Hydrogen peroxide activates p70(S6k) signaling pathway

We investigated a possible role of reactive oxygen species (ROS) in p70(S6k) activation, which plays an important role in the progression of cells from G₀/G₁ to S phase of the cell cycle by translational up- regulation of a family of mRNA transcripts that encode for components of the protein synthetic machinery. Treatment of mouse epidermal cell JB6 with H₂O₂ generated extracellularly by glucose/glucose oxidase led to the activation of p70(S6k) and p90(Rsk) and to phosphorylation of p42(MAPK)/p44(MAPK). The activation of p70(S6k) and p90(Rsk) was dose- dependent and transient, maximal activities being in extracts treated for 15 and 30 min, respectively. Further characterization of ROS-induced activation of p70(s6k) using specific inhibitors for p70(S6k) signaling pathway, rapamycin, and wortmannin revealed that ROS acted upstream of the rapamycin- sensitive component FRAP/RAFT and wortmannin-sensitive component phosphatidylinositol 3-kinase, because both inhibitors caused the inhibition of ROS-induced p70(S6k) activity. In addition, Ca²⁺ chelation also inhibited ROS-induced activation of p70(S6k), indicating that Ca²⁺ is a mediator of p70(S6k) activation by ROS. However, down-regulation of 12-O- tetradecanoylphorbol-13-acetate (TPA)-responsive protein kinase C (PKC) by chronic pretreatment with TPA or a specific PKC inhibitor Ro-31-8220 did not block the activation of p70(S6k) by ROS, indicating that the activation of TPA-responsive PKC was not required for stimulation of p70(S6k) activity by H₂O₂ in JB6 cells. Exposure of JB6 cells to platelet-derived growth factor or epidermal growth factor led to a rapid increase in H₂O₂, phosphorylation, and activation of p70S6k, which were antagonized by the pretreatment of catalase. Taken together, the results suggest that ROS act as a messenger in growth factor-induced p70(S6k) signaling pathway.