Sphingosine‐1‐phosphate is a mediator of TNF‐α action on the Na+/K+ ATPase in HepG2 cells

We showed previously that TNF-α down-regulates the Na+/K+ ATPase in HepG2 cells. This work was undertaken to study the role of ceramide and its metabolites in TNF-α action. Treating HepG2 cells with the cytokine in presence of an inhibitor of sphingomyelinase, abrogated the effect of TNF-α on the ATPase. To confirm the involvement of ceramide or its metabolites, cells were incubated with exogenous ceramide. Ceramide reduced time-dependently the activity of the ATPase and its effect disappeared in presence of CAY 10466 or SHKI, respective inhibitors of ceramidase and spingosine kinase, suggesting that ceramide acts via sphingosine or sphingosine-1-phosphate (S1P). However, HepG2 cells treated with exogenous sphingosine showed a higher Na+/K+ ATPase activity inferring that S1P is the one responsible for the down-regulatory effect of TNF-α and ceramide. This hypothesis was confirmed by the observed inhibitory effect of exogenous S1P on the pump, which was maintained when JNK and NF-κB were inhibited separately or simultaneously. The concurrent, but not individual inhibition of the kinase and transcription factor in the absence of S1P imitated the effect of S1P. It was concluded that S1P down-regulates the ATPase by inhibiting both JNK and NF-κB. This conclusion was supported by the observed decrease in the phosphorylation of c-jun and the enhanced protein expression of IκB and lower NK-KB activity.