Abstract

FTY720-P is a non-selective Sphingosine-1-phosphate (S1P) receptor agonist thought to be devoid of activity at the S1P₂ receptor subtype. However, we have recently shown that FTY720-P displays significant S1P₂ receptor agonist activity in recombinant cells and in fibroblasts expressing endogenous S1P₂ receptors. To elucidate the S1P₂-dependent signaling pathways that were activated by FTY720-P, we employed second messenger assays and impedance-based assays in combination with pharmacological and siRNA-based pathway inhibition in recombinant CHO-S1P₂ cells as well as human lung myofibroblasts generated in vitro. In CHO-S1P₂ cells, FTY720-P did not modulate cAMP or calcium levels. However, reporter-gene assays, impedance-based assays with a selective Rho-associated kinase (ROCK) inhibitor, Gα_12/13 knock-down and activated Rho-pull-down assays demonstrated that FTY720-P potently activated Gα_12/13/ Rho/ ROCK signaling. S1P similarly activated Gα_12/13/ Rho/ ROCK signaling via S1P₂ receptors, while the two selective S1P₁ receptor agonists ponesimod and SEW2871 were inactive. In lung myofibroblasts, which mainly expressed the S1P₂ receptor subtype, we showed that FTY720-P selectively activated the Gα_12/13/ Rho/ ROCK pathway via the S1P₂ receptor. Moreover, the activation of the Gα_12/13/ Rho/ ROCK pathway in myofibroblasts by FTY720-P caused potent myofibroblast contraction similar to that induced by the natural ligand S1P. Thus, complementing second messenger assays with unbiased label-free assays or with phenotypic assays in native expression systems can uncover activation of additional pathways such as Gα_12/13/ Rho/ ROCK signaling.