Ceramide and its metabolites modulate time-dependently the activity of the Na⁺/K⁺ ATPase in HepG2 cells.

Ceramide is involved in the regulation of many cellular processes including cell proliferation and apoptosis, which are accompanied respectively with a decrease and an increase in the activity of the Na(+)/K(+) ATPase. These antagonistic effects may be time-dependent and due to different signaling pathways requiring different time intervals to be activated. While we showed previously a ceramide-induced inhibition of the ATPase in HepG2 cells during the first hour, we study here the effect of ceramide thereafter. Ceramide stimulated the Na(+)/K(+) ATPase between 1 and 4h with a peak at 2h. This stimulation was maintained in the simultaneous presence of an inhibitor of ceramidase (CAY 10466) but disappeared when ceramide kinase was inhibited, suggesting a role of ceramide-1-phosphate (cer-1-P) in the observed effect. Exogenous cer-1-P caused a similar stimulation of the ATPase which was not affected by an inhibition of JNK but changed into a decrease in presence of PDTC, a specific inhibitor of NF-κB, and disappeared when NF-κB and JNK were inhibited simultaneously. It was concluded that cer-1-P activates both JNK and NF-κB. While JNK exerts an inhibitory effect on the ATPase, NF-κB increases its activity and abrogates the stimulatory effect of the sphingolipid on JNK leading thus to an additional increase in the ATPase activity.

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.

Signaling pathway underlying the up-regulatory effect of TNF-alpha on the Na(+)/K(+) ATPase in HepG2 cells.

The activity of the Na(+)/K(+) ATPase was shown to be reduced during apoptosis and enhanced during cell proliferation. This work investigated whether TNF-alpha exerts also opposite effects on the Na(+)/K(+) ATPase in HepG2 cells and whether these effects are time-dependent. A time response study demonstrated that the activity and protein expression of the ATPase are decreased at 1h and increased at 4, 6 and 8h. This work focused on the up-regulatory 4h-response. TNF-alpha was shown to exert a stimulatory effect on cJNK and NF-kappaB and an inhibitory effect on caspases which, in the basal state, down-regulate the ATPase. The cytokine was found to target the caspases by activating JNK which in turn activates NF-kappaB. The activated transcription factor inhibits the caspases and frees the ATPase from their inhibitory action leading thus to its up-regulation.