Effect of FTY720P on lipid accumulation in HEPG2 cells.

Nonalcoholic fatty liver disease (NAFLD) is characterized by an increase in hepatic lipid accumulation due to impaired lipid metabolism. Although a correlation was found between NAFLD and sphingosine-1-phosphate (S1P), the role of the sphingolipid remains controversial. The aim of this study was to investigate any involvement of S1P in steatosis using its analog FTY720P and HepG2 cells. Lipid accumulation was induced by incubating the cells in a mixture of oleic and palmitic acid, and was quantified using Oil Red O. The involvement of signaling mediators was studied using pharmacological inhibitors and western blot analysis. FTY720P increased lipid accumulation, but this increase wasn't maintained in the presence of inhibitors of S1PR3, Gq, SREBP, mTOR, PI3K, and PPARγ indicating their involvement in the process. The results revealed that FTY720P binds to S1PR3 which activates sequentially Gq, PI3K, and mTOR leading to an increase in SREBP expression and PPARγ activation. It was concluded that in presence of a high level of fatty acids, lipid accumulation is increased in hepatocytes by the exogenously added FTY720P.

Adverse effect of FTY720P on colonic Na+ /K+ ATPase is mediated via ERK, p38MAPK, PKC, and PI3K.

FTY720P, an analogue of sphingosine 1-phosphate, has emerged lately as a potential causative agent of inflammatory bowel disease, in which electrolytes movements driven by the sodium gradient established by the Na+ /K+ ATPase are altered. We showed previously in Caco-2 cells, a 50% FTY720P-induced decrease in the ATPase activity, mediated via S1PR2 and PGE2. This work aims at delineating the mechanism underlying PGE2 release and at investigating if the ATPase inhibition is due to changes in its abundance. The activity of the ATPase and the localization of a GFP-tagged Na+ /K+ -ATPase α1 -subunit were assessed in cells treated with 7.5 nM FTY720P. The involvement of ERK, p38 MAPK, PKC, and PI3K was studied in cells treated with 7.5 nM FTY720P or 1 nM PGE2 in presence of their inhibitors, or by determining changes in the protein expression of their activated phosphorylated forms. Imaging data showed ∼30% reduction in the GFP-tagged Na+ /K+ ATPase at the plasma membrane. Both FTY720P and PGE2 showed, respectively, 50% and 60% reduction in ATPase activity that disappeared when p38 MAPK, PKC, and PI3K were inhibited individually but not with ERK inhibition. The effect of FTY720P was imitated by PMA, an activator of PKC. Western blotting revealed inhibition of ERK by FTY720P. It was concluded that FTY720P, through activation of S1PR2, downregulates the Na+ /K+ ATPase by inhibiting ERK, which in turn activates p38 MAPK leading to the sequential activation of PKC and PI3K, PGE2 release, and a decrease in the Na+ /K+ ATPase activity and membrane abundance.

PGE2 upregulates the Na+/K+ ATPase in HepG2 cells via EP4 receptors and intracellular calcium.

The Na+/K+ ATPase is a key regulator of the hepatocytes ionic homeostasis, which when altered may lead to many liver disorders. We demonstrated recently, a significant stimulation of the Na+/K+ ATPase in HepG2 cells treated with the S1P analogue FTY 720P, that was mediated through PGE2. The mechanism by which the prostaglandin exerts its effect was not investigated, and is the focus of this work. The type of receptors involved was determined using pharmacological inhibitors, while western blot analysis, fluorescence imaging of GFP-tagged Na+/K+ ATPase, and time-lapse imaging on live cells were used to detect changes in membrane abundance of the Na+/K+ ATPase. The activity of the ATPase was assayed by measuring the amount of inorganic phosphate liberated in the presence and absence of ouabain. The enhanced activity of the ATPase was not observed when EP4 receptors were blocked but still appeared in presence inhibitors of EP1, EP2 and EP3 receptors. The involvement of EP4 was confirmed by the stimulation observed with EP4 agonist. The stimulatory effect of PGE2 did not appear in presence of Rp-cAMP, an inhibitor of PKA, and was imitated by db-cAMP, a PKA activator. Chelating intracellular calcium with BAPTA-AM abrogated the effect of db-cAMP as well as that of PGE2, but PGE2 treatment in a calcium-free PBS medium did not, suggesting an involvement of intracellular calcium, that was confirmed by the results obtained with 2-APB treatment. Live cell imaging showed movement of GFP-Na+/K+ ATPase-positive vesicles to the membrane and increased abundance of the ATPase at the membrane after PGE2 treatment. It was concluded that PGE2 acts via EP4, PKA, and intracellular calcium.

FTY720P inhibits the Na+/K+ ATPase in Caco-2 cells via S1PR2: PGE2 and NO are along the signaling pathway.

AIMS: Sphingosine-1-phosphate (S1P) has been implicated lately in inflammatory bowel disease which has diarrhea as one of its symptoms. Diarrhea is due to altered water movements as a result of altered electrolyte transport, and in particular sodium. Sodium movements are geared by the sodium gradient established by the Na+/K+ ATPase. The aim of this work was to investigate if S1P can modulate the activity of the ATPase, using Caco-2 cells as a model and the S1P analogue, FTY720P. MATERIALS AND METHODS: The activity of the ATPase was assayed by measuring the amount of inorganic phosphate liberated in presence and absence of ouabain. Protein expression of the various S1P receptors was studied by western blot analysis. KEY FINDINGS: Caco-2 cells were found to express mainly S1PR2 and S1PR3. FTY720P (7.5 nM) reduced significantly the activity of the Na+/K+ ATPase when applied for 15 min. This inhibitory effect disappeared in presence of JTE-013, a specific blocker of S1PR2, and indomethacin, an inhibitor of cyclooxygenase enzymes, and was mimicked by CYM5520, a S1PR2 agonist and by exogenous PGE2. The inhibitory effect of PGE2 did not appear when EP3 receptors were blocked or when a nitric oxide scavenger was added. RpcAMP, a PKA inhibitor, reduced the activity of the Na+/K+ ATPase, while dbcAMP, a PKA activator was without any effect and when added, abrogated the effect of PGE2. SIGNIFICANCE: It was concluded that FTY720P inhibits the Na+/K+ ATPase via activation of S1PR2 and generation of PGE2 nitric oxide.