The expression of the GLUT2 glucose transporter gene in liver is suppressed in cultured hepatoma cell lines and primary cultured hepatocytes. Earlier report showed that CCAAT/enhancer binding protein (C/EBP) regulates the promoter activity of the rat GLUT2 glucose transporter gene in liver cells. C/EBPa and C/EBPb activated the promoter activity by binding to at least two regions of the promoter and one of the C/EBP binding sites, named as site F, also has the AP-1 binding consensus. In this study, we investigated whether the AP-1 can influence on C/EBP binding to this site. The addition of recombinant c-Jun protein with liver extract caused the attenuation of C/EBP binding to site F with the appearance of a new shifted band. The shifted band was competed out with the addition of unlabeled AP-1 consensus oligonucleotide, indicating that c-Jun also can bind to site F. Another C/EBP site on GLUT2 promoter, site H, did not bind AP-1. Analysis of the DNA-protein complex revealed that C/EBP and c-Jun bind to site F in mutually exclusive manner rather than form heterodimeric complex with each other. From these results, it is suggested that the transcriptional activation of C/EBP may be influenced by c-Jun protein in certain status of the liver cells, such as acute phase response, as well as hepatocarcinogenesis.
Animals ; Base Sequence ; Binding Sites ; CCAAT-Enhancer-Binding Proteins/*metabolism ; Cell Nucleus/metabolism ; Cells, Cultured ; Liver/cytology/metabolism ; Male ; Molecular Sequence Data ; Monosaccharide Transport Proteins/*genetics/metabolism ; Promoter Regions (Genetics)/*physiology ; Proto-Oncogene Proteins c-jun/genetics/*metabolism ; Rats ; Rats, Sprague-Dawley ; Recombinant Proteins/genetics/metabolism ; Transcription Factor AP-1/genetics/metabolism

OBJECTIVETo observe activating protein-1 (AP- 1) members in response to changes of wall-shear stress in osteoblastic cells in vitro.

METHODSIsolated and purified osteoblastic cells from the calvaria of newborn SD rats were cultured and subcultured. The third generation cells were subjected to wall-shear stress of 0.8 Pa, 1.2 Pa, 1.4 Pa and 1.6 Pa separately. Gene expression of the seven AP-1 members were studied before (0 h) and 10 min, 15 min, 30 min, 60 min after treated with wall-shear stress.

RESULTSThe expression of FosB, c-Fos, c-Jun, JunD and JunB mRNA increased transiently after application of 1.2 Pa wall-shear stress in osteoblastic cells compared to 0.8 Pa , 1.4 Pa and 1.6 Pa stress, and peaked at 15 min.

CONCLUSIONMechanical environment changes in osteoblastic cells induced a dramatic induction of most of the AP-1 members.

Animals ; Proto-Oncogene Proteins c-fos ; Proto-Oncogene Proteins c-jun ; RNA, Messenger ; Rats ; Rats, Sprague-Dawley ; Stress, Mechanical ; Transcription Factor AP-1

BACKGROUND: High mobility group box 1 (HMGB1) is a novel, late mediator of inflammation. This study compared the pro-inflammatory effects of LPS and HMGB1. The transcriptional factors that play an important role in mediating the HMGB1-induced stimulation of IL-8 were also evaluated. METHODS: RAW264.7 cells were stimulated with either LPS (100 ng/ml) or HMGB1 (500 ng/ml). The TNF-alpha, MIP-2 and IL-1beta levels in the supernatant were evaluated by ELISA at 0, 2, 4, 8, 12 and 24h after stimulation. An acute lung injury was induced by an injection of LPS (5 mg/kg) or HMGB1 (2.5 mg/kg) into the peritoneum of the Balb/c mice. The lung cytokines and MPO activity were measured at 4h (for LPS) or 24h (for HMGB1) after the injection. The transcriptional factor binding sites for NF-IL6, NF-kappaB and AP-1 in the IL-8 promoter region were artificially mutated. Each mutant was ligated with pIL-6luc and transfected into the RAW264.7 cells. One hour after stimulation with HMGB1 (500 ng/ml), the cell lysate was analyzed for the luciferase activity. RESULTS: The expression of MIP-2, which peaked at 8h with LPS stimulation, increased sequentially until 24h after HMGB1 stimulation. An intraperitoneal injection of HMGB1, which induced a minimal increased in IL-1beta expression, provoked the accumulation of neutrophils the lung. A mutation of AP-1 as well as NF-kappaB in the IL-8 promoter region resulted in a lower luciferase activity after HMGB1 stimulation. CONCLUSION: The proinflammatory effects of HMGB1, particularly on IL-8, are mediated by both NF-kappaB and AP-1.
Acute Lung Injury ; Animals ; Binding Sites ; CCAAT-Enhancer-Binding Protein-beta ; Cytokines ; Enzyme-Linked Immunosorbent Assay ; HMGB1 Protein* ; Inflammation ; Injections, Intraperitoneal ; Interleukin-8* ; Luciferases ; Lung ; Mice ; Negotiating ; Neutrophils ; NF-kappa B ; Peritoneum ; Promoter Regions, Genetic ; Transcription Factor AP-1 ; Tumor Necrosis Factor-alpha

OBJECTIVETo investigate whether the decrease in expression of interleukin-2 (IL-2) after trauma is associated with changes in DNA binding activity of nuclear factor of activated T cells (NFAT) and activator protein-1 (AP-1).

METHODSMice with closed impact injury with fracture in both hind limbs were adopted as the trauma model. Spleen lymphocytes were isolated from traumatized mice and stimulated with Con-A. Culture supernatants were assayed for IL-2 activity, and total RNA was extracted from spleen lymphocytes and assayed for IL-2 mRNA. DNA binding activity of NFAT and AP-1 were measured by electrophoretic mobility shift assay (EMSA). The expression of c-Fos, c-Jun and JunB proteins was determined by the Western blot analysis.

RESULTSDNA binding activity of NFAT and AP-1 gradually decreased to a minimum of 41% and 49%, respectively, of the control on the 4th day after injury, which was closely followed by the decline in IL-2 activity and IL-2 mRNA. A decrease in the expression of c-Fos on the 1st and 4th day after trauma had no significant effect on c-Jun expression; the increase in expression of JunB was only on the 1st day after injury.

CONCLUSIONDecreased IL-2 expression is, at least in part, due to a decline in the activation of NFAT and AP-1 in traumatized mice. The decline in DNA binding activity of NFAT and AP-1 is partly due to a trauma-induced block in the expression of c-Fos.

Animals ; Cell Nucleus ; chemistry ; DNA ; metabolism ; DNA-Binding Proteins ; metabolism ; Electrophoretic Mobility Shift Assay ; Female ; Interleukin-2 ; analysis ; genetics ; Male ; Mice ; NFATC Transcription Factors ; Nuclear Proteins ; Proto-Oncogene Proteins c-fos ; analysis ; Proto-Oncogene Proteins c-jun ; analysis ; RNA, Messenger ; analysis ; Transcription Factor AP-1 ; metabolism ; Transcription Factors ; metabolism

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

OBJECTIVETo study the role of activator protein-1 (AP-1) in the up-regulation expression of tumor necrosis factor-alpha (TNF-alpha) and transforming growth factor-beta1(TGF-beta(1)) in silica-stimulated macrophage cells (RAW264.7).

METHODSRAW264.7 cells were treated with AP-1 inhibitor Curcumin. The expression of c-jun and c-fos in nuclear protein was detected by western blotting. The level of TNF-alpha and TGF-beta(1) protein in the cell supernatant was measured using enzyme-linked immunoadsorbent assay (ELISA). Meanwhile the expression of TNF-alpha and TGF-beta(1) mRNA was also monitored by reverse transcriptase-polymerase chain reaction (RT-PCR).

RESULTSThe nucleoprotein expression of c-jun and c-fos in 10 and 20 micromol/L Curcumin prevention group (1.150 +/- 0.020, 1.010 +/- 0.108, 80.430 +/- 0.023, 0.256 +/- 0.015) were lower than those in silica-stimulated group (1.550 +/- 0.029, 0.860 +/- 0.036) (P < 0.01). In 20 micromol/L Curcumin prevention group and silica stimulated group, the expression of TNF-alpha protein were 23.58 +/- 45.78 and 32.12 +/- 5.34, and the expression of TGF-beta(1) protein were 1582.18 +/- 437.52 and 55.60 +/- 5.51 (P < 0.05 =; the expression of TNF-alpha, TGF-beta(1) mRNA were 0.74 +/- 0.01, 0.22 +/- 0.04 and 2.27 +/- 0.33, 2.96 +/- 0.15 (P < 0.05 =.

CONCLUSIONThe expression of TNF-alpha, TGF-beta(1) mRNA and proteins is associated with activation of AP-1 in silica-stimulated macrophage cells.

Animals ; Cell Line ; Curcumin ; pharmacology ; Macrophages ; drug effects ; metabolism ; Mice ; Proto-Oncogene Proteins c-fos ; metabolism ; Proto-Oncogene Proteins c-jun ; metabolism ; RNA, Messenger ; genetics ; Silicon Dioxide ; pharmacology ; Transcription Factor AP-1 ; antagonists & inhibitors ; metabolism ; Transforming Growth Factor beta1 ; metabolism ; Tumor Necrosis Factor-alpha ; metabolism

OBJECTIVETo investigate the role of c-Jun N-terminal kinase (JNK)-c-Jun/activator protein-1 (AP-1) signal pathway in expression of monocyte chemoattractant protein-1 (MCP-1) in experimental rat glomerulonephritis.

METHODSNephrotoxic sera nephritis (NTN) was induced by injection of anti-GBM antibody into the tail veins of rats. Electrophoretic mobility shift assay (EMSA) and non-radioactive kinase assay were used to detect the activity of AP-1 and JNK in kidneys and angiotensin II-stimulated human mesangial cells. Ribonuclear protection assay was used to detect MCP-1 expression in cultured human mesangial cells.

RESULTSSignificant up-regulation of JNK and AP-1 was observed in NTN rats (3.82 +/- 0.58) folds and (5.36 +/- 0.61) folds, as compared with the controls. Supershift assay demonstrated that c-Jun and c-Fos were the predominant subunits involved. Activation of JNK and AP-1 significantly correlated with MCP-1 expression in NTN rats. Angiotensin II enhanced the expression of MCP-1 and activation of JNK and AP-1 in cultured human mesangial cells in a dose-dependent manner, with maximal stimulation seen at 100 nmol/L (20.99 +/- 4.71) folds, (6.91 +/- 1.65) folds and (7.82 +/- 1.32) folds respectively. Significant down-regulation of AP-1 activation and MCP-1 expression were observed in angiotensin II-induced human mesangial cells pretreated with JNK specific inhibitor SP600125.

CONCLUSIONSAngiotensin II and MCP-1 may play an important role in glomerulosclerosis via the JNK-c-Jun/AP-1 signal pathway.

Angiotensin II ; pharmacology ; Animals ; Cells, Cultured ; Chemokine CCL2 ; metabolism ; Glomerular Mesangium ; cytology ; metabolism ; Glomerulonephritis ; metabolism ; Humans ; JNK Mitogen-Activated Protein Kinases ; metabolism ; Male ; Proto-Oncogene Proteins c-fos ; metabolism ; Proto-Oncogene Proteins c-jun ; metabolism ; Rats ; Rats, Sprague-Dawley ; Signal Transduction ; drug effects ; Transcription Factor AP-1 ; metabolism

The heterodimeric c-Jun/c-Fos, an activator protein-1 (AP-1) has been implicated in mesoderm induction (Dong et al., 1996; Kim et al., 1998) whereas the homodimer of c-Jun was reported to be involved in neural inhibition during the early development of Xenopus embryos. During the early vertebrate development AP-1 involvement in the neural induction is still not clearly understood. We report here that AP-1 has a role in Zic3 expression, a critical proneural gene and a primary regulator of neural and neural crest development (Nakata et al., 1997; Nakata et al., 1998). AP-1 was able to induce the Zic3 gene in a dose dependent manner but other homo- or hetero-dimeric proteins, such as c-Jun/c-Jun, JunD/FosB or JunD/Fra-1 were not. The inhibition of AP-1 activity using morpholino antisenses of c-jun mRNAs blocked the Zic3 expression induced by activin. In addition, co-injection of c-jun mRNA rescued the down-regulated Zic3 expression. The promoter region of isolated Zic3 genomic DNA was found to possess several consensus-binding site of AP-1. Thus, in the functional assays, AP-1 could increase promoter activity of Zic3 gene. These findings suggest that proneural gene, Zic3 may be regulated by heterodimeric AP-1(c-Jun/c-Fos) and it may have a role in activin signaling for the regulation of neural specific gene, Zic3.
Activins/pharmacology/physiology ; Animals ; Base Sequence ; Binding Sites/genetics ; Consensus Sequence/genetics ; Dimerization ; Embryo, Nonmammalian/metabolism ; *Gene Expression Regulation, Developmental ; Homeodomain Proteins/*genetics ; Molecular Sequence Data ; Promoter Regions (Genetics)/genetics ; Proto-Oncogene Proteins c-fos/genetics/physiology ; Proto-Oncogene Proteins c-jun/genetics/physiology ; RNA, Antisense/genetics ; Research Support, Non-U.S. Gov't ; Transcription Factor AP-1/genetics/*physiology ; Transcription Factors/*genetics ; *Transcription, Genetic ; Up-Regulation ; Xenopus Proteins/*genetics ; Xenopus laevis/*embryology/*genetics

In the present study, we first examined whether the changes in the DNA binding activities of the transcription factors, cAMP response element binding protein (CREB) and activator protein-1 (AP-1) mediate the long-term effects of morphine in differentiated SH-SY5Y human neuroblastoma cells. The increases in CREB and AP-1 DNA binding activities were time-dependent up to 6 days of morphine treatment (1, 4, and 6 days). However, the significant reduction in the DNA binding activities of CREB and AP-1 was observed after 10 days of chronic morphine (10 muM) administration. Secondly, we examined whether the changes of CREB and AP-1 DNA binding activities could be modulated by dopamine and haloperidol. Dopamine cotreatment moderately increased the levels of the CREB and AP-1 DNA binding activities induced by 10 days of chronic morphine treatment, and haloperidol cotreatment also resulted in a moderate increase of the CREB and AP-1 DNA binding activities. However, dopamine or haloperidol only treatment showed a significant increase or decrease of the CREB and AP-1 DNA binding activities, respectively. In the case of acute morphine treatment, the CREB and AP-1 DNA binding activities were shown to decrease in a time-dependent manner (30, 60, 90, and 120 min). Taken these together, in differentiated SH-SY5Y cells, morphine tolerance seems to involve simultaneous changes of the CREB and AP-1 DNA binding activities. Our data also suggest the possible involvement of haloperidol in prevention or reversal of morphine tolerance at the transcriptional level.
Cyclic AMP Response Element-Binding Protein ; DNA* ; Dopamine* ; Haloperidol* ; Humans* ; Morphine ; Neuroblastoma* ; Transcription Factor AP-1* ; Transcription Factors

To investigate the molecular mechanisms of all-trans retinoic acid (ATRA)-induced rig-g gene expression and to better understand the signal transduction of ATRA during acute promyelocytic leukemia (APL) cell differentiation, the luciferase reporter assay, co-immunoprecipitation and chromatin immunoprecipitation were used to clarify the basic transcriptional factors, which directly initiated the expression of rig-g gene. The results showed that the expression of STAT2, IRF-9 and IRF-1 could be upregulated by ATRA with different kinetics in NB4 cells. IRF-9 was able to interact with STAT2 to form a complex, which could bind the rig-g gene promoter and trigger the rig-g expression. IRF-1 alone could also activate the reporter gene containing rig-g gene promoter, but C/EBPalpha could strongly inhibit this transcription activity of IRF-1. It is concluded that during ATRA-induced APL cell differentiation, IRF-1 is first upregulated by ATRA, and then IRF-1 increases the protein levels of IRF-9 and STAT2 with the downregulation of C/EBPalpha. The complex of IRF-9 and STAT2 is the primary transcriptional factor for rig-g gene induction. This study will be helpful for better understanding the signal transduction networks of ATRA during the course of APL cell differentiation.
CCAAT-Enhancer-Binding Protein-alpha ; metabolism ; Gene Expression Regulation, Leukemic ; drug effects ; Genes, Regulator ; drug effects ; Humans ; Interferon Regulatory Factor-1 ; metabolism ; Interferon-Stimulated Gene Factor 3, gamma Subunit ; metabolism ; Intracellular Signaling Peptides and Proteins ; genetics ; Leukemia, Promyelocytic, Acute ; genetics ; STAT2 Transcription Factor ; metabolism ; Signal Transduction ; Tretinoin ; pharmacology ; Tumor Cells, Cultured