Decreased superoxide production in macrophages of long-lived p66Shc knock-out mice.
A decrease in reactive oxygen species (ROS) production has been associated with extended life span in animal models of longevity. Mice deficient in the p66Shc gene are long-lived, and their cells are both resistant to oxidative stress and produce less ROS. Our microarray analysis of p66Shc(-/-) mouse tissues showed alterations in transcripts involved in heme and superoxide production and insulin signaling. Thus, we carried out analysis of ROS production by NADPH oxidase (PHOX) in macrophages of control and p66Shc knock-out mice. p66Shc(-/-) mice had a 40% reduction in PHOX-dependent superoxide production. To confirm whether the defect in superoxide production was a direct consequence of p66Shc deficiency, p66Shc was knocked down with siRNA in the macrophage cell line RAW264, and a 30% defect in superoxide generation was observed. The pathway of PHOX-dependent superoxide generation was investigated. PHOX protein levels were not decreased in mutant macrophages; however, the rate and extent of phosphorylation of p47phox was decreased in mutants, as was membrane translocation of the complex. Consistently, phosphorylation of protein kinase Cdelta, Akt, and ERK (the kinases responsible for phosphorylation of p47phox) was decreased. Thus, p66Shc deficiency causes a defect in activation of the PHOX complex that results in decreased superoxide production. p66Shc-deficient mice have recently been observed to be resistant to atherosclerosis and to oxidant injury in kidney and brain. Because phagocyte-derived superoxide is often a component of oxidant injury and inflammation, we suggest that the decreased superoxide production by PHOX in p66Shc-deficient mice could contribute significantly to their relative protection from oxidant injury and consequent longevity.
Tomilov
Alexey A
AA
Department of Molecular Biosciences, University of California, Davis, California 95616, USA.
Bicocca
Vincent
V
Schoenfeld
Robert A
RA
Giorgio
Marco
M
Migliaccio
Enrica
E
Ramsey
Jon J
JJ
Hagopian
Kevork
K
Pelicci
Pier Giuseppe
PG
Cortopassi
Gino A
GA
D000818:Animals; D002460:Cell Line; D004789:Enzyme Activation / Q000235:genetics; D048049:Extracellular Signal-Regulated MAP Kinases / Q000235:genetics / Q000378:metabolism; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation / Q000235:genetics; D055785:Gene Knockdown Techniques; D008136:Longevity; D051379:Mice; D018345:Mice, Knockout; D019255:NADPH Oxidases / Q000235:genetics / Q000378:metabolism*; D020411:Oligonucleotide Array Sequence Analysis; D018384:Oxidative Stress; D051745:Protein Kinase C-delta / Q000235:genetics / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt / Q000235:genetics / Q000378:metabolism; D055765:Shc Signaling Adaptor Proteins; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1; D013481:Superoxides / Q000378:metabolism*
D055765:Shc Signaling Adaptor Proteins; C528034:Shc1 protein, mouse; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1; D013481:Superoxides; D019255:NADPH Oxidases; C060505:neutrophil cytosolic factor 1; D051057:Proto-Oncogene Proteins c-akt; D051745:Protein Kinase C-delta; D048049:Extracellular Signal-Regulated MAP Kinases