title: Functional characterization of TNF-α in grass carp head kidney leukocytes: induction and involvement in the regulation of NF-κB signaling.
abstract: Tumor necrosis factor-alpha (TNF-α) is a potent regulatory cytokine, which serves as a key mediator of inflammation, immunity and apoptosis in mammals. Identification, expression and regulatory effects of TNF-α have been reported in various fish species, showing the structural and functional similarity or discrepancy between each other. In this study, TNF-α was identified from grass carp (Ctenopharyngodon idella) and the deduced grass carp TNF-α (gcTNF-α) protein possessed the TNF family signature motifs, a protease cleavage site, a transmembrane domain and two conserved cysteine residues. Further studies showed that gcTNF-α expression was induced with a rapid kinetics by immune challenge in vitro and in vivo. To characterize the function of gcTNF-α, recombinant gcTNF-α (rgcTNF-α) was prepared by using the Escherichia coli expression system. It was shown to enhance the mRNA expression of gcTNF-α and gcIL-1β in head kidney leukocytes (HKLs), confirming the biological activity of rgcTNF-α. In the same model, NF-κB inhibitor (PDTC) was able to attenuate rgcTNF-α-induced gcTNF-α mRNA expression, implying the involvement of NF-κB pathway in fish TNF-α action. This notion was reinforced by the finding that rgcTNF-α could induce the phosphorylation of IκBα in a time-dependent oscillation in HKLs, indicating a dynamical variation of NF-κB activity as seen in mammals. In addition, rgcTNF-α could up-regulate the expression of two TNF receptor-associated factors (TRAF), TRAF1 and TRAF2, in a time- and dose-dependent manner, suggesting that gcTNF-α may function as a regulator of fish NF-κB pathway. These results for the first time reveal the link of gcTNF-α to the NF-κB pathway and provide a better understanding of TNF-α signaling in teleost immunity.
authors.affiliation: School of Life Science and Technology, University of Electronic Science and Technology of China, Chengdu 610054, People's Republic of China.
authors.forename: Anying
authors.initials: A
authors.lastname: Zhang
authors.forename: Danyan
authors.initials: D
authors.lastname: Chen
authors.forename: He
authors.initials: H
authors.lastname: Wei
authors.forename: Linyong
authors.initials: L
authors.lastname: Du
authors.forename: Taiqiang
authors.initials: T
authors.lastname: Zhao
authors.forename: Xinyan
authors.initials: X
authors.lastname: Wang
authors.forename: Hong
authors.initials: H
authors.lastname: Zhou
mesh_terms: D000704:Analysis of Variance; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D002347:Carps / Q000276:immunology* / Q000378:metabolism; D003001:Cloning, Molecular; D016000:Cluster Analysis; D018076:DNA, Complementary / Q000235:genetics; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli; D005786:Gene Expression Regulation / Q000187:drug effects / Q000276:immunology*; D060226:Head Kidney / Q000166:cytology* / Q000378:metabolism; D053583:Interleukin-1beta / Q000378:metabolism; D007962:Leukocytes / Q000378:metabolism*; D008969:Molecular Sequence Data; D016328:NF-kappa B / Q000037:antagonists & inhibitors / Q000378:metabolism*; D010802:Phylogeny; D011392:Proline / Q000031:analogs & derivatives / Q000494:pharmacology; D060888:Real-Time Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA; D015398:Signal Transduction / Q000276:immunology*; D047990:TNF Receptor-Associated Factor 1 / Q000378:metabolism; D047992:TNF Receptor-Associated Factor 2 / Q000378:metabolism; D013859:Thiocarbamates / Q000494:pharmacology; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics / Q000378:metabolism*
chemical_list: D018076:DNA, Complementary; D053583:Interleukin-1beta; D016328:NF-kappa B; D011994:Recombinant Proteins; D047990:TNF Receptor-Associated Factor 1; D047992:TNF Receptor-Associated Factor 2; D013859:Thiocarbamates; D014409:Tumor Necrosis Factor-alpha; C066229:prolinedithiocarbamate; D011392:Proline