PLD Activity Research Articles

Development and Evaluation of Indole-Based Phospholipase D Inhibitors for Lung Cancer Immunotherapy.

This study explored novel immunomodulatory approaches for cancer treatment, with a specific focus on lung cancer, the leading cause of cancer-related deaths worldwide. We synthesized indole-based phospholipase D (PLD) inhibitors with various substituents to improve anticancer efficacy. Through structure-activity relationship studies, the key compound was identified that significantly inhibiting PLD, suppressing cell growth, viability, and migration in vitro, while inducing apoptosis of lung cancer cells. In silico docking studies confirmed its binding to the PLD1 active site, highlighting the role of specific residues in inhibiting PLD1 activity. The inhibitor modulated oncogenic pathways and immune evasion in lung cancer cells, showing potential for immunotherapy. In vivo experiments in a mouse model showed tumor reduction and immune response alteration. Combining these inhibitors with gemcitabine, an anticancer drug, synergistically enhanced inhibition of lung cancer cell apoptosis and proliferation. This research offers new insights into PLD inhibitor as potential cancer therapeutics.

Melatonin Mitigates Lead-Induced Oxidative Stress and Modifies Phospholipid Profile in Tobacco BY-2 Suspension Cells.

Many studies have shown that melatonin (an indoleamine) is an important molecule in plant physiology. It is known that this indoleamine is crucial during plant stress responses, especially by counteracting secondary oxidative stress (efficient direct and indirect antioxidant) and switching on different defense plant strategies. In this report, we present exogenous melatonin's potential to protect lipid profile modification and membrane integrity in Nicotiana tabacum L. line Bright Yellow 2 (BY-2) cell culture exposed to lead. There are some reports of the positive effect of melatonin on animal cell membranes; ours is the first to report changes in the lipid profile in plant cells. The experiments were performed in the following variants: LS: cells cultured on unmodified LS medium-control; (ii) MEL: BY-2 cells cultured on LS medium with melatonin added from the beginning of culture; (iii) Pb: BY-2 cells cultured on LS medium with Pb2+ added on the 4th day of culture; (iv) MEL+Pb: BY-2 cells cultured on LS medium with melatonin added from the start of culture and stressed with Pb2+ added on the 4th day of culture. Lipidomic analysis of BY-2 cells revealed the presence of 40 different phospholipids. Exposing cells to lead led to the overproduction of ROS, altered fatty acid composition and increased PLD activity and subsequently elevated the level of phosphatidic acid at the cost of dropping the phosphatidylcholine. In the presence of lead, double-bond index elevation, mainly by higher quantities of linoleic (C18:2) and linolenic (C18:3) acids in the log phase of growth, was observed. In contrast, cells exposed to heavy metal but primed with melatonin showed more similarities with the control. Surprisingly, the overproduction of ROS caused of lipid peroxidation only in the stationary phase of growth, although considerable changes in lipid profiles were observed in the log phase of growth-just 4 h after lead administration. Our results indicate that the pretreatment of BY-2 with exogenous melatonin protected tobacco cells against membrane dysfunctions caused by oxidative stress (lipid oxidation), but also findings on a molecular level suggest the possible role of this indoleamine in the safeguarding of the membrane lipid composition that limited lead-provoked cell death. The presented research indicates a new mechanism of the defense strategy of plant cells generated by melatonin.

Lysosomal endonuclease RNase T2 and PLD exonucleases cooperatively generate RNA ligands for TLR7 activation

Toll-like receptor 7 (TLR7) is essential for recognition of RNA viruses and initiation of antiviral immunity. TLR7 contains two ligand-binding pockets that recognize different RNA degradation products: pocket 1 recognizes guanosine, while pocket 2 coordinates pyrimidine-rich RNA fragments. We found that the endonuclease RNase T2, along with 5' exonucleases PLD3 and PLD4, collaboratively generate the ligands for TLR7. Specifically, RNase T2 generated guanosine 2',3'-cyclic monophosphate-terminated RNA fragments. PLD exonuclease activity further released the terminal 2',3'-cyclic guanosine monophosphate (2',3'-cGMP) to engage pocket 1 and was also needed to generate RNA fragments for pocket 2. Loss-of-function studies in cell lines and primary cells confirmed the critical requirement for PLD activity. Biochemical and structural studies showed that PLD enzymes form homodimers with two ligand-binding sites important for activity. Previously identified disease-associated PLD mutants failed to form stable dimers. Together, our data provide a mechanistic basis for the detection of RNA fragments by TLR7.

Acidic electrolyzed-oxidizing water treatment mitigated the disease progression in Phomopsis longanae Chi-infected longans by modulating ROS and membrane lipid metabolism

Postharvest harmful pathogenic infestation leads to rapid decay in longan fruit. Compared with P. longanae-infected longans, AEOW alleviated fruit disease severity and diminished the O2-. production rate and MDA content. It also increased APX, CAT, and SOD activities, delayed the decrease in the levels of GSH and AsA, as well as the reducing power and DPPH radical scavenging ability, which resulted in a decline in membrane lipid peroxidation in P. longanae-infected longans. Additionally, AEOW reduced LOX, lipase, PI-PLC, PC-PLC, and PLD activities, maintained higher levels of PC, PI, IUFA, USFAs, and U/S, while reducing levels of PA, DAG, SFAs, and CMP. These effects alleviated membrane lipid degradation and peroxidation in P. longanae-infected longans. Consequently, AEOW effectively maintained membrane integrity via improving antioxidant capacity and suppressing membrane lipid peroxidation. This comprehensive coordination of ROS and membrane lipid metabolisms improved fruit resistance and delayed disease development in longans.

Abstract 2084: Autocrine regulation of laryngeal cancer tumorigenesis by 24R,25(OH)2D3

Abstract Vitamin D3 supplementation is becoming widely accepted as a therapeutic in many common estrogen responsive cancers. Previous studies in breast cancer showed that 24R,25-dihydroxyvitamin D3 (24,25) regulates tumorigenesis via a caveolae membrane-associated signaling pathway involving phospholipase D [PLD] and palmitoylation of an unknown receptor. The goal of this study was to examine the effects of 24,25 in estrogen responsive laryngeal cancer tumorigenesis, investigate the mechanisms involved, and determine the ability of the tumor cells to produce 24,25 locally. Expression of CYP24A1 and CYP27B1 was measured in ERα66+ UM-SCC-12 (UM12) and ERα66- UM-SCC-11A (UM11A) laryngeal cancer cells. Cells were treated with 25(OH)D3, and production of the vitamin D3 metabolites 1,25(OH)2D3, 24,25(OH)2D3, and 1,24,25(OH)3D3 was measured using HPLC. The therapeutic potential of 24,25 was then assessed by treating the cells with 24,25 and measuring proliferation and apoptosis. In vivo, UM12 and UM11A cells were implanted into the cheek of NSG mice; the mice were treated with 24,25, and evaluated for survival and tumor volume. The signaling pathways involving PLD, palmitoylation, and caveolin-1 were investigated as driving mechanisms. CYP24A1 and CYP27B1 were present in ERα66+ UM12 and ERα66- UM11A cells, both of which are responsive to estrogen. When treated with 25(OH)D3, UM11A cells produced more 24,25 than UM12s, comparable to the physiologically relevant levels in blood. Both cells produced similar levels of 1,25. 24,25 increased proliferation and inhibited apoptosis in ERα66+ UM12 cells in vitro, but had the opposite effect in ERα66- UM11A cells. 24,25 increased PLD activity in UM12 cells but decreased PLD activity in UM11A cells. Inhibition of PLD activity or palmitoylation, as well as silencing expression of caveolin-1, reduced the effects of 24,25 on P53 in both cell types, indicating they played a role. Mice with UM11A tumors given 24,25 exhibited reduced tumor burden and increased survival compared to vehicle-treated controls. In contrast, mice with UM12 tumors given 24,25 had an increased tumor burden and reduced survival compared to controls. The presence of CYP24A1 and CYP27B1, local production of 24,25, and demonstration that laryngeal cancer cells respond to 24,25, indicate that 24,25 acts in an autocrine/paracrine manner. 24,25 was pro-tumorigenic in ERα66+ laryngeal cancer cells while having the opposite effect in ERα66- cells, both in vitro and in vivo, demonstrating a dependence on ERα66 expression. 24,25 acts via a mechanism involving PLD, caveolae, and palmitoylation. These data show that 24,25 regulates laryngeal cancer tumorigenesis, which is mediated by membrane associated signaling pathways that may depend on ERα66 level, and suggest a major role of 24R,25-dihydroxyvitamin D3 in laryngeal cancer. Citation Format: Cydney D. Dennis, D. Joshua Cohen, Jonathan T. Dillon, Barbara D. Boyan, Zvi Schwartz. Autocrine regulation of laryngeal cancer tumorigenesis by 24R,25(OH)2D3 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2084.

Abstract 3356: Inhibiting phospholipase D1 reduces pancreatic carcinogenesis in mice

Abstract New therapeutic targets are needed for pancreatic ductal adenocarcinoma (PDAC), a leading cause of cancer-related death worldwide. Phospholipase D (PLD) is a lipid-signaling enzyme that appears to play a critical role in cell proliferation, survival/apoptosis, and downstream oncogenic transformation. Elevated PLD1 or PLD2 (the two classical mammalian isoforms) activity has been shown to promote tumor progression in various cancer types, however, the role and mechanisms of PLD in pancreatic cancer are not yet understood. Thus, the objective is to determine the role of PLD1 regulation in pancreatic carcinogenesis through genetic and pharmacological inhibition. To evaluate the role of PLD1 in tumor growth, we subcutaneously transplanted human MIA PaCa-2 pancreatic cancer cells expressing endogenous PLD1 levels (Ctr KD cells), or MIA PaCa-2 cells in which PLD1 was knocked down (Pld1 KD cells), into immunodeficient nude mice. After 18 days post implantation, tumors that arose from Pld1-KD cells presented a significantly smaller tumor size (p<0.05) and had reduced levels of Bcl-xL and Bcl-2, compared to controls (Ctr KD). Then, to assess the role of PLD1 in the tumor microenvironment, we implanted KPC pancreatic cancer cells into C57BL/6 wild-type (WT) or C57BL/6 PLD1 knockout (Pld1−/−) mice with and without treatment with gemcitabine. Fourteen days after tumor implantation, the growth of KPC tumors was attenuated in Pld1−/− mice by 49% (p<0.05). Treatment with gemcitabine reduced tumor growth by 51.6% (p<0.05) in WT mice and by 79.5% (p<0.03) in Pld1−/− mice. Moreover, we evaluated the effects of PLD1 genetic ablation by crossing LSL-KrasG12D/+; Ptf1Cre/+ (KC) mice into PLD1 knockout (Pld1−/−) mice to generate KC; Pld1−/− mice. At 8 months of age, KC; Pld1−/− mice had significantly smaller pancreases (p<0.05) compared to KC mice. Interestingly, PLD1 ablation increased phosphorylation of ERK and c-Jun in KC; Pld1−/− mice compared to KC mice. Finally, to assess whether pharmacological inhibition of PLD can inhibit pancreatic carcinogenesis, KC mice received daily intraperitoneal injections of a small molecule inhibitor of PLD1 and PLD2 (FIPI) at a dosage of 3 mg/kg of body weight for a duration of 5 weeks. Treatment with FIPI reduced acinar-to-ductal metaplasia (p<0.05) and reduced cell proliferation (p<0.01), when compared to vehicle-treated KC controls. Additionally, FIPI treatment was safe demonstrating no significant differences in body weight between the groups and no significant alteration in the serum levels of liver enzymes, including aspartate aminotransferase, alanine aminotransferase and alkaline phosphatase. In conclusion, these findings reveal PLD1 plays a critical role in pancreatic carcinogenesis and might represent a novel therapeutic target. Citation Format: Hala Amer Addassi, Karen Matsukuma, Gerardo G. Mackenzie. Inhibiting phospholipase D1 reduces pancreatic carcinogenesis in mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3356.

Characterization of Lysophospholipase D Activity in Mammalian Cell Membranes.

Lysophosphatidic acid (LPA) is a lipid mediator that binds to G-protein-coupled receptors, eliciting a wide variety of responses in mammalian cells. Lyso-phospholipids generated via phospholipase A2 (PLA2) can be converted to LPA by a lysophospholipase D (lyso-PLD). Secreted lyso-PLDs have been studied in more detail than membrane-localized lyso-PLDs. This study utilized in vitro enzyme assays with fluorescent substrates to examine LPA generation in membranes from multiple mammalian cell lines (PC12, rat pheochromocytoma; A7r5, rat vascular smooth muscle; Rat-1, rat fibroblast; PC-3, human prostate carcinoma; and SKOV-3 and OVCAR-3, human ovarian carcinoma). The results show that membranes contain a lyso-PLD activity that generates LPA from a fluorescent alkyl-lyso-phosphatidylcholine, as well as from naturally occurring acyl-linked lysophospholipids. Membrane lyso-PLD and PLD activities were distinguished by multiple criteria, including lack of effect of PLD2 over-expression on lyso-PLD activity and differential sensitivities to vanadate (PLD inhibitor) and iodate (lyso-PLD inhibitor). Based on several lines of evidence, including siRNA knockdown, membrane lyso-PLD is distinct from autotaxin, a secreted lyso-PLD. PC-3 cells express GDE4 and GDE7, recently described lyso-PLDs that localize to membranes. These findings demonstrate that membrane-associated lyso-D activity, expressed by multiple mammalian cell lines, can contribute to LPA production.

Effect of oxyresveratrol on the quality and membrane lipid metabolism of shiitake mushroom (Lentinus edodes) during storage.

The effect of oxyresveratrol on postharvest quality and membrane lipid metabolism of shiitake mushroom was investigated. The result exhibited that oxyresveratrol retarded browning, maintained firmness and alleviated occurrence of decay of shiitake mushroom. The oxidation and hydrolysis of membrane phospholipids were suppressed by oxyresveratrol treatment, which was associated with reduced LOX and PLD activities and increased SOD and CAT activities. The membrane lipidomics of shiitake mushroom was determined by LC-MS. 385 lipid species and 13 fatty acids in membrane lipids were identified by multiple reaction monitoring method. Compared with control group, the phospholipic acid and lysophospholipid reduced by 29.24% and 21.29% in oxyresveratrol-treated group, respectively, which alleviated hydrolysis of phospholipid. Meanwhile, oxyresveratrol maintained the unsaturation of fatty acids and alleviated oxidation of phospholipid. These results demonstrated that oxyresveratrol could play a dual role of inhibiting the oxidation and hydrolysis of phospholipids to mitigate cellular damage of shiitake mushroom.

Phospholipase D1 activity is crucial for cytosolic phospholipase A2 –dependent prostaglandin E2 formation in murine osteoblastic MC3T3-E1 cells

In bone, prostaglandin E2 (PGE2) is highly osteogenic and formed by osteoblasts, a key modulatory event in the regulation of bone cell activity. MC3T3-E1 cells are widely used as an in vitro model of osteoblast function. It is still not clear which pathways contribute to the release of AA in these cells. In this study we have focussed on the contribution of phospholipase D (PLD) enzymes to osteoblastic PGE2 formation after stimulation with endothelin-1 (ET-1). Using specific inhibitors of PLD1 and PLD2 we could show that PGE2 formation was strictly dependent on PLD1 but not PLD2 activity and cytosolic phospholipase A2 (cPLA2) was activated by triggering through PLD1. We have identified diacyl glycerol (DAG) as a possible effector molecule which may serve as a triggering signal for PKC activation and subsequent cPLA2 phosphorylation.

The atypical 'hippocampal' glutamate receptor coupled to phospholipase D that controls stretch-sensitivity in primary mechanosensory nerve endings is homomeric purely metabotropic GluK2.

A metabotropic glutamate receptor coupled to phospholipase D (PLD-mGluR) was discovered in the hippocampus over three decades ago. Its pharmacology and direct linkage to PLD activation are well established and indicate it is a highly atypical glutamate receptor. A receptor with the same pharmacology is present in spindle primary sensory terminals where its blockade can totally abolish, and its activation can double, the normal stretch-evoked firing. We report here the first identification of this PLD-mGluR protein, by capitalizing on its expression in primary mechanosensory terminals, developing an enriched source, pharmacological profiling to identify an optimal ligand, and then functionalizing it as a molecular tool. Evidence from immunofluorescence, western and far-western blotting indicates PLD-mGluR is homomeric GluK2, since GluK2 is the only glutamate receptor protein/receptor subunit present in spindle mechanosensory terminals. Its expression was also found in the lanceolate palisade ending of hair follicle, also known to contain the PLD-mGluR. Finally, in a mouse model with ionotropic function ablated in the GluK2 subunit, spindle glutamatergic responses were still present, confirming it acts purely metabotropically. We conclude the PLD-mGluR is a homomeric GluK2 kainate receptor signalling purely metabotropically and it is common to other, perhaps all, primary mechanosensory endings.

Functional role of PLD1 signalosome in Tauopathies

AbstractBackgroundPhospholipase D (PLD), a lipolytic enzyme for membrane phospholipids, exists in two isoforms of mammalian phosphatidyl choline (PC) specific PLD, inducible PC‐PLD1 and constitutive PC‐PLD2. Elevated PLD activity is well documented in clinical studies involving Alzheimer’s disease (AD). Our lab was the first to show elevated PLD1 facilitates synaptic dysfunction and underlying memory deficits driven by amyloidogenic insults by Aß/tau. We observed preservation of memory in response to PLD1 specific small molecule inhibitor in transgenic mouse models and documented preservation of dendritic spines as a underlying basis. In this study, we explored a role for PLD1 in Frontotemporal Dementia in an attempt to explore its pathological role in this tauopathy‐driven dementia.MethodWe studied altered expression/activity of key F‐actin signaling elements common to PLD1 signalosome in synaptosomal fractions of human post‐mortem FTD brain regions. Synaptic deficits associated with elevated PLD1 expression in human FTD samples was studied by Fluorescence Analysis of Single Synapse Long Term Potentiation (FASS‐LTP). Using immunofluorescence, we studied the association of PLD1 with tau, presynaptic and post‐synaptic receptors, and neuroinflammation markers.ResultElevated PLD1 expression in synaptosomal fractions of post‐mortem FTD brain tissues was corroborated by synaptic deficits and association of PLD1 with postsynaptic density protein (PSD95). FASS‐LTP assay indicated relatively lower long‐term potentiation in synaptosomal fractions of FTD brains as compared to the control brains implying synaptic deficits in FTD subjects.ConclusionIn the light of these observations, we conclude elevated PLD1 expression is associated with toxicity of tau causing memory deficits in FTD patients. This PLD1‐tau interaction alters PLD signaling partners such as mTOR (mammalian target of rapamycin), cofilin and phosphocofilin. This would implicate the actin‐dependent remodeling of synapse to be a key step in the role of PLD1 signalosome in FTD. Delineating the effects of PLD1 signalosome in tauopathies such as FTD will hugely be beneficial in tau‐targeting therapeutics for a multipronged approach in the treatment of neurodegenerative disorders.

Coupling Lipid Nanoparticle Structure and Automated Single-Particle Composition Analysis to Design Phospholipase-Responsive Nanocarriers.

Lipid nanoparticles (LNPs) are versatile structures with tunable physicochemical properties that are ideally suited as a platform for vaccine delivery and RNA therapeutics. A key barrier to LNP rational design is the inability to relate composition and structure to intracellular processing and function. Here Single Particle Automated Raman Trapping Analysis (SPARTA) is combined with small-angle X-ray and neutron scattering (SAXS/SANS) techniques to link LNP composition with internal structure and morphology and to monitor dynamic LNP-phospholipase D (PLD) interactions. This analysis demonstrates that PLD, a key intracellular trafficking mediator, can access the entire LNP lipid membrane to generate stable, anionic LNPs. PLD activity on vesicles with matched amounts of enzyme substrate isan order of magnitude lower, indicating that the LNP lipid membrane structure can be used to control enzyme interactions. This represents an opportunity to design enzyme-responsive LNP solutions for stimuli-responsive delivery and diseases where PLD is dysregulated.

Effects of postharvest deastringency and 1-methylcyclopropene treatments on membrane permeability, membrane-degrading enzymes and their encoding genes in persimmon (Diospyros kaki, cv Mopanshi) fruit

Astringent persimmon fruit softens rapidly after harvest deastringency treatment. The purpose of this study was to understand the characteristics of membrane deterioration and its mechanism during rapid softening. The experiment included three treatments: (1) control, (2) CO2 treatment (≈100% CO2, 24 h), and (3) CO2 plus 1-methylcyclopropene (1-MCP, 1.5 μL∙L-1) treatment. CO2 treatment can effectively remove the astringency of persimmon fruit and cause a sharp drop in fruit firmness. The addition of 1-MCP was beneficial for firmness maintenance, but the process of astringency removal was delayed by one day. The fruit respiration rate and ethylene evolution rate were significantly increased by CO2 treatment, regardless of the presence or absence of 1-MCP, but the addition of 1-MCP could reduce the peak value and delay the occurrence of ethylene peaks by one day. The deastringency treatment resulted in a significant increase in membrane permeability and malondialdehyde content, and 1-MCP could significantly reduce membrane damage. CO2 treatment induced an enormous increase in LOX activity and the transcription of DkLOX3; however, 1-MCP significantly inhibited these increases. The PLD activity and DkPLDα1 expression level of the CO2-treated fruit were significantly promoted, and the addition of 1-MCP had less inhibition effect on the PLD activity and no significant inhibition on the DkPLDα1 expression. These results demonstrate that CO2 deastringency treatment could cause severe damage to the membrane integrity by promoting LOX and PLD activities and related gene expression. 1-MCP may ameliorate membrane damage mainly by inhibiting LOX activity and DkLOX3 gene expression and partly by suppressing PLDα1 expression.

Aldolase A and Phospholipase D1 Synergistically Resist Alkylating Agents and Radiation in Lung Cancer.

Exposure to alkylating agents and radiation may cause damage and apoptosis in cancer cells. Meanwhile, this exposure involves resistance and leads to metabolic reprogramming to benefit cancer cells. At present, the detailed mechanism is still unclear. Based on the profiles of several transcriptomes, we found that the activity of phospholipase D (PLD) and the production of specific metabolites are related to these events. Comparing several particular inhibitors, we determined that phospholipase D1 (PLD1) plays a dominant role over other PLD members. Using the existing metabolomics platform, we demonstrated that lysophosphatidylethanolamine (LPE) and lysophosphatidylcholine (LPC) are the most critical metabolites, and are highly dependent on aldolase A (ALDOA). We further demonstrated that ALDOA could modulate total PLD enzyme activity and phosphatidic acid products. Particularly after exposure to alkylating agents and radiation, the proliferation of lung cancer cells, autophagy, and DNA repair capabilities are enhanced. The above phenotypes are closely related to the performance of the ALDOA/PLD1 axis. Moreover, we found that ALDOA inhibited PLD2 activity and enzyme function through direct protein–protein interaction (PPI) with PLD2 to enhance PLD1 and additional carcinogenic features. Most importantly, the combination of ALDOA and PLD1 can be used as an independent prognostic factor and is correlated with several clinical parameters in lung cancer. These findings indicate that, based on the PPI status between ALDOA and PLD2, a combination of radiation and/or alkylating agents with regulating ALDOA-PLD1 may be considered as a new lung cancer treatment option.

Phospholipase D1-generated phosphatidic acid modulates secretory granule trafficking from biogenesis to compensatory endocytosis in neuroendocrine cells.

Calcium-regulated exocytosis is a multi-step process that allows specialized secretory cells to release informative molecules such as neurotransmitters, neuropeptides, and hormones for intercellular communication. The biogenesis of secretory vesicles from the Golgi cisternae is followed by their transport towards the cell periphery and their docking and fusion to the exocytic sites of the plasma membrane allowing release of vesicular content. Subsequent compensatory endocytosis of the protein and lipidic constituents of the vesicles maintains cell homeostasis. Despite the fact that lipids represent the majority of membrane constituents, little is known about their contribution to these processes. Using a combination of electrochemical measurement of single chromaffin cell catecholamine secretion and electron microscopy of roof-top membrane sheets associated with genetic, silencing and pharmacological approaches, we recently reported that diverse phosphatidic acid (PA) species regulates catecholamine release efficiency by controlling granule docking and fusion kinetics. The enzyme phospholipase D1 (PLD1), producing PA from phosphatidylcholine, seems to be the major responsible of these effects in this model. Here, we extended this work using spinning disk confocal microscopy showing that inhibition of PLD activity also reduced the velocity of granules undergoing a directed motion. Furthermore, a dopamine β-hydroxylase (DβH) internalization assay revealed that PA produced by PLD is required for an optimal recovery of vesicular membrane content by compensatory endocytosis. Thus, among numerous roles that have been attributed to PA our work gives core to the key regulatory role in secretion that has been proposed in different cell models. Few leads to explain these multiple functions of PA along the secretory pathway are discussed.

RvD2 stimulates conjunctival goblet cell secretion by activating multiple Ca 2+ ‐dependent intracellular signaling pathways

Background Resolvin (Rv)D2 is biosynthesized from docosahexaenoic acid (DHA) and was identified from murine self-resolving exudates during the resolution phase of self-limited acute inflammation in vivo. RvD2 was identified in non-diseased human tears, which indicates the potential physiological role of RvD2 on the ocular surface. We determined the action of RvD2 on conjunctival goblet cells and the Ca2+- dependent signaling pathways involved. Method Goblet cells were cultured from male rat conjunctiva. Signaling pathways were studied by measuring intracellular [Ca2+] ([Ca2+]i) using fura 2/AM. Mucin secretion was determined using an enzyme-linked lectin assay (ELLA). Results Administration of RvD2 at 10-10M-10-7M to conjunctival goblet cells increased both the [Ca2+]i and the secretion of high molecular weight glycoprotein including the secretory mucin MUC5AC in a dose-dependent manner. The depletion of extracellular calcium significantly blocked the RvD2-induced [Ca2+]i increase. Pre-treatment with phospholipase (PL) C inhibitor U73122 (10-6M), significantly blocked the [Ca2+]i increase and mucin secretion induced by RvD2 10-8M, U73343 as the negative control failed to lower the RvD2-induced [Ca2+]i increase and mucin secretion. Treatment with 2APB 10-6M, the inositol trisphosphate (IP)3 receptor inhibitor, significantly reduced the RvD2-induced [Ca2+]i increase and the mucin secretion. Pre-treatment with 1-butanol 0.3%, the inhibitor of PLD, significantly blocked the [Ca2+]i increase induced by RvD2 10-8M, but not mucin secretion. The PLA2 inhibitor aristolochic acid 10-5M did not block the RvD2-induced [Ca2+]i increase, but successfully downregulated the RvD2-induced mucin secretion. Conclusion RvD2 induces an increase in [Ca2+]i level and mucin secretion in conjunctival goblet cells. The [Ca2+]i increase is dependent upon activation of PLC and PLD, but not PLA2 pathways. The mucin secretion is dependent on PLA2 and PLC, but not PLD pathways. RvD2 may act as a regulator of conjunctival goblet cells physiologically.

Pleckstrin homology domain of phospholipase D2 is a negative regulator of focal adhesion kinase

Phospholipase D2 (PLD2) has been implicated in the tyrosine kinase-mediated signaling pathways, but the regulation events are yet to be identified. Herein, we demonstrate that pleckstrin homology (PH) domain of PLD2 (PLD2-PH) exerts an antitumorigenic effect via the suppression of PLD2 and focal adhesion kinase (FAK). The kinase domain of FAK interacts with PLD2-PH and induces tyrosine phosphorylation and activation of PLD2. Furthermore, PLD2 increased tyrosine phosphorylation of FAK. However, ectopic expression of the PLD2-PH competes for binding to FAK and reduces the interaction between PLD2 and FAK, thereby suppressing FAK-induced PLD activation and tyrosine phosphorylation of FAK. The PLD2-PH suppressed the migration and invasion of glioblastoma cells, as well as tumor formation in a xenograft mouse model. This study uncovers a novel role of PLD2-PH as a negative regulator of PLD2 and FAK.

Interrelation between ABA and phospholipases D, C and A2 in early responses of citrus fruit to Penicillium digitatum infection

We investigated whether phospholipases play a role in citrus fruit susceptibility to be infected by Penicillium digitatum, and whether a connection exists between hormone abscisic acid (ABA) and phospholipases in the citrus fruit-P. digitatum interaction. Changes in both the activity of enzymes PLD, PLC and PLA2 and the expression of a set of genes encoding them in response to infection in Navelate (Citrus sinensis (L.) Osbeck) orange and its ABA-deficient mutant Pinalate, which is less resistant to infection, were compared. The results showed the activation of PLD and PLC in infected Navelate fruit before disease development, and this activation was attenuated in the mutant, which suggests that both enzymes play a protective role in citrus fruit to cope with P. digitatum infection and the participation of ABA in their regulation. The transcriptional analyses further demonstrated a differential activation of various phospholipases-encoding genes by the fungus. Of the CsPLD genes (CsPLDα, CsPLDβ, CsPLDδ, CsPLDγ, CsPLDζ), the fungus had a stronger effect on CsPLDγ and CsPLDζ. This is the first report to suggest the participation of a PLDζ isoform in the plant-microbe interaction, and to indicate that this gene may be modulated by ABA in response to infection. The results also revealed that the CsPLC isoforms encoding both non-specific PLC (NPC) and phosphoinositide-specific PLCs (PI-PLC) may participate in the citrus fruit-P. digitatum interaction, and that ABA action occurs upstream of CsPI-PLC gene activation in infected citrus fruit. The changes induced by the fungus in PLA2 activity and gene expression were less relevant.

NME3 Binds to Phosphatidic Acid and Tethers Mitochondria for Fusion

The fission and fusion processes determine mitochondrial morphology and function, in which signaling lipids play pivotal roles. Phosphatidic acid (PA) generated by PLD6, a mitochondrial phospholipase D, is essential for mitochondrial fusion; however, the underlying mechanism is still unclear. We previously discovered that a nucleoside diphosphate kinase, NME3, is critical for mitochondrial fusion yet through a kinase-independent activity. Here we demonstrate that NME3 functions as a mitochondrial tethering factor through binding to PA with its N-terminal amphipathic helix, enriching at the tip of mitochondria, and clustering membranes via its oligomeric structure. Optogenetic induction of PLD activity enhances NME3 recruitment to mitochondria in a catalytic-dependent manner, and NME3 is required for PLD6-mediated mitochondrial fusion. Together, PLD6-generated PA on mitochondria promotes NME3 binding, thereby increasing mitochondrial tethering to facilitate membrane fusion.

D-series Resolvins activate Phospholipase D in phagocytes during inflammation and resolution.

A successful acute inflammatory response results in the elimination of infectious agents by neutrophils and monocytes, followed by resolution and repair through tissue-resident and recruited macrophages. Resolvins (D-series and E-series) are pro-resolving lipid mediators involved in resolution and tissue repair, whose intracellular signaling remains of interest. Here, we report that D-series resolvins (RvD1- RvD5) activate phospholipase D (PLD), a ubiquitously expressed membrane lipase enzyme activity in modulating phagocyte functions. The mechanism for PLD-mediated actions of Resolvin-D5 (RvD5) in polarizing macrophages (M1-like toward M2-like) was found to be two-pronged: (a) RvD5 inhibits post-transcriptional modifications, by miRs and 3'exonucleases that process PLD2 mRNA, thus increasing PLD2 expression and activity; and (b) RvD5 enhances PLD2-S6Kinase signaling required for membrane expansion and efferocytosis. In an in vivo model of second organ reflow injury, we found that RvD5 did not reduce lung neutrophil myeloperoxidase levels in PLD2-/- mice compared to WT and PLD1-/- mice, confirming a novel role of PLD2 as the isoform in RvD5-mediated resolution processes. These results demonstrate that RvD5-PLD2 are attractive targets for therapeutic interventions in vascular inflammation such as ischemia-reperfusion injury and cardiovascular diseases.