HaRas activates the NADPH oxidase complex in human neuroblastoma cells via extracellular signal-regulated kinase 1/2 pathway.
In this study we have investigated the effects of the small GTP-binding-protein Ras on the redox signalling of the human neuroblastoma cell line, SK-N-BE stably transfected with HaRas(Val12). The levels of reactive oxygen species (ROS) and superoxide anions were significantly higher in HaRas(Val12) expressing (SK-HaRas) cells than in control cells. The treatment of cells with 4-(2-aminoethyl) benzenesulfonylfluoride, a specific inhibitor of the membrane superoxide generating system NADPH oxidase, suppressed the rise in ROS and significantly reduced superoxide levels produced by SK-HaRas cells. Moreover, HaRas(Val12) induced the translocation of the cytosolic components of the NADPH oxidase complex p67(phox) and Rac to the plasma membrane. These effects depended on the mitogen-activated protein kinase kinase/extracellular signal-regulated kinase (MEK/ERK1/2) pathway, as the specific MEK inhibitor, PD98059, prevented HaRas-mediated increase in ROS and superoxide anions. In contrast, the specific phosphoinositide 3-kinase (PI3K) inhibitors LY294002 and wortmannin were unable to reverse the effects of HaRas(Val12). Moreover, cholinergic stimulation of neuroblastoma cells by carbachol, which activated endogenous Ras/ERK1/2, induced a significant increase in ROS levels and elicited membrane translocation of p67(phox) and Rac. ROS generation induced by carbachol required the activation of ERK1/2 and PI3K. Hence, these data indicate that HaRas-induced ERK1/2 signalling selectively activates NADPH oxidase system in neuroblastoma cells.
Dipartimento di Neuroscienze e di Scienze del Comportamento, Sezione di Fisiologia, Università Federico II di Napoli, 80131 Napoli, Italy.
Rosalba
R
Serù
Paolo
P
Mondola
Simona
S
Damiano
Silvia
S
Svegliati
Savina
S
Agnese
Enrico V
EV
Avvedimento
Mariarosaria
M
Santillo
D019943:Amino Acid Substitution; D002217:Carbachol / Q000494:pharmacology; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000201:enzymology; D018679:Cholinergic Agonists / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects / Q000235:genetics; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism*; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism*; D019255:NADPH Oxidases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D009447:Neuroblastoma / Q000188:drug therapy / Q000235:genetics / Q000378:metabolism*; D010084:Oxidation-Reduction; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D021381:Protein Transport / Q000235:genetics; D017382:Reactive Oxygen Species / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D013481:Superoxides / Q000378:metabolism; D014162:Transfection; D020744:rac GTP-Binding Proteins / Q000378:metabolism; D018631:ras Proteins / Q000235:genetics / Q000378:metabolism*
D018679:Cholinergic Agonists; D004791:Enzyme Inhibitors; D010750:Phosphoproteins; D017382:Reactive Oxygen Species; C079789:neutrophil cytosol factor 67K; D013481:Superoxides; D002217:Carbachol; D019255:NADPH Oxidases; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020744:rac GTP-Binding Proteins; D018631:ras Proteins