Abstract
Sphingosine-1-phosphate (S1P) regulates a broad spectrum of fundamental cellular processes like proliferation, death, migration and cytokine production. Therefore, elevated levels of S1P may be causal to various pathologic conditions including cancer, fibrosis, inflammation, autoimmune diseases and aberrant angiogenesis. Here we report that S1P lyase from the prokaryote Symbiobacterium thermophilum (StSPL) degrades extracellular S1P in vitro and in blood. Moreover, we investigated its effect on cellular responses typical of fibrosis, cancer and aberrant angiogenesis using renal mesangial cells, endothelial cells, breast (MCF-7) and colon (HCT 116) carcinoma cells as disease models. In all cell types, wild-type StSPL, but not an inactive mutant, disrupted MAPK phosphorylation stimulated by exogenous S1P. Functionally, disruption of S1P receptor signaling by S1P depletion inhibited proliferation and expression of connective tissue growth factor in mesangial cells, proliferation, migration and VEGF expression in carcinoma cells, and proliferation and migration of endothelial cells. Upon intravenous injection of StSPL in mice, plasma S1P levels rapidly declined by 70% within 1 h and then recovered to normal 6 h after injection. Using the chicken chorioallantoic membrane model we further demonstrate that also under in vivo conditions StSPL, but not the inactive mutant, inhibited tumor cell-induced angiogenesis as an S1P-dependent process. Our data demonstrate that recombinant StSPL is active under extracellular conditions and holds promise as a new enzyme therapeutic for diseases associated with increased levels of S1P and S1P receptor signaling.
Highlights
Sphingolipids are essential constituents of cellular membranes and serve as signalling molecules involved in various physiological and pathophysiological processes
Biochemical characterization of recombinant S1P lyase from the prokaryote Symbiobacterium thermophilum (StSPL) The previously cloned full-length STH1274 gene was expressed in E. coli and the StSPL was purified to homogeneity as described [32]
Based on our previous work which resolved the structure of WT StSPL at 2.0 Aresolution [32], we conclude that StSPL is a typical type I-fold dimeric pyridoxal 59-phosphate (PLP)-dependent enzyme (Fig. 1 B) where residues from both subunits contribute to the active site
Summary
Sphingolipids are essential constituents of cellular membranes and serve as signalling molecules involved in various physiological and pathophysiological processes. S1P is present in blood at high nanomolar concentrations due to the S1P-producing activity of sphingosine kinases (SK1) in various cell types including mast cells, erythrocytes and vascular endothelial cells [6,7,8,9]. Five receptor subtypes have been identified and denoted as S1P1–5 [17,18,19]. Their activation triggers downstream signaling via mitogen-activated protein kinases (MAPK), phosphoinositide 3-kinase, cyclic AMP and other mediators of cellular responses. Subsequent biological effects include cytoskeletal rearrangements, cell proliferation and migration, invasion, vascular development, platelet aggregation and lymphocyte trafficking [14,20]
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