Deoxycholate induces the preferential hydrolysis of polyphosphoinositides by human platelet and rat corneal phospholipase C

Deoxycholate promotes phospholipase C degradation of endogenous phosphatidyl[3H]inositol (Pl), phosphatidyl[3H]inositol monophosphate (PIP) and phosphatidyl[3H]inositol bisphosphate (PIP2) in rat cornea and human platelets. Hydrolysis of phosphatidyl[3H]inositol significantly lags polyphospho[3H]inositide degradation. Concomitantly, formation of [3H]inositol monophosphate (IP1) lags behind [3H]inositol bisphosphate (IP2) and [3H]inositol trisphosphate (IP3) production. These results demonstrate that rat cornea and human platelet phospholipase C cause a preferential hydrolysis of the endogenous polyphosphoinositides rather than phosphatidylinositol.     

Association of alpha- and beta-subunits during the biosynthesis of beta-hexosaminidase in cultured human fibroblasts

Subunit association of beta-hexosaminidase was studied in intact fibroblasts using antisera that discriminate between free and associated alpha-chains. These were anti-beta-hexosaminidase A (anti-alpha beta), which precipitated all alpha-chains, free or associated; anti-beta-hexosaminidase B (anti-beta beta), which precipitated those alpha-chains that were associated with beta; and anti-alpha-chains, which recognized only monomeric alpha-chains. After biosynthetic labeling, beta-hexosaminidase or its free alpha-subunit were immuno-precipitated from extracts of cells and medium with the aid of protein A-bearing Staphylococcus aureus, subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and visualized by fluorography. Pulse-chase labeling showed that the alpha-chains existed predominantly in the monomeric precursor form during the first 5 h, and then began to accumulate in the mature (lysosomal) associated alpha beta form. Precursor alpha beta complexes were secreted, along with some precursor alpha monomers; the latter were catalytically inert. Both alpha- and beta-chains were phosphorylated (a Golgi modification) prior to association. Thus alpha-beta association probably occurred in the Golgi area before transfer to lysosomes and before secretion. Cycloheximide inhibited the association and subsequent maturation of preformed alpha-chains, perhaps by causing a depletion of a pool of beta-chain precursor upstream from the site of subunit association. In fibroblasts from a patient with Sandhoff disease, that produced no beta-chains, the alpha-chains self-associated but their maturation was markedly decreased. We suggest that association with beta-chains is necessary not only for acquisition of catalytic activity but also for transport of alpha-chains to lysosomes.     

Sphingosine-1-phosphate Phosphatase 1 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.     

Kidney sulfatides in mouse models of inherited glycosphingolipid disorders: determination by nano-electrospray ionization tandem mass spectrometry

Sulfatides show structural, and possibly physiological similarities to gangliosides. Kidney dysfunction might be correlated with changes in sulfatides, the major acidic glycosphingolipids in this organ. To elucidate their in vivo metabolic pathway these compounds were analyzed in mice afflicted with inherited glycosphingolipid disorders. The mice under study lacked the genes encoding either beta-hexosaminidase alpha-subunit (Hexa-/-), the beta-hexosaminidase beta-subunit (Hexb-/-), both beta-hexosaminidase alpha and beta-subunits (Hexa-/- and Hexb-/-), GD3 synthase (GD3S-/-), GD3 synthase and GalNAc transferase (GD3S-/- and GalNAcT-/-), GM2 activator protein (Gm2a-/-), or arylsulfatase A (ASA-/-). Quantification of the sulfatides, I(3)SO(3)(-)-GalCer (SM4s), II(3)SO(3)(-)-LacCer (SM3), II(3)SO(3)(-)-Gg(3)Cer (SM2a), and IV(3,) II(3)-(SO(3)(-))(2)-Gg(4)Cer (SB1a), was performed by nano-electrospray tandem mass spectrometry. We conclude for the in vivo situation in mouse kidneys that: 1) a single enzyme (GalNAc transferase) is responsible for the synthesis of SM2a and GM2 from SM3 and GM3, respectively. 2) In analogy to GD1a, SB1a is degraded via SM2a. 3) SM2a is hydrolyzed to SM3 by beta-hexosaminidase S (Hex S) and Hex A, but not Hex B. Both enzymes are supported by GM2-activator protein. 4) Arylsulfatase A is required to degrade SB1a. It is probably the sole sphingolipid-sulfatase cleaving the galactosyl-3-sulfate bond. In addition, a human Tay-Sachs patient's liver was investigated, which showed accumulation of SM2a along with GM2 storage. The different ceramide compositions of both compounds indicated they were probably derived from different cell types. These data demonstrate that in vivo the sulfatides of the ganglio-series follow the same metabolic pathways as the gangliosides with the replacement of sulfotransferases and sulfatases by sialyltransferases and sialidases. Furthermore, a novel neutral GSL, IV(6)GlcNAcbeta-Gb(4)Cer, was found to accumulate only in Hexa-/- and Hexb-/- mouse kidneys. From this we conclude that Hex S also efficiently cleaves terminal beta1-6-linked HexNAc residues from neutral GSLs in vivo.     

Sphingosine-1-phosphate receptors and the development of the vascular system

Extracellular sphingolipid signaling has been implicated as an essential event in vascular development. Sphingosine-1-phosphate (S1P), through interactions with G protein-coupled receptors, regulates functions of endothelial and smooth muscle cells (SMCs)-the major cell types of the vasculature. The knockout of the gene encoding the S1P1 receptor (formally known as Edg-1) in mice blocks vascular maturation, the process where SMCs and pericytes envelop nascent endothelial tubes. The question that remains is how stimulation of S1P receptors controls this critical event in the developmental sequence leading to the formation of functional blood vessels.     

Proteome analysis of the rat cornea during angiogenesis

Angiogenesis is the formation of new blood vessels from the pre-existing vasculature. However, the study of this complex process is often hampered by the lack of a suitable cell-based model and the tools to study the biochemical events that lead to new blood vessel growth. The most widely accepted model for angiogenesis is the in vivo rat corneal model. In this model, the cornea, which is normally an avascular tissue, is stimulated to undergo angiogenesis in response to silver nitrate cauterization or to the implantation of an exogenous angiogenic agent. The physical changes associated with the new vessel growth can be readily monitored visually, but the regulated biochemical events that result in the growth and remodeling of the new vessels are much more challenging to decipher. In this report, a proteomics approach is evaluated for its utility in deciphering the biochemical events during a time course of angiogenic stimulation. At various time points post-silver nitrate cautery, corneas were harvested, solubilized, and analyzed by two-dimensional gel electrophoresis. Protein expression profiles at the various stages of angiogenesis were compared to those of control corneas. One hundred and eleven differentially-expressed proteins were identified by either matrix-assisted laser desorption/ionization-time of flight mass spectrometry or liquid chromatography-coupled electrospray ionization tandem mass spectrometry. Many of the proteins up-regulated during the angiogenesis process were identified as blood-related proteins, thus validating the development of functional blood vessels in the normally avascular tissue of the cornea. Furthermore, detection of differentially-regulated proteins in cauterized versus control tissue clearly validated the utility of a proteomics approach to study this model of angiogenesis. However, in order to get at the key regulatory proteins in the angiogenesis process, it is clear that additional scale-up and enrichment approaches will be required.     

Depletion of ceramides with very long chain fatty acids causes defective skin permeability barrier function, and neonatal lethality in ELOVL4 deficient mice

Very long chain fatty acids (VLCFA), either free or as components of glycerolipids and sphingolipids, are present in many organs. Elongation of very long chain fatty acids-4 (ELOVL4) belongs to a family of 6 members of putative fatty acid elongases that are involved in the formation of VLCFA. Mutations in ELOVL4 were found to be responsible for an autosomal dominant form of Stargardt's-like macular dystrophy (STGD3) in human. We have previously disrupted the mouse Elovl4 gene, and found that Elovl4+/- mice were developmentally normal, suggesting that haploinsufficiency of ELOVL4 is not a cause for the juvenile retinal degeneration in STGD3 patients. However, Elovl4-/- mice died within several hours of birth for unknown reason(s). To study functions of ELOVL4 further, we have explored the causes for the postnatal lethality in Elovl4-/- mice. Our data indicated that the mutant mice exhibited reduced thickness of the dermis, delayed differentiation of keratinocytes, and abnormal structure of the stratum corneum. We showed that all Elovl4-/- mice exhibited defective skin water permeability barrier function, leading to the early postnatal death. We further showed that the absence of ELOVL4 results in depletion in the epidermis of ceramides with omega-hydroxy very long chain fatty acids (> or = C28) and accumulation of ceramides with non omega-hydroxy fatty acids of C26, implicating C26 fatty acids as possible substrates of ELOVL4. These data demonstrate that ELOVL4 is required for VLCFA synthesis that is essential for water permeability barrier function of skin.     

Plexin-B1 activates NF-κB and IL-8 to promote a pro-angiogenic response in endothelial cells

Background

The semaphorins and their receptors, the plexins, are proteins related to c-Met and the scatter factors that have been implicated in an expanding signal transduction network involving co-receptors, RhoA and Ras activation and deactivation, and phosphorylation events. Our previous work has demonstrated that Semaphorin 4D (Sema4D) acts through its receptor, Plexin-B1, on endothelial cells to promote angiogenesis in a RhoA and Akt-dependent manner. Since NF-κB has been linked to promotion of angiogenesis and can be activated by Akt in some contexts, we wanted to examine NF-κB in Sema4D treated cells to determine if there was biological significance for the pro-angiogenic phenotype observed in endothelium.

Methods/Principal Findings

Using RNA interference techniques, gel shifts and NF-κB reporter assays, we demonstrated NF-κB translocation to the nucleus in Sema4D treated endothelial cells occurring downstream of Plexin-B1. This response was necessary for endothelial cell migration and capillary tube formation and protected endothelial cells against apoptosis as well, but had no effect on cell proliferation. We dissected Plexin-B1 signaling with chimeric receptor constructs and discovered that the ability to activate NF-κB was dependent upon Plexin-B1 acting through Rho and Akt, but did not involve its role as a Ras inhibitor. Indeed, inhibition of Rho by C3 toxin and Akt by {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 blocked Sema4D-mediated endothelial cell migration and tubulogenesis. We also observed that Sema4D treatment of endothelial cells induced production of the NF-κB downstream target IL-8, a response necessary for angiogenesis. Finally, we could show through co-immunofluorescence for p65 and CD31 that Sema4D produced by tumor xenografts in nude mice activated NF-κB in vessels of the tumor stroma.

Conclusion/Significance

These findings provide evidence that Sema4D/Plexin-B1-mediated NF-κB activation and IL-8 production is critical in the generation a pro-angiogenic phenotype in endothelial cells and suggests a new therapeutic target for the anti-angiogenic treatment of some cancers.     

Conditional LoxP-flanked glucosylceramide synthase allele controlling glycosphingolipid synthesis

Glycosphingolipids are organizational building blocks of plasma membranes that participate in key cellular functions, such as signaling and cell-to-cell interactions. Glucosylceramide synthase--encoded by the Ugcg gene--controls the first committed step in the major pathway of glycosphingolipid synthesis. Global disruption of the Ugcg gene in mice is lethal during gastrulation. We have now established a Ugcg allele flanked by loxP sites (floxed). When cre recombinase was expressed in the nervous system under control of the nestin promoter, the floxed gene underwent recombination, resulting in a substantial reduction of Ugcg expression and of glycosphingolipid ganglio-series levels. The mice deficient in Ugcg expression in the nervous system show a striking loss of Purkinje cells and abnormal neurologic behavior. The floxed Ugcg allele will facilitate analysis of the function of glycosphingolipids in development, physiology, and in diseases such as diabetes and cancer.     

Regulation of limb development by the sphingosine 1-phosphate receptor S1p1/EDG-1 occurs via the hypoxia/VEGF axis

Angiogenesis, also known as new blood vessel formation, is regulated coordinately with other tissue differentiation events during limb development. Although vascular endothelial cell growth factor (VEGF) is important in the regulation of angiogenesis, chondrogenesis and osteogenesis during limb development, the role of other angiogenic factors is not well understood. Sphingosine 1-phosphate, a platelet-derived lipid mediator, regulates angiogenesis and vascular maturation via its action on the G-protein-coupled receptor S1P(1) (also known as EDG-1). In addition to vascular defects, abnormal limb development was also observed in S1p(1)(-/-) mice. Here we show that strong induction of S1P(1) expression is observed in the blood vessels and the interdigital mesenchymal cells during limb development. Deletion of S1P(1) results in aberrant chondrocyte condensation and defective digit morphogenesis. Interestingly, the vasculature in the S1p(1)(-/-) limbs was hyperplastic and morphologically altered. In addition, the hypoxia inducible factor (HIF)-1 alpha and its response gene VEGF were induced in S1p(1)(-/-) limbs. However, aberrant regulation of HIF-1 alpha and VEGF were not observed in embryonic fibroblasts derived from S1p(1)(-/-) mice, suggesting a non-cell autonomous effect of S1P(1) on VEGF expression. Indeed, similar limb defects were observed in endothelium-specific S1P(1) null mice in vivo. These data suggest that the function of S1P(1) in the developing vasculature is essential for proper limb development.