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Sphingolipid and cholesterol metabolism are closely associated at the structural, biochemical, and functional levels. Although HDL-associated sphingosine-1-phosphate (S1P) contributes to several HDL functions, and S1P signaling regulates glucose and lipid metabolism, no study has addressed the involvement of S1P in cholesterol efflux. Here, we show that sphingosine kinase (Sphk) activity was induced by the LXR agonist 22(R)-hydroxycholesterol and required for the stimulation of ABCA1-mediated cholesterol efflux to apolipoprotein A-I. In support, pharmacological Sphk inhibition and Sphk2 but not Sphk1 deficiency abrogated efflux. The involved mechanism included stimulation of both transcriptional and functional ABCA1 regulatory pathways and depended for the latter on the S1P receptor 3 (S1P3). Accordingly, S1P3-deficient macrophages were resistant to 22(R)-hydroxycholesterol-stimulated cholesterol efflux. The inability of excess exogenous S1P to further increase efflux was consistent with tonic S1P3 signaling by a pool of constitutively generated Sphk-derived S1P dynamically regulating cholesterol efflux. In summary, we have established S1P as a previously unrecognized intermediate in LXR-stimulated ABCA1-mediated cholesterol efflux and identified S1P/S1P3 signaling as a positive-feedback regulator of cholesterol efflux. This constitutes a novel regulatory mechanism of cholesterol efflux by sphingolipids.

Sphingosine-1-phosphate (S1P) signaling influences bone metabolism, but its therapeutic potential in bone disorders has remained unexplored. We show that raising S1P levels in adult mice through conditionally deleting or pharmacologically inhibiting S1P lyase, the sole enzyme responsible for irreversibly degrading S1P, markedly increased bone formation, mass and strength and substantially decreased white adipose tissue. S1P signaling through S1P2potently stimulated osteoblastogenesis at the expense of adipogenesis by inversely regulating osterix and PPAR-γ, and it simultaneously inhibited osteoclastogenesis by inducing osteoprotegerin through newly discovered p38–GSK3β–β-catenin and WNT5A–LRP5 pathways. Accordingly, S1P2-deficient mice were osteopenic and obese. In ovariectomy-induced osteopenia, S1P lyase inhibition was as effective as intermittent parathyroid hormone (iPTH) treatment in increasing bone mass and was superior to iPTH in enhancing bone strength. Furthermore, lyase inhibition in mice successfully corrected severe genetic osteoporosis caused by osteoprotegerin deficiency. Human data from 4,091 participants of the SHIP-Trend population-based study revealed a positive association between serum levels of S1P and bone formation markers, but not resorption markers. Furthermore, serum S1P levels were positively associated with serum calcium , negatively with PTH , and curvilinearly with body mass index. Bone stiffness, as determined through quantitative ultrasound, was inversely related to levels of both S1P and the bone formation marker PINP, suggesting that S1P stimulates osteoanabolic activity to counteract decreasing bone quality. S1P-based drugs should be considered as a promising therapeutic avenue for the treatment of osteoporotic diseases.

The hepatocyte nuclear factor (HNF) family regulates complex networks of metabolism and organ development. Human mutations in its prototypical member HNF1A cause maturity-onset diabetes of the young (MODY) type 3. In this study, we identified an important role for HNF1A in the preservation of erythrocyte membrane integrity, calcium homeostasis, and osmotic resistance through an as-yet unrecognized link of HNF1A to sphingolipid homeostasis. HNF1A−/−mice displayed microcytic hypochromic anemia with reticulocytosis that was partially compensated by avid extramedullary erythropoiesis at all erythroid stages in the spleen thereby excluding erythroid differentiation defects. Morphologically, HNF1A−/−erythrocytes resembled acanthocytes and displayed increased phosphatidylserine exposure, high intracellular calcium, and elevated osmotic fragility. Sphingolipidome analysis by mass spectrometry revealed substantial and tissue-specific sphingolipid disturbances in several tissues including erythrocytes with the accumulation of sphingosine as the most prominent common feature. All HNF1A−/−erythrocyte defects could be simulated by exposure of wild-type (WT) erythrocytes to sphingosine in vitro and attributed in part to sphingosine-induced suppression of the plasma-membrane Ca2+-ATPase activity. Bone marrow transplantation rescued the anemia phenotype in vivo, whereas incubation with HNF1A−/−plasma increased the osmotic fragility of WT erythrocytes in vitro. Our data suggest a non–cell-autonomous erythrocyte defect secondary to the sphingolipid changes caused by HNF1A deficiency. Transcriptional analysis revealed 4 important genes involved in sphingolipid metabolism to be deregulated in HNF1A deficiency: Ormdl1, sphingosine kinase-2, neutral ceramidase, and ceramide synthase-5. The considerable erythrocyte defects in murine HNF1A deficiency encourage clinical studies to explore the hematological consequences of HNF1A deficiency in human MODY3 patients.

The hepatocyte nuclear factor (HNF) family regulates complex networks of metabolism and organ development. Human mutations in its prototypical member HNF1A cause maturity-onset diabetes of the young (MODY) type 3. In this study, we identified an important role for HNF1A in the preservation of erythrocyte membrane integrity, calcium homeostasis, and osmotic resistance through an as-yet unrecognized link of HNF1A to sphingolipid homeostasis. HNF1A−/− mice displayed microcytic hypochromic anemia with reticulocytosis that was partially compensated by avid extramedullary erythropoiesis at all erythroid stages in the spleen thereby excluding erythroid differentiation defects. Morphologically, HNF1A−/− erythrocytes resembled acanthocytes and displayed increased phosphatidylserine exposure, high intracellular calcium, and elevated osmotic fragility. Sphingolipidome analysis by mass spectrometry revealed substantial and tissue-specific sphingolipid disturbances in several tissues including erythrocytes with the accumulation of sphingosine as the most prominent common feature. All HNF1A−/− erythrocyte defects could be simulated by exposure of wild-type (WT) erythrocytes to sphingosine in vitro and attributed in part to sphingosine-induced suppression of the plasma-membrane Ca2+-ATPase activity. Bone marrow transplantation rescued the anemia phenotype in vivo, whereas incubation with HNF1A−/− plasma increased the osmotic fragility of WT erythrocytes in vitro. Our data suggest a non–cell-autonomous erythrocyte defect secondary to the sphingolipid changes caused by HNF1A deficiency. Transcriptional analysis revealed 4 important genes involved in sphingolipid metabolism to be deregulated in HNF1A deficiency: Ormdl1, sphingosine kinase-2, neutral ceramidase, and ceramide synthase-5. The considerable erythrocyte defects in murine HNF1A deficiency encourage clinical studies to explore the hematological consequences of HNF1A deficiency in human MODY3 patients.