Selectivity and Specificity of Sphingosine-1-Phosphate Receptor Ligands: Caveats and Critical Thinking in Characterizing Receptor-Mediated Effects

Adaptive immunity depends on T-cell exit from the thymus and T and B cells travelling between secondary lymphoid organs to survey for antigens. After activation in lymphoid organs, T cells must again return to circulation to reach sites of infection; however, the mechanisms regulating lymphoid organ exit are unknown. An immunosuppressant drug, FTY720, inhibits lymphocyte emigration from lymphoid organs, and phosphorylated FTY720 binds and activates four of the five known sphingosine-1-phosphate (S1P) receptors. However, the role of S1P receptors in normal immune cell trafficking is unclear. Here we show that in mice whose haematopoietic cells lack a single S1P receptor (S1P1; also known as Edg1) there are no T cells in the periphery because mature T cells are unable to exit the thymus. Although B cells are present in peripheral lymphoid organs, they are severely deficient in blood and lymph. Adoptive cell transfer experiments establish an intrinsic requirement for S1P1 in T and B cells for lymphoid organ egress. Furthermore, S1P1-dependent chemotactic responsiveness is strongly upregulated in T-cell development before exit from the thymus, whereas S1P1 is downregulated during peripheral lymphocyte activation, and this is associated with retention in lymphoid organs. We find that FTY720 treatment downregulates S1P1, creating a temporary pharmacological S1P1-null state in lymphocytes, providing an explanation for the mechanism of FTY720-induced lymphocyte sequestration. These findings establish that S1P1 is essential for lymphocyte recirculation and that it regulates egress from both thymus and peripheral lymphoid organs.

Sphingosine 1-phosphate (S1P), a potent sphingolipid mediator produced by sphingosine kinase isoenzymes (SphK1 and SphK2), regulates diverse cellular processes important for breast cancer progression acting in an autocrine and/or paracrine manner. Here we show that SphK1, but not SphK2, increased S1P export from MCF-7 cells. Whereas for both estradiol (E(2)) and epidermal growth factor-activated SphK1 and production of S1P, only E(2) stimulated rapid release of S1P and dihydro-S1P from MCF-7 cells. E(2)-induced S1P and dihydro-S1P export required estrogen receptor-alpha, not GPR30, and was suppressed either by pharmacological inhibitors or gene silencing of ABCC1 (multidrug resistant protein 1) or ABCG2 (breast cancer resistance protein). Inhibiting these transporters also blocked E(2)-induced activation of ERK1/2, indicating that E(2) activates ERK via downstream signaling of S1P. Taken together, our findings suggest that E(2)-induced export of S1P mediated by ABCC1 and ABCG2 transporters and consequent activation of S1P receptors may contribute to nongenomic signaling of E(2) important for breast cancer pathophysiology.

Sphingosine kinase 1 (SK1) is an enzyme that catalyses the phosphorylation of sphingosine to produce the bioactive lipid sphingosine 1-phosphate (S1P). We demonstrate here that FTY720 (Fingolimod™) and (S)-FTY720 vinylphosphonate are novel inhibitors of SK1 catalytic activity and induce the proteasomal degradation of this enzyme in human pulmonary artery smooth muscle cells, MCF-7 breast cancer cells and androgen-independent LNCaP-AI prostate cancer cells. Proteasomal degradation of SK1 in response to FTY720 and (S)-FTY720 vinylphosphonate is associated with the down-regulation of the androgen receptor in LNCaP-AI cells. (S)-FTY720 vinylphosphonate also induces the apoptosis of these cells. These findings indicate that SK1 is involved in protecting LNCaP-AI from apoptosis. This protection might be mediated by so-called ‘inside-out’ signalling by S1P, as LNCaP-AI cells exhibit increased expression of S1P2/3 receptors and reduced lipid phosphate phosphatase expression (compared with androgen-sensitive LNCaP cells) thereby potentially increasing the bioavailability of S1P at S1P2/3 receptors.