OBJECTIVES: Systemic lupus erythematosus (SLE) is a kind of autoimmune inflammatory connective tissue disease which seriously endangers human health. Genetic factors play a key role in the pathogenesis of SLE. This study aims to investigate a novel phospholipase D2 (PLD2) mutation associated with familial SLE, and further explore the underlying mechanism of the mutation in SLE. METHODS: The blood samples from a SLE patient, the patient's parents, and 147 normal controls were collected and DNA was extracted. Whole genome high-throughput sequencing was performed in the patient and her parents and the results were further analyzed by various bioinformatics methods. The wild type (wt), mutant type (mu), and negative control PLD2 plasmids were further constructed and transfected into 293 cells. The expression level of HRAS protein in 293 cells was detected by Western blotting. RESULTS: In this SLE family, the female SLE patient and her mother, 1 in generation II and 1 in generation III had typical clinical manifestations of SLE, and all of them had lupus nephritis at early stage. The genetic characteristics are consistent with autosomal dominant inheritance. A novel PLD2 heterozygous mutation (c.2722C>T) was found in the patient and her mother, but not in her father and other normal controls. Compared with wtPLD2 plasmid and negative control PLD2 plasmid, the expression of HRAS in 293 cells transfected with muPLD2 plasmid was significantly up-regulated (both CONCLUSIONS PLD2 c.2722C>T mutation may be one of the pathogeny of SLE in this family.
Case-Control Studies ; Female ; High-Throughput Nucleotide Sequencing ; Humans ; Lupus Erythematosus, Systemic/genetics* ; Lupus Nephritis ; Mutation ; Phospholipase D

OBJECTIVETo assess the association of polymorphisms of N-acyl-phosphatidylethanolamine-phospholipase D (DAPE-PLD) and fatty acid amide hydrolase (FAAH) genes, as well as their interaction, with schizophrenia.

METHODSPolymorphisms of NAPE-PLD rs12540583 and FAAH rs324420, rs2295633, and rs6429600 were determined with PCR - restriction fragment length polymorphism assay and Sanger sequencing. The genotypes of 345 subjects of Han Chinese origin diagnosed with schizophrenia and a 403 controls were compared. The results were analyzed with SPSS 17.0, and the interaction of the two genes was analyzed using a multifactor dimensionality reduction (MDR) method.

RESULTSThe frequency of NAPE-PLD rs12540583 polymorphism was significantly different between the two groups under both dominant and additive models (χ2=17.18 vs. χ2=18.94, P<0.0125). The frequencies of AC genotype and C allele of the patient group at rs12540583 were higher than those of the controls, and the interaction of NAPE-PLD and FAAH was associated with schizophrenia. A four-loci model (rs12540583, rs324420, rs2295633 and rs6429600) can best model the interaction between NAPE-PLD and FAAH.

CONCLUSIONThe AC genotype and C allele of NAPE-PLD rs12540583 locus are risk factors for schizophrenia, and the interaction between NAPE-PLD rs12540583 and FAAH rs324420, rs2295633 and rs6429600 is associated with schizophrenia.

Adult ; Amidohydrolases ; genetics ; Asian Continental Ancestry Group ; genetics ; China ; ethnology ; Female ; Genotype ; Humans ; Male ; Middle Aged ; Phospholipase D ; genetics ; Polymorphism, Genetic ; Schizophrenia ; genetics

OBJECTIVE: To investigate the effect of overexpression of glycosylphosphatidyl-inositol-specific phospholipase D (GPI-PLD) on the biological character of hepatocellular carcinoma cell line HepG2. METHODS: The GPI-PLD gene eukaryon expression vector pcDNA3.1(+)/ GPI-PLD was transiently transfected into HepG2 cell by lipid-media transfection. The untransfected HepG2 and HepG2 transfected with pcDNA3.1(+) were used as controls. After screening with G418, the single clone was obtained. The expression level of GPI-PLD mRNA in HepG2 was identified by reverse transcription polymerase chain reaction (RT-PCR). GPI-PLD activities were analyzed quantitatively by triton-X-114 partition with GPI anchored placental alkaline phosphatase (PLAP) as a substrate. Cell count was used to detect the proliferation of the 3 groups, and complement dependent cytotoxicity (CDC) effects were observed by the staining of trypan blue. Apoptosis cells were analyzed by flow cytometry. Carcinoembryonic antigen (CEA)was detected by enzyme linked immunosorbent assay (ELISA). RESULTS: Compared with HepG2 and pcDNA3.1(+)/HepG2 cell, the levels of GPI-PLD activities and its mRNA from pcDNA3.1(+)/GPI-PLD/HepG2 were increased with almost 2 to 5 times,respectively. The GPI anchored PLAP and CEA released into the medium by GPI-PLD, and the rate of CDC killing on the cells were significantly increased. However, the proliferative capacity was obviously decreased, and the typical apoptosis cells were presented in positive clones and its apoptosis rates were increased significantly. CONCLUSION The stable cell line with overexpression of GPI-PLD has been constructed. The overexpression of GPI-PLD in these cells increases the sensitivity of these cells to CDC killing and impairs the proliferative capacity of cells, and promotes the apoptosis.
Apoptosis ; genetics ; Carcinoma, Hepatocellular ; genetics ; pathology ; Complement Activation ; genetics ; Cytotoxicity, Immunologic ; genetics ; Eukaryotic Cells ; metabolism ; Genetic Vectors ; genetics ; metabolism ; Humans ; Liver Neoplasms ; genetics ; pathology ; Phospholipase D ; biosynthesis ; genetics ; RNA, Messenger ; biosynthesis ; genetics ; Transfection ; Tumor Cells, Cultured ; Up-Regulation

MicroRNAs (miRNAs) are a type of small non-coding RNAs that are often play important roles in carcinogenesis, but the carcinogenic mechanism of miRNAs is still unclear. This study will investigate the function and the mechanism of miR-638 in carcinoma (GC). The expression of miR-638 in GC and the DNA copy number of miR-638 were detected by real-time PCR. The effect of miR-638 on cell proliferation was measured by counting kit-8 assay. Different assays, including bioinformatics algorithms (TargetScan and miRanda), luciferase report assay and Western blotting, were used to identify the target gene of miR-638 in GC. The expression of miR-638 target gene in clinical CRC tissues was also validated by immunohistochemical assay. From this research, we found that miR-638 was downregulated in GC tissues compared with corresponding noncancerous tissues (NCTs), and the DNA copy number of miR-638 was lower in GC than NCTs, which may induce the corresponding downregulation of miR-638 in GC. Ectopic expression of miR-638 inhibited GC cell growth in vitro. Subsequently, we identified that PLD1 is the target gene of miR-638 in GC, and silencing PLD1 expression phenocopied the inhibitory effect of miR-638 on GC cell proliferation. Furthermore, we observed that PLD1 was overexpressed in GC tissues, and high expression of PLD1 in GC predicted poor overall survival. In summary, we revealed that miR-638 functions as a tumor suppressor in GC through inhibiting PLD1.
3' Untranslated Regions ; genetics ; Apoptosis ; genetics ; Base Sequence ; Cell Line, Tumor ; Cell Proliferation ; genetics ; Down-Regulation ; genetics ; Humans ; MicroRNAs ; genetics ; Phospholipase D ; genetics ; Prognosis ; Stomach Neoplasms ; diagnosis ; enzymology ; genetics ; pathology

Phospholipase Ds (PLDs) exist in many plants. PLDs catalyse the hydrolysis of phospholipids (e.g. phosphatidylcholine) in cell membrane into phosphatidic acid (PA) and polar free heads (e.g. choline). Two PLD members from rice, OsPLD3 and OsPLD4, were studied by reverse genetics approaches. The results showed that the promoters of OsPLD3 and OsPLD4 could drive the expression of the reporter gene in various tissues of the rice flower organs at different levels. The expression of both genes was induced by wounding and methyl jasmonate (MeJA), but with different intensity at different time intervals. No prominent phenotypes were observed by RNA interference with the gene-specific artificial miRNAs or over-expression of the target genes in rice plants, implying the functional redundancy among different members of the rice PLD family.
Acetates ; pharmacology ; Base Sequence ; Cyclopentanes ; pharmacology ; Flowers ; genetics ; metabolism ; Genes, Plant ; genetics ; Genes, Reporter ; genetics ; Molecular Sequence Data ; Oryza ; enzymology ; genetics ; Oxylipins ; pharmacology ; Phospholipase D ; genetics ; metabolism ; Promoter Regions, Genetic ; genetics

Ceramide, a product of sphingomyelin hydrolysis, is now recognized as an intracellular lipid messenger, which mediates the effects of extracellular agents on cellular growth, differentiation and apoptosis. Recently, ceramide has been implicated in the regulation of phospholipase D (PLD). In this study, we examined the effects of ceramide on the activity and mRNA level of PLD during apoptotic process in FRTL-5 thyroid cells. C2-ceramide (N-acetyl sphingosine) induced apoptosis in FRTL-5 thyroid cells. Fluorescent staining showed that ceramide induced the typical features of apoptosis including condensed or fragmented nuclei. DNA fragmentation was also observed by agarose gel electrophoresis. Flow cytometric cell cycle analysis showed more clearly that ceramide induced apoptotic cell death in FRTL-5 thyroid cells. The treatment of FRTL-5 thyroid cells with thyroid-stimulating hormone (TSH) resulted in an increased PLD activity in a dose- and time-dependent manner. However, the TSH-induced increase in PLD activity was down-regulated within 2 h after ceramide treatment. Furthermore, the levels of PLD mRNA were found to be decreased throughout apoptotic process as inferred by reverse transcription-polymerase chain reaction. However, the decreases in PLD mRNA levels were not correlated with those in PLD activities after ceramide treatment. Taken together, these data suggest that ceramide inhibits the PLD activity in an early apoptotic phase and down-regulation of the levels of PLD mRNA may be implicated in apoptotic process in FRTL-5 thyroid cells.
Animal ; Apoptosis/drug effects* ; Cells, Cultured ; DNA Fragmentation ; Enzyme Activation/drug effects ; Flow Cytometry ; Gene Expression Regulation, Enzymologic/drug effects ; Phospholipase D/metabolism* ; Phospholipase D/genetics ; RNA, Messenger/genetics ; Rats ; Rats, Inbred Strains ; Sphingosine/pharmacology ; Sphingosine/analogs & derivatives* ; Thyroid Gland/enzymology ; Thyroid Gland/drug effects* ; Thyrotropin/pharmacology

The pathogenesis and pathophysiology of Acanthamoeba infections remain incompletely understood. Phos-pholipases are known to cleave phospholipids, suggesting their possible involvement in the host cell plasma membrane disruption leading to host cell penetration and lysis. The aims of the present study were to determine phospholipase activities in Acanthamoeba and to determine their roles in the pathogenesis of Acanthamoeba. Using an encephalitis isolate (T1 genotype), a keratitis isolate (T4 genotype), and an environmental isolate (T7 genotype), we demonstrated that Acanthamoeba exhibited phospholipase A2 (PLA2) and phospholipase D (PLD) activities in a spectrophotometry-based assay. Interestingly, the encephalitis isolates of Acanthamoeba exhibited higher phospholipase activities as compared with the keratitis isolates, but the environmental isolates exhibited the highest phospholipase activities. Moreover, Acanthamoeba isolates exhibited higher PLD activities compared with the PLA2. Acanthamoeba exhibited optimal phospholipase activities at 37degrees C and at neutral pH indicating their physiological relevance. The functional role of phospholipases was determined by in vitro assays using human brain microvascular endothelial cells (HBMEC), which constitute the blood-brain barrier. We observed that a PLD-specific inhibitor, i.e., compound 48/80, partially inhibited Acanthamoeba encephalitis isolate cytotoxicity of the host cells, while PLA2-specific inhibitor, i.e., cytidine 5'-diphosphocholine, had no effect on parasite-mediated HBMEC cytotoxicity. Overall, the T7 exhibited higher phospholipase activities as compared to the T4. In contract, the T7 exhibited minimal binding to, or cytotoxicity of, HBMEC.
Acanthamoeba/*enzymology/genetics/*isolation & purification/physiology ; Cell Adhesion ; Cells, Cultured ; Endothelial Cells/parasitology ; Humans ; Keratitis/*parasitology ; Phospholipase D/genetics/*metabolism ; Phospholipases A2/genetics/*metabolism ; Protozoan Proteins/genetics/*metabolism ; Soil/*parasitology

Autophagy is a conserved lysosomal self-digestion process used for the breakdown of long-lived proteins and damaged organelles, and it is associated with a number of pathological processes, including cancer. Phospholipase D (PLD) isozymes are dysregulated in various cancers. Recently, we reported that PLD1 is a new regulator of autophagy and is a potential target for cancer therapy. Here, we investigated whether PLD2 is involved in the regulation of autophagy. A PLD2-specific inhibitor and siRNA directed against PLD2 were used to treat HT29 and HCT116 colorectal cancer cells, and both inhibition and genetic knockdown of PLD2 in these cells significantly induced autophagy, as demonstrated by the visualization of light chain 3 (LC3) puncta and autophagic vacuoles as well as by determining the LC3-II protein level. Furthermore, PLD2 inhibition promoted autophagic flux via the canonical Atg5-, Atg7- and AMPK-Ulk1-mediated pathways. Taken together, these results suggest that PLD2 might have a role in autophagy and that its inhibition might provide a new therapeutic basis for targeting autophagy.
Autophagy/*drug effects ; Cell Line, Tumor ; Colorectal Neoplasms/enzymology/*genetics/*therapy ; Genetic Therapy ; HCT116 Cells ; Humans ; Phospholipase D/*antagonists & inhibitors/*genetics/metabolism ; Quinolines/*pharmacology ; *RNA Interference ; RNA, Small Interfering/genetics/pharmacology ; Signal Transduction/drug effects ; Spiro Compounds/*pharmacology

Growth factor-stimulated phospholipase D (PLD) catalyzes the hydrolysis of phosphatidylcholine (PC), generating phosphatidic acid (PA) which may act as a second messenger during cell proliferation and survival. Therefore, PLD is believed to play an important role in tumorigenesis. In this study, a potential mechanism for PLD-mediated tumorigenesis was explored. Ectopic expression of PLD1 or PLD2 in human glioma U87 cells increased the expression of hypoxia-inducible factor-1alpha (HIF-1alpha) protein. PLD-induced HIF-1 activation led to the secretion of vascular endothelial growth factor (VEGF), a HIF-1 target gene involved in tumorigenesis. PLD induction of HIF-1alpha was significantly attenuated by 1-butanol which blocks PA production by PLD, and PA per se was able to elevate HIF-1alpha protein level. Inhibition of mTOR, a PA-responsive kinase, reduced the levels of HIF-1alpha and VEGF in PLD-overexpressed cells. Epidermal growth factor activated PLD and increased the levels of HIF-1alpha and VEGF in U87 cells. A specific PLD inhibitor abolished expression of HIF-1alpha and secretion of VEGF. PLD may utilize HIF-1-VEGF pathway for PLD-mediated tumor cell proliferation and survival.
Cell Line, Tumor ; Epidermal Growth Factor/metabolism ; Gene Expression Regulation, Neoplastic ; Glioma/genetics/*metabolism ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit/genetics/metabolism ; Phosphatidic Acids/*metabolism ; Phospholipase D/genetics/*metabolism ; *Signal Transduction ; Transfection ; Vascular Endothelial Growth Factor A/*metabolism

In spite of the importance of phospholipase D (PLD) in cell proliferation and tumorigenesis, little is known about the molecules regulating PLD expression. Thus, identification of small molecules inhibiting PLD expression would be an important advance for PLD-mediated physiology. We examined one such here, denoted "Triptolide", which was identified in a chemical screen for inhibitors of PLD expression using cell assay system based on measurement of PLD promoter activity. Triptolide significantly suppressed the expression of both PLD1 and PLD2 with sub-microM potency in MDA-MB-231 breast cancer cells as analyzed by promoter assay and RT-PCR. Moreover, triptolide abolished the protein level of PLD in a time and dose-dependent manner. Triptolide-induced PLD1 downregulation was also observed in all the cancer cells examined, suggesting a general phenomenon detected in various cancer cells. Decrease of PLD expression by triptolide suppressed both basal and PMA-induced PLD activity. In addition, triptolide inhibited activation of NFkappaB which increased PLD1 expression. Ultimately, downregulation of PLD by triptolide inhibited proliferation of breast cancer cells. Taken together, we demonstrate that triptolide suppresses the expression of PLD via inhibition of NFkappaB activation and then decreases cell proliferation.
Antineoplastic Agents, Alkylating/*pharmacology ; Breast Neoplasms/drug therapy/enzymology ; Cell Line, Tumor ; Cell Proliferation/drug effects ; Diterpenes/*pharmacology ; Epoxy Compounds/pharmacology ; Female ; Gene Expression Regulation, Neoplastic/*drug effects ; Humans ; NF-kappa B/genetics/metabolism ; Phenanthrenes/*pharmacology ; Phospholipase D/*genetics/metabolism