title: The Rho GTPase Rac1 is required for recycling endosome-mediated secretion of TNF in macrophages.
abstract: Rho GTPases are required for many cellular events such as adhesion, motility, and membrane trafficking. Here we show that in macrophages, the Rho GTPases Rac1 and Cdc42 are involved in lamellipodia and filopodia formation, respectively, and that both of these Rho GTPases are essential for the efficient surface delivery of tumor necrosis factor (TNF) to the plasma membrane following TLR4 stimulation. We have previously demonstrated intracellular trafficking of TNF via recycling endosomes in lipopolysaccharide (LPS)-activated macrophages. Here, we further define a specific role for Rac1 in intracellular TNF trafficking, demonstrating impairment in TNF release following TLR4 stimulation in the presence of a Rac inhibitor, in cells expressing a dominant negative (DN) form of Rac1, and following small interfering RNA (siRNA) knockdown of Rac1. Rac1 activity was required for TNF trafficking but not for TLR4 signaling following LPS stimulation. Reduced TNF secretion was due to a defect in Rac1 activity, but not of the closely related Rho GTPase Rac2, demonstrated by the additional use of macrophages derived from Rac2-deficient mice. Labeling recycling endosomes by the uptake of fluorescent transferrin enabled us to show that Rac1 was required for the final stages of TNF trafficking and delivery from recycling endosomes to the plasma membrane. Thus, actin remodeling by the Rho GTPase Rac1 is required for TNF cell surface delivery and release from macrophages.
authors.affiliation: Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland, Australia.
authors.forename: Amanda C
authors.initials: AC
authors.lastname: Stanley
authors.affiliation: Institute of Medical Biology, Immunos, Singapore.
authors.forename: Colin X
authors.initials: CX
authors.lastname: Wong
authors.affiliation: College of Life and Environmental Sciences, University of Exeter, Exeter, UK.
authors.forename: Massimo
authors.initials: M
authors.lastname: Micaroni
authors.affiliation: Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland, Australia.
authors.forename: Juliana
authors.initials: J
authors.lastname: Venturato
authors.affiliation: Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland, Australia.
authors.forename: Tatiana
authors.initials: T
authors.lastname: Khromykh
authors.affiliation: Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland, Australia.
authors.forename: Jennifer L
authors.initials: JL
authors.lastname: Stow
authors.affiliation: Pulmonary Research Group, Department of Medicine, University of Alberta, Edmonton, Alberta, Canada.
authors.forename: Paige
authors.initials: P
authors.lastname: Lacy
mesh_terms: D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D004705:Endocytosis / Q000187:drug effects; D011992:Endosomes / Q000187:drug effects / Q000378:metabolism*; D017353:Gene Deletion; D055785:Gene Knockdown Techniques; D049452:Green Fluorescent Proteins / Q000378:metabolism; D008264:Macrophages / Q000187:drug effects / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D021381:Protein Transport / Q000187:drug effects; D011554:Pseudopodia / Q000187:drug effects / Q000378:metabolism; D011753:Pyrones / Q000494:pharmacology; D011804:Quinolines / Q000494:pharmacology; D034741:RNA, Small Interfering / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism*; D020764:cdc42 GTP-Binding Protein / Q000378:metabolism; D020744:rac GTP-Binding Proteins / Q000378:metabolism; D020830:rac1 GTP-Binding Protein / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000097766:RAC2 GTP-Binding Protein
chemical_list: D049452:Green Fluorescent Proteins; D011753:Pyrones; D011804:Quinolines; D034741:RNA, Small Interfering; D011993:Recombinant Fusion Proteins; D014409:Tumor Necrosis Factor-alpha; D020764:cdc42 GTP-Binding Protein; D020744:rac GTP-Binding Proteins; D020830:rac1 GTP-Binding Protein; D000097766:RAC2 GTP-Binding Protein; C506907:EHT 1864