OBJECTIVEIt has been known that the intrahepatic renin-angiotensin-aldosterone system (RAAS) plays a key role in the fibrogenesis in livers. Aldosterone (Aldo), the principal effector molecule of the RAAS, exerts local effects on cell growth and fibrogenesis. However, the signal transduction mechanisms underlying the effects of Aldo on hepatic fibrogenesis remain to be fully elucidated. The present study aims to investigate the signal transduction mechanism underlying the effects of Aldo on the signal passageway of active protein-1 (AP-1).

METHODSIn vitro, HSCs-T6 cell line was treated with Aldo for 10 min, 30 min, 60 min, 120 min and 180 min, and protein expression of Phospho-P42/44 was detected by Western blot. In addition, HSCs-T6 cell line was preincubated for 60 min or not with U0126 (an inhibitor of the MAPK/ERK kinase), and also with antioxidant-N-acetylcysteine (NAC) prior to exposure to Aldo for the indicated times. Protein expression of Phospho-P42/44 was measured by Western blot. DNA biding activity of AP-1 was analyzed by electrophoretic gel mobility shift assay (EMSA). By means of RT-PCR, expression of alpha1(1) procollagen mRNA was detected.

RESULTSAldo stimulated HSC via extracellular signal-regulated kinase (ERK1/2) pathway. Time course experiments showed that Aldo induced Phospho-P42/44 expression, which was abrogated by U0126, reaching a maximum at 10 minutes, and then declined progressively. NAC inhibited the Phospho-P42/44 expression. EMSA showed that stimulation of HSC by Aldo markedly increased AP-1 DNA binding activity. U0126 markedly reduced AP-1 DNA binding activity induced by Aldo; NAC partly decreased AP-1 activity induced by Aldo. Aldo up-regulated expression of alpha1(1) procollagen mRNA, which was attenuated by U0126 and NAC.

CONCLUSIONStimulation of HSC by Aldo results in activation of AP-1 via ERK1/2 pathway, leading to up-regulation of AP-1 target gene alpha1(1) procollagen mRNA expression.

Aldosterone ; pharmacology ; Cell Line ; Collagen Type I ; biosynthesis ; genetics ; Hepatocytes ; cytology ; metabolism ; Humans ; Mitogen-Activated Protein Kinase 3 ; metabolism ; RNA, Messenger ; biosynthesis ; genetics ; Transcription Factor AP-1 ; metabolism

OBJECTIVETo investigate the effect of activator protein-1 (AP-1) decoy-oligodeoxynucleotides (Decoy-ODNs) on the expression of fibroblast alpha2 type I collagen, so as to explore the gene therapy of pathologic scar.

METHODSDecoy-ODNs targeting AP-1 were designed and synthesized. NIH3T3 cells were transfected by cationic liposomes. The distribution of Decoy-ODNs in the cells was investigated. The inhibiting effects of Decoy-ODNs on AP-1 were determined by electrophoretic mobility shift assay (EMSA). And the effects of Decoy-ODNs on the collagen synthesis in the cells were analyzed by RT-PCR.

RESULTSAP-1 Decoy-ODNs could competitively inhibit the AP-1 in vitro activity. Cationic liposomes could play roles by effectively transfecting Decoy-ODNs into the plasma and nucleus. The mRNA expression of fibroblast alpha2 type I collagen decreased evidently after 24 hours of Decoy-ODNs action.

CONCLUSIONDecoy-ODNs could inhibit the mRNA expression of fibroblast alpha2 type I collagen by antagonizing AP-1.

Animals ; Collagen Type I ; biosynthesis ; Fibroblasts ; drug effects ; metabolism ; Mice ; NIH 3T3 Cells ; Oligodeoxyribonucleotides ; genetics ; pharmacology ; RNA, Messenger ; metabolism ; Transcription Factor AP-1 ; genetics ; Transfection

OBJECTIVETo study the effect of nuclear transcription factor AP-1 on tumor necrosis factor alpha (TNF-alpha) or minimal modified low density lipoprotein (mmLDL)-induced expression of plasminogen activator inhibitor-1 (PAI-1) in human vascular endothelial cells.

METHODSUsing gene recombination techniques, four luciferase reporter gene plasmids containing different length of human PAI-1 gene promoter were constructed. Through the transient transfection analysis, the roles of AP-1 element (from -823 bp to -553 bp) in PAI-1 promoter have been determined. In order to further verify the role of AP-1 element, the three site-directed mutants were recovered using PCR and sequencing assay.

RESULTSThe induction by TNF-alpha or mmLDL were decreased markedly when the three AP-1 elements in PAI-1 promoter had been mutated respectively.

CONCLUSIONSThese results indicate that the AP-1 element in PAI-1 promoter may have important role in PAI-1 gene transcriptions in endothelial cells induced by TNF-alpha or mmLDL.

Base Sequence ; Cells, Cultured ; Endothelium, Vascular ; cytology ; metabolism ; Humans ; Molecular Sequence Data ; Plasminogen Activator Inhibitor 1 ; biosynthesis ; genetics ; Promoter Regions, Genetic ; Transcription Factor AP-1 ; genetics ; metabolism ; Tumor Necrosis Factor-alpha ; genetics ; metabolism ; Umbilical Veins ; cytology

OBJECTIVETo clarify if Epstein-Barr virus encoded LMP1 induces matrix metalloproteinase 9 expression via NF-kappa B or AP-1 signaling pathway, which gives evidence to the elucidation of the mechanism of LMP1- mediated carcinogenesis.

METHODSTo determine whether LMP1 or its mutants contribute to MMP9 production via NF-kappa B or AP-1 transcription factor, MMP9-chloramphenicol acetyl transferase (CAT), NF-kappa B mut 9-CAT, AP-1 mut MMP9-CAT were transfected into human nasopharyngeal carcinoma cells stably expressing LMP1 (HNE2-LMP1) or its mutants, [HNE2-LMP1 (1-185), HNE2-LMP1 (1-231), HNE2-LMP1 delta 187-351] by electroporation technic. The difference of MMP9 reporter activity among those cell lines was detected by CAT assay and expression of MMP9 was determined in nasopharyngeal carcinoma cells stably expressing LMP1 or its mutants by zymographic analysis. In the meantime, efforts were made to demonstrate if LMP1 regulates NF-kappa B or AP-1 activation using reporter gene analysis.

RESULTSIn contrast with vector-transfected cells, MMP9 CAT activity in HNE2-LMP1, HNE2-LMP1 (1-185), HNE2-LMP1(1-231), HNE2-LMP1 delta 187-351 increased 7.2, 1.3, 3.3, 4.0 times respectively. Zymographic analysis demonstrated that the 92 kDa MMP9 expression was induced in HNE2-LMP1, HNE2-LMP1(1-231) and HNE2-LMP1 delta 187-351 cells, whereas it was negative in HNE2-pSG5 and HNE2-LMP1 (1-185) cells. As compared to the HNE2 cells, NF-kappa B or AP-1 reporter activity in HNE2-LMP1 cells were increased 13.8, 8.4 fold respectively. Moreover, In contrast with MMP9 CAT-transfected cells, MMP9 CAT activity in NF-kappa B mut MMP9-CAT or AP-1 mut MMP9-CAT transfected HNE2-LMP1, HNE2-LMP1 (1-185), HNE2-LMP1(1-231) and HNE2-LMP1 delta 187-351 cells were significantly decreased by 18.1% or 16.3%, 35.0% or 33.3%, 29.1% or 26.1% from the original level. However, there was no difference in NF-kappa B mut MMP9-CAT or AP-1 mut MMP9-CAT transfected HNE2-pSG5, HNE2-LMP1 (1-185) cells.

CONCLUSIONIn nasophargyngeal carcinoma, Epstein-Barr virus-encoded LMP1 induces MMP9 transcription and enzymatic activity via an NF-kappa B or AP-1 signaling pathway, which may contribute to invasiveness and metastasis.

Gene Expression ; drug effects ; Herpesvirus 4, Human ; chemistry ; Humans ; Matrix Metalloproteinase 9 ; biosynthesis ; NF-kappa B ; metabolism ; Nasopharyngeal Neoplasms ; pathology ; Signal Transduction ; Transcription Factor AP-1 ; metabolism ; Tumor Cells, Cultured ; Viral Matrix Proteins ; pharmacology

BACKGROUNDSolar ultraviolet (UV) irradiation induces the production of matrix metalloproteinases (MMPs) by activating cellular signalling transduction pathways. MMPs are responsible for the degradation and/or inhibition of synthesis of collagenous extracellular matrix in connective tissues. We mimicked the action of environmental ultraviolet on skin and investigated the effects of UVB-irradiated human keratinocytes HaCaT and IL-1alpha on mitogen activated protein (MAP) kinase activation, c-Jun and c-Fos (AP-1 is composed of Jun and Fos proteins) mRNA expression and MMP-1 production in UVA-irradiated dermal fibroblasts.

METHODSFollowing UVA irradiation, the culture medium of fibroblasts was replaced by culture medium from UVB-irradiated HaCaT, or replaced by the complete culture medium with interleukin (IL)-1alpha. MAP kinase activity expression in fibroblasts was detected by Western blot. c-Jun and c-Fos mRNA expressions were determined by reverse transcriptional polymerase chain reaction (RT-PCR); MMP-1 production in culture medium was detected by enzyme-linked immunosorbent assay (ELISA).

RESULTSCulture medium from UVB-irradiated keratinocytes increased MAP kinase activity and c-Jun mRNA expression in UVA-irradiated fibroblasts. IL-1alpha increased MAP kinase activity and c-Jun mRNA expression, IL-1alpha also increased c-Fos mRNA expression. Both culture media from UVB-irradiated human keratinocytes and externally applied IL-1alpha increased MMP-1 production in UVA-irradiated fibroblasts.

CONCLUSIONSUVB-irradiated keratinocytes and IL-1alpha indirectly promote MMP-1 production in UVA-irradiated fibroblasts by increasing MAP kinase/AP-1 activity. IL-1 may play an important role in the paracrine activation and dermal collagen excessive degradation leading to skin photoaging.

Cell Line ; Enzyme Activation ; Fibroblasts ; enzymology ; radiation effects ; Humans ; Interleukin-1 ; pharmacology ; Keratinocytes ; physiology ; radiation effects ; Matrix Metalloproteinase 1 ; biosynthesis ; Mitogen-Activated Protein Kinases ; metabolism ; Proto-Oncogene Proteins c-fos ; genetics ; Proto-Oncogene Proteins c-jun ; genetics ; RNA, Messenger ; analysis ; Skin ; radiation effects ; Skin Aging ; Transcription Factor AP-1 ; metabolism ; Ultraviolet Rays

Aldosterone has been shown to stimulate renal TGF-beta1 expression. However, the mechanisms for aldosterone-induced TGF-beta1 expression have not been clearly determined in mesangial cells. We examined the role of extracellular-signal regulated kinase 1 and 2 (ERK1/2), c-Jun N-terminal kinase (JNK) and activator protein- 1 (AP-1) in the aldosterone-induced TGF-beta1 expression in rat mesangial cells. TGF-beta1 protein in the conditioned medium released from rat mesangial cells was measured by sandwich ELISA, TGF-beta1 mRNA expression was analyzed by Northern blotting, AP-1 DNA binding activity was measured by EMSA and the ERK1/2, JNK activity was analyzed by western blotting. Aldosterone significantly stimulated TGF-beta1 protein production and TGF-beta1 mRNA expression in mesangial cells in a dose-dependent manner. Aldosterone significantly increased AP-1 DNA binding activity in mesangial cells. Pre-treatment of cells with AP-1 inhibitor, curcumin, blocked aldosterone-induced AP-1 DNA binding activity as well as aldosterone-induced TGF-beta1 production. Aldosterone increased phosphorylation of ERK1/2 and JNK in mesangial cells. Pre-treatment of cells with ERK1/2 inhibitor, PD98059, or JNK inhibitor, SP600125 significantly inhibited aldosterone-induced ERK1/2 and JNK activity and subsequently TGF-beta1 production, respectively. We conclude that aldosteroneinduced TGF-beta1 expression in mesangial cells is regulated by the ERK1/ 2, JNK and AP-1 intracellular signaling pathways.
Aldosterone/*pharmacology ; Animals ; Culture Media, Conditioned/pharmacology ; DNA/metabolism ; Extracellular Signal-Regulated MAP Kinases/metabolism ; *Gene Expression Regulation, Enzymologic ; Humans ; *MAP Kinase Signaling System ; Mesangial Cells/*metabolism ; Models, Biological ; Phosphorylation ; Protein Binding ; Rats ; Transcription Factor AP-1/*metabolism ; Transforming Growth Factor beta1/*biosynthesis

PURPOSE: In the gastric mucosa of Helicobacter pylori (H. pylori)-infected patients with gastritis or adenocarcinoma, proliferation of gastric epithelial cells is increased. Hyperproliferation is related to induction of oncogenes, such as β-catenin and c-myc. Even though transcription factors NF-κB and AP-1 are activated in H. pylori-infected cells, whether NF-κB or AP-1 regulates the expression of β-catenein or c-myc in H. pylori-infected cells has not been clarified. The present study was undertaken to investigate whether H. pylori-induced activation of NF-κB and AP-1 mediates the expression of oncogenes and hyperproliferation of gastric epithelial cells. MATERIALS AND METHODS: Gastric epithelial AGS cells were transiently transfected with mutant genes for IκBα (MAD3) and c-Jun (TAM67) or treated with a specific NF-κB inhibitor caffeic acid phenethyl ester (CAPE) or a selective AP-1 inhibitor SR-11302 to suppress activation of NF-κB or AP-1, respecively. As reference cells, the control vector pcDNA was transfected to the cells. Wild-type cells or transfected cells were cultured with or without H. pylori. RESULTS: H. pylori induced activation of NF-κB and AP-1, cell proliferation, and expression of oncogenes (β-catenein, c-myc) in AGS cells, which was inhibited by transfection of MAD3 and TAM67. Wild-type cells and the cells transfected with pcDNA showed similar activities of NF-κB and AP-1, proliferation, and oncogene expression regardless of treatment with H. pylori. Both CAPE and SR-11302 inhibited cell proliferation and expression of oncogenes in H. pylori-infected cells. CONCLUSION: H. pylori-induced activation of NF-κB and AP-1 regulates transcription of oncogenes and mediates hyperproliferation in gastric epithelial cells.
Blotting, Western ; Caffeic Acids ; Cell Line, Tumor ; Cell Proliferation ; DNA, Bacterial/analysis/genetics ; DNA-Binding Proteins/*metabolism ; Epithelial Cells/*metabolism ; Gastric Mucosa/*metabolism/pathology ; Gastritis/pathology ; Gene Expression Regulation, Bacterial ; Helicobacter Infections/metabolism/pathology/physiopathology ; Helicobacter pylori/pathogenicity/physiology ; Humans ; NF-kappa B/antagonists & inhibitors/*biosynthesis/metabolism ; Peptide Fragments ; Phenylethyl Alcohol/analogs & derivatives ; Proto-Oncogene Proteins c-jun ; Repressor Proteins ; Transcription Factor AP-1/*biosynthesis ; Transcription Factors/*metabolism ; beta Catenin/*metabolism

OBJECTIVETo investigate the inhibitory effects of AP-1 decoy oligodeoxynucleotides (ODNs) on angiotensin II (AngII)-induced proliferation and collagen synthesis in neonatal rat cardiac fibroblasts (CFs).

METHODSThe CFs of neonatal SD rats were cultured in serum-free medium for 24 h and stimulated with 10(-7) mol/L AngII in the presence of AP-1 decoy ODNs or mutational AP-1 decoy ODNs at varied concentrations. MTT assay was employed for quantitative evaluation of the CF proliferation. Collagen synthesis in the CFs was assessed with hydroxyproline, and the cell cycle distribution determined with flow cytometry (FCM).

RESULTSWith the increase of the concentration of AP-1 decoy ODNs, the absorbance at 490 nm (OD490) of the CFs decreased gradually as shown by MTT assay. Treatment with 100 or 200 nmol/L AP-1 decoy ODNs resulted in significantly lowered OD490 of the CFs as compared with that of AngII group. The concentration of hydroxyproline increased significantly after treatment with 10(-7) mol/L AngII in comparison with the control group (P<0.05). Hydroxyproline concentration in cells treated with 100 or 200 nmol/L AP-1 decoy ODNs was significantly lower than that in the 10(-7) mol/L AngII-treated cells. AP-1 decoy ODNs decreased the cell percentage in S phase and increased hydroxyproline concentration, but increased the percentage of cells in G0/G1 phase. AP-1 decoy ODNs at 100 and 200 nmol/L did not obviously affect AngII-induced CF proliferation and collagen synthesis (P<0.01).

CONCLUSIONAP-1 decoy can inhibit AngII-induced rat CF proliferation and collagen synthesis possibly by affecting the cell cycle distribution.

Angiotensin II ; pharmacology ; Animals ; Animals, Newborn ; Cell Cycle ; drug effects ; Cell Proliferation ; drug effects ; Cell Survival ; drug effects ; Cells, Cultured ; Collagen ; biosynthesis ; Dose-Response Relationship, Drug ; Fibroblasts ; cytology ; drug effects ; metabolism ; Flow Cytometry ; Mutation ; Myocardium ; cytology ; metabolism ; Oligodeoxyribonucleotides ; genetics ; pharmacology ; Rats ; Rats, Sprague-Dawley ; Transcription Factor AP-1 ; genetics

DNase I footprinting assay using liver nuclear extracts revealed six protected regions between nucleotide -600 and +110 and hence named Box I-VI. Upstream promoter element (UPE), a DNA element playing crucial role in transcriptional control of the tissue specific expression of pancreatic beta-cell, has been detected within the proximal region of rat GLUT2 promoter. This region is included in Box VI. The protein-DNA interaction in this region (Box VI) was confirmed by mobility shift assay using liver nuclear extracts. Deletion of the region between -585 bp and -146 bp resulted in dramatic changes in promoter activity when they were expressed in liver and beta-cell derived cell line. When -585/-146 construct was expressed in liver, the activity was decreased to 46%, whereas the activity in beta-cell line, HIT-T15 cell, was increased by 84% when compared to -146/+190 construct. These opposing phenomena can be explained by the fact that beta-cell specifically expresses the UPE binding protein. Assuming that there may be Box VI-binding protein playing negative roles both in hepatocyte and beta-cell, and that the protein acts as a negative regulator of GLUT2 gene, the UPE binding protein in the beta-cell may overcome the inhibition by binding to the protein.
Animal ; Binding Sites ; Cell Line ; Comparative Study ; DNA Footprinting ; Deoxyribonuclease I ; Gene Expression Regulation ; Islets of Langerhans/metabolism* ; Islets of Langerhans/cytology ; Liver/metabolism* ; Liver/cytology ; Monosaccharide Transport Proteins/genetics ; Monosaccharide Transport Proteins/biosynthesis* ; Promoter Regions (Genetics)* ; Protein Binding ; Rats ; Transcription Factor AP-1