title: Small GTPases Rap1 and RhoA regulate superoxide formation by Rac1 GTPases activation during the phagocytosis of IgG-opsonized zymosans in macrophages.
abstract: Phagocytic NADPH oxidase plays a critical role in superoxide generation in macrophage cells. Small GTPases, including Rac1 and Rac2, have been implicated in the regulation of NADPH oxidase activity. Rap1, which has no effect in a cell-free system of oxidase activation, recently has been proven to colocalize with cytochrome b(558). In addition, neutrophils from rap1A(-/-) mice reduce fMLP-stimulated superoxide production. Here, we tried to determine whether Rap1 also plays a role in the production of superoxide. IgG-opsonized zymosan (IOZ) particles treatment induced Rap1 activation and superoxide generation. Knock-down of Rap1 by si-Rap1 suppressed IOZ-induced superoxide formation. Sh-RhoA also reduced superoxide levels, but 8CPT-2Me-cAMP, an activator of Epac1 (a guanine nucleotide exchange factor (GEF) of Rap1), could recover the levels to the control value. When cells were stimulated by IOZ, Rap1 and Rac1 were translocated to the membrane, and then interacted with p22(phox). 8CPT-2Me-cAMP rescued sh-RhoA-induced reduction of the interaction between Rac1 and p22(phox), and enhanced lysophosphatidic acid (LPA)-induced increase of their interaction. Moreover, Rac1 activity was increased by both LPA and 8CPT-2Me-cAMP when treated with IOZ particles. Si-Vav2 impaired GTP-Rac1 levels in response to 8CPT-2Me-cAMP/IOZ. Phosphorylation of RhoA activates Rac1 in response to IOZ by the enhanced binding of phospho-RhoA to RhoGDI, leading to the release of Rac1 from the Rac1-RhoGDI complex. In conclusion, IOZ treatment induces Rap1 activation and phosphorylation of RhoA, which in turn cause Rac1 activation and promote Rac1 translocation to the membrane leading to binding with p22(phox) that activates NADPH oxidase and produces superoxide.
authors.affiliation: Department of Biochemistry, Hallym University College of Medicine, Chuncheon, Kangwon-Do, 200-702, Republic of Korea.
authors.forename: Yi
authors.initials: Y
authors.lastname: Li
authors.forename: Jae-Gyu
authors.initials: JG
authors.lastname: Kim
authors.forename: Hee-Jun
authors.initials: HJ
authors.lastname: Kim
authors.forename: Mi-Young
authors.initials: MY
authors.lastname: Moon
authors.forename: Jae-Yong
authors.initials: JY
authors.lastname: Lee
authors.forename: Jaebong
authors.initials: J
authors.lastname: Kim
authors.forename: Sung-Chan
authors.initials: SC
authors.lastname: Kim
authors.forename: Dong-Keun
authors.initials: DK
authors.lastname: Song
authors.forename: Yong-Sun
authors.initials: YS
authors.lastname: Kim
authors.forename: Jae-Bong
authors.initials: JB
authors.lastname: Park
mesh_terms: D000818:Animals; D002460:Cell Line; D004789:Enzyme Activation; D007074:Immunoglobulin G / Q000378:metabolism*; D047468:Immunoprecipitation; D008264:Macrophages / Q000378:metabolism*; D051379:Mice; D020559:Monomeric GTP-Binding Proteins / Q000502:physiology*; D010587:Phagocytosis; D013481:Superoxides / Q000378:metabolism*; D015054:Zymosan / Q000378:metabolism*; D020830:rac1 GTP-Binding Protein / Q000378:metabolism*; D020827:rap1 GTP-Binding Proteins / Q000502:physiology*; D020742:rhoA GTP-Binding Protein / Q000502:physiology*
chemical_list: D007074:Immunoglobulin G; D013481:Superoxides; D015054:Zymosan; D020559:Monomeric GTP-Binding Proteins; D020830:rac1 GTP-Binding Protein; D020827:rap1 GTP-Binding Proteins; D020742:rhoA GTP-Binding Protein