsutsumi
Yasuo
Y
D017667:Adipocytes / Q000378:metabolism*; D000818:Animals; D000893:Anti-Inflammatory Agents / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects; D001681:Biological Assay; D002462:Cell Membrane / Q000378:metabolism; D002470:Cell Survival; D002478:Cells, Cultured; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D053200:Fas-Associated Death Domain Protein / Q000378:metabolism; D005822:Genetic Vectors; D006801:Humans; D016086:Lentivirus / Q000235:genetics; D051379:Mice; D017434:Protein Structure, Tertiary; D047888:Receptors, Tumor Necrosis Factor, Type I / Q000172:deficiency / Q000235:genetics; D047889:Receptors, Tumor Necrosis Factor, Type II / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism*; D019014:fas Receptor / Q000235:genetics / Q000378:metabolism*
D000893:Anti-Inflammatory Agents; C505534:Fadd protein, mouse; C491899:Fas protein, mouse; D053200:Fas-Associated Death Domain Protein; D047888:Receptors, Tumor Necrosis Factor, Type I; D047889:Receptors, Tumor Necrosis Factor, Type II; D011993:Recombinant Fusion Proteins; C415902:TNFRSF1B protein, human; C506236:Tnfrsf1a protein, mouse; D014409:Tumor Necrosis Factor-alpha; D019014:fas Receptor