Dissecting Gq/11-Mediated Plasma Membrane Translocation of Sphingosine Kinase-1.

Diverse extracellular signals induce plasma membrane translocation of sphingosine kinase-1 (SphK1), thereby enabling inside-out signaling of sphingosine-1-phosphate. We have shown before that Gq-coupled receptors and constitutively active Gαq/11 specifically induced a rapid and long-lasting SphK1 translocation, independently of canonical Gq/phospholipase C (PLC) signaling. Here, we further characterized Gq/11 regulation of SphK1. SphK1 translocation by the M3 receptor in HEK-293 cells was delayed by expression of catalytically inactive G-protein-coupled receptor kinase-2, p63Rho guanine nucleotide exchange factor (p63RhoGEF), and catalytically inactive PLCß3, but accelerated by wild-type PLCß3 and the PLCδ PH domain. Both wild-type SphK1 and catalytically inactive SphK1-G82D reduced M3 receptor-stimulated inositol phosphate production, suggesting competition at Gαq. Embryonic fibroblasts from Gαq/11 double-deficient mice were used to show that amino acids W263 and T257 of Gαq, which interact directly with PLCß3 and p63RhoGEF, were important for bradykinin B2 receptor-induced SphK1 translocation. Finally, an AIXXPL motif was identified in vertebrate SphK1 (positions 100-105 in human SphK1a), which resembles the Gαq binding motif, ALXXPI, in PLCß and p63RhoGEF. After M3 receptor stimulation, SphK1-A100E-I101E and SphK1-P104A-L105A translocated in only 25% and 56% of cells, respectively, and translocation efficiency was significantly reduced. The data suggest that both the AIXXPL motif and currently unknown consequences of PLCß/PLCδ(PH) expression are important for regulation of SphK1 by Gq/11.

Characterization of cholesterol homeostasis in sphingosine-1-phosphate lyase-deficient fibroblasts reveals a Niemann-Pick disease type C-like phenotype with enhanced lysosomal Ca2+ storage.

Sphingosine-1-phosphate (S1P) lyase irreversibly cleaves S1P, thereby catalysing the ultimate step of sphingolipid degradation. We show here that embryonic fibroblasts from S1P lyase-deficient mice (Sgpl1-/--MEFs), in which S1P and sphingosine accumulate, have features of Niemann-Pick disease type C (NPC) cells. In the presence of serum, overall cholesterol content was elevated in Sgpl1-/--MEFs, due to upregulation of the LDL receptor and enhanced cholesterol uptake. Despite this, activation of sterol regulatory element-binding protein-2 was increased in Sgpl1-/--MEFs, indicating a local lack of cholesterol at the ER. Indeed, free cholesterol was retained in NPC1-containing vesicles, which is a hallmark of NPC. Furthermore, upregulation of amyloid precursor protein in Sgpl1-/--MEFs was mimicked by an NPC1 inhibitor in Sgpl1+/+-MEFs and reduced by overexpression of NPC1. Lysosomal pH was not altered by S1P lyase deficiency, similar to NPC. Interestingly, lysosomal Ca2+ content and bafilomycin A1-induced [Ca2+]i increases were enhanced in Sgpl1-/--MEFs, contrary to NPC. These results show that both a primary defect in cholesterol trafficking and S1P lyase deficiency cause overlapping phenotypic alterations, and challenge the present view on the role of sphingosine in lysosomal Ca2+ homeostasis.

Upregulation of ABC transporters contributes to chemoresistance of sphingosine 1-phosphate lyase-deficient fibroblasts.

Sphingosine 1-phosphate (S1P) is an extra- and intracellular mediator that regulates cell growth, survival, migration, and adhesion in many cell types. S1P lyase is the enzyme that irreversibly cleaves S1P and thereby constitutes the ultimate step in sphingolipid catabolism. It has been reported previously that embryonic fibroblasts from S1P lyase-deficient mice (Sgpl1(-/-)-MEFs) are resistant to chemotherapy-induced apoptosis through upregulation of B cell lymphoma 2 (Bcl-2) and Bcl-2-like 1 (Bcl-xL). Here, we demonstrate that the transporter proteins Abcc1/MRP1, Abcb1/MDR1, Abca1, and spinster-2 are upregulated in Sgpl1(-/-)-MEFs. Furthermore, the cells efficiently sequestered the substrates of Abcc1 and Abcb1, fluo-4 and doxorubicin, in subcellular compartments. In line with this, Abcb1 was localized mainly at intracellular vesicular structures. After 16 h of incubation, wild-type MEFs had small apoptotic nuclei containing doxorubicin, whereas the nuclei of Sgpl1(-/-)-MEFs appeared unchanged and free of doxorubicin. A combined treatment with the inhibitors of Abcb1 and Abcc1, zosuquidar and MK571, respectively, reversed the compartmentalization of doxorubicin and rendered the cells sensitive to doxorubicin-induced apoptosis. It is concluded that upregulation of multidrug resistance transporters contributes to the chemoresistance of S1P lyase-deficient MEFs.

Evidence for a link between histone deacetylation and Ca²+ homoeostasis in sphingosine-1-phosphate lyase-deficient fibroblasts.

Embryonic fibroblasts from S1P (sphingosine-1-phosphate) lyase-deficient mice [Sgpl1-/- MEFs (mouse embryonic fibroblasts)] are characterized by intracellular accumulation of S1P, elevated cytosolic [Ca2+]i and enhanced Ca2+ storage. Since S1P, produced by sphingosine kinase 2 in the nucleus of MCF-7 cells, inhibited HDACs (histone deacetylases) [Hait, Allegood, Maceyka, Strub, Harikumar, Singh, Luo, Marmorstein, Kordula, Milstein et al. (2009) Science 325, 1254-1257], in the present study we analysed whether S1P accumulated in the nuclei of S1P lyase-deficient MEFs and caused HDAC inhibition. Interestingly, nuclear concentrations of S1P were disproportionally elevated in Sgpl1-/- MEFs. HDAC activity was reduced, acetylation of histone 3-Lys9 was increased and the HDAC-regulated gene p21 cyclin-dependent kinase inhibitor was up-regulated in these cells. Furthermore, the expression of HDAC1 and HDAC3 was reduced in Sgpl1-/- MEFs. In wild-type MEFs, acetylation of histone 3-Lys9 was increased by the S1P lyase inhibitor 4-deoxypyridoxine. The non-specific HDAC inhibitor trichostatin A elevated basal [Ca2+]i and enhanced Ca2+ storage, whereas the HDAC1/2/3 inhibitor MGCD0103 elevated basal [Ca2+]i without influence on Ca2+ storage in wild-type MEFs. Overexpression of HDAC1 or HDAC2 reduced the elevated basal [Ca2+]i in Sgpl1-/- MEFs. Taken together, S1P lyase-deficiency was associated with elevated nuclear S1P levels, reduced HDAC activity and down-regulation of HDAC isoenzymes. The decreased HDAC activity in turn contributed to the dysregulation of Ca2+ homoeostasis, particularly to the elevated basal [Ca2+]i, in Sgpl1-/- MEFs.

Cis-4-methylsphingosine is a sphingosine-1-phosphate receptor modulator.

Sphingosine-1-phosphate (S1P) acts as high affinity agonist at specific G-protein-coupled receptors, S1P(1-5), that play important roles e.g. in the cardiovascular and immune systems. A S1P receptor modulating drug, FTY720 (fingolimod), has been effective in phase III clinical trials for multiple sclerosis. FTY720 is a sphingosine analogue and prodrug of FTY720-phosphate, which activates all S1P receptors except S1P(2) and disrupts lymphocyte trafficking by internalizing the S1P(1) receptor. Cis-4-methylsphingosine (cis-4M-Sph) is another synthetic sphingosine analogue that is readily taken up by cells and phosphorylated to cis-4-methylsphingosine-1-phosphate (cis-4M-S1P). Therefore, we analysed whether cis-4M-Sph interacted with S1P receptors through its metabolite cis-4M-S1P in a manner similar to FTY720. Indeed, cis-4M-Sph caused an internalization of S1P receptors, but differed from FTY720 as it acted on S1P(2) and S1P(3) and only weakly on S1P(1), while FTY720 internalized S1P(1) and S1P(3) but not S1P(2). Consequently, pre-incubation with cis-4M-Sph specifically desensitized S1P-induced [Ca(2+)](i) increases, which are mediated by S1P(2) and S1P(3), in a time- and concentration-dependent manner. This effect was not shared by sphingosine or FTY720, indicating that metabolic stability and targeting of S1P(2) receptors were important. The desensitization of S1P-induced [Ca(2+)](i) increases was dependent on the expression of SphKs, predominantly of SphK2, and thus mediated by cis-4M-S1P. In agreement, cis-4M-S1P was detected in the supernatants of cells exposed to cis-4M-Sph. It is concluded that cis-4M-Sph, through its metabolite cis-4M-S1P, acts as a S1P receptor modulator and causes S1P receptor internalization and desensitization. The data furthermore help to define requirements for sphingosine kinase substrates as S1P receptor modulating prodrugs.

Enhanced Ca2+ storage in sphingosine-1-phosphate lyase-deficient fibroblasts.

Sphingosine-1-phosphate (S1P) regulates cell growth and survival, migration and adhesion in many cell types. S1P is generated by sphingosine kinases (SphKs), and dephosphorylated by phosphatases or cleaved by S1P lyase. Extracellular S1P activates specific G protein-coupled receptors while intracellular S1P can mobilize Ca2+ from thapsigargin-sensitive stores. Here, we have studied Ca2+ signalling in mouse embryonic fibroblasts (MEFs) deficient in S1P lyase. In these cells, S1P and sphingosine concentrations were elevated about 6-fold and 2-fold, respectively, as measured by liquid chromatography/tandem mass spectrometry. Measurements with fura-2-loaded cells in suspension revealed that resting [Ca2+]i was elevated and agonist-induced [Ca2+]i increases were augmented in S1P lyase-deficient MEFs both in the presence and absence of extracellular Ca2+. Importantly, [Ca2+]i increases and Ca2+ mobilization induced by the SERCA inhibitor, thapsigargin, were augmented, indicating enhanced Ca2+ storage in S1P lyase-deficient MEFs. Measurements with single cells expressing the calmodulin-based Ca2+ sensor, cameleon, revealed that at least two cell types could be distinguished in both MEF cell populations, one with a rapid and transient [Ca2+]i increase and the other with a slower and prolonged [Ca2+]i elevation upon stimulation with thapsigargin. The area under the time course of thapsigargin-induced [Ca2+]i increases, reflecting overall Ca2+ release, was significantly increased by more than 50% in both rapidly and slowly responding S1P lyase-deficient cells. It is concluded that elevated concentrations of S1P and/or sphingosine lead to enhanced Ca2+ storage and elevated basal [Ca2+]i. S1P metabolism thus plays a role not only in acute Ca2+ mobilization but also in long-term regulation of Ca2+ homeostasis.