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Inflammation is a critically important barrier found in innate immunity. However, severe and sustained inflammatory conditions are regarded as causes of many different serious diseases, such as cancer, atherosclerosis, and diabetes. Although numerous studies have addressed how inflammatory responses proceed and what kinds of proteins and cells are involved, the exact mechanism and protein components regulating inflammatory reactions are not fully understood. In this paper, to determine the regulatory role of mixed lineage kinase 3 (MLK3), which functions as mitogen-activated protein kinase kinase kinase (MAP3K) in cancer cells in inflammatory response to macrophages, we employed an overexpression strategy with MLK3 in HEK293 cells and used its inhibitor URMC-099 in lipopolysaccharide (LPS)-treated RAW264.7 cells. It was found that overexpressed MLK3 increased the mRNA expression of inflammatory genes (COX-2, IL-6, and TNF-α) via the activation of AP-1, according to a luciferase assay carried out with AP-1-Luc. Overexpression of MLK3 also induced phosphorylation of MAPKK (MEK1/2, MKK3/6, and MKK4/7), MAPK (ERK, p38, and JNK), and AP-1 subunits (c-Jun, c-Fos, and FRA-1). Phosphorylation of MLK3 was also observed in RAW264.7 cells stimulated by LPS, Pam3CSK, and poly(I:C). Finally, inhibition of MLK3 by URMC-099 reduced the expression of COX-2 and CCL-12, phosphorylation of c-Jun, luciferase activity mediated by AP-1, and phosphorylation of MAPK in LPS-treated RAW264.7 cells. Taken together, our findings strongly suggest that MLK3 plays a central role in controlling AP-1-mediated inflammatory responses in macrophages and that this enzyme can serve as a target molecule for treating AP-1-mediated inflammatory diseases. [ABSTRACT FROM AUTHOR]

Penaeidins are members of an antimicrobial peptide (AMP) family that have broad anti-microbial activities only found in penaeid shrimps. The LvBigPEN, a member of penaeidins from shrimp Litopenaeus vannamei, has showed antiviral activity against white spot syndrome virus (WSSV) in our previous report. However, whether LvBigPEN possesses potential anti-bacterial activities is still unknown. Herein, we found that the LvBigPEN played an important role in restricting the infection of Vibrio parahaemolyticus, a natural and Gram-negative bacteria pathogen in shrimp. The transcription of LvBigPEN was strongly induced after V. parahaemolyticus challenge. RNA interference (RNAi) mediated knockdown of LvBigPEN showed that LvBigPEN had a potential antibacterial function against V. parahaemolyticus. Microorganism binding assays indicated that rLvBigPEN could bind to both Gram-negative bacteria and Gram-positive bacteria. Transmission electron microscopy (TEM) analysis showed its ability to destroy bacterial cells in vitro. Besides, in a gel retardation assay, rLvBigPEN could bind to plasmid DNA and bacteria (V. parahaemolyticus) genomic DNA in a concentration-dependent manner. Moreover, the AP-1 pathway could participate in the transcription of LvBigPEN by the dual luciferase reporter assays. Taken together, these results suggested that LvBigPEN possessed the antibacterial activity against V. parahaemolyticus and may be alternative agents for the prevention and treatment of diseases caused by V. parahaemolyticus.

The c-Jun NH2-terminal kinases (JNKs) are an evolutionarily conserved family of serine/threonine protein kinases that play critical roles in the pathological process in species ranging from insects to mammals. However, the function of JNKs in bacteria-induced intestinal inflammation is still poorly understood. In this study, a fish JNK (CiJNK) pathway was identified, and its potential roles in bacterial muramyl dipeptide (MDP)-induced intestinal inflammation were investigated in Ctenopharyngodon idella. The present CiJNK was found to possess a conserved dual phosphorylation motif (TPY) in a serine/threonine protein kinase (S_TKc) domain and to contain several potential immune-related transcription factor binding sites, including nuclear factor kappa B (NF-κB), activating protein 1 (AP-1), and signal transducer and activator of downstream transcription 3 (STAT3), in its 5′ flanking regions. Quantitative real-time PCR results revealed that the mRNA levels of the JNK pathway genes in the intestine were significantly upregulated after challenge with a bacterial pathogen (Aeromonas hydrophila) and MDP in a time-dependent manner. Additionally, the JNK pathway was found to be involved in regulating the MDP-induced expression levels of inflammatory cytokines (IL-6, IL-8, and TNF-α) in the intestine of grass carp. Moreover, the nutritional dipeptide carnosine and Ala–Gln could effectively alleviate MDP-induced intestinal inflammation by regulating the intestinal expression of JNK pathway genes and inflammatory cytokines in grass carp. Finally, fluorescence microscopy and dual-reporter assays indicated that CiJNK could associate with CiMKK4 and CiMKK7 involved in the regulation of the AP-1 signaling pathway. Overall, these results provide the first experimental demonstration that the JNK signaling pathway is involved in the intestinal immune response to MDP challenge in C. idella, which may provide new insight into the pathogenesis of inflammatory bowel disease.

The c-Jun NH2-terminal kinases (JNKs) are an evolutionarily conserved family of serine/threonine protein kinases that play critical roles in the pathological process in species ranging from insects to mammals. However, the function of JNKs in bacteria-induced intestinal inflammation is still poorly understood. In this study, a fish JNK (Ci JNK) pathway was identified, and its potential roles in bacterial muramyl dipeptide (MDP)-induced intestinal inflammation were investigated in Ctenopharyngodon idella. The present Ci JNK was found to possess a conserved dual phosphorylation motif (TPY) in a serine/threonine protein kinase (S_TKc) domain and to contain several potential immune-related transcription factor binding sites, including nuclear factor kappa B (NF-κB), activating protein 1 (AP-1), and signal transducer and activator of downstream transcription 3 (STAT3), in its 5′ flanking regions. Quantitative real-time PCR results revealed that the mRNA levels of the JNK pathway genes in the intestine were significantly upregulated after challenge with a bacterial pathogen (Aeromonas hydrophila) and MDP in a time-dependent manner. Additionally, the JNK pathway was found to be involved in regulating the MDP-induced expression levels of inflammatory cytokines (IL-6, IL-8, and TNF-α) in the intestine of grass carp. Moreover, the nutritional dipeptide carnosine and Ala–Gln could effectively alleviate MDP-induced intestinal inflammation by regulating the intestinal expression of JNK pathway genes and inflammatory cytokines in grass carp. Finally, fluorescence microscopy and dual-reporter assays indicated that Ci JNK could associate with Ci MKK4 and Ci MKK7 involved in the regulation of the AP-1 signaling pathway. Overall, these results provide the first experimental demonstration that the JNK signaling pathway is involved in the intestinal immune response to MDP challenge in C. idella , which may provide new insight into the pathogenesis of inflammatory bowel disease. [ABSTRACT FROM AUTHOR]

Prasiola japonica possesses several biological activities. However, reports on the anti-inflammatory activities and molecular mechanisms of its different solvent fractions remain limited. In this study, we investigated the potential anti-inflammatory activities of P. japonica ethanol extract (Pj-EE) and four solvent fractions of Pj-EE made with hexane (Pj-EE-HF), chloroform (Pj-EE-CF), butanol (Pj-EE-BF), or water (Pj-EE-WF) in both in vitro (LPS-induced macrophage-like RAW264.7 cells) and in vivo (carrageenan-induced acute paw edema mouse models) experiments. The most active solvent fraction was selected for further analysis. Various in vitro and in vivo assessments, including nitric oxide (NO), cytokines, luciferase assays, real-time polymerase chain reactions, and immunoblotting analyses were performed to evaluate the underlying mechanisms. In addition, the phytochemical constituents were characterized by Liquid chromatography-tandem mass spectrometry. In in vitro studies, the highest inhibition of NO production was observed in Pj-EE-CF. Further examination revealed that Pj-EE-CF decreased the expression of inflammation-related cytokines in LPS-induced RAW264.7 cells and suppressed subsequent AP-1-luciferase activity by inhibition of phosphorylation events in the AP-1 signaling pathway. Pj-EE-CF treatment also demonstrated the strongest reduction in thickness and volume of carrageenan-induced paw edema, while Pj-EE-BF showed the lowest activity. Furthermore, Pj-EE-CF also reduced gene expression and cytokines production in tissue lysates of carrageenan-induced paw edema. These findings support and validate the evidence that Pj-EE, and especially Pj-EE-CF, could be a good natural source for an anti-inflammatory agent that targets the AP1 pathway.

IL-13Rα2 is a high-affinity binding protein for its ligand IL-13 and a cancer-testis antigen as it is expressed in the testis. IL-13Rα2 is highly expressed in various cancers, including pancreatic cancer, and consists of three domains: extracellular, transmembrane, and cytoplasmic. The extracellular domain binds to the ligand to form a biologically active complex, which initiates signaling through AP-1 and other pathways. IL-13Rα2 is also expressed in diseased cells such as fibroblasts that are involved in various inflammatory diseases, including cancer. We have reported that IL-13Rα2 is a prognostic biomarker for malignant glioma, adrenocortical cancer, and pancreatic cancer. In pancreatic cancer, a small sample of tissue could be examined for the expression of IL-13Rα2 by using the endoscopic ultrasound-fine needle aspiration technique (EUS-FNA). In addition, a peptide-based targeted approach using Pep-1L peptide could be used to study the biodistribution and whole-body cancer imaging for the screening of pancreatic cancer in suspected subjects.

• Transcript levels of p38MAPK pathway genes could be significantly induced by bacterial challenge. • The MDP-induced expression levels of intestinal immune cytokines were significantly inhibited by SB203580. • The stimulating effects of MDP on intestinal p38MAPK and immune cytokines were inhibited by dipeptide nutritional. • Ci p38α/ Ci p38β could associate with Ci MKK6 involved in the regulation of the AP-1 pathway. The p38 mitogen-activated protein kinases (MAPKs) are essential cytoplasmic signal molecules of innate immune pathways that play a vital role in host immune defense responses to pathogenic challenges. In this study, two fish p38 genes (Ci p38α and Ci p38β) were characterized for the first time from the grass carp Ctenopharyngodon idella. Similar to other reported p38MAPKs, both Ci p38α and Ci p38β contained a conserved phosphorylation motif (Thr-Gly-Tyr, TGY) and a substrate binding site (Ala-Thr-Arg-Trp, ATRW) in the serine/threonine protein kinase (S_TKc) domain. Expression profile analysis showed that Ci p38α and Ci p38β mRNAs were broadly expressed in all of the examined tissues and developmental stages of C. idella. In addition, in vivo injection experiments directly revealed that Ci p38α and Ci p38β showed strong responsiveness to Aeromonas hydrophila and muramyl dipeptide (MDP) challenges, and their expression levels were significantly upregulated in the intestine of grass carp. Additionally, the MDP-induced expression levels of intestinal inflammatory cytokines (TNF-α and IL-15) and an antimicrobial peptide (β-defensin) were significantly inhibited by the p38MAPK-specific inhibitor SB203580. Moreover, the nutritional dipeptide carnosine and Ala-Gln were found to significantly suppress the bacterial MDP-induced expression of p38MAPK pathway genes and inflammatory cytokines in the intestine of grass carp. Finally, overexpression analysis demonstrated that Ci p38α and Ci p38β could act as efficient activators in the regulation of AP-1 signaling pathways through interaction with Ci MKK6. Altogether, this study provided experimental evidence of the presence of a functional p38 pathway in grass carp, which revealed its involvement in the intestinal immune response to bacterial challenges in bony fish. [ABSTRACT FROM AUTHOR]

In this study, we investigated the functional role of isoprenylcysteine carboxyl methyltransferase (ICMT) and its methylatable substrate Ras in Toll-like receptor (TLR)-activated macrophages and in mouse inflammatory disease conditions. ICMT and RAS expressions were strongly increased in macrophages under the activation conditions of TLRs by lipopolysaccharide (LPS, a TLR4 ligand), pam3CSK (TLR2), or poly(I:C) (TLR3) and in the colons, stomachs, and livers of mice with colitis, gastritis, and hepatitis. The inhibition and activation of ICMT and Ras through genetic and pharmacological approaches significantly affected the activation of interleukin-1 receptor-associated kinase (IRAK)s, tumor necrosis factor receptor associated factor 6 (TRAF6), transforming growth factor-β-activated kinase 1 (TAK1), mitogen-activated protein kinase (MAPK), and MAPK kinases (MAPKKs); translocation of the AP-1 family; and the expressions of inflammation-related genes that depend on both MyD88 and TRIF. Interestingly, the Ras/ICMT-mediated inflammatory reaction critically depends on the TIR domains of myeloid differentiation primary response 88 (MyD88) and TIR-domain-containing adapter-inducing interferon-β (TRIF). Taken together, these results suggest that ICMT and its methylated Ras play important roles in the regulation of inflammatory responses through cooperation with the TIR domain of adaptor molecules.

Ring1 and YY1 Binding Protein (RYBP) induces tumor-specific cell apoptosis, but the underlying molecular mechanism has not been fully understood. Here we conducted a yeast two hybrid screen and identified FANK1 (Fibronectin type III and ankyrin repeat domains 1) as a novel RYBP-interacting protein. This interaction was confirmed by coimmunoprecipitation, GST pulldown and immunofluorescence assays. We mapped that the FNIII domain at the N-terminal of FANK1 binds to the Serine/Threonine-rich region at the C-terminal of RYBP. Further studies showed that overexpression of RYBP stabilized, whereas knockdown of RYBP by its specific shRNAs reduced, the expression of FANK1. Mechanistic studies revealed that RYBP inhibited the proteasome degradation of polyubiquitinated FANK1, thus prolonging the half-life of FANK1 protein. Functional studies indicated that RYBP activates FANK1-mediated activator protein 1 (AP-1) signaling pathway which contributes to tumor cell apoptosis. Taken together, our current study uncovered a new mechanism which RYBP utilizes to exert its pro-apoptotic activity in human tumor cells. [ABSTRACT FROM AUTHOR]

The transcription factor Fos is a member of one of the best-studied AP-1 sub-families and has been implicated in a wide variety of biological processes, including the regulation of apoptosis, immune responses and cytokine production. In this report, a novel mollusk Fos (referred to as Ch Fos) gene was cloned and characterized from the Hong Kong oyster, Crassostrea hongkongensis . The deduced Ch Fos protein sequence comprised 333 amino acids and shared significant homology with invertebrate homologs. Phylogenetic analysis revealed that Ch Fos clusters with Fos from Crassostrea gigas and Crassostrea ariakensis . Quantitative real-time PCR analysis revealed that ChFos mRNA was broadly expressed in all tested tissues and during different stages of the oyster's embryonic and larval development. In addition, the expression of ChFos mRNA was significantly up-regulated under challenge with microorganisms ( Vibrio alginolyticus , Staphylococcus haemolyticus and Saccharomyces cerevisiae ) and pathogen-associated molecular patterns (PAMPs: LPS, PGN and polyI:C). Moreover, fluorescence microscopy showed that Ch Fos protein is localized in the nucleus in HEK293T cells. Reporter assays suggested that Ch Fos may act as an efficient transcription activator in the regulation of AP-1-responsive gene expression through interaction with Ch Jun. Overall, this study presents the first experimental evidence of the presence and functional characteristics of Fos in mollusks, which reveals its involvement in host protection against immune challenge in the oyster. [ABSTRACT FROM AUTHOR]

Abstract: Shuang-Huang-Lian (SHL) is a traditional Chinese compound formula prepared from Lonicerae Japonicae Flos, Scutellariae Radix and Fructus Forsythiae. In this study, we demonstrate the anti-inflammatory and anti-oxidative activities of SHL in lipopolysaccharide (LPS)-stimulated murine alveolar macrophages (MH-S). SHL significantly reduces the transcriptional and translational levels of iNOS and COX-2 as well as the production of NO and prostaglandin E2 (PGE2). It also suppresses the transcription and translation of inflammatory cytokines production, such as TNF-α, IL-1β and IL-6. These inhibitory effects mainly act via ERK1/2- and p38-mediated AP-1 rather than the NF-κB pathway. In parallel with the anti-inflammatory activity, SHL suppresses LPS-induced intracellular total ROS levels by weakening NADPH oxidase activity, enhancing SOD activity and increasing GSH content. In addition, SHL directly scavenges OH and O2 −. Thus, our study elucidates the anti-inflammatory and anti-oxidative mechanisms of SHL in LPS-stimulated MH-S. [Copyright &y& Elsevier]

The transcription factor p53 and AP-1 play an important role in cellular proliferation, transformation and death. In this study, we investigated the role of a novel human gene, TTC5 (tetratricopeptide repeat domain 5), in the regulation of cell signaling pathway and cell viability. TTC5 is a member of the TTC family of proteins and has previously been shown to participate in cellular stress response. Here we demonstrate for the first time that TTC5 significantly activates p53 pathway and inhibits AP-1 transcriptional activity. Further investigation revealed that overexpression of TTC5 up-regulated p53 and p21 expression, and significantly inhibited transcriptional activity, expression and phosphorylation of c-Jun. As for the upstream of signaling pathway of AP-1, our study demonstrated that overexpression of TTC5 significantly down-regulated the expression and phosphorylation of JNK/SAPK. Moreover, overexpression of TTC5 repressed cell proliferation and induced S phase cell cycle arrest. These results indicated that TTC5 may regulate cell viability by p53 and AP-1 signaling pathway. [ABSTRACT FROM AUTHOR]

Abstract: Ormeloxifene is a selective estrogen receptor modulator that exerts antiestrogenic effects and thereby inhibits growth in uterus. The present study was undertaken to examine the AP-1 protein interaction with AP-1 enhancer DNA elements in rat uterus in vivo and in vitro with a view to explore the modulation of estrogen action mediated via alternative pathway under the influence of ormeloxifene (Orm). In addition, the changes in expression of c-fos and c-jun transcription factors and mRNA expression of growth factor (IGF-1) were investigated with a view to assess the AP-1 mediated transcription. Ovariectomizedoung adult rats were administered with estradiol-17β (5μg/100g body weight) or Orm (200μg/100g body weight) or vehicle for 3 days and sacrificed on fourth day. Electrophoretic mobility shift assay using uterine nuclear fraction from various treatment groups demonstrated that Orm caused a significant reduction in E 2 induced AP-1 DNA binding. In vitro study revealed that Orm promotes AP-1 complex formation whereas its 7-hydroxy derivative inhibits it significantly. Uterine expression of c-fos and c-jun was increased significantly in Orm treated rats as compared to vehicle treated rats. However, the expression of c-fos and c-jun was decreased in rats receiving Orm plus E 2. Semi-quantitative RT-PCR analysis revealed that mRNA expression of IGF-1 was increased in E 2 treated group as compared to control group whereas reduced expression was observed in Orm treated rats as compared to E 2 treated rats. The uterine weight and IGF-1 mRNA showed similar pattern, indicating that IGF-1 is involved in regulation of uterine weight. These results indicate that 7-hydroxy ormeloxifene (an active metabolite of Orm) is a potent antagonist at AP-1 sites. It inhibits the function of AP-1 transcription factors rather than their expression as evident by downregulation of mRNA expression of AP-1 regulated gene IGF-1, thereby inhibits proliferation in rat uterus. Study suggested a non-classicalregulation of estrogen action on uterus by ormeloxifene. [Copyright &y& Elsevier]

Prasiola japonica possesses several biological activities. However, reports on the anti-inflammatory activities and molecular mechanisms of its different solvent fractions remain limited. In this study, we investigated the potential anti-inflammatory activities of P. japonica ethanol extract (Pj-EE) and four solvent fractions of Pj-EE made with hexane (Pj-EE-HF), chloroform (Pj-EE-CF), butanol (Pj-EE-BF), or water (Pj-EE-WF) in both in vitro (LPS-induced macrophage-like RAW264.7 cells) and in vivo (carrageenan-induced acute paw edema mouse models) experiments. The most active solvent fraction was selected for further analysis. Various in vitro and in vivo assessments, including nitric oxide (NO), cytokines, luciferase assays, real-time polymerase chain reactions, and immunoblotting analyses were performed to evaluate the underlying mechanisms. In addition, the phytochemical constituents were characterized by Liquid chromatography-tandem mass spectrometry. In in vitro studies, the highest inhibition of NO production was observed in Pj-EE-CF. Further examination revealed that Pj-EE-CF decreased the expression of inflammation-related cytokines in LPS-induced RAW264.7 cells and suppressed subsequent AP-1-luciferase activity by inhibition of phosphorylation events in the AP-1 signaling pathway. Pj-EE-CF treatment also demonstrated the strongest reduction in thickness and volume of carrageenan-induced paw edema, while Pj-EE-BF showed the lowest activity. Furthermore, Pj-EE-CF also reduced gene expression and cytokines production in tissue lysates of carrageenan-induced paw edema. These findings support and validate the evidence that Pj-EE, and especially Pj-EE-CF, could be a good natural source for an anti-inflammatory agent that targets the AP1 pathway. [ABSTRACT FROM AUTHOR]

IL-13Rα2 is a high-affinity binding protein for its ligand IL-13 and a cancer-testis antigen as it is expressed in the testis. IL-13Rα2 is highly expressed in various cancers, including pancreatic cancer, and consists of three domains: extracellular, transmembrane, and cytoplasmic. The extracellular domain binds to the ligand to form a biologically active complex, which initiates signaling through AP-1 and other pathways. IL-13Rα2 is also expressed in diseased cells such as fibroblasts that are involved in various inflammatory diseases, including cancer. We have reported that IL-13Rα2 is a prognostic biomarker for malignant glioma, adrenocortical cancer, and pancreatic cancer. In pancreatic cancer, a small sample of tissue could be examined for the expression of IL-13Rα2 by using the endoscopic ultrasound-fine needle aspiration technique (EUS-FNA). In addition, a peptide-based targeted approach using Pep-1L peptide could be used to study the biodistribution and whole-body cancer imaging for the screening of pancreatic cancer in suspected subjects. [ABSTRACT FROM AUTHOR]

In this study, we investigated the functional role of isoprenylcysteine carboxyl methyltransferase (ICMT) and its methylatable substrate Ras in Toll-like receptor (TLR)-activated macrophages and in mouse inflammatory disease conditions. ICMT and RAS expressions were strongly increased in macrophages under the activation conditions of TLRs by lipopolysaccharide (LPS, a TLR4 ligand), pam3CSK (TLR2), or poly(I:C) (TLR3) and in the colons, stomachs, and livers of mice with colitis, gastritis, and hepatitis. The inhibition and activation of ICMT and Ras through genetic and pharmacological approaches significantly affected the activation of interleukin-1 receptor-associated kinase (IRAK)s, tumor necrosis factor receptor associated factor 6 (TRAF6), transforming growth factor-β-activated kinase 1 (TAK1), mitogen-activated protein kinase (MAPK), and MAPK kinases (MAPKKs); translocation of the AP-1 family; and the expressions of inflammation-related genes that depend on both MyD88 and TRIF. Interestingly, the Ras/ICMT-mediated inflammatory reaction critically depends on the TIR domains of myeloid differentiation primary response 88 (MyD88) and TIR-domain-containing adapter-inducing interferon-β (TRIF). Taken together, these results suggest that ICMT and its methylated Ras play important roles in the regulation of inflammatory responses through cooperation with the TIR domain of adaptor molecules. [ABSTRACT FROM AUTHOR]