Generation of mouse macrophages expressing membrane-bound TNF variants with selectivity for TNFR1 or TNFR2.
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.
Shibata
Hiroko
H
National Institute of Biomedical Innovation, Ibaraki, Osaka, Japan.
Abe
Yasuhiro
Y
Yoshioka
Yasuo
Y
Nomura
Tetsuya
T
Sato
Masaki
M
Kayamuro
Hiroyuki
H
Kawara
Tomoyuki
T
Arita
Shuhei
S
Furuya
Tsuyoshi
T
Nagano
Kazuya
K
Yoshikawa
Tomoaki
T
Kamada
Haruhiko
H
Tsunoda
Shin-ichi
S
Tsutsumi
Yasuo
Y
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*
D050505:Mutant Proteins; D047888:Receptors, Tumor Necrosis Factor, Type I; D047889:Receptors, Tumor Necrosis Factor, Type II; D014409:Tumor Necrosis Factor-alpha