Stimulated Phospholipase D Activity Research Articles

Production of Nitric Oxide and Phosphatidic Acid is Involved in Activation of Plasma Membrane H+-ATPase in Maize Root Tips in Simulated Drought Stress

Nitric oxide (NO), phospholipase D (PLD) and plasma membrane (PM) H+-ATPase play crucial roles in plant response to drought stress, but the mechanisms of their function and their relationships in this process are still unclear. Here, we investigated how NO regulate PM H+-ATPase activity in maize (Zea mays L.) root tips in drought stress via inducing PLD-derived phosphatidic acid (PA) formation. Our results showed that drought stress stimulated NO production, up-regulated ZmPLD gene expression and stimulated PLD activity, coupled with PA production. With the elevation of PA content in a specific period (6 to 24 h after treatment), the PM H+-ATPase was stimulated. The NO scavenger impaired drought-induced PLD activation, PA formation and PM H+-ATPase activation at 12 h after treatment. The PLD inhibitor impaired drought-induced PA formation and PM H+-ATPase activation at 12 h after treatment. The NO scavenger, PLD inhibitor and PM H+-ATPase inhibitor aggravated the injury caused by drought stress. Exogenous NO alleviated the injury through a further elevation and a longer maintenance of ZmPLD gene expression, PLD activity, PA content and PM H+-ATPase activity in drought stress. The stimulation effect of exogenous NO on PA formation and PM H+-ATPase activation at 12 h after treatment were partly inhibited by the PLD inhibitor. The results suggested that as an early signal, NO triggers maize plant response to drought stress through increasing PM H+-ATPase activity in root tips. PLD-derived PA acts downstream of NO in this process.

Role of curcumin in PLD activation by Arf6-cytohesin1 signaling axis in U46619-stimulated pulmonary artery smooth muscle cells.

Phospholipase D (PLD) catalyzes the hydrolysis of phosphatidylcholine to produce phosphatidic acid (PA) which in some cell types play a pivotal role in agonist-induced increase in NADPH oxidase-derived [Formula: see text]production. Involvement of ADP ribosylation factor (Arf) in agonist-induced activation of PLD is known for smooth muscle cells of systemic arteries, but not in pulmonary artery smooth muscle cells (PASMCs). Additionally, role of cytohesin in this scenario is unknown in PASMCs. We, therefore, determined the involvement of Arf and cytohesin in U46619-induced stimulation of PLD in PASMCs, and the probable mechanism by which curcumin, a natural phenolic compound, inhibits the U46619 response. Treatment of PASMCs with U46619 stimulated PLD activity in the cell membrane, which was inhibited upon pretreatment with SQ29548 (Tp receptor antagonist), FIPI (PLD inhibitor), SecinH3 (inhibitor of cytohesins), and curcumin. Transfection of the cells with Tp, Arf-6, and cytohesin-1 siRNA inhibited U46619-induced activation of PLD. Upon treatment of the cells with U46619, Arf-6 and cytohesin-1 were translocated and associated in the cell membrane, which were not inhibited upon pretreatment of the cells with curcumin. Cytohesin-1 appeared to be necessary for in vitro binding of GTPγS with Arf-6; however, addition of curcumin inhibited binding of GTPγS with Arf-6 even in the presence of cytohesin-1. Our computational study suggests that although curcumin to some extent binds with Tp receptor, yet the inhibition of Arf6GDP to Arf6GTP conversion appeared to be an important mechanism by which curcumin inhibits U46619-induced increase in PLD activity in PASMCs.

Light induces lettuce seed germination through promoting nitric oxide production and phospholipase D-derived phosphatidic acid formation

Nitric oxide (NO) and phospholipase D (PLD) are essential regulators of seed germination in many plant species, but their roles and relationships in seeds that require light for germination are poorly understood. Here, we investigated how NO and PLD regulate lettuce (Lactuca sativa) seed germination under conditions that either promote (i.e., 23°C in light) or restrict (i.e., 28°C in light or 23°C in darkness) germination. Our results showed that light induced NO production, increased PLD activity and phosphatidic acid (PA) levels in lettuce seeds, which resulted in seed germination. These processes were impaired at 23°C in darkness or at 28°C in light. The NO scavenger(s) inhibited light-induced PLD activation, PA formation and seed germination. Inhibiting PLD prevented light-induced PA formation and seed germination, whereas inhibiting phospholipase C (PLC) did not. Exogenous NO stimulated PLD activity, PA formation and seed germination at 23°C in darkness or at 28°C in light and its effects were offset by the NO scavenger and the PLD inhibitor, rather than by the PLC inhibitor. The results suggested that during lettuce seed germination, light-induced NO production stimulated germination through inducing PA formation via PLD, whereas darkness or high temperature inhibited light-induced germination by impairing NO generation, PLD activation and PA formation.

Role of phospholipases D1 and 2 in astroglial proliferation: effects of specific inhibitors and genetic deletion

Phospholipase D (PLD) activity has been linked to proliferation in many cell types including tumor cells. In the present study, we investigated the effects of genetic deletion of PLD1 and PLD2 and of specific PLD1 and PLD2 inhibitors on PLD activity and cell proliferation in primary mouse astrocytes. Basal and stimulated PLD activity was negligible in PLD1/2 double knockouts. PLD activity was significantly reduced in PLD1-deficient cells when fetal calf serum (FCS), insulin-like growth factor 1 (IGF-1) or phorbol ester was used as a stimulant. The specificity of PLD inhibitors VU0359595 and VU0285655-1 at 500nM was confirmed in phorbol ester-stimulated cells. Significant reductions of cell proliferation were observed in PLD-deficient cell lines under basal and stimulated conditions. At 500nM, the PLD1 inhibitor VU0359595 reduced proliferation in PLD2-deficient cells, but also in PLD1-deficient cells stimulated by IGF-1 or phorbol ester. Vice versa, at 500nM, the PLD2 inhibitor VU0285655-1 reduced proliferation in PLD1-deficient cells, but also in PLD2-deficient cells exposed to IGF-1. At 5µM, both inhibitors showed non-specific effects because they inhibited cell proliferation even in PLD1/2 double knockouts. Summarizing, inhibition of PLD occurs in parallel with reduced cell proliferation in astrocytes which are deficient in PLD1 or PLD2. Synthetic PLD inhibitors show high specificity for PLD in low (nanomolar) concentrations, but have additional, non-specific effects on cell proliferation when used at high (micromolar) concentrations.

Role of phospholipase D in adrenal glomerulosa cell function

ObjectiveTo study the role of agonist‐induced phospholipase D (PLD) activation in modulating aldosterone secretion in human adrenocortical carcinoma cells (HAC15).MethodsHAC15 cells were treated with elevated extracellular potassium (K+) and angiotensin II (AngII) in the presence and absence of 5‐fluoro‐2‐indolyl des‐chlorohalopemide (FIPI), a selective PLD inhibitor. To verify that FIPI was not cytotoxic, we used 22(R)‐hydroxycholesterol [22(R)OH‐cholesterol], a cholesterol analog that can bypass signal transduction pathways and be directly used for aldosterone production. Supernatants were collected and assayed for aldosterone. [3H]Oleate‐prelabeled HAC15 cells were stimulated with AngII in the presence of ethanol, and lipids extracted and assayed for the PLD product, phosphatidylethanol.ResultsFIPI inhibited agonist‐induced aldosterone secretion in a manner unrelated to cytotoxicity, as shown with 22(R)OH‐cholesterol. Furthermore, AngII stimulated PLD activity in HAC15 cells.ConclusionAldosterone secretion in HAC15 cells in response to secretagogues was inhibited by FIPI, suggesting that PLD plays a role not only in AngII‐ but also in K+‐induced aldosterone secretion. Experiments are in progress to determine the time course and dose dependence of the PLD response to AngII and lastly, to define its mechanism of action. Funded by NIH award#HL70046 and a VA Merit award.

Protein Kinase B (AKT) Mediates Phospholipase D Activation via ERK1/2 and Promotes Respiratory Burst Parameters in Formylpeptide-stimulated Neutrophil-like HL-60 Cells

Phospholipase D (PLD), a major source of lipid second messengers (phosphatidic acid, diglycerides) in many cell types, is tightly regulated by protein kinases, but only a few of them have been identified. We show here that protein kinase B (AKT) is a novel major signaling effector of PLD activity induced by the formylpeptide f-Met-Leu-Phe (fMLP) in human neutrophil-like HL-60 cells (dHL-60 cells). AKT inhibition with the selective antagonist AKTib1/2 almost completely prevented fMLP-mediated activity of PLD, its upstream effector ERK1/2, but not p38 MAPK. Immunoprecipitation studies show that phosphorylated AKT, ERK, and PLD2 form a complex induced by fMLP, which can be prevented by AKTib1/2. In cell-free systems, AKT1 stimulated PLD activity via activation of ERK. AKT1 actually phosphorylated ERK2 as a substrate (K(m) 1 μm). Blocking AKT activation with AKTib1/2 also prevented fMLP- but not phorbol 12-myristate 13-acetate-mediated NADPH oxidase activation (respiratory burst, RB) of dHL-60 cells. Impaired RB was associated with defective membrane translocation of NADPH oxidase components p67(phox) and p47(phox), ERK, AKT1, AKT2, but not AKT3. Depletion of AKT1 or AKT2 with antisense oligonucleotides further indicates a partial contribution of both isoforms in fMLP-induced activation of ERK, PLD, and RB, with a predominant role of AKT1. Thus, formylpeptides induce sequential activation of AKT, ERK1/2, and PLD, which represents a novel signaling pathway. A major primarily role of this AKT signaling pathway also emerges in membrane recruitment of NOX2 components p47(phox), p67(phox), and ERK, which may contribute to assembly and activation of the RB motor system, NADPH oxidase.

Protein Kinase C-η and Phospholipase D2 Pathway Regulates Foam Cell Formation via Regulator of G Protein Signaling 2

Regulator of G protein signaling 2 (RGS2) is a GTPase-activating protein for Galpha(q), which is involved in regulating various vascular functions. To understand how RGS2 regulates foam cell formation, the present study identified signaling pathways controlled by lipopolysaccharide (LPS) and discovered new mechanisms whereby protein kinase C (PKC)-eta and phospholipase D (PLD) 2 regulate RGS2 expression. The toll-like receptor (TLR) 4 agonist LPS caused foam cell formation of Raw264.7 macrophages and dramatically decreased RGS2 mRNA expression. RGS2 down-regulation by LPS was partially recovered by TLR4 small interfering RNA (siRNA). Peritoneal macrophages were separated from wild-type and TLR4 mutant mice, and treatment with LPS showed RGS2 expression decrease in wild-type macrophages but no change in TLR4 mutant macrophages. RGS2 overexpression was suppressed, whereas RGS2 down-regulation accelerated foam cell formation by LPS. Treatment of PKC-eta pseudosubstrate weakened foam cell formation and recovered RGS2 down-regulation by LPS. In addition, LPS or phorbol 12-myristate 13-acetate stimulated PLD activity, and the pretreatment of PLD inhibitor weakened foam cell formation and recovered RGS2 down-regulation. Inhibition of PLD2 expression by siRNA also weakened foam cell formation and partially recovered LPS-mediated RGS2 down-regulation. On the other hand, PLD2 overexpression intensified RGS2 down-regulation and foam cell formation by LPS. These results suggest that LPS causes foam cell formation by increasing PKC-eta and PLD2 activity by down-regulating RGS2 expression via TLR4 dependently.

The Coffin‐Lowry syndrome‐associated protein RSK2 is implicated in calcium‐regulated exocytosis through the regulation of PLD1

Exocytosis of neurotransmitters and hormones occurs through the fusion of secretory vesicles with the plasma membrane. This highly regulated process involves key proteins such as SNAREs, and also specific lipids at the site of membrane fusion. Phospholipase D (PLD) has recently emerged as a promoter of membrane fusion in various exocytotic events potentially by providing fusogenic cone‐shaped phosphatidic acid. We show here that PLD1 is regulated by ribosomal S6 kinase 2 (RSK2)‐dependent phosphorylation. RSK2 is activated by a high K+induced rise in cytosolic calcium. Expression of inactive RSK2 mutants or selective knock down of endogenous RSK2 dramatically affects the different kinetic components of the exocytotic response in chromaffin cells. RSK2 physically interacts with and stimulates PLD activity through the phosphorylation of Thr147 in the PLD1 amino‐terminal PX‐domain. Expression of PLD1 phosphomimetic mutants fully restores secretion in cells depleted of RSK2, suggesting that RSK2 is a critical upstream signaling element in the activation of PLD1 to produce the lipids required for exocytosis. We propose that PLD‐related defects in neuronal and endocrine activities could contribute to the effect observed after the loss of function mutations in Rsk2 that leads to Coffin‐Lowry syndrome (CLS), an X‐linked form of growth and mental retardation.

CtBP1/BARS is an activator of phospholipase D1 necessary for agonist-induced macropinocytosis

Vesicular trafficking such as macropinocytosis is a dynamic process that requires coordinated interactions between specialized proteins and lipids. A recent report suggests the involvement of CtBP1/BARS in epidermal growth factor (EGF)-induced macropinocytosis. Detailed mechanisms as to how lipid remodelling is regulated during macropinocytosis are still undefined. Here, we show that CtBP1/BARS is a physiological activator of PLD1 required in agonist-induced macropinocytosis. EGF-induced macropinocytosis was specifically blocked by 1-butanol but not by 2-butanol. In addition, stimulation of cells by serum or EGF resulted in the association of CtBP1/BARS with PLD1. Finally, CtBP1/BARS activated PLD1 in a synergistic manner with other PLD activators, including ADP-ribosylation factors as demonstrated by in vitro and intact cell systems. The present results shed light on the molecular basis of how the ‘fission protein' CtBP1/BARS controls vesicular trafficking events including macropinocytosis.

The Coffin–Lowry Syndrome‐associated Protein RSK2 Controls Neuroendocrine Secretion through the Regulation of Phospholipase D1 at the Exocytotic Sites

Together with the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins, fusogenic cone-shaped lipids, such as phosphatidic acid (PA), have been recently shown to be important actors in membrane fusion during exocytosis. Phospholipase D (PLD) appears to be the main provider of PA at the exocytotic site in neuroendocrine cells. We show here that ribosomal S6 kinase 2 (RSK2) stimulates PLD activity through the phosphorylation of Thr147 in the PLD1 amino-terminal Phox-homology domain. In PC12 cells, depletion of RSK2 dramatically prevents PA synthesis at exocytotic sites and inhibits hormone release. Expression of PLD1 phosphomimetic mutants fully restores secretion in cells depleted of RSK2, suggesting that RSK2 is a critical upstream signaling element in the activation of PLD1 to produce the lipids required for exocytosis.

The Coffin–Lowry syndrome-associated protein RSK2 is implicated in calcium-regulated exocytosis through the regulation of PLD1

Exocytosis of neurotransmitters and hormones occurs through the fusion of secretory vesicles with the plasma membrane. This highly regulated process involves key proteins, such as SNAREs, and specific lipids at the site of membrane fusion. Phospholipase D (PLD) has recently emerged as a promoter of membrane fusion in various exocytotic events potentially by providing fusogenic cone-shaped phosphatidic acid. We show here that PLD1 is regulated by ribosomal S6 kinase 2 (RSK2)-dependent phosphorylation. RSK2 is activated by a high K(+)-induced rise in cytosolic calcium. Expression of inactive RSK2 mutants or selective knockdown of endogenous RSK2 dramatically affects the different kinetic components of the exocytotic response in chromaffin cells. RSK2 physically interacts with and stimulates PLD activity through the phosphorylation of Thr-147 in the PLD1 amino-terminal phox homology domain. Expression of PLD1 phosphomimetic mutants fully restores secretion in cells depleted of RSK2, suggesting that RSK2 is a critical upstream signaling element in the activation of PLD1 to produce the lipids required for exocytosis. We propose that PLD-related defects in neuronal and endocrine activities could contribute to the effect observed after the loss-of-function mutations in Rsk2 that lead to Coffin-Lowry syndrome, an X-linked form of growth and mental retardation.

Up-regulation of cyclooxygenase-2 by cobalt chloride-induced hypoxia is mediated by phospholipase D isozymes in human astroglioma cells

Cyclooxygenase-2 (COX-2) is an isoform of prostaglandin H synthase induced by hypoxia and has been implicated in the growth and progression of a variety of human cancers. In the present study, we investigated the role of phospholipase D (PLD) isozymes in cobalt chloride (CoCl 2)-induced hypoxia-driven COX-2 expression in U87 MG human astroglioma cells. CoCl 2 stimulated PLD activity and synthesis of COX-2 protein in a dose and time-dependent manner. Moreover, elevated expression of PLD1 and PLD2 increased hypoxia-induced COX-2 expression and prostaglandin E2 (PGE 2) production. Pretreatment of cells with 1-butanol, but not 3-butanol, suppressed CoCl 2-induced COX-2 expression and PGE 2 formation. In addition, evidence that PLD activity was involved in the stimulation of COX-2 expression was provided by the observations that overexpression of wild type PLD isozymes, but not catalytically inactive PLD isozymes, stimulated CoCl 2-induced COX-2 expression and PGE 2 production. PLD1 enhanced COX-2 expression by CoCl 2 via reactive oxygen species (ROS), p38 MAPK kinase, PKC-δ, and PKA, but not ERK, whereas PLD2 enhanced CoCl 2-induced COX-2 expression via ROS and p38 MAPK, but not ERK, PKC-δ, and PKA. Differential regulation of COX-2 expression mediated through PLD isozymes was comparable with that of CoCl 2-induced PLD activity in these two PLD isozymes. Taken together, our results demonstrate for the first time that PLD1 and PLD2 isozymes enhance CoCl 2-induced COX-2 expression through differential signaling pathways in astroglioma cells.

Rottlerin inhibits P2X7 receptor‐stimulated phospholipase D activity in chronic lymphocytic leukaemia B‐lymphocytes

Phospholipase D (PLD) is a ubiquitous enzyme that can be activated by extracellular adenosine 5'-triphosphate (ATP) or phorbol 12-myristate 13-acetate (PMA) in B-lymphocytes from subjects with chronic lymphocytic leukaemia (CLL). In this study, ATP- but not PMA-induced PLD stimulation in CLL B-lymphocytes was abolished in the presence of an anti-P2X(7) receptor monoclonal antibody, as well as in B-lymphocytes from CLL subjects homozygous for the Glu(496) to Ala loss-of-function P2X(7) polymorphism. Rottlerin, an inhibitor of novel protein kinase C (PKC) isoforms, but not GF 109203X, an inhibitor of conventional PKC isoforms, impaired the ATP-stimulated PLD activity in CLL B-lymphocytes. In contrast, both inhibitors impaired PLD activity stimulated by PMA, a known mediator of PKC activation. The inhibition of P2X(7)-stimulated PLD activity by rottlerin was attributed to a target downstream of P2X(7) activation, as the ATP-mediated (86)Rb(+) efflux from CLL B-lymphocytes was not altered in the presence of rottlerin. Our results indicate a possible role for novel PKC isoforms in the regulation of P2X(7)-mediated PLD activity.

Phospholipase D and extracellular signal-regulated kinase in hepatic stellate cells: effects of platelet-derived growth factor and extracellular nucleotides

We have previously provided evidence suggesting that phosphatidic acid, possibly derived from the hydrolysis of phosphatidylcholine by phospholipase D (PLD), is involved in platelet-derived growth factor (PDGF)-mediated increases in extracellular signal-regulated kinase (ERK) activity and DNA synthesis in rat hepatic stellate cells (HSC), the primary fibrogenic cells of the liver. A recent study has shown the presence of P2Y nucleotide receptors on HSC that are coupled to contraction and synthesis of the matrix component, alpha1-procollagen, leading to the suggestion that they may represent a new therapeutic target in the treatment of liver fibrosis. However, although extracellular nucleotides have been shown to stimulate both PLD and ERK, and to elicit proliferation of fibrogenic cells outside the liver, their effect on these parameters in HSC have not yet been investigated. PLD activity was determined by [3H]choline release and [3H]phosphatidylbutanol production, ERK activity by Western blotting, and DNA synthesis by [3H]thymidine incorporation. We report here, for the first time in HSC, that extracellular nucleotides stimulate PLD activity and a sustained activation of ERK. However, in contrast to PDGF, nucleotides had negligible effects on DNA synthesis. Moreover, the effects of PDGF and nucleotides on PLD and ERK were not additive, suggesting activation of the same PLD isoform and pool of ERK. The data demonstrate that nucleotide-stimulated PLD and ERK activities are not coupled to DNA synthesis in HSC. Instead, these responses may be linked to other phenotypic changes associated with activated HSC such as increases in contraction, motility, or extracellular matrix deposition.

인간 교세포주에서 CoCl2에 의한 phospholipase D의 조절기전

본 연구에서 최근 세포내 신호전달을 매개하는 중요한 효소로써 PLD 동위효소에 대하여, <TEX>$CoCl_2$</TEX>가 PLD 동위효소의 활성을 증가시킨다는 사실을 밝혔으며, 중간에 매개되는 단백질로써, PLD1은 p38 MAP kinase, PKA와 <TEX>$PKC-{\delta}$</TEX>의 조절을 받고 PLD2는 p38 MAP kinase와 PLC의 조절을 받으므로 그 활성 기전이 각각 다르다는 사실을 확인하였다. 그리고 <TEX>$CoCl_2$</TEX>에 의해 생성되는 활성산소 종에 의한 염증상태가 유도될 것이라고 예상하였고 <TEX>$CoCl_2$</TEX>가 PLD 동위효소를 매개로 하여 염증상태에서만 특이적으로 발현되고 염증반응을 매개하는 COX-2 단백질에 어떠한 영향을 미칠 것인가를 조사하였다. 결과적으로 <TEX>$CoCl_2$</TEX>에 의해 PLD 효소 활성이 증가됨으로써 COX-2의 발현이 증가한다는 것을 발견하였을 뿐만 아니라 COX-2의 발현에 대하여 COX-2 promoter의 활성도 증가한다는 사실을 확인함으로써 전사수준에서의 결과도 이를 뒷받침 해 주고 있었다. Phospholipase D (PLD) is known to play an important role in a variety of cells. However, little is known about <TEX>$CoCl_2-mediated$</TEX> PLD signaling. In this study we demonstrated for the first time that <TEX>$CoCl_2$</TEX> stimulates PLD activity and increases expression of cyclooxygenase-2 (COX-2), which is known to mediate inflammatory reaction. <TEX>$CoCl_2-induced$</TEX> PLD activity was assessed by measuring the formation of <TEX>$[^3H]$</TEX> phosphatidylbutanol (PtdBut), the product of PLD-mediated transphosphatidylation, in the presence of 1-butanol. To study mechanism of PLD signaling induced by <TEX>$CoCl_2$</TEX>, U87 human glioblastoma cells were stimulated by <TEX>$CoCl_2$</TEX> and regulators of PLD activity induced by <TEX>$CoCl_2$</TEX> were investigated using several inhibitors of signaling proteins. Moreover, PLD activation by <TEX>$CoCl_2$</TEX> increased not only expression of COX-2 protein but also COX-2 promoter activity. In summary, these results suggest that <TEX>$CoCl_2$</TEX> increases expression of COX-2 protein via PLD in human U87 glioblastoma cells.

PACAP, VIP, and PHI

Pituitary adenylate cyclase-activating polypeptide (PACAP), vasoactive intestinal peptide (VIP), and peptide histidine-isoleucine (PHI) are members of a superfamily of structurally related peptides widely distributed in the body and displaying pleiotropic biological activities. All these peptides are known to act via common receptors-VPAC1 and VPAC2. In addition, the effects of PACAP are mediated through its specific receptor named PAC1. The main signal transduction pathway of the mentioned receptors is adenylyl cyclase (AC)-->cAMP system. PACAP and VIP may also signal through receptor-linked phospholipase C (PLC)-->IP3/DAG-->PKC and phospholipase D (PLD)-->phosphatidic acid (PA) pathways. In the present article, we have studied the effects of PACAP, VIP, and PHI (0.001-5000 nM) on the AC-, PLC-, and PLD-driven signaling pathways in rat primary glial cell (astrocytes) cultures. All tested peptides dose-dependently and strongly stimulated cyclic adenosine 3',5'-monophosphate (cAMP) production in this experimental model, displaying the following rank order of potency: PACAP >> VIP > or = PHI. Their effects on PLC-IP3/DAG were weaker, while only PACAP and VIP (0.1-5 microM) significantly stimulated PLD activity. The obtained results showed that rat cerebral cortex-derived astrocytes are responsive to PACAP, VIP and PHI/PHM and possess PAC1 and likely VPAC-type receptors linked to activation of AC-cAMP-, PLC-IP3/DAG-, and PLD-PA signaling systems.

Phospholipase D1 is associated with amyloid precursor protein in Alzheimer's disease

Amyloid precursor protein (APP) is a widely expressed transmembrane protein of unknown function that is involved in the pathogenesis of Alzheimer's disease (AD). We investigated the involvement of phospholipase D (PLD) in the pathophysiology of AD. We showed dramatic upregulation of PLD1 immunoreactivity in reactive astroglial cells in brain tissue sections from authentic AD patients. Expression and activity of PLD1 were up-regulated in brain tissues from AD patients, especially caveolae membrane fraction, compared with those of control brains. Interestingly, PLD1 physically interacts and colocalizes with APP and caveolin-3. We found that APP was associated with the pleckstrin homology domain of PLD1, and the amyloid region of APP interacted with PLD. Elevated expression of APP stimulated PLD activity in human astroglioma cells. These results suggest that up-regulation of PLD might have a role in the neuronal pathology associated with AD.

Phospholipase D1 Regulates Phagocyte Adhesion

Adhesion is a fundamental cellular response that is essential to the physiologic processes of development, differentiation, proliferation, and motility, as well as to the pathology of inflammation, transformation, and metastasis. Adhesion of phagocytic leukocytes is a critical modulator of antimicrobial and cytotoxic functions, including the respiratory burst, secretion, and apoptosis. Because phospholipase D (PLD) is linked to several signaling pathways implicated in these processes, we tested the hypothesis that PLD regulates phagocyte adhesion. Adhesion of primary human neutrophils and monocyte-derived macrophages to fibronectin was accompanied by marked stimulation of PLD activity. Similarly, adhesion of both human (PLB, THP-1) and murine (RAW) myeloid-macrophage cell lines to fibronectin, fibrinogen, collagen, or plastic resulted in significant activation of PLD. Stimulation of PLD activity was rapid and persisted for at least 90 min. Confocal microscopy indicated that PLD1 exhibited partial colocalization with actin filaments at the adherent interface, in proximity to the focal adhesion protein, paxillin. Reductions in PLD activity by chemical inhibitors or specific short-interfering RNA-induced knockdown of PLD1 resulted in significant inhibition of phagocyte adhesion and was accompanied by reductions in total cellular F-actin. These data support the hypotheses that adhesion stimulates PLD activity, and that PLD1 regulates the initial stages of phagocyte adhesion. Stimulation of PLD activity may promote adhesion-dependent phagocyte effector responses.

Phospholipase D1 Regulates Phagocyte Adhesion

Adhesion is a fundamental cellular response that is essential to the physiologic processes of development, differentiation, proliferation, and motility, as well as to the pathology of inflammation,transformation, and metastasis. Adhesion of phagocytic leukocytes is a critical modulator of antimicrobial and cytotoxic functions, including the respiratory burst, secretion, and apoptosis. Since phospholipase D (PLD) is linked to several signaling pathways implicated in these processes, we tested the hypothesis that PLD regulates phagocyte adhesion. Using the fluorescent cellular label, calcein, we found that adhesion of primary human neutrophils and monocyte-derived macrophages to fibronectin was accompanied by marked stimulation of PLD activity. Similarly, adhesion of both human (PLB, THP-1) and murine (RAW) myeloid-macrophage cell lines to fibronectin, fibrinogen, collagen or plastic resulted in significant activation of PLD. Stimulation of PLD activity was rapid and persisted for at least 90 min. Confocal microscopy demonstrated that PLD1 colocalized with actin filaments in adherent phagocytes, in proximity to the focal adhesion proteins, paxillin and talin. Reductions in PLD activity by chemical inhibitors or specific siRNA-induced knockdown of PLD1 resulted in significant inhibition of phagocyte adhesion. This was accompanied by decreased polymerization of actin at the adhesion interface and reductions in total cellular F-actin, quantitated using TRITC- phalloidin. These data support the hypothesis that PLD1 regulates the initial stages of phagocyte adhesion via promotion of actin polymerization and suggest that stimulation of PLD activity may promote adhesion-dependent effector responses. Studies supported by NIH GM62302 and AI0559126, and VA Merit Review grants to DJK.

Phospholipase D is activated and phosphorylated by casein kinase-II in human U87 astroglioma cells

Elevated expression of protein casein kinase II (CKII) stimulated basal phospholipase D (PLD) activity as well as PMA-induced PLD activation in human U87 astroglioma cells. Moreover, CKII-selective inhibitor, emodin and apigenin suppressed PMA-induced PLD activation in a dose-dependent manner as well as basal PLD activity, suggesting the involvement of CKII in the activation of both PLD1 and PLD2. CKII was associated with PLD1 and PLD2 in co-transfection experiments. Furthermore, CKII induced serine/threonine phosphorylation of PLD2 in vivo, and the multiple regions of PLD2 were phosphorylated by CKII in vitro kinase assay using glutathione S-transferase-PLD2 fusion protein fragments. Elevated expression of CKII or PLD increased cell proliferation but pretreatment of cells with 1-butanol suppressed CKII-induced cell proliferation. These results suggest that CKII is involved in proliferation of U87 cells at least in part, through stimulation of PLD activity.