Toxoplasma gondii: role of the phosphatidylcholine-specific phospholipase C during cell invasion and intracellular development

The effect of D609, a specific inhibitor of phosphatidylcholine-specific phospholipase C, was investigated on cyst development of the Prugniaud strain of Toxoplasma gondii in vitro. Following treatment with the inhibitor 24 h after cell infection, cyst development was affected as assessed by staining with the bradyzoite-specific mAb CC2: the CC2-reactive antigen was shown to be differently located (in the wall versus the matrix under control conditions). This correlated with a decrease in parasite multiplication induced by D609. Pretreatment of the parasites with D609 inhibited their entry into the host cells, whereas pretreatment of the host cells enhanced the intracellular multiplication of the para sites, without any effect on cell invasion or cyst formation. Our results suggest a crucial role for phosphatidylcholine-specific phospholipase C in the pathophysiology of toxoplasmosis.     

Fas-mediated activation of phospholipase D is coupled to the stimulation of phosphatidylcholine-specific phospholipase C in A20 cells.

The activation of phospholipase D in murine B cell lymphoma A20 cells treated with anti-Fas monoclonal antibody has been investigated. Fas cross-linking resulted in a both dose- and time-dependent increases in phospholipase D activity. There was a nearly maximum saturated rise in phospholipase D activity at the dose of 200 ng/ml anti-Fas monoclonal antibody showing a fourfold increase within 3 h. Fas activation also caused an approximately twofold increase of phosphatidylcholine-specific phospholipase C activity and 1,2-diacylglycerol release, which could be blocked by 30 min pretreatment with the phosphatidylcholine-specific phospholipase C inhibitor D609 (50 microgram/ml). Pretreatment of D609 also effectively inhibited the translocation of protein kinase C betaI and betaII from the cytosol to the membrane and the activation of phospholipase D induced by Fas cross-linking, suggesting that 1, 2-diacylglycerol released from the cellular phosphatidylcholine pool through phosphatidylcholine-specific phospholipase C plays a major role in protein kinase C/phospholipase D activation. Anti-Fas monoclonal antibody failed to elicit phosphoinositide-specific phospholipase C activation and any changes in the intracellular Ca2+ level in A20 cells, indicating that the phosphoinositide-mediated pathway is not involved in this Fas signaling. Therefore, these results suggest that Fas-mediated phospholipase D activation may be a consequence of primary stimulation of phosphatidylcholine-specific phospholipase C and that phospholipase D may play a role in Fas cross-linking signaling downstream from phosphatidylcholine-specific phospholipase C.     

Lipopolysaccharide activated phosphatidylcholine-specific phospholipase C and induced IL-8 and MCP-1 production in vascular endothelial cells

Secretion of proinflammatory cytokines by lipopolysaccharide (LPS) activated vascular endothelial cells (VECs) contributes substantially to the pathogenesis of several inflammatory diseases such as atherosclerosis and septic shock. However, the mechanisms involved in this process are not well understood. Here, we investigated the role of phosphatidylcholine-specific phospholipase C (PC-PLC) in LPS-induced IL-8 and MCP-1 production in VECs. The results showed that LPS elevated the level of PC-PLC and the production of IL-8 and MCP-1 in Human umbilical vein vascular endothelial cells (HUVECs). Blocking the function of PC-PLC by exploiting the neutralization antibody of PC-PLC or tricyclodecan-9-yl-xanthogenate (D609), an inhibitor of PC-PLC, significantly inhibited LPS-induced production of IL-8 and MCP-1 in HUVECs. Furthermore, the in vivo experimental results showed that the levels of PC-PLC, IL-8, and MCP-1 in the aortic endothelium and serum were increased in mice injected with LPS. The increased levels of these molecules were also inhibited by the treatment with D609. The data suggested that blocking PC-PLC function significantly inhibited LPS-induced IL-8 and MCP-1 production in cultured HUVECs and in vivo. PC-PLC might be a potential target for therapy in inflammation associated-diseases such as atherosclerosis.     

Isolation and properties of a phosphatidylcholine-specific phospholipase C from bull seminal plasma

A phospholipase C which hydrolyzes [14C]phosphatidylcholine has been purified 1782-fold from 70% ammonium sulfate extract of bull seminal plasma. Purification steps included acid precipitation, chromatography on DEAE-Sephacel, concanavalin A, octyl-Sepharose 4B and Ultrogel AcA 34. The final step provided homogeneous phospholipase C as determined by polyacrylamide gel electrophoresis. The enzyme comprised two subunits, Mr 69,000 and Mr 55,000, respectively. The enzyme had an optimum at pH 7.2 and pI 5.0. EDTA, Cd2+, Pb2+, Ni2+, Fe2+, and Zn2+ inhibited phospholipase C activity. Km and Vmax on p-nitrophenyl phosphorylcholine and phosphatidylcholine substrates were 20 mM and 17 mumol/min/mg of the purified enzyme and 100 microM and 18 mumol/min/mg of the purified enzyme, respectively. The enzyme appeared to be localized in the acrosome as judged by the binding of anti-phospholipase C to the acrosome. This phospholipase C, unlike other known phospholipases (C), did not hydrolyze [1-14C]phosphatidylinositol. The testicular extract of the guinea pig contained inactive phospholipase C which was activated on incubation with acrosin and trypsin but not chymotrypsin.     

Involvement of phosphatidylcholine-specific phospholipase C in thromboxane A2-induced activation of mitogen-activated protein kinase in astrocytoma cells

Thromboxane A2 (TXA2) receptor-mediated signal transduction was investigated in 1321N1 human astrocytoma cells. 9,11-Epithio-11,12-methano-TXA2 (STA2), a TXA2 receptor agonist, induced Ca2+ mobilization and phosphoinositide hydrolysis in a concentration-dependent manner. These responses were inhibited by treatment with U73122, an inhibitor of phosphatidylinositol-specific phospholipase C, or by culturing in 0.5% fetal calf serum containing 0.5 mM dibutyryladenosine 3',5'-cyclic monophosphate (dbcAMP) for 2 days. However, the dbcAMP treatment augmented the TXA2 receptor-mediated phosphorylation of mitogen-activated protein kinase (MAPK). These results were confirmed by a functional MAPK assay measuring the incorporation of 32P into the MAPK substrate peptide. The TXA2 receptor-mediated MAPK activation was inhibited by SQ29548, a TXA2 receptor antagonist, and GF109203X, an inhibitor of protein kinase C. Although U73122 did not inhibit or only slightly inhibited the activation of MAPK, D-609, an inhibitor of phosphatidylcholine-specific phospholipase C, potently attenuated the activation in a concentration-dependent manner. Furthermore, STA2 accelerated the release of [3H]choline metabolites from the cells prelabeled with [3H]choline chloride. This release was inhibited by treatment with D-609. These results suggest that phosphatidylcholine-specific phospholipase C and protein kinase C, but not phosphatidylinositol-specific phospholipase C, are involved in TXA2 receptor-mediated MAPK activation in 1321N1 human astrocytoma cells.     

Cooperation of phosphatidylcholine-specific phospholipase C and basic fibroblast growth factor in the neural differentiation of mesenchymal stem cells in vitro

Previously, we found that suppressing phosphatidylcholine-specific phospholipase C could induce neuronal differentiation of rat mesenchymal stem cells in the absence of serum and fibroblast growth factor. It is well known that basic fibroblast growth factor plays an important role in mesenchymal stem cell neuronal differentiation. In this study, our purpose was to understand the cooperation of phosphatidylcholine-specific phospholipase C and basic fibroblast growth factor in controlling mesenchymal stem cell neuronal differentiation. Our results showed that suppressing phosphatidylcholine-specific phospholipase C in the presence of basic fibroblast growth factor could induce cell neuronal differentiation and the viability of the differentiated cells was obviously increased. Furthermore, we found that the resting membrane potential of the differentiated cells gradually decreased, but the mitochondrial membrane potential rose with increasing treatment time and these characteristics were similar to cultured neurons from mouse embryo forebrains. To determine the possible mechanism by which this combination controls cell neuronal differentiation, we measured changes in the mitochondrial membrane potential and in the levels of reactive oxygen species. The results showed that both the mitochondrial membrane potential and reactive oxygen species levels decreased when basic fibroblast growth factor was added. The data suggested that lower phosphatidylcholine-specific phospholipase C activity was required for mesenchymal stem cell neuronal differentiation and basic fibroblast growth factor was necessary for maintaining the neuronal differentiation state. Moreover, basic fibroblast growth factor could contribute to rescuing the differentiated cells from death through decreasing overly high mitochondrial membrane potentials and reactive oxygen species levels.     

D609, an inhibitor of phosphatidylcholine-specific phospholipase C, inhibits group IV cytosolic phospholipase A2

As an inhibitor of phosphatidylcholine-specific phospholipase C (PC-PLC), D609 has been widely used to explain the role of PC-PLC in various signal transduction pathways. This study shows that D609 inhibits group IV cytosolic phospholipase A2 (cPLA2), but neither secretory PLA2 nor a Ca2+ -dependent PLA2. Dixon plot analysis shows a mixed pattern of noncompetitive and uncompetitive inhibition with Ki = 86.25 microM for the cPLA2 purified from bovine spleen. D609 also time- and dose-dependently reduces the release of arachidonic acid from a Ca2+- ionophore A23187-stimulated MDCK cells. In the AA release experiment, IC50 of D609 was approximately 375 microM, suggesting that this reagent may not enter the cells easily. The present study indicates that the inhibitory effects of D609 on various cellular responses may be partially attributable to the inhibition of cPLA2.     

MicroRNA在自然杀伤细胞发育分化及功能调控过程中的作用

自然杀伤(natural killer, NK)细胞是固有免疫系统中重要的效应细胞,在宿主抗感染、抗肿瘤中发挥重要作用,并参与免疫调节。NK细胞主要由骨髓造血干细胞发育分化而来,NK细胞的发育、成熟及功能依赖于微环境、胞内转录因子及转录后水平的调节。MicroRNA作为一种小非编码RNA,主要在转录后水平上调节靶基因的表达,在NK细胞的生命过程中发挥重要作用。研究发现年龄会影响NK细胞的miRNA表达谱,进而影响NK细胞的发育与功能。基于miRNA对NK细胞发育及功能的调节作用,miRNA可能作为潜在的靶标来保护或恢复患者NK细胞功能,从而治疗相关疾病。为此,文中将对microRNA在NK细胞发育分化及功能调控过程中的研究进展做初步概述。     

Suppressing phosphatidylcholine-specific phospholipase C and elevating ROS level, NADPH oxidase activity and Rb level induced neuronal differentiation in mesenchymal stem cells

In the previous research, we found that D609 (tricyclodecan-9-yl-xanthogenate) could induce human marrow stromal cell (hMSC) differentiation to neuron-like cells. In this study, to understand the possible mechanism, we sequentially investigated the changes of phosphatidylcholine-specific phospholipase C (PC-PLC) activity, the expression of Rb, the intracellular reactive oxygen species (ROS) levels, NADPH oxidase and superoxide dismutase (SOD) activities when D609 induced neuronal differentiation in rat mesenchymal stem cells (MSCs). The results showed that D609 obviously inhibited the activity of PC-PLC when it induced neuronal differentiation in rat MSCs. Simultaneously, ROS level and the activity of NADPH oxidase increased significantly, but the MnSOD and Cu/ZnSOD activities were not altered. Furthermore, the level of Rb protein was evidently elevated. Our data suggested that PC-PLC mediated neuronal differentiation of rat MSCs by elevating NADPH oxidase activity, ROS level, and up-regulating the expression of Rb protein.     

Cellular localization of group IIA phospholipase A2 in rats.

It has been known that group II phospholipase A2 (PLA2) mRNA and protein are present in the homogenates of the spleen, lung, liver, and kidney in normal rats, but the cellular origin of this enzyme has not been yet identified. At present, five subtypes of group II PLA2 have been identified in mammals. Antibodies or mRNA probes previously used for detecting group II PLA2 need to be evaluated to identify the subtypes of group II PLA2. In this study we tried to identify group IIA PLA2-producing cells in normal rat tissues by in situ hybridization (ISH) using an almost full-length RNA probe for rat group IIA enzyme. Group IIA PLA2 mRNA was detected in megakaryocytes in the spleen and Paneth cells in the intestine by ISH. These cells were also immunopositive for an antibody raised against group IIA PLA(2) isolated from rat platelets. Group IIA PLA2 mRNA-positive cells were not detected in lung, liver, kidney, and pancreas. Under normal conditions, group IIA PLA2-producing cells are splenic megakaryocytes and intestinal Paneth cells in rats.     

Evidence that phosphatidylcholine-specific phospholipase C is a key molecule mediating insulin-induced enhancement of gene expression from human cytomegalovirus promoter in CHO cells

The signal transduction from insulin to its receptors and Ras has been extensively studied, while little has been reported beyond these steps. We found that the expression of human interleukin 6 gene under the control of immediate early gene promoter of human cytomegalovirus was enhanced by insulin sitmulation in Chinese hamster ovary cells. The induction effect of insulin was not significantly affected by inhibitors or activators of conventional protein kinase C, cAMP dependent protein kinase and phosphoinositide -3 kinase, however, pre-incubation of the cells with D609, a specific inhibitors of phosphatidylcholine-specific phospholipase C completely abolished the induction effect. These results clearly demonstrate that phosphatidylcholine-specific phospholipase C is a key molecule mediating insulin-induced enhancement of hIL-6 expression from the human cytomegalovirus promoter in Chinese hamster ovary cells and strongly suggest that it plays an important role in the insulin signaling pathways.Abbreviations CHO – Chinese hamster ovary; hCMV promoter – immediate early gene promoter of human cytomegalovirus; hIL-6 – human interleukin 6; PC-PLC-phosphatidylcholine-specific phospholipase C; PI-3 kinase – phosphoinositide 3 kinase; PKA – cAMP dependent protein kinase; PKC – protein kinase C.     

Induction of Toxoplasma gondii cystogenesis and multiplication arrest by treatments with a phosphatidylcholine-specific phospholipase C inhibitor.

A model of tissue cyst formation was developed using D609, a specific inhibitor of phosphatidylcholine specific-phospholipase C. The phospholipase inhibitor induced a decrease in Toxoplasma gondii multiplication and several successive treatments could lead to an arrest in parasite multiplication and full encystment of the parasites. This could be a first step towards an in vitro model of T. gondii reactivation.     

Cellular localization and functional expression of P-glycoprotein in rat astrocyte cultures

We investigated the cellular/subcellular localization and functional expression of P-glycoprotein, an ATP-dependent membrane-associated efflux transporter, in astrocytes, a brain parenchyma compartment that is poorly characterized for the expression of membrane drug transporters. Analyses were carried out on primary cultures of astrocytes isolated from the cerebral cortex of neonatal Wistar rats and CTX TNA2, an immortalized rat astrocyte cell line. Both cell cultures display morphological features typical of type I astrocytes. RT-PCR analysis revealed mdr1a and mdr1b mRNA in primary cultures of astrocytes and in CTX TNA2 cells. Western blot analysis using the P-glycoprotein monoclonal C219 antibody detected a single band of appropriate size in both cell systems. Immunocytochemical analysis using the monoclonal antibodies C219 and MRK16 labeled P-glycoprotein along the plasma membrane, caveolae, coated vesicles and nuclear envelope. Immunoprecipitation studies using the caveolin-1 polyclonal H-97 antibody demonstrated that P-glycoprotein is physically associated with caveolin-1 in both cell culture systems. The accumulation of [(3)H]digoxin (an established P-glycoprotein substrate) by the astrocyte cultures was significantly enhanced in the presence of standard P-glycoprotein inhibitors and an ATP depleting agent. These results demonstrate the cellular/subcellular location and functional expression of P-glycoprotein in rat astrocytes and suggest that this glial compartment may play an important role in the regulation of drug transport in the CNS.     

Expression, localization, and functional role for synaptotagmins in pancreatic acinar cells

Secretagogue-induced changes in intracellular Ca(2+) play a pivotal role in secretion in pancreatic acini yet the molecules that respond to Ca(2+) are uncertain. Zymogen granule (ZG) exocytosis is regulated by soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes. In nerve and endocrine cells, Ca(2+)-stimulated exocytosis is regulated by the SNARE-associated family of proteins termed synaptotagmins. This study examined a potential role for synaptotagmins in acinar secretion. RT-PCR revealed that synaptotagmin isoforms 1, 3, 6, and 7 are present in isolated acini. Immunoblotting and immunofluorescence using three different antibodies demonstrated synaptotagmin 1 immunoreactivity in apical cytoplasm and ZG fractions of acini, where it colocalized with vesicle-associated membrane protein 2. Synaptotagmin 3 immunoreactivity was detected in membrane fractions and colocalized with an endolysosomal marker. A potential functional role for synaptotagmin 1 in secretion was indicated by results that introduction of synaptotagmin 1 C2AB domain into permeabilized acini inhibited Ca(2+)-dependent exocytosis by 35%. In contrast, constructs of synaptotagmin 3 had no effect. Confirmation of these findings was achieved by incubating intact acini with an antibody specific to the intraluminal domain of synaptotagmin 1, which is externalized following exocytosis. Externalized synaptotagmin 1 was detected exclusively along the apical membrane. Treatment with CCK-8 (100 pM, 5 min) enhanced immunoreactivity by fourfold, demonstrating that synaptotagmin is inserted into the apical membrane during ZG fusion. Collectively, these data indicate that acini express synaptotagmin 1 and support that it plays a functional role in secretion whereas synaptotagmin 3 has an alternative role in endolysosomal membrane trafficking.     

Protein kinase C activation by interleukin (IL)-1 limits IL-1-induced IL-6 synthesis in osteoblast-like cells: Involvement of phosphatidylcholine-specific phospholipase C

We investigated the regulatory mechanism of interleukin-6 (IL-6) synthesis induced by interleukin-1 (IL-1) in osteoblast-like MC3T3-E1 cells. IL-1 stimulated the secretion of IL-6 in a dose-dependent manner in the range between 0.1 and 100 ng/ml. Staurosporine and calphostin C, inhibitors of protein kinase C (PKC), significantly enhanced the IL-1-induced secretion of IL-6. The stimulative effect of IL-1 was markedly amplified in PKC down-regulated MC3T3-E1 cells. IL-1 produced diacylglycerol in MC3T3-E1 cells. IL-1 had little effect on the formation of inositol phosphates and choline. On the contrary, IL-1 significantly stimulated the formation of phosphocholine dose-dependently. D-609, an inhibitor of phosphatidylcholine-specific phospholipase C, suppressed the IL-1-induced diacylglycerol production. The IL-1-induced IL-6 secretion was significantly enhanced by D-609. These results indicate that IL-1 activates PKC via phosphatidylcholine-specific phospholipase C in osteoblast-like cells, and the PKC activation then limits IL-6 synthesis induced by IL-1 itself. J. Cell. Biochem. 67:103–111, 1997. © 1997 Wiley-Liss, Inc.     

Subcellular localization of phospholipase C isoforms in vascular smooth muscle

The phospholipase C (PLC) isoform most important during agonist-activated IP(3) production in vascular smooth muscle is still unknown. When PLC activity in rat tail artery homogenate was determined, this activity was shown to be inhibited by an antibody directed against PLCbeta2. Antibodies directed against the gamma1, beta1, beta3 and delta1 isoforms of PLC failed to inhibit PLC activity in this tissue. Both PLCbeta2 and PLCgamma1 were isolated from rat tail artery by DEAE column chromatography and PLCbeta2 activity was shown to be 3-fold greater than PLCgamma1 activity. When rat tail artery was treated with norepinephrine (10 mM), PLCbeta2 was shown to translocate from cytosol to membranes. When subcellular fractions of rat tail artery were isolated by sucrose density gradient centrifugation, including nuclei, plasma membrane, and cytosol, PLCbeta2 was detected in the plasma membrane and the cytosol but not in the nuclei. PLCdelta1 and PLCgamma1 were found only in cytosol. This evidence is consistent with the model wherein an agonist such as norepinephrine can activate smooth muscle contraction via interaction with a plasma membrane receptor which can easily interact with a plasma membrane-associated isoform of PLC, such as PLCbeta2.     

Intracellular localization of phospholipase D1 in mammalian cells

Phospholipase D (PLD) hydrolyzes phosphatidylcholine to generate phosphatidic acid. In mammalian cells this reaction has been implicated in the recruitment of coatomer to Golgi membranes and release of nascent secretory vesicles from the trans-Golgi network. These observations suggest that PLD is associated with the Golgi complex; however, to date, because of its low abundance, the intracellular localization of PLD has been characterized only indirectly through overexpression of chimeric proteins. We have used highly sensitive antibodies to PLD1 together with immunofluorescence and immunogold electron microscopy as well as cell fractionation to identify the intracellular localization of endogenous PLD1 in several cell types. Although PLD1 had a diffuse staining pattern, it was enriched significantly in the Golgi apparatus and was also present in cell nuclei. On fragmentation of the Golgi apparatus by treatment with nocodazole, PLD1 closely associated with membrane fragments, whereas after inhibition of PA synthesis, PLD1 dissociated from the membranes. Overexpression of an hemagglutinin-tagged form of PLD1 resulted in displacement of the endogenous enzyme from its perinuclear localization to large vesicular structures. Surprisingly, when the Golgi apparatus collapsed in response to brefeldin A, the nuclear localization of PLD1 was enhanced significantly. Our data show that the intracellular localization of PLD1 is consistent with a role in vesicle trafficking from the Golgi apparatus and suggest that it also functions in the cell nucleus.