The signaling pathway mediating the stimulatory effect of FTY720-P on hepatic Na+-K+ ATPase -

Abstract

Alterations in hepatocytes’ volume contribute to the pathophysiology of several hepatic disorders including liver insufficiency, diabetic ketoacidosis, hyper-catabolism and infection. The Na+-K+ ATPase is a key regulator of ionic homeostasis in hepatocytes and consequently of cell volume. Emerging evidence ascribes a role for sphingosine 1-phosphate (S1P) in the development and progression of liver diseases. Previous studies in our lab showed that S1P modulates time-dependently the activity of the Na+-K+ ATPase in HepG2 cells, with an inhibitory effect appearing at 15min and a stimulatory one at 2hrs. This study focuses on the effect of S1P at 2 hours using the S1P analogue FTY720P. HepG2 cells were incubated with FTY720P for 2 hrs and the activity of the pump was assayed by measuring the amount of inorganic-phosphate liberated in presence and absence of ouabain, a specific inhibitor of the Na+-K+ ATPase. FTY720P induced a 2.5 fold increase in the activity of the ATPase which was maintained in the presence of JTE-013, a specific blocker of S1PR2, but disappeared completely in presence of CAY 10444, a specific S1PR3 antagonist. The involvement of S1PR3 was confirmed by the stimulatory effect observed with Cym5541, a S1PR3 agonist. FTY720P increased the expression level of COX2, an enzyme involved in PGE2 synthesis, and its effect on the ATPase disappeared in presence of indomethacin, an inhibitor of COX enzymes, suggesting that FTY720P acts by promoting PGE2 production. The involvement of PGE2 was confirmed by the ATPase stimulation induced by exogenous PGE2. Inhibiting PKC and ERK with respectively calphostin and PD98059 abolished the effect of FTY720P on the Na+-K+ ATPase, but not that of exogenous PGE2 indicating that the two kinases are upstream of PGE2.The PKC activator PMA increased the activity of the Na+-K+ ATPase as well as the expression levels of phopho-ERK inferring that PKC is upstream of ERK. The effect of PGE2 disappeared in presence of PF-04418948 (blocker of EP2 receptor), RpcAMP (PKA inh