Activator Protein-1 Signaling Pathways Research Articles

Inhibitory effect of emodin on tumor invasion through suppression of activator protein-1 and nuclear factor-κB

3-Methyl-1,6,8-trihydroxyanthraquinone (emodin) is an active component from the rhizome of Rheum palmatum, a widely used traditional Chinese herb. In this study, we found that emodin significantly inhibited 12- O-tetradecanoylphorbol-13-acetate (TPA)-induced in vitro invasion of human cancer cells including HSC5 and MDA-MB-231 cells. Matrix metalloproteinases (MMPs) are known to be associated with cancer invasion. Zymographic analysis showed that emodin suppressed TPA-induced MMP-9 activity in a concentration-dependent manner. We further demonstrated that emodin reduced the transcriptional activity of activator protein-1 (AP-1) and nuclear factor kappaB (NF-κB), two important nuclear transcription factors involved in MMP-9 expression. Emodin suppressed the phosphorylation of two mitogen-activated protein kinases, extracellular signal-regulated protein kinase and c-Jun N-terminal kinase, but not p38 kinase, leading to reduced c-Jun phosphorylation and AP-1 DNA-binding. Moreover, emodin inhibited TPA-induced degradation of inhibitor of kappaBα, nuclear translocation of p65, and NF-κB DNA-binding activity. Taken together, these results suggest that emodin inhibits the invasiveness of human cancer cells by suppressing MMP-9 expression through inhibiting AP-1 and NF-κB signaling pathways.

Heterodimer formation between c-Jun and Jun B proteins mediated by Epstein–Barr virus encoded latent membrane protein 1

Epstein–Barr virus (EBV) encoded latent membrane protein 1 (LMP1) is essential for the immortalization of human B cells and is linked etiologically to several human tumors. LMP1 is an integral membrane protein which acts like a constitutively active receptor. It binds tumor necrosis factor (TNF)-receptor-associated factors (TRAFs), activates NFκB and triggers the transcription factor activating protein-1 (AP-1) via the c-Jun N-terminal kinase (JNK) cascade, but its specific contribution to AP-1 has not been elucidated fully. Members of AP-1 family, the Jun and fos related protein, have been shown to directly interact and form heterodimeric complexes. In this report, using a Tet-on LMP1 HNE2 cell line which is a dual-stable LMP1 integrated nasopharyngeal carcinoma (NPC) cell line and the expression of LMP1 in which could be regulated by Tet-on system, we show that Jun B can efficiently form a new heterodimeric complex with the c-Jun protein under the regulation of LMP1, phosphorylation of c-Jun (ser63, ser73) and Jun B involved in the process of the new heterodimeric form. We also find that this heterodimeric form can bind to the AP-1 consensus sequence. Transfection studies suggest that JNK interaction protein (JIP) could inhibit the heterodimer form of c-Jun and Jun B through blocking the AP-1 signaling pathway triggered by LMP1. The interaction and function between c-Jun protein and Jun B protein increase the repertoire of possible regulatory complexes by LMP1 that could play an important role in the regulation of transcription of specific cellular genes in the process of genesis of nasopharyngeal carcinoma.

Inhibitory effect of emodin on tumor invasion through suppression of activator protein-1 and nuclear factor-$kappa;B

3-Methyl-1,6,8-trihydroxyanthraquinone (emodin) is an active component from the rhizome of Rheum palmatum, a widely used traditional Chinese herb. In this study, we found that emodin significantly inhibited 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced in vitro invasion of human cancer cells including HSC5 and MDA-MB-231 cells. Matrix metalloproteinases (MMPs) are known to be associated with cancer invasion. Zymographic analysis showed that emodin suppressed TPA-induced MMP-9 activity in a concentration-dependent manner. We further demonstrated that emodin reduced the transcriptional activity of activator protein-1 (AP-1) and nuclear factor kappaB (NF-κB), two important nuclear transcription factors involved in MMP-9 expression. Emodin suppressed the phosphorylation of two mitogen-activated protein kinases, extracellular signal-regulated protein kinase and c-Jun N-terminal kinase, but not p38 kinase, leading to reduced c-Jun phosphorylation and AP-1 DNA-binding. Moreover, emodin inhibited TPA-induced degradation of inhibitor of kappaBα, nuclear translocation of p65, and NF-κB DNA-binding activity. Taken together, these results suggest that emodin inhibits the invasiveness of human cancer cells by suppressing MMP-9 expression through inhibiting AP-1 and NF-κB signaling pathways.

Porphyromonas gingivalis induces receptor activator of NF-kappaB ligand expression in osteoblasts through the activator protein 1 pathway.

Porphyromonas gingivalis, an important periodontal pathogen, is closely associated with inflammatory alveolar bone resorption, and several components of the organism such as lipopolysaccharides have been reported to stimulate production of cytokines that promote inflammatory bone destruction. We investigated the effect of infection with viable P. gingivalis on cytokine production by osteoblasts. Reverse transcription-PCR and real-time PCR analyses revealed that infection with P. gingivalis induced receptor activator of nuclear factor kappaB (NF-kappaB) ligand (RANKL) mRNA expression in mouse primary osteoblasts. Production of interleukin-6 was also stimulated; however, osteoprotegerin was not. SB20350 (an inhibitor of p38 mitogen-activated protein kinase), PD98059 (an inhibitor of classic mitogen-activated protein kinase kinase, MEK1/2), wortmannin (an inhibitor of phosphatidylinositol 3 kinase), and carbobenzoxyl-leucinyl-leucinyl-leucinal (an inhibitor of NF-kappaB) did not prevent the RANKL expression induced by P. gingivalis. Degradation of inhibitor of NF-kappaB-alpha was not detectable; however, curcumin, an inhibitor of activator protein 1 (AP-1), prevented the RANKL production induced by P. gingivalis infection. Western blot analysis revealed that phosphorylation of c-Jun, a component of AP-1, occurred in the infected cells, and an analysis of c-Fos binding to an oligonucleotide containing an AP-1 consensus site also demonstrated AP-1 activation in infected osteoblasts. Infection with P. gingivalis KDP136, an isogenic deficient mutant of arginine- and lysine-specific cysteine proteinases, did not stimulate RANKL production. These results suggest that P. gingivalis infection induces RANKL expression in osteoblasts through AP-1 signaling pathways and cysteine proteases of the organism are involved in RANKL production.

New Players from Genomic-Scale Screens

The availability of large collections of cDNAs to human genes has enabled genome-scale screens for new components of signaling pathways. Iourgenko et al. transfected cells with each of some 20,000 full-length cDNAs. They then monitored the cells for increased expression of a reporter gene linked to the interleukin 8 (IL-8) promoter. Although not previously implicated in regulation of IL-8, cyclic AMP response element-binding (CREB) proteins were found to be strong activators of the IL-8 reporter. The screen also identified TORC1 [for transducer of regulated CREB]. TORC1 is a member of a family of evolutionarily conserved, previously uncharacterized proteins that appears to function as coactivator proteins with CREB. Chanda et al. conducted a similar screen for genes whose expression would activate AP-1 (activator protein-1). Although AP-1-mediated transcription has been highly studied as a key component of growth and mitogenic response pathways, new regulators were identified along with known ones in the screen. Because such transient expression might produce nonphysiological outcomes, the authors further analyzed candidate genes by other methods, including elimination of the potential modulators by expression of short hairpin RNA. The latter manipulation inhibited activation of AP-1 promoters in response to mitogenic stimuli in cultured human embryonic kidney cells. The authors propose that their combination of "integrative genomics approaches will allow for increasingly sophisticated models that extend our knowledge of the complex relationships between gene products, signal transduction pathways, and their underlying biological functions." V. Iourgenko, W. Zhang, C. Mickanin, I. Daly, C. Jiang, J. M. Hexham, A. P. Orth, L. Miraglia, J. Meltzer, D. Garza, G.-W. Chirn, E. McWhinnie, D. Cohen, J. Skelton, R. Terry, Y. Yu, D. Bodian, F. P. Buxton, J. Zhu, C. Song, M. A. Labow, Identification of a family of cAMP response element-binding protein coactivators by genome-scale functional analysis in mammalian cells. Proc. Natl. Acad. Sci. U.S.A. 100 , 12147-12152 (2003). [Abstract] [Full Text] S. K. Chanda, S. White, A. P. Orth, R. Reisdorph, L. Miraglia, R. S. Thomas, P. DeJesus, D. E. Mason, Q. Huang, R. Vega, D.-H. Yu, C. G. Nelson, B. M. Smith, R. Terry, A. S. Linford, Y. Yu, G. Chirn, C. Song, M. A. Labow, D. Cohen, F. J. King, E. C. Peters, P. G. Schultz, P. K. Vogt, J. B. Hogenesch, J. S. Caldwell, Genome-scale functional profiling of the mammalian AP-1 signaling pathway. Proc. Natl. Acad. Sci. U.S.A. 100 , 12153-12158 (2003). [Abstract] [Full Text]

Genome-scale functional profiling of the mammalian AP-1 signaling pathway

Large-scale functional genomics approaches are fundamental to the characterization of mammalian transcriptomes annotated by genome sequencing projects. Although current high-throughput strategies systematically survey either transcriptional or biochemical networks, analogous genome-scale investigations that analyze gene function in mammalian cells have yet to be fully realized. Through transient overexpression analysis, we describe the parallel interrogation of approximately 20,000 sequence annotated genes in cancer-related signaling pathways. For experimental validation of these genome data, we apply an integrative strategy to characterize previously unreported effectors of activator protein-1 (AP-1) mediated growth and mitogenic response pathways. These studies identify the ADP-ribosylation factor GTPase-activating protein Centaurin alpha1 and a Tudor domain-containing hypothetical protein as putative AP-1 regulatory oncogenes. These results provide insight into the composition of the AP-1 signaling machinery and validate this approach as a tractable platform for genome-wide functional analysis.

Down-regulation of c-jun N-terminal kinase-activator protein-1 signaling pathway by Ginkgo biloba extract in human peripheral blood T cells

The activation of T lymphocytes contributes to inflammatory process of cardiovascular and cerebrovascular diseases. We investigated the effects of the extract of Ginkgo biloba (EGb), an ancient plant preserving antioxidant property, on phorbol 12-myristate 13-acetate+ionomycin or anti-CD3+anti-CD28 monoclonal antibodies-activated T cells. Human peripheral blood T cells were negatively selected from whole blood. Cytokines were measured by ELISA, cell surface markers by flow cytometry and the activities of transcription factors and kinases were determined by electrophoresis mobility shift assays, kinase assays and transfection assays. We showed that EGb inhibited several cytokines, including tumor necrosis factor-alpha, interleukin (IL)-2, IL-4 and interferon-gamma production from activated T cells. Electrophoresis mobility shift assay analysis indicated that EGb down-regulated activator protein-1 (AP-1) but not nuclear factor kappa B DNA-binding activity. In addition, EGb inhibited c-jun N-terminal kinase but not extracellular signal regulated protein kinase activity. The inhibitory specificity on AP-1 by EGb was also demonstrated in transfection assays. The inhibition of AP-1 signaling pathway in T cells by EGb provides a support for its efficacy in cardiovascular and cerebrovascular diseases and raises a therapeutic potential for this drug in activated T cell-mediated pathologies.

Homocysteine decreases endothelin-1 expression by interfering with the AP-1 signaling pathway

The increased cardiovascular risk associated with hyperhomocysteinemia has been linked to homocysteine-induced endothelial cell (EC) dysfunction. Endothelin-1 is a vasoactive peptide, synthesized mainly by vascular ECs. We have previously shown that homocysteine decreases endothelin-1 biosynthesis. Here we addressed the molecular mechanism of endothelin-1 regulation by homocysteine. Experiments with the transcription inhibitor actinomycin D indicated that the decrease in preproendothelin-1 mRNA content in homocysteine-treated cells did not result from transcript destabilization. Transient transfection assays demonstrated that homocysteine downregulated endothelin-1 at the transcriptional level by decreasing preproendothelin-1 promoter activity. Mutation of the activator protein-1 (AP-1) site of the promoter eliminated the repression induced by homocysteine. Western blot analysis showed that the homocysteine-induced decrease in promoter activity was not associated with reduced expression of the AP-1 components c-Fos and c-Jun. The inhibitory action of homocysteine on preproendothelin-1 mRNA expression was not prevented by cycloheximide. Electrophoretic mobility shift assays demonstrated that homocysteine reduced the binding activity of ECs nuclear extracts to an AP-1 consensus site. These results indicate that homocysteine downregulates endothelin-1 synthesis by inhibiting AP-1 activity, and that the AP-1 signaling pathway may be of major importance in homocysteine-induced endothelial dysfunction.

Quercetin glucuronide prevents VSMC hypertrophy by angiotensin II via the inhibition of JNK and AP-1 signaling pathway

We previously reported that quercetin, a bioflavonoid belonging to polyphenols, inhibited Angiotensin II (Ang II)-induced vascular smooth muscle cell (VSMC) hypertrophy through the inhibition of c-Jun N-terminal kinase (JNK) activation. However, we recently found that orally administered quercetin appeared in plasma as glucuronide-conjugated forms in rats and humans. Therefore we examined the effect of chemically synthesized quercetin glucuronide on Ang II-induced mitogen-activated protein (MAP) kinase activation and hypertrophy of cultured rat aortic smooth muscle cells (RASMC). Ang II activated extracellular signal-regulated kinase (ERK)1/2, JNK, and p38 in RASMC. Ang II-induced JNK activation was inhibited by quercetin 3- O-β- d-glucuronide (Q3GA) whereas ERK1/2 and p38 activations were not affected. Q3GA scavenged 1,1-diphenyl-2-picrylhydrazyl radical measured by a method of electron paramagnetic resonance. Q3GA also inhibited Ang II-induced increases in activator protein-1 (AP-1) DNA binding, a downstream transcription factor of JNK. Finally, Ang II-induced [ 3 H] leucine incorporation into RASMC was abolished by Q3GA. These findings suggest that the preventing effect of Q3GA on Ang II-induced VSMC hypertrophy is attributable in part to its inhibitory effect on JNK and the AP-1 signaling pathway. Q3GA would be an active metabolite of quercetin in plasma and may possess a preventing effect for cardiovascular diseases relevant to VSMC growth.

Peroxisome proliferator-activated receptor activators inhibit thrombin-induced endothelin-1 production in human vascular endothelial cells by inhibiting the activator protein-1 signaling pathway.

Endothelin-1 (ET-1), a 21-amino acid vasoactive peptide mainly produced by vascular endothelial cells, is involved in the regulation of vascular tone and smooth muscle cell proliferation. Peroxisome proliferator-activated receptors (PPARs), key players in lipid and glucose metabolism, have been implicated in metabolic disorders that are predisposing to atherosclerosis. Because of the potential role of ET-1 in vascular disorders such as hypertension and atherosclerosis, we investigated the regulation of ET-1 expression by PPAR activators. Western blot and reverse transcription-polymerase chain reaction analyses demonstrated that both PPARalpha and PPARgamma are expressed in human coronary artery endothelial cells as well as in endothelial cell lines such as HMEC-1 and ECV304. In bovine aortic endothelial cells and HMEC-1 cells, both PPARalpha and PPARgamma ligands inhibited thrombin-induced ET-1 secretion, whereas basal ET-1 secretion was only slightly suppressed. Reverse transcription-polymerase chain reaction experiments showed that this inhibition of ET-1 production occurs at the gene expression level. Using transient transfection assays, we demonstrated that PPARs downregulate thrombin-activated transcription of the human ET-1 promoter. Transactivation studies with c-Jun and c-Fos expression plasmids indicated that PPARs negatively interfere with the activator protein-1 signaling pathway, which mediates thrombin activation of ET-1 gene transcription. Furthermore, electrophoretic mobility shift assays demonstrated that PPAR activators reduce the thrombin-stimulated binding activity of bovine aortic endothelial cell nuclear extracts as well as c-Jun binding to an activator protein-1 consensus site. Taken together, these data indicate that (1) both PPARalpha and PPARgamma are expressed in human vascular endothelial cells and (2) PPAR activators inhibit thrombin-induced ET-1 biosynthesis, indicating a novel role for PPARs in vascular endothelial function.

The Activity of a Highly Promiscuous AP-1 Element Can Be Confined to Neurons by a Tissue-Selective Repressive Element

Tissue-specific gene transcription can be determined by the use of either positive-acting or negative-acting DNA regulatory elements. We have analyzed a promoter from the growth-associated protein 43 (GAP-43) gene and found that it uses both of these mechanisms to achieve its high degree of neuron-specific activity. Two novel transcription factor binding sites, designated Cx1 and Cx2, drive promoter activity in neurons from developing cerebral cortex but not in several other cell types. The promoter also contains an activator protein 1 (AP-1) site that contributes to activity in neurons. The AP-1 site can drive promoter activity in a wide range of non-neuronal cells that express little or no endogenous GAP-43, but only in the absence of a tissue-specific repressive element located downstream of the GAP-43 TATA box. These findings suggest that the GAP-43 repressive element plays an important role in allowing AP-1 signaling pathways to modulate activity of the GAP-43 gene in neurons, without also causing inappropriate activation by AP-1 transcription factors in other cell types.