title: Generation of mouse macrophages expressing membrane-bound TNF variants with selectivity for TNFR1 or TNFR2.
abstract: Tumor necrosis factor-alpha (TNF) is expressed on the cell surface as a transmembrane form (tmTNF), that can be released as a soluble form (solTNF) via proteolytic cleavage. These two types of TNF exert their biological functions by binding to one of two TNF receptors, TNFR1 or TNFR2. However, the biological function of tmTNF through these two receptors remains to be determined. Here, we generated macrophages that expressed tmTNF mutants with selectivity for either TNFR1 or TNRF2 as a tool to evaluate signaling through these receptors. Wild-type TNF (wtTNF), TNFR1-selective mutant TNF (mutTNF-R1) or TNFR2-selective mutant TNF (mutTNF-R2) were individually expressed on the TNFR1(-/-)R2(-/-) mouse macrophages (Mphi) as the tmTNF forms. tm-mutTNF-R1-expressing Mphi exhibited significant selectivity for binding to TNFR1, whereas tm-mutTNF-R2-expressing Mphi only showed a slight selectivity for binding to TNFR2. Signaling by tm-mutTNF-R1-expressing Mphi through the hTNFR2 was weaker than that of tm-wtTNF-expressing Mphi, suggesting that the binding selectivity correlated with functional selectivity. Interestingly, signaling by tm-mutTNF-R2-expressing Mphi through TNFR2 was much stronger than signaling by tm-wtTNF-expressing Mphi, whereas signaling by the corresponding soluble form was weaker than that mediated by wtTNF. These results indicate tmTNF variants might prove useful for the functional analysis of signaling through TNF receptors.
authors.affiliation: National Institute of Biomedical Innovation, Ibaraki, Osaka, Japan.
authors.forename: Hiroko
authors.initials: H
authors.lastname: Shibata
authors.forename: Yasuhiro
authors.initials: Y
authors.lastname: Abe
authors.forename: Yasuo
authors.initials: Y
authors.lastname: Yoshioka
authors.forename: Tetsuya
authors.initials: T
authors.lastname: Nomura
authors.forename: Masaki
authors.initials: M
authors.lastname: Sato
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Kayamuro
authors.forename: Tomoyuki
authors.initials: T
authors.lastname: Kawara
authors.forename: Shuhei
authors.initials: S
authors.lastname: Arita
authors.forename: Tsuyoshi
authors.initials: T
authors.lastname: Furuya
authors.forename: Kazuya
authors.initials: K
authors.lastname: Nagano
authors.forename: Tomoaki
authors.initials: T
authors.lastname: Yoshikawa
authors.forename: Haruhiko
authors.initials: H
authors.lastname: Kamada
authors.forename: Shin-ichi
authors.initials: S
authors.lastname: Tsunoda
authors.forename: Yasuo
authors.initials: Y
authors.lastname: Tsutsumi
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D016923:Cell Death; D002462:Cell Membrane / Q000378:metabolism*; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D007700:Kinetics; D016086:Lentivirus / Q000235:genetics; D008264:Macrophages / Q000166:cytology* / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D047888:Receptors, Tumor Necrosis Factor, Type I / Q000378:metabolism*; D047889:Receptors, Tumor Necrosis Factor, Type II / Q000378:metabolism*; D012995:Solubility; D014409:Tumor Necrosis Factor-alpha / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D050505:Mutant Proteins; D047888:Receptors, Tumor Necrosis Factor, Type I; D047889:Receptors, Tumor Necrosis Factor, Type II; D014409:Tumor Necrosis Factor-alpha