Induced Activation Of Activator Protein-1 Research Articles

Magnesium Sulfate Inhibits Activator Protein-1 Upregulation in Endotoxin-activated Murine Macrophages

Objective The expression of inflammatory molecules is regulated by the transcription factor activator protein-1 (AP-1), a heterodimeric protein that consists of proteins from various families, including c-Jun and c-Fos. We sought to elucidate whether MgSO 4 regulates the activation of AP-1 in endotoxin-activated RAW264.7 cells, a murine macrophage-like cell line. The possible roles of the L-type calcium channels in this process were also elucidated. Materials and Methods RAW264.7 cells were treated with phosphate buffered saline, MgSO 4, lipopolysaccharide (LPS), LPS plus MgSO 4 (20 mM), or LPS plus MgSO 4 plus the L-type calcium channel activator BAY-K8644 (1 μM). After harvesting, expression of AP-1 was evaluated. Results LPS induced AP-1 activation based on the fact that the nuclear protein concentrations of AP-1 components, including c-Jun and c-Fos, as well as the AP-1 DNA-binding activity, were significantly increased in LPS-treated RAW264.7 cells. MgSO 4, in contrast, significantly inhibited LPS-induced AP-1 activation in activated RAW264.7 cells. Moreover, the effect of MgSO 4 on AP-1 was reversed by BAY-K8644. Conclusion MgSO 4 inhibited AP-1 activation in LPS-treated macrophages and the mechanism may involve the L-type calcium channels.

MicroRNA-101 regulates expression of the v-fos FBJ murine osteosarcoma viral oncogene homolog (FOS) oncogene in human hepatocellular carcinoma

MicroRNAs (miRNAs) have recently been proposed as a versatile class of molecules involved in regulation of various biological processes. Although there is emerging evidence that some microRNAs can function as oncogenes or tumor suppressors, the specific role of miRNA in human hepatocellular carcinoma (HCC) is unclear at this point. In this study, we examined the microRNA expression profiles in a set of 20 human HCC specimens by miRNA microarray and quantitative real-time polymerase chain reaction. The results showed that among the 20 HCC samples analyzed, microRNA-101 was significantly down-regulated twofold or more (twofold to 20-fold) in 16 samples compared with the matching nontumoral liver tissues. Using both a luciferase reporter assay and Western blot analysis, we showed that microRNA-101 repressed the expression of v-fos FBJ murine osteosarcoma viral oncogene homolog (FOS) oncogene, a key component of the activator protein-1 (AP-1) transcription factor. Moreover, using a luciferase expression vector (pAP-1-Luc) driven by seven copies of an AP-1 cis-element, we observed that microRNA-101 expression inhibited phorbol 12-myristate 13-acetate (PMA)-induced AP-1 activity. In in vitro Matrigel invasion and Transwell migration assays, enhanced microRNA-101 expression inhibited the invasion and migration of cultured HCC cells, respectively. These findings suggest that microRNA-101 may play an important role in HCC. MicroRNA-101, which is aberrantly expressed in HCC, could repress the expression of the FOS oncogene.

Abstract B83: Sulfiredoxin is an AP-1 target gene that is required for transformation and shows elevated expression in human skin tumors

Abstract B83 Previous studies from others and our lab have shown that activator protein 1 (AP-1) plays a pivotal role in tumorigenesis in vitro and in vivo. Expression of a dominant negative form c-Jun, TAM67, dramatically inhibits tumor promoter induced skin carcinogenesis. In this study, our goals are to identify target genes that are specifically induced by tumor promoter and inhibited by TAM67, and to investigate their functional significances in tumor promotion and progression. First, we demonstrated that Sulfiredoxin (Srx) is a novel target of AP-1 activation and TAM67 inhibition. In mouse epidermis, expression of Srx was upregulated by tumor promoter and inhibited by TAM67 as revealed by DNA microarray. This was further confirmed in mouse epithelial-derived JB6 cells by RT-PCR, Western blotting, luciferase reporter and chromatin immunoprecipitation assays at transcript and protein levels. Next, we demonstrated that Srx is important for maintaining of cellular redox homeostasis through reduction of overoxidized Peroxiredoxins (Prxs). Knockdown of Srx led to a much slower reduction rate of overoxidized Prxs, whereas overexpression of Srx resulted in an increased reduction rate of overoxidized Prxs. Then we found that knockdown of Srx dramatically inhibited tumor promoter induced neoplastic transformation as indicated by anchorage-independent growth in soft agar. This effect was not due to the inhibition of cell proliferation as depletion of Srx stimulated cell proliferation under adherent conditions. Depletion of Srx also led to impaired AP-1 activation and increased cell death under oxidative conditions, which indicated that Srx might be involved in the regulation of AP-1 activity through a novel positive feedback loop. Finally, we examined the expression profiling of Srx in normal skin and tissues from patients with various skin diseases by tissue microarray. We found strong positive expression of Srx in skin malignant tumors. In summary, we identified Srx as a novel target of tumor promoter induced AP-1 activation and is inhibited by TAM67. We demonstrated that Srx is important for maintaining of cellular redox balance and is required for tumor promoter induced neoplastic transformation. We also found strong expression of Srx in human skin malignant tumors. Therefore, we propose that Srx can be used as a marker for skin malignancy, and manipulation of Srx expression may be of interest in cancer prevention and treatment. Citation Information: Cancer Prev Res 2008;1(7 Suppl):B83.

MG132 proteasome inhibitor modulates proinflammatory cytokines production and expression of their receptors in U937 cells: involvement of nuclear factor‐κB and activator protein‐1

In response to inflammatory stimuli, monocytes/macrophages secrete greater quantities of the proinflammatory cytokines tumour necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta) and IL-6. The inflammatory process and the innate immune response are related to the activation of several transcription factors, such as nuclear factor kappaB (NF-kappaB) and activator protein 1 (AP-1). The proteasome is a multimeric protease complex, which plays a vital role in several cellular functions, including the regulation of transcription factors like NF-kappaB. In this study, we used the human monocyte cell line U937 stimulated with lipopolysaccharide (LPS) and phorbol 12-myristate 13-acetate (PMA) as a model to investigate the in vitro effects of MG132, a proteasome inhibitor, on the release of TNF-alpha, IL-1beta and IL-6 and on the expression of their membrane and soluble receptors TNF-R1, IL-1R1 and IL-6R. We also analysed the effects of MG132 on the activation of NF-kappaB and AP-1 and on the IkappaB molecule. MG132 significantly inhibited the secretion of those proinflammatory cytokines. MG132 increased the release of the soluble receptors TNF-R1 and IL-1R1 from U937 cells and decreased their cell-surface expression. MG132 also increased IL-6R cell-surface expression and decreased its release. Proteasome inhibition also led to an increase in LPS+PMA-induced AP-1 activation and the attenuation of LPS+PMA-induced IkappaB degradation, resulting in the abolition of NF-kappaB activation. Our experiments strongly suggest that the proteasome is an important factor in the regulation of proinflammatory cytokines and their receptors.

Acetaldehyde induces matrix metalloproteinase-9 gene expression via nuclear factor-κB and activator protein 1 signaling pathways in human hepatocellular carcinoma cells: Association with the invasive potential

Alcohol consumption is a significant risk factor for hepatocellular carcinoma (HCC). Alcohol also increases the prevalence of invasion in HCC patients. However, the molecular mechanism on the metastatic effect of alcohol is unclear so far. Herein we demonstrated that acetaldehyde, the primary metabolite of ethanol, increased matrix metalloproteinase-9 (MMP-9) gelatinolytic activity and promoted cell invasion through the up-regulation of MMP-9 gene transcription in HepG2 cells. The transcription of MMP-9 gene was regulated by 10 μM acetaldehyde via inductions of nuclear factor-κB (NF-κB) and activator protein 1 (AP-1) activities. Acetaldehyde stimulated the translocation of NF-κB into nucleus through inhibitory κB-α (IκB-α) and c-Jun N-terminal kinase (JNK)/β-transducin repeat-containing protein (β-TrCP) signaling pathways. Acetaldehyde also induced AP-1 activity via the phosphorylation of p38 kinase. In conclusion, our findings demonstrated for the first time that acetaldehyde activated NF-κB and AP-1 activities via IκB, JNK/β-TrCP, and p38 signaling pathways, resulting in MMP-9 gene expression and hepatocarcinoma cells invasion. These results suggested that acetaldehyde might be a potential factor involved in the invasiveness of HCC in alcoholic patients.

Modulation of activator protein-1 (AP-1) and MAPK pathway by flavonoids in human prostate cancer PC3 cells

In last couple of decades the use of natural compounds like flavonoids as chemopreventive agents has gained much attention. Our current study focuses on identifying chemopreventive flavonoids and their mechanism of action on human prostate cancer cells. Human prostate cancer cells (PC3), stably transfected with activator protein 1 (AP-1) luciferase reporter gene were treated with four main classes of flavonoids namely flavonols, flavones, flavonones, and isoflavones. The maximum AP-1 luciferase induction of about 3 fold over control was observed with 20 microM concentrations of quercetin, chrysin and genistein and 50 microM concentration of kaempferol. At higher concentrations, most of the flavonoids demonstrated inhibition of AP-1 activity. The MTS assay for cell viability at 24 h showed that even at a very high concentration (500 microM), cell death was minimal for most of the flavonoids. To determine the role of MAPK pathway in the induction of AP-1 by flavonoids, Western blot of phospho MAPK proteins was performed. Four out of the eight flavonoids namely kaempferol, apigenin, genistein and naringenin were used for the Western Blot analysis. Induction of phospho-JNK and phospho-ERK activity was observed after two hour incubation of PC3-AP1 cells with flavonoids. However no induction of phospho-p38 activity was observed. Furthermore, pretreating the cells with specific inhibitors of JNK reduced the AP-1 luciferase activity that was induced by genistein while pretreatment with MEK inhibitor reduced the AP-1 luciferase activity induced by kaempferol. The pharmacological inhibitors did not affect the AP-1 luciferase activity induced by apigenin and naringenin. These results suggest the possible involvement of JNK pathway in genistein induced AP-1 activity while the ERK pathway seems to play an important role in kaempferol induced AP-1 activity.

ERK and JNK signaling pathways are involved in the regulation of activator protein 1 and cell death elicited by three isothiocyanates in human prostate cancer PC-3 cells

Many isothiocyanates (ITCs) such as sulforaphane (SFN), phenethyl isothiocyanate (PEITC) and allyl isothiocyanate (AITC) are highly effectively in chemoprevention or reduction of the risk of cancer and possess antitumor activities in vitro and in vivo. The activator protein 1 (AP-1) and MAPK signaling pathways are believed to play an important role in cancer chemoprevention and chemotherapy due to their involvement in tumor cell growth, proliferation, apoptosis and survival. In the present study, we determined the effects of SFN, PEITC and AITC on AP-1 activation, and investigated the roles of extracellular signal-regulated protein kinase (ERK) and c-Jun N-terminal kinase (JNK) signaling pathways in the regulation of AP-1 activation and cell death elicited by these ITCs in human prostate cancer PC-3 cells. SFN, PEITC and AITC each induced AP-1 activity potently and caused a significant elevation in the phosphorylation of ERK1/2, JNK1/2, Elk-1 and c-Jun. Transfection with ERK2 and upstream kinase DNEE-MEK1 activated AP-1 activity, and transfection with dominant-negative mutant ERK2 (dnERK2) potently decreased AP-1 activation induced by SFN, PEITC and AITC. Transfection with JNK1 and upstream kinase MKK7 activated AP-1 activity, and transfection with dominant-negative mutant JNK1-APF significantly attenuated AP-1 activation induced by SFN, PEITC and AITC. Pretreatment with MEK1-ERK inhibitor U0126 and JNK inhibitor SP600125 substantially attenuated the decrease in cell viability induced by SFN, PEITC and AITC. Transfection with dnERK2 and JNK1-APF significantly reversed the decrease of Bcl-2 expression elicited by these ITCs. Furthermore, transfection with dnERK2 and JNK1-APF blocked the apoptosis induced by these ITCs in PC-3 cells. Taken together, our results indicate that the activation of the ERK and JNK signaling pathways is important for transcriptional activity of AP-1 and is involved in the regulation of cell death elicited by ITCs in PC-3 cells.

Apoptosis signal-regulating kinase 1 in leukotriene D 4-induced activator protein-1 activation in airway smooth muscle cells

Cysteinyl leukotrienes (LTs) are involved in allergic disorders including bronchial asthma. Transcription factor activator protein-1 (AP-1) activation is essential for cell proliferation and differentiation. LTD 4 is shown to promote human airway smooth muscle cell proliferation; however, the effect of LTD 4 on AP-1 activation in airway smooth muscle cells and the molecular mechanism in regulating AP-1 activation have not been determined. We examined the effect LTD 4 on AP-1 activation in human airway smooth muscle cells and analyzed a role of apoptosis signal-regulating kinase1 (ASK1), an upstream kinase kinase of c- Jun-NH 2-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK) in LTD 4-induced AP-1 activation to clarify the signaling molecule regulating AP-1 activation. The results showed that LTD 4 induced AP-1 activation determined by AP-1-dependent luciferase gene activity and ASK1 phosphorylation. Transient transfection of the dominant negative form of ASK1 attenuated LTD 4-induced AP-1 activation. In addition, LTD 4-induced AP-1 activity was depressed in the dominant negative form of ASK1-stably transfected porcine artery endothelial cells compared to that in the parental porcine artery endothelial cells. These results indicate that LTD 4 is capable of inducing AP-1 activation and ASK1 regulates AP-1 activation in LTD 4-stimulated airway smooth muscle cells.

Activation of activator protein-1 DNA binding activity due to low level manganese exposure in pheochromocytoma cells

Low-level exposure of manganese (Mn 2+) induces adverse neurological effects. We have previously shown that low-level Mn 2+ exposure induces cell death in dopaminergic neurons, in which the redox-sensitive transcription factor nuclear factor kappa-B (NF-κB) and associated mitogen-activated protein kinase (MAPK) were stimulated. In continuation of this work, we evaluated the activator protein-1 (AP-1). DNA binding activity and N-terminal c-Jun kinase (JNK) activation due to low-level exposure to manganese. Rat catecholaminergic-rich pheochromocytoma (PC12) cells were exposed to low concentrations of Mn 2+ (0–10 μM) for 120 min and examined changes in AP-1 DNA binding activity by electrophoretic mobility shift assay (EMSA). Mn 2+-exposed cells produced a eightfold increase in AP-1 DNA binding activity compared to untreated controls. This significant increase was seen within 60 min after Mn 2+ exposure. In addition, Mn 2+ activated N-terminal c-Jun kinase, suggesting that the JNK pathway is involved in Mn 2+-induced AP-1 activation. Thus, JNK activation may in turn stimulate AP-1 via c-Jun phosphorylation. Since the transcription factors NF-κB and AP-1 have been shown to play an essential role in oxidative stress related toxicity, we suggest that induction of the signaling factor AP-1 during Mn 2+ exposure in the present study may have a significant cytotoxic effect on neuronal function.

Apoptosis signal-regulating kinase 1-mediated signaling pathway regulates nitric oxide-induced activator protein-1 activation in human bronchial epithelial cells.

Exhaled nitric oxide (NO) is increased in individuals with bronchial asthma. NO may have antiinflammatory and proinflammatory effects; however, its role in bronchial asthma is unclear. In the present study, to clarify this issue we examined the effect of NO in inducing activator protein-1 (AP-1) activation in human bronchial epithelial cells (BEC) and a role of apoptosis signal-regulating kinase1 (ASK1), an upstream kinase kinase of c-Jun-NH2-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK) in NO-mediated AP-1 activation. The results showed that (1) the reactive nitrogen generating species NOR-1(+/--(E)-methyl-2-[(E)-hydroxykmino]-5-nitro-6-methoxy-3-hexeneamide]) induced AP-1 activation determined by AP-1-dependent luciferase gene activity, and an NO scavenger, carboxyl-PTIO, attenuated NOR-1-induced AP-1 activation; (2) NOR-1 phosphorylated ASK1, JNK, and p38 MAPK; and (3) transient transfection of the dominant negative form of AKS1 attenuated NOR-1-induced AP-1 activation in BEC. To further characterize the role of ASK-1 cascade, the dominant negative form of ASK1-stable transfected porcine artery endothelial (PAE) cells were used. AP-1 activity and JNK and p38 MAPK phosphorylation were depressed in the dominant-negative form of ASK1-stable transfected PAE cells. These results indicate that NO is capable of inducing AP-1 activation, and that ASK1-p38 MAPK/JNK cascade regulates AP-1 activation in NO-stimulated BEC.

Differential requirement of EGF receptor and its tyrosine kinase for AP-1 transactivation induced by EGF and TPA.

The transcription factor activator protein-1 (AP-1) has been implicated in a large variety of biological processes including cell differentiation, proliferation, apoptosis and oncogenic transformation. It is thought that the 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced AP-1 activity is because of the activation of the PKC/MAPK/AP-1 pathway, although the detailed molecular mechanism has not been fully characterized. The tyrosine kinases of epidermal growth factor receptor (EGFR) lie at the head of a complex of signal transduction cascade that modulates cell proliferation, survival, adhesion, migration and differentiation. Currently, little is known about whether EGFR or its tyrosine kinase is necessary for TPA-induced AP-1 activation. In the present study, we investigated this issue using a well-characterized mouse fibroblast B82 cell line, which is devoid of the EGFR, and its stable transfectants with either wild-type EGFR (B82L) or tyrosine kinase-deficient EGFR (mutation at Lys-721) (B82M721). We demonstrated that the TPA or epidermal growth factor (EGF) induced AP-1 activation in the B82L cells that express wild-type EGFR, but not in the B82 cell, whereas autophosphorylation at tyrosine(1173) of EGFR in B82L cells was only induced by EGF, but not TPA. The expression of tyrosine kinase-deficient EGFR (mutation at Lys-721) (B82M721) resulted in deficiency of AP-1 induction in cellular response to EGF, while TPA treatment led to fully AP-1 activation. Furthermore, the mutation at Lys-721 of EGFR resulted in impairing of EGFR autophosphorylation at tyrosine(1173) induced by EGF. Based on these results, we conclude that TPA-induced AP-1 activation requires the basal level-EGFR protein, but not EGFR tyrosine kinase and EGFR autophosphorylation at tyrosine(1173), whereas both EGFR tyrosine kinase and EGFR autophosphorylation at Y(1173) play a critical role in EGF-induced AP-1 activation.

Suppression of insulin-induced AP-1 transactivation by menin accompanies inhibition of c-Fos induction.

The translation product of the MEN1 gene, menin, has been reported to suppress JunD-mediated activator protein-1 (AP-1) transactivation and inhibit Ras-mediated tumor formation, but its molecular mechanisms and physiologic significance have been poorly elucidated. To better understand the function of menin as a tumor suppressor, we examined the effect of menin on physiologically induced AP-1 activity. Overexpression of menin strongly suppressed insulin-induced AP-1 activity in CHO-IR cells, which express high levels of insulin receptor. We found that menin suppressed c-Fos induction at the transcriptional level, although that cannot explain the entire mechanism of AP-1 suppression by menin. Menin did not alter the expression levels of AP-1 proteins except c-Fos, phosphorylation of c-Jun and JunD and DNA binding properties of AP-1 proteins. Suppression of AP-1 activation by menin may be exerted through 2 independent mechanisms, direct inhibition on AP-1-mediated transcription and suppression of c-Fos induction. The molecular mechanism of inhibition of AP-1 function by menin needs further elucidation.

Role of nitric oxide in the activation of NF-kappaB, AP-1 and NOS II expression in articular chondrocytes.

Determine the sources of nitric oxide (NO) and evaluate its role in the activation of nuclear Factor-kappaB (NF-kappaB) and activator protein-1 (AP-1) and in the expression of NO synthase II (NOS II), induced by interleukin-1beta (IL-1). Primary cultures of bovine articular chondrocytes. The cells were treated with IL-1, 5 ng/ml with or without the NO donor S-nitroso-N-acetylpenicillamine (SNAP), in concentrations ranging from 10 to 300 microM. NF-kappaB and AP-1 activation were evaluated by electrophoretic mobility shift assay. Northern blot was used to detect NOS II mRNA levels and western blot to evaluate IkappaB-alpha, NOS I and NOS II protein levels. Under basal conditions, chondrocytes expressed NOS I, which was lost upon IL-I treatment. SNAP inhibited IL-I-induced NF-kappaB activation and NOS II expression. When added alone, SNAP induced AP-1 activation to approximately the same extent as IL-I. These results suggest that, in chondrocytes, NO is a key regulator of the signaling pathways leading from IL-I to NF-kappaB and AP-1 activation and to the expression of genes that are involved in the pathophysiology of arthritic diseases.

Contribution of extracellular signal-regulated kinase to angiotensin II-induced transforming growth factor-beta1 expression in vascular smooth muscle cells.

We have previously demonstrated that angiotensin II (Ang II) contributes to the increase in aortic transforming growth factor-beta(1) (TGF-beta(1)) mRNA levels in hypertensive rats. However, the molecular mechanism whereby Ang II promotes TGF-beta(1) expression in vascular smooth muscle cells (VSMCs) is poorly understood. In this study, we examined the role of extracellular signal-regulated kinase (ERK) in Ang II-mediated TGF-beta(1) expression in VSMCs and the role of Ang II in aortic ERK activity of stroke-prone spontaneously hypertensive rats. Treatment of quiescent VSMCs with 100 nmol/L Ang II induced rapid phosphorylation and activation of ERK1 and ERK2 with a peak at 5 minutes followed by an increase in activator protein-1 (AP-1) DNA binding activity, as shown by gel mobility shift assay. An increase in TGF-beta(1) mRNA was shown by Northern blot analysis. Treatment of VSMCs with PD98059, a specific inhibitor of the ERK pathway, attenuated both the activation of AP-1 and the increase in TGF-beta(1) mRNA induced by Ang II. Inhibition of Ang II-induced AP-1 activation with c-fos antisense oligodeoxynucleotide led to a significant reduction of TGF-beta(1) mRNA in VSMCs. Furthermore, in vivo treatment of stroke-prone spontaneously hypertensive rats with losartan, an Ang II type 1 receptor antagonist, decreased aortic ERK activity. Thus, we show that ERK, through AP-1 activation, is involved in Ang II-induced TGF-beta(1) mRNA expression in VSMCs and suggest that ERK may participate in vascular remodeling of hypertension. However, it remains to be determined whether the increase in TGF-beta(1) mRNA leads to the increase in its active protein.