ABSTRACT
Phospholipase D (PLD)2 via its enzymatic activity regulates cell proliferation and migration and thus is implicated in cancer. However, the role of PLD2 in obesity and type 2 diabetes has not previously been investigated. Here, we show that during diet-induced thermogenesis and obesity, levels of PLD2 but not PLD1 in adipose tissue are inversely related with uncoupling protein 1, a key thermogenic protein. We demonstrate that the thermogenic program in adipose tissue is significantly augmented in mice with adipocyte-specific Pld2 deletion or treated with a PLD2-specific inhibitor and that these mice are resistant to high fat diet-induced obesity, glucose intolerance, and insulin resistance. Mechanistically, we show that Pld2 deletion in adipose tissue or PLD2 pharmacoinhibition acts via p62 to improve mitochondrial quality and quantity in adipocytes. Thus, PLD2 inhibition is an attractive therapeutic approach for obesity and type 2 diabetes by resolving defects in diet-induced thermogenesis.
Subject(s)
Adipocytes/metabolism , Mitochondria/genetics , Mitochondria/metabolism , Phospholipase D/genetics , Thermogenesis/genetics , Animals , Biomarkers , Blood Glucose , Diet, High-Fat , Energy Metabolism , Enzyme Inhibitors/pharmacology , Gene Expression Regulation , Immunohistochemistry , Insulin Resistance , Male , Mice , Mice, Knockout , Mitochondria/ultrastructure , Obesity/etiology , Obesity/metabolism , Phospholipase D/antagonists & inhibitors , Phospholipase D/metabolism , Proteasome Endopeptidase Complex/metabolism , Uncoupling Protein 1/genetics , Uncoupling Protein 1/metabolismABSTRACT
Although Krüppel-associated box domain-containing zinc-finger proteins (K-ZNFs) may be associated with sophisticated gene regulation in higher organisms, the physiological functions of most K-ZNFs remain unknown. The Zfp212 protein was highly conserved in mammals and abundant in the brain; it was mainly expressed in the cerebellum (Cb). Zfp212 (mouse homolog of human ZNF212) knockout (Zfp212-KO) mice showed a reduction in survival rate compared to wild-type mice after 20 months of age. GABAergic Purkinje cell degeneration in the Cb and aberrant locomotion were observed in adult Zfp212-KO mice. To identify genes related to the ataxia-like phenotype of Zfp212-KO mice, 39 ataxia-associated genes in the Cb were monitored. Substantial alterations in the expression of ataxin 10, protein phosphatase 2 regulatory subunit beta, protein kinase C gamma, and phospholipase D3 (Pld3) were observed. Among them, Pld3 alone was tightly regulated by Flag-tagged ZNF212 overexpression or Zfp212 knockdown in the HT22 cell line. The Cyclic Amplification and Selection of Targets assay identified the TATTTC sequence as a recognition motif of ZNF212, and these motifs occurred in both human and mouse PLD3 gene promoters. Adeno-associated virus-mediated introduction of human ZNF212 into the Cb of 3-week-old Zfp212-KO mice prevented Purkinje cell death and motor behavioral deficits. We confirmed the reduction of Zfp212 and Pld3 in the Cb of an alcohol-induced cerebellar degeneration mouse model, suggesting that the ZNF212-PLD3 relationship is important for Purkinje cell survival.
Subject(s)
Ataxia/pathology , DNA-Binding Proteins/metabolism , Gait Disorders, Neurologic/pathology , Nerve Tissue Proteins/physiology , Phospholipase D/antagonists & inhibitors , Purkinje Cells/pathology , Animals , Ataxia/etiology , DNA-Binding Proteins/administration & dosage , DNA-Binding Proteins/genetics , Gait Disorders, Neurologic/etiology , Humans , Male , Mice , Mice, Knockout , Nerve Tissue Proteins/administration & dosage , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Purkinje Cells/metabolismABSTRACT
PLD3 and PLD4 have recently been revealed to be endosomal exonucleases that regulate the innate immune response by digesting the ligands of nucleic acid sensors. These enzymes can suppress RNA and DNA innate immune sensors like toll-like receptor 9, and PLD4-deficent mice exhibit inflammatory disease. Targeting these immunoregulatory enzymes presents an opportunity to indirectly regulate innate immune nucleic acid sensors that could yield immunotherapies, adjuvants, and nucleic acid drug stabilizers. To aid in delineating the therapeutic potential of these targets, we have developed a high-throughput fluorescence enzymatic assay to identify modulators of PLD3 and PLD4. Screening of a diversity library (N = 17952) yielded preferential inhibitors of PLD3 and PLD4 in addition to a PLD3 selective activator. The modulation models of these compounds were delineated by kinetic analysis. This work presents an inexpensive and simple method to identify modulators of these immunoregulatory exonucleases.
Subject(s)
Enzyme Activators/chemistry , Enzyme Inhibitors/chemistry , Exodeoxyribonucleases/antagonists & inhibitors , Phospholipase D/antagonists & inhibitors , Enzyme Assays , Fluorescent Dyes/chemistry , High-Throughput Screening Assays , Humans , Nitrophenols/chemistry , Thymine Nucleotides/chemistry , Umbelliferones/chemistryABSTRACT
BACKGROUND: Phosphotyrosine kinase (PTK)-mediated phospholipase C-γ1 (PLC-γ1) signaling plays a crucial role in the release of the universal second messenger calcium from intracellular stores, which is mandatory for several cellular processes, including cell migration. However, PLC-γ1 could also be activated in a PTK-independent manner by phospholipase D (PLD)-derived phosphatidic acid (PA). Because both higher PLD expression levels and PLD activity have also been associated with breast cancer cell invasion and migration, we wondered whether there might be a link between PLD and PLC-γ1, which was investigated in this study. MATERIALS: MDA-MB-468-NEO (EGFR positive) and MDA-MB-468-HER2 (EGFR and HER2 positive) human breast cancer cells were used in this study. The migratory behavior of the cells in the presence of epidermal growth factor (EGF) and the PLD inhibitor 5-fluoro-2-indolyl-des-chlorohalopemide (FIPI) was analyzed using the 3D collagen matrix migration assay. Changes in cytosolic calcium levels in the presence of EGF, FIPI and Sig-1R agonists and antagonists as well as in PLD1 siRNA knockdown cells were determined by flow cytometry. Western blot analyses were performed to determine the basal expression levels and phosphorylation patterns of EGFR, HER2, AKT, MAPKp42/44, PLC-γ1 and Sig-1R. RESULTS: The EGF-induced migration of MDA-MB-468-NEO and MDA-MB-468-HER2 cells was significantly impaired by FIPI. Likewise, FIPI also significantly abolished EGF-induced calcium release in both cell lines. However, neither the expression levels nor the phosphorylation patterns of EGFR, HER2, AKT, MAPKp42/44 and PLC-γ1 were markedly changed by FIPI. Knockdown of PLD1 expression by siRNA also significantly impaired EGF-induced calcium release in both cell lines. Targeting Sig-1R, which interacts with IP3R, with the antagonist BD1047 also abrogated EGF-induced calcium release. However, EGF-induced calcium release was also impaired if cells were treated with the Sig-1R agonists PRE084 and PPBP maleate. CONCLUSION: In summary, blocking PLD activity with the specific inhibitor FIPI or knocking down PDL1 expression by siRNA significantly impaired EGF-induced calcium release in MDA-MB-468-NEO and MDA-MB-468-HER2 cells, likely indicating a connection between PLD activity and PLC-γ1-mediated calcium signaling. However, how PLD activity interferes with the release of calcium from intracellular stores remains unclear. Video Abstract.
Subject(s)
Breast Neoplasms/metabolism , Calcium Signaling , Domperidone/analogs & derivatives , Enzyme Inhibitors/pharmacology , Epidermal Growth Factor/pharmacology , Indoles/pharmacology , Phospholipase D/antagonists & inhibitors , Breast Neoplasms/pathology , Calcium/metabolism , Calcium Signaling/drug effects , Cell Line, Tumor , Cell Movement/drug effects , Domperidone/pharmacology , ErbB Receptors/metabolism , Female , Humans , Phospholipase D/metabolism , Phosphorylation/drug effects , RNA, Small Interfering/metabolism , Receptors, sigma/metabolism , Sigma-1 ReceptorABSTRACT
Mammalian phospholipase D (PLD) generates phosphatidic acid, a dynamic lipid secondary messenger involved with a broad spectrum of cellular functions including but not limited to metabolism, migration, and exocytosis. As a promising pharmaceutical target, the biochemical properties of PLD have been well characterized. This has led to the recent crystal structures of human PLD1 and PLD2, the development of PLD specific pharmacological inhibitors, and the identification of cellular regulators of PLD. In this review, we discuss the PLD1 and PLD2 structures, PLD inhibition by small molecules, and the regulation of PLD activity by effector proteins and lipids.
Subject(s)
Phospholipase D/chemistry , Phospholipase D/metabolism , Animals , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/metabolism , Humans , Phosphatidic Acids/metabolism , Phospholipase D/antagonists & inhibitors , Phospholipase D/genetics , Signal TransductionABSTRACT
Significance: Cardiometabolic disorders (CMD) are composed of a plethora of metabolic dysfunctions such as dyslipidemia, nonalcoholic fatty liver disease, insulin resistance, and hypertension. The development of these disorders is highly linked to inflammation and oxidative stress (OxS), two metabolic states closely related to physiological and pathological conditions. Given the drastically rising CMD prevalence, the discovery of new therapeutic targets/novel nutritional approaches is of utmost importance. Recent Advances: The tremendous progress in methods/technologies and animal modeling has allowed the clarification of phospholipase D (PLD) critical roles in multiple cellular processes, whether directly or indirectly via phosphatidic acid, the lipid product mediating signaling functions. In view of its multiple features and implications in various diseases, PLD has emerged as a drug target. Critical Issues: Although insulin stimulates PLD activity and, in turn, PLD regulates insulin signaling, the impact of the two important PLD isoforms on the metabolic syndrome components remains vague. Therefore, after outlining PLD1/PLD2 characteristics and functions, their role in inflammation, OxS, and CMD has been analyzed and critically reported in the present exhaustive review. The influence of functional foods and nutrients in the regulation of PLD has also been examined. Future Directions: Available evidence supports the implication of PLD in CMD, but only few studies emphasize its mechanisms of action and specific regulation by nutraceutical compounds. Therefore, additional investigations are first needed to clarify the functional role of nutraceutics and, second, to elucidate whether targeting PLDs with food compounds represents an appropriate therapeutic strategy to treat CMD. Antioxid. Redox Signal. 34, 252-278.
Subject(s)
Metabolic Syndrome/metabolism , Phospholipase D/metabolism , Animals , Humans , Metabolic Syndrome/drug therapy , Molecular Structure , Phosphatidylcholines/chemistry , Phosphatidylcholines/metabolism , Phospholipase D/antagonists & inhibitorsABSTRACT
The membrane lipids diacylglycerol (DAG) and phosphatidic acid (PA) are important second messengers that can regulate membrane transport by recruiting proteins to the membrane and by altering biophysical membrane properties. DAG and PA are involved in the transport from the Golgi apparatus to endosomes, and we have here investigated whether changes in these lipids might be important for regulation of transport to the Golgi using the protein toxin ricin. Modulation of DAG and PA levels using DAG kinase (DGK) and phospholipase D (PLD) inhibitors gave a strong increase in retrograde ricin transport, but had little impact on ricin recycling or degradation. Inhibitor treatment strongly affected the endosome morphology, increasing endosomal tubulation and size. Furthermore, ricin was present in these tubular structures together with proteins known to regulate retrograde transport. Using siRNA to knock down different isoforms of PLD and DGK, we found that several isoforms of PLD and DGK are involved in regulating ricin transport to the Golgi. Finally, by performing lipidomic analysis we found that the DGK inhibitor gave a weak, but expected, increase in DAG levels, while the PLD inhibitor gave a strong and unexpected increase in DAG levels, showing that it is important to perform lipidomic analysis when using inhibitors of lipid metabolism.
Subject(s)
Diacylglycerol Kinase/metabolism , Endosomes/metabolism , Golgi Apparatus/metabolism , Phospholipase D/metabolism , Cell Line, Tumor , Diacylglycerol Kinase/antagonists & inhibitors , Diacylglycerol Kinase/genetics , Diglycerides/metabolism , Endocytosis/drug effects , Enzyme Inhibitors/pharmacology , Humans , Lipidomics/methods , Lipids/analysis , Phospholipase D/antagonists & inhibitors , Phospholipase D/genetics , Protein Isoforms/antagonists & inhibitors , Protein Isoforms/genetics , Protein Isoforms/metabolism , Protein Transport/drug effects , Proteolysis/drug effects , Pyrimidinones/pharmacology , RNA Interference , RNA, Small Interfering/metabolism , Ricin/metabolism , Thiazoles/pharmacologyABSTRACT
Phospholipase D (PLD) is a ubiquitous enzyme that cleaves the distal phosphoester bond of phospholipids generating phosphatidic acid (PA). In plants, PA is involved in numerous cell responses triggered by stress. Similarly, in mammals, PA is also a second messenger involved in tumorigenesis. PLD is nowadays considered as a therapeutic target and blocking its activity with specific inhibitors constitutes a promising strategy to treat cancers. Starting from already described PLD inhibitors, this study aims to investigate the effect of their structural modifications on the enzyme's activity, as well as identifying new potent inhibitors of eukaryotic PLDs. Being able to purify the plant PLD from Vigna unguiculata (VuPLD), we obtained a SAXS model of its structure. We then used a fluorescence-based test suitable for high-throughput screening to review the effect of eukaryotic PLD inhibitors described in the literature. In this regard, we found that only few molecules were in fact able to inhibit VuPLD and we confirmed that vanadate is the most potent of all with an IC50 around 58 µM. Moreover, the small-scale screening of a chemical library of 3120 compounds allowed us to optimize the different screening's steps and paved the way towards the discovery of new potent inhibitors.
Subject(s)
Drug Evaluation, Preclinical , Enzyme Inhibitors/analysis , Enzyme Inhibitors/pharmacology , Phospholipase D/antagonists & inhibitors , Alcohols/pharmacology , High-Throughput Screening Assays , Humans , Hydrolysis , Phospholipase D/metabolism , Salts/pharmacology , Scattering, Small Angle , Vanadates/pharmacology , Vigna/enzymology , X-Ray DiffractionABSTRACT
OBJECTIVE: Phospholipase D1 (PLD1) is an enzyme of the phospholipase D (PLD) superfamily. It is involved in the occurrence of various tumors. However, its role in multiple myeloma (MM) remained undefined. This study aimed to investigate the mechanism of PLD1 in the therapy of myeloma disease. MATERIAL AND METHODS: Cell lines U266 and H929 were treated with PLD1 specific inhibitor VU0359595 combined bortezomib, a proteasome inhibitor. Their effects on MM cell proliferation, apoptosis, and relevant signal pathways of apoptosis were determined by cell counting kit-8 (CCK-8), real-time polymerase reaction chain (RT-PCR), ATP assay, and western blot. RESULTS: PLD1 was highly expressed in U266 and H929 cells. VU0359595 didn't affect the proliferation and apoptosis of MM cells. However, VU0359595 could enhance growth inhibition, decreasing mitochondrial membrane potentials (MMPs) and ATP levels of bortezomib treated MM cells. VU0359595 also strengthened bortezomib-induced apoptosis via activating caspase-8, caspase-9, caspase-3; and down-regulating the expressions of anti-apoptosis proteins BCL-2. In addition, the bortezomib-induced cytotoxicity on MM cells was significantly augmented by VU0359595 through efficient suppression of the mTOR/NF-κB signal pathway. CONCLUSION: PLD1 inhibition can remarkably exert antitumor effects with bortezomib on MM, which is a novel potentially targeting therapeutic agent, especially for drug-resistant MM patients.
Subject(s)
Bortezomib/pharmacology , Multiple Myeloma , NF-kappa B/metabolism , Neoplasm Proteins , Phospholipase D , Signal Transduction/drug effects , TOR Serine-Threonine Kinases/metabolism , Cell Line, Tumor , Humans , Multiple Myeloma/drug therapy , Multiple Myeloma/enzymology , Neoplasm Proteins/antagonists & inhibitors , Neoplasm Proteins/metabolism , Phospholipase D/antagonists & inhibitors , Phospholipase D/metabolismABSTRACT
Resistance of glioblastoma to the chemotherapeutic compound temozolomide is associated with the presence of glioblastoma stem cells in glioblastoma and is a key obstacle for the poor prognosis of glioblastoma. Here, we show that phospholipase D1 is elevated in CD44High glioblastoma stem cells and in glioblastoma, especially recurring glioblastoma. Phospholipase D1 elevation positively correlated with the level of CD44 and poor prognosis in glioblastoma patients. Temozolomide significantly upregulated the expression of phospholipase D1 in the low and moderate CD44 populations of glioblastoma stem cells, but not in the CD44High population in which phospholipase D1 is highly expressed. Phospholipase D1 conferred resistance to temozolomide in CD44High glioblastoma stem cells and increased their self-renewal capacity and maintenance. Phospholipase D1 expression significantly correlated with levels of temozolomide resistance factors, which were suppressed by microRNA-320a and -4496 induced by phospholipase D1 inhibition. Genetic and pharmacological targeting of phospholipase D1 attenuated glioblastoma stem cell-derived intracranial tumors of glioblastoma using the microRNAs, and improved survival. Treatment solely with temozolomide produced no benefits on the glioblastoma, whereas in combination, phospholipase D1 inhibition sensitized glioblastoma stem cells to temozolomide and reduced glioblastoma tumorigenesis. Together, these findings indicate that phospholipase D1 inhibition might overcome resistance to temozolomide and represents a potential treatment strategy for glioblastoma. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.
Subject(s)
Antineoplastic Agents, Alkylating/therapeutic use , Brain Neoplasms/drug therapy , Drug Resistance, Neoplasm/drug effects , Glioblastoma/drug therapy , MicroRNAs/pharmacology , Phospholipase D/antagonists & inhibitors , Temozolomide/therapeutic use , Animals , Biomarkers, Tumor/antagonists & inhibitors , Brain Neoplasms/metabolism , Carcinogenesis/drug effects , Cell Line, Tumor , Down-Regulation , Glioblastoma/metabolism , Humans , Hyaluronan Receptors/metabolism , Male , Mice , Mice, Inbred C57BL , Mice, Inbred NOD , Mice, SCID , MicroRNAs/therapeutic use , Neoplasm Transplantation , Up-RegulationABSTRACT
Phospholipase D1 (PLD1) plays a crucial role in various inflammatory and autoimmune diseases. Rheumatoid arthritis (RA) is a chronic and systemic autoimmune disease. However, the role of PLD1 in the pathogenesis of RA remains unknown. Here, we first investigated the role and effects of PLD1 in collagen-induced arthritis (CIA) and found that genetic and pharmacological inhibition of PLD1 in DBA1/J mice with CIA reduced the incidence of CIA, decreased the clinical score, and abrogated disease symptoms including infiltration of leukocytes, synovial inflammation, bone erosion, and cartilage destruction. Moreover, ablation and inhibition of PLD1 suppressed the production of type II collagen-specific IgG2a autoantibody and proinflammatory cytokines, accompanied by an increase in the regulatory T (Treg) cell population and a decrease in the Th17 cell population in CIA mice. The PLD1 inhibitor also promoted differentiation of Treg cells and suppressed differentiation of Th17 cells in vitro. Furthermore, the PLD1 inhibitor attenuated pathologic bone destruction in CIA mice by suppressing osteoclastogenesis and bone resorption. Thus, our findings indicate that the targeting of PLD1 can ameliorate CIA by modulating the imbalance of Treg and Th17 cells and suppressing osteoclastogenesis, which might be a novel strategy to treat autoimmune diseases, such as RA.
Subject(s)
Arthritis, Experimental/prevention & control , Benzimidazoles/pharmacology , Osteogenesis/drug effects , Phospholipase D/antagonists & inhibitors , Piperidines/pharmacology , T-Lymphocytes, Regulatory/drug effects , Th17 Cells/drug effects , Animals , Arthritis, Experimental/immunology , Arthritis, Experimental/metabolism , Arthritis, Rheumatoid/immunology , Arthritis, Rheumatoid/metabolism , Arthritis, Rheumatoid/prevention & control , Cell Differentiation/drug effects , Cell Differentiation/immunology , Cytokines/blood , Disease Models, Animal , Knee Joint/drug effects , Knee Joint/metabolism , Knee Joint/pathology , Male , Mice, Inbred C57BL , Mice, Inbred DBA , Mice, Knockout , Osteogenesis/genetics , Phospholipase D/genetics , Phospholipase D/metabolism , T-Lymphocytes, Regulatory/immunology , T-Lymphocytes, Regulatory/metabolism , Th17 Cells/immunology , Th17 Cells/metabolism , X-Ray MicrotomographyABSTRACT
N-acylethanolamines (NAEs), which include the endocannabinoid anandamide, represent an important family of signaling lipids in the brain. The lack of chemical probes that modulate NAE biosynthesis in living systems hamper the understanding of the biological role of these lipids. Using a high-throughput screen, chemical proteomics and targeted lipidomics, we report here the discovery and characterization of LEI-401 as a CNS-active N-acylphosphatidylethanolamine phospholipase D (NAPE-PLD) inhibitor. LEI-401 reduced NAE levels in neuroblastoma cells and in the brain of freely moving mice, but not in NAPE-PLD KO cells and mice, respectively. LEI-401 activated the hypothalamus-pituitary-adrenal axis and impaired fear extinction, thereby emulating the effect of a cannabinoid CB1 receptor antagonist, which could be reversed by a fatty acid amide hydrolase inhibitor. Our findings highlight the distinctive role of NAPE-PLD in NAE biosynthesis in the brain and suggest the presence of an endogenous NAE tone controlling emotional behavior.
Subject(s)
Behavior, Animal/drug effects , Enzyme Inhibitors/chemistry , Lipid Metabolism/drug effects , Phosphatidylethanolamines/metabolism , Phospholipase D/antagonists & inhibitors , Amidohydrolases/metabolism , Animals , Blood Proteins/metabolism , Brain/metabolism , Cannabinoid Receptor Antagonists/metabolism , Cell Line, Tumor , Drug Evaluation, Preclinical , Enzyme Inhibitors/metabolism , Enzyme Inhibitors/pharmacokinetics , Fear/drug effects , Humans , Male , Mice , Mice, Inbred C57BL , Molecular Structure , Receptors, Cannabinoid/metabolism , Signal TransductionABSTRACT
N-Acyl-phosphatidylethanolamine phospholipase D (NAPE-PLD) (EC 3.1.4.4) catalyzes the final step in the biosynthesis of N-acyl-ethanolamides. Reduced NAPE-PLD expression and activity may contribute to obesity and inflammation, but a lack of effective NAPE-PLD inhibitors has been a major obstacle to elucidating the role of NAPE-PLD and N-acyl-ethanolamide biosynthesis in these processes. The endogenous bile acid lithocholic acid (LCA) inhibits NAPE-PLD activity (with an IC50 of 68 µm), but LCA is also a highly potent ligand for TGR5 (EC50 0.52 µm). Recently, the first selective small-molecule inhibitor of NAPE-PLD, ARN19874, has been reported (having an IC50 of 34 µm). To identify more potent inhibitors of NAPE-PLD, here we used a quenched fluorescent NAPE analog, PED-A1, as a substrate for recombinant mouse Nape-pld to screen a panel of bile acids and a library of experimental compounds (the Spectrum Collection). Muricholic acids and several other bile acids inhibited Nape-pld with potency similar to that of LCA. We identified 14 potent Nape-pld inhibitors in the Spectrum Collection, with the two most potent (IC50 = â¼2 µm) being symmetrically substituted dichlorophenes, i.e. hexachlorophene and bithionol. Structure-activity relationship assays using additional substituted dichlorophenes identified key moieties needed for Nape-pld inhibition. Both hexachlorophene and bithionol exhibited significant selectivity for Nape-pld compared with nontarget lipase activities such as Streptomyces chromofuscus PLD or serum lipase. Both also effectively inhibited NAPE-PLD activity in cultured HEK293 cells. We conclude that symmetrically substituted dichlorophenes potently inhibit NAPE-PLD in cultured cells and have significant selectivity for NAPE-PLD versus other tissue-associated lipases.
Subject(s)
Dichlorophen , Enzyme Inhibitors , Phospholipase D , Animals , Bacterial Proteins/antagonists & inhibitors , Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Bithionol/chemistry , Bithionol/pharmacology , Dichlorophen/chemistry , Dichlorophen/pharmacology , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , HEK293 Cells , Hexachlorophene/chemistry , Hexachlorophene/pharmacology , Humans , Mice , Phospholipase D/antagonists & inhibitors , Phospholipase D/chemistry , Phospholipase D/metabolism , Quinazolines/chemistry , Quinazolines/pharmacology , Streptomyces/enzymology , Sulfonamides/chemistry , Sulfonamides/pharmacologyABSTRACT
BACKGROUND: Breast cancer is the most common cancer in women. Despite high survival rates in Western countries, treatments are less effective in metastatic cases and triple-negative breast cancer (TNBC) patient survival is the shortest across breast cancer subtypes. High expression levels of stearoyl-CoA desaturase-1 (SCD1) have been reported in breast cancer. The SCD1 enzyme catalyzes the formation of oleic acid (OA), a lipid stimulating the migration of metastatic breast cancer cells. Phospholipase activity is also implicated in breast cancer metastasis, notably phospholipase D (PLD). METHODS: Kaplan-Meier survival plots generated from gene expression databases were used to analyze the involvement of SCD1 and PLD in several cancer subtypes. SCD1 enzymatic activity was modulated with a pharmaceutical inhibitor or by OA treatment (to mimic SCD1 over-activity) in three breast cancer cell lines: TNBC-derived MDA-MB-231 cells as well as non-TNBC MCF-7 and T47D cells. Cell morphology and migration properties were characterized by various complementary methods. RESULTS: Our survival analyses suggest that SCD1 and PLD2 expression in the primary tumor are both associated to metastasis-related morbid outcomes in breast cancer patients. We show that modulation of SCD1 activity is associated with the modification of TNBC cell migration properties, including changes in speed, direction and cell morphology. Cell migration properties are regulated by SCD1 activity through a PLD-mTOR/p70S6K signaling pathway. These effects are not observed in non-TNBC cell lines. CONCLUSION: Our results establish a key role for the lipid desaturase SCD1 and delineate an OA-PLD-mTOR/p70S6K signaling pathway in TNBC-derived MDA-MB-231 cell migration.
Subject(s)
Cell Movement , Stearoyl-CoA Desaturase/metabolism , Triple Negative Breast Neoplasms/pathology , Cell Line, Tumor , Datasets as Topic , Disease-Free Survival , Female , Humans , Kaplan-Meier Estimate , Neoplasm Metastasis , Oleic Acid/metabolism , Phospholipase D/antagonists & inhibitors , Phospholipase D/metabolism , Ribosomal Protein S6 Kinases, 70-kDa/metabolism , Signal Transduction/drug effects , TOR Serine-Threonine Kinases/antagonists & inhibitors , TOR Serine-Threonine Kinases/metabolism , Triple Negative Breast Neoplasms/mortality , Triple Negative Breast Neoplasms/surgeryABSTRACT
Despite being discovered over 60 years ago, the precise role of phospholipase D (PLD) is still being elucidated. PLD enzymes catalyze the hydrolysis of the phosphodiester bond of glycerophospholipids producing phosphatidic acid and the free headgroup. PLD family members are found in organisms ranging from viruses, and bacteria to plants, and mammals. They display a range of substrate specificities, are regulated by a diverse range of molecules, and have been implicated in a broad range of cellular processes including receptor signaling, cytoskeletal regulation and membrane trafficking. Recent technological advances including: the development of PLD knockout mice, isoform-specific antibodies, and specific inhibitors are finally permitting a thorough analysis of the in vivo role of mammalian PLDs. These studies are facilitating increased recognition of PLD's role in disease states including cancers and Alzheimer's disease, offering potential as a target for therapeutic intervention.
Subject(s)
Alzheimer Disease/drug therapy , Alzheimer Disease/metabolism , Neoplasms/drug therapy , Neoplasms/metabolism , Phospholipase D/antagonists & inhibitors , Phospholipase D/metabolism , Animals , Humans , Phospholipase D/deficiencyABSTRACT
Phospholipase D (PLD) hydrolyzes the phosphodiester bond of glycerophospholipids and produces phosphatidic acid (PA), which acts as a second messenger in many living organisms. A large number of PLDs have been identified in eukaryotes, and are viewed as promising targets for drug design because these enzymes are known to be tightly regulated and to function in the pathophysiology of many human diseases. However, the underlying molecular mechanisms of catalysis and regulation of eukaryotic PLD remain elusive. Here, we determined the crystal structure of full-length plant PLDα1 in the apo state and in complex with PA. The structure shows that the N-terminal C2 domain hydrophobically interacts with the C-terminal catalytic domain that features two HKD motifs. Our analysis reveals the catalytic site, substrate-binding mechanism, and a new Ca2+-binding site that is required for the activation of PLD. In addition, we tested several efficient small-molecule inhibitors against PLDα1, and suggested a possible competitive inhibition mechanism according to structure-based docking analysis. This study explains many long-standing questions about PLDs and provides structural insights into PLD-targeted inhibitor/drug design.
Subject(s)
Arabidopsis Proteins/chemistry , Phospholipase D/chemistry , Arabidopsis Proteins/antagonists & inhibitors , Arabidopsis Proteins/metabolism , Catalytic Domain , Crystallography, X-Ray , Enzyme Activation , Enzyme Inhibitors/chemistry , Enzymes , Models, Molecular , Phospholipase D/antagonists & inhibitors , Phospholipase D/metabolism , Protein Conformation , Protein DomainsABSTRACT
Lipids are key building blocks of biological membranes and are involved in complex signaling processes such as metabolism, proliferation, migration, and apoptosis. Extracellular signaling by growth factors, stress, and nutrients is transmitted through receptors that activate lipid-modifying enzymes such as the phospholipases, sphingosine kinase, or phosphoinositide 3-kinase, which then modify phospholipids, sphingolipids, and phosphoinositides. One such important enzyme is phospholipase D (PLD), which cleaves phosphatidylcholine to yield phosphatidic acid and choline. PLD isoforms have dual role in cells. The first involves maintaining cell membrane integrity and cell signaling, including cell proliferation, migration, cytoskeletal alterations, and invasion through the PLD product PA, and the second involves protein-protein interactions with a variety of binding partners. Increased evidence of elevated PLD expression and activity linked to many pathological conditions, including cancer, neurological and inflammatory diseases, and infection, has motivated the development of dual- and isoform-specific PLD inhibitors. Many of these inhibitors are reported to be efficacious and safe in cells and mouse disease models, suggesting the potential for PLD inhibitors as therapeutics for cancer and other diseases. Current knowledge and ongoing research of PLD signaling networks will help to evolve inhibitors with increased efficacy and safety for clinical studies.
Subject(s)
Enzyme Inhibitors/pharmacology , Neoplasms/enzymology , Phosphatidic Acids , Phospholipase D/antagonists & inhibitors , Animals , Humans , Mice , Signal TransductionABSTRACT
Functions for phospholipase D1 and D2 (PLD1 and PLD2), the canonical isoforms of the PLD superfamily in mammals, have been explored using cell biological and animal disease models for two decades. PLD1 and PLD2, which are activated as a consequence of extracellular signaling events and generate the second messenger signaling lipid phosphatidic acid (PA), have been reported to play roles in settings ranging from platelet activation to the response to cardiac ischemia, viral infection, neurodegenerative disease, and cancer. Of these, the most tractable as therapeutic targets may be thrombotic disease and cancer, as will be discussed here in the context of ongoing efforts to develop small molecule PLD inhibitors.
Subject(s)
Enzyme Inhibitors/pharmacology , Neoplasms/enzymology , Phospholipase D/antagonists & inhibitors , Thrombosis/enzymology , Animals , Humans , Isoenzymes/antagonists & inhibitors , Signal TransductionABSTRACT
Current approaches in treatment of Alzheimer's disease (AD) is focused on early stages of cognitive decline. Identifying therapeutic targets that promote synaptic resilience during early stages may prevent progressive memory deficits by preserving memory mechanisms. We recently reported that the inducible isoform of phospholipase D (PLD1) was significantly increased in synaptosomes from post-mortem AD brains compared to age-matched controls. Using mouse models, we reported that the aberrantly elevated neuronal PLD1 is key for oligomeric amyloid driven synaptic dysfunction and underlying memory deficits. Here, we demonstrate that chronic inhibition using a well-tolerated PLD1 specific small molecule inhibitor is sufficient to prevent the progression of synaptic dysfunction during early stages in the 3xTg-AD mouse model. Firstly, we report prevention of cognitive decline in the inhibitor-treated group using novel object recognition (NOR) and fear conditioning (FC). Secondly, we provide electrophysiological assessment of better synaptic function in the inhibitor-treated group. Lastly, using Golgi staining, we report that preservation of dendritic spine integrity as one of the mechanisms underlying the action of the small molecule inhibitor. Collectively, these studies provide evidence for inhibition of PLD1 as a potential therapeutic strategy in preventing progression of cognitive decline associated with AD and related dementia.
Subject(s)
Alzheimer Disease/drug therapy , Brain/diagnostic imaging , Phospholipase D/genetics , Small Molecule Libraries/pharmacology , Alzheimer Disease/diagnostic imaging , Alzheimer Disease/physiopathology , Amyloid beta-Peptides/genetics , Animals , Brain/drug effects , Brain/physiopathology , Cognitive Dysfunction/drug therapy , Cognitive Dysfunction/genetics , Cognitive Dysfunction/pathology , Disease Models, Animal , Humans , Mice , Neurons/drug effects , Neurons/pathology , Phospholipase D/antagonists & inhibitors , Synaptosomes/drug effects , Synaptosomes/pathology , tau Proteins/geneticsABSTRACT
N-acylphosphatidylethanolamine phospholipase D (NAPE-PLD) catalyzes the cleavage of membrane NAPEs into bioactive fatty-acid ethanolamides (FAEs). Along with this precursor role, NAPEs might also serve autonomous signaling functions. Here, we report that injections of 6-hydroxydopamine (6-OHDA) into the mouse striatum cause a local increase in NAPE and FAE levels, which precedes neuronal cell death. NAPE, but not FAE, accumulation is enhanced in mice lacking NAPE-PLD, which display a substantial reduction in 6-OHDA-induced neurotoxicity, as shown by increased survival of substantia nigra dopamine neurons, integrity of striatal dopaminergic fibers, and striatal dopamine metabolite content. Reduced damage is accompanied by attenuation of the motor response evoked by apomorphine. Furthermore, NAPE-PLD silencing protects cathecolamine-producing SH-SY5Y cells from 6-OHDA-induced reactive oxygen species formation, caspase-3 activation and death. Mechanistic studies in mice suggest the existence of multiple molecular contributors to the neuroprotective effects of NAPE-PLD deletion, including suppression of Rac1 activity and attenuated transcription of several genes (Cadps, Casp9, Egln1, Kcnj6, Spen, and Uchl1) implicated in dopamine neuron survival and/or Parkinson's disease. The findings point to a previously unrecognized role for NAPE-PLD in the regulation of dopamine neuron function, which may be linked to the control of NAPE homeostasis in membranes.