OBJECTIVETo investigate the changes in plasma level of interleukin-13 (IL-13) and the changes in the pulmonary IL-13 mRNA content and the pulmonary activator protein-1 (AP-1) activity of the rats inflicted with acute lung injury (ALI) induced by lipopolysaccharide (LPS), so as to explore the relationship between IL-13 expression and AP-1 activity.

METHODSOne hundred and twenty Wistar rats were employed in the study and were randomly divided into A (2 mg/kg), B (4 mg/kg), C (6 mg/kg) and D (8 mg/kg) groups according to different dosage of LPS administration and a control group (NS group) at each observing time point. The rats were observed at 1, 2, 4 and 6 postburn hours (PBHs) and every 6 rats were deployed in every group and each time points. A model of systemic inflammatory response syndrome-acute lung injury (SIRS-ALI) was replicated in Wistar rats. The plasma content of IL-13 was assayed by enzyme-linked immunosorbent assay (ELISA), and the pulmonary tissue content of IL-13 mRNA and AP-1 activity by reverse transcriptase-polymerase chain reaction (RT-PCR) and electrophoretic mobility shift assays (EMSA).

RESULTSThe plasma content of IL-13, pulmonary content of IL-13 mRNA and AP-1 activity increased simultaneously after LPS administration. All the above indices were significantly different statistically between the LPS groups and the control group (P < 0.05 - 0.01). The plasma level of IL-13 and pulmonary tissue mRNA content and AP-1 activity in A, B, C and D groups were increased significantly with peak levels at 2 PBHs.

CONCLUSIONThe pulmonary AP-1 activity increased with the enhanced expression of IL-13, which was related to the development of SIRS-ALI.

Acute Lung Injury ; metabolism ; Animals ; Endotoxins ; toxicity ; Female ; Interleukin-13 ; blood ; genetics ; physiology ; Lung ; chemistry ; Male ; Rats ; Rats, Wistar ; Transcription Factor AP-1 ; analysis ; physiology

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

BACKGROUNDHow the transcriptional factors regulated the innate and adaptive immune system in pregnancy and pre-eclampsia are less understood. Nevertheless, what the plasma work in the development of this disease was not sure. The present study was design to evaluate what the transcriptional factors change in innate and adaptive immune system and what the plasma do in this filed.

METHODSPeripheral blood mononuclear cells (PBMC) from non-pregnant women (n = 18), women with clinically normal pregnancies (n = 23) and women with pre-eclampsia (n = 20) were separated from peripheral blood to isolate monocytes and T cells. The purity of monocytes and T cells were analysed by flow cytometry. Monocytes and T cells were stimulated in either lipopolysaccharides (LPS) or phorbol-myristate-acetate (PMA), respectively. Transcription Factor Arrays were used to screen the transcription factors of interest in comparing of different groups. PBMC were isolated from another 8 non-pregnant samples were co-incubated with different groups of plasma. Polymerase chain reaction (PCR) was performed using whole cell extractions of the samples.

RESULTSNuclear factor of activated T-cells-1 (NFAT-1), signal transducers and activators of transcription-1 (STAT-1) and activator protein-1 (AP-1) are up-regulated in monocytes in pregnancy and more so in pre-eclampsia. On the the contrary, NFAT-1, STAT-1 and AP-1 are down-regulated in T cells in pregnancy and more so in pre-eclampsia. A reduction was observed in interferon (IFN)-γ, interleukin (IL)-12 and IL-4 expression in T cells incubated with pre-eclamptic plasma. An elevation was observed in tumor necrosis factor (TNF)-α, IL-1 and IL-12 expression in monocytes incubated with pre-eclamptic plasma.

CONCLUSIONSInnate immunity is over activated and adaptive immunity is over suppressed in the development of pre-eclampsia. NFAT-1, STAT-1 and AP-1 might be the central transcription factors in the pathogenesis of pre-eclampsia. They induced some changes in plasma and "educate" the monocytes and T cells for relevant cytokine production. Successful completion of this study will enhance our understanding of pre-eclampsia and will discover new knowledge beyond pregnancy. The work will inform future therapies for the treatment of a wide range of condition such as transplantation immunology and a wide range of immune and inflammatory conditions.

Adult ; Female ; Humans ; Immunity, Innate ; physiology ; Interferon-gamma ; metabolism ; Interleukin-12 ; metabolism ; Interleukin-4 ; metabolism ; Leukocytes, Mononuclear ; metabolism ; Male ; NFATC Transcription Factors ; genetics ; metabolism ; Pre-Eclampsia ; immunology ; metabolism ; Pregnancy ; STAT1 Transcription Factor ; genetics ; metabolism ; Transcription Factor AP-1 ; genetics ; metabolism ; Transcription Factors ; genetics ; metabolism ; Young Adult

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

To detect effects of B7 co-stimulation on cytokines, especially on IL-2 mRNA and transcription factors NF-kappa B and AP-1, antiB7-1 McAb, antiB7-2 McAb and C TLA-4 Ig were added into mixture lymphocyte reaction (MLR) system to block B7/C D28 signal transduction, IL-2 mRNA and IL-4 mRNA were determined by using competitive PCR and IFN-gamma mRNA by using semi-quantitative PCR. MHC class II molecules and B7 transfectants were used to stimulate CD28(+) T cell, effects of B7 on NF-kappa B and AP-1 were detected by DNA-protein binding assay. The results showed that IL-2, IL-4 and IFN-gamma mRNA were significantly lower when blockade of B7-2 in MLR than blockade of B7-1. Synergistic effects could be seen with combination of two or three antibodies. One to six hours after MLR, tDR7 alone induced NF-kappa B binding activity; cotransfecting B7 no significantly difference at early time point. After 6 hours, induction of tDR7 was decreased whereas B7 prolonged the induction of NF-kappa B till 72 hours. tDR7 alone also upregulated AP-1 binding activity, on the contrary to NF-kappa B, co-transfecting B7-1 and B7-2 decreased AP-1 binding activity within 24 hours. But during 48 - 72 hours, B7 also prolonged the AP-1 binding activity. It is concluded that after MLR, B 7 increased IL-2 secretion by decreasing the degradation of IL-2 mRNA and upregulating IL-2 transcription factors. B7 also induced several kinds of cytokines secretion. Effects of B7-1 and B7-2 had no significant difference on transcription factors. It is suggested that the different functions between B7-1 and B7-2 maybe related to the difference of cell expression and kinetics. To study the molecular mechanism of B7 mediated T cell immune tolerance can help us to design a better clinic schema to prevent transplantation rejection and GVHD.
3T3 Cells ; Animals ; Antigens, CD ; physiology ; B7-1 Antigen ; physiology ; B7-2 Antigen ; CD28 Antigens ; physiology ; Gene Expression Regulation ; Humans ; Interferon-gamma ; genetics ; Interleukin-2 ; genetics ; Interleukin-4 ; genetics ; Lymphocyte Culture Test, Mixed ; Membrane Glycoproteins ; physiology ; Mice ; NF-kappa B ; metabolism ; RNA, Messenger ; analysis ; Transcription Factor AP-1 ; metabolism

Oxidative stress is critical for causing cardiac injuries during ischemia-reperfusion (IR), yet the molecular mechanism for this remains unclear. In the present study, we observe that hypoxia and reoxygenation, a component of ischemia, effectively induces apoptosis in the cardiac myocytes from neonatal rats and it concomitantly leads to induction of GADD153, an apoptosis-related gene. Furthermore, IR injury of rat heart showed a GADD153 overexpression in the ischemic area where the TUNEL reaction was positive. A downregulation of cardiac ankyrin repeat protein (CARP) was also observed in this ischemic area. Promoter deletion and reporter analysis revealed that hypoxia transcriptionally activates a GADD153 promoter through the AP-1 element in neonatal cardiomyocytes. Ectopic overexpression of GADD153 resulted in the downregulation of CARP expression. Accordingly, the induction of GADD153 mRNA were followed by the CARP down-regulation in an in vivo rat coronary ischemia/reperfusion injury model. These results suggest that GADD153 over-expression and the resulting downregulation of CARP may have causative roles in apoptotic cell death during cardiac IR injury.
Animals ; Animals, Newborn ; Anoxia ; Apoptosis/physiology ; Cells, Cultured ; Humans ; Male ; *Myocardial Reperfusion Injury/metabolism/pathology ; *Myocardium/metabolism/pathology ; Myocytes, Cardiac/cytology/metabolism ; Nuclear Proteins/genetics/*metabolism ; Promoter Regions, Genetic ; Rats ; Rats, Sprague-Dawley ; Repressor Proteins/genetics/*metabolism ; Transcription Factor AP-1/genetics/metabolism ; Transcription Factor CHOP/genetics/*metabolism

The equilibrium between deposition and degradation of extracellular matrix(ECM) is essential to normal tissue development and repair of wound or inflammatory responses. It has recently become apparent that several cytokines and growth factors are capable of modulating fibroblast proliferation and biosynthetic activity. To understand the role of these factors in connective tissue regulation, we examined the effect of interferon-gamma (IFN-gamma) on stromelysin-1 gene expression in cultured human dermal fibroblasts. The steady-state levels of stromelysin-1 mRNA were increased in IFN-gamma treated cultured dermal fibroblasts. In the CAT assay, the stromelysin-1 promoter activity was increased 2.8-fold compared with untreated control. Therefore IFN-gamma stimulates the stromelysin-1 promoter activity, resulting in transcriptional enhancement of gene expression. Transforming growth factor-beta (TGF-beta) showed the antagonistic action to the effects of IFN-gamma in cultured dermal fibroblasts. Furthermore, gel mobility shift assays demonstrated enhanced AP-1 binding activities in nuclear extracts from cells incubated with IFN-gamma. These data suggest that IFN-gamma is an up-regulator and TGF-beta is a down regulator on the stromelysin-1 gene expression, respectively, and the AP-1 binding site may be necessary for gene response.
Cell Nucleus ; Cells, Cultured ; Chloramphenicol O-Acetyltransferase/metabolism ; Chloramphenicol O-Acetyltransferase/genetics ; Collagenases/genetics ; Collagenases/drug effects ; Fibroblasts/metabolism ; Fibroblasts/drug effects* ; Gene Expression Regulation/drug effects ; Human ; Interferon Type II/pharmacology* ; Promoter Regions (Genetics) ; Recombinant Proteins/metabolism ; Recombinant Proteins/genetics ; Skin/cytology* ; Stromelysin 1/metabolism* ; Stromelysin 1/genetics* ; Stromelysin 1/drug effects ; Transcription Factor AP-1/metabolism ; Transcription, Genetic ; Transforming Growth Factor beta/pharmacology ; Up-Regulation (Physiology)

Using forward and reverse genetics and global gene expression analyses, we explored the crosstalk between the IκB kinase β (IKKβ) and the transforming growth factor β (TGFβ) signaling pathways. We show that in vitro ablation of Ikkβ in fibroblasts led to progressive ROS accumulation and TGFβ activation, and ultimately accelerated cell migration, fibroblast-myofibroblast transformation and senescence. Mechanistically, the basal IKKβ activity was required for anti-oxidant gene expression and redox homeostasis. Lacking this activity, IKKβ-null cells showed ROS accumulation and activation of stress-sensitive transcription factor AP-1/c-Jun. AP-1/c-Jun activation led to up-regulation of the Tgfβ2 promoter, which in turn further potentiated intracellular ROS through the induction of NADPH oxidase (NOX). These data suggest that by blocking the autocrine amplification of a ROS-TGFβ loop IKKβ plays a crucial role in the prevention of fibroblast-myofibroblast transformation and senescence.
Adenoviridae ; genetics ; Animals ; Autocrine Communication ; physiology ; Cell Line ; Cell Movement ; Cellular Senescence ; Genetic Vectors ; genetics ; metabolism ; I-kappa B Kinase ; deficiency ; genetics ; metabolism ; JNK Mitogen-Activated Protein Kinases ; metabolism ; Mice ; Myofibroblasts ; cytology ; metabolism ; NADPH Oxidases ; metabolism ; Oxidative Stress ; Promoter Regions, Genetic ; Reactive Oxygen Species ; metabolism ; Signal Transduction ; Superoxide Dismutase ; genetics ; metabolism ; Transcription Factor AP-1 ; metabolism ; Transforming Growth Factor beta ; genetics ; metabolism ; Up-Regulation

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

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