Salmonid Tollip and MyD88 factors can functionally replace their mammalian orthologues in TLR-mediated trout SAA promoter activation.
Many functional details of the piscine Toll-like receptor (TLR) signal-mediated activation of immune defense are still elusive. We used an established reconstitution system of mammalian TLR signaling to examine if this system would allow for pathogen-dependent promoter activation of the serum amyloid A (SAA)-encoding gene from rainbow trout (Oncorhynchus mykiss) and if the key mediators MyD88 and Tollip from trout can functionally substitute for their mammalian orthologues. Cells of the established human embryonic kidney line HEK-293 were transiently co-transfected with vectors expressing bovine TLR2 or TLR4 factors and a reporter gene driven by the promoter of the trout SAA gene. Escherichia coli stimulation increased reporter gene expression more than 3-fold. Deletion series and point mutations identified in the proximal SAA promoter a composite overlapping binding site for NF-κB and CEBP factors as crucial for promoter activation. Overexpression of NF-κB p65, but not of p50 or different members of the CEBP factor family proved this factor as an essential driver for SAA expression. Overexpression of a transdominant-negative mutant of the trout MyD88 factor reduced TLR-mediated SAA promoter activation confirming functional conservation of its TIR domain. Overexpression of the Tollip factor from trout also quenched TLR-mediated NF-κB and TLR4-mediated SAA promoter activation. The MyD88 mutant and Tollip expression studies confirm the functional homology of both piscine factors and their mammalian counterparts. We provide for the first time evidence that also the Tollip-mediated negative loop of TLR signaling may be conserved in non-mammalian organisms.
Rebl
Alexander
A
Leibniz Institute for Farm Animal Biology (FBN), Molecular Biology Research Unit, Wilhelm-Stahl-Allee 2, 18196 Dummerstorf, Germany.
Rebl
Henrike
H
Liu
Shuzhen
S
Goldammer
Tom
T
Seyfert
Hans-Martin
HM
D000818:Animals; D002417:Cattle; D017124:Conserved Sequence; D004926:Escherichia coli / Q000276:immunology; D004927:Escherichia coli Infections / Q000276:immunology; D057809:HEK293 Cells; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000276:immunology; D053594:Myeloid Differentiation Factor 88 / Q000235:genetics / Q000276:immunology; D011401:Promoter Regions, Genetic; D012487:Salmonidae / Q000235:genetics* / Q000276:immunology; D000685:Serum Amyloid A Protein / Q000235:genetics* / Q000378:metabolism; D015398:Signal Transduction; D015854:Up-Regulation
D047908:Intracellular Signaling Peptides and Proteins; D053594:Myeloid Differentiation Factor 88; D000685:Serum Amyloid A Protein