Phosphorylation of muramyl peptides by NAGK is required for NOD2 activation.

Bacterial cell wall components provide various unique molecular structures that are detected by pattern recognition receptors (PRRs) of the innate immune system as non-self. Most bacterial species form a cell wall that consists of peptidoglycan (PGN), a polymeric structure comprising alternating amino sugars that form strands cross-linked by short peptides. Muramyl dipeptide (MDP) has been well documented as a minimal immunogenic component of peptidoglycan1-3. MDP is sensed by the cytosolic nucleotide-binding oligomerization domain-containing protein 24 (NOD2). Upon engagement, it triggers pro-inflammatory gene expression, and this functionality is of critical importance in maintaining a healthy intestinal barrier function5. Here, using a forward genetic screen to identify factors required for MDP detection, we identified N-acetylglucosamine kinase (NAGK) as being essential for the immunostimulatory activity of MDP. NAGK is broadly expressed in immune cells and has previously been described to contribute to the hexosamine biosynthetic salvage pathway6. Mechanistically, NAGK functions upstream of NOD2 by directly phosphorylating the N-acetylmuramic acid moiety of MDP at the hydroxyl group of its C6 position, yielding 6-O-phospho-MDP. NAGK-phosphorylated MDP-but not unmodified MDP-constitutes an agonist for NOD2. Macrophages from mice deficient in NAGK are completely deficient in MDP sensing. These results reveal a link between amino sugar metabolism and innate immunity to bacterial cell walls.

Loss of phosphatidylinositol-4-phosphate 5-kinase type-1 gamma (Pip5k1c) in mesenchymal stem cells leads to osteopenia by impairing bone remodeling.

Phosphatidylinositol-4-phosphate 5-kinase type-1 gamma (Pip5k1c) is a lipid kinase that plays a pivotal role in the regulation of receptor-mediated calcium signaling in multiple tissues; however, its role in the skeleton is not clear. Here, we show that while deleting Pip5k1c expression in the mesenchymal stem cells using Prx1-Cre transgenic mice does not impair the intramembranous and endochondral ossification during skeletal development, it does cause osteopenia in adult mice, but not rapidly growing young mice. We found Pip5k1c loss dramatically decreases osteoblast formation and osteoid and mineral deposition, leading to reduced bone formation. Furthermore, Pip5k1c loss inhibits osteoblastic, but promotes adipogenic, differentiation of bone marrow stromal cells. Pip5k1c deficiency also impairs cytoplasmic calcium influx and inactivates the calcium/calmodulin-dependent protein kinase, which regulates levels of transcription factor Runx2 by modulating its stability and subsequent osteoblast and bone formation. In addition, Pip5k1c loss reduces levels of the receptor activator of nuclear factor-κB ligand, but not that of osteoprotegerin, its decoy receptor, in osteoblasts in bone and in sera. Finally, we found Pip5k1c loss impairs the ability of bone marrow stromal cells to support osteoclast formation of bone marrow monocytes and reduces the osteoclast precursor population in bone marrow, resulting in reduced osteoclast formation and bone resorption. We conclude Pip5k1c deficiency causes a low-turnover osteopenia in mice, with impairment of bone formation being greater than that of bone resorption. Collectively, we uncover a novel function and mechanism of Pip5k1c in the control of bone mass and identify a potential therapeutic target for osteoporosis.

Deficiency of the sedoheptulose kinase (Shpk) does not alter the ability of hematopoietic stem cells to rescue cystinosis in the mouse model.

Cystinosis is an autosomal recessive lysosomal storage disorder caused by mutations in the CTNS gene encoding the lysosomal cystine transporter, cystinosin, and leading to multi-organ degeneration including kidney failure. A clinical trial for cystinosis is ongoing to test the safety and efficacy of transplantation of autologous hematopoietic stem and progenitor cells (HSPCs) ex vivo gene-modified to introduce functional CTNS cDNA. Preclinical studies in Ctns-/- mice previously showed that a single HSPC transplantation led to significant tissue cystine decrease and long-term tissue preservation. The main mechanism of action involves the differentiation of the transplanted HSPCs into macrophages within tissues and transfer of cystinosin-bearing lysosomes to the diseased cells via tunneling nanotubes. However, a major concern was that the most common cystinosis-causing mutation in humans is a 57-kb deletion that eliminates not only CTNS but also the adjacent sedopheptulose kinase SHPK/CARKL gene encoding a metabolic enzyme that influences macrophage polarization. Here, we investigated if absence of Shpk could negatively impact the efficiency of transplanted HSPCs to differentiate into macrophages within tissues and then to prevent cystinosis rescue. We generated Shpk knockout mouse models and detected a phenotype consisting of perturbations in the pentose phosphate pathway (PPP), the metabolic shunt regulated by SHPK. Shpk-/- mice also recapitulated the urinary excretion of sedoheptulose and erythritol found in cystinosis patients homozygous for the 57-kb deletion. Transplantation of Shpk-/--HSPCs into Ctns-/- mice resulted in significant reduction in tissue cystine load and restoration of Ctns expression, as well as improved kidney architecture comparable to WT-HSPC recipients. Altogether, these data demonstrate that absence of SHPK does not alter the ability of HSPCs to rescue cystinosis, and then patients homozygous for the 57-kb deletion should benefit from ex vivo gene therapy and can be enrolled in the ongoing clinical trial. However, because of the limits inherent to animal models, outcomes of this patient population will be carefully compared to the other enrolled subjects.

Inositol polyphosphate multikinase in adipocytes is dispensable for regulating energy metabolism and whole body metabolic homeostasis.

Adipose tissue plays a central role in regulating whole body energy and glucose homeostasis at both organ and systemic levels. Inositol polyphosphates, such as 5-diphosphoinositol pentakisphosphate, reportedly control adipocyte functions and energy expenditure. However, the physiological roles of the inositol polyphosphate (IP) pathway in the adipose tissue are not yet fully defined. The aim of the present study was to test the hypothesis that inositol polyphosphate multikinase (IPMK), a key enzyme in the IP metabolism, plays a critical role in adipose tissue biology and obesity. We generated adipocyte-specific IPMK knockout (Ipmk AKO) mice and evaluated metabolic phenotypes by measuring fat accumulation, glucose homeostasis, and insulin sensitivity in adult mice fed either a regular-chow diet or high-fat diet (HFD). Despite substantial reduction of IPMK, Ipmk AKO mice exhibited normal glucose tolerance and insulin sensitivity and did not show changes in fat accumulation in response to HFD-feeding. In addition, loss of IPMK had no major impact on thermogenic processes in response to cold exposure. Collectively, these findings suggest that adipocyte IPMK is dispensable for normal adipose tissue and its physiological functions in whole body metabolism, suggesting the complex roles that inositol polyphosphate metabolism has in the regulation of adipose tissue.

Physiological relevance of the neuronal isoform of inositol-1,4,5-trisphosphate 3-kinases in mice.

Inositol-1,4,5-trisphosphate 3-kinase-A (ITPKA) is the neuronal isoform of ITPKs and exhibits both actin bundling and InsP3kinase activity. In addition to neurons, ITPKA is ectopically expressed in tumor cells, where its oncogenic activity increases tumor cell malignancy. In order to analyze the physiological relevance of ITPKA, here we performed a broad phenotypic screening of itpka deficient mice. Our data show that among the neurobehavioral tests analyzed, itpka deficient mice reacted faster to a hotplate, prepulse inhibition was impaired and the accelerating rotarod test showed decreased latency of itpka deficient mice to fall. These data indicate that ITPKA is involved in the regulation of nociceptive pathways, sensorimotor gating and motor learning. Analysis of extracerebral functions in control and itpka deficient mice revealed significantly reduced glucose, lactate, and triglyceride plasma concentrations in itpka deficient mice. Based on this finding, expression of ITPKA was analyzed in extracerebral tissues and the highest level was found in the small intestine. However, functional studies on CaCo-2 control and ITPKA depleted cells showed that glucose, as well as triglyceride uptake, were not significantly different between the cell lines. Altogether, these data show that ITPKA exhibits distinct functions in the central nervous system and reveal an involvement of ITPKA in energy metabolism.

The lipogenic LXR-SREBF1 signaling pathway controls cancer cell DNA repair and apoptosis and is a vulnerable point of malignant tumors for cancer therapy.

Cancer cells are defective in DNA repair, so they experience increased DNA strand breaks, genome instability, gene mutagenesis, and tumorigenicity; however, multiple classic DNA repair genes and pathways are strongly activated in malignant tumor cells to compensate for the DNA repair deficiency and gain an apoptosis resistance. The mechanisms underlying this phenomenon in cancer are unclear. We speculate that a key DNA repair gene or signaling pathway in cancer has not yet been recognized. Here, we show that the lipogenic liver X receptor (LXR)-sterol response element binding factor-1 (SREBF1) axis controls the transcription of a key DNA repair gene polynucleotide kinase/phosphatase (PNKP), thereby governing cancer cell DNA repair and apoptosis. Notably, the PNKP levels were significantly reduced in 95% of human pancreatic cancer (PC) patients, particularly deep reduction for sixfold in all of the advanced-stage PC cases. PNKP is also deficient in three other types of cancer that we examined. In addition, the expression of LXRs and SREBF1 was significantly reduced in the tumor tissues from human PC patients compared with the adjacent normal tissues. The newly identified LXR-SREBF1-PNKP signaling pathway is deficient in PC, and the defect in the pathway contributes to the DNA repair deficiency in the cancer. Strikingly, further diminution of the vulnerable LXR-SREBF1-PNKP signaling pathway using a small molecule triptonide, a new LXR antagonist identified in this investigation, at a concentration of 8 nM robustly activated tumor-suppressor p53 and readily elevated cancer cell DNA strand breaks over an apoptotic threshold, and selectively induced PC cell apoptosis, resulting in almost complete elimination of tumors in xenograft mice without obvious complications. Our findings provide new insight into DNA repair and apoptosis in cancer, and offer a new platform for developing novel anticancer therapeutics.

Decrease in Multidrug Resistance-associated Protein 2 Activities by Knockdown of Phosphatidylinositol 4-phosphate 5-kinase in Hepatocytes and Cancer Cells.

PURPOSE: The plasma membrane localization and transport activity of multidrug resistance- associated protein 2 (MRP2/ABCC2) and P-glycoprotein (P-gp/ABCB1) efflux transporters are governed by transporter-associated proteins. Phosphatidylinositol 4,5-bisphosphate (PIP2) formed by phosphatidylinositol 4-phosphate 5-kinase type 1 (PIP5K1) activates the linker function of radixin for efflux transporters. Radixin is involved in the plasma membrane localization of efflux transporters. We examined whether PIP5K1 could be a target for the modulation of transporter activities in hepatocytes and cancer cells. METHODS: The effects of PIP5K1 depletion by siRNA in mouse primary hepatocytes, PANC1 human pancreatic carcinoma cells, and HepG2 human hepatocellular carcinoma cells on the intracellular accumulation of MRP2 and P-gp substrates were examined. RESULTS: PIP5K1A depletion resulted in increased intracellular accumulation of carboxydichlorofluorescein, a MRP2 fluorescent substrate, in mouse primary hepatocytes, PANC1 cells, and HepG2 cells. In PANC1 and HepG2 cells, the transport activities of MRP2 were significantly decreased by PIP5K1C depletion. However, the transport activities of P-gp were unchanged by PIP5K1 depletion. PIP2 levels were unchanged between control and PIP5K1A- or PIP5K1C-depleted HepG2 cells. MRP2 mRNA levels showed few changes in HepG2 cells following PIP5K1A or PIP5K1C depletion. The expression of phosphorylated radixin was decreased by PIP5K1A and PIP5K1C depletion, although total radixin levels were unchanged. CONCLUSIONS: These data suggest that PIP5K1A and PIP5K1C could be target proteins for modulating MRP2 function, partly because of the resulting changes of the linker function of radixin.

Targeting GNE Myopathy: A Dual Prodrug Approach for the Delivery of N-Acetylmannosamine 6-Phosphate.

ProTides comprise an important class of prodrugs currently marketed and developed as antiviral and anticancer therapies. The ProTide technology employs phosphate masking groups capable of providing more favorable druglike properties and an intracellular activation mechanism for enzyme-mediated release of a nucleoside monophosphate. Herein, we describe the application of phosphoramidate chemistry to 1,3,4-O-acetylated N-acetylmannosamine (Ac3ManNAc) to deliver ManNAc-6-phosphate (ManNAc-6-P), a critical intermediate in sialic acid biosynthesis. Sialic acid deficiency is a hallmark of GNE myopathy, a rare congenital disorder of glycosylation (CDG) caused by mutations in GNE that limit the production of ManNAc-6-P. Synthetic methods were developed to provide a library of Ac3ManNAc-6-phosphoramidates that were evaluated in a series of studies for their potential as a treatment for GNE myopathy. Prodrug 12b showed rapid activation in a carboxylesterase (CPY) enzymatic assay and favorable ADME properties, while also being more effective than ManNAc at increasing sialic acid levels in GNE-deficient cell lines. These results provide a potential platform to address substrate deficiencies in GNE myopathy and other CDGs.

Inositol polyphosphate multikinase deficiency leads to aberrant induction of synaptotagmin-2 in the forebrain.

Inositol polyphosphate multikinase (IPMK), the key enzyme responsible for the synthesis of higher inositol polyphosphates and phosphatidylinositol 3, 4, 5-trisphosphate, is known to mediate various biological events, such as cellular growth and metabolism. Conditional deletion of IPMK in excitatory neurons of the mouse postnatal forebrain results in enhanced extinction of fear memory accompanied by activation of p85 S6 kinase 1 signaling in the amygdala; it also facilitates hippocampal long-term potentiation. However, the molecular changes triggered by IPMK deletion in the brain have not been fully elucidated. In the present study, we investigated gene expression changes in the hippocampal region of IPMK conditional knockout (cKO) mice by performing genome-wide transcriptome analyses. Here we show that expression of synaptotagmin 2 (Syt2), a synaptic vesicle protein essential for Ca2+-dependent neurotransmitter release, is robustly upregulated in the forebrain of IPMKcKO mice. Compared to wild-type mice, in which weak Syt2 expression was detected in the forebrain, IPMKcKO mice showed marked increases in both Syt2 mRNA and protein expression in the hippocampus as well as the amygdala. Collectively, our results suggest a physiological role for IPMK in regulating expression of Syt2, providing a potential underlying molecular mechanism to explain IPMK-mediated neural functions.

Sphingosine kinase 1 knockout alleviates hepatic ischemia/reperfusion injury by attenuating inflammation and oxidative stress in mice.

BACKGROUND: Hepatic ischemia/reperfusion (I/R) injury remains a significant problem in clinical practice. Sphingosine kinase 1 (SphK1) phosphorylates sphingosine to sphingosine-1-phosphate (S1P) which participates in multiple bioactive processes. However, little is known about the role of SphK1 in hepatic I/R injury. This study aimed to investigate the effect of SphK1 knockout on liver I/R injury and to explore underlying mechanisms. METHODS: SphK1 knockout and wild type mice were subjected to 70% partial hepatic I/R. Serum alanine aminotransferase was determined to indicate the degree of liver damage. Hematoxylin-eosin staining and TUNEL assay were used to assess histological changes and hepatocellular apoptosis, respectively. Immunohistochemistry was performed to detect the expression and translocation of phosphorylated p65 and signal transducer and activator of transcription 3 (STAT3). Western blotting was used to determine the expression of S1P receptor 1 (S1PR1), phosphorylated p65 and STAT3. Real-time PCR was used to demonstrate the changes of proinflammatory cytokines. Oxidative stress markers were also determined through biochemical assays. RESULTS: SphK1 knockout significantly ameliorated I/R-induced liver damage, mitigated liver tissue necrosis and apoptosis compared with wild type control. I/R associated inflammation was alleviated in SphK1 knockout mice as demonstrated by attenuated expression of S1PR1 and reduced phosphorylation of nuclear factor kappa B p65 and STAT3. The proinflammatory cytokines interleukin-1ß, interleukin-6 and tumor necrosis factor-α were also inhibited by SphK1 genetic deletion. The oxidative stress markers were lower in SphK1 knockout mice after I/R injury than wild type mice. CONCLUSIONS: Knockout of SphK1 significantly alleviated damage after hepatic I/R injury, possibly through inhibiting inflammation and oxidative stress. SphK1 may be a novel and potent target in clinical practice in I/R-related liver injury.

A virus-packageable CRISPR screen identifies host factors mediating interferon inhibition of HIV.

Interferon (IFN) inhibits HIV replication by inducing antiviral effectors. To comprehensively identify IFN-induced HIV restriction factors, we assembled a CRISPR sgRNA library of Interferon Stimulated Genes (ISGs) into a modified lentiviral vector that allows for packaging of sgRNA-encoding genomes in trans into budding HIV-1 particles. We observed that knockout of Zinc Antiviral Protein (ZAP) improved the performance of the screen due to ZAP-mediated inhibition of the vector. A small panel of IFN-induced HIV restriction factors, including MxB, IFITM1, Tetherin/BST2 and TRIM5alpha together explain the inhibitory effects of IFN on the CXCR4-tropic HIV-1 strain, HIV-1LAI, in THP-1 cells. A second screen with a CCR5-tropic primary strain, HIV-1Q23.BG505, described an overlapping, but non-identical, panel of restriction factors. Further, this screen also identifies HIV dependency factors. The ability of IFN-induced restriction factors to inhibit HIV strains to replicate in human cells suggests that these human restriction factors are incompletely antagonized. Editorial note: This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that all the issues have been addressed (see decision letter).

HldE Is Important for Virulence Phenotypes in Enterotoxigenic Escherichia coli.

Enterotoxigenic Escherichia coli (ETEC) is one of the most common causes of diarrheal illness in third world countries and it especially affects children and travelers visiting these regions. ETEC causes disease by adhering tightly to the epithelial cells in a concerted effort by adhesins, flagella, and other virulence-factors. When attached ETEC secretes toxins targeting the small intestine host-cells, which ultimately leads to osmotic diarrhea. HldE is a bifunctional protein that catalyzes the nucleotide-activated heptose precursors used in the biosynthesis of lipopolysaccharide (LPS) and in post-translational protein glycosylation. Both mechanisms have been linked to ETEC virulence: Lipopolysaccharide (LPS) is a major component of the bacterial outer membrane and is needed for transport of heat-labile toxins to the host cells, and ETEC glycoproteins have been shown to play an important role for bacterial adhesion to host epithelia. Here, we report that HldE plays an important role for ETEC virulence. Deletion of hldE resulted in markedly reduced binding to the human intestinal cells due to reduced expression of colonization factor CFA/I on the bacterial surface. Deletion of hldE also affected ETEC motility in a flagella-dependent fashion. Expression of both colonization factors and flagella was inhibited at the level of transcription. In addition, the hldE mutant displayed altered growth, increased biofilm formation and clumping in minimal growth medium. Investigation of an orthogonal LPS-deficient mutant combined with mass spectrometric analysis of protein glycosylation indicated that HldE exerts its role on ETEC virulence both through protein glycosylation and correct LPS configuration. These results place HldE as an attractive target for the development of future antimicrobial therapeutics.

Elevating Endogenous Sphingosine-1-Phosphate (S1P) Levels Improves Endothelial Function and Ameliorates Atherosclerosis in Low Density Lipoprotein Receptor-Deficient (LDL-R-/-) Mice.

BACKGROUND: Sphingosine-1-phosphate (S1P) is a bioactive lysosphingolipid and a constituent of high-density lipoprotein (HDL) exerting several atheroprotective effects in vitro. However, the few studies addressing anti-atherogenic effects of S1P in vivo have led to disparate results. We here examined atherosclerosis development in low-density lipoprotein receptor (LDL-R)-deficient (LDL-R-/-) mice with elevated endogenous S1P levels. METHODS AND RESULTS: Sub-lethally irradiated LDL-R-/- mice were transplanted with bone marrow deficient in sphingosine kinase 2 (SphK2), which led to the elevation of S1P concentrations in erythrocytes, plasma and HDL by approximately 1.5- to 2.0-fold in SphK2-/-/LDL-R-/- mice. Afterwards, mice were fed a Western diet for 14 weeks. Elevation of endogenous S1P significantly reduced atherosclerotic lesion formation by approximately half without affecting the plasma lipid profile. Furthermore, the macrophage content of atherosclerotic lesions and lipopolysaccharide-induced monocyte recruitment to the peritoneal cavity were reduced in SphK2-/-/LDL-R-/- mice. Studies using intra-vital microscopy revealed that endogenous S1P lowered leukocyte adhesion to capillary wall and decreased endothelial permeability to fluorescently labelled LDL. Moreover, SphK2-/-/LDL-R-/- mice displayed decreased levels of vascular cell adhesion molecule 1 in atherosclerotic lesions and in plasma. Studies in vitro demonstrated reduced monocyte adhesion and transport across an endothelial layer exposed to increasing S1P concentrations, murine plasma enriched in S1P or plasma obtained from SphK2-deficient animals. In addition, decreased permeability to fluorescence-labelled dextran beads or LDL was observed in S1P-treated endothelial cells. CONCLUSION: We conclude that raising endogenous S1P levels exerts anti-atherogenic effects in LDL-R-/- mice that are mediated by favourable modulation of endothelial function.

The fucose salvage pathway inhibits invadopodia formation and extracellular matrix degradation in melanoma cells.

The fucose salvage pathway is a two-step process in which mammalian cells transform L-fucose into GDP-L-fucose, a universal fucose donor used by fucosyltransferases to modify glycans. Emerging evidence indicates the fucose salvage pathway and the fucosylation of proteins are altered during melanoma progression and metastasis. However the underlying mechanisms are not completely understood. Here, we report that the fucose salvage pathway inhibits invadopodia formation and extracellular matrix degradation by promoting α-1,2 fucosylation. Chemically or genetically increasing the fucose salvage pathway decreases invadopodium numbers and inhibits the proteolytic activity of invadopodia in WM793 melanoma cells. Inhibiting fucosylation by depleting fucokinase abrogates L-fucose-mediated inhibition of invadopodia, suggesting dependence on the fucose salvage pathway. The inhibition of invadopodium formation by L-fucose or ectopically expressed FUK could be rescued by treatment with α-1,2, but not α-1,3/α-1,4 fucosidase, implicating an α-1,2 fucose linkage-dependent anti-metastatic effect. The expression of FUT1, an α-1,2 fucosyltransferase, is remarkably down-regulated during melanoma progression, and the ectopic expression of FUT1 is sufficient to inhibit invadopodium formation and ECM degradation. Our findings indicate that the fucose salvage pathway can inhibit invadopodium formation, and consequently, invasiveness in melanoma via α-1,2 fucosylation. Re-activation of this pathway in melanoma could be useful for preventing melanoma invasion and metastasis.

Cell-intrinsic sphingosine kinase 2 promotes macrophage polarization and renal inflammation in response to unilateral ureteral obstruction.

Sphingosine Kinase-2 (Sphk2) is responsible for the production of the bioactive lipid Sphingosine-1 Phosphate, a key regulator of tissue repair. Here we address the in vivo significance of Sphingosine Kinase -2 in renal inflammation/fibrosis in response to unilateral ureteral obstruction using both genetic and pharmacological strategies. Obstructed kidneys of Sphk2-/- mice showed reduced renal damage and diminished levels of the renal injury markers TGFß1 and αSMA when compared to wild type controls. We found a consistently significant increase in anti-inflammatory (M2) macrophages in obstructed Sphk2-/- kidneys by flow cytometry and a decrease in mRNA levels of the inflammatory cytokines, MCP1, TNFα, CXCL1 and ILß1, suggesting an anti-inflammatory bias in the absence of Sphk2. Indeed, metabolic profiling showed that the pro-inflammatory glycolytic pathway is largely inactive in Sphk2-/- bone marrow-derived macrophages. Furthermore, treatment with the M2-promoting cytokines IL-4 or IL-13 demonstrated that macrophages lacking Sphk2 polarized more efficiently to the M2 phenotype than wild type cells. Bone marrow transplant studies indicated that expression of Sphk2-/- on either the hematopoietic or parenchymal cells did not fully rescue the pro-healing phenotype, confirming that both infiltrating M2-macrophages and the kidney microenvironment contribute to the damaging Sphk2 effects. Importantly, obstructed kidneys from mice treated with an Sphk2 inhibitor recapitulated findings in the genetic model. These results demonstrate that reducing Sphk2 activity by genetic or pharmacological manipulation markedly decreases inflammatory and fibrotic responses to obstruction, resulting in diminished renal injury and supporting Sphk2 as a novel driver of the pro-inflammatory macrophage phenotype.

Fosmetpantotenate (RE-024), a phosphopantothenate replacement therapy for pantothenate kinase-associated neurodegeneration: Mechanism of action and efficacy in nonclinical models.

In cells, phosphorylation of pantothenic acid to generate phosphopantothenic acid by the pantothenate kinase enzymes is the first step in coenzyme A synthesis. Pantothenate kinase 2, the isoform localized in neuronal cell mitochondria, is dysfunctional in patients with pantothenate kinase-associated neurodegeneration. Fosmetpantotenate is a phosphopantothenic acid prodrug in clinical development for treatment of pantothenate kinase-associated neurodegeneration, which aims to replenish phosphopantothenic acid in patients. Fosmetpantotenate restored coenzyme A in short-hairpin RNA pantothenate kinase 2 gene-silenced neuroblastoma cells and was permeable in a blood-brain barrier model. The rate of fosmetpantotenate metabolism in blood is species-dependent. Following up to 700 mg/kg orally, blood exposure to fosmetpantotenate was negligible in rat and mouse, but measurable in monkey. Consistent with the difference in whole blood half-life, fosmetpantotenate dosed orally was found in the brains of the monkey (striatal dialysate) but was absent in mice. Following administration of isotopically labeled-fosmetpantotenate to mice, ~40% of liver coenzyme A (after 500 mg/kg orally) and ~50% of brain coenzyme A (after 125 µg intrastriatally) originated from isotopically labeled-fosmetpantotenate. Additionally, 10-day dosing of isotopically labeled-fosmetpantotenate, 12.5 µg, intracerebroventricularly in mice led to ~30% of brain coenzyme A containing the stable isotopic labels. This work supports the hypothesis that fosmetpantotenate acts to replace reduced phosphopantothenic acid in pantothenate kinase 2-deficient tissues.

Inositol Polyphosphate Multikinase Inhibits Angiogenesis via Inositol Pentakisphosphate-Induced HIF-1α Degradation.

RATIONALE: Inositol polyphosphate multikinase (IPMK) and its major product inositol pentakisphosphate (IP5) regulate a variety of cellular functions, but their role in vascular biology remains unexplored. OBJECTIVE: We have investigated the role of IPMK in regulating angiogenesis. METHODS AND RESULTS: Deletion of IPMK in fibroblasts induces angiogenesis in both in vitro and in vivo models. IPMK deletion elicits a substantial increase of VEGF (vascular endothelial growth factor), which mediates the regulation of angiogenesis by IPMK. The regulation of VEGF by IPMK requires its catalytic activity. IPMK is predominantly nuclear and regulates gene transcription. However, IPMK does not apparently serve as a transcription factor for VEGF. HIF (hypoxia-inducible factor)-1α is a major determinant of angiogenesis and induces VEGF transcription. IPMK deletion elicits a major enrichment of HIF-1α protein and thus VEGF. HIF-1α is constitutively ubiquitinated by pVHL (von Hippel-Lindau protein) followed by proteasomal degradation under normal conditions. However, HIF-1α is not recognized and ubiquitinated by pVHL in IPMK KO (knockout) cells. IP5 reinstates the interaction of HIF-1α and pVHL. HIF-1α prolyl hydroxylation, which is prerequisite for pVHL recognition, is interrupted in IPMK-deleted cells. IP5 promotes HIF-1α prolyl hydroxylation and thus pVHL-dependent degradation of HIF-1α. Deletion of IPMK in mouse brain increases HIF-1α/VEGF levels and vascularization. The increased VEGF in IPMK KO disrupts blood-brain barrier and enhances brain blood vessel permeability. CONCLUSIONS: IPMK, via its product IP5, negatively regulates angiogenesis by inhibiting VEGF expression. IP5 acts by enhancing HIF-1α hydroxylation and thus pVHL-dependent degradation of HIF-1α.

Expression profiling of genes regulated by sphingosine kinase1 signaling in a murine model of hyperoxia induced neonatal bronchopulmonary dysplasia.

BACKGROUND: Sphingosine- 1-Phosphate (S1P) is a bioactive lipid and an intracellular as well as an extracellular signaling molecule. S1P ligand specifically binds to five related cell surface G-protein-coupled receptors (S1P1-5). S1P levels are tightly regulated by its synthesis catalyzed by sphingosine kinases (SphKs) 1 & 2 and catabolism by S1P phosphatases, lipid phosphate phosphatases and S1P lyase. We previously reported that knock down of SphK1 (Sphk1 -/- ) in a neonatal mouse BPD model conferred significant protection against hyperoxia induced lung injury. To better understand the underlying molecular mechanisms, genome-wide gene expression profiling was performed on mouse lung tissue using Affymetrix MoGene 2.0 array. RESULTS: Two-way ANOVA analysis was performed and differentially expressed genes under hyperoxia were identified using Sphk1 -/- mice and their wild type (WT) equivalents. Pathway (PW) enrichment analyses identified several signaling pathways that are likely to play a key role in hyperoxia induced lung injury in the neonates. These included signaling pathways that were anticipated such as those involved in lipid signaling, cell cycle regulation, DNA damage/apoptosis, inflammation/immune response, and cell adhesion/extracellular matrix (ECM) remodeling. We noted hyperoxia induced downregulation of the expression of genes related to mitotic spindle formation in the WT which was not observed in Sphk1 -/- neonates. Our data clearly suggests a role for SphK1 in neonatal hyperoxic lung injury through elevated inflammation and apoptosis in lung tissue. Further, validation by RT-PCR on 24 differentially expressed genes showed 83% concordance both in terms of fold change and vectorial changes. Our findings are in agreement with previously reported human BPD microarray data and completely support our published in vivo findings. In addition, the data also revealed a significant role for additional unanticipitated signaling pathways involving Wnt and GADD45. CONCLUSION: Using SphK1 knockout mice and differential gene expression analysis, we have shown here that S1P/SphK1 signaling plays a key role in promoting hyperoxia induced DNA damage, inflammation, apoptosis and ECM remodeling in neonatal lungs. It also appears to suppress pro-survival cellular responses involved in normal lung development. We therefore propose SphK1 as a therapeutic target for the development drugs to combat BPD.

Sphingosine-1-phosphate signalling-a key player in the pathogenesis of Angiotensin II-induced hypertension.

AIMS: Hypertension is a complex condition involving functional and structural alterations of the microvasculature and an activation of the immune system. T-lymphocytes play a crucial role during the development of hypertension in experimental models, yet the underlying mechanisms remain elusive. Lymphocyte egress from lymph nodes is controlled by sphingosine-1-phosphate (S1P), a natural lipid mediator regulating immune cell and vascular function in health and disease. We therefore investigated the involvement of S1P signalling in the pathogenesis of hypertension. METHODS AND RESULTS: Angiotensin-II (AngII) treatment resulted in high blood pressure (BP) associated to increased plasma S1P and circulating T-cell counts. T-cell egress from lymph nodes was found to be a critical initial step for the onset of hypertension as fingolimod, a S1P-receptor agonist sequestering lymphocytes in the lymph nodes and inducing lymphopenia, blunted BP responses to AngII. Furthermore, activity of S1P-generating enzyme type 2 (SphK2) in haematopoietic cells critically contributed to AngII-induced lymphocyte mobilization from the lymph nodes as SphK2-/- mice and mice where SphK2 was ablated only in the haematopoietic system presented an accumulation of T-cells in mesenteric lymph nodes and a blunted BP response. In addition, deregulation of vascular SphK2 expression associated to a thrombo-inflammatory phenotype of the microvasculature, and to functional alterations of small resistance arteries. CONCLUSION: The presented results point to a critical involvement of S1P and its signalling axis in the pathogenesis of hypertension. Specifically, SphK2 evolves as key player in immune cell trafficking and vascular dysfunction contributing to the development of overt hypertension.

Host sphingosine kinase 1 worsens pancreatic cancer peritoneal carcinomatosis.

BACKGROUND: There are no effective treatments for pancreatic cancer peritoneal carcinomatosis (PC) or cancer dissemination in abdominal cavity. Sphingosine-1-phosphate (S1P), a bioactive lipid mediator produced by sphingosine kinases (SphK1 and SphK2), plays critical roles in cancer progression. We reported that SphK1, but not SphK2, is responsible for S1P export from breast cancer cells and recently discovered that S1P is linked to inflammation and cancer in colitis-associated cancer progression. Given the fact that inflammation is known to be essential for the establishment and progression of PC, we hypothesized that SphK1 in the host animals is involved in progression of pancreatic cancer PC. METHODS: Murine pancreatic adenocarcinoma panc02-luc cells were intraperitoneally injected into wildtype or SphK1 knockout (KO) mice to generate a syngeneic PC model. Cell proliferation and apoptosis were determined by Ki67 and TUNEL staining, respectively. RESULTS: All the animals developed panc02-luc PC. SphK1 KO mice developed significantly less tumor burden, less total tumor weight, and fewer number of PC nodules at 14 d after implantation. Histologically, less inflammatory cell infiltration and less cancer cell proliferation were observed in the tumors. There was no difference in apoptosis. CONCLUSIONS: Our results raise an intriguing possibility that S1P generated by SphK1 in the host promotes pancreatic cancer PC progression by stimulation of proliferation of cancer cells.