PURPOSE: Increased lipoprotein-associated phospholipase A2 (Lp-PLA2) activity and Rho kinase activity may be associated with atherosclerosis. The principal aim of this study was to examine whether darapladib (a selective Lp-PLA2 inhibitor) could reduce the elevated Lp-PLA2 and Rho kinase activity in atherosclerosis. MATERIALS AND METHODS: Studies were performed in male Sprague-Dawley rats. The atherosclerosis rats were prepared by feeding them with a high-cholesterol diet for 10 weeks. Low-dose darapladib (25 mg.kg-1.d-1) and high-dose darapladib (50 mg.kg-1.d-1) interventions were then administered over the course of 2 weeks. RESULTS: The serum levels of triglycerides, total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), high-sensitivity C-reactive protein (hs-CRP), and Lp-PLA2, significantly increased in atherosclerosis model groups, as did Rho kinase activity and cardiomyocyte apoptosis (p<0.05 vs. sham group), whereas nitric oxide (NO) production was reduced. Levels of TC, LDL-C, CRP, Lp-PLA2, and Rho kinase activity were respectively reduced in darapladib groups, whereas NO production was enhanced. When compared to the low-dose darapladib group, the reduction of the levels of TC, LDL-C, CRP, and Lp-PLA2 was more prominent in the high-dose darapladib group (p<0.05), and the increase of NO production was more prominent (p<0.05). Cardiomyocyte apoptosis of the high-dose darapladib group was also significantly reduced compared to the low-dose darapladib group (p<0.05). However, there was no significant difference in Rho kinase activity between the low-dose darapladib group and the high-dose darapladib group (p>0.05). CONCLUSION: Darapladib, a Lp-PLA2 inhibitor, leads to cardiovascular protection that might be mediated by its inhibition of both Rho kinase and Lp-PLA2 in atherosclerosis.
1-Alkyl-2-acetylglycerophosphocholine Esterase/*antagonists & inhibitors/blood/drug effects ; Animals ; Atherosclerosis/blood/*drug therapy/*enzymology ; *Benzaldehydes ; C-Reactive Protein/metabolism ; Cholesterol/blood ; Cholesterol, HDL/blood ; Cholesterol, LDL/blood ; Dose-Response Relationship, Drug ; Male ; *Oximes ; Phospholipase A2 Inhibitors/*administration & dosage/adverse effects ; Rats ; Rats, Sprague-Dawley ; Triglycerides/blood ; rho-Associated Kinases/*metabolism

OBJECTIVETo explore the role of phospholipase C-gamma1 (PLC-gamma1) signaling pathway in H(2)O(2)-induced apoptosis of PC12 cells.

METHODSPC12 cells were exposed to 50 micromol/L H(2)O(2) after pretreatment with 10 micromol/L U73122, a specific PLC-gamma1 inhibitor. Hoechst/PI double staining was performed to observe the morphological changes of the cells under light microscope. MTT assay was used to evaluate the cell viability, and the percentage of apoptotic cells was analyzed by flow cytometry. DNA fragmentation assay was carried out to characterize the cell apoptosis.

RESULTSAfter inhibition of the PLC-gamma1 signaling pathway with 10 micromol/L U73122, PC12 cells showed obvious apoptotic morphology, the viable cells decreased significantly, and the percentage of apoptotic cells rose to 35.7%. PC12 cells treated with U73122 presented with a distinct DNA ladder on electrophoresis resulting from DNA cleavage in the apoptotic cells.

CONCLUSIONPLC-gamma1 signaling pathway plays an important protective role in H(2)O(2)-induced PC12 cell apoptosis.

Animals ; Apoptosis ; drug effects ; Estrenes ; pharmacology ; Hydrogen Peroxide ; pharmacology ; PC12 Cells ; Phospholipase C gamma ; antagonists & inhibitors ; metabolism ; Pyrrolidinones ; pharmacology ; Rats ; Signal Transduction

BACKGROUNDIn most colorectal carcinomas, the level of phospholipase C (PLC)-gamma 1 expression is greatly elevated. Increased expression of PLC-gamma 1 may play an important role in colon carcinogenesis, but the mechanism is not well known. The aim of this study was to evaluate the role of PLC-gamma 1 in colon carcinogenesis by using recombinant lentivirus that stably suppressed the PLC-gamma 1 expression in human colorectal carcinoma LoVo cells.

METHODSRecombinant lentivirus producing PLC-gamma 1 siRNA were prepared. After LoVo cells were transduced by each lentivirus, stably transduced cells were selected by Blasticidin. The protein and mRNA expression of PLC-gamma 1 were examined by Western-blot and reverse transcription-polymerase chain reaction (RT-PCR) analysis, and the effects of the lentivirus on the cell adhesion, migration and apoptosis were analyzed.

RESULTSStable LoVo cell line deficient in PLC-gamma 1, was established. Notably, PLC-gamma 1 was silenced without affecting the levels of other subtypes of PLC so that the role of PLC-gamma 1 in colon carcinogenesis could be examined. Silencing of endogenous PLC-gamma 1 resulted in efficient inhibition of the adhesion and migration of LoVo cells in vitro and a great increase of 5-fluorouracil induced apoptosis (30%-40%) of LoVo cells.

CONCLUSIONSPLC-gamma 1 may play an important role in metastasis and anti-apoptosis in human colorectal carcinomas.

Apoptosis ; drug effects ; Cell Adhesion ; Cell Line, Tumor ; Colorectal Neoplasms ; pathology ; therapy ; Fluorouracil ; pharmacology ; Humans ; Laminin ; antagonists & inhibitors ; genetics ; Lentivirus ; genetics ; Phospholipase C gamma ; antagonists & inhibitors ; genetics ; physiology ; RNA, Small Interfering ; therapeutic use

Phosphoinositide-specific phospholipase C-gamma1 (PLC-gamma1) has two pleckstrin homology (PH) domains: an amino-terminal domain (PH1) and a split PH domain (PH2). Here, we show that overlay assay of bovine brain tubulin pool with glutathione-S-transferase (GST)-PLC-gamma1 PH domain fusion proteins, followed by matrix-assisted laser-desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), identified 68-kDa neurofilament light chain (NF-L) as a binding protein of amino-terminal PH domain of PLC-gamma1. NF-L is known as a component of neuronal intermediate filaments, which are responsible for supporting the structure of myelinated axons in neuron. PLC-gamma1 and NF-L colocalized in the neurite in PC12 cells upon nerve growth factor stimulation. In vitro binding assay and immunoprecipitation analysis also showed a specific interaction of both proteins in differentiated PC12 cells. The phosphatidylinositol 4, 5-bisphosphate [PI(4,5)P2] hydrolyzing activity of PLC-gamma1 was slightly decreased in the presence of purified NF-L in vitro, suggesting that NF-L inhibits PLC-gamma1. Our results suggest that PLC-gamma1-associated NF-L sequesters the phospholipid from the PH domain of PLC-gamma1.
Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization ; Rats ; Protein Interaction Mapping ; Protein Biosynthesis/drug effects ; Protein Binding/drug effects ; Phosphoproteins/chemistry/*metabolism ; Phospholipase C gamma/antagonists & inhibitors/chemistry/*metabolism ; Phosphatidylinositol 4,5-Diphosphate/metabolism ; Peptides/chemistry/metabolism ; PC12 Cells ; Neurofilament Proteins/chemistry/*metabolism ; Nerve Growth Factor/pharmacology ; Molecular Weight ; Molecular Sequence Data ; Microtubules/metabolism ; Microscopy, Fluorescence ; Isoenzymes/metabolism/pharmacology/physiology ; Glutathione Transferase/metabolism ; Blotting, Far-Western ; Blood Proteins/chemistry/*metabolism ; Binding Sites ; Animals ; Amino Acid Sequence

OBJECTIVETo explore the role of phospholipase C-gamma1 (PLC-gamma1) in tumor necrosis factor-alpha (TNF-alpha)-induced apoptosis of human glioma SWO cells.

METHODSThe PLC-gamma1 pathway was blocked by U73122 in SWO cells, and the inhibitory effect of TNF-alpha on SWO glioma cell proliferation with or without U73122 treatment was investigated by MTT assay. The cell apoptosis induced by TNF-alpha along or in combination with U73122 was detected by flow cytometry with PI staining. The expression of caspase-3 and Bcl-2 was detected by Western blotting.

RESULTS AND CONCLUSIONU73122 can sensitize SWO glioma cells to TNF-alpha-induced apoptosis. Blocking the PLC-gamma1 pathway may not induce apoptosis of SWO glioma cells, but can sensitize SWO glioma cells to small-dose TNF-alpha-induced apoptosis, the mechanism of which may involve down-regulation of bcl-2.

Apoptosis ; drug effects ; Blotting, Western ; Caspase 3 ; metabolism ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Cell Survival ; drug effects ; Dose-Response Relationship, Drug ; Down-Regulation ; drug effects ; Estrenes ; pharmacology ; Flow Cytometry ; Glioma ; enzymology ; pathology ; Humans ; Phosphodiesterase Inhibitors ; pharmacology ; Phospholipase C gamma ; antagonists & inhibitors ; metabolism ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Pyrrolidinones ; pharmacology ; Signal Transduction ; drug effects ; Tumor Necrosis Factor-alpha ; pharmacology

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

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

Phospholipase Cgamma1 (PLCgamma1) plays an important role in controlling cellular proliferation and differentiation. PLCgamma1 is overexpressed in some tumors, and its overexpression induces solid tumors in nude mice. However, the regulatory mechanisms underlying PLCgamma1-induced cell proliferation are not fully understood. Here we show that overexpression of PLCgamma1 highly phosphorylated glycogen synthase kinase-3beta (GSK-3beta) at serine-9 in 3Y1 fibroblasts. Inhibition of protein kinase C (PKC)s with GF109203X abrogated GSK-3beta phosphorylation by PLCgamma1. We also found that steady-state level of cyclin D1 protein, but not cyclin D1 mRNA, was highly elevated in response to serum stimulation in PLCgamma1-transfected cells as compared with vector-transfected cells. Since GSK-3beta is involved in cyclin D1 proteolysis in response to mitogenic stimulation, PLCgamma1-mediated GSK-3beta phosphorylation may function as a regulation of cyclin D1 accumulation in PLCgamma1-overexpressing cells.
Animals ; Cyclin D1/metabolism ; Epidermal Growth Factor/pharmacology ; Fibroblasts ; Gene Expression ; Glycogen Synthase Kinase 3/chemistry/*metabolism ; Mitogens/pharmacology ; Phospholipase C/genetics/*metabolism ; Phosphorylation/drug effects ; Phosphoserine/*metabolism ; Protein Kinase C/antagonists & inhibitors/*metabolism ; Rats ; Signal Transduction

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