1.Effects of protein kinase C activity on CD44 expression and subcellular distribution in human erythrocytes.
Yi-Wen HAO ; Da-Ye CHENG ; Jin-Tao CHEN
Journal of Experimental Hematology 2009;17(4):933-937
The study was aimed to investigate the effects of protein kinase C (PKC) on standard type CD44 expression and subcellular distribution in human erythrocytes. PKC activity was detected by the incorporation of [gamma-(32)P]-ATP into exogenous substrate, phosphorylation of CD44 was determined by autoradiograph, distribution of CD44 was observed by indirect immunofluorescence, and expression of CD44 was analyzed by flow cytometry. The results showed that PKC activity reached the maximal level at 30 minutes after treatment with phorbol-myristate-acetate (PMA), and the peak of CD44 phosphorylation and CD44 expression appeared at the same time, which all increased significantly as compared with control group (p < 0.001). PKC activation resulted in CD44 aggregation on membrane and colocalization of PKC and CD44. Calphostin C could inhibit the above reaction resulted from PKC activation. It is concluded that PKC activation can up-regulate CD44 expression by phosphorylation, and result in the coherent migration and colocalization of CD44 and PKC in human erythrocytes.
Erythrocyte Count
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Erythrocytes
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enzymology
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metabolism
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Humans
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Hyaluronan Receptors
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metabolism
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Membrane Proteins
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metabolism
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Phosphorylation
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Protein Kinase C
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metabolism
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Tetradecanoylphorbol Acetate
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analogs & derivatives
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Up-Regulation
2.Relaxin inhibit cardiac fibrosis induced by phorbol 12-myristate 13-acetate.
Yu Peng WANG ; Ping WANG ; Lei DONG ; Hui CHEN ; Yong Quan WU ; Hong Wei LI ; Min LI
Biomedical and Environmental Sciences 2014;27(2):138-141
Relaxin is known to inhibit cardiac fibrosis. However, it is unclear whether relaxin could regulate the effects of Phorbol 12-myristate 13-acetate (PMA, PKC activator) on cardiac fibrosis. So the influence of relaxin on the cell proliferation and collagen expression induced by PMA in cultured cardiac fibroblasts was studied. It showed that PMA significantly increased cardiac fibroblasts proliferation, Type I pro-collagen protein expression, Type I pro-collagen mRNA expression, and rhRLX absolutely significantly decreased PMA induced effects on cardiac fibroblasts proliferation and Type I pro-collagen expressions, indicating that relaxin could inhibit cardiac fibrosis induced by PMA.
Animals
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Animals, Newborn
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Cells, Cultured
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Fibroblasts
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pathology
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Fibrosis
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Heart Diseases
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chemically induced
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pathology
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prevention & control
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Rats
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Rats, Sprague-Dawley
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Relaxin
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therapeutic use
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Tetradecanoylphorbol Acetate
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analogs & derivatives
3.Effects of budesonide, desloratadine and dexamethasone on interleukine-4 release and expression from human mast cell line.
Yu ZHAO ; C Andrew van HASSELT ; Kang-Sang WOO ; Yeuk-Oi WONG ; Chuan-Yu LIANG ; Ping-Chung LEUNG
Chinese Journal of Otorhinolaryngology Head and Neck Surgery 2005;40(2):124-127
OBJECTIVESince human mast cell is an important source of cytokines, it is of importance to understand the effects of anti-allergic drugs on cytokines modulation in mast cells. In the present study, we aimed at observing whether IL-4 could be released from human mast cell line (HMC-1) after the stimulation of PMA + A23187, and the effects of systemic glucocorticosteroid, dexamethasone, topical glucocorticosteroid, budesonide and H1 antagonist, desloratadine on IL-4 release and mRNA expression.
METHODSHMC-1 was stimulated with 25 ng/ml phorbol 12-myristate 13-acetate (PMA) and 2.5 x 10(-7) mol/L ionomycin (A23187) and cultured for 6 hours, 12 hours and 24 hours respectively in the presence or absence of 10(-6)-10(-10) mol/L concentrations of test drugs. Culture supernatants were collected and the levels of IL-4 were assayed by enzyme-linked immunosorbent assays (ELISA). The mRNA expression of IL-4 was measured by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR).
RESULTSHMC-1 expressed IL-4 mRNA and the resulting protein production of IL-4 released after being stimulated with PMA plus A23187. Dexamethasone, budesonide and desloratadine had potent inhibitory effect on IL-4 release at any concentrations and time points, with significant deference (P < 0.05) compared to the control cells. The inhibitory effect did not show time-dependent and concentration-dependent manner. Desloratadine and budesonide showed neither up-regulatory nor down-regulatory effects on IL-4 mRNA expression at the test concentrations, however, desloratadine could down-regulate IL-4 mRNA expression.
CONCLUSIONSHMC-1 could express and produce IL4 after stimulation. Dexamethasone, budesonide and desloratadine all had inhibitory effects on IL-4 release from HMC-1. In addition, desloratadine could also inhibit the IL-4 mRNA expression.
Budesonide ; pharmacology ; Cell Line ; Dexamethasone ; pharmacology ; Humans ; Interleukin-4 ; biosynthesis ; Loratadine ; analogs & derivatives ; pharmacology ; Mast Cells ; drug effects ; metabolism ; Tetradecanoylphorbol Acetate ; pharmacology
4.Protein kinase C agonist phorbol 12-Myristate-Acetate induces nociception and enhances nitric oxide production in the spinal cord of rats.
Xin-Hua GUO ; Qing-Jun LI ; Ling-Yun LIU ; Wen-Bin LI ; Li-Li REN
Chinese Journal of Applied Physiology 2005;21(3):256-259
AIMTo determine the involvement of NO signal pathway in the development of hyperalgesia induced by activation of protein kinase C (PKC ), nociceptive responses and nitric oxide synthase(NOS) expression and nitric oxide (NO) content in the spinal cord were observed after administration of Phorbol 12-Myristate-Acetate (PMA), a PKC agonist, in rats.
METHODSNociceptive response was observed by behavioral approach. Pain threshold was assayed using thermal tail-flick test. NADPH-d histochemistry was used to investigate the changes of NOS expression. Nitrate/nitrite (NO3-/NO2-) was assayed to represent NO content of lumbar enlargement of spinal cord.
RESULTSNociceptive response was induced and pain threshold decreased after intrathecal injection of PMA. The number of NADPH-d positive cells increased significantly in the superficial layer of the spinal cord dorsal horn (Laminae I - II ) and the grey matter surrounding the central canal (Laminae X), and the reactive degree of NADPH-d positive soma and processes and NO content of the lumbar enlargement of the spinal cord increased significantly after intrathecal injection of PMA. Pretreatment of PKC inhibitor chelerythrine chloride blocked the changes induced by PMA.
CONCLUSIONThe activation of PKC in the spinal cord neurons might induce spontaneous nociceptive responses and hyperalgesia in rats, as well as promote NOS expression and NO production, suggesting that increase in NO production is one of mechanisms of hyperalgesia induced by activation of PKC.
Animals ; Enzyme Activators ; pharmacology ; Nitric Oxide ; biosynthesis ; Nociception ; drug effects ; Pain Threshold ; drug effects ; Protein Kinase C ; Rats ; Rats, Sprague-Dawley ; Signal Transduction ; drug effects ; Spinal Cord ; drug effects ; metabolism ; Tetradecanoylphorbol Acetate ; analogs & derivatives ; pharmacology
5.DADLE suppresses the proliferation of human liver cancer HepG2 cells by activation of PKC pathway and elevates the sensitivity to cis-diammine dichloridoplatium.
Bo TANG ; Jian DU ; Zhen-ming GAO ; Rui LIANG ; De-guang SUN ; Xue-li JIN ; Li-ming WANG
Chinese Journal of Oncology 2012;34(6):425-429
OBJECTIVETo investigate the effect of DADLE, a δ-opioid receptor agonist, on the proliferation of human liver cancer HepG2 cells and explore the mechanism involving PKC pathway.
METHODSHepG2 cells were treated with DADLE at different doses (0.01, 0.1, 1.0 and 10 µmol/L). Cell viability was determined using methyl thiazolyl terazolium (MTT) assay. The expression of PKC mRNA and p-PKC protein were examined by RT-PCR and Western blot assay. After treated separately with DADLE plusing NAL or PMA, the cell cycle of HepG2 cells was analyzed by flow cytometer. MTT was used to detect their proliferation capacity and Western blot was used to examine the p-PKC expression. The growth inhibitory rate of HepG2 cells treated with DADLE and cis-diammine dichloridoplatinum (CDDP) was analyzed.
RESULTSDADLE at different concentrations showed an inhibitory effect on the proliferation of HepG2 cells though inhibiting the expression of PKC mRNA and p-PKC protein. The results of flow cytometry showed that compared with the control group, the percentage of S + G(2)/M cells in DADLE-treated group was lowered by 3.94% (P < 0.01). Meanwhile, after treated with NAL and PMA, the percentage was elevated by 3.22% and 3.63%, respectively (P < 0.01). The MTT and Western blot assays showed that compared with the control group, the values of A570 and p-PKC protein levels in the HepG2 cells of DADLE-treated group were significantly decreased (P < 0.01). After treatment with NAL and PMA, the values of A570 and p-PKC protein levels were elevated significantly (P < 0.01). The growth inhibitory rate of DADLE + CDDP group was 79.9%, significantly lower than 25.2% and 43.2% of the DADLE and CDDP groups, respectively.
CONCLUSIONSActivation of δ-opioid receptor by DADLE inhibits the apoptosis of human liver cancer HepG2 cells. The underlying mechanism may be correlated with PKC pathway. DADLE can enhance the chemosensitivity of HepG2 cells to CDDP.
Antineoplastic Agents ; pharmacology ; Cell Cycle ; drug effects ; Cell Proliferation ; drug effects ; Cisplatin ; pharmacology ; Dose-Response Relationship, Drug ; Drug Resistance, Neoplasm ; Enkephalin, Leucine-2-Alanine ; administration & dosage ; pharmacology ; Hep G2 Cells ; Humans ; Naltrexone ; analogs & derivatives ; pharmacology ; Phosphorylation ; Protein Kinase C ; genetics ; metabolism ; RNA, Messenger ; metabolism ; Receptors, Opioid, delta ; agonists ; Signal Transduction ; Tetradecanoylphorbol Acetate ; analogs & derivatives ; pharmacology
6.Effect of sphingosine kinase 1 on the apoptosis, migration and invasion of colon cancer HT-29 cells and its molecular mechanisms.
Shi-quan LIU ; Meng-bin QIN ; Jie-an HUANG ; Yue-yuan ZHONG ; Guo-du TANG ; Hai-xing JIANG
Chinese Journal of Oncology 2011;33(3):178-182
OBJECTIVETo investigate the effect of sphingosine kinase 1 (SphK1) on the proliferation, apoptosis, migration and invasion of colon cancer TH-29 cells and to explore its molecular mechanisms.
METHODSPhorbol 12-myristate 13-acetate (PMA) was used to induce the activity of SphK1 and N, N-dimethylsphingosine (DMS) was used to suppress the activity of SphK1. Cell prolieration and apoptosis were detected by MTT assay and flow cytometry, respectively. The migration and invasion capabilities of the cells were assessed in Transwell chambers. The activity of SphK1 was assayed by autoradiography. Western blot was used to evaluate the protein expression of SphK1, p38, phosphorylated p38 (p-p38) and SAPK/JNK.
RESULTSPMA and DMS were able to induce and suppress the activity and protein expression of SphK1 in a time-dependent manner, respectively. PMA enhanced and DMS suppressed the cell viability in a time- and dose-dependent manner. Being treated with 100 nmol/L PMA or 50 µmol/L DMS for 0, 6, 12, 24 h, the cell apoptosis rates of PMA group were (9.35 ± 0.84)%, (7.61 ± 0.48)%, (5.53 ± 0.76)% and (0.56 ± 0.33)%, contrastly, that of DMS group were (9.18 ± 0.94)%, (12.06 ± 1.41)%, (19.80 ± 2.36)% and (31.85 ± 3.60)%, respectively. Compared with the control group, the cell migration and invasion capabilities of the PMA group were significantly enhanced, and that of the DMS group were significantly suppressed. The migration cell number of control, PMA and DMS groups were 68.75 ± 6.15, 109.33 ± 11.63 and 10.83 ± 2.48, the invasion cell number of control, PMA and DMS groups were 55.42 ± 4.50, 90.58 ± 7.06 and 9.58 ± 2.39, respectively. With the elevating activity and expression of SphK1, the protein expressions of p38, p-p38 and SAPK/JNK were strikingly suppressed. On the contrary, after treating with DMS the protein expressions of p38, p-p38 and SAPK/JNK were enhanced.
CONCLUSIONSSphK1 potently enhances the prolieration, migration and invasion of colon cancer HT-29 cells, meanwhile suppresses the cell apoptosis. The suppressing of the p38 and SAPK/JNK signalling pathways may be one of its molecular mechanisms.
Apoptosis ; drug effects ; Carcinogens ; administration & dosage ; pharmacology ; Cell Movement ; drug effects ; Cell Proliferation ; drug effects ; Dose-Response Relationship, Drug ; Enzyme Inhibitors ; administration & dosage ; pharmacology ; HT29 Cells ; Humans ; MAP Kinase Kinase 4 ; metabolism ; Neoplasm Invasiveness ; Phosphorylation ; Phosphotransferases (Alcohol Group Acceptor) ; metabolism ; physiology ; Sphingosine ; administration & dosage ; analogs & derivatives ; pharmacology ; Tetradecanoylphorbol Acetate ; administration & dosage ; analogs & derivatives ; pharmacology ; Time Factors ; p38 Mitogen-Activated Protein Kinases ; metabolism
7.Influence of signal transduction modulators on the secretory function of T lymphocytes in severely scalded mice and its mechanism.
Jun-song ZHENG ; Dai-zhi PENG ; Wen-hua HUANG ; Gao-xing LUO ; Hui HUANG ; Jing LIU ; Xin ZHOU
Chinese Journal of Burns 2006;22(6):411-414
OBJECTIVETo investigate the influence of various signal transduction modulators on the splenic T lymphocytes secretion of IL-2 and IL-10 in severely scalded mice, and to explore its mechanism.
METHODSThe mice were inflicted with 18% TBSA full-thickness scald by high-pressure heat vapour, and T lymphocytes were isolated from murine splenocytes through nylon wool column at 12 and 96 post-scald hours (PSH). Then the cells were divided into following groups: i. e. control, scald, scald and modulator [1 ml of 50 micromol/L PKC inhibitor ( H-7) , 30 micromol/L tetradecanoylphorbol-13-acetate (TPA) , 10micromol/L nonreceptor tyrosine protein kinase inhibitor (herbimycin) , 25 microg/ml of mitogen activated protein kinase kinase inhibitor (PD098059) , 100 nmol/L Calcium ionophore ( A23187) were added to the cells, respectively] groups. The scald group was subdivided into S1 (with scald at 12 PSH) and S2 (with scald at 96 PSH) groups. The modulator group was subdivided into modulator, S1 and modulator( the modulators were added into cells at 12 PSH) , and S2 and modulator( the modulators were added to cells at 96 PSH) groups. The influence of modulators to T lymphocyte secretion of IL-2 and IL-10 were observed.
RESULTSAfter the addition of H-7, the IL-2 and IL-10 levels in each group were obviously lower than that in controls( P <0. 05 or 0.01) , and that in S1 and H7 group, S2 and H7 group were obviously lower than that in scald group at corresponding time-points( P <0.01). The levels of IL-10, and especially IL-2 were elevated by TPA, but they were markedly lower than that in control group after PD098059 pretreatment. The secretion of IL-2 and IL-10 was significantly suppressed by herbimycin in S1 and herbimycin, and S2 and herbimycin groups, but those in Sl and A21387[ (2 417+/-39) pg/ml, (2 793+/-25)pg/ml] , S2 and A21387 [ (921+/-50) pg/ml, (2 633+/-35)pg/ml] groups were evidently higher than those in S1[ (1 542+/-40)pg/ml, (2 390+/-15)pg/ml] , S2 [(328+/-19)pg/ml, (1 618+/-21)pg/ml,( P <0.05 or <0.01)]groups.
CONCLUSIONPKC, calcium, MAPKK and TPK play critical roles in the dysfunction of splenic T lymphocyte secretion of IL-2 and IL-10 in severely scalded mice, among which TPK and PKC are mainly targeted to IL-2 secretion, and MAPKK is targeted to IL-10 secretion. TPA and A23187 can markedly rectify the disturbance of IL-2/IL-10 secretion ratio by increasing the IL-2 secretion after scald.
Animals ; Benzoquinones ; pharmacology ; Burns ; metabolism ; Calcimycin ; pharmacology ; Calcium ; metabolism ; Cells, Cultured ; Female ; Flavonoids ; pharmacology ; Interleukin-10 ; metabolism ; Interleukin-2 ; metabolism ; Lactams, Macrocyclic ; pharmacology ; Lymphocyte Activation ; Male ; Mice ; Mice, Inbred Strains ; Mitogen-Activated Protein Kinase Kinases ; antagonists & inhibitors ; Protein Kinase C ; metabolism ; Protein-Tyrosine Kinases ; metabolism ; Rifabutin ; analogs & derivatives ; Signal Transduction ; Spleen ; cytology ; T-Lymphocytes ; drug effects ; metabolism ; Tetradecanoylphorbol Acetate ; pharmacology
8.Tyrosine phosphatase and cytochrome P450 activity are critical in regulating store-operated calcium channels in human fibroblasts.
Experimental & Molecular Medicine 2006;38(6):703-717
Diverse signaling pathways have been proposed to regulate store-operated calcium entry (SOCE) in a wide variety of cell types. However, it still needs to be determined if all of these known pathways operate in a single cell type. In this study, we examined involvement of various signaling molecules in SOCE using human fibroblast cells (HSWP). Bradykinin (BK)-stimulated Ca2+ entry, previously shown to be via SOCE, is enhanced by the addition of vanadate, an inhibitor of tyrosine phosphatases. Furthermore, SOCE is regulated by cytochrome P-450, as demonstrated by the fact that the products of cytochrome P-450 activity (14,15 EET) stimulated SOCE while econazole, an inhibitor of cytochrome P450, suppressed BK-stimulated Ca2+ entry. In contrast, Ca2+ entry was unaffected by the guanylate cyclase inhibitor LY83583, or the membrane permeant cyclic GMP analog 8-bromo-cyclic GMP (8-Br-cGMP). Neither nitric oxide donors nor phorbol esters affected BK-stimulated Ca2+ entry. SOCE in HSWP cells is primarily regulated by tyrosine phosphorylation and the cytochrome P-450 pathway, but not by cyclic GMP, nitric oxide, or protein kinase C. Thus, multiple pathways do operate in a single cell type leading to the activation of Ca2+ entry and some of these signaling pathways are more prominently involved in regulating calcium entry in different cell types.
Vanadates/pharmacology
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Tetradecanoylphorbol Acetate/pharmacology
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Protein-Tyrosine-Phosphatase/*metabolism
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Phosphotyrosine/metabolism
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Phosphorylation/drug effects
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Nitric Oxide/metabolism
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Humans
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Fibroblasts
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Epidermal Growth Factor/pharmacology
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Enzyme Inhibitors/pharmacology
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Econazole/pharmacology
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Cytochrome P-450 Enzyme System/antagonists & inhibitors/*metabolism
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Cyclic GMP/analogs & derivatives/metabolism
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Cells, Cultured
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Calcium Channels/*metabolism
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Calcium/metabolism
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Bradykinin/pharmacology
9.Der f 2 activates phospholipase D in human T lymphocytes from Dermatophagoides farinae specific allergic individuals: Involvement of protein kinase C-alpha.
Jae Won OH ; Eun Young KIM ; Bon Suk KOO ; Ha Baik LEE ; Ki Sung LEE ; Yong Seok KIM ; Joong Soo HAN
Experimental & Molecular Medicine 2004;36(5):486-492
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
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Adult
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Animals
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Antigens, Dermatophagoides/*immunology
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Dermatophagoides farinae/*immunology
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Female
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Humans
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Hypersensitivity, Immediate/*enzymology/*immunology
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Male
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Phospholipase D/*metabolism
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Protein Kinase C/antagonists & inhibitors/*physiology
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Research Support, Non-U.S. Gov't
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Skin Tests
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T-Lymphocytes/*enzymology/immunology
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Tetradecanoylphorbol Acetate/*analogs & derivatives/pharmacology
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Up-Regulation
10.Phospholipase D activity is elevated in hepatitis C virus core protein-transformed NIH3T3 mouse fibroblast cells.
Joonmo KIM ; Bok Hee CHOI ; Kyung Lib JANG ; Do Sik MIN
Experimental & Molecular Medicine 2004;36(5):454-460
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
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Cell Line, Transformed
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*Cell Transformation, Viral
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Fibroblasts/enzymology/virology
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Hepacivirus/genetics/*physiology
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Mice
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NIH 3T3 Cells
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Phospholipase D/*metabolism
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Protein Kinase C/antagonists & inhibitors/physiology
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Protein Transport/drug effects
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Research Support, Non-U.S. Gov't
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Tetradecanoylphorbol Acetate/*analogs & derivatives/pharmacology
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Transfection
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Up-Regulation
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Viral Core Proteins/genetics/*metabolism
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p38 Mitogen-Activated Protein Kinases/physiology