Abstract Sphingolipids (SL) are both membrane building blocks and potent signaling molecules regulating a variety of cellular functions in both physiological and pathological conditions. Under normal physiology, sphingolipid levels are tightly regulated, whereas disruption of sphingolipid homeostasis and signaling has been implicated in diabetes, cancer, cardiovascular and autoimmune diseases. Yet, mechanisms governing cellular sensing of SL, and according regulation of their biosynthesis remain largely unknown. In yeast, serine palmitoyltransferase (SPT), catalyzing the first and rate limiting step of sphingolipid de novo biosynthesis, is negatively regulated by Orosomucoid 1 and 2 (Orm) proteins. Lowering sphingolipid levels triggers Orms phosphorylation, resulting in the removal of the inhibitory brake on SPT to enhance sphingolipid de novo biosynthesis. However, mammalian orthologs ORMDLs lack the N-terminus hosting the phosphosites. Thus, which sphingolipid(s) are sensed by the cells, and mechanisms of homeostasis remain largely unknown. This study is aimed at filling this knowledge gap. Here, we identify sphingosine-1-phosphate (S1P) as the key sphingolipid sensed by endothelial cells via S1PRs. The increase of S1P-S1PR signaling stabilizes ORMDLs, which downregulates SPT activity to maintain SL homeostasis. These findings reveal the S1PR/ORMDLs axis as the sensor-effector unit regulating SPT activity accordingly. Mechanistically, the hydroxylation of ORMDLs at Pro137 allows a constitutive degradation of ORMDLs via ubiquitin-proteasome pathway, therefore preserving SPT activity at steady state. The disruption of the S1PR/ORMDL axis results in ceramide accrual, mitochondrial dysfunction, and impaired signal transduction, all leading to endothelial dysfunction, which is an early event in the onset of cardio- and cerebrovascular diseases. The disruption of S1P-ORMDL-SPT signaling may be implicated in the pathogenesis of conditions such as diabetes, cancer, cardiometabolic disorders, and neurodegeneration, all characterized by deranged sphingolipid metabolism. Our discovery may provide the molecular basis for a therapeutic intervention to restore sphingolipid homeostasis.

S1P controls endothelial sphingolipid homeostasis via ORMDL