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.
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.
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 PLCβ3, but accelerated by wild-type PLCβ3 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 PLCβ3 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 PLCβ 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 PLCβ/PLCδ(PH) expression are important for regulation of SphK1 by Gq/11.
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