Partial Deficiency of Sphingosine-1-Phosphate Lyase Confers Protection in Experimental Autoimmune Encephalomyelitis

Background

Sphingosine-1-phosphate (S1P) regulates the egress of T cells from lymphoid organs; levels of S1P in the tissues are controlled by S1P lyase (Sgpl1). Hence, Sgpl1 offers a target to block T cell-dependent inflammatory processes. However, the involvement of Sgpl1 in models of disease has not been fully elucidated yet, since Sgpl1 KO mice have a short life-span.

Methodology

We generated inducible Sgpl1 KO mice featuring partial reduction of Sgpl1 activity and analyzed them with respect to sphingolipid levels, T-cell distribution, and response in models of inflammation.

Principal Findings

The partially Sgpl1 deficient mice are viable but feature profound reduction of peripheral T cells, similar to the constitutive KO mice. While thymic T cell development in these mice appears normal, mature T cells are retained in thymus and lymph nodes, leading to reduced T cell numbers in spleen and blood, with a skewing towards increased proportions of memory T cells and T regulatory cells. The therapeutic relevance of Sgpl1 is demonstrated by the fact that the inducible KO mice are protected in experimental autoimmune encephalomyelitis (EAE). T cell immigration into the CNS was found to be profoundly reduced. Since S1P levels in the brain of the animals are unchanged, we conclude that protection in EAE is due to the peripheral effect on T cells, leading to reduced CNS immigration, rather than on local effects in the CNS.

Significance

The data suggest Sgpl1 as a novel therapeutic target for the treatment of multiple sclerosis.     

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.     

SGPL1 (sphingosine phosphate lyase 1) modulates neuronal autophagy via phosphatidylethanolamine production

Macroautophagy/autophagy defects have been identified as critical factors underlying the pathogenesis of neurodegenerative diseases. The roles of the bioactive signaling lipid sphingosine-1-phosphate (S1P) and its catabolic enzyme SGPL1/SPL (sphingosine phosphate lyase 1) in autophagy are increasingly recognized. Here we provide in vitro and in vivo evidence for a previously unidentified route through which SGPL1 modulates autophagy in neurons. SGPL1 cleaves S1P into ethanolamine phosphate, which is directed toward the synthesis of phosphatidylethanolamine (PE) that anchors LC3-I to phagophore membranes in the form of LC3-II. In the brains of SGPL1^fl/fl/Nes mice with developmental neural specific SGPL1 ablation, we observed significantly reduced PE levels. Accordingly, alterations in basal and stimulated autophagy involving decreased conversion of LC3-I to LC3-II and increased BECN1/Beclin-1 and SQSTM1/p62 levels were apparent. Alterations were also noticed in downstream events of the autophagic-lysosomal pathway such as increased levels of lysosomal markers and aggregate-prone proteins such as APP (amyloid β [A4] precursor protein) and SNCA/α-synuclein. In vivo profound deficits in cognitive skills were observed. Genetic and pharmacological inhibition of SGPL1 in cultured neurons promoted these alterations, whereas addition of PE was sufficient to restore LC3-I to LC3-II conversion, and control levels of SQSTM1, APP and SNCA. Electron and immunofluorescence microscopy showed accumulation of unclosed phagophore-like structures, reduction of autolysosomes and altered distribution of LC3 in SGPL1^fl/fl/Nes brains. Experiments using EGFP-mRFP-LC3 provided further support for blockage of the autophagic flux at initiation stages upon SGPL1 deficiency due to PE paucity. These results emphasize a formerly overlooked direct role of SGPL1 in neuronal autophagy and assume significance in the context that autophagy modulators hold an enormous therapeutic potential in the treatment of neurodegenerative diseases.     

Potential Link between the Sphingosine-1-Phosphate (S1P) System and Defective Alveolar Macrophage Phagocytic Function in Chronic Obstructive Pulmonary Disease (COPD)

Introduction

We previously reported that alveolar macrophages from patients with chronic obstructive pulmonary disease (COPD) are defective in their ability to phagocytose apoptotic cells, with a similar defect in response to cigarette smoke. The exact mechanisms for this defect are unknown. Sphingolipids including ceramide, sphingosine and sphingosine-1-phosphate (S1P) are involved in diverse cellular processes and we hypothesised that a comprehensive analysis of this system in alveolar macrophages in COPD may help to delineate the reasons for defective phagocytic function.

Methods

We compared mRNA expression of sphingosine kinases (SPHK1/2), S1P receptors (S1PR1-5) and S1P-degrading enzymes (SGPP1, SGPP2, SGPL1) in bronchoalveolar lavage-derived alveolar macrophages from 10 healthy controls, 7 healthy smokers and 20 COPD patients (10 current- and 10 ex-smokers) using Real-Time PCR. Phagocytosis of apoptotic cells was investigated using flow cytometry. Functional associations were assessed between sphingosine signalling system components and alveolar macrophage phagocytic ability in COPD. To elucidate functional effects of increased S1PR5 on macrophage phagocytic ability, we performed the phagocytosis assay in the presence of varying concentrations of suramin, an antagonist of S1PR3 and S1PR5. The effects of cigarette smoking on the S1P system were investigated using a THP-1 macrophage cell line model.

Results

We found significant increases in SPHK1/2 (3.4- and 2.1-fold increases respectively), S1PR2 and 5 (4.3- and 14.6-fold increases respectively), and SGPL1 (4.5-fold increase) in COPD vs. controls. S1PR5 and SGPL1 expression was unaffected by smoking status, suggesting a COPD “disease effect” rather than smoke effect per se. Significant associations were noted between S1PR5 and both lung function and phagocytosis. Cigarette smoke extract significantly increased mRNA expression of SPHK1, SPHK2, S1PR2 and S1PR5 by THP-1 macrophages, confirming the results in patient-derived macrophages. Antagonising SIPR5 significantly improved phagocytosis.

Conclusion

Our results suggest a potential link between the S1P signalling system and defective macrophage phagocytic function in COPD and advise therapeutic targets.     

Erythrocytes serve as a reservoir for cellular and extracellular sphingosine 1‐phosphate

Sphingosine 1‐phosphate (S1P) in blood is phosphorylated, stored, and transported by red blood cells (RBC). Release of S1P from RBC into plasma is a regulated process that does not occur in plasma‐ or serum‐free media. Plasma fractionation and incubations with isolated and recombinant proteins identified high density lipoprotein (HDL) and serum albumin (SA) as non‐redundant endogenous triggers for S1P release from RBC. S1P bound to SA and HDL was able to stimulate the S1P₁ receptor in calcium flux experiments. The binding capability of acceptor molecules triggers S1P release, as demonstrated with the anti‐S1P antibody Sphingomab?. More S1P was extracted from RBC membranes by HDL than by SA. Blood samples from anemic patients confirmed a reduced capacity for S1P release in plasma. In co‐cultures of RBC and endothelial cells (EC), we observed transcellular transportation of S1P as a second function of RBC‐associated S1P in the absence of SA and HDL and during tight RBC‐EC contact, mimicking conditions in tissue interstitium and capillaries. In contrast to S1P bound to SA and HDL, RBC‐associated S1P was significantly incorporated by EC after S1P lyase (SGPL1) inhibition. RBC‐associated S1P, therefore, has two functions: (1) It contributes to the cellular pool of SGPL1‐sensitive S1P in tissues after transcellular transportation and (2) it helps maintain extracellular S1P levels via SA and HDL independently from SGPL1 activity. J. Cell. Biochem. 109: 1232–1243, 2010. © 2010 Wiley‐Liss, Inc.     

S1P-lyase deficiency uncouples ganglioside formation – Potential contribution to tumorigenic capacity

Sphingosine-1-phosphate (S1P) is not only a catabolic intermediate of all sphingolipids but also an evolutionary conserved bioactive lipid with critical functions in cell survival, differentiation, and migration as well as in immunity and angiogenesis. S1P-lyase (SGPL1) irreversibly cleaves S1P in the final step of sphingolipid catabolism. As sphingoid bases and their 1-phosphates are not only metabolic intermediates but also highly bioactive lipids that modulate a wide range of physiological processes, it would be predicted that their elevation might induce adjustments in other facets of sphingolipid metabolism and/or alter cell behavior. We actually found in a previous study that in terminally differentiated neurons SGPL1 deficiency increases sphingolipid formation via recycling at the expense of de novo synthesis. We now investigated whether and how SGPL1 deficiency affects the metabolism of (glyco)sphingolipids in mouse embryonic fibroblasts (MEFs). According to our previous experiments in neurons, we found a strong accumulation of S1P in SGPL1-deficient MEFs. Surprisingly, a completely different situation arose as we analyzed sphingolipid metabolism in this non-differentiated cell type. The production of biosynthetic precursors of complex glycosphingolipids including ceramide, glucosylceramide and also ganglioside GM3 via de novo synthesis and recycling pathway was substantially increased whereas the amount of more complex gangliosides dropped significantly.     

Regulation of GDF9 and CDKN1B expression in Tibetan sheep testes during different stages of maturity

Normal spermatogenesis is heavily dependent on the balance of germ cell proliferation, differentiation and apoptosis. Growth differentiation factor 9 (GDF9) and cyclin-dependent kinase inhibitor 1 B (CDKN1B) are strongly associated with cell cycle transition from G0/G1 to S and G2/M phase and hence regulating the growth and development of testicular germ cells and somatic cells. The current study was aimed at seeking out scientific evidence to determine if GDF9 and CDKN1B gene expression functions in the development of Tibetan sheep testes. To this end, developmental testes were derived from three-month-old (pre-puberty), one-year-old (sexual maturity), and three-year-old (adult) Tibetan sheep and then the expression and localization patterns of GDF9 and CDKN1B in these testes were evaluated using quantitative real-time PCR (qRT-PCR), Western blot and immunofluorescence. qRT-PCR and Western blot results showed that GDF9 and CDKN1B were detected in the testes throughout the different developmental stages. The abundance of GDF9 mRNA and protein in the testes of one- and three-year-old Tibetan sheep were higher than that in the testes of three-month-old Tibetan sheep; the mRNA and protein abundance of the CDKN1B gene in three-month-old Tibetan sheep testes were higher than that in the testes of the one-and three-year-old sheep. Moreover, immunofluorescence results suggested that the GDF9 protein was expressed in spermatogonia and Leydig cells, and that the CDKN1B protein was localized mainly in Leydig cells with some in the seminiferous epithelium throughout developmental stages. This indicated a novel role of the GDF9 and CDKN1B genes in Leydig cell development over and above their known roles in germ cell development. These findings have significant implications for our understanding of the molecular mechanisms of GDF9 and CDKN1B genes in Tibetan sheep spermatogenesis.     

Legionella pneumophila restrains autophagy by modulating the host's sphingolipid metabolism

Sphingolipids are bioactive molecules playing a key role as membrane components, but they are also central regulators of many intracellular processes including macroautophagy/autophagy. In particular, sphingosine-1-phosphate (S1P) is a critical mediator that controls the balance between sphingolipid-induced autophagy and cell death. S1P levels are adjusted via S1P synthesis, dephosphorylation or degradation, catalyzed by SGPL1 (sphingosine-1-phosphate lyase 1). Intracellular pathogens are able to modulate many different host cell pathways to allow their replication. We have found that infection of eukaryotic cells with the human pathogen Legionella pneumophila triggers a change in the host cell sphingolipid metabolism and specifically affects the levels of sphingosine. Indeed, L. pneumophila secretes a protein highly homologous to eukaryotic SGPL1 (named LpSPL). We solved the crystal structure of LpSPL and showed that it encodes lyase activity, targets the host's sphingolipid metabolism, and plays a role in starvation-induced autophagy during L. pneumophila infection to promote intracellular survival.     

Sphingosine-1-phosphate and ceramide are associated with health and atresia of bovine ovarian antral follicles

The follicle destiny towards ovulation or atresia is multi-factorial in nature and involves outcries, paracrine and endocrine factors that promote cell proliferation and survival (development) or unchain apoptosis as part of the atresia process. In several types of cells, sphingosine-1-phospate (S1P) promotes cellular proliferation and survival, whereas ceramide (CER) triggers cell death, and the S1P/CER ratio may determine the fate of the cell. The aim of present study was to quantify S1P and CER concentrations and their ratio in bovine antral follicles of 8 to 17 mm classified as healthy and atretic antral follicles. Follicles were dissected from cow ovaries collected from a local abattoir. The theca cell layer, the granulosa cells and follicular fluid were separated, and 17β-estradiol (E2) and progesterone (P4) concentrations were measured in the follicular fluid by radioimmunoassay. Based on the E2/P4 ratio, the follicles were classified as healthy (2.2±0.3) or atretic (0.2±0.3). In both follicular compartments (granulosa and theca cell layer), sphingolipids were extracted and S1P and CER concentrations were quantified by HPLC (XTerra RP18; 5 µm, 3.0×150 mm column). Results showed that in both follicular compartments, S1P concentrations were higher in healthy antral follicles than in atretic antral follicles (P<0.05). The concentration of CER in the granulosa cells was higher in atretic antral follicles than in healthy antral follicles, but no differences were observed in the theca cell layer. The S1P/CER ratio in both follicular compartments was also higher in healthy antral follicles. Interestingly, in these follicles, there was a 45-fold greater concentration of S1P than CER in the granulosa cells (P<0.05), whereas in the theca cell layer, S1P had only a 14-fold greater concentration than CER when compared with atretic antral follicles. These results suggest that S1P plays a role in follicle health, increasing cellular proliferation and survival. In contrast, reduction of S1P and the S1P/CER in the antral follicle could trigger cellular death and atresia.     

Specific Disruption of Tsc1 in Ovarian Granulosa Cells Promotes Ovulation and Causes Progressive Accumulation of Corpora Lutea

Tuberous sclerosis complex 1 (Tsc1) is a tumor suppressor negatively regulating mammalian target of rapamycin complex 1 (mTORC1). It is reported that mice lacking Tsc1 gene in oocytes show depletion of primordial follicles, resulting in premature ovarian failure and subsequent infertility. A recent study indicated that deletion of Tsc1 in somatic cells of the reproductive tract caused infertility of female mice. However, it is not known whether specific disruption of Tsc1 in granulosa cells influences the reproductive activity of female mice. To clarify this problem, we mated Tsc1^flox/flox mice with transgenic mice strain expressing cyp19-cre which exclusively expresses in granulosa cells of the ovary. Our results demonstrated that Tsc1^flox/flox; cyp19-cre mutant mice were fertile, ovulating more oocytes and giving birth to more pups than control Tsc1^flox/flox mice. Progressive accumulation of corpora lutea occurred in the Tsc1^flox/flox; cyp19-cre mutant mice with advanced age. These phenotypes could be explained by the elevated activity of mTORC1, as indicated by increased phosphorylation of rpS6, a substrate of S6 in the Tsc1^flox/flox; cyp19-cre mutant granulosa cells. In addition, rapamycin, a specific mTORC1 inhibitor, effectively rescued the phenotype caused by increased mTORC1 activity in the Tsc1^cko ovaries. Our data suggest that conditional knockout of Tsc1 in granulosa cells promotes reproductive activity in mice.     

Genome-wide analysis of germ cell proliferation in C.elegans identifies VRK-1 as a key regulator of CEP-1/p53

Proliferating germ cells in Caenorhabditiselegans provide a useful model system for deciphering fundamental mechanisms underlying the balance between proliferation and differentiation. Using gene expression profiling, we identified approximately 200 genes upregulated in the proliferating germ cells of C. elegans. Functional characterization using RNA-mediated interference demonstrated that over forty of these factors are required for normal germline proliferation and development. Detailed analysis of two of these factors defined an important regulatory relationship controlling germ cell proliferation. We established that the kinase VRK-1 is required for normal germ cell proliferation, and that it acts in part to regulate CEP-1(p53) activity. Loss of cep-1 significantly rescued the proliferation defects of vrk-1 mutants. We suggest that VRK-1 prevents CEP-1 from triggering an inappropriate cell cycle arrest, thereby promoting germ cell proliferation. This finding reveals a previously unsuspected mechanism for negative regulation of p53 activity in germ cells to control proliferation.     

Seasonal changes in the expression of PACAP,VPAC1, VPAC2, PAC1 and testicular activity in the testis of the muskrat (Ondatra zibethicus)

Pituitary adenylate cyclase-activating polypeptide (PACAP) plays an important role in the steroidogenesis and spermatogenesis in the testis through its receptors PAC1, VPAC1 and VPAC2. In this study, we investigated the seasonal expressions of PACAP, PAC1, VPAC1, VPAC2, luteinizing hormone receptor (LHR), follicle stimulating hormone receptor (FSHR), steroidogenic acute regulatory protein (StAR), 3β-hydroxysteroid dehydrogenase (3β-HSD) and CYP17A1 in the testis of male muskrat during the breeding season and non-breeding season, respectively. Histologically, we observed the presence of Leydig cells, Sertoli cells and various types of germ cells in the testis during the breeding season, yet only Leydig cells, Sertoli cells, spermatogonia and primary spermatocyte during the non-breeding season. In addition, the immunohistochemical localizations of PACAP and VPAC1 were identified in the Leydig cells, spermatogonia and spermatozoa during the breeding season, while only in the Leydig cells and spermatogonia during the non-breeding season, and PAC1 and VPAC2 were localized in the Leydig cells in both seasons, among which LHR, StAR, 3β-HSD and CYP17A1 were also expressed. Meanwhile, the protein and mRNA expression levels of PACAP, PAC1, VPAC1, VPAC2, LHR, FSHR, StAR, 3β-HSD and CYP17A1 in the testis during the breeding season were significantly higher than those during the non-breeding season. These results suggested that PACAP is involved in the regulation of steroidogenesis and spermatogenesis via an endocrine, autocrine or paracrine manner in the testis of muskrat.Key words: Pituitary adenylate cyclase-activating peptide (PACAP), PACAP receptors, steroidogenesis, testis, Ondatra zibethicus.     

Sphingosine-1-Phosphate Lyase Deficient Cells as a Tool to Study Protein Lipid Interactions

Cell membranes contain hundreds to thousands of individual lipid species that are of structural importance but also specifically interact with proteins. Due to their highly controlled synthesis and role in signaling events sphingolipids are an intensely studied class of lipids. In order to investigate their metabolism and to study proteins interacting with sphingolipids, metabolic labeling based on photoactivatable sphingoid bases is the most straightforward approach. In order to monitor protein-lipid-crosslink products, sphingosine derivatives containing a reporter moiety, such as a radiolabel or a clickable group, are used. In normal cells, degradation of sphingoid bases via action of the checkpoint enzyme sphingosine-1-phosphate lyase occurs at position C2-C3 of the sphingoid base and channels the resulting hexadecenal into the glycerolipid biosynthesis pathway. In case the functionalized sphingosine looses the reporter moiety during its degradation, specificity towards sphingolipid labeling is maintained. In case degradation of a sphingosine derivative does not remove either the photoactivatable or reporter group from the resulting hexadecenal, specificity towards sphingolipid labeling can be achieved by blocking sphingosine-1-phosphate lyase activity and thus preventing sphingosine derivatives to be channeled into the sphingolipid-to-glycerolipid metabolic pathway. Here we report an approach using clustered, regularly interspaced, short palindromic repeats (CRISPR)-associated nuclease Cas9 to create a sphingosine-1-phosphate lyase (SGPL1) HeLa knockout cell line to disrupt the sphingolipid-to-glycerolipid metabolic pathway. We found that the lipid and protein compositions as well as sphingolipid metabolism of SGPL1 knock-out HeLa cells only show little adaptations, which validates these cells as model systems to study transient protein-sphingolipid interactions.     

Seasonal Expression of Androgen Receptor,Aromatase, and Estrogen Receptor Alpha and Beta in the Testis of the Wild Ground Squirrel (Citellus Dauricus Brandt)

The aim of this study was to investigate the seasonal expression of androgen receptor (AR), estrogen receptors α and β (ERα and ERβ) and aromatase cytochrome P450 (P450arom) mRNA and protein by real-time PCR and immunohistochemistry in the wild ground squirrel (WGS) testes. Histologically, all types of spermatogenic cells including mature spermatozoa were identified in the breeding season (April), while spermatogonia and primary spermatocytes were observed in the nonbreeding season (June), and spermatogonia, primary spermatocytes and secondary spermatocytes were found in pre-hibernation (September). AR was present in Leydig cells, peritubular myoid cells and Sertoli cells in the breeding season and pre-hibernation with more intense staining in the breeding season, whereas AR was only found in Leydig cells in the nonbreeding season; P450arom was expressed in Leydig cells, Sertoli cells and germ cells during the breeding season, whereas P450arom was found in Leydig cells and Sertoli cells during pre-hibernation, but P450arom was not present in the nonbreeding season; Stronger immunohistochemical signal for ERα was present in Sertoli cells and Leydig cells during the breeding season; ERβ was only expressed in Leydig cells of the breeding season. Consistent with the immunohistochemical results, the mean mRNA level of AR, P450arom, ERα and ERβ were higher in the testes of the breeding season when compared to pre-hibernation and the nonbreeding season. These results suggested that the seasonal changes in spermatogenesis and testicular recrudescence and regression process in WGSs might be correlated with expression levels of AR, P450arom and ERs, and that estrogen and androgen may play an important autocrine/paracrine role to regulate seasonal testicular function.Key words: Wild ground squirrels, testes, seasonal expression, androgen and estrogen receptors, aromatase cytochrome P450, Citellus dauricus Brandt