PLD Activity Research Articles

Phospholipase D inhibition by hexanal is associated with calcium signal transduction events in raspberry

Raspberry (Rubus spp.) is an economically important crop with a restricted growing season and very limited fruit shelf-life due to its extreme tenderness. In order to prolong its shelf life, an aqueous composition containing hexanal as the key active ingredient (HC) was applied as a preharvest spray during fruit development. The effects of HC were assessed using physiological, biochemical and anatomical parameters on the treated fruits and compared with the effects of mock inoculation which lacked hexanal. Sugars and acidity did not show a significant change in response to HC treatment, while the pulling force (the tension required to detach the berry from the receptacle) significantly improved in the HC-treated fruits, compared to control. Scanning electron microscope (SEM) analysis revealed a high correlation between the presence of rigid epidermal hairs and a stronger degree of attachment between berries and their receptacle in the HC treated fruits. Further, electron micrographs also showed abnormal crystalline depositions on the epidermal drupelets of the treated berries. Energy Dispersive X-ray Spectroscopy (EDS) analysis showed those crystals to be largely composed of calcium. HC treatment also resulted in the reduction of transcript level of three phospholipase D genes, as well as altered expression pattern of five members of the annexin gene family, and four calmodulin-binding transcription activators. Quantification of PLD activity showed that hexanal inhibited PLD activity in treated berries. The potential crosstalk between hexanal, phospholipase D activity and calcium and this crosstalk’s role in delaying fruit softening and in prolonging storage life of fruits shelf life is discussed.

Inhibition of PLD1 activity causes ER stress via regulation of COPII vesicle formation

Phospholipase D (PLD) plays a crucial role in the regulation of some cellular processes, including autophagy and apoptosis. Accumulation of protein in the endoplasmic reticulum (ER) lumen causes ER stress. Although ER stress is a principal cause of apoptosis and autophagy, the relationship between PLD activity and ER stress remains unclear. Protein transport from the ER to the Golgi apparatus is conducted by coat complex II (COPII) transport vesicles. Here, we demonstrated that inhibition of PLD1 activity or PLD1 knockdown suppressed COPII vesicle transport in normal rat kidney (NRK) cells. COPII vesicle coat proteins are composed of Sar1 as well as Sec23/24 and Sec13/31 complexes. For COPII vesicle formation on the ER membrane, Sar1, Sec23/24, and Sec13/31 are sequentially recruited from the cytosol to the ER membrane. Using a cell-free COPII coat protein recruitment assay, we demonstrated that inhibition of PLD1 activity suppressed Sec13/31 recruitment from the cytosol to the ER membrane in COPII vesicle formation. PLD1 knockdown in NRK cells was associated with increased expression of the ER stress marker GRP78 and apoptosis. Taken together, these results suggest that PLD1 activity regulates COPII vesicle transport from the ER to the Golgi apparatus by regulating Sec13/31 recruitment from the cytosol to the ER membrane during COPII vesicle formation.

A snake venom group IIA PLA2 with immunomodulatory activity induces formation of lipid droplets containing 15-d-PGJ2 in macrophages

Crotoxin B (CB) is a catalytically active group IIA sPLA2 from Crotalus durissus terrificus snake venom. In contrast to most GIIA sPLA2s, CB exhibits anti-inflammatory effects, including the ability to inhibit leukocyte functions. Lipid droplets (LDs) are lipid-rich organelles associated with inflammation and recognized as a site for the synthesis of inflammatory lipid mediators. Here, the ability of CB to induce formation of LDs and the mechanisms involved in this effect were investigated in isolated macrophages. The profile of CB-induced 15-d-PGJ2 (15-Deoxy-Delta-12,14-prostaglandin J2) production and involvement of LDs in 15-d-PGJ2 biosynthesis were also investigated. Stimulation of murine macrophages with CB induced increased number of LDs and release of 15-d-PGJ2. LDs induced by CB were associated to PLIN2 recruitment and expression and required activation of PKC, PI3K, MEK1/2, JNK, iPLA2 and PLD. Both 15-d-PGJ2 and COX-1 were found in CB-induced LDs indicating that LDs contribute to the inhibitory effects of CB by acting as platform for synthesis of 15-d-PGJ2, a pro-resolving lipid mediator. Together, our data indicate that an immunomodulatory GIIA sPLA2 can directly induce LD formation and production of a pro-resolving mediator in an inflammatory cell and afford new insights into the roles of LDs in resolution of inflammatory processes.

Identification of differentially expressed genes associated with clinical response after treatment of breast cancer skin metastases with imiquimod.

e12541 Background: Imiquimod, a Toll-like receptor 7 agonist, activates innate immunity and has a response rate of 20% in breast cancer skin metastases. Tumor regression is associated with increased tumor infiltrating lymphocytes and increased Th1 cytokines in tumor supernatant, however gene expression changes are unknown. This is the first report of gene expression changes induced by imiquimod treatment in breast cancer skin metastases. Methods: FFPE-extracted mRNA from 8 patients pre and post 8 weeks of imiquimod treatment was analyzed using Nanostring PanCancer Immune Profiling Panel. Gene expression and pathway analysis was conducted with nSolver 3.0 with clinical response as outcome, controlling for HER2 ER/PR status, with 5% significance level used. Results: In all patients, imiquimod was associated with upregulation of genes in innate immunity such as TLR7, IL-1, T-cell, B-cell, and NK related genes. Three patients had a clinical response after imiquimod. Forty-two differentially expressed genes were identified in responders vs. non-responders after imiquimod. Reponders had up-regulation of T cell (CD2, SPN), NK cell (KLRC2), B cell (CD48), lymphocyte (LY96), and IL-2 (IL2RG) related genes. The top upregulated pathways were cytotoxicity, NK cell function, and antigen processing. Pre-treatment, the responders had upregulation of TNF and IL-17 related genes (TNFRSF10B, IL17RA) compared to non-responders. Comparing pre-treatment to post-treatment, Imiquimod was associated with mRNA expression changes in 72 genes in responders vs non responders. The top upregulated genes involved activation of lymphocytes (SH2D1A), NK cells (KLRC2), DCs (CLEC4C), several chemokines, IL7R and immune regulators PLD1 and IRF5. Conclusions: Clinical response to Imiquimod treatment of breast cancer skin metastases is associated with upregulation of genes in innate immunity suggestive of an anti-tumor immune response. Inhibition of negative feedback by addition of PLD1 inhibitors may enhance treatment response. Understanding the genetic signature of imiquimod can enhance prediction of response and inform possible combination therapies in the future.

Enzymatic methods for choline-containing water soluble phospholipids based on fluorescence of choline oxidase: Application to lyso-PAF

In this paper we present methods to determine water soluble phospholipids containing choline (wCh-PL). The analytes were hydrolyzed by the enzyme phospholipase D and the choline formed was oxidized by the enzyme Choline Oxidase (ChOx); the fluorescence changes of the ChOx are followed during the enzymatic reaction, avoiding the necessity of an indicating step. Both reactions (hydrolysis and oxidation) can be combined in two different ways: 1) a two-step process (TSP) in which the hydrolysis reaction takes place during an incubation time and then the oxidation reaction is carried out, the analytical signal being provided by the intrinsic fluorescence of ChOx due to tryptophan; 2) a one-step process (OSP) in which both enzymatic reactions are carried out simultaneously in the same test; in this case the analytical signal is provided by the ChOx extrinsic fluorescence due to a fluorescent probe (Ru (II) chelate) linked to the enzyme (ChOx-RuC).The analytical capabilities of these methods were studied using 1,2-dioctanoyl-sn-glycero-3-phosphocholine (C8PC), a water soluble short alkyl chain Ch-PL as a substrate, and 1-O-hexadecyl-sn-glyceryl-3-phosphorylcholine (lyso-PAF).The analytical features of merit for both analytes using both methods were obtained. The TSP gave a 10-fold sensitivity and lower quantification limit (1.0*10−5 M for lyso-PAF), but OSP reduced the determination time and permitted to use the same enzyme aliquot for several measurements. Both methods gave similar precision (RSD 7%, n = 5). The TSP was applied to the determination of C8PC and lyso-PAF in spiked synthetic serum matrix using the standard addition method.The application of this methodology to PLD activity determination is also discussed.

HS1BP3 negatively regulates autophagy by modulation of phosphatidic acid levels.

A fundamental question is how autophagosome formation is regulated. Here we show that the PX domain protein HS1BP3 is a negative regulator of autophagosome formation. HS1BP3 depletion increased the formation of LC3-positive autophagosomes and degradation of cargo both in human cell culture and in zebrafish. HS1BP3 is localized to ATG16L1- and ATG9-positive autophagosome precursors and we show that HS1BP3 binds phosphatidic acid (PA) through its PX domain. Furthermore, we find the total PA content of cells to be significantly upregulated in the absence of HS1BP3, as a result of increased activity of the PA-producing enzyme phospholipase D (PLD) and increased localization of PLD1 to ATG16L1-positive membranes. We propose that HS1BP3 regulates autophagy by modulating the PA content of the ATG16L1-positive autophagosome precursor membranes through PLD1 activity and localization. Our findings provide key insights into how autophagosome formation is regulated by a novel negative-feedback mechanism on membrane lipids.

Kinetic disruption of lipid rafts is a mechanosensor for phospholipase D.

The sensing of physical force, mechanosensation, underlies two of five human senses—touch and hearing. How transduction of force in a membrane occurs remains unclear. We asked if a biological membrane could employ kinetic energy to transduce a signal absent tension. Here we show that lipid rafts are dynamic compartments that inactivate the signalling enzyme phospholipase D2 (PLD2) by sequestering the enzyme from its substrate. Mechanical disruption of the lipid rafts activates PLD2 by mixing the enzyme with its substrate to produce the signalling lipid phosphatidic acid (PA). We calculate a latency time of <650 μs for PLD activation by mixing. Our results establish a fast, non-tension mechanism for mechanotransduction where disruption of ordered lipids initiates a mechanosensitive signal for cell growth through mechanical mixing.

PLD1 regulates adipogenic differentiation through mTOR - IRS-1 phosphorylation at serine 636/639.

Phospholipase D1 (PLD1) plays a known role in several differentiation processes, but its role in adipogenic differentiation remains unknown. In the present study, we identified PLD1 as a negative regulator of adipogenic differentiation. We showed that PLD activity was downregulated by both 3-Isobutyl-1-methylxanthine (IBMX) and insulin upon induction of differentiation in 3T3-L1 adipogenic cells. In line with this observation, PLD activity decreased in both high fat diet (HFD)-fed mice and ob/ob mice. We also found that differentiation of 3T3-L1 preadipocytes was enhanced by the depletion of PLD1 levels or inhibition of PLD1 activity by VU0155069, a PLD1-specific inhibitor. Conversely, treatment with phosphatidic acid (PA), a PLD product, and overexpression of PLD1 both caused a decrease in adipogenic differentiation. Moreover, the elevated differentiation in PLD1-knockdown 3T3-L1 cells was reduced by either PA treatment or PLD1 expression, confirming negative roles of PLD1 and PA in adipogenic differentiation. Further investigation revealed that PA displaces DEP domain-containing mTOR-interacting protein (DEPTOR) from mTORC1, which subsequently phosphorylates insulin receptor substrate-1 (IRS-1) at serine 636/639 in 3T3-L1 cells. Taken together, our findings provide convincing evidence for a direct role of PLD1 in adipogenic differentiation by regulating IRS-1 phosphorylation at serine 636/639 through DEPTOR displacement and mTOR activation.

PLD-Specific Small-Molecule Inhibitors Decrease Tumor-Associated Macrophages and Neutrophils Infiltration in Breast Tumors and Lung and Liver Metastases.

Phospholipase D-2 (PLD2) has a key role in breast cancer formation and metastasis formation with PLD small inhibitors reducing primary tumor growth. This study aimed to evaluate the importance of targeting PLD on the tumor microenvironment. We provide evidence about the beneficial effect of PLD inhibitors [FIPI (dual PLD1/PLD2) or VU0155072-2 (PLD2 inhibitor)] on avoiding infiltration of tumor-helping macrophages and neutrophils. Tumor growth and metastasis within the primary tumors had low (<20% over controls) PLD enzyme activity. Unexpectedly, we found that the inhibitors also affected PLD2 gene expression and protein albeit at a lesser extent. The later could indicate that targeting both the actual PLD enzyme and its activity could be beneficial for potential cancer treatments in vivo. F4/80 and Ly6G staining of macrophages and neutrophils, respectively, and Arg1 staining data were consistent with M2 and N2 polarization. NOS2 staining increased in xenotransplants upon treatment with PLD2 inhibitors suggesting the novel observation that an increased recruitment of M1 macrophages occurred in primary tumors. PLD inhibitor-treated primary tumors had large, fragile, necrotic areas that were Arg1+ for M2 macrophages. The xenotransplants also caused the formation of large F4/80+ and Ly6G+ (>100 μm) clusters in lungs. However, PLD inhibitors, particularly FIPI, were able to diminish leukocyte presence. Ex vivo chemotaxis and PLD activity of peripheral blood neutrophils (PMN) and peritoneal macrophages was also determined. Whereas PMN had impaired functionality, macrophages did not. This significantly increased (“emboldened”) macrophage function was due to PLD inhibition. Since tumor-associated leukocytes in primary tumors and metastases were targeted via PLD inhibition, we posit that these inhibitors have a key role in cancer regression, while still affording an appropriate inflammatory response at least from off-site innate immunity macrophages.

위암치료제로서 rebamipide의 잠재적 효능

Rebamipoide는 위궤양과 위염치료에 쓰이는 위보호제로 임상에서 사용되고 있지만 위암에서의 역할에 대해서는 알려지바가 거의 없다. 헬리코박토 파일로리(Helicobacter pylori)의 주요독성인자인 CagA는 위암의 발생과 관련이 있다. CagA는 위암세포에서 NF&#x3BA;B의 활성을 증가시켜 PLD1 단백질의 발현을 증가시킴으로써 위암세포의 증식과 침윤을 유도한다. Rebamipide는 NF&#x3BA;B의 활성을 억제함으로써 H. pylori의 CagA에 의해 유도되는 PLD1의 발현을 저해하는것으로 규명되었다. 게다가, rebamipide는 H. pylori의 CagA에 의해 유도되는 &#x3B2;-catenin과 그 표적 유전자로서 인 암줄기세포 마커 단백질의 발현을 증가시킴으로써 위암줄기세포의 자가재생능력을 감소시킨다. 또한 rebamipide는 항암제 내성을 극복하는 화학감수성을 증가시켜서 위암생성을 감소시키는 것으로 나타났다. 그래서 rebamipide는 위암치료제로서의 잠재적 효능을 가지고 있는 약물로서의 가능성이 제시되고 있다. Rebamipide is a mucosal-protective antiulcer drug, but its mechanism of action in gastric cancer remains elusive. CagA, a major virulence factor of Helicobacter pylori (H. pylori), is associated with the risk of gastric cancer. CagA protein is injected into gastric epithelial cells and deregulates a variety of cellular signaling molecules. CagA from H. pylori induces phospholipase D1 (PLD1) expression through NF&#x3BA;B activation in gastric epithelial cells, followed by invasion and proliferation of gastric epithelial cancer cells. Infection with cagA-positive H. pylori and expression of CagA enhances the binding of NF&#x3BA;B to the PLD1 promoter. Rebamipide abolishes H. pylori cagA-induced PLD1 expression via inhibition of binding of NF&#x3BA;B to the PLD1 promoter and also inhibits PLD activity. Moreover, rebamipide abolishes H. pylori CagA-induced &#x3B2;-catenin and the expression of a target cancer stem cell (CSC) marker gene via upregulation of miRNA-320a and -4496, followed by attenuation of self-renewal capacity of H. pylori CagA-infected gastric CSCs. In addition, rebamipide increases the chemosensitivity of CagA-expressed gastric CSCs and suppresses gastric carcinogenesis. Thus, it is speculated that rebamipide might show a potent efficacy as chemotherapeutic drug against gastric cancer cells. In this review, we summarizes recent results regarding the novel insights for the efficacy of rebamipide in gastric cancer cells.

Phosphatidic Acid Produced by RalA-activated PLD2 Stimulates Caveolae-mediated Endocytosis and Trafficking in Endothelial Cells

Caveolae are the primary route for internalization and transendothelial transport of macromolecules, such as insulin and albumin. Caveolae-mediated endocytosis is activated by Src-dependent caveolin-1 (Cav-1) phosphorylation and subsequent recruitment of dynamin-2 and filamin A (FilA), which facilitate vesicle fission and trafficking, respectively. Here, we tested the role of RalA and phospholipase D (PLD) signaling in the regulation of caveolae-mediated endocytosis and trafficking. The addition of albumin to human lung microvascular endothelial cells induced the activation of RalA within minutes, and siRNA-mediated down-regulation of RalA abolished fluorescent BSA uptake. Co-immunoprecipitation studies revealed that albumin induced the association between RalA, Cav-1, and FilA; however, RalA knockdown with siRNA did not affect FilA recruitment to Cav-1, suggesting that RalA was not required for FilA and Cav-1 complex formation. Rather, RalA probably facilitates caveolae-mediated endocytosis by activating downstream effectors. PLD2 was shown to be activated by RalA, and inhibition of PLD2 abolished Alexa-488-BSA uptake, indicating that phosphatidic acid (PA) generated by PLD2 may facilitate caveolae-mediated endocytosis. Furthermore, using a PA biosensor, GFP-PASS, we observed that BSA induced an increase in PA co-localization with Cav-1-RFP, which could be blocked by a dominant negative PLD2 mutant. Total internal reflection fluorescence microscopy studies of Cav-1-RFP also showed that fusion of caveolae with the basal plasma membrane was dependent on PLD2 activity. Thus, our results suggest that the small GTPase RalA plays an important role in promoting invagination and trafficking of caveolae, not by potentiating the association between Cav-1 and FilA but by stimulating PLD2-mediated generation of phosphatidic acid.

Leucyl-tRNA Synthetase Activates Vps34 in Amino Acid-Sensing mTORC1 Signaling

Amino acid availability activates signaling by the mammalian target of rapamycin (mTOR) complex 1, mTORC1, a master regulator of cell growth. The class III PI-3-kinase Vps34 mediates amino acid signaling to mTORC1 by regulating lysosomal translocation and activation of the phospholipase PLD1. Here, we identify leucyl-tRNA synthetase (LRS) as a leucine sensorfor the activation of Vps34-PLD1 upstream ofmTORC1. LRS is necessary for amino acid-induced Vps34 activation, cellular PI(3)P level increase, PLD1activation, and PLD1 lysosomal translocation. Leucine binding, but not tRNA charging activity of LRS, is required for this regulation. Moreover, LRS physically interacts with Vps34 in amino acid-stimulatable non-autophagic complexes. Finally, purified LRS protein activates Vps34 kinase invitro in a leucine-dependent manner. Collectively, our findings provide compelling evidence for a direct role of LRS in amino acid activation of Vps34 via a non-canonical mechanism and fill a gap in the amino acid-sensing mTORC1 signaling network.

Phospholipases D1 and D2 Suppress Appetite and Protect against Overweight.

Obesity is a major risk factor predisposing to the development of peripheral insulin resistance and type 2 diabetes (T2D). Elevated food intake and/or decreased energy expenditure promotes body weight gain and acquisition of adipose tissue. Number of studies implicated phospholipase D (PLD) enzymes and their product, phosphatidic acid (PA), in regulation of signaling cascades controlling energy intake, energy dissipation and metabolic homeostasis. However, the impact of PLD enzymes on regulation of metabolism has not been directly determined so far. In this study we utilized mice deficient for two major PLD isoforms, PLD1 and PLD2, to assess the impact of these enzymes on regulation of metabolic homeostasis. We showed that mice lacking PLD1 or PLD2 consume more food than corresponding control animals. Moreover, mice deficient for PLD2, but not PLD1, present reduced energy expenditure. In addition, deletion of either of the PLD enzymes resulted in development of elevated body weight and increased adipose tissue content in aged animals. Consistent with the fact that elevated content of adipose tissue predisposes to the development of hyperlipidemia and insulin resistance, characteristic for the pre-diabetic state, we observed that Pld1-/- and Pld2-/- mice present elevated free fatty acids (FFA) levels and are insulin as well as glucose intolerant. In conclusion, our data suggest that deficiency of PLD1 or PLD2 activity promotes development of overweight and diabetes.

Therapeutic inhibition of phospholipase D1 suppresses hepatocellular carcinoma

Hepatocellular carcinoma (HCC) represents a leading cause of deaths worldwide. Novel therapeutic targets for HCC are needed. Phospholipase D (PD) is involved in cell proliferation and migration, but its role in HCC remains unclear. In the present study, we show that PLD1, but not PLD2, was overexpressed in HCC cell lines (HepG2, Bel-7402 and Bel-7404) compared with the normal human L-02 hepatocytes. PLD1 was required for the proliferation, migration and invasion of HCC cells without affecting apoptosis and necrosis, and PLD1 overexpression was sufficient to promote those effects. By using HCC xenograft models, we demonstrated that therapeutic inhibition of PLD1 attenuated tumour growth and epithelial-mesenchymal transition (EMT) in HCC mice. Moreover, PLD1 was found to be highly expressed in tumour tissues of HCC patients. Finally, mTOR (mechanistic target of rapamycin) and Akt (protein kinase B) were identified as critical pathways responsible for the role of PLD1in HCC cells. Taken together, the present study indicates that PLD1 activation contributes to HCC development via regulation of the proliferation, migration and invasion of HCC cells, as well as promoting the EMT process. These observations suggest that inhibition of PLD1 represents an attractive and novel therapeutic modality for HCC.

CaMKII prevents spontaneous acrosomal exocytosis in sperm through induction of actin polymerization

In order to interact with the egg and undergo acrosomal exocytosis or the acrosome reaction (AR), mammalian spermatozoa must undergo a series of biochemical changes in the female reproductive tract, collectively called capacitation. We showed that F-actin is formed during sperm capacitation and fast depolymerization occurs prior to the AR. We hypothesized that F-actin protects the sperm from undergoing spontaneous-AR (sAR) which decreases fertilization rate. We show that activation of the actin-severing protein gelsolin induces a significant increase in sAR. Moreover, inhibition of CaMKII or PLD during sperm capacitation, caused an increase in sAR and inhibition of F-actin formation. Spermine, which leads to PLD activation, was able to reverse the effects of CaMKII inhibition on sAR-increase and F-actin-decrease. Furthermore, the increase in sAR and the decrease in F-actin caused by the inactivation of the PLD-pathway, were reversed by activation of CaMKII using H2O2 or by inhibiting protein phosphatase 1 which enhance the phosphorylation and oxidation states of CaMKII. These results indicate that two distinct pathways lead to F-actin formation in the sperm capacitation process which prevents the occurrence of sAR.

Phospholipase D enzymes facilitate IL-17- and TNFα-induced expression of proinflammatory genes in rheumatoid arthritis synovial fibroblasts (RASF)

In rheumatoid arthritis, the synovium exhibits fibroblast hyperplasia and dynamic infiltration of activated T cells. Interaction between rheumatoid arthritis synovial fibroblasts (RASF) and T cell subsets such as Th17 cells can stimulate RASF to express IL-6, IL-8, CCL20, and other proinflammatory mediators of joint destruction. PLD enzymes specifically cleave phosphatidyl choline (PC) producing phosphatidic acid (PA) and choline. Agonist-induced PLD activation results in PA synthesis, which is thought to be involved in a variety of rapid cellular responses such as cytokine secretion. Furthermore, the cellular response to TNF-mediated signaling in myeloid cells is in part mediated by PLD1. However, very few studies have examined the role of PLD enzymes in pro-inflammatory responses of RASF to key pathogenic cytokines such as TNF and IL-17. Microarray analysis of RASF showed that phospholipase D1 (PLD1) is among genes significantly induced by IL-17. We therefore hypothesized that PLD1 might have a role in RASF responses to proinflammatory cytokines. We used 1-butanol, PLD1-specific siRNAs, and small molecule inhibitors specific for PLD1 or PLD2, to investigate the possible role of PLD enzymes in basal, IL-17-, and/or TNFα-evoked expression of proinflammatory cytokines and chemokines by RASF. We studied the in vitro responses of RASF to IL-17A and/or TNFα, with particular attention to effects on IL-6, IL-8 and CCL20 mRNA and secretion as determined by RT-QPCR and ELISA, respectively. Transcriptional and prominent post-transcriptional effects were demonstrated, with robust decreases in RASF secretion of IL-6, IL-8, and CCL20 when both PLD isoforms were inhibited together. Moreover, RA synovial biopsy explants cultured in media containing PLD isoform-specific inhibitors showed significantly reduced constitutive secretion of IL-6 and IL-8. PLD enzymes could be promising targets for controlling proinflammatory gene expression in the treatment of RA in view of roles for PLD in cytokine-evoked transcription and secretion/exocytosis.

Target-controlled gating liposome "off-on" cascade amplification for sensitive and accurate detection of phospholipase D in breast cancer cells with a low-background signal.

Here we developed a simple, sensitive and accurate PLD detection method based on a target-controlled gating liposome (TCGL) "off-on" cascade amplified strategy and personal glucose meters (PGMs). It showed excellent sensitivity with a detection limit of 0.005 U L(-1) and well performed PLD activity analysis in breast cancer cells and inhibitor drug screening.

Phospholipase D2 Modulates the Secretory Pathway in RBL-2H3 Mast Cells.

Phospholipase D (PLD) hydrolyses phosphatidylcholine to produce phosphatidic acid (PA) and choline. It has two isoforms, PLD1 and PLD2, which are differentially expressed depending on the cell type. In mast cells it plays an important role in signal transduction. The aim of the present study was to clarify the role of PLD2 in the secretory pathway. RBL-2H3 cells, a mast cell line, transfected to overexpress catalytically active (PLD2CA) and inactive (PLD2CI) forms of PLD2 were used. Previous observations showed that the Golgi complex was well organized in CA cells, but was disorganized and dispersed in CI cells. Furthermore, in CI cells, the microtubule organizing center was difficult to identify and the microtubules were disorganized. These previous observations demonstrated that PLD2 is important for maintaining the morphology and organization of the Golgi complex. To further understand the role of PLD2 in secretory and vesicular trafficking, the role of PLD2 in the secretory process was investigated. Incorporation of sialic acid was used to follow the synthesis and transport of glycoconjugates in the cell lines. The modified sialic acid was subsequently detected by labeling with a fluorophore or biotin to visualize the localization of the molecule after a pulse-chase for various times. Glycoconjugate trafficking was slower in the CI cells and labeled glycans took longer to reach the plasma membrane. Furthermore, in CI cells sialic acid glycans remained at the plasma membrane for longer periods of time compared to RBL-2H3 cells. These results suggest that PLD2 activity plays an important role in regulating glycoconjugate trafficking in mast cells.

PLD1 participates in BDNF-induced signalling in cortical neurons.

The brain-derived neurotrophic factor BDNF plays a critical role in neuronal development and the induction of L-LTP at glutamatergic synapses in several brain regions. However, the cellular and molecular mechanisms underlying these BDNF effects have not been firmly established. Using in vitro cultures of cortical neurons from knockout mice for Pld1 and Rsk2, BDNF was observed to induce a rapid RSK2-dependent activation of PLD and to stimulate BDNF ERK1/2-CREB and mTor-S6K signalling pathways, but these effects were greatly reduced in Pld1−/− neurons. Furthermore, phospho-CREB did not accumulate in the nucleus, whereas overexpression of PLD1 amplified the BDNF-dependent nuclear recruitment of phospho-ERK1/2 and phospho-CREB. This BDNF retrograde signalling was prevented in cells silenced for the scaffolding protein PEA15, a protein which complexes with PLD1, ERK1/2, and RSK2 after BDNF treatment. Finally PLD1, ERK1/2, and RSK2 partially colocalized on endosomal structures, suggesting that these proteins are part of the molecular module responsible for BDNF signalling in cortical neurons.

The TLR4-associated phospholipase D1 activation is crucial for Der f 2-induced IL-13 production.

House dust mites (HDMs) are the most important source of indoor aeroallergens that contribute to the rising incidence of allergic diseases such as allergic asthma. The major HDM, Der f 2, induces inflammatory cytokine expression. Little is known about the signaling pathway involved. We wanted to define the Der f 2 signaling pathway from its receptor to the transcription factor responsible for IL-13 expression and production. Human bronchial epithelial cells were stimulated with Der f 2. The release and gene expression of IL-13 were measured by means of ELISA and RT-PCR, respectively. In the airway inflammation mouse model, airway responses were assessed using ELISA, histology, BAL fluid, and methacholine responsiveness. Here, we show that Der f 2 binds to TLR4 and induces IL-13 expression and production. In the airway inflammation mouse model, Der f 2-induced IL-13 production significantly decreased with treatment of TAK-242, a novel TLR4 inhibitor. Activation of TLR4 by Der f 2 requires the recruitment and activation of Syk, which leads to phosphorylation of PLCγ and membrane translocation of PKCα. p38 MAPK is then activated by PKCα and stimulates PLD1 activity by phosphorylating the Thr147 residue of PLD1. PLD1 activation enhanced binding of ROCK1 to ATF-2 and leads to increased expression of IL-13. Our data extend the knowledge for a variety of possible roles of PLD1 in allergic disorders including asthma pathogenesis and suggest possible candidacy of PLD1 as a molecular target for novel therapeutic approaches.