AP-1 Binding Activity Research Articles

Reduced AP-1-mediated transcription of interleukin-11 gene in marrow stromal cells as a mechanism of senile osteoporosis: lessons from SAMP6

SAMP6 is a substrain of senescence-accelerated mice (SAM) that exhibits decreased bone mass due to impaired bone formation. Bipotential bone marrow stromal cells from SAMP6 showed enhanced adipogenesis and suppressed osteoblastogenesis. These abnormalities of stromal cells were cured by addition of an anti-adipogenic cytokine, interleukin (IL)-11, expression of which was decreased in SAMP6. We further demonstrated that IL-11 transcription was largely dependent on AP-1 transcription factors and that DNA binding activity of AP-1, especially Jun D, was reduced in SAMP6 marrow stromal cells. These results suggest that reduced AP-1-mediated transcription of IL-11 is a mechanism causing impaired bone formation in SAMP6. Interestingly, reduced AP-1 activity and a resultant decrease in IL-11 expression was also observed in aged mice of ordinary ICR strain. Moreover, transgenic mice over-expressing IL-11 showed increased bone mass due to enhanced bone formation and were protected from aging-associated bone loss. Based on these findings, we propose that IL-11 is a physiological stimulator of bone formation and that the aging-associated decline in stromal expression of IL-11 may be a general mechanism of impaired bone formation in the aged.

Molecular Basis for the Direct Inhibition of AP-1 DNA Binding by 15-Deoxy-Δ12,14-prostaglandin J2

Cyclopentenone prostaglandins may interfere with cellular functions by multiple mechanisms. The cyclopentenone 15-deoxy-Delta 12,14-prostaglandin J2 (15d-PGJ2) has been reported to inhibit the activity of the transcription factor AP-1 in several experimental settings. We have explored the possibility of a direct interaction of 15d-PGJ2 with AP-1 proteins. Here we show that 15d-PGJ2 covalently modifies c-Jun and directly inhibits the DNA binding activity of AP-1. The modification of c-Jun occurs both in vitro and in intact cells as detected by labeling with biotinylated 15d-PGJ2 and mass spectrometry analysis. Attachment of the cyclopentenone prostaglandin occurs at cysteine 269, which is located in the c-Jun DNA binding domain. In addition, 15d-PGJ2 can promote the oligomerization of a fraction of c-Jun through the formation of intermolecular disulfide bonds or 15d-PGJ2-bonded dimers. Our results identify a novel site of interaction of 15d-PGJ2 with the AP-1 activation pathway that may contribute to the complex effects of cyclopentenone prostaglandins on the cellular response to pro-inflammatory agents. They also show the first evidence for the induction of protein cross-linking by 15d-PGJ2.

Glycoprotein isolated from Ulmus davidiana Nakai inhibits TPA-induced apoptosis through nuclear factor-kappa B in NIH/3T3 cells

Glycoprotein of Ulmus davidiana Nakai (UDN glycoprotein) was isolated and identified using SDS-PAGE. UDN glycoprotein was shown to have strong scavenging activities against oxygen free radicals, as detected by different oxygen-radical formation assays. To investigate the anti-apoptotic effects of UDN glycoprotein, we investigated the activity of protein kinase C alpha (PKCα), the DNA-binding activation of nuclear factor-kappa B (NF-κB), the production of nitric oxide (NO) and apoptosis in 12- O-tetradecanoylphorbol 13-acetate (TPA)-stimulated NIH/3T3 cells using a western blot analysis, electrophoretic mobility shift assays (EMSA) and NO assays. Results in this experiment showed that 100 μg/ml of UDN glycoprotein has inhibitory effects on PKCα translocation, NF-κB DNA binding activity, NO production, and apoptosis in TPA (61.68 ng/ml)-stimulated NIH/3T3 cells. Interestingly however, it could not regulate the DNA binding activity of AP-1. Therefore, UDN glycoprotein, a natural anti-oxidant, is a potential modulator of apoptotic signal pathways in NIH/3T3 cells.

Zinc Modulates PPARγ Signaling and Activation of Porcine Endothelial Cells

Dietary zinc has potent antioxidant and anti-inflammatory properties and is a critical component of peroxisome proliferator-activated receptor (PPAR) gene expression and regulation. To assess the protective mechanisms of PPARgamma in endothelial cell dysfunction and the role of zinc in the modulation of PPARgamma signaling, cultured porcine pulmonary artery endothelial cells were exposed to the membrane-permeable zinc chelator N,N,N'N'-tetrakis (2-pyridylmethyl)-ethylene diamine (TPEN), thiazolidinedione (TZD; PPARgamma agonist) or bisphenol A diglycidyl ether (BADGE; PPARgamma antagonist). Subsequently, endothelial cells were activated by treatment with linoleic acid (90 micro mol/L) for 6 h. Zinc chelation by TPEN increased the DNA binding activity of nuclear factor (NF)-kappaB and activator protein (AP)-1, decreased PPARgamma expression and activation as well as up-regulated interleukin (IL)-6 expression and production. These effects were fully reversed by zinc supplementation. In addition, exposure to TZD down-regulated linoleic acid-induced DNA binding activity of NF-kappaB and AP-1, whereas BADGE further induced activation of these oxidative stress-sensitive transcription factors. Most importantly, the TZD-mediated down-regulation of NF-kappaB and AP-1 and reduced inflammatory response were impaired during zinc chelation. These data suggest that zinc plays a critical role in PPARgamma signaling in linoleic acid-induced endothelial cell activation and indicate that PPARgamma signaling is impaired during zinc deficiency.

Cyclosporin A enhances interleukin-8 expression by inducing activator protein-1 in human aortic smooth muscle cells.

Cyclosporin A (CsA) and tacrolimus (FK506) are widely used as immunosuppressants. However, their use has been hampered by various adverse effects, such as acceleration of atherosclerosis. Interleukin (IL)-8, a chemotactic cytokine, plays an important role in pathogenesis of atherosclerosis. We thus investigated whether synthesis of IL-8 from primary human aortic smooth muscle cells is influenced by CsA and FK506. Northern blot analysis revealed that CsA increased IL-8 mRNA level and enhanced its increase by epidermal growth factor or tumor necrosis factor-alpha. In contrast, FK506 had no effect on the mRNA level. IL-8 accumulation in culture media was also increased by CsA. Stability of IL-8 mRNA was not affected by CsA, whereas luciferase reporter gene assay using the human IL-8 promoter revealed that CsA significantly augmented the promoter activity. Electrophoretic mobility shift assay showed that binding activity of activator protein (AP)-1 was increased by CsA, and introduction of a mutation into the AP-1 site in the promoter abolished its CsA-dependent activation. The increased AP-1 binding activity was accompanied by c-Fos synthesis. CsA stimulates synthesis of IL-8 via activation of AP-1 in human aortic smooth muscle cells, providing a novel aspect of biological effects of CsA on the cells.

Hepatitis C Virus and HIV Envelope Proteins Collaboratively Mediate Interleukin-8 Secretion through Activation of p38 MAP Kinase and SHP2 in Hepatocytes

Hepatitis C virus (HCV) infects approximately 40% of human immunodeficiency virus (HIV) patients, and the resulting hepatic dysfunction that occurs is the primary cause of death in patients with co-infection. We hypothesized that hepatocytes exposed to HCV and HIV proteins might be susceptible to injury via an "innocent bystander" mechanism. To assess this, we studied the effects of envelope proteins, E2 of HCV and gp120 of HIV, in model HepG2 cells. Upon co-stimulation with HCV-E2 and HIV-gp120, we observed a potent proinflammatory response with the induction of IL-8. Furthermore, our studies revealed that HCV-E2 and HIV-gp120 act collaboratively to trigger a specific set of downstream signaling pathways that include activation of p38 mitogen-activated protein (MAP) kinase and the tyrosine phosphatase, SHP2. Both specific inhibitors of p38 MAP kinase and sodium vanadate, a potent protein-tyrosine phosphatase inhibitor, blocked IL-8 production in a dose-dependent manner. The role of p38 MAP kinase and SHP2 was further defined by transiently overexpressing dominant negative mutants of these proteins into HepG2 cells. These studies revealed that overexpression of an inactive p38 MAP kinase or SHP2 mutant partially abrogated HCV-E2- and HIV-gp120-induced IL-8 production. Further studies revealed that IL-8 induction was not mediated through activation of the NF-kappa B pathway. However, HCV-E2 plus HIV-gp120 was shown to increase the DNA binding activity of AP-1. These results emphasize that expression of the proinflammatory chemokine IL-8, induced by HCV-E2 and HIV-gp120, may be mediated through p38 MAP kinase and SHP2 in an NF-kappa B-independent manner, albeit through AP-1-driven processes.

Hypoxia increases AP-1 binding activity by enhancing capacitative Ca2+ entry in human pulmonary artery endothelial cells.

Activating protein (AP)-1 transcription factors modulate expression of genes involved in cell proliferation and migration. Chronic hypoxia increases pulmonary artery smooth muscle cell proliferation by upregulating AP-1-responsive genes encoding for endothelium-derived vasoactive and mitogenic factors implicated in pulmonary hypertension development. The expression of AP-1 transcription factors is sensitive to changes in cytosolic free [Ca2+] ([Ca2+]cyt). Capacitative Ca2+ entry (CCE) via store-operated Ca2+ channels (SOC) is an important mechanism for raising [Ca2+]cyt in pulmonary artery endothelial cells (PAEC). Using combined molecular biological, fluorescence microscopy, and biophysical approaches, we examined the effect of chronic hypoxia (3% O2, 72 h) on AP-1 DNA binding activity, CCE, and transient receptor potential (TRP) gene expression in human (h) PAEC. EMSA showed that AP-1 binding to hPAEC nuclear protein extracts was significantly enhanced by hypoxia, the increase being dependent on store-operated Ca2+ influx and sensitive to La3+, an SOC inhibitor. Hypoxia also increased basal [Ca2+]cyt, the amount of CCE produced by store depletion with cyclopiazonic acid, and the amplitude of SOC-mediated currents (ISOC). The increases of CCE amplitude and ISOC current density by hypoxia were paralleled by enhanced TRPC4 mRNA and protein expression. Hypoxia-enhanced CCE and TRPC4 expression were also attenuated by La3+. These data suggest that hypoxia increases AP-1 binding activity by enhancing Ca2+ influx via La3+-sensitive TRP-encoded SOC channels in hPAEC. The Ca2+-mediated increase in AP-1 binding may play an important role in upregulating AP-1-responsive gene expression, in stimulating pulmonary vascular cell proliferation and, ultimately, in pulmonary vascular remodeling in patients with hypoxia-mediated pulmonary hypertension.

Induction of matrix metalloproteinases-14 and -2 by cyclical mechanical stretch is mediated by tumor necrosis factor-alpha in cultured human umbilical vein endothelial cells.

Mechanical forces have profound effects on endothelial cells. This study was undertaken to examine the hypothesis that tumor necrosis factor-alpha (TNF-alpha) is a potential mediator of stretch-induced effects on matrix metalloproteinase (MMP). Human umbilical vein endothelial cells (HUVECs) grown on a flexible membrane base were stretched by vacuum to 20% of maximum elongation, at 60 cycles/min. We used the TNF-alpha monoclonal antibody and c-Jun N-terminal kinase (JNK) inhibitor, SP600125, to investigate the cyclical stretch-induced expression of MMP-14 and -2 in cultured HUVECs. Cyclical mechanical stretch significantly increased protein synthesis and mRNA expression for MMP-14 and -2 from 2 to 24 h. The increased MMP-14 and-2 proteins after stretch were completely blocked after the addition of TNF-alpha monoclonal antibody (5 microg/ml) or SP600125 (20 microM) 30 min before stretch. By zymography, MMP-2 expression was induced by cyclical stretch and was attenuated by TNF-alpha monoclonal antibody and SP600125. Cyclical stretch increased the immunohistochemical labeling of MMP-14 and -2 and significantly increased release of TNF-alpha into the culture media from 120+/-2 to 331+/-2 pg/ml (P<0.001) after stretch for 12 h. Cyclical stretch increased and SP600125 decreased the phosphorylated JNK. Gel-shifting assay showed that DNA-protein binding activity of AP-1 increased after cyclical stretch and TNF-alpha monoclonal antibody and SP600125 abolished the binding activity induced by cyclical stretch. These findings indicate that cyclical stretch augments TNF-alpha production and MMP genes expression in HUVECs. TNF-alpha mediates the stretch-induced MMP genes expression, at least in part, through the JNK pathway.

Hydrogen peroxide mediates arsenite activation of p70 s6k and extracellular signal-regulated kinase

To define the mechanism of arsenite-induced tumor promotion, we examined the role of reactive oxygen species (ROS) in the signaling pathways of cells exposed to arsenite. Arsenite treatment resulted in the persistent activation of p70 s6k and extracellular signal-regulated kinase 1/2 (ERK1/2) which was accompanied by an increase in intracellular ROS production. The predominant produced appeared to be H 2O 2, because the arsenite-induced increase in dichlorofluorescein (DCF) fluorescence was completely abolished by pretreatment with catalase but not with heat-inactivated catalase. Elimination of H 2O 2 by catalase or N-acetyl- l-cysteine inhibited the arsenite-induced activation of p70 s6k and ERK1/2, indicating the possible role of H 2O 2 in the arsenite activation of the p70 s6k and the ERK1/2 signaling pathways. A specific inhibitor of p70 s6k, rapamycin, and calcium chelators significantly blocked the activation of p70 s6k induced by arsenite. While the phosphatidylinositol 3-kinase (PI3K) inhibitors wortmannin and LY294002 completely abrogated arsenite activation of p70 s6k, ERK1/2 activation by arsenite was not affected by these inhibitors, indicating that H 2O 2 might act as an upstream molecule of PI3K as well as ERK1/2. Consistent with these results, none of the inhibitors impaired H 2O 2 production by arsenite. DNA binding activity of AP-1, downstream of ERK1/2, was also inhibited by catalase, N-acetyl- l-cysteine, and the MEK inhibitor PD98059, which significantly blocked arsenite activation of ERK1/2. Taken together, these studies provide insight into mechanisms of arsenite-induced tumor promotion and suggest that H 2O 2 plays a critical role in tumor promotion by arsenite through activation of the ERK1/2 and p70 s6k signaling pathways.

Essential role for IKKgamma/NEMO in TCR-induced IL-2 expression in Jurkat T cells.

The control of IL-2 gene expression in T cells by multiple transcriptional factors has been extensively explored, however, the role of the NF-kappaB signaling pathway in TCR-dependent IL-2 production still remains unclear. In this study, we used a somatic cell genetics approach to address this question. Triggering TCR in mutant Jurkat T cells lacking IKKgamma/NEMO failed to induce IL-2 due to a selective loss in I-kappaB kinase activity, I-kappaBalpha degradation and NF-kappaB DNA-binding activity. The AP-1 and NF-AT binding activities in the IL-2 promoter were comparable between wild-type and mutant T cells. These defects in the mutant cell line were rescued by the reintroduction of exogenous IKKgamma. Taken together, our data demonstrate that IKKgamma plays an essential role in TCR-induced signaling pathways leading to IL-2 expression.

Suppression of IL-8 gene transcription by resveratrol in phorbol ester treated human monocytic cells

Resveratrol (3,4′,5-trihydroxy-trans-stilbene), a natural phytoalexin found in grapes and other food products, has promising anti-inflammatory and anticancer effects. To observe the modulation of interleukin-8 (IL-8) production in human monocytic cells by resveratrol and explore its mechanism at the gene transcription level, U937 cells were stimulated with phorbol 12-myristate 13-acetate (PMA) for 24 h. IL-8 protein in supernatants was measured by radioimmunoassay. The cytotoxicity of PMA, dexamethasone and resveratrol was accessed by MTT cell proliferation assay. The RNA level of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and IL-8 were detected by RT-PCR using specific primers. DNA binding activities of NF-κB and AP-1 were examined by electrophoretic mobility shift assay (EMSA). 0.01–100 nM PMA could significantly induce IL-8 production in U937 cells; 10 μM Dexamethasone and 10, 1, 0.1 μM resveratrol could inhibit PMA-induced IL-8 protein production and mRNA accumulation. The cytotoxicity did not contribute to their inhibitory effect. The DNA binding activity of AP-1 was inhibited by dexamethasone and resveratrol, but resveratrol has little effect on PMA-induced NF-κB activation. Resveratrol could inhibit PMA-induced IL-8 production in U937 cells at protein and mRNA levels. The suppression of IL-8 gene transcription by resveratrol was, at least partly, due to inhibition of AP-1 activation.

Mediation of interleukin-1beta-induced transforming growth factor beta1 expression by activator protein 4 transcription factor in primary cultures of bovine articular chondrocytes: possible cooperation with activator protein 1.

Interleukin-1 (IL-1) and transforming growth factor beta1 (TGFbeta1) play major roles in osteoarticular diseases, exerting opposite effects on both the catabolism and anabolism of cartilage matrix. Previous findings suggest that IL-1 and TGFbeta1 could function in a feedback interaction. However, the effect exerted by IL-1 on expression of TGFbeta by articular chondrocytes is, so far, poorly understood. The present study was carried out to determine the influence of IL-1beta on the expression of TGFbeta1 by bovine articular chondrocytes (BACs) in primary culture. BAC primary cultures were treated with IL-1beta, and TGFbeta1 messenger RNA (mRNA) steady-state levels and protein expression were measured by real-time reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. Transient transfection of TGFbeta1 gene promoter constructs was performed to delineate the DNA sequences that mediate the IL-1beta effect. Electrophoretic mobility shift assays (EMSAs) and supershift analysis were used to characterize the transcription factors binding to these sequences. Cultured BACs responded to IL-1beta exposure by exhibiting an increase of TGFbeta1 expression at both the mRNA and protein levels. The effect was found to be mediated by a major 80-bp sequence located between -732 and -652 upstream of the transcription initiation site. EMSA and supershift analysis revealed that the transcription factors activator protein 4 (AP-4) and AP-1 specifically bound to the -720/-696 part of this sequence under IL-1beta treatment. Overexpression of AP-4 in the BAC cultures resulted in stimulation of the transcriptional activity of the -732/+11 TGFbeta1 promoter construct through the same IL-1beta-responsive element. IL-1beta induces an increase of TGFbeta1 in articular chondrocytes through activation of AP-4 and AP-1 binding to the TGFbeta1 gene promoter. These findings may help us understand the role of IL-1beta in the disease process. Notwithstanding its deleterious effect on cartilage, IL-1 could initiate the repair response displayed by injured cartilage in the early stages of osteoarthritis through its ability to enhance TGFbeta1 expression by local chondrocytes.

Activation of c-Jun-N-terminal-kinase is crucial for the induction of a cell cycle arrest in human colon carcinoma cells caused by flurbiprofen enantiomers.

The unselective cyclooxygenase (COX) inhibitor S-flurbiprofen and its-in terms of COX-inhibition-"inactive" enantiomer R-flurbiprofen have been previously found to inhibit tumor development and growth in various animal models. The underlying mechanisms are unknown. Here, we show that both R- and S-flurbiprofen reduce survival of three colon cancer cell lines, which differ in the expression of COX-2 (HCT-15, no COX-2; Caco-2, inducible COX-2; and HT-29, constitutive COX-2). The IC50 for S- and R-flurbiprofen ranged from 250 to 450 microM. Both flurbiprofen enantiomers induced apoptosis in all three cell lines as indicated by DNA- and PARP-cleavage. In addition, R- and S-flurbiprofen caused a G1-cell cycle block. The latter was associated with an activation of c-Jun N-terminal kinase (JNK), an increase of the DNA binding activity of the transcription factor AP-1 and down-regulation of cyclin D1 expression. Western blot analysis, as well as supershift experiments, revealed that the AP-1 activation was associated with a change of AP-1 composition toward an increase of JunB. The JNK inhibitor SP600125 antagonized R- and S-flurbiprofen-induced AP-1 DNA binding, suppression of cyclin D1 expression, and the G1-cell cycle block. However, JNK inhibition had no effect on flurbiprofen-induced apoptosis. Hence, the cell cycle arrest is obviously mediated, at least in part, through JNK-activation, whereas R- and S-flurbiprofen-induced apoptosis is largely independent of JNK. Although in vitro effects of R- and S-flurbiprofen were indistinguishable, only R-flurbiprofen inhibited HCT-15 tumor growth in nude mice, suggesting the involvement of additional in vivo targets, which are differently affected by R- and S-flurbiprofen.

Amyloid peptide-induced cytokine and chemokine expression in THP-1 monocytes is blocked by small inhibitory RNA duplexes for early growth response-1 messenger RNA.

In Alzheimer's disease (AD) one finds increased deposition of A beta and also an increased presence of monocytes/macrophages in the vessel wall and activated microglial cells in the brain. AD patients show increased levels of proinflammatory cytokines by activated microglia. Here we used a human monocytic THP-1 cell line as a model for microglia to delineate the cellular signaling mechanism involved in amyloid peptides (A beta(1-40) and A beta(1-42))-induced expression of inflammatory cytokines and chemokines. We observed that A beta peptides at physiological concentrations (125 nM) increased mRNA expression of cytokines (TNF-alpha, and IL-1 beta) and chemokines (monocyte chemoattractant protein-1 (MCP-1), IL-8, and macrophage inflammatory protein-1 beta (MIP-1 beta)). The cellular signaling involved activation of c-Raf, extracellular signal-regulated kinase-1 (ERK-1)/ERK-2, and c-Jun N-terminal kinase, but not p38 mitogen-activated protein kinase. This is further supported by the data showing that A beta causes phosphorylation of ERK-1/ERK-2, which, in turn, activates Elk-1. Furthermore, A beta mediated a time-dependent increase in DNA binding activity of early growth response-1 (Egr-1) and AP-1, but not of NF-kappa B and CREB. Moreover, A beta-induced Egr-1 DNA binding activity was reduced >60% in THP-1 cells transfected with small interfering RNA duplexes for Egr-1 mRNA. We show that A beta-induced expression of TNF-alpha, IL-1 beta, MCP-1, IL-8, and MIP-1 beta was abrogated in Egr-1 small inhibitory RNA-transfected cells. Our results indicate that A beta-induced expression of cytokines (TNF-alpha and IL-1 beta) and chemokines (MCP-1, IL-8, and MIP-1 beta) in THP-1 monocytes involves activation of ERK-1/ERK-2 and downstream activation of Egr-1. The inhibition of Egr-1 by Egr-1 small inhibitory RNA may represent a potential therapeutic target to ameliorate the inflammation and progression of AD.

CCAAT/Enhancer-binding Protein and Activator Protein-1 Transcription Factors Regulate the Expression of Interleukin-8 through the Mitogen-activated Protein Kinase Pathways in Response to Mechanical Stretch of Human Airway Smooth Muscle Cells

Here we investigated the mechanisms by which mechanical stretch regulates the production of IL-8 in primary human airway smooth muscle cells (HASMC). Bronchial HASMC were subjected to cyclic mechanical stretch (12%, 1 Hz) using the computer-controlled Flexcell Strain system. Mechanical stretch increased IL-8 mRNA expression and protein production. Cyclic stretch of HASMC also increased the kinase activities of ERK1/2, JNK1, p38, and the DNA binding activities of AP-1 and C/EBP transcription factors with little effect on NF-kappa B. The inhibition of AP-1 and C/EBP transcriptional activities blocked the production of IL-8 in culture supernatants. Furthermore, the inhibition of ERK1/2 and p38 but not JNK1 caused a significant down-regulation in the expression and production of IL-8 in response to cyclic stretch. Although protein tyrosine kinases were required for the activation of both ERK1/2 and p38 kinase, stretch-activated channels, small GTPase proteins, and extracellular Ca2+ influx were required only for the activation of p38 kinase whereas phosphoinositide 3-kinase was needed for ERK1/2 activation. In addition, the phosphorylation of ERK1/2 was essential for the activation of AP-1 whereas p38 MAP kinase was needed for the activation of C/EBP. Our data demonstrate that the cyclic stretch of HASMC causes the increased production of IL-8 by activating the AP-1 and C/EBP transcription factors through the activation of ERK1/2 and p38 kinase signaling pathways.

Selective modulation of lipopolysaccharide-stimulated cytokine expression and mitogen-activated protein kinase pathways by dibutyryl-cAMP in BV2 microglial cells

Cyclic AMP is a very important regulator in a wide range of biological processes, including inflammatory reactions. To investigate the role of cAMP in microglia, we examined the effect of dibutyryl-cAMP (dbcAMP) on lipopolysaccharide (LPS)-stimulated cytokine expression and signaling pathways in murine BV2 microglial cells. DbcAMP strongly suppressed LPS-induced TNF-α expression, without affecting NO, IL-6 or TGF-β1 expression. In contrast, LPS-induced IL-1β or IL-10 expressions were dramatically increased by dbcAMP. We further examined the effect of elevated cAMP on signaling molecules such as MAP kinases (p38 MAPK, ERK and JNK), NF-κB and AP1, which are involved in the regulation of inflammatory responses. DbcAMP decreased the LPS-induced phosphorylation of p38 MAPK, while it modestly enhanced the ERK activity. JNK phosphorylation was slightly reduced by dbcAMP only at the later time point. Electrophoretic mobility shift assay revealed that the elevated cAMP potentiated AP-1 binding activity by enhancing c-fos binding. On the other hand, dbcAMP repressed NF-κB-mediated transcription without affecting NF-κB binding. Treatment with H89, a selective inhibitor of protein kinase A, completely reversed cAMP-induced IL-10 and IL-1β upregulation but only partially reversed the cAMP-induced repression of TNF-α. Thus, the effect of dbcAMP in BV2 cells appears to be mediated through both protein kinase A-dependent and -independent pathways. Taken together, our results demonstrate that cAMP modulates microglia activation in a diverse and complex manner.

Alcohol consumption decreases IL-2-induced NF-kappaB activity in enriched NK cells from C57BL/6 mice.

Previously we showed that ethanol (EtOH) consumption suppressed IL-2-induced cytolytic activity of murine splenic natural killer (NK) cells. Although IL-2 receptor signaling is involved in activation of NK cells, neither the mechanism for this activation nor the role of EtOH consumption in modulating activation is completely understood. In this study we show by electrophoretic mobility-shift assay (EMSA) that enriched splenic NK cells from EtOH-consuming C57BL/6 mice exhibit reduced NF-kappaB and AP-1 binding activity in response to IL-2 stimulation as compared to the water-drinking mice. Semiquantitative RT-PCR and real-time PCR analyses indicated that EtOH consumption inhibits the induction of perforin, granzyme A, and granzyme B in response to IL-2. Pyrrolidine dithiocarbamate (PDTC) and N-tosyl-L-phenylalanine chloromethyl ketone (TPCK) blocked NFkappaB and AP-1 binding activity in nuclear extracts of IL-2-stimulated NK cells in an EMSA and also inhibited the IL-2-induced expression of perforin, granzyme A, and granzyme B gene expression in enriched NK cells. These inhibitors dramatically suppressed IL-2-stimulated NK cytolytic activity against YAC-1 lymphoma target cells. Taken together, these results suggest that NFkappaB and AP-1 are important regulators of NK cell cytolytic function through regulation of perforin, granzyme A, and granzyme B gene expression. The findings further suggest that the decreased cytolytic activity of IL-2-stimulated NK cytolytic activity in EtOH-consuming mice is due at least in part to impaired transactivation of these and possibly other genes involved in control of NK-cell target lysis.

Effect of intra-ischemic hypothermia on the expression of c-Fos and c-Jun, and DNA binding activity of AP-1 after focal cerebral ischemia in rat brain

It is unknown whether immediate early gene (IEG) induction and subsequent late gene regulation after ischemia is beneficial or deleterious. The aim of this study was to examine the effect of hypothermia on expression of c-Fos and c-Jun, and AP-1 DNA binding activity, after transient focal cerebral ischemia in rat brain, and clarify the role of IEGs and AP-1 after insults. Male Wistar rats underwent right middle cerebral artery occlusion for 1 h with the intraluminal suture method. During ischemia, animals were assigned to either normothermic (NT) or hypothermic (HT) groups. In the NT group, brain temperature was observed to spontaneously increase to 40 °C during ischemia. In the HT group, brain temperature decreased to 30 °C. Infarct volume in cortex was decreased in the HT group, compared with that in the NT group ( P<0.001). Increased c-Fos immunoreactivity in the cortex was observed at 3 h after reperfusion in the HT, but not the NT group, while c-Jun expression was not affected by HT treatment. There was also a significant increase in AP-1 DNA binding activity at 3 h in the HT group when compared to the NT group ( P<0.01). In conclusion, hypothermia decreased cerebral infarction in association with early increases in c-Fos expression and AP-1 DNA binding activity in peri-infarct cortex. It remains to be established whether such responses are a cause or consequence of cell survival, but these results clearly establish that altered transcription is a key feature of tissue spared following hypothermic focal ischemia.

Heat shock response downregulates IL-18 expression in the murine macrophage cell line, RAW264.7

Heat shock response is a self-defense mechanism for protection of cells and organisms from a wide range of harmful Stressors. Recent studies revealed that it is involved in the regulation of cytokines expression. IL-18 is an important cytokine in mediating immune response. We studied LPS-induced IL-18 expression in heat shock treated RAW264.7 murine macrophages. Our results show that the heat shock response significantly inhibited the expression of LPS-induced pro-inflammatory cytokine IL-18. Further research on the down-regulation mechanism shows that this inhibitory effect is correlated to the great suppression of the binding activity of AP-1, which is a transcription factor binding to the promoter of IL-18 (−1120 to −1083) and regulates the transcription of IL-18. Meanwhile, we observed that the phosphorylation of JNK, which is AP-1 upstream kinase, was greatly decreased. These results confirmed that the down-regulation effect on IL-18 production in heat shock response is related to the suppression of the JNK/AP-1 signaling pathway.

Oxidized LDL induces transcription factor activator protein-1 in rat mesangial cells.

It has been shown that oxidized low-density lipoprotein (ox-LDL), through the activation of glomerular cells, stimulates pathobiological processes involved in monocyte infiltration into the mesangium. The underlying molecular mechanisms are not fully understood. The present study showed that ox-LDL strongly induced AP-1 binding activity in rat mesangial cells (RMCs) in a dose- and time-dependent manner, reaching the maximal activation at 250 microg ml(-1) within 24 h. The results from mobility shift assays and Western blotting analysis revealed that this AP-1 binding increase involved c-Jun, but not c-Fos. Moreover, this ox-LDL-increased AP-1 binding was inhibited by several protein kinase (PK) inhibitors: the protein kinase C (PKC) inhibitor Bisindolylmaleimide I, the cAMP-dependent PK (PKA) inhibitor H89, and the tyrosine PK (PTK) inhibitor genistein. Protein phosphorylation represents mitogen-activated protein kinase (MAPK) activity. Therefore, we examined the role of ox-LDL on the activation of mesangial cell JNK/SAPK, the only recognized protein kinase that catalyses phosphorylation of c-Jun. The incubation of mesangial cells with ox-LDL induced phosphorylation of JNK1/SAPK dose dependently, with the maximal response at 150 microg ml(-1). This study demonstrates that multiple kinase activities are involved in the mechanism of ox-LDL-induced AP-1 activation in mesangial cells, and ox-LDL stimulates AP-1 through JNK-c-Jun other than MEK-c-Fos signalling pathway.