BACKGROUNDNuclear factor kappaB (NF-kappaB) overactivation, requiring phosphorylation and degradation of its inhibitor IkappaBalpha, is the basis for chronicity of airway inflammation in asthma. Based on our previous plasmid pShuttle-IkappaBalpha, carrying an IkappaBalpha gene from human placenta, we optimized a novel IkappaBalpha mutant (IkappaBalphaM) gene, constructed and characterized its replication-deficient recombinant adenovirus (AdIkappaBalphaM), and tested whether AdIkappaBalphaM-mediated overexpression of IkappaBalphaM could inhibit the NF-kappaB activation in endothelial cells.

METHODSIkappaBalphaM gene (203 - 1003 bp) encoding 267 amino acids, acquired by site-directed deleting N-terminal phosphorylation sites of serine 32/36, was subcloned into the pShuttle and pGEM-T vectors for further polymerase chain reaction (PCR), restriction digestion, deoxyribonucleic acid (DNA) sequencing and homology analyses. Subsequent to inserting the expression unit of pShuttle-IkappaBalphaM, containing cytomegalovirus (CMV) promoter, IkappaBalphaM complementary DNA (cDNA) and polyadenylic acid (PolyA) signals, into the type 5 adenovirus (Ad5) vector, the resultant AdIkappaBalphaM was packaged in human embryonic kidney (HEK) 293 cells by cotransfection with lipofectamine. Western blot analysis and electrophoretic mobility shift assay were utilized to detect the AdIkappaBalphaM-mediated overexpression of IkappaBalphaM in HEK293 cells and its suppressive effect on phorbol 12-myristate 13-acetate (PMA)-induced NF-kappaB activation in human umbilical vein endothelial (ECV304) cells, respectively.

RESULTSThe relevant nucleotides and deduced amino acids of 801 bp IkappaBalphaM gene were consistent with those of IkappaBalpha gene (GenBank accession number: M69043). The titer of the prepared AdIkappaBalphaM was 4.0 x 10 (12) plaque-forming units (pfu)/L. Moreover, the IkappaBalphaM gene was overexpressed in HEK293 cells, and potently inhibited the PMA-induced NF-kappaB activation in ECV304 cells dose-dependently.

CONCLUSIONSAdIkappaBalphaM is a novel vector for both efficient transfer and specific overexpression of IkappaBalphaM gene, as well as potent inhibition of NF-kappaB activity, providing a promising strategy for gene therapy of asthma.

Adenoviridae ; genetics ; Cell Line ; Endothelial Cells ; metabolism ; Genetic Therapy ; Humans ; I-kappa B Proteins ; genetics ; Mutation ; NF-KappaB Inhibitor alpha ; NF-kappa B ; antagonists & inhibitors ; Tetradecanoylphorbol Acetate ; pharmacology

BACKGROUNDThe CKLF-like MARVEL transmembrane domain-containing family (CMTM) is a novel family of proteins linking chemokines and TM4SF. Different members exhibit diverse biological functions. In this study, the effect of intracellular CMTM2 on regulating human immunodeficiency virus type-1 (HIV-1) transcription was evaluated.

METHODSThe effects of CMTM2 on regulating full-length HIV-1 provirus and the HIV-1 long terminal repeat (LTR)-directed transcription were assessed by luciferase assay. Transcription factor assays, using the luciferase reporter plasmids of AP-1, CRE, and NF-κB were conducted to explore the signaling pathway(s) that may be regulated by CMTM2. The potential relationship between CMTM2 and the transcription factor AP-1 was further analyzed by Western blotting analyses to investigate the effect of CMTM2 on PMA-induced ERK1/2 phosphorylation.

RESULTSThe results from the current study revealed that CMTM2 acts as a negative regulator of HIV-1 transcription. CMTM2 exerted a suppressive action on both full-length HIV-1 provirus and HIV-1 LTR-directed transcription. Transcription factor assays showed that CMTM2 selectively inhibited basal AP-1 and CREB activity. Co-expression of HIV-1 Tat, a potent AP-1 and CREB activator, can not reverse CMTM2-mediated AP-1 and CREB inhibition, suggesting a potent and specific effect of CMTM2 on negatively regulating these two signaling pathways.

CONCLUSIONIntracellular CMTM2 can negatively regulate HIV-1 transcription, at least in part, by targeting the AP-1 and CREB pathways. Exploring the mechanisms further may lead to new ways to control HIV-1 replication.

Chemokines ; physiology ; Cyclic AMP Response Element-Binding Protein ; antagonists & inhibitors ; HIV Long Terminal Repeat ; HIV-1 ; genetics ; Humans ; Intracellular Space ; metabolism ; Jurkat Cells ; MARVEL Domain-Containing Proteins ; Tetradecanoylphorbol Acetate ; pharmacology ; Transcription Factor AP-1 ; antagonists & inhibitors ; Transcription, Genetic ; U937 Cells

Arginine vasopressin (AVP), a neurohormone and hemodynamic factor implicated in the pathophysiology of hypertension and congestive heart failure, can also act as a growth-stimulating factor. Our previous work demonstrated that AVP is a mitogen for neonatal rat cardiac fibroblasts (CFs). In the present study, we extended our investigations to adult rat CFs to explore whether AVP could induce adult rat CF proliferation and, if so, to identify the mechanism involved. Adult rat CFs were isolated, cultured and subjected to AVP treatment. DNA synthesis and cell cycle distribution were analyzed by [(3)H]-thymidine incorporation and flow cytometry. Cellular extracellular signal-regulated kinase 1/2 (ERK1/2) activity was measured by in vitro kinase assay using myelin basic protein (MBP) as a substrate. Protein expressions of total- and phospho-ERK1/2, p27(Kip1), cyclins D1, A, E were assessed by Western blot. The results showed that AVP stimulated DNA synthesis in adult rat CFs, and the effect was abolished by a V1 receptor antagonist, d(CH(2))(5)[Tyr(2)(Me), Arg(8)]-vasopressin (0.1 μmol/L), but not by a V2 receptor antagonist, desglycinamide-[d(CH(2))(5), D-Ile(2), Ile(4), Arg8]-vasopressin (0.1 μmol/L). AVP induced an activation of ERK1/2, which could be mimicked by the protein kinase C (PKC) activator, phorbol 12-myristate 13-acetate (PMA, 30 nmol/L, 5 min), but abolished by depletion of PKC via chronic PMA incubation (2.5 μmol/L, 24 h). In addition, AVP down-regulated protein expression of p27(Kip1), increased protein expressions of cyclins D1, A and E, and induced cell cycle progression from G(0)/G(1) into S stage. Inhibition of ERK1/2 activation by PD98059 (30 μmol/L) abolished the effect of AVP on DNA synthesis, protein expressions of p27(Kip1), cyclins D1, A and E as well as cell cycle progression. These results suggest that AVP is also a growth factor for adult rat CFs. The mitogenic effect of AVP is mediated via V1 receptors and PKC-ERK1/2 pathway. Moreover, AVP modulates the expressions of cell cycle regulatory proteins p27(Kip1) and cyclins D1, A and E, which lie downstream of ERK1/2 activation, and induces cell cycle progression in adult rat CFs.
Animals ; Antidiuretic Hormone Receptor Antagonists ; pharmacology ; Arginine Vasopressin ; pharmacology ; Cell Cycle ; Cell Cycle Proteins ; metabolism ; Cell Proliferation ; Fibroblasts ; cytology ; drug effects ; Mitogen-Activated Protein Kinase 3 ; metabolism ; Myocardium ; cytology ; Phosphorylation ; Protein Kinase C ; metabolism ; Rats ; Signal Transduction ; Tetradecanoylphorbol Acetate ; pharmacology

During chronic inflammatory response, mono- cytes/macrophages produce 92-kDa matrix metalloproteinase-9 (MMP-9), which may contribute to their extravasation, migration and tissue remodeling. Activation of peroxisome proliferator- activated factor receptor-gamma (PPAR-gamma) has been shown to inhibit MMP-9 activity. To evaluate whether ox-LDL, a PPAR-gamma activator, inhibits PMA-induced MMP-9 expression and activity, and if so, whether CD36 and PPAR-gamma are involved in this process, we investigated the effect of ox-LDL on MMP-9 expression and activity in PMA-activated human monocytic cell line U937. PMA-induced MMP-9 expression and activity were suppressed by the treatment with ox-LDL (50 micrigram/ml) or PPAR-gamma activators such as troglitazone (5 micrometer), ciglitazone (5 micrometer), and 15d- PGJ2 (1 micrometer) for 24 h. This ox-LDL or PPAR-gamma activator-mediated inhibition of micrometer P-9 activity was diminished by the pre-treatment of cells with a blocking antibody to CD36, or PGF2a (0.3 micrometer), which is a PPAR-gamma inhibitor, as well as overexpression of a dominant-negative form of CD36. Taken together, these results suggest that ox-LDL suppresses PMA-induced MMP-9 expression and activity through CD36-mediated activation of PPAR-gamma.
Antibodies, Blocking/pharmacology ; Antigens, CD36/immunology/*physiology ; Cells, Cultured ; Chromans/pharmacology ; Gelatinase B/antagonists & inhibitors/genetics/*metabolism ; Humans ; Lipoproteins, LDL/pharmacology/*physiology ; Monocytes/drug effects/*enzymology/metabolism ; NF-kappa B/antagonists & inhibitors ; PPAR gamma/*metabolism ; Prostaglandin D2/*analogs & derivatives/pharmacology ; RNA, Messenger/analysis/metabolism ; Research Support, Non-U.S. Gov't ; Tetradecanoylphorbol Acetate/antagonists & inhibitors/pharmacology ; Thiazolidinediones/pharmacology ; Transcription, Genetic/drug effects

OBJECTIVETo investigate the protective effects of protein kinase C (PKC) and mitogen-activated protein kinases (MAPKs) their and mechanisms in liver ischemic preconditioning.

METHODSIn rat models of liver ischemia-reperfusion (IR) and ischemic preconditioning (IP), the liver function was evaluated by examining serum alanine aminotransferase and aspartate aminotransferase levels, and the morphological changes of the liver cells were observed under microscope. PKC activator phorbol 12-myristate 13-acetate(PMA) and inhibitor chelerythrine(CHE), as well as MEK inhibitor PD98059, were utilized to analyze the phosphorylation of PKC and P44/42 MAPKs.

RESULTSCompared with the control rats, the liver function was best protected in rats of IP group, but not in those of IP group with PD98059 or CHE treatment. The rats in IR group showed improved liver function after PMA treatment. Similarly, the phosphorylation of PKC and P44/42 MAPKs was correlated with the liver function, and highly enhanced PKC and P44/42 MAPKs activity was observed in IP and IR+PMA groups, but decreased activity in IR and IP+CHE groups.

CONCLUSIONPhosphorylation of PKC and MAPKs plays a pivotal role in the preservation of the hepatocytes during IP.

Alanine Transaminase ; blood ; Alkaloids ; pharmacology ; Animals ; Aspartate Aminotransferases ; blood ; Benzophenanthridines ; pharmacology ; Enzyme Inhibitors ; pharmacology ; Flavonoids ; pharmacology ; Ischemic Preconditioning ; Liver ; blood supply ; enzymology ; Male ; Mitogen-Activated Protein Kinases ; antagonists & inhibitors ; metabolism ; Phosphorylation ; drug effects ; Protein Kinase C ; antagonists & inhibitors ; metabolism ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Reperfusion Injury ; blood ; physiopathology ; Tetradecanoylphorbol Acetate ; pharmacology

Telomerase, a ribonucleoprotein reverse transcriptase that extends telomeres of eukaryotic chromosomes is repressed in normal somatic cells but is activated during development and neoplasia. The regulation mechanism of telomerase activity in cancer cells is not clearly known. In this report, a possible affect of PKC on telomerase activity was examined using HeLa and CUMC-6 cervical cancer cell lines. Exposure of cells to PKC inhibitor, bisindolylmaleimide I and Go6976, and high levels of PKC activator, 12-O-tetradecanoyl phorbol 13-acetate (TPA) resulted in the inhibition of PKC activity in both cells. Telomerase activities were also inhibited by bisindolyl-maleimide I and Go6976, respectively, in a time-dependent manner. As PKC activity changes in TPA-treated cervical cancer cells, telomerase activities were increased at low dose of TPA and decreased at high dose. The expression levels of human telomerase subunits, human telomerase RNA (hTR) were not influenced by PKC modulating drugs. In contrast, the expression of full-length human telomerase reverse transcriptase (hTERT) was decreased after exposure to bisindolylmaleimide I and Go6976 in a time-dependent manner. hTERT expression was not affected by low dose of TPA. In contrast, high dose of TPA inhibited hTERT expression level. But the expression patterns of beta-deletion transcript of hTERT after 72 h of treatment with PKC inhibitors or high dose of TPA exposure were not discernable as compared with those of full-length hTERT transcripts to PKC modulating drugs. These results suggest that PKC-modulating drugs altered telomerase activities by affecting full-length hTERT expression profile in human cervical cancers.
Alternative Splicing ; Carbazoles/pharmacology ; Catalytic Domain ; Cervix Neoplasms/*enzymology ; Enzyme Inhibitors/metabolism ; Female ; Hela Cells ; Human ; Indoles/pharmacology ; Maleimides/pharmacology ; Protein Kinase C/antagonists & inhibitors/*metabolism ; RNA, Messenger/metabolism ; Support, Non-U.S. Gov't ; Telomerase/antagonists & inhibitors/genetics/*metabolism ; Tetradecanoylphorbol Acetate/pharmacology ; Tumor Cells, Cultured

Breast Neoplasms ; metabolism ; pathology ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Down-Regulation ; drug effects ; Female ; Guanine Nucleotide Exchange Factors ; antagonists & inhibitors ; genetics ; metabolism ; Humans ; Indoles ; pharmacology ; MCF-7 Cells ; Maleimides ; pharmacology ; Protein Isoforms ; genetics ; metabolism ; Protein Kinase C ; antagonists & inhibitors ; genetics ; metabolism ; RNA Interference ; RNA, Small Interfering ; metabolism ; Receptor, ErbB-2 ; genetics ; metabolism ; Tetradecanoylphorbol Acetate ; toxicity

OBJECTIVETo explore the mechanism of reversal of multidrug resistance in renal carcinoma cells by protein kinase C inhibitor.

METHODSRT-PCR, Western blot and inverted fluorescent microscopy were used to determine the expression of PKCalpha and MDR related gene MDR1, MRP1, LRP in RCC cells transferred by PKCalpha cDNA. Also effects of activator and inhibitor of PKC in combination with adriamycin on multidrug resistance in RCC cells were evaluated by MTT.

RESULTSThe results of semi-quantitative RT-PCR analysis showed that the expression level of MDR1 was higher in RCC cells transferred by PKCalpha cDNA than in RCC cells, the reversal effectiveness of PKC inhibitors in combination with adriamycin (ADM) was apparently favorable. IC(50) of ADM in 786 - 0 cells was 7.8015e(-7) (5.7046e(-7) to 1.0669e(-6)); IC(50) of ADM in PKCalpha/786 - 0 cells was 1.6588e(-6) (1.1621e(-6) to 2.3677e(-6)); IC(50) of ADM in combination with PMA in PKCalpha/786 - 0 cells was 2.6794e(-6) (2.0521e(-6) to 3.4983e(-6)); IC(50) of ADM in combination with calphostin C in PKCalpha/786 - 0 cells was 9.2506e(-8) (5.9337e(-8) to 1.4422e(-7)).

CONCLUSIONPKC inhibitors can reverse multidrug resistance in renal carcinoma cells in vitro via changes of expression of MDR1.

ATP-Binding Cassette, Sub-Family B, Member 1 ; metabolism ; Antibiotics, Antineoplastic ; pharmacology ; Cell Line, Tumor ; DNA, Complementary ; genetics ; Doxorubicin ; pharmacology ; Drug Resistance, Multiple ; Drug Resistance, Neoplasm ; Genetic Vectors ; Humans ; Inhibitory Concentration 50 ; Kidney Neoplasms ; metabolism ; pathology ; Multidrug Resistance-Associated Proteins ; metabolism ; Naphthalenes ; pharmacology ; Protein Kinase C ; antagonists & inhibitors ; Protein Kinase C-alpha ; genetics ; metabolism ; Tetradecanoylphorbol Acetate ; pharmacology ; Transfection

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

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