title: Toll-like receptor 4 signalling is specifically TGF-beta-activated kinase 1 independent in synovial fibroblasts.
abstract: OBJECTIVE
  Activated synovial fibroblasts are key players in the pathogenesis of RA by driving inflammation and joint destruction. Numerous molecules including cytokines and Toll-like receptor (TLR) ligands induce pro-inflammatory signalling and gene expression through a hierarchical network of kinases. Upstream mitogen-activated protein kinase kinase kinases (MAP3Ks) represent an attractive target for RA treatment. In this study, we sought to determine the role of the MAP3K TGF-β-activated kinase 1 (TAK1) in cytokine and TLR-mediated signalling.
  
  
  METHODS
  TAK1 activity was inhibited using either a small molecule inhibitor or lentivirally overexpressed kinase-inactive TAK1-K63W mutant in murine embryonic and human dermal and synovial fibroblasts. Fibroblasts were stimulated with IL-1, TNF, TLR2 or TLR4 agonists and responses were evaluated using transcriptional reporters, western blotting and analysis of gene expression of collagenases (MMP3 and MMP13), cytokines (IL-1β and IL-6) and chemokines (IL-8 and MCP-1).
  
  
  RESULTS
  TAK1 inhibition abrogated cytokine- and TLR-induced nuclear factor-κB (NF-κB) and Saa3-promoter reporter activation in murine and human dermal fibroblasts. In synovial fibroblasts, TAK1 regulated IL-1 and TNF-mediated NF-κB, but not Saa3-promoter reporter activation. Inducible mRNA expression of cytokines, collagenases and chemokines, except MCP-1, was TAK1 dependent for IL-1, TNF and TLR2 signalling. Unexpectedly, TLR4-mediated NF-κB reporter activation and inducible mRNA expression was fully TAK1 independent. Accordingly, NF-κB p65 and p38 MAPK phosphorylation was unaffected by TAK1 inhibition.
  
  
  CONCLUSIONS
  In general, TAK1 crucially regulates IL-1 and TNF signalling in fibroblasts. Interestingly, TLR4 signalling is specifically TAK1 independent in synovial fibroblasts. Consequently, therapeutic TAK1 inhibition in arthropathies may not dampen the damage-associated molecular pattern-mediated TLR4 activation of synovial fibroblasts.
authors.affiliation: Department of Rheumatology, Rheumatology Research and Advanced Therapeutics, Radboud University Nijmegen Medical Centre, PO Box 9101, 6500 HB Nijmegen, The Netherlands. a.vandeloo@reuma.umcn.nl.
authors.forename: Jeroen
authors.initials: J
authors.lastname: Geurts
authors.forename: Ben T
authors.initials: BT
authors.lastname: van den Brand
authors.forename: Alexander
authors.initials: A
authors.lastname: Wolf
authors.forename: Shahla
authors.initials: S
authors.lastname: Abdollahi-Roodsaz
authors.forename: Onno J
authors.initials: OJ
authors.lastname: Arntz
authors.forename: Michael
authors.initials: M
authors.lastname: Kracht
authors.forename: Wim B
authors.initials: WB
authors.lastname: van den Berg
authors.forename: Fons A J
authors.initials: FA
authors.lastname: van de Loo
mesh_terms: D000704:Analysis of Variance; D000818:Animals; D001172:Arthritis, Rheumatoid / Q000378:metabolism; D015153:Blotting, Western; D002478:Cells, Cultured; D004789:Enzyme Activation; D005260:Female; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism*; D020930:MAP Kinase Kinase Kinases / Q000378:metabolism*; D008297:Male; D051379:Mice; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D015398:Signal Transduction; D013583:Synovial Membrane / Q000166:cytology* / Q000378:metabolism*; D051197:Toll-Like Receptor 4 / Q000032:analysis / Q000378:metabolism*; D053773:Transforming Growth Factor beta1 / Q000378:metabolism*; D000099151:MAP Kinase Kinase Kinase 7
chemical_list: D020930:MAP Kinase Kinase Kinases; D019950:Mitogen-Activated Protein Kinase 1; D051197:Toll-Like Receptor 4; D053773:Transforming Growth Factor beta1; D000099151:MAP Kinase Kinase Kinase 7; C535148:Mapk1 protein, mouse