Molecular cloning and functional characterization of TRAF6 and TAK1 in rainbow trout, Oncorhynchus mykiss

Abstract

TRAF6 and TAK1 are known to play important roles in vertebrate innate immunity as molecular bridge, linking upstream toll-like receptors (TLRs) with the downstream MAPK and NF-kappa Beta signalling pathways. However, their roles in TLR signalling pathway have yet to be fully described in fish. Here we identified genes encoding TRAF6 (OmTRAF6) and TAK1 (OmTAK1) from rainbow trout, Oncorhynchus mykiss, and examined their roles during pathogenic infections. The deduced amino acid sequences of OmTRAF6 and OmTAK1 contained the characteristic domains conserved in the TRAF and TAK1 families, respectively (OmTRAF6: RING, two TRAF-type zinc fingers, CCR and MATH domains; OmTAK1: STKc and CCR domains). In RTH-149 cells, the expression of OmTRAF6 and OmTAK1 was increased by stimulation with Edwardsieila tarda and LPS. Silencing of OmTRAF6 and OmTAK1 in RTH-149 cells negatively regulated the LPS-induced phosphorylation of p38 MAPK and JNK. TAK1 inhibitor (5z)-7-Oxozeaenol significantly decreased the LPS-induced activation of NF-kappa B in RTH-149 cells. In addition, silencing of OmTRAF6 and OmTAK1 significantly decreased the expression of MAPK.s and NF-kappa Beta downstream target genes induced by LPS in RTH-149 cells. These findings suggest that OmTRAF6 and OmTAK1 might function like those of mammals to regulate bacteria-triggered signalling pathway in fish.