Phosphatidylinositol 3-kinase regulates glycosylphosphatidylinositol hydrolysis through PLC-gamma(2) activation in erythropoietin-stimulated cells

Erythropoietin (Epo)-induced glycosylphosphatidylinositol (GPI) hydrolysis was previously described to be correlated with phospholipase C-gamma 2 (PLC-gamma2) activation. Here, we analyzed the involvement of phosphatidylinositol (PtdIns) 3-kinase in GPI hydrolysis through PLC-gamma2 tyrosine phosphorylation in response to Epo in FDC-P1 cells transfected with a wild type (WT) erythropoietin-receptor (Epo-R). We showed that phosphatidylinositol 3-kinase (PtdIns 3-kinase) inhibitor LY294002 inhibits Epo-induced hydrolysis of endogenous GPI and Epo-induced PLC-gamma2 tyrosine phosphorylation in a dose-dependent manner. Wortmannin, another PtdIns 3-kinase inhibitor, also suppressed Epo-induced PLC-gamma2 tyrosine phosphorylation. We also present evidence that PLC-gamma2 translocation to the membrane fraction on Epo stimulation is completely inhibited by LY294002. Upon Epo stimulation, the tyrosine-phosphorylated PLC-gamma2 was found to be associated with the tyrosine-phosphorylated Grb2-associated binder (GAB)2, SHC and SHP2 proteins. LY294002 cell preincubation did not affect GAB2, SHC and SHP2 tyrosine phosphorylation but inhibited the binding of PLC-gamma2 to GAB2 and SHP2. Taken together, these results show that PtdIns 3-kinase controls Epo-induced GPI hydrolysis through PLC-gamma2.     

Possible involvement of poly(A) in protein synthesis.

The experiments of this paper have re-evaluated the possibility that poly(A) is involved in protein synthesis by testing whether purified poly(A) might competitively inhibit in vitro protein synthesis in rabbit reticulocyte extracts. We have found that poly(A) inhibits the rate of translation of many different poly(A)+ mRNAs and that comparable inhibition is not observed with other ribopolymers. Inhibition by poly(A) preferentially affects the translation of adenylated mRNAs and can be overcome by increased mRNA concentrations or by translating mRNPs instead of mRNA. The extent of inhibition is dependent on the size of the competitor poly(A) as well as on the translation activity which a lysate has for poly(A)+ RNA. In light of our results and numerous experiments in the literature, we propose that poly(A) has a function in protein synthesis and that any role in the determination of mRNA stability is indirect.     

Structure and expression of the human glycosylphosphatidylinositol phospholipase D1 (GPLD1) gene

Here we report the structure of the human glycosylphosphatidylinositol phospholipase D1 gene, which covers at least 80 kb on chromosome 6p22. The gene comprises 25 exons and encodes a 5.8 kb mRNA, which was detected only in the liver. Southern blot analysis shows that the human genome contains only one GPLD gene and we could only detect one of the two previously reported cDNAs.     

Alpha 1-adrenergic receptor-mediated phosphoinositide hydrolysis and prostaglandin E2 formation in Madin-Darby canine kidney cells. Possible parallel activation of phospholipase C and phospholipase A2

alpha 1-Adrenergic receptors mediate two effects on phospholipid metabolism in Madin-Darby canine kidney (MDCK-D1) cells: hydrolysis of phosphoinositides and arachidonic acid release with generation of prostaglandin E2 (PGE2). The similarity in concentration dependence for the agonist (-)-epinephrine in eliciting these two responses implies that they are mediated by a single population of alpha 1-adrenergic receptors. However, we find that the kinetics of the two responses are quite different, PGE2 production occurring more rapidly and transiently than the hydrolysis of phosphoinositides. The antibiotic neomycin selectively decreases alpha 1-receptor-mediated phosphatidylinositol 4,5-bisphosphate hydrolysis without decreasing alpha 1-receptor-mediated arachidonic acid release and PGE2 generation. In addition, receptor-mediated inositol trisphosphate formation is independent of extracellular calcium, whereas release of labeled arachidonic acid is largely calcium-dependent. Moreover, based on studies obtained with labeled arachidonic acid, receptor-mediated generation of arachidonic acid cannot be accounted for by breakdown of phosphatidylinositol monophosphate, phosphatidylinositol bisphosphate, or phosphatidic acid. Further studies indicate that epinephrine produces changes in formation or turnover of several classes of membrane phospholipids in MDCK cells. We conclude that alpha 1-adrenergic receptors in MDCK cells appear to regulate phospholipid metabolism by the parallel activation of phospholipase C and phospholipase A2. This parallel activation of phospholipases contrasts with models described in other systems which imply sequential activation of phospholipase C and diacylglycerol lipase or phospholipase A2.     

Tumour necrosis factor (cachectin) induces phospholipase A2 activity and synthesis of a phospholipase A2-activating protein in endothelial cells.

Tumour necrosis factor (TNF) is an important mediator of endotoxin-induced vascular collapse and other inflammatory reactions. Eicosanoids have been implicated in the pathogeensis of these responses. In order to explore further the potential interactions between TNF and eicosanoid metabolism in eliciting vascular responses, we studied the effects of TNF on the bovine endothelial cell line CPAE. TNF induced cellular retraction observed by light microscope. This morphological change was monitored by the passage of iodinated protein A between adjacent cells and by release of [3H]arachidonic acid metabolites from cells. Both the morphological and functional responses were abrogated by inhibition of eicosanoid synthesis with BW755c. The release of [3H]arachidonic acid metabolites appeared to be mediated by a transient increase in phospholipase A2 activity. Phospholipase C activity was not affected by TNF. The maximal increase in phospholipase A2 activity occurred at 5 min following the addition of TNF. Phospholipase A2 activation, [3H]arachidonic acid-metabolite synthesis and passage of iodinated protein A, required both RNA and protein synthesis and were associated with an increase in the synthesis of a recently described phospholipase A2-activating protein. The Bordetella pertussis toxin, islet-activating protein, also inhibited the increase in phospholipase A2 activity, the release of [3H]arachidonic acid metabolites and the passage of iodinated protein A, suggesting that the TNF receptor-ligand interaction resulting in cellular retraction, phospholipase A2 activation and eicosanoid synthesis, is coupled through the Ni guanine nucleotide regulatory protein in these cells.     

Lag-burst kinetics in phospholipase A(2) hydrolysis of DPPC bilayers visualized by atomic force microscopy.

The lag-burst phenomenon in the phospholipase A(2) mediated hydrolysis of phospholipid bilayers is for the first time demonstrated in an atomic force microscopy (AFM) study. Simultaneous AFM measurements of the degree of bilayer degradation and the physical-chemical state of the membrane reveals growing nanoscale indentations in the membrane during the lag phase. It is argued that these indentations are domains of hydrolysis products (lysoPC/PC) which eventually trigger the burst. The rate of the rapid hydrolysis following the burst is found to be proportional to the length of the edge between membrane adsorbed and desorbed to the mica base. The observed maximal rate of membrane degradation is approx. 0.2 mmol lipid/min/mol lipase in solution.     

Clearance of group X secretory phospholipase A(2) via mouse phospholipase A(2) receptor.

Given the potent hydrolyzing activity toward phosphatidylcholine, group X secretory phospholipase A(2) (sPLA(2)-X) elicits a marked release of arachidonic acid linked to the potent production of lipid mediators in various cell types. We have recently shown that sPLA(2)-X can also act as a ligand for mouse phospholipase A(2) receptor (PLA(2)R). Here, we found that sPLA(2)-X was internalized and degraded via binding to PLA(2)R associated with the diminished prostaglandin E(2) (PGE(2)) formation in PLA(2)R-expressing Chinese hamster ovary (CHO) cells compared to CHO cells. Indirect immunocytochemical analysis revealed that internalized sPLA(2)-X was co-localized with PLA(2)R in the punctate structures in PLA(2)R-expressing CHO cells. Moreover, in mouse osteoblastic MC3T3-E(1) cells that endogenously express the PLA(2)R, the internalized sPLA(2)-X was localized in lysosomes. These findings demonstrate that PLA(2)R acts as a clearance receptor for sPLA(2)-X to suppress its strong enzymatic activity.     

Secretory phospholipase A(2) induces apoptosis via a mechanism involving ceramide generation

Secretory phospholipase A(2) (sPLA(2)) plays important roles in cellular signaling and various biological events. In this study, we examined the biological effects and the potential signaling mechanism of purified sPLA(2) in MV1Lu cells. Three types of snake venom sPLA(2) were purified and their enzymatic activities were characterized by using various lipid substrates prepared from [3H]-myristate-labeled cells and by determining their effects on the induction of arachidonic acid (AA) release. The purified sPLA(2) induced apoptosis in Mv1Lu cells in a dose- and time-dependent manner, and was associated with a rapid increase in the intracellular ceramide level. Similar apoptotic effects were observed in Mv1Lu cells treated with exogenous ceramide analog, C(2)- and C(8)-ceramide. Moreover, treatment of cells with sphingomyelinase (SMase), which reduced the intracellular SM level, enhanced the apoptotic response to sPLA(2)s. sPLA(2)s also displayed an inhibitory effect on bradykinin-induced phospholipase D (PLD) activity, which can be imitated by exogenous ceramide. Our data indicate that sPLA(2) induces cell apoptosis via a mechanism involving increased ceramide generation.     

Possible involvement of the p57(Kip2) gene in bone metabolism

We previously uncovered that growth stimulation of rat primary osteoblasts by transforming growth factor-beta1 (TGF-beta1) resulted in a dramatic decrease in p57(Kip2), a member of cyclin-dependent kinase (CDK) inhibitors, through the proteasomal degradation pathway (Urano et al., J. Biol. Chem. 274, 12197-12200, 1999). Here we demonstrated that the amount of p57 protein increases markedly, when rat calvarial primary osteoblasts treated with 1,25-dihydroxyvitamin D3 transit from proliferation toward differentiation. Next, we have analyzed the association of four amino acids deletion polymorphism of p57 and bone mineral density (BMD). The p57 genotype was determined in 154 postmenopausal Japanese women. When we separated the subjects into two groups, one having one or two copies of deletion polymorphism and the other without the deletion, the former subjects had higher BMD (Z score of total body, 0.67 +/- 0.93 vs 0. 23 +/- 0.90, mean +/- standard deviation; P = 0.021). Taken together, these findings suggest that the p57 regulated in the osteoblast proliferation and differentiation may play a role in determination of bone mineral density and pathogenesis of osteoporosis.     

Phosphatidic and lysophosphatidic acid production in phospholipase C-and thrombin-treated platelets. Possible involvement of a platelet lipase.

Incubation of 32P-labelled platelets with Clostridium welchii phospholipase C greatly stimulates 32P-incorporation into phosphatidic and lysophosphatidic acids. A net synthesis is demonstrated for both phospholipids, which exhibit identical specific radioactivities. Phosphatidic acid production roughly parallels the phospholipase C-induced aggregation, whereas lysophosphatidic acid appears secondarily during cell lysis. The same qualitative variations are observed during thrombin-induced aggregation. At the physiological pH used throughout the incubations, platelets display no phospholipase A activity towards phosphatidic acid, whereas diglycerides are deacylated by platelet lysates. On the basis of these findings, a mechanism for phosphatidic and lysophosphatidic acid production is proposed, involving a phosphorylation of the di- and monoglycerides formed upon phospholipase C and lipase action. The possible role of such a pathway in regulating arachidonic acid release from phospholipids during platelet activation is discussed.     

Distribution of reaction products in phospholipase A2 hydrolysis

We have monitored the composition of supported phospholipid bilayers during phospholipase A(2) hydrolysis using specular neutron reflection and ellipsometry. Porcine pancreatic PLA(2) shows a long lag phase of several hours during which the enzyme binds to the bilayer surface, but only 5+/-3% of the lipids react before the onset of rapid hydrolysis. The amount of PLA(2), which resides in a 21+/-1 A thick layer at the water-bilayer interface, as well as its depth of penetration into the membrane, increase during the lag phase, the length of which is also proportional to the enzyme concentration. Hydrolysis of a single-chain deuterium labelled d(31)-POPC reveals for the first time that there is a significant asymmetry in the distribution of the reaction products between the membrane and the aqueous environment. The lyso-lipid leaves the membrane while the number of PLA(2) molecules bound to the interface increases with increasing fatty acid content. These results constitute the first direct measurement of the membrane structure and composition, including the location and amount of the enzyme during hydrolysis. These are discussed in terms of a model of fatty-acid mediated activation of PLA(2).     

NGF induces activation of phospholipase C (membrane bound) in PC12 cells.

The aim of this work is to examine if the membrane-bound Phospholipase C system is correlated with the differentiative action of Nerve Growth Factor in PC12 cells. We found that the activity of membrane-bound Phospholipase C system increased with the presence of Nerve Growth Factor at two different phases. The early phase occurs during the first minutes after the formation of Nerve Growth Factor-receptor complex and it is completed within one hour. The later phase starts two hours after Nerve Growth Factor introduction and lasts for at least a total of 48 hours. The inositol-triphosphate levels measured in intact cells by radioimmunoassay show the same pattern of activation. We think that this dual response to Nerve Growth Factor could be due to either a double separated activation of the same enzyme or the presence of two different forms of membrane-bound Phospholipase C.     

Possible involvement of proteasomes (prosomes) in AUUUA-mediated mRNA decay

We have identified a cellular target for proteasomal endonuclease activity. Thus, 20 S proteasomes interact with the 3'-untranslated region of certain cytoplasmic mRNAs in vivo, and 20 S proteasomes isolated from Friend leukemia virus-infected mouse spleen cells were found to be associated with a mRNA fragment showing great homology to the 3'-untranslated region of tumor necrosis factor-beta mRNA that contains AUUUA sequences. We furthermore demonstrate that 20 S proteasomes destabilize oligoribonucleotides corresponding to the 3'-untranslated region of tumor necrosis factor-alpha, creating a specific cleavage pattern. The cleavage reaction is accelerated with increasing number of AUUUA motifs, and major cleavage sites are localized at the 5' side of the A residues. These results strongly suggest that 20 S proteasomes could be involved in the destabilization of cytokine mRNAs such as tumor necrosis factor mRNAs and other short-lived mRNAs containing AUUUA sequences.     

Serotonin 5-HT(2A) receptor activation induces 2-arachidonoylglycerol release through a phospholipase c-dependent mechanism

To date, several studies have demonstrated that phospholipase C-coupled receptors stimulate the production of endocannabinoids, particularly 2-arachidonoylglycerol. There is now evidence that endocannabinoids are involved in phospholipase C-coupled serotonin 5-HT(2A) receptor-mediated behavioral effects in both rats and mice. The main objective of this study was to determine whether activation of the 5-HT(2A) receptor leads to the production and release of the endocannabinoid 2-arachidonoylglycerol. NIH3T3 cells stably expressing the rat 5-HT(2A) receptor were first incubated with [(3)H]-arachidonic acid for 24 h. Following stimulation with 10 mum serotonin, lipids were extracted from the assay medium, separated by thin layer chromatography, and analyzed by liquid scintillation counting. Our results indicate that 5-HT(2A) receptor activation stimulates the formation and release of 2-arachidonoylglycerol. The 5-HT(2A) receptor-dependent release of 2-arachidonoylglycerol was partially dependent on phosphatidylinositol-specific phospholipase C activation. Diacylglycerol produced downstream of 5-HT(2A) receptor-mediated phospholipase D or phosphatidylcholine-specific phospholipase C activation did not appear to contribute to 2-arachidonoylglycerol formation in NIH3T3-5HT(2A) cells. In conclusion, our results support a functional model where neuromodulatory neurotransmitters such as serotonin may act as regulators of endocannabinoid tone at excitatory synapses through the activation of phospholipase C-coupled G-protein coupled receptors.     

Screening and modulation of extracellular signals by mucous barrier. Serum glycosylphosphatidylinositol phospholipase D (GPI-PLD) releases protective mucous barrier from oral mucosa.

BACKGROUND: Performance of mucosal epithelial barrier is modified by numerous agents that exert effects on mucin- Mucin Binding Protein (MBP) complex. The aim ofthe studies described was to determine the nature of the damage or modification of oral mucous barrier by the short-term exposure to ethanol. METHODS: Culture of rat buccal mucosa in the presence of ethanol and [3H]-labeled proline and palmitate revealed substantial decrease in MBP synthesis and the release of MBPto the medium. The radioscanning of the samples prepared from the culture medium and the apical epithelial membranes subjected to SDS-PAGE and western blotting disclosed that the released, water soluble 97kDa MBP glycopeptide was labeled with proline and palmitate. When the experiments were conducted in the presence of 5mM EDTA, the GPI-PLD inhibitor, the majority of radiolabeled MBP remained in the membrane-bound form and was extractable with Triton X- 114. The results on the purified GPI-linked MBP degradation by serum enzyme, by the saliva containing serum transudate, and the suppression of the process by inclusion of GPI-PLD-specific inhibitor support our contention that membrane MBP is released to medium by GPI-PLD-like activity. RESULTS: The release of MBP from apical epithelial surfaces was induced by depletion of mucin and the presence of serum-derived GPI-PLD in the tissue homogenate. Strong likelihood exists that under in situ conditions ethanol-induced transudation of serum to saliva provides the vehicle for the transfer of GPI-PLD activity to salivary contents. Defacement of the oral surfaces from mucous barrier signals prospect of lumenal agent influence on the unprotected epithelial exterior, and allows ingression of microbes and untoward acting substances into the organism.     

Insulin-receptor interactions. Possible involvement of metals

Addition of Zn2+ or Cu2+ ions to plasma membrane preparations or to purified insulin receptors from rat liver resulted in an increase of specific insulin binding; no effect was observed with the addition of Fe3+, Ca2+ or Na+. Dialysis of membrane preparations, or of purified receptors, against chelating agents such as zincon (2-carboxy-2'-hydroxy-5'-sulfoformazyl-benzene) or 1,10-phenantroline resulted in a decrease in specific binding of insulin. With the readdition of Zn2+ or Cu2+ to the medium an increase in specific binding was observed, and values much higher than those of the original preparations were obtained; the addition of Ca2+, Fe3+ or Na+ to dialyzed preparations did not cause any effect on the specific binding. Dialysis of purified receptors against chelating agents resulted in a decrease in the content of Zn2+ and Cu2+. Zincon has been found to be a competitive inhibitor of insulin interfering with the specific binding to the receptor, and noncompetitive with the nonspecific binding. These results suggest the possible involvement of a metal ion present in the receptor in the formation of the insulin-receptor complex.     

Possible involvement of phospholipase A2 and cyclooxygenase in stimulatory action of L-histidine on protein synthesis in L6 myotubes

Effects of L-histidine and related compounds on protein synthesiswere studied in cultured L6 myotubes. L-Histidine specifically stimulated protein synthesis, whereas D-histidine, histamine, L-arginine and L-lysine did not. Inhibitors of phospholipase A₂, phospholipase C and cyclooxygenase intercepted the stimulatory action of L-histidine on protein synthesis, while inhibitors of protein kinase C and 5-lipoxygenase did not. These results suggest an involvement of phospholipase A₂ and cyclooxygenase in the stimulatory action of L-histidine on protein synthesis in L6 myotubes. This revised version was published online in August 2006 with corrections to the Cover Date.     

Clustering of sialylated glycosylphosphatidylinositol anchors mediates PrP-induced activation of cytoplasmic phospholipase A2 and synapse damage

Precisely how the accumulation of PrP^Sc causes the neuronal degeneration that leads to the clinical symptoms of prion diseases is poorly understood. Our recent paper showed that the clustering of specific glycosylphosphatidylinositol (GPI) anchors attached to PrP proteins triggered synapse damage in cultured neurons. First, we demonstrated that small, soluble PrP^Sc oligomers caused synapse damage via a GPI-dependent process. Our hypothesis, that the clustering of specific GPIs caused synapse damage, was supported by observations that cross-linkage of PrP^C, either chemically or by monoclonal antibodies, also triggered synapse damage. Synapse damage was preceded by an increase in the cholesterol content of synapses and activation of cytoplasmic phospholipase A₂ (cPLA₂). The presence of a terminal sialic acid moiety, a rare modification of mammalian GPI anchors, was essential in the activation of cPLA₂ and synapse damage induced by cross-linked PrP^C. We conclude that the sialic acid modifies local membrane microenvironments (rafts) surrounding clustered PrP molecules resulting in aberrant activation of cPLA₂ and synapse damage. A recent observation, that toxic amyloid-β assemblies cross-link PrP^C, suggests that synapse damage in prion and Alzheimer diseases is mediated via a common molecular mechanism, and raises the possibility that the pharmacological modification of GPI anchors might constitute a novel therapeutic approach to these diseases.     

Specificity reversal in phospholipase A2 hydrolysis of lipid mixtures

The activity and specificity of phospholipase A₂ from cobra venom ($\text{Naja naja naja}$) toward binary mixtures of phosphatidylcholine and phosphatidylethanolamine in mixed micelles with the nonionic surfactant Triton X-100 were examined. In mixtures containing 5–50 mol % phosphatidylcholine, the rate for phosphatidylethanolamine hydrolysis was enhanced greatly over that for phosphatidylcholine. This is in marked contrast to previous studies with individual phospholipid species in mixed micelles where phosphatidylcholine was found to be the preferred substrate and phosphatidylethanolamine was found to be a very poor substrate. Possible explanations for this specificity reversal are considered.