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

Abstract

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.

Identified and potential internalization signals involved in trafficking and regulation of Na+/K+ ATPase activity

“…Consistent with these results, we demonstrated in a separate study using Caco-2 cells that FTY720P, an analogue of sphingosine 1-phosphate, mediates the inhibition of Na + /K + ATPase activity by activating S1PR2 and inducing PGE2 release [ 125 ]. Recently, we showed, using confocal imaging of live Caco-2 cells co-expressing GFP-tagged alpha Na + /K + ATPase and mcherry-tagged membrane marker, that FTY720P-mediated inhibition of the NKA pump results from a decrease in its abundance in the plasma membrane [ 13 ]. In addition to PGE2, FTY720P was found to induce NO release in these cells.…”

“…The Na + /K + ATPase is known to traffic between the cell membrane and intracellular stores, thus the regulation of its spatial and temporal distribution in the cell is crucial for the maintenance of homeostasis. Although previous studies reported trafficking as a mechanism of NKA regulation in several cell types [ 8 – 13 ], the molecular determinants of this process are still unclear. The pump residence in the plasma membrane depends on the rate of endocytosis and exocytosis.…”

Effect of FTY720P on lipid accumulation in HEPG2 cells

“…Extracellular FTY720P mimics S1P and binds to S1PRs which are coupled to Gi/o, G12/13, or Gq proteins. The same receptor may couple to more than one type of G protein and activate various downstream signaling molecules including PI3K and Akt 19 , 20 which are also involved in lipid metabolism. FTY720 has been approved for the treatment of Multiple Sclerosis 21 .…”