Galina Tuymetova

S1P1 receptor directs the release of immature B cells from bone marrow into blood

S1P1 receptor expression is required for the egress of newly formed T cells from the thymus and exit of mature T and B cells from secondary lymphoid organs. In this study, we deleted the expression of the S1P1 receptor gene (S1pr1) in developing B cells in the bone marrow. Although B cell maturation within the bone marrow was largely normal in the B cell–specific S1pr1 knockout (B-S1pr1KO) mice, their newly generated immature B cells appeared in the blood at abnormally low numbers as compared with control mice. In the bone marrow of B-S1pr1KO mice, immature B cells in contact with the vascular compartment displayed increased apoptosis as compared with control mice. Forced expression of CD69, a negative regulator of S1P1 receptor expression, in developing bone marrow B cells also reduced the number of immature B cells in the blood. Attenuation of CXCR4 signaling, which is required for the proper retention of developing B cells in bone marrow, did not release immature B cells into the blood of B-S1pr1KO mice as effectively as in control mice. Our results indicate that the S1P1 receptor provides a signal necessary for the efficient transfer of newly generated immature B cells from the bone marrow to the blood.

Sphingosine-1-phosphate Lyase Deficiency Produces a Pro-inflammatory Response While Impairing Neutrophil Trafficking

Sphingosine-1-phosphate (S1P) lyase catalyzes the degradation of S1P, a potent signaling lysosphingolipid. Mice with an inactive S1P lyase gene are impaired in the capacity to degrade S1P, resulting in highly elevated S1P levels. These S1P lyase-deficient mice have low numbers of lymphocytes and high numbers of neutrophils in their blood. We found that the S1P lyase-deficient mice exhibited features of an inflammatory response including elevated levels of pro-inflammatory cytokines and an increased expression of genes in liver associated with an acute-phase response. However, the recruitment of their neutrophils into inflamed tissues was impaired and their neutrophils were defective in migration to chemotactic stimulus. The IL-23/IL-17/granulocyte-colony stimulating factor (G-CSF) cytokine-controlled loop regulating neutrophil homeostasis, which is dependent on neutrophil trafficking to tissues, was disturbed in S1P lyase-deficient mice. Deletion of the S1P4 receptor partially decreased the neutrophilia and inflammation in S1P lyase-deficient mice, implicating S1P receptor signaling in the phenotype. Thus, a genetic block in S1P degradation elicits a pro-inflammatory response but impairs neutrophil migration from blood into tissues.

Sphingosine kinase 1 and sphingosine-1-phosphate receptor 2 are vital to recovery from anaphylactic shock in mice

Sphingosine kinase 1 (SphK1) and SphK2 are ubiquitous enzymes that generate sphingosine-1-phosphate (S1P), a ligand for a family of G protein-coupled receptors (S1PR1-S1PR5) with important functions in the vascular and immune systems. Here we explore the role of these kinases and receptors in recovery from anaphylaxis in mice. We found that Sphk2-/- mice had a rapid recovery from anaphylaxis. In contrast, Sphk1-/- mice showed poor recovery from anaphylaxis and delayed histamine clearance. Injection of S1P into Sphk1-/- mice increased histamine clearance and promoted recovery from anaphylaxis. Adoptive cell transfer experiments demonstrated that SphK1 activity was required in both the hematopoietic and nonhematopoietic compartments for recovery from anaphylaxis. Mice lacking the S1P receptor S1PR2 also showed a delay in plasma histamine clearance and a poor recovery from anaphylaxis. However, S1P did not promote the recovery of S1pr2-/- mice from anaphylaxis, whereas S1pr2+/- mice showed partial recovery. Unlike Sphk2-/- mice, Sphk1-/- and S1pr2-/- mice had severe hypotension during anaphylaxis. Thus, SphK1-produced S1P regulates blood pressure, histamine clearance, and recovery from anaphylaxis in a manner that involves S1PR2. This suggests that specific S1PR2 agonists may serve to counteract the vasodilation associated with anaphylactic shock.

S1P1 receptor expression regulates emergence of NKT cells in peripheral tissues

The S1P1 receptor, on the surface of lymphocytes and endothelial cells, regulates the unique trafficking behavior of certain lymphocyte populations. We have examined whether the S1P1 receptor also dictates the distinctive tissue distribution of Vα14‐Jα18 natural killer T (NKT) cells, whose trafficking pattern is not well understood. Mice (TCS1P1KO) were established with a conditional deletion of the S1P1 receptor in thymocytes that included precursors of NKT cells. Within the thymus, NKT cells were found at normal or increased levels, indicating that S1P1 receptor expression was dispensable for NKT cell development. However, substantially reduced numbers of NKT cells were detected in the peripheral tissues of the TCS1P1KO mice. Short‐term S1P1 deletion after NKT cells had established residence in the periphery did not substantially alter their distribution in tissues, except for a partial decrease in the spleen. FTY720, a S1P1 receptor ligand that has potent effects on the trafficking of conventional T cells, did not alter the preexisting distribution of NKT cells within peripheral tissues of wild‐type mice. Our results indicate that the S1P1 receptor expression on NKT cells is dispensable for development within thymus but is essential for the establishment of their tissue residency in the periphery.— Allende, M. L., Zhou, D., Kalkofen, D. N., Benhamed, S., Tuymetova, G., Borowski, C., Bendelac, A., Proia, R. L. S1P1 receptor expression regulates emergence of NKT cells in peripheral tissues. FASEB J. 22, 307–315 (2008)

Design of drug-resistant alleles of type-III phosphatidylinositol 4-kinases using mutagenesis and molecular modeling.

Molecular modeling and site directed mutagenesis were used to analyze the structural features determining the unique inhibitor sensitivities of type-III phosphatidylinositol 4-kinase enzymes (PI4Ks). Mutation of a highly conserved Tyr residue that provides the bottom of the hydrophobic pocket for ATP yielded a PI4KIIIbeta enzyme that showed greatly reduced wortmannin sensitivity and was catalytically still active. Similar substitutions were not tolerated in the type-IIIalpha enzyme rendering it catalytically inactive. Two conserved Cys residues located in the active site of PI4KIIIalpha were found responsible for the high sensitivity of this enzyme to the oxidizing agent, phenylarsine oxide. Mutation of one of these Cys residues reduced the phenylarsine oxide sensitivity of the enzyme to the same level observed with the PI4KIIIbeta protein. In search of inhibitors that would discriminate between the closely related PI4KIIIalpha and -IIIbeta enzymes, the PI3Kgamma inhibitor, PIK93, was found to inhibit PI4KIIIbeta with significantly greater potency than PI4KIIIalpha. These studies should aid development of subtype-specific inhibitors of type-III PI4Ks and help to better understand the significance of localized PtdIns4P production by the various PI4Ks isoforms in specific cellular compartments.

Autophagy regulates sphingolipid levels in the liver

Sphingolipid levels are tightly regulated to maintain cellular homeostasis. During pathologic conditions such as in aging, inflammation, and metabolic and neurodegenerative diseases, levels of some sphingolipids, including the bioactive metabolite ceramide, are elevated. Sphingolipid metabolism has been linked to autophagy, a critical catabolic process in both normal cell function and disease; however, the in vivo relevance of the interaction is not well-understood. Here, we show that blocking autophagy in the liver by deletion of the Atg7 gene, which is essential for autophagosome formation, causes an increase in sphingolipid metabolites including ceramide. We also show that overexpression of serine palmitoyltransferase to elevate de novo sphingolipid biosynthesis induces autophagy in the liver. The results reveal autophagy as a process that limits excessive ceramide levels and that is induced by excessive elevation of de novo sphingolipid synthesis in the liver. Dysfunctional autophagy may be an underlying mechanism causing elevations in ceramide that may contribute to pathogenesis in diseases.

Sphingosine-1-phosphate Phosphatase 1 Regulates Keratinocyte Differentiation and Epidermal Homeostasis

Background: Sphingosine-1-phosphate phosphatase 1, encoded by Sgpp1, dephosphorylates the signaling lysophospholipid, S1P; however, its in vivo functions are not well understood. Results: Deletion of Sgpp1 causes epidermal defects with intrinsic keratinocyte abnormalities. Conclusion: Sgpp1 deficiency in keratinocytes causes S1P elevation and accelerates their differentiation. Significance: Intracellular S1P metabolism regulates keratinocyte differentiation and epidermal homeostasis. Sphingosine 1-phosphate (S1P) is a bioactive lipid whose levels are tightly regulated by its synthesis and degradation. Intracellularly, S1P is dephosphorylated by the actions of two S1P-specific phosphatases, sphingosine-1-phosphate phosphatases 1 and 2. To identify the physiological functions of S1P phosphatase 1, we have studied mice with its gene, Sgpp1, deleted. Sgpp1−/− mice appeared normal at birth, but during the 1st week of life they exhibited stunted growth and suffered desquamation, with most dying before weaning. Both Sgpp1−/− pups and surviving adults exhibited multiple epidermal abnormalities. Interestingly, the epidermal permeability barrier developed normally during embryogenesis in Sgpp1−/− mice. Keratinocytes isolated from the skin of Sgpp1−/− pups had increased intracellular S1P levels and displayed a gene expression profile that indicated overexpression of genes associated with keratinocyte differentiation. The results reveal S1P metabolism as a regulator of keratinocyte differentiation and epidermal homeostasis.

Sphingosine-1-phosphate phosphatase 2 regulates pancreatic islet β-cell endoplasmic reticulum stress and proliferation

Sphingosine-1-phosphate (S1P) is a sphingolipid metabolite that regulates basic cell functions through metabolic and signaling pathways. Intracellular metabolism of S1P is controlled, in part, by two homologous S1P phosphatases (SPPases), 1 and 2, which are encoded by the Sgpp1 and Sgpp2 genes, respectively. SPPase activity is needed for efficient recycling of sphingosine into the sphingolipid synthesis pathway. SPPase 1 is important for skin homeostasis, but little is known about the functional role of SPPase 2. To identify the functions of SPPase 2 in vivo, we studied mice with the Sgpp2 gene deleted. In contrast to Sgpp1−/− mice, Sgpp2−/− mice had normal skin and were viable into adulthood. Unexpectedly, WT mice expressed Sgpp2 mRNA at high levels in pancreatic islets when compared with other tissues. Sgpp2−/− mice had normal pancreatic islet size; however, they exhibited defective adaptive β-cell proliferation that was demonstrated after treatment with either a high-fat diet or the β-cell-specific toxin, streptozotocin. Importantly, β-cells from untreated Sgpp2−/− mice showed significantly increased expression of proteins characteristic of the endoplasmic reticulum stress response compared with β-cells from WT mice, indicating a basal islet defect. Our results show that Sgpp2 deletion causes β-cell endoplasmic reticulum stress, which is a known cause of β-cell dysfunction, and reveal a juncture in the sphingolipid recycling pathway that could impact the development of diabetes.

De novo sphingolipid biosynthesis is required for adipocyte survival and metabolic homeostasis

Sphingolipids are a diverse class of essential cellular lipids that function as structural membrane components and as signaling molecules. Cells acquire sphingolipids by both de novo biosynthesis and recycling of exogenous sphingolipids. The individual importance of these pathways for the generation of essential sphingolipids in differentiated cells is not well understood. To investigate the requirement for de novo sphingolipid biosynthesis in adipocytes, a cell type with highly regulated lipid metabolism, we generated mice with an adipocyte-specific deletion of Sptlc1. Sptlc1 is an obligate subunit of serine palmitoyltransferase, the enzyme responsible for the first and rate-limiting step of de novo sphingolipid biosynthesis. These mice, which initially developed adipose tissue, exhibited a striking age-dependent loss of adipose tissue accompanied by evidence of adipocyte death, increased macrophage infiltration, and tissue fibrosis. Adipocyte differentiation was not affected by the Sptlc1 deletion. The mice also had elevated fasting blood glucose, fatty liver, and insulin resistance. Collectively, these data indicate that de novo sphingolipid biosynthesis is required for adipocyte cell viability and normal metabolic function and that reduced de novo sphingolipid biosynthesis within adipocytes is associated with adipocyte death, adipose tissue remodeling, and metabolic dysfunction.