Themis2/ICB1 is a signaling scaffold that selectively regulates macrophage Toll-like receptor signaling and cytokine production.
BACKGROUND
Thymocyte expressed molecule involved in selection 1 (Themis1, SwissProt accession number Q8BGW0) is the recently characterised founder member of a novel family of proteins. A second member of this family, Themis2 (Q91YX0), also known as ICB1 (Induced on contact with basement membrane 1), remains unreported at the protein level despite microarray and EST databases reporting Themis2 mRNA expression in B cells and macrophages.


RESULTS
Here we characterise Themis2 protein for the first time and show that it acts as a macrophage signalling scaffold, exerting a receptor-, mediator- and signalling pathway-specific effect on TLR responses in RAW 264.7 macrophages. Themis2 over-expression enhanced the LPS-induced production of TNF but not IL-6 or Cox-2, nor TNF production induced by ligands for TLR2 (PAM3) or TLR3 (poly IratioC). Moreover, LPS-induced activation of the MAP kinases ERK and p38 was enhanced in cells over-expressing Themis2 whereas the activation of JNK, IRF3 or NF-kappaB p65, was unaffected. Depletion of Themis2 protein by RNA inteference inhibited LPS-induced TNF production in primary human macrophages demonstrating a requirement for Themis2 in this event. Themis2 was inducibly tyrosine phosphorylated upon LPS challenge and interacted with Lyn kinase (P25911), the Rho guanine nucleotide exchange factor, Vav (P27870), and the adaptor protein Grb2 (Q60631). Mutation of either tyrosine 660 or a proline-rich sequence (PPPRPPK) simultaneously interrupted this complex and reduced by approximately 50% the capacity of Themis2 to promote LPS-induced TNF production. Finally, Themis2 protein expression was induced during macrophage development from murine bone marrow precursors and was regulated by inflammatory stimuli both in vitro and in vivo.


CONCLUSIONS
We hypothesise that Themis2 may constitute a novel, physiological control point in macrophage inflammatory responses.
Peirce
Matthew J
MJ
Kennedy Institute of Rheumatology Division, Imperial College London, London, UK. m.pierce@imperial.ac.uk
Brook
Matthew
M
Morrice
Nicholas
N
Snelgrove
Robert
R
Begum
Shajna
S
Lanfrancotti
Alessandra
A
Notley
Clare
C
Hussell
Tracy
T
Cope
Andrew P
AP
Wait
Robin
R
D000818:Animals; D002460:Cell Line; D016207:Cytokines / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D051380:GRB2 Adaptor Protein / Q000235:genetics / Q000378:metabolism; D047468:Immunoprecipitation; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism*; D008070:Lipopolysaccharides / Q000494:pharmacology; D051379:Mice; D008810:Mice, Inbred C57BL; D011485:Protein Binding; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D051567:Proto-Oncogene Proteins c-vav / Q000235:genetics / Q000378:metabolism; D034622:RNA Interference; D015398:Signal Transduction / Q000187:drug effects / Q000235:genetics; D053719:Tandem Mass Spectrometry; D051197:Toll-Like Receptor 4 / Q000378:metabolism; D051193:Toll-Like Receptors / Q000235:genetics / Q000378:metabolism*; D014409:Tumor Necrosis Factor-alpha
D016207:Cytokines; D051380:GRB2 Adaptor Protein; C495949:Grb2 protein, mouse; D047908:Intracellular Signaling Peptides and Proteins; D008070:Lipopolysaccharides; D011506:Proteins; D051567:Proto-Oncogene Proteins c-vav; C575530:Themis2 protein, mouse; D051197:Toll-Like Receptor 4; D051193:Toll-Like Receptors; D014409:Tumor Necrosis Factor-alpha