Abstract
Phagocytosis is accompanied by the production of superoxide by the NADPH oxidase complex, for which GTP-bound Rac is essential. We wanted to determine whether Rho is also involved in the production of superoxide during phagocytosis. Inhibition of Rho by Tat-C3 exoenzyme (Tat-C3) blocked superoxide formation and curtailed the phagocytosis of serum- (SOZ), C3bi- (COZ), and IgG-opsonized zymosan (IOZ) particles. Tat-C3 did not affect superoxide formation in response to phorbol myristate acetate (PMA), formyl Met-Leu-Phe (fMLP), or macrophage colony-stimulating factor (M-CSF). Superoxide formation was also reduced in J774 cells transfected with a cDNA expressing dominant-negative form of RhoA (N19RhoA). However, purified prenylated recombinant RhoA did not activate NADPH oxidase in vitro, suggesting that Rho does not interact directly with NADPH oxidase. Tat-C3 inhibited the activity of RhoA, but did not affect that of Rac in vitro or in vivo. It also inhibited the phosphorylation of p47(PHOX), one of the cytosolic components of NADPH oxidase. Taken together, these results suggest that Rho plays an important role in superoxide formation during phagocytosis of SOZ, COZ, and IOZ via phosphorylation of p47(PHOX).
Highlights
Phagocytosis is accompanied by the production of superoxide by the NADPH oxidase complex, for which GTP-bound Rac is essential
Transduction of Tat-C3 into Cells—In an attempt to establish whether Rho is involved in phagocytosis and superoxide formation in macrophages, J774 cells were treated with TatC3, which is readily transduced into cells and inhibits Rho
Rho Is Involved in Superoxide Formation by Macrophages— In the present study, we found that Tat-C3, virtually abolished opsonized zymosan particles
Summary
Phagocytosis is accompanied by the production of superoxide by the NADPH oxidase complex, for which GTP-bound Rac is essential. Tat-C3 inhibited the activity of RhoA, but did not affect that of Rac in vitro or in vivo It inhibited the phosphorylation of p47PHOX, one of the cytosolic components of NADPH oxidase. Taken together, these results suggest that Rho plays an important role in superoxide formation during phagocytosis of SOZ, COZ, and IOZ via phosphorylation of p47PHOX. Rac is a critical regulator of NADPH oxidase in response to formyl methionylleucyl-phenylalanine (fMLP) and antibody-coated sheep red blood cells (SRBC) (IgG-SRBC), whereas it is not responsible for superoxide formation induced by serum-opsonized zymosans (SOZ) [18]. Another Ras-related GTPase, is rapidly and transiently activated upon stimulation by fMLP, platelet-
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