Implication of the small GTPase Rac1 in the generation of reactive oxygen species in response to beta-amyloid in C6 astroglioma cells.
Exogenous application of beta-amyloid (Abeta(25-35), a fragment of Abeta(1-42)) significantly elevated levels of reactive oxygen species (ROS) in C6 astroglioma cells, as measured by confocal microscopic analysis of H(2)O(2)-sensitive 2',7'-dichlorofluorescin fluorescence. Subsequent characterization of the signalling pathway revealed that expression of RacN17, a dominant-negative Rac1 mutant, completely blocked Abeta(25-35)-induced generation of ROS, which is indicative of the crucial role played by Rac GTPase in this process. To better understand the downstream mediators affected by Rac, we assessed the degree to which inhibition of cytosolic phospholipase A(2) (cPLA(2)) and 5-lipoxygenase (5-LO) contributed to the response and found that inhibition of either enzyme completely blocked Abeta(25-35)-induced ROS generation, indicating its dependence on arachidonic acid synthesis and metabolism to leukotrienes (e.g. leukotriene B(4)). Consistent with those findings, Abeta(25-35) Rac-dependently stimulated translocation of 5-LO to the nuclear envelope and increased intracellular levels of leukotriene B(4), while exogenous application of leukotriene B(4) increased intracellular H(2)O(2) via BLT, its cell-surface receptor. In addition to the aforementioned downstream mediators, inhibition of phosphoinositide 3-kinase (PI 3-kinase), an enzyme situated upstream of Rac, also completely blocked Abeta(25-35)-induced H(2)O(2) generation. Our findings thus demonstrate that PI 3-kinase, Rac, cPLA(2) and 5-LO are all essential components of the beta-amyloid signaling cascade leading to generation of ROS.
Lee
Mina
M
Graduate School of Biotechnology, Korea University, 5-1 Anam-dong, Sungbuk-gu, Seoul, 136-701, Korea.
You
Hye-Jin
HJ
Cho
Sung-Hoon
SH
Woo
Chang-Hoon
CH
Yoo
Min-Hyuk
MH
Joe
Eun-Hye
EH
Kim
Jae-Hong
JH
D016229:Amyloid beta-Peptides / Q000378:metabolism*; D001094:Arachidonate 5-Lipoxygenase / Q000378:metabolism; D001254:Astrocytoma / Q000378:metabolism; D015153:Blotting, Western; D001932:Brain Neoplasms / Q000378:metabolism; D003600:Cytosol / Q000201:enzymology; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D007975:Leukotriene B4 / Q000494:pharmacology; D019255:NADPH Oxidases / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010741:Phospholipases A / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D017382:Reactive Oxygen Species; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D013347:Subcellular Fractions / Q000378:metabolism; D013997:Time Factors; D014162:Transfection; D014407:Tumor Cells, Cultured; D020830:rac1 GTP-Binding Protein / Q000737:chemistry* / Q000378:metabolism*
D016229:Amyloid beta-Peptides; D017382:Reactive Oxygen Species; D007975:Leukotriene B4; D006861:Hydrogen Peroxide; D001094:Arachidonate 5-Lipoxygenase; D019255:NADPH Oxidases; D019869:Phosphatidylinositol 3-Kinases; D010741:Phospholipases A; D020830:rac1 GTP-Binding Protein