Expression and translocation of Rac2 in eosinophils during superoxide generation.
Eosinophils induce tissue injury by releasing granule-associated cytotoxic proteins, lipid mediators and superoxide anions in response to appropriate stimuli. Superoxide generation associated with respiratory burst is largely dependent on the assembly of the NADPH oxidase complex in the membrane, consisting of membrane-bound cytochrome b558 and translocated p47phox and p67phox. The activation of this complex is critically dependent on the translocation of GTP-bound Rac1, or its homologue Rac2, from the cytosol to the membrane in neutrophils. Rac expression has not yet been fully characterized in eosinophils. We proposed that eosinophils translate and express Rac2 and its GDP-dissociation inhibitor, RhoGDI. Furthermore, we hypothesized that Rac2 translocates along with p47phox and p67phox proteins from the cytosol to the plasma membrane during respiratory burst. By reverse transcription-polymerase chain reaction analysis and sequencing of the amplified product, guinea-pig eosinophils were found to express Rac2 mRNA, exhibiting 93% homology with the human Rac2 sequence. Rac1 mRNA was also detected in eosinophils but not its translated product. In contrast, Rac2 protein expression was detected using a specific antibody. In subcellular fractions, Rac2 was found to translocate, along with p47phox and p67phox, from cytosol to plasma membrane-associated fractions following phorbol myristate acetate stimulation, while RhoGDI remained within cytosolic fractions. These findings suggest that Rac2 is preferentially expressed and activated in eosinophils, and is likely to be a crucial regulator of the release of reactive oxygen species from these cells during inflammatory reactions.
Lacy
P
P
Pulmonary Research Group, Department of Medicine, University of Alberta, Edmonton, AB, Canada.
Mahmudi-Azer
S
S
Bablitz
B
B
Gilchrist
M
M
Fitzharris
P
P
Cheng
D
D
Man
S F
SF
Bokoch
G M
GM
Moqbel
R
R
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D002478:Cells, Cultured; D004804:Eosinophils / Q000378:metabolism*; D006168:Guinea Pigs; D006801:Humans; D008297:Male; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012689:Sequence Homology, Nucleic Acid; D013481:Superoxides / Q000378:metabolism*; D020744:rac GTP-Binding Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*
D012333:RNA, Messenger; D013481:Superoxides; D020744:rac GTP-Binding Proteins