Aim: 

Reactive Oxygen Species (ROS) are signal molecules produced by different cell types upon stimulation with various growth factors. The aim of the present study was to analyze the molecular mechanisms involved in ROS induction by PDGF receptor/Ras/ERK1/2 signalling.

Methods: 

Human neuroblastoma cells, SK-N-BE, and human primary fibroblasts were used. ROS levels were measured using the oxidation-sensitive fluorescent probe 2',7'-dichlorofluorescein diacetate (DCFH-DA). NADPH oxidase activation was evaluated by Western blotting as membrane translocation of Rac and p67^phox. Subcellular localization and interaction of PDGF receptor, HaRas and the catalytic subunit of NADPH oxidase, gp91^phox, were assessed by confocal immunofluorescence microscopy and immunoprecipitation-Western blotting assay.

Results: 

Stimulation of both cell systems with PDGF_BB strongly stimulates ROS production by activating membrane NADPH oxidase complex. Using the specific inhibitor of the NADPH oxidase, 4-(2-aminoethyl) benzenesulfonylfluoride (AEBSF), we found that NADPH oxidase-derived ROS sustain thyrosine phosphorylation of PDGF receptor and ERK1/2 activation. We also found that gp91^phox and PDGF receptor, upon stimulation with PDGF_BB, form a stable membrane complex. This complex is located in the lipid compartment of the plasma membrane (lipid rafts) and contains also Ha-Ras. Disruption of lipid rafts with 2-hydroxypropyl-b-cyclodextrin, which depletes cell membrane of cholesterol, blocks PDGF signal transmission to ERK1/2.

Conclusions: 

These data demonstrate that ROS produced by PDGF receptor-activated NADPH oxidase amplify downstream signalling through a loop involving the formation, in the lipid rafts, of an active complex between the PDGF receptor and gp91^phox.