A signaling cascade mediated by ceramide, src and PDGFRβ coordinates the activation of the redox-sensitive neutral sphingomyelinase-2 and sphingosine kinase-1

Stress-inducing agents, including oxidative stress, generate the sphingolipid mediators ceramide (Cer) and sphingosine-l-phosphate (S1P) that are involved in stress-induced cellular responses. The two redox-sensitive neutral sphingomyelinase-2 (nSMase2) and sphingosine kinase-1 (SKI) participate in transducing stress signaling to ceramide and SIP, respectively; however, whether these key enzymes are coordinately regulated is not known. We investigated whether a signaling link coordinates nSMase2 and SKI activation by H2O2. In mesenchymal cells, H2O2 elicits a dose-dependent biphasic effect, mitogenic at low concentration (5 mu M), and anti-proliferative and toxic at high concentration (100 mu M). Low H2O2 concentration triggered activation of nSMase2 and SKI through a nSMase2/Cer-dependent signaling pathway that acted upstream of activation of SK1. Further results implicated src and the trans-activation of PDGFR beta, as supported by the blocking effect of specific siRNAs, pharmacological inhibitors, and genetically deficient cells for nSMase2, src and SKI. The H2O2-induced src/PDGFR beta/SK1 signaling cascade was impaired in nSMase2-deficient fro/fro cells and was rescued by exogenous C2Cer that activated src/PDGFR beta/SK1. Thus, the results define a nSMase2/SK1 signaling pathway implicated in the mitogenic response to low oxidative stress. On the other hand, high oxidative stress induced inhibition of SK1. The results also showed that the toxicity of high H2O2 concentration was comparable in control and nSMase2-deficient cells. Taken together the results identify a tightly coordinated nSMase2/SK1 pathway that mediates the mitogenic effects of H2O2 and may sense the degree of oxidative stress. (C) 2013 Elsevier B.V. All rights reserved.