Modulation of Nod2-dependent NF-kappaB signaling by the actin cytoskeleton.
Actin disruption by CytochalasinD (CytD) and LatrunculinB (LatB) induced NF-kappaB activation in myelomonocytic and intestinal epithelial cells. In an attempt to elucidate the mechanism by which actin disruption induced IKK activation, we studied the human Nod2 protein, which was able to induce NF-kappaB activation and whose expression was restricted to myelomonocytic and intestinal epithelial cells. Nod2 is thought to play key roles in pathogen defence through sensing bacteria and generating an inflammatory immune response. We showed that actin disruption by CytD significantly and specifically increased Nod2-mediated NF-kappaB signaling. Nod2 was fully partitioned in the Triton-X-100-insoluble fraction but translocated into the soluble fraction after CytD treatment, demonstrating that the presence of Nod2 in the detergent-insoluble pellet was specific to actin cytoskeleton. Confocal analysis also revealed a Nod2 colocalization with membrane-associated F-actin. Colocalization and co-immunoprecipitation assays with endogenous Rac1 have shown that Nod2 associated with activated Rac1 in membrane ruffles through both its N-terminal caspase recruitment domains (CARD) and C-terminal leucine-rich repeats (LRRs). Membrane ruffle disruption by a Rac1 dominant negative form primed Nod2-dependent NF-kappaB signaling. The recruitment of Nod2 in Rac-induced dynamic cytoskeletal structures could be a strategy to both repress the Nod2-dependent NF-kappaB signaling in unstimulated cells and rapidly mobilize Nod2 during bacterial infection.
Legrand-Poels
Sylvie
S
Laboratory of Virology and Immunology, CBIG-GIGA, University of Liège, Liège, Belgium. S.Legrand@ulg.ac.be
Kustermans
Gaelle
G
Bex
Françoise
F
Kremmer
Elisabeth
E
Kufer
Thomas A
TA
Piette
Jacques
J
D000199:Actins / Q000378:metabolism*; D000818:Animals; D018929:Cell Culture Techniques; D002457:Cell Extracts / Q000737:chemistry; D002460:Cell Line; D002478:Cells, Cultured; D003599:Cytoskeleton / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay; D016650:Fluorescein-5-isothiocyanate; D005456:Fluorescent Dyes; D017930:Genes, Reporter; D019073:HT29 Cells; D006801:Humans; D015151:Immunoblotting; D047468:Immunoprecipitation; D008156:Luciferases / Q000032:analysis / Q000378:metabolism; D008264:Macrophages / Q000166:cytology / Q000378:metabolism; D051379:Mice; D008856:Microscopy, Fluorescence; D016328:NF-kappa B / Q000378:metabolism*; D053473:Nod2 Signaling Adaptor Protein / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction; D014162:Transfection
D000199:Actins; D002457:Cell Extracts; D005456:Fluorescent Dyes; D016328:NF-kappa B; C429018:NOD2 protein, human; D053473:Nod2 Signaling Adaptor Protein; C495945:Nod2 protein, mouse; D008156:Luciferases; D016650:Fluorescein-5-isothiocyanate