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

Radiation and drug resistance remain the major challenges and causes of mortality in the treatment of locally advanced, recurrent and metastatic breast cancer. Dysregulation of phospholipase D (PLD) has been found in several human cancers and is associated with resistance to anticancer drugs. In the present study, we evaluated the effects of PLD inhibition on cell survival, cell death and DNA damage after exposure to ionizing radiation (IR). Combined IR treatment and PLD inhibition led to an increase in the radiation-induced apoptosis of MDA-MB-231 metastatic breast cancer cells. The selective inhibition of PLD1 and PLD2 led to a significant decrease in the IR-induced colony formation of breast cancer cells. Moreover, PLD inhibition suppressed the radiation-induced activation of extracellular signal-regulated kinase and enhanced the radiation-stimulated phosphorylation of the mitogen-activated protein kinases p38 and c-Jun N-terminal kinase. Furthermore, PLD inhibition, in combination with radiation, was very effective at inducing DNA damage, when compared with radiation alone. Taken together, these results suggest that PLD may be a useful target molecule for the enhancement of the radiotherapy effect.
Breast Neoplasms/*drug therapy/*enzymology/pathology ; Cell Death/drug effects/radiation effects ; Cell Line, Tumor ; Cell Proliferation/drug effects/radiation effects ; DNA Damage ; Enzyme Activation/drug effects/radiation effects ; Enzyme Inhibitors/*pharmacology/*therapeutic use ; Extracellular Signal-Regulated MAP Kinases/metabolism ; Female ; Humans ; JNK Mitogen-Activated Protein Kinases/metabolism ; Phospholipase D/*antagonists & inhibitors/metabolism ; Radiation Tolerance/*drug effects ; Radiation, Ionizing ; p38 Mitogen-Activated Protein Kinases/metabolism

As glucose is known to induce insulin secretion in pancreatic beta cells, this study investigated the role of a phospholipase D (PLD)-related signaling pathway in insulin secretion caused by high glucose in the pancreatic beta-cell line MIN6N8. It was found that the PLD activity and PLD1 expression were both increased by high glucose (33.3 mM) treatment. The dominant negative PLD1 inhibited glucose-induced Beta2 expression, and glucose-induced insulin secretion was blocked by treatment with 1-butanol or PLD1-siRNA. These results suggest that high glucose increased insulin secretion through a PLD1-related pathway. High glucose induced the binding of Arf6 to PLD1. Pretreatment with brefeldin A (BFA), an Arf inhibitor, decreased the PLD activity as well as the insulin secretion. Furthermore, BFA blocked the glucose-induced mTOR and p70S6K activation, while mTOR inhibition with rapamycin attenuated the glucose induced Beta2 expression and insulin secretion. Thus, when taken together, PLD1 would appear to be an important regulator of glucose-induced insulin secretion through an Arf6/PLD1/mTOR/p70S6K/Beta2 pathway in MIN6N8 cells.
ADP-Ribosylation Factors/metabolism/physiology ; Animals ; Basic Helix-Loop-Helix Transcription Factors/metabolism/physiology ; Cells, Cultured ; Gene Expression Regulation, Enzymologic/drug effects ; Glucose/*pharmacology ; Insulin/*secretion ; Insulin-Secreting Cells/*drug effects/enzymology/metabolism/secretion ; Intracellular Signaling Peptides and Proteins/metabolism/physiology ; Mice ; Models, Biological ; Oligodeoxyribonucleotides, Antisense/pharmacology ; Phospholipase D/antagonists & inhibitors/genetics/metabolism/*physiology ; Protein-Serine-Threonine Kinases/metabolism/physiology ; Ribosomal Protein S6 Kinases, 70-kDa/metabolism/physiology ; Signal Transduction/drug effects/genetics

Lysophosphatidylcholine (LPC) is a bioactive lipid generated by phospholipase A2-mediated hydrolysis of phosphatidylcholine. In the present study, we demonstrate that LPC stimulates phospholipase D2 (PLD2) activity in rat pheochromocytoma PC12 cells. Serum deprivation induced cell death of PC12 cells, as demonstrated by decreased viability, DNA fragmentation, and increased sub-G1 fraction of cell cycle. LPC treatment protected PC12 cells partially from the cell death and induced neurite outgrowth of the cells. Overexpression of PLD2 drastically enhanced the LPC-induced inhibition of apoptosis and neuritogenesis. Pretreatment of the cells with 1-butanol, a PLD inhibitor, completely abrogated the LPC-induced inhibition of apoptosis and neurite outgrowth in PC12 cells overexpressing PLD2. These results indicate that LPC possesses the neurotrophic effects, such as anti-apoptosis and neurite outgrowth, through activation of PLD2.
Starvation ; Rats ; Phospholipase D/antagonists & inhibitors/*metabolism ; PC12 Cells ; Neurites/*drug effects ; Lysophosphatidylcholines/*pharmacology ; Cell Survival/drug effects ; Apoptosis/*drug effects ; Animals

The major house-dust mite allergen, Der f 2, stimulates the phospholipase D (PLD) in T lymphocytes from Dermatophagoides farinae specific allergic individuals. PLD activity increased more than two-fold in T cells from allergic patients compared with those cells from normal controls with maximal responses within 30 min after exposure of Der f 2. A well-known PLD activator PKC-alpha was found to be translocated to membrane from cytosol in Der f 2-treated T cells from Dermatophagoides farinae specific allergic individuals. Down-regulation of PKC-alpha with phorbol myristate acetate pretreatment for 24 h abolished Der f 2-induced PLD activation. Ro 320432, PKC inhibitor also reduced the effects of Der f 2-induced PLD activation suggesting that PKC-alpha acts as upstream activator of PLD in Der f 2-treated T cells. Taken together, the present data suggest that Der f 2 can stimulate PLD activity through the PKC-alpha activation in T cells from Dermatophagoides farinae allergic individuals
Adolescent ; Adult ; Animals ; Antigens, Dermatophagoides/*immunology ; Dermatophagoides farinae/*immunology ; Female ; Humans ; Hypersensitivity, Immediate/*enzymology/*immunology ; Male ; Phospholipase D/*metabolism ; Protein Kinase C/antagonists & inhibitors/*physiology ; Research Support, Non-U.S. Gov't ; Skin Tests ; T-Lymphocytes/*enzymology/immunology ; Tetradecanoylphorbol Acetate/*analogs & derivatives/pharmacology ; Up-Regulation

Hepatitis C Virus (HCV) is associated with a severe liver disease and increased frequency in the development of hepatocellular carcinoma. Overexpression of HCV core protein is known to transform fibroblast cells. Phospholipase D (PLD) activity is commonly elevated in response to mitogenic signals, and has also been overexpressed and hyperactivated in some human cancer cells. The aim of this study was to understand how PLD was regulated in the HCV core protein-transformed NIH3T3 mouse fibroblast cells. We observed that PLD activity was elevated in the NIH3T3 cells overexpressing HCV core protein over the vector alone-transfected control cells, however, expression levels of PLD protein and protein kinase C (PKC) in the HCV core protein-transformed cells was similar to the control cells. Phorbol 12-myristate 13-acetate (PMA), which is known to activate PKC, stimulated PLD activity significantly more in the core protein-transformed cells, in comparison with that of the control cells. PLD activity assay using PKC isozyme-specific inhibitor and PKC translocation experiment showed that PKC-delta was mainly involved in the PMA- induced PLD activation in the core-transformed cells. Moreover, in cells overexpressing HCV core protein, PMA also stimulated p38 kinase more potently than that of the control cells, and an inhibitor of p38 kinase abolished PMA-induced PLD activation in cells overexpressing HCV core protein. Taken together, these results suggest that PLD might be implicated in core protein-induced transformation.
Animals ; Cell Line, Transformed ; *Cell Transformation, Viral ; Fibroblasts/enzymology/virology ; Hepacivirus/genetics/*physiology ; Mice ; NIH 3T3 Cells ; Phospholipase D/*metabolism ; Protein Kinase C/antagonists & inhibitors/physiology ; Protein Transport/drug effects ; Research Support, Non-U.S. Gov't ; Tetradecanoylphorbol Acetate/*analogs & derivatives/pharmacology ; Transfection ; Up-Regulation ; Viral Core Proteins/genetics/*metabolism ; p38 Mitogen-Activated Protein Kinases/physiology

Agents that elevate cellular cAMP are known to inhibit the activation of phospholipase D (PLD). We investigated whether PLD can be phosphorylated by cAMP-dependent protein kinase (PKA) and PKA-mediated phosphorylation affects the interaction between PLD and RhoA, a membrane regulator of PLD. PLD1, but not PLD2 was found to be phosphorylated in vivo by the treatment of dibutyryl cAMP (dbcAMP) and in vitro by PKA. PKA inhibitor (KT5720) abolished the dbcAMP-induced phosphorylation of PLD1, but dibutyryl cGMP (dbcGMP) failed to phosphorylate PLD1. The association between PLD1 and Val14RhoA in an immunoprecipitation assay was abolished by both dbcAMP and dbcGMP. Moreover, RhoA but not PLD1 was dissociated from the membrane to the cytosolic fraction in dbcAMP-treated cells. These results suggest that both PLD1 and RhoA are phosphorylated by PKA and the interaction between PLD1 and RhoA is inhibited by the phosphorylation of RhoA rather than by the phosphorylation of PLD1.
Bucladesine/pharmacology ; Carbazoles/pharmacology ; Cell Line, Tumor ; Cyclic AMP-Dependent Protein Kinases/antagonists & inhibitors/*metabolism ; Dibutyryl Cyclic GMP/pharmacology ; Enzyme Inhibitors/pharmacology ; Humans ; Indoles/pharmacology ; Phospholipase D/*metabolism ; Phosphorylation/drug effects ; Pyrroles/pharmacology ; Research Support, Non-U.S. Gov't ; rhoA GTP-Binding Protein/*metabolism

Oxidative stress has been implicated in mediation of vascular disorders. In the presence of vanadate, H2O2 induced tyrosine phosphorylation of PLD1, protein kinase C-a (PKC-a), and other unidentified proteins in rat vascular smooth muscle cells (VSMCs). Interestingly, PLD1 was found to be constitutively associated with PKC-a in VSMCs. Stimulation of the cells by H2O2 and vanadate showed a concentration-dependent tyrosine phosphorylation of the proteins in PLD1 immunoprecipitates and activation of PLD. Pretreatment of the cells with the protein tyrosine kinase inhibitor, genistein resulted in a dose-dependent inhibition of H2O2-induced PLD activation. PKC inhibitor and down-regulation of PKC abolished H2O2-stimulated PLD activation. The cells stimulated by oxidative stress (H2O2) caused increased cell migration. This effect was prevented by the pretreatment of cells with tyrosine kinase inhibitors, PKC inhibitors, and 1-butanol, but not 3-butanol. Taken together, these results suggest that PLD might be involved in oxidative stress-induced migration of VSMCs, possibly via tyrosine phosphorylation and PKC activation.
Animals ; Cell Movement/drug effects/*physiology ; Cells, Cultured ; Enzyme Activation/drug effects ; Enzyme Inhibitors/pharmacology ; Genistein/pharmacology ; Hydrogen Peroxide/pharmacology ; Muscle, Smooth, Vascular/cytology/*physiology ; *Oxidative Stress/drug effects ; Phospholipase D/*metabolism ; Phosphorylation/drug effects ; Protein Kinase C/*metabolism ; Protein-Tyrosine Kinase/antagonists & inhibitors ; Rats ; Rats, Sprague-Dawley ; Research Support, Non-U.S. Gov't ; Signal Transduction/drug effects ; Vanadates/pharmacology ; Vascular Diseases/metabolism

Extracellular ATP has been known to modulate various cellular responses including mitogenesis, secretion and morphogenic activity in neuronal cells. In the ATP-induced morphogenic activity, focal adhesion kinase(s) such as Fak have been suggested to play a critical role. Binding of ATP to its specific cell surface receptor in PC12 cells induces phospholipase D (PLD) activity. However, the role of PLD on ATP-induced Fak activation in PC12 cells remains unclear. In this study, we investigated the role of PLD on the ATP-induced Fak activation and paxillin phosphorylation using two established cell lines: wild type PLD2- and lipase-inactive mutant PLD2-inducible PC12 cells. Stimulation of cells with ATP caused PLD2 activation via classical protein kinase C activation. ATP also induced Fak activation, and paxillin phosphorylation, and were dramatically reduced by wild type PLD2 overexpression but not by lipase-inactive mutant PLD2 overexpression. When the PC12 cells were pretreated with propranolol, a specific inhibitor for phosphatidic acid phosphohydrolase resulting in the accumulation of PA, ATP-induced Fak activation and paxillin phosphorylation were also reduced. We found that inhibition of tyrosine phosphatases by pervanadate completely blocked PLD2-dependent Fak and paxillin dephosphorylation. Taken together, we suggest that PLD2 activity might play a negative role in ATP-induced Fak and paxillin phosphorylation possibly through tyrosine phosphatases.
Adenosine Triphosphate/*metabolism ; Animal ; Culture Media, Serum-Free ; Cytoskeletal Proteins/metabolism ; Enzyme Activation/drug effects ; Enzyme Inhibitors/pharmacology ; Focal Adhesions/metabolism ; PC12 Cells ; Phospholipase D/*metabolism ; Phosphoproteins/metabolism ; Phosphorylation ; Propranolol/pharmacology ; Protein Kinase C/antagonists & inhibitors ; Protein-Tyrosine Kinase/*metabolism ; Rats ; Support, Non-U.S. Gov't ; Vasodilator Agents/pharmacology

Both Fas and PMA can activate phospholipase D via activation of protein kinase Cbeta in A20 cells. Phospholipase D activity was increased 4 fold in the presence of Fas and 2.5 fold in the presence of PMA. The possible involvement of tyrosine phosphorylation in Fas-induced activation of phospholipase D was investigated. In five minute after Fas cross-linking, there was a prominent increase in tyrosine phosphorylated proteins, and it was completely inhibited by D609, a specific inhibitor of phosphatidylcholine-specific phospholipase C (PC-PLC). A tyrosine kinase inhibitor, genistein, can partially inhibit Fas-induced phospholipase D activation. There were no effects of genistein on Fas-induced activation of PC-PLC and protein kinase C. These results strongly indicate that tyrosine phosphorylation may in part account for the increase in phospholipase D activity by Fas cross-linking and D609 can block not only PC-PLC activity but also tyrosine phosphorylation involved in Fas-induced phospholipase D activation.
Animal ; Antibodies, Monoclonal/immunology/*pharmacology ; Antigens, CD95/immunology/*metabolism ; Bridged Compounds/*pharmacology ; Cell Line ; Cross-Linking Reagents ; Dose-Response Relationship, Immunologic ; Enzyme Activation ; Genistein/pharmacology ; Hydrolysis ; Lymphoma/pathology ; Mice ; Phospholipase C/*antagonists & inhibitors ; Phospholipase D/*metabolism ; Phosphorylation ; Phosphorylcholine/metabolism ; Solubility ; Thiones/*pharmacology ; Tumor Cells, Cultured ; Tyrosine/*metabolism ; Water/chemistry