Rationale A significant and sustained increase in vascular permeability is a hallmark of acute inflammatory diseases such as acute lung injury (ALI), but effective therapies for preserving or reconstituting the vascular barrier are lacking. Prior work has demonstrated that FTY720 S-phosphonate (Tysiponate/Tys), an analog of sphingosine 1-phosphate (S1P) and FTY720, has more potent pulmonary barrier protective effects than these agents in vitro and in the LPS- and bleomycin-induced models of mouse ALI. Moreover, Tys preserves expression of the barrier promoting S1P1 receptor (S1PR1), whereas S1P and FTY720 induce its ubiquitination and degradation. In this report, we further characterize the mechanism of preservation of S1PR1 expression by Tys in cultured human pulmonary endothelial cells (EC).
Results P-FTY720 significantly induced the association of S1PR1 and GRK2 and increased the p-serine content of S1PR1, which are critical for S1PR1 internalization and degradation, but Tys failed to do so. In contrast, both p-FTY720 and Tys induced significant tyrosine phosphorylation of S1PR1. Tys also preserves expression of S1PR2 and S1PR3. Although prior work reported that E3 ubiquitin-protein ligase WWP2 is critical for ubiquitination and degradation of S1PR1, neither p-FTY720 nor Tys changed its association with S1PR1 in human lung EC. Moreover, ubiquitin activating enzyme E1 inhibitor significantly inhibited S1PR1 degradation induced by p-FTY or S1P. Pharmacological inhibition of PKA, PKG, PKC, GSK3, PI3K, ERK, Src, or c-Abl activity, as well as inhibition of calcium flux, all fail to inhibit p-FTY720-induced S1PR1 degradation.
Conclusion Unlike p-FTY720, Tys fails to activate the GRK2-mediated ubiquitination pathway and thus preserves S1PR1 expression. These results provide additional mechanistic insights into the barrier-regulatory effects of this potential ALI therapy.