Enhancement of phospholipase D activity by overexpression of amyloid precursor protein in P19 mouse embryonic carcinoma cells.

It has been shown that phospholipase D (PLD) activity is stimulated by the beta-amyloid protein in neuronal cells. The aim of this study was to determine whether overexpression of the amyloid precursor protein (APP) affects the activity and the level of PLD expression in P19 embryonic carcinoma cells. We observed that the unstimulated basal PLD activity was higher in wild-type APP(695)-transfected cells than in non-transfected control cells. The protein kinase C (PKC) activator, phorbol 12-myristate 13-acetate (PMA), has been shown to activate PLD. PMA-stimulated PLD activity was 3-fold higher in the APP overexpressing cells than in the control cells. P19 cells express two distinct PLD isozymes, PLD1 and PLD2. The level of PLD2 expression was increased by APP overexpression. Although the PKC inhibitor, GF109203X, inhibited PMA-stimulated PLD activity, it did not affect the high basal PLD activity induced by APP overexpression. Neuronal differentiation of the P19 cells by retinoic acid did not affect the basal or PMA stimulated-PLD activity. Interestingly, APP overexpression in the differentiated P19 cells also led to an increase in PLD activity. The PLD activity of the P19 cells is apparently regulated by amyloid protein through both PKC-dependent and -independent mechanisms.

Increased expression of serine palmitoyltransferase (SPT) in balloon-injured rat carotid artery.

The response to vascular injury is a complex wound healing response involving cell proliferation, migration, remodeling and inflammation. In the present studies we employed a rat balloon angioplasty model of vascular injury to investigate the potential role of sphingolipid signaling in the response to vascular injury. The enzyme serine palmitoyltransferase (SPT) catalyzes the first committed step in de novo sphingolipid biosynthesis. We observed marked upregulation of expression of both SPT subunits in actively proliferating cells in injured vessels. This enhanced SPT expression occurs in de-differentiated fibroblasts and proliferating vascular smooth muscle cells. The upregulation is particularly apparent in the proliferating luminal edge of the neointima and the adventitial de-differentiated fibroblasts and may serve as a hallmark of this process. The possible functional consequences of this enzyme upregulation and its role in the response to vascular injury are suggested but remain to be determined.

Dexamethasone enhances phospholipase D activity in M-1 cells.

Phospholipase D (PLD) is an enzyme involved in signal transduction and widely distributed in mammalian cells. The signal transduction pathways and role for phospholipid metabolism during hormonal response in cortical collecting duct remain partly undefined. It has been reported that dexamethasone increases transepithelial transport in M-1 cells that are derived from the mouse cortical collecting duct. We investigated the expression and activity of PLD in M-1 cells. Basal PLD activity of M-1 cells cultured in the presence of dexamethasone (5 microM) was higher than in the absence of dexamethasone. Dexamethasone and ATP activated PLD in M-1 cells but phorbol ester did not stimulate PLD activity. Vasopressin, bradykinin, dibutyryl cyclic AMP, and ionomycin were ineffective in activating PLD of the cells. The PLD2 isotype was detected by immunoprecipitation but PLD1 was not detected in M-1 cells. Addition of GTPgammaS and ADP-ribosylation factor or phosphatidylinositiol 4,5-bisphosphate to digitonin-permeabilized cells did not augment PLD activity. In intact cells PLD activity was increased by sodium oleate but there was no significant change between dexamethasone treated- and untreated cells by oleate. These results suggest that at least two types of PLD are present in M-1 cells and PLD plays a role in the corticosteroid-mediated response of cortical collecting duct cells.

Serine palmitoyltransferase regulates de novo ceramide generation during etoposide-induced apoptosis.

The de novo pathway of sphingolipid synthesis has been identified recently as a novel means of generating ceramide during apoptosis. Furthermore, it has been suggested that the activation of dihydroceramide synthase is responsible for increased ceramide production through this pathway. In this study, accumulation of ceramide mass in Molt-4 human leukemia cells by the chemotherapy agent etoposide was found to occur primarily due to activation of the de novo pathway. However, when the cells were labeled with a substrate for dihydroceramide synthase in the presence of etoposide, there was no corresponding increase in labeled ceramide. Further investigation using a labeled substrate for serine palmitoyltransferase, the rate-limiting enzyme in the pathway, resulted in an accumulation of label in ceramide upon etoposide treatment. This result suggests that the activation of serine palmitoyltransferase is the event responsible for increased ceramide generation during de novo synthesis initiated by etoposide. Importantly, the ceramide generated from de novo synthesis appears to have a distinct function from that induced by sphingomyelinase action in that it is not involved in caspase-induced poly (ADP-ribose)polymerase proteolysis but does play a role in disrupting membrane integrity in this model system. These results implicate serine palmitoyltransferase as the enzyme controlling de novo ceramide synthesis during apoptosis and begin to define a unique function of ceramide generated from this pathway.

Downregulation of phospholipase D by protein kinase A in a cell-free system of human neutrophils.

Agents which elevate cellular cAMP are known to inhibit the activation of phospholipase D (PLD) in human neutrophils. The PLD activity of human neutrophils requires protein factors in both membrane and cytosolic fractions. We have studied the regulation of PLD by the catalytic subunit of protein kinase A (cPKA) in a cell-free system. cPKA significantly inhibited GTPgammaS-stimulated PLD activity but had no effect on phorbol ester-activated PLD activity. Pretreatment of plasma membranes with cPKA and ATP inhibited subsequent PLD activation upon reconstitution with untreated cytosol. RhoA, which is known to be a plasma membrane activator of PLD, was dissociated from PKA-treated plasma membrane by addition of cytosol. Plasma membrane-associated RhoA in human neutrophils was phosphorylated by cPKA. The PKA-phosphorylated form of RhoA was more easily extracted from membranes by RhoGDI than the unphosphorylated form. These results suggest that inhibition of neutrophil PLD by PKA may be due to phosphorylation of RhoA on the plasma membrane.

Calyculin A modulates activation of the NADPH-oxidase in Me2SO-differentiated HL-60 cells.

Human promyelocytic leukemia cells (HL-60) have been used as a model system in which to study the effects of protein phosphatase inhibitors on NADPH-oxidase activation. Since O2- is generated by NADPH-oxidase, we examined the effect of calyculin A pretreatment on oxidase activation in response to various agonists. When Me2SO-differentiated HL-60 cells were treated with calyculin A prior to the addition of phorbol 12-myristate 13-acetate (PMA), O2- production was inhibited; however, calyculin A enhanced O2- production by N-formyl-methionyl-leucyl-phenylalanine (FMLP). The decreased O2- production seen with calyculin A pretreatment followed by PMA may be due to diminished translocation of the p47-phox and p67-phox, cytosolic components of the oxidase, and inhibition of arachidonic acid release. Interestingly calyculin A pretreatment followed by either agonist significantly enhanced mitogen-activated-protein kinase (MAPK) activity. The differential effects of pretreatment with calyculin A on subsequent oxidase stimulation elicited by FMLP or PMA provide further evidence for substantial heterogeneity in the activation of the respiratory burst.

Lipase activities of p37, the major envelope protein of vaccinia virus.

p37, the major protein of the extracellular enveloped form of vaccinia virus, is involved in the biogenesis of the viral double membrane and in egress of virus from the cell. p37 was expressed as a glutathione S-transferase fusion protein and was purified to homogeneity by silver staining using glutathione-agarose, Sephacryl S-200, and DEAE-cellulose chromatography. Incubation of p37 with phosphatidylcholine labeled in the fatty acyl side chains resulted in the production of multiple lipid products that were identified by thin layer chromatography and mass spectrometry as diacylglycerol, free fatty acid, monoacylglycerol, and lysophosphatidylcholine. Lipid-metabolizing activities colocalized with p37-containing fractions throughout the chromatographic steps. p37 also metabolized phosphatidylethanolamine efficiently, but it had less activity toward phosphatidylinositol and little or no activity toward phosphatidylserine. The purified enzyme also metabolized triacylglycerol to diacylglycerol but was inactive toward sn-1, 2-diacylglycerol. p37 was also expressed in insect cells as a poly-His fusion protein; cell lysates and partially purified proteins also generated products expected from phospholipase C and A activities. Thus, p37 is a broad specificity lipase with phospholipase C, phospholipase A, and triacylglycerol lipase activities.

Spermine suppresses the activation of human neutrophil NADPH oxidase in cell-free and semi-recombinant systems.

Spermine, a cellular polyamine, down-regulates O2- generation in human neutrophils stimulated by receptor-linked agonist [Ogata, Tamura and Takeshita (1992) Biochem. Biophys. Res. Commun. 182, 20-26]. In this study, to elucidate the mechanism for the inhibition, the effect of spermine on cell-free activation of the O2- generating enzyme (NADPH oxidase) was examined. Spermine suppressed the SDS-induced activation of NADPH oxidase in a dose-dependent manner with an IC50 of 18 microM. The inhibition was specific for spermine over its precursor amines, spermidine and putrescine. Spermine did not alter the Km for NADPH or the optimal concentration of SDS for activation. The amine was inhibitory only when added before activation, indicating that it affects the activation process rather than the enzyme's activity. An increased concentration of cytosol partly prevented the inhibition by spermine. In semi-recombinant cell-free system, spermine inhibited the activation of NADPH oxidase as effectively as in the cell-free system (IC50 = 13 microM). Pretreatment of each recombinant cytosolic component with spermine revealed that they (especially p67phox) are sensitive to spermine. These results suggest that spermine interacts with cytosolic component(s) and impairs the assembly of NADPH oxidase.

RhoA and a cytosolic 50-kDa factor reconstitute GTP gamma S-dependent phospholipase D activity in human neutrophil subcellular fractions.

Receptor activation of phospholipase D has been implicated in signal transduction in a variety of cells. Reconstitution of cell-free guanosine 5'-O-(3-thiotriphosphate)(GTP gamma S)-dependent phospholipase D activity from human neutrophils requires protein factors in both the plasma membrane and the cytosol. We previously proposed that one of the factors is a Ras-family small molecular weight GTPase of the Rho subtype (Bowman, E. P., Uhlinger, D. J., and Lambeth, J. D. (1993) J. Biol. Chem. 268, 21509-21512). Herein, we have used RhoGDI (GDP dissociation inhibitor), an inhibitory Rho-binding protein, to selectively extract Rho-type GTPases from the plasma membrane, and have used immunoprecipitation as well as chromatographic methods to remove cytosolic Rho. Depletion of RhoA from either the plasma membrane or the cytosol resulted in a partial loss in GTP gamma S dependent activity, while removal of RhoA from both fractions resulted in a nearly complete loss in activity. Activity was nearly completely restored by adding purified recombinant RhoA, which showed an EC50 of 52 nM, while Rac1 showed little activity. Cytosol fractionated using DEAE-cellulose chromatography separated ADP-ribosylation factor and Rho from the major activating fraction. Gel exclusion chromatography of this fraction revealed an activating factor of 50 kDa apparent molecular mass. Using RhoA-depleted membranes, reconstitution of phospholipase D activity required both RhoA and the 50-kDa factor. Thus, RhoA along with a non-Rho, non-ADP-ribosylation factor 50-kDa cytosolic factor are both required to reconstitute GTP gamma S-dependent phospholipase D activity by neutrophil plasma membranes.

ADP-ribosylation factor functions synergistically with a 50-kDa cytosolic factor in cell-free activation of human neutrophil phospholipase D.

Proteins in both the cytosol and plasma membrane are needed to reconstitute cell-free phospholipase D activity from phagocytes (Olson, S., Bowman, E. P., and Lambeth, J. D. (1991) J. Biol. Chem. 266, 17236-17242); membrane factors include a small GTP-binding protein in the Rho family (Bowman, E., Uhlinger, D. J., and Lambeth, J. D. (1993) J. Biol. Chem. 268, 21509-21512). ADP-ribosylation factor (ARF) was recently implicated as the cytosolic factor, as it activates phospholipase D in HL-60 membranes. Herein, we show that ion exchange chromatography separates ARF from the major phospholipase D-stimulating cytosolic factor. Both bovine brain ARF and recombinant human ARF-1 stimulated a small amount of phospholipase D activity in the absence of cytosol (about 10% of the response seen with cytosol). With a high concentration of ARF-depleted cytosol, ARF did not further activate. However, at low cytosol, ARF caused marked activation. Thus, ARF synergizes with the cytosolic factor in phospholipase D activation.

On the mechanism of inhibition of the neutrophil respiratory burst oxidase by a peptide from the C-terminus of the large subunit of cytochrome b558.

A peptide (RGVHFIF) from near the carboxyl terminus (residues 559-565) of gp91-phox, the large subunit of cytochrome b558, was previously shown to inhibit activation of the respiratory burst oxidase [Kleinberg, M. E., Malech, H. L., & Rotrosen, D. (1990) J. Biol. Chem. 265, 15577-15583]. The peptide has been proposed to compete with gp91-phox binding to p47-phox, one of the cytosolic oxidase components. In the present studies, we have used a semirecombinant system consisting of recombinant cytosolic factors (p47-phox, p67-phox, and Rac1) along with isolated plasma membrane to investigate the mechanism by which the peptide inhibits oxidase activation. In an in vitro translocation model, the peptide inhibited arachidonate-activated translocation of both p47-phox and p67-phox to the plasma membrane. The kinetic mechanism of inhibition was examined. Inhibition was noncompetitive or mixed with respect to not only Rac and p67-phox but also to p47-phox. We suggest that the peptide, rather than competing for cytochrome-p47-phox interactions, inhibits indirectly, perhaps by binding to and altering the conformation of cytochrome b558.

Ras effector-homologue region on Rac regulates protein associations in the neutrophil respiratory burst oxidase complex.

Rac, a small molecular weight GTPase in the Ras superfamily, participates in the activation of the multicomponent superoxide-generating NADPH oxidase of human neutrophils. Rac is 30% identical to Ras overall, but is 75% identical within the sequence corresponding to the effector region of Ras, which regulates mitogenesis through interactions with the protein kinase Raf1. We investigated the role of this region in Rac1 using site-directed mutagenesis. In a cell-free semirecombinant NADPH oxidase system, mutants in the 26, 33, 38, and 45 amino acids showed 20-110-fold reduced binding to the oxidase complex as judged by EC50 values and reduced (44-80%) maximal activities in superoxide generation. Only the GTP gamma S-bound form associated, since the GDP-bound form of Rac neither activated alone nor competed with GTP gamma S-Rac. EC50 values for neither p47-phox nor p67-phox were affected when mutant Racs were used in place of Rac. Data indicate direct binding of the Rac effector region to one or more components of the respiratory burst oxidase. Results indicate a general role for conserved effector-equivalent regions in small GTPases in the regulation of protein-protein interactions.

p67-phox enhances the binding of p47-phox to the human neutrophil respiratory burst oxidase complex.

The neutrophil respiratory burst oxidase consists of both the plasma membrane-associated flavocytochrome b558 and cytosolic regulatory proteins including p47-phox, p67-phox, and a small GTP-binding protein (Rac1 and/or Rac2). Oxidase activation is thought to result from the assembly of the cytosolic components on the cytochrome. A model has been proposed in which p47-phox binds directly to the cytochrome, while p67-phox binding occurs indirectly by binding to p47-phox. We have carried out a steady state kinetic analysis using a cell-free semirecombinant activation system (isolated plasma membrane plus recombinant cytosolic factors) to analyze the effects of p47-phox and p67-phox on one another's association in the active oxidase complex. As predicted from the model, increasing p47-phox concentration markedly lowered the EC50 for p67-phox, indicating that p67-phox is dependent upon p47-phox for binding. Unexpectedly, increasing p67-phox concentration also produced a significant enhancement of p47-phox binding. Thus, a more complex binding model must be invoked in which p47-phox and p67-phox mutually enhance one another's binding.

Expression of p47-phox and p67-phox proteins in murine bone marrow-derived macrophages: enhancement by lipopolysaccharide and tumor necrosis factor alpha but not colony stimulating factor 1.

We have investigated the relationship between the expression of the p47-phox and p67-phox cytosolic components of the NADPH oxidase and priming of the macrophage respiratory burst. Western blot analysis revealed that murine bone marrow-derived macrophages (BMM) contain immunoreactive proteins detected by antisera raised against recombinant human p47-phox and p67-phox. Priming BMM by exposure to tumor necrosis factor alpha (TNF-alpha) or lipopolysaccharide (LPS) increased the levels of p47-phox and p67-phox. Colony-stimulating factor 1 (CSF-1), which we previously found to have a negative effect on the priming of murine macrophages, had no effect on the level of p47-phox but down-regulated that of p67-phox. Our results suggest that the regulatory effects of LPS, TNF-alpha, and CSF-1 on the respiratory burst of BMM may be due to modulation of the expression of the p47-phox and p67-phox cytosolic components of the NADPH oxidase.

Participation of the small molecular weight GTP-binding protein Rac1 in cell-free activation and assembly of the respiratory burst oxidase. Inhibition by a carboxyl-terminal Rac peptide.

NADPH-dependent superoxide generation was activated by anionic amphiphiles plus GTP gamma S in a cell-free system consisting of plasma membranes plus recombinant p47-phox, p67-phox, and the small GTP-binding protein Rac1. Rac1 was expressed in Escherichia coli both as the native form and as a mutant form (Rac1(C189S)) lacking the prenylation site. When preloaded with GTP gamma S, both Rac proteins supported activity to a level comparable to that seen using cytosol. A peptide corresponding to the carboxyl-terminal region of Rac1 was used to investigate oxidase assembly and activation. Rac1(178-188), but not several control peptides, inhibited activity. The peptide inhibited competitively (Ki = 15 microM) with respect to Rac1(C189S), while inhibition was noncompetitive or mixed with respect to p47-phox and p67-phox. This indicated specific inhibition of the interaction of the Rac protein with its target, possibly cytochrome b558. The peptide was effective only when added prior to activation with arachidonic acid, suggesting that it affects assembly rather than activity. Consistent with this possibility, the peptide prevented translocation of p47-phox and p67-phox to the plasma membrane. Thus, Rac plays a central role in the assembly of the neutrophil NADPH oxidase.

Neutrophil phospholipase D is activated by a membrane-associated Rho family small molecular weight GTP-binding protein.

Phospholipase D in human neutrophil lysates is activated by GTP gamma S (guanosine 5'-O-(3-thiotriphosphate)), implying the participation of a GTP-binding protein. Reconstitution of GTP gamma S-dependent activity requires protein factors in both the plasma membrane and the cytosol (Olson, S. C., Bowman, E. P., and Lambeth, J. D. (1991) J. Biol. Chem. 266, 17236-17242). The location of the GTP-binding protein was investigated by preincubating either cytosol or plasma membrane with GTP gamma S, followed by removal of all but tightly bound nucleotide and reconstituting activity with the complementing untreated subcellular fraction. This approach indicated that the GTP-binding protein was membrane-associated. A low magnesium requirement for GTP gamma S prebinding, as well as a failure of aluminum fluoride to activate, suggested a Ras-like small M(r) GTP-binding protein. smg GDP dissociation stimulator, which stimulates the exchange of GDP for GTP on a variety of small GTP-binding proteins, stimulated GTP-dependent phospholipase D activity. Rho GDP dissociation inhibitor, a regulatory protein that binds specifically to and inhibits the functions of Rho family small GTP-binding proteins, inhibited GTP gamma S-dependent activity. Thus, neutrophil phospholipase D is regulated by a membrane-associated small molecular weight GTP-binding protein, likely to be a member of the Rho family.

The respiratory burst oxidase of human neutrophils. Guanine nucleotides and arachidonate regulate the assembly of a multicomponent complex in a semirecombinant cell-free system.

We recently characterized a "semirecombinant" cell-free NADPH-oxidase system, comprised of plasma membrane plus the recombinant cytosolic proteins p47-phox and p67-phox, wherein superoxide generation was activated by an anionic amphiphile plus guanosine 5'-O-(2-thiotriphosphate) (GTP gamma S) (Uhlinger, D. J., Inge, K. L., Kreck, M. L., Tyagi, S. R., Neckelmann, N., and Lambeth, J. D. (1992) Biochem. Biophys. Res. Commun. 186, 509-516). Based on preincubation with guanine nucleotides, we show that plasma membrane contains G protein(s) that support oxidase activation at submaximal rates. By varying p47-phox and p67-phox concentrations, kinetic parameters (EC50 and Vmax) for each were determined. For both, GTP gamma S increased the Vmax and decreased the EC50, whereas guanosine 5'-O-(2-thiodiphosphate) (GDP beta S) produced the opposite effect, consistent with the participation of a G protein in an activation complex containing p47-phox and p67-phox. Using [35S]methionine-labeled p47-phox and p67-phox, we investigated the association of these components with both normal plasma membranes and chronic granulomatous disease membranes lacking cytochrome b558. p47-phox translocation was stimulated by arachidonate but not GTP gamma S, was about 50% cytochrome-dependent, and occurred independently of p67-phox. Arachidonate-stimulated translocation of p67-phox required both cytochrome and p47-phox and was enhanced by GTP gamma S. The mass of p47-phox and p67-phox which assembled with cytochrome b558 indicated a ternary complex with a 1:1:1 stoichiometry.

Requirement for posttranslational processing of Rac GTP-binding proteins for activation of human neutrophil NADPH oxidase.

Rac1 and Rac2 are closely related, low molecular weight GTP-binding proteins that have both been implicated in regulation of phagocyte NADPH oxidase. This enzyme system is composed of multiple membrane-bound and cytosolic subunits and when activated catalyzes the one-electron reduction of oxygen to superoxide. Superoxide and its highly reactive derivatives are essential for killing microorganisms. Rac proteins undergo posttranslational processing, primarily the addition of an isoprenyl group to a carboxyl-terminal cysteine residue. We directly compared recombinant Rac1 and Rac2 in a human neutrophil cell-free NADPH oxidase system in which cytosol was replaced by purified recombinant cytosolic components (p47-phox and p67-phox). Processed Rac1 and Rac2 were both highly active in this system and supported comparable rates of superoxide production. Under different cell-free conditions, however, in which suboptimal amounts of cytosol were present in the assay mixture, processed Rac2 worked much better than Rac1 at all but the lowest concentrations. This suggests that a factor in the cytosol may suppress the activity of Rac1 but not of Rac2. Unprocessed Rac proteins were only weakly able to support superoxide generation in either system, but preloading of Rac1 or Rac2 with guanosine 5'-O-(3-thio-triphosphate) (GTP gamma S) restored activity. These results indicate that processing is required for nucleotide exchange but not for interaction with oxidase components.

Expression of recombinant myeloperoxidase using a baculovirus expression system.

Myeloperoxidase (MPO) is a glycosylated heme-containing enzyme present in the azurophilic granules of normal human polymorphonuclear neutrophils. This enzyme plays a major role in the microbicidal activity of the host defense system by catalyzing the formation of the potent oxidant, hypochlorous acid. Although the amino acid sequence of MPO has been deduced from the cDNA, the structural basis for the observed heterogeneity of this enzyme is not known. Furthermore, the nature of the prosthetic group and its mode of linkage to the apoprotein has not been determined. To address questions regarding the structural features of MPO, which arise during the complex posttranslational processing of this enzyme, we utilized a baculovirus system to express MPO in Sf9 insect cells. Two glycosylated, single-chain precursor species of MPO were observed: an 84 kDa species that was secreted and a 74 kDa species that was cell-associated. This is the first report of an expression system in which a cell-associated MPO precursor undergoes posttranslational proteolytic processing.