Differential expression of choline kinase isoforms in skeletal muscle explains the phenotypic variability in the rostrocaudal muscular dystrophy mouse

Choline kinase in mammals is encoded by two genes, Chka and Chkb. Disruption of murine Chka leads to embryonic lethality, whereas a spontaneous genomic deletion in murine Chkb results in neonatal forelimb bone deformity and hindlimb muscular dystrophy. Surprisingly, muscular dystrophy isn't significantly developed in the forelimb. We have investigated the mechanism by which a lack of choline kinase β, encoded by Chkb, results in minimal muscular dystrophy in forelimbs. We have found that choline kinase β is the major isoform in hindlimb muscle and contributes more to choline kinase activity, while choline kinase α is predominant in forelimb muscle and contributes more to choline kinase activity. Although choline kinase activity is decreased in forelimb muscles of Chkb^−/− mice, the activity of CTP:phosphocholine cytidylyltransferase is increased, resulting in enhanced phosphatidylcholine biosynthesis. The activity of phosphatidylcholine phospholipase C is up-regulated while the activity of phospholipase A₂ in forelimb muscle is not altered. Regeneration of forelimb muscles of Chkb^−/− mice is normal when challenged with cardiotoxin. In contrast to hindlimb muscle, mega-mitochondria are not significantly formed in forelimb muscle of Chkb^−/− mice. We conclude that the relative lack of muscle degeneration in forelimbs of Chkb^−/− mice is due to abundant choline kinase α and the stable homeostasis of phosphatidylcholine.     

Phosphatidylcholine metabolism and choline kinase in human osteoblasts

There is a paucity of information about phosphatidylcholine (PC) biosynthesis in bone formation. Thus, we characterized PC metabolism in both primary human osteoblasts (HOB) and human osteosarcoma MG-63 cells. Our results show that the CDP-choline pathway is the only de novo route for PC biosynthesis in both HOB and MG-63 cells. Both CK activity and CKα expression in MG-63 cells were significantly higher than those in HOB cells. Silencing of CKα in MG-63 cells had no significant effect on PC concentration but decreased the amount of phosphocholine by approximately 80%. The silencing of CKα also reduced cell proliferation. Moreover, pharmacological inhibition of CK activity impaired the mineralization capacity of MG-63 cells. Our data suggest that CK and its product phosphocholine are required for the normal growth and mineralization of MG-63 cells.     

Phosphatidylcholine metabolism in human endothelial cells: modulation by phosphocholine

Phosphatidylcholine is the principal phospholipid in mammalian tissues, and a major source for the production of arachidonic acid. In this study, the effect of exogenous phosphocholine, a precursor of phosphatidylcholine biosynthesis, on the metabolism of phosphatidylcholine in human umbilical vein endothelial cells was investigated. Incubation of endothelial cells with exogenous phosphocholine at concentrations of 1 to 5 mM was found to inhibit choline uptake and its subsequent incorporation into phosphatidylcholine. Phosphocholine appeared to inhibit choline uptake in a competitive manner. Since phosphatidylcholine is metabolized mainly by the action of phospholipase A₂, with the release of arachidonic acid and other fatty acids, the effect of phosphocholine on arachidonic acid release in endothelial cells was also examined. The induction of arachidonic acid release by ATP was enhanced in cells treated with 1 mM phosphocholine. In vitro assays of phospholipase A₂ activity in cells incubated with phosphocholine, however, did not produced any significant change in the activity of this enzyme. The results of this study show that phosphocholine modulates the biosynthesis and catabolism of phosphatidylcholine in an indirect manner.     

Elucidation of human choline kinase crystal structures in complex with the products ADP or phosphocholine

Choline kinase, responsible for the phosphorylation of choline to phosphocholine as the first step of the CDP-choline pathway for the biosynthesis of phosphatidylcholine, has been recognized as a new target for anticancer therapy. Crystal structures of human choline kinase in its apo, ADP and phosphocholine-bound complexes, respectively, reveal the molecular details of the substrate binding sites. ATP binds in a cavity where residues from both the N and C-terminal lobes contribute to form a cleft, while the choline-binding site constitutes a deep hydrophobic groove in the C-terminal domain with a rim composed of negatively charged residues. Upon binding of choline, the enzyme undergoes conformational changes independently affecting the N-terminal domain and the ATP-binding loop. From this structural analysis and comparison with other kinases, and from mutagenesis data on the homologous Caenorhabditis elegans choline kinase, a model of the ternary ADP.phosphocholine complex was built that reveals the molecular basis for the phosphoryl transfer activity of this enzyme.     

Degumming of vegetable oils by a novel phospholipase B from Pseudomonas fluorescens BIT-18

Pseudomonas fluorescens BIT-18 was isolated from soil near a vegetable oil factory and shown to produce a B-type phospholipase. The enzyme was partially purified by ammonium sulfate precipitation. Gas chromatography demonstrated that the enzyme preparation hydrolyzed both the 1- and 2-ester bonds of phosphatidylcholine. When degumming of soybean, rapeseed, and peanut oil was performed with this enzyme preparation, oils with phosphorous contents lower than 5 mg/kg were obtained after 5 h of enzyme treatment at 40 °C. The enzyme preparation did not show lipase activity, thus free fatty acids were only generated from the phospholipids. Therefore, this novel phospholipase B is potentially useful for the refining of high-quality oils with attractive yields.     

Characterization of eicosanoid synthesis in a genetic ablation model of ceramide kinase

Multiple reports have demonstrated a role for ceramide kinase (CERK) in the production of eicosanoids. To examine the effects of the genetic ablation of CERK on eicosanoid synthesis, primary mouse embryonic fibroblasts (MEFs) and macrophages were isolated from CERK^−/− and CERK^+/+ mice, and the ceramide-1-phosphate (C1P) and eicosanoid profiles were investigated. Significant decreases were observed in multiple C1P subspecies in CERK−/− cells as compared to CERK^+/+ cells with overall 24% and 48% decreases in total C1P. In baseline experiments, the levels of multiple eicosanoids were significantly lower in the CERK^−/− cells compared with wild-type cells. Importantly, induction of eicosanoid synthesis by calcium ionophore was significantly reduced in the CERK^−/− MEFs. Our studies also demonstrate that the CERK^−/− mouse has adapted to loss of CERK in regards to airway hyper-responsiveness as compared with CERK siRNA treatment. Overall, we demonstrate that there are significant differences in eicosanoid levels in ex vivo CERK^−/− cells compared with wild-type counterparts, but the effect of the genetic ablation of CERK on eicosanoid synthesis and the serum levels of C1P was not apparent in vivo.     

Choline kinase beta is required for normal endochondral bone formation

Background

Choline kinase has three isoforms encoded by the genes Chka and Chkb. Inactivation of Chka in mice results in embryonic lethality, whereas Chkb^−/− mice display neonatal forelimb bone deformations.

Methods

To understand the mechanisms underlying the bone deformations, we compared the biology and biochemistry of bone formation from embryonic to young adult wild-type (WT) and Chkb^−/− mice.

Results

The deformations are specific to the radius and ulna during the late embryonic stage. The radius and ulna of Chkb^−/− mice display expanded hypertrophic zones, unorganized proliferative columns in their growth plates, and delayed formation of primary ossification centers. The differentiation of chondrocytes of Chkb^−/− mice was impaired, as was chondrocyte proliferation and expression of matrix metalloproteinases 9 and 13. In chondrocytes from Chkb^−/− mice, phosphatidylcholine was slightly lower than in WT mice whereas the amount of phosphocholine was decreased by approximately 75%. In addition, the radius and ulna from Chkb^−/− mice contained fewer osteoclasts along the cartilage/bone interface.

Conclusions

Chkb has a critical role in the normal embryogenic formation of the radius and ulna in mice.

General Significance

Our data indicate that choline kinase beta plays an important role in endochondral bone formation by modulating growth plate physiology.     

Correlation between the phospholipids domains of the target cell membrane and the extent of Naja kaouthia PLA2-induced membrane damage: Evidence of distinct catalytic and cytotoxic sites in PLA2 molecules

Two phospholipase A₂ (PLA₂) enzymes (NK-PLA₂-A and NK-PLA₂-B) were purified from the venom of the monocled cobra Naja kaouthia. The molecular weights of NK-PLA₂-A and NK-PLA₂-B, as estimated by mass spectrometry, were 13,619 and 13,303 Da respectively. Both phospholipases were highly thermostable, had maximum catalytic activity at basic pH, and showed preferential hydrolysis of phosphatidylcholine. Intravenous injection of either PLA₂ up to a dose of 10 mg/kg body weight was non-toxic to mice and did not show neurotoxic symptoms. The N. kaouthia PLA₂s displayed anticoagulant and cytotoxic activity, but poor hemolytic activity. Both the PLA₂s were more toxic to Sf9 and Tn cells compared to VERO cells. NK-PLA₂ exhibited selective lysis of wild-type baculovirus-infected Sf9 cells compared to normal cells. Amino acid modification studies and heating experiments suggest that separate sites in the NK-PLA₂ molecules are responsible for their catalytic, anticoagulant and cytotoxic activities.     

Expression of recombinant human antibody fragments capable of inhibiting the phospholipase and myotoxic activities of Bothrops jararacussu venom

Phospholipases A₂ are components of Bothrops venoms responsible for disruption of cell membrane integrity via hydrolysis of its phospholipids. This study used a large nonimmune human scFv library named Griffin.1 (MRC, Cambridge, UK) for selection of recombinant antibodies against antigens present in Bothrops jararacussu venom and identification of specific antibodies able to inhibit phospholipase activity. Four clones were identified as capable of inhibiting this activity in vitro. These clones were able to reduce in vivo the myotoxic activity of BthTX-I and BthTX-II PLA₂, but had no effect on the in vitro anticoagulant activity of BthTX-II. This work shows the potential of using recombinant scFv libraries in the search for antibodies that neutralize relevant venom components.     

Plant low-molecular-weight phospholipase A2s (PLA2s) are structurally related to the animal secretory PLA2s and are present as a family of isoforms in rice (Oryza sativa)

Recently, we purified to homogeneity and characterized a low-molecular-weight calcium-dependent phospholipase A₂ (PLA₂) from developing elm seed endosperm. This represented the first purified and characterized PLA₂ from a plant tissue. The full sequences of two distinct but homologous rice (Oryza sativa) cDNAs are given here. These encode mature proteins of 119 amino acids (PLA₂-I, preceded by a 19 amino acid signal peptide) and 128 amino acids (PLA₂-II, preceded by a 25 amino acid signal peptide), and were derived from four expressed sequence tag (EST) clones. Both proteins were homologous to the N-terminal amino acid sequence of the elm PLA₂. They contained twelve conserved cysteine residues and sequences that are likely to represent the Ca²⁺-binding loop and active-site motif, which are characteristic of animal secretory PLA₂s. A soluble PLA₂ activity was purified 145 000-fold from green rice shoots. This had the same biochemical characteristics as the elm and animal secretory PLA₂s. The purified rice PLA₂ consisted of two proteins, with a molecular weight of 12 440 and 12 920, that had identical N-terminal amino acid sequences. This sequence was different from but homologous to the PLA₂-I and PLA₂-II sequences. Taken together, the results suggest that at least three different low-molecular-weight PLA₂s are expressed in green rice shoots. Southern blot analysis suggested that multiple copies of such genes are likely to occur in the rice and in other plant genomes.     

Dynamics of arachidonic acid mobilization by inflammatory cells

The development of mass spectrometry-based techniques is opening new insights into the understanding of arachidonic acid (AA) metabolism. AA incorporation, remodeling and release are collectively controlled by acyltransferases, phospholipases and transacylases that exquisitely regulate the distribution of AA between the different glycerophospholipid species and its mobilization during cellular stimulation. Traditionally, studies involving phospholipid AA metabolism were conducted by using radioactive precursors and scintillation counting from thin layer chromatography separations that provided only information about lipid classes. Today, the input of lipidomic approaches offers the possibility of characterizing and quantifying specific molecular species with great accuracy and within a biological context associated to protein and/or gene expression in a temporal frame. This review summarizes recent results applying mass spectrometry-based lipidomic approaches to the identification of AA-containing glycerophospholipids, phospholipid AA remodeling and synthesis of oxygenated metabolites.     

Enhanced expression of the alpha 7 beta 1 integrin reduces muscular dystrophy and restores viability in dystrophic mice

Muscle fibers attach to laminin in the basal lamina using two distinct mechanisms: the dystrophin glycoprotein complex and the alpha 7 beta 1 integrin. Defects in these linkage systems result in Duchenne muscular dystrophy (DMD), alpha 2 laminin congenital muscular dystrophy, sarcoglycan-related muscular dystrophy, and alpha 7 integrin congenital muscular dystrophy. Therefore, the molecular continuity between the extracellular matrix and cell cytoskeleton is essential for the structural and functional integrity of skeletal muscle. To test whether the alpha 7 beta 1 integrin can compensate for the absence of dystrophin, we expressed the rat alpha 7 chain in mdx/utr(-/-) mice that lack both dystrophin and utrophin. These mice develop a severe muscular dystrophy highly akin to that in DMD, and they also die prematurely. Using the muscle creatine kinase promoter, expression of the alpha 7BX2 integrin chain was increased 2.0-2.3-fold in mdx/utr(-/-) mice. Concomitant with the increase in the alpha 7 chain, its heterodimeric partner, beta 1D, was also increased in the transgenic animals. Transgenic expression of the alpha 7BX2 chain in the mdx/utr(-/-) mice extended their longevity by threefold, reduced kyphosis and the development of muscle disease, and maintained mobility and the structure of the neuromuscular junction. Thus, bolstering alpha 7 beta 1 integrin-mediated association of muscle cells with the extracellular matrix alleviates many of the symptoms of disease observed in mdx/utr(-/-) mice and compensates for the absence of the dystrophin- and utrophin-mediated linkage systems. This suggests that enhanced expression of the alpha 7 beta 1 integrin may provide a novel approach to treat DMD and other muscle diseases that arise due to defects in the dystrophin glycoprotein complex. A video that contrasts kyphosis, gait, joint contractures, and mobility in mdx/utr(-/-) and alpha 7BX2-mdx/utr(-/-) mice can be accessed at http://www.jcb.org/cgi/content/full/152/6/1207.     

Kinetic and structural characterization of triacylglycerol lipases possessing phospholipase A1 activity

The pancreatic lipase gene family displays various substrate selectivities for triglycerides and phospholipids. The structural basis for this difference in substrate specificity has not been definitively established. Based on a kinetic comparative study between various pancreatic lipase family members, we showed here that porcine pancreatic lipase (PPL), which was so far classified as “classical lipase”, was able to hydrolyze phosphatidylcholine (PC). Amino acid sequence alignments revealed that Val260 residue in PPL lid could be critical for the interaction with lipid substrate. Molecular dynamics was applied to investigate PC binding modes within the catalytic cavity of PPL and human pancreatic lipase (HPL), aiming to explain the difference of specificity of these enzymes towards phospholipids. Results showed that with HPL, the oxyanion hole was not able to accommodate the PC molecule, suggesting that no activity could be obtained. With PPL, the formation of a large pocket involving Val260 allowed the PC molecule to come near the catalytic residues, suggesting that it could be hydrolyzed. One more interesting finding is that human pancreatic lipase related protein 2 could hydrolyze phospholipids through its PLA₁ and PLA₂ activities. Overall, our study shed the light on new structural features of the phospholipase activity of pancreatic lipase family members.     

Interleukin-1 and tumor necrosis factor are not synergistic for human synovial fibroblast PLA2 activation and PGE2 production

Patterns of arachidonic acid release and metabolism were altered in human synovial fibroblasts following exposure to cytokines. Recombinant interleukin-1 induced an approximate 3-fold in crease in [³H]-AA release, a 7-fold increase in PGE₂ production and a 2-fold increase in PLA₂ activity in human synovial fibroblasts. Recombinant tumor necrosis factor induced similar responses, however, the magnitude was less than that mediated by interleukin-1. A combination of the two cytokines had an additive effect on [³H]-AA release and PLA₂ activity while PGE₂ production was similar to that detected using interleukin-1 alone. [³H]-AA, was released in substantial amounts when sodium fluoride was used as a stimulus but PGE₂ was not. These data show that tumor necrosis factor and interleukin-1 can both activate synovial cell PLA₂ and induce generation of PGE₂, but act in an additive rather than a synergistic fashion. Furthermore, the data show that PGE₂ production is not always concordant with [³H]-AA release, suggesting that appropriate enzyme(s) must be activated.     

Simultaneous activation of p38 and JNK by arachidonic acid stimulates the cytosolic phospholipase A2-dependent synthesis of lipid droplets in human monocytes

Exposure of human peripheral blood monocytes to free arachidonic acid (AA) results in the rapid induction of lipid droplet (LD) formation by these cells. This effect appears specific for AA in that it is not mimicked by other fatty acids, whether saturated or unsaturated. LDs are formed by two different routes: (i) the direct entry of AA into triacylglycerol and (ii) activation of intracellular signaling, leading to increased triacylglycerol and cholesteryl ester formation utilizing fatty acids coming from the de novo biosynthetic route. Both routes can be dissociated by the arachidonyl-CoA synthetase inhibitor triacsin C, which prevents the former but not the latter. LD formation by AA-induced signaling predominates, accounting for 60–70% of total LD formation, and can be completely inhibited by selective inhibition of the group IVA cytosolic phospholipase A₂α (cPLA₂α), pointing out this enzyme as a key regulator of AA-induced signaling. LD formation in AA-treated monocytes can also be blocked by the combined inhibition of the mitogen-activated protein kinase family members p38 and JNK, which correlates with inhibition of cPLA₂α activation by phosphorylation. Collectively, these results suggest that concomitant activation of p38 and JNK by AA cooperate to activate cPLA₂α, which is in turn required for LD formation possibly by facilitating biogenesis of this organelle, not by regulating neutral lipid synthesis.     

Role of phosphorylation sites and the C2 domain in regulation of cytosolic phospholipase A2.

Cytosolic phospholipase A2 (cPLA2) mediates agonist-induced arachidonic acid release, the first step in eicosanoid production. cPLA2 is regulated by phosphorylation and by calcium, which binds to a C2 domain and induces its translocation to membrane. The functional roles of phosphorylation sites and the C2 domain of cPLA2 were investigated. In Sf9 insect cells expressing cPLA2, okadaic acid, and the calcium-mobilizing agonists A23187 and CryIC toxin induce arachidonic acid release and translocation of green fluorescent protein (GFP)-cPLA2 to the nuclear envelope. cPLA2 is phosphorylated on multiple sites in Sf9 cells; however, only S505 phosphorylation partially contributes to cPLA2 activation. Although okadaic acid does not increase calcium, mutating the calcium-binding residues D43 and D93 prevents arachidonic acid release and translocation of cPLA2, demonstrating the requirement for a functional C2 domain. However, the D93N mutant is fully functional with A23187, whereas the D43N mutant is nearly inactive. The C2 domain of cPLA2 linked to GFP translocates to the nuclear envelope with calcium-mobilizing agonists but not with okadaic acid. Consequently, the C2 domain is necessary and sufficient for translocation of cPLA2 to the nuclear envelope when calcium is increased; however, it is required but not sufficient with okadaic acid.     

Bone marrow transplantation improves outcome in a mouse model of congenital muscular dystrophy

We examined whether pathogenesis in dystrophin-deficient (mdx) mice and laminin-alpha2-deficient (dy) mice is ameliorated by bone marrow transplantation (BMT). Green fluorescent protein (GFP) mice were used as donors. In mdx mice, BMT failed to produce any significant differences in muscle pathology, although some GFP-positive fibers with restored dystrophin expression were observed. In contrast, in the dy mice, BMT led to a significant increase in lifespan and an increase in growth rate, muscle strength, and respiratory function. We conclude that BMT improved outcome in dy mice but not mdx mice.     

Alterations in phospholipid and fatty acid lipid profiles in primary neocortical cells during oxidant-induced cell injury

Specific phospholipids and fatty acids altered during oxidant-induced neuronal cell injury were determined using electrospray ionization mass spectrometry (ESI-MS) and ion trapping. The oxidants hydrogen peroxide (H(2)O(2), 0-1000 microM) and tert-butylhydroperoxide (TBHP, 0-400 microM) induced time- and concentration-dependent increases in reactive oxygen species in primary cultures of mouse neocortical cells as determined by 2',7'-dichlorofluorescein diacetate staining and thiobarbituric acid formation. ESI-MS analysis of 26 m/z values, representing 42 different phospholipids, demonstrated that H(2)O(2) and TBHP increased the abundance of phospholipids containing polyunsaturated fatty acids, but had minimal affect on those containing mono- or di-unsaturated fatty acids. These increases correlated to time-dependent increase in 16:1-20:4, 16:0-20:4, 18:1-20:4 and 18:0-20:4 phosphatidylcholine. Oxidant exposure also increased mystric (14:0), palmitic (16:0), and stearic (18:0) acid twofold, oleic acid (18:1) two- to threefold, and arachidonic acid (20:4) fourfold, compared to controls. Increases in arachidonic acid levels occurred prior to increases in the phospholipids, but after increases in ROS, and correlated to increases in oxidized arachidonic acid species, specifically [20:4-OOH]-H(2)O-, 20:4-OH-, and Tri-OH-20:4-arachidonic acid. Treatment of cells with methyl arachidonyl flourophosphonate an inhibitor of Group IV and VI PLA(2), decreased oxidant-induced arachidonic acid release, while bromoenol lactone, an inhibitor of Group VI PLA(2), did not. Collectively, these data identify phospholipids and fatty acids altered during oxidant treatment of neurons and suggest differential roles for Group IV and VI PLA(2) in oxidant-induced neural cell injury.     

Regulation of KCNQ2/KCNQ3 current by G protein cycling: the kinetics of receptor-mediated signaling by Gq

Receptor-mediated modulation of KCNQ channels regulates neuronal excitability. This study concerns the kinetics and mechanism of M1 muscarinic receptor-mediated regulation of the cloned neuronal M channel, KCNQ2/KCNQ3 (Kv7.2/Kv7.3). Receptors, channels, various mutated G-protein subunits, and an optical probe for phosphatidylinositol 4,5-bisphosphate (PIP2) were coexpressed by transfection in tsA-201 cells, and the cells were studied by whole-cell patch clamp and by confocal microscopy. Constitutively active forms of Galphaq and Galpha11, but not Galpha13, caused a loss of the plasma membrane PIP2 and a total tonic inhibition of the KCNQ current. There were no further changes upon addition of the muscarinic agonist oxotremorine-M (oxo-M). Expression of the regulator of G-protein signaling, RGS2, blocked PIP2 hydrolysis and current suppression by muscarinic stimulation, confirming that the Gq family of G-proteins is necessary. Dialysis with the competitive inhibitor GDPbetaS (1 mM) lengthened the time constant of inhibition sixfold, decreased the suppression of current, and decreased agonist sensitivity. Removal of intracellular Mg2+ slowed both the development and the recovery from muscarinic suppression. When combined with GDPbetaS, low intracellular Mg2+ nearly eliminated muscarinic inhibition. With nonhydrolyzable GTP analogs, current suppression developed spontaneously and muscarinic inhibition was enhanced. Such spontaneous suppression was antagonized by GDPbetaS or GTP or by expression of RGS2. These observations were successfully described by a kinetic model representing biochemical steps of the signaling cascade using published rate constants where available. The model supports the following sequence of events for this Gq-coupled signaling: A classical G-protein cycle, including competition for nucleotide-free G-protein by all nucleotide forms and an activation step requiring Mg2+, followed by G-protein-stimulated phospholipase C and hydrolysis of PIP2, and finally PIP2 dissociation from binding sites for inositol lipid on the channels so that KCNQ current was suppressed. Further experiments will be needed to refine some untested assumptions.