Antigen presentation capability and AP-1 activation accompany methotrexate-induced colon cancer cell senescence in the context of aberrant ß-catenin signaling.

Reversible cellular senescence was demonstrated previously to constitute colon cancer cell response to methotrexate. The current study presents a comparison of two senescent states of colon cancer cells, arrested and reversing, resulting from respectively, 120 h exposure to the drug, and 48 h exposure followed by 96 h regrowth in drug-free media. The upregulation of immunoproteasome subunit-coding genes and the increase in human leukocyte antigen HLA-A/B/C membrane level indicated MHC-I-restricted antigen presentation as common to both senescent states. Nuclear factor NF-κB p65 level decreased and activating protein AP-1: c-Jun, Fra2 and JunB nuclear levels increased in both senescent cell populations. Notably, the increase in AP-1- dependent transcription occurred after 48 h exposure to methotrexate. ß-catenin nuclear level increased after 48 h exposure to the drug and remained as such only in senescence-arrested cells. ß-catenin level was found uncoupled from the protein phosphorylation status indicating the deregulation of ß-catenin signaling in colon cancer cells employed in the study. These findings carry implications for both, a general mechanism of senescence establishment and putative advantages for colon cancer treatment.

Nc886, a Novel Suppressor of the Type I Interferon Response Upon Pathogen Intrusion.

Interferons (IFNs) are a crucial component in the innate immune response. Especially the IFN-ß signaling operates in most cell types and plays a key role in the first line of defense upon pathogen intrusion. The induction of IFN-ß should be tightly controlled, because its hyperactivation can lead to tissue damage or autoimmune diseases. Activation of the IFN-ß promoter needs Interferon Regulatory Factor 3 (IRF3), together with Nuclear Factor kappa-light-chain-enhancer of activated B cells (NF-κB) and Activator Protein 1 (AP-1). Here we report that a human noncoding RNA, nc886, is a novel suppressor for the IFN-ß signaling and inflammation. Upon treatment with several pathogen-associated molecular patterns and viruses, nc886 suppresses the activation of IRF3 and also inhibits NF-κB and AP-1 via inhibiting Protein Kinase R (PKR). These events lead to decreased expression of IFN-ß and resultantly IFN-stimulated genes. nc886's role might be to restrict the IFN-ß signaling from hyperactivation. Since nc886 expression is regulated by epigenetic and environmental factors, nc886 might explain why innate immune responses to pathogens are variable depending on biological settings.

The Clusters of Transcription Factors NFATC2, STAT5, GATA2, AP1, RUNX1 and EGR2 Binding Sites at the Induced Il13 Enhancers Mediate Il13 Gene Transcription in Response to Antigenic Stimulation.

IL-13 plays a critical role in mediating many biological processes responsible for allergic inflammation. Mast cells express Il13 mRNA and produce IL-13 protein in response to antigenic stimulation. Enhancers are essential in promoting gene transcription and are thought to activate transcription by delivering essential accessory cofactors to the promoter to potentiate gene transcription. However, enhancers mediating Il13 have not been identified. Furthermore, which Il13 enhancers detect signals triggered by antigenic stimulation have not yet been defined. In this study, we identified potential mouse Il13 enhancers using histone modification monomethylation at lysine residue 4 on histone 3 (H3K4me1) chromatin immunoprecipitation sequencing and acetylation at lysine residue 27 on histone 3 (H3K27ac) chromatin immunoprecipitation sequencing. We used Omni-assay for transposase-accessible chromatin sequencing to determine which accessible regions within the potential Il13 enhancers that responded to IgE receptor crosslinking. We also demonstrated that the transcription factor cluster consisting of the NFATC2, STAT5, GATA2, AP1, and RUNX1 binding sites at the proximal Il13 enhancer and the transcription factor cluster consisting of the EGR2 binding site at the distal Il13 E+6.5 enhancer are critical in sensing the signals triggered by antigenic stimulation. Those enhancers, which are responsive to antigenic stimulation and are constitutively active, cooperate to generate greater transcriptional outputs. Our study reveals a novel mechanism underlying how antigenic stimulation induces robust Il13 mRNA expression in mouse mast cells.

Isodeoxyelephantopin, a sesquiterpene lactone from Elephantopus scaber Linn., inhibits pro-inflammatory mediators' production through both NF-κB and AP-1 pathways in LPS-activated macrophages.

Isodeoxyelephantopin (IDET) has been identified as an anti-tumor natural constituent whose anti-tumor activity and mechanism have been widely investigated. Since the occurrence and development of cancer usually accompany with inflammation, and tumor signaling shares many components with inflammation signaling, the agents with anti-tumor activity are likely to possess anti-inflammation potential. Thus, the current study aims to demonstrate the anti-inflammatory activity along with the underlying mechanism of IDET in lipopolysaccharide (LPS)-primed macrophages. By using Griess method and ELISA, we found that in both bone marrow derived macrophages and alveolar macrophage cell line, IDET, at relatively low concentrations (0.75, 1.5 and 3 µM), could inhibit LPS-induced expression of various pro-inflammatory mediators including nitric oxide (NO) generated by inducible nitric oxide synthase (iNOS), interleukin (IL)-6, monocyte chemotactic protein-1 (MCP-1) and IL-1ß. Meanwhile, in activated MH-S cells, the inhibitory action of IDET on mRNA expression levels of these cytokines was also detected using qPCR. Mechanistically, the effects of IDET on two key inflammatory signalings, nuclear factor-κB (NF-κB) and activator protein-1 (AP-1) pathways, were determined in LPS-activated MH-S cells by reporter gene along with western blot assays. On the one hand, IDET suppressed NF-κB signaling via down-regulating phosphorylation and degradation of inhibitor of NF-κB (IκB)-α and the subsequent p65 translocation. On the other hand, IDET dampened AP-1 signaling through attenuating phosphorylation of both c-jun N-terminal kinase 1/2 (JNK1/2) and extracellular signal regulated kinase 1/2 (ERK1/2). Our study indicates that IDET might be a promising constituent from the anti-inflammatory herb Elephantopus scaber Linn. in mitigating inflammatory conditions, especially respiratory inflammation.

c-Jun overexpression in CAR T cells induces exhaustion resistance.

Chimeric antigen receptor (CAR) T cells mediate anti-tumour effects in a small subset of patients with cancer1-3, but dysfunction due to T cell exhaustion is an important barrier to progress4-6. To investigate the biology of exhaustion in human T cells expressing CAR receptors, we used a model system with a tonically signaling CAR, which induces hallmark features of exhaustion6. Exhaustion was associated with a profound defect in the production of IL-2, along with increased chromatin accessibility of AP-1 transcription factor motifs and overexpression of the bZIP and IRF transcription factors that have been implicated in mediating dysfunction in exhausted T cells7-10. Here we show that CAR T cells engineered to overexpress the canonical AP-1 factor c-Jun have enhanced expansion potential, increased functional capacity, diminished terminal differentiation and improved anti-tumour potency in five different mouse tumour models in vivo. We conclude that a functional deficiency in c-Jun mediates dysfunction in exhausted human T cells, and that engineering CAR T cells to overexpress c-Jun renders them resistant to exhaustion, thereby addressing a major barrier to progress for this emerging class of therapeutic agents.

Insight into the molecular function and transcriptional regulation of activator protein 1 (AP-1) components c-Jun/c-Fos ortholog in red lip mullet (Liza haematocheila).

The transcription factor, activator protein-1 (AP-1), is a dimeric protein and a downstream member of the mitogen-activated protein kinase (MAPK) signaling pathway. It regulates a wide array of functions including, cell proliferation, survival, differentiation, response to UV-irradiation, immune responses, and inflammatory conditions. AP-1 belongs to the basic leucine zipper (bZIP) protein family, which consists of members from Jun, Fos, Maf, and ATF subfamilies. In the present study, c-Jun and c-Fos homologs were identified from a transcriptome database of Liza haematocheila and designated as Lhc-Jun and Lhc-Fos. In both sequences, the signature bZIP domain was identified and also the DNA binding sites, dimerization sites, as well as the phosphorylation sites, were found to be highly conserved through evolution. Tissue distribution analysis revealed that both Lhc-Jun and Lhc-Fos transcripts were ubiquitously expressed in all examined tissues of healthy mullets. In order to determine the transcriptional modulations of Lhc-Jun and Lhc-Fos, challenge experiments were carried out using LPS, poly I:C, and L. garvieae. The qRT-PCR analysis revealed significant upregulation of Lhc-Jun and Lhc-Fos in blood, gill, liver, and spleen. This is the first study that explores the correlation between UV-irradiation and AP-1 ortholog expression in teleosts. Also, this is the first time that the functional characterization of the teleost c-Fos ortholog has been carried out. Sub-cellular localization of Lhc-Jun and Lhc-Fos was observed in the nucleus. AP-1-Luc reporter assays revealed significant higher luciferase activities in both Lhc-Jun and Lhc-Fos proteins compared to mock controls. These results strongly suggest that Lhc-Jun and Lhc-Fos might play a significant role in Liza haematocheila immunity by regulating AP-1 promoter sequences in immune and stress-related genes.

NLRX1 Facilitates Histoplasma capsulatum-Induced LC3-Associated Phagocytosis for Cytokine Production in Macrophages.

LC3-associated phagocytosis (LAP) is an emerging non-canonical autophagy process that bridges signaling from pattern-recognition receptors (PRRs) to autophagic machinery. LAP formation results in incorporation of lipidated LC3 into phagosomal membrane (termed LAPosome). Increasing evidence reveals that LAP functions as an innate defense mechanism against fungal pathogens. However, the molecular mechanism involved and the consequence of LAP in regulating anti-fungal immune response remain largely unexplored. Here we show that Histoplasma capsulatum is taken into LAPosome upon phagocytosis by macrophages. Interaction of H. capsulatum with Dectin-1 activates Syk and triggers subsequent NADPH oxidase-mediated reactive oxygen species (ROS) response that is involved in LAP induction. Inhibiting LAP induction by silencing LC3α/ß or treatment with ROS inhibitor impairs the activation of MAPKs-AP-1 pathway, thereby reduces macrophage proinflammatory cytokine response to H. capsulatum. Additionally, we unravel the importance of NLRX1 in fungus-induced LAP. NLRX1 facilitates LAP by interacting with TUFM which associates with autophagic proteins ATG5-ATG12 for LAPosome formation. Macrophages from Nlrx1-/- mice or TUFM-silenced cells exhibit reduced LAP induction and LAP-mediated MAPKs-AP-1 activation for cytokine response to H. capsulatum. Furthermore, inhibiting ROS production in Nlrx1-/- macrophages almost completely abolishes H. capsulatum-induced LC3 conversion, indicating that both Dectin-1/Syk/ROS-dependent pathway and NLRX1-TUFM complex-dependent pathway collaboratively contribute to LAP induction. Our findings reveal new pathways underlying LAP induction by H. capsulatum for macrophage cytokine response.

Holding All the CARDs: How MALT1 Controls CARMA/CARD-Dependent Signaling.

The scaffold proteins CARMA1-3 (encoded by the genes CARD11, -14 and -10) and CARD9 play major roles in signaling downstream of receptors with immunoreceptor tyrosine activation motifs (ITAMs), G-protein coupled receptors (GPCR) and receptor tyrosine kinases (RTK). These receptors trigger the formation of oligomeric CARMA/CARD-BCL10-MALT1 (CBM) complexes via kinases of the PKC family. The CBM in turn regulates gene expression by the activation of NF-κB and AP-1 transcription factors and controls transcript stability. The paracaspase MALT1 is the only CBM component having an enzymatic (proteolytic) activity and has therefore recently gained attention as a potential drug target. Here we review recent advances in the understanding of the molecular function of the protease MALT1 and summarize how MALT1 scaffold and protease function contribute to the transmission of CBM signals. Finally, we will highlight how dysregulation of MALT1 function can cause pathologies such as immunodeficiency, autoimmunity, psoriasis, and cancer.

Mutant Cellular AP-1 Proteins Promote Expression of a Subset of Epstein-Barr Virus Late Genes in the Absence of Lytic Viral DNA Replication.

Epstein-Barr virus (EBV) ZEBRA protein activates the EBV lytic cycle. Cellular AP-1 proteins with alanine-to-serine [AP-1(A/S)] substitutions homologous to ZEBRA(S186) assume some functions of EBV ZEBRA. These AP-1(A/S) mutants bind methylated EBV DNA and activate expression of some EBV genes. Here, we compare expression of 67 viral genes induced by ZEBRA versus expression induced by AP-1(A/S) proteins. AP-1(A/S) activated 24 genes to high levels and 15 genes to intermediate levels; activation of 28 genes by AP-1(A/S) was severely impaired. We show that AP-1(A/S) proteins are defective at stimulating viral lytic DNA replication. The impairment of expression of many late genes compared to that of ZEBRA is likely due to the inability of AP-1(A/S) proteins to promote viral DNA replication. However, even in the absence of detectable viral DNA replication, AP-1(A/S) proteins stimulated expression of a subgroup of late genes that encode viral structural proteins and immune modulators. In response to ZEBRA, expression of this subgroup of late genes was inhibited by phosphonoacetic acid (PAA), which is a potent viral replication inhibitor. However, when the lytic cycle was activated by AP-1(A/S), PAA did not reduce expression of this subgroup of late genes. We also provide genetic evidence, using the BMRF1 knockout bacmid, that these genes are true late genes in response to ZEBRA. AP-1(A/S) binds to the promoter region of at least one of these late genes, BDLF3, encoding an immune modulator.IMPORTANCE Mutant c-Jun and c-Fos proteins selectively activate expression of EBV lytic genes, including a subgroup of viral late genes, in the absence of viral DNA replication. These findings indicate that newly synthesized viral DNA is not invariably required for viral late gene expression. While viral DNA replication may be obligatory for late gene expression driven by viral transcription factors, it does not limit the ability of cellular transcription factors to activate expression of some viral late genes. Our results show that expression of all late genes may not be strictly dependent on viral lytic DNA replication. The c-Fos A151S mutation has been identified in a human cancer. c-Fos A151S in combination with wild-type c-Jun activates the EBV lytic cycle. Our data provide proof of principle that mutant cellular transcription factors could cause aberrant regulation of viral lytic cycle gene expression and play important roles in EBV-associated diseases.

The Antioxidant and Anti-Inflammatory Activities of 8-Hydroxydaidzein (8-HD) in Activated Macrophage-Like RAW264.7 Cells.

8-Hydroxydaidzein (8-HD) is a daidzein metabolite isolated from soybeans. This compound has been studied for its anti-proliferation, depigmentation, and antioxidant activities. However, the anti-inflammatory activities of 8-HD are not well-understood. Through its antioxidant effects in ABTS and DPPH assays, 8-HD reduces the production of sodium nitroprusside (SNP)-induced radical oxygen species (ROS). By triggering various Toll-like receptors (TLRs), 8-HD suppresses the inflammatory mediator nitric oxide (NO) without cytotoxicity. We examined the regulatory mechanism of 8-HD in lipopolysaccharide (LPS)-induced conditions. We found that 8-HD diminishes inflammatory gene expression (e.g., inducible nitric oxide synthase (iNOS), cyclooxygenase (COX)-2, and tumor necrosis factor (TNF)-α) by regulating the transcriptional activities of nuclear factor (NF)-κB and activator protein 1 (AP-1). To find the potential targets of 8-HD, signaling pathways were investigated by immunoblotting analyses. These analyses revealed that 8-HD inhibits the activation of TAK1 and that phosphorylated levels of downstream molecules decrease in sequence. Together, our results demonstrate the antioxidant and anti-inflammatory actions of 8-HD and suggest its potential use in cosmetics or anti-inflammatory drugs.

JunB promotes Th17 cell identity and restrains alternative CD4+ T-cell programs during inflammation.

T helper 17 (Th17) cell plasticity contributes to both immunity and autoimmunity; however, the factors that control lineage flexibility are mostly unknown. Here we show the activator protein-1 (AP-1) factor JunB is an essential regulator of Th17 cell identity. JunB activates expression of Th17 lineage-specifying genes and coordinately represses genes controlling Th1 and regulatory T-cell fate. JunB supports Th17 cell identity by regulating key AP-1 complex constituents. In particular, JunB limits the expression of the subset repressor IRF8, and impedes access of JunD to regulatory regions of alternative effector loci. Although dispensable for homeostatic Th17 cell development, JunB is required for induction and maintenance of Th17 effector responses in the inflammatory contexts of both acute infection and chronic autoimmunity in mice. Through regulatory network analysis, we show that JunB is a core regulator of global transcriptional programs that promote Th17 cell identity and restrict alternative CD4+ T-cell potential.AP-1 family transcription factors regulate CD4+ T helper cell differentiation. Here the authors show that the AP-1 member JunB is a nonredundant regulator of transcriptional programs that support Th17 cell identity and restrain alternative Th1 and Treg cell fates in inflammatory contexts of acute fungal infection and chronic autoimmunity.

Total tanshinones exhibits anti-inflammatory effects through blocking TLR4 dimerization via the MyD88 pathway.

Tanshinones belong to a group of lipophilic constituents of Salvia miltiorrhiza Bunge (Danshen), which is widely used in traditional Chinese medicine. A deluge of studies demonstrated that tanshinones exert anti-inflammatory effects, but the underlying mechanisms remain unclear to date. This study investigated the anti-inflammatory effects and mechanisms of total tanshinones (TTN). TTN suppressed the expression of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) and the secretion of TNF-α, IL-6, and IL-1ß in RAW264.7 cells, bone marrow-derived macrophages, and THP-1 cells. TTN attenuated the LPS-induced transcriptional activity of NF-κB and decreased IκB-α and IKK phosphorylation and NF-κB/p65 nuclear translocation. Furthermore, TTN inhibited the LPS-induced transcriptional activity of AP-1, which was induced by the reduction of JNK1/2, ERK1/2, and p38MAPK phosphorylation. TTN blocked LPS-induced Toll-like receptor 4 (TLR4) dimerization, which consequently decreased MyD88 recruitment and TAK1 phosphorylation. In addition, TTN pretreatment effectively inhibited xylene-induced ear edema and LPS-induced septic death and improved LPS-induced acute kidney injury in mice. TTN exerts anti-inflammatory effects in vitro and in vivo by blocking TLR4 dimerization to activate MyD88-TAK1-NF-κB/MAPK signaling cascades, which provide the molecular basis of the anti-inflammatory effect of Danshen and suggest that TTN is a potential agent for the treatment of inflammatory diseases.

Didox (3,4-dihydroxybenzohydroxamic acid) suppresses IL-33-induced cytokine production in primary mouse mast cells.

While IgE is considered the primary mediator of mast cell activation, IL-33 contributes substantially in asthma, allergic rhinitis, and atopic dermatitis. To develop effective treatments for allergic disease, it is important to understand the role of therapeutic agents on IL-33 activation. We examined the effect of Didox (3,4-dihydroxybenzohydroxamic acid), an antioxidant and ribonucleotide reductase (RNR) inhibitor, on IL-33-mediated mast cell activation. Didox suppressed IL-6, IL-13, TNF, and MIP-1α (CCL3) production in bone marrow derived mast cells following IL-33 activation. This suppression was observed in different genetic backgrounds and extended to peritoneal mast cells. The antioxidant N-acetylcysteine mimicked the suppression of Didox, albeit at a much higher dose, while the RNR inhibitor hydroxyurea had no effect. Didox substantially suppressed IL-33-mediated NFκB and AP-1 transcriptional activities. These results suggest that Didox attenuates IL-33-induced mast cell activation and should be further studied as a potential therapeutic agent for inflammatory diseases involving IL-33.

Scaffold protein JLP mediates TCR-initiated CD4+T cell activation and CD154 expression.

CD4+ T-cell activation and its subsequent induction of CD154 (CD40 ligand, CD40L) expression are pivotal in shaping both the humoral and cellular immune responses. Scaffold protein JLP regulates signal transduction pathways and molecular trafficking inside cells, thus represents a critical component in maintaining cellular functions. Its role in regulating CD4+ T-cell activation and CD154 expression, however, is unclear. Here, we demonstrated expression of JLP in mouse tissues of lymph nodes, thymus, spleen, and also CD4+ T cells. Using CD4+ T cells from jlp-deficient and jlp-wild-type mice, we demonstrated that JLP-deficiency impaired T-cell proliferation, IL-2 production, and CD154 induction upon TCR stimulations, but had no impacts on the expression of other surface molecules such as CD25, CD69, and TCR. These observed impaired T-cell functions in the jlp-/- CD4+ T cells were associated with defective NF-AT activation and Ca2+ influx, but not the MAPK, NF-κB, as well as AP-1 signaling pathways. Our findings indicated that, for the first time, JLP plays a critical role in regulating CD4+ T cells response to TCR stimulation partly by mediating the activation of TCR-initiated Ca2+/NF-AT.

Heterotrimeric complex of p38 MAPK, PKCδ, and TIRAP is required for AP1 mediated inflammatory response.

Inflammation could be described as a physiological response of the body to tissue injury, pathogen invasion, and irritants. During the inflammatory phase, cells of both the innate as well as adaptive immune system are activated and recruited to the site of inflammation. These mediators are downstream targets for the transcription factors; activator protein-1 (AP1), nuclear factor kappa-light-chain-enhancer (NF-κB), signal transducers and activators of transcription factors (STAT1), as well as interferon regulatory factors (IRFs), which control the expression of most immunomodulatory genes. There is a significant increase in active p38 mitogen-activated protein kinase (p38MAK) immediately after lipopolysaccharide (LPS) stimulation, which results in the activation of AP-1 transcription factor and expression of proinflammatory cytokines, IL-12 and IL-23. We studied the novel mechanism of p38 MAPK activation through the formation of a heterotrimeric complex of Protein kinase C delta type (PKCδ), Toll-Interleukin 1 Receptor (TIR) Domain Containing Adaptor Protein (TIRAP), and p38 proteins. TIRAP serves as an adaptor molecule which brings PKCδ and p38 in close proximity. The complex facilitates the activation of p38MAPK by PKCδ. Therefore, we propose that disruption of the heterotrimeric complex may be a good strategy to dampen the inflammatory response. Structure-based design of small molecules or peptides targetting PKCδ-TIRAP or TIRAP-p38 interfaces would be beneficial for therapy in AP1 mediated inflammatory diseases.

Transmissible gastroenteritis virus infection induces NF-κB activation through RLR-mediated signaling.

Transmissible gastroenteritis virus (TGEV) is a porcine enteric coronavirus which causes lethal severe watery diarrhea in piglets. The pathogenesis of TGEV is strongly associated with inflammation. In this study, we found that TGEV infection activates transcription factors NF-κB, IRF3 and AP-1 in a time- and dose-dependent manner in porcine kidney cells. Treatment with the NF-κB-specific inhibitor BAY11-7082 significantly decreased TGEV-induced proinflammatory cytokine production, but did not affect virus replication. Phosphorylation of NF-κB subunit p65 and proinflammatory cytokine production were greatly decreased after knockdown of retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) or its adaptors MAVS and STING, while only slight reduction was observed in cells following silencing of Toll-like receptor adaptors, MyD88 and TRIF. Furthermore, TGEV infection significantly upregulated mRNA expression of RIG-I and MDA5. Taken together, our results indicate that the RLR signaling pathway is involved in TGEV-induced inflammatory responses.

The AP-1 Transcription Factor c-Jun Promotes Arthritis by Regulating Cyclooxygenase-2 and Arginase-1 Expression in Macrophages.

Activation of proinflammatory macrophages is associated with the inflammatory state of rheumatoid arthritis. Their polarization and activation are controlled by transcription factors such as NF-κB and the AP-1 transcription factor member c-Fos. Surprisingly, little is known about the role of the AP-1 transcription factor c-Jun in macrophage activation. In this study, we show that mRNA and protein levels of c-Jun are increased in macrophages following pro- or anti-inflammatory stimulations. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment cluster analyses of microarray data using wild-type and c-Jun-deleted macrophages highlight the central function of c-Jun in macrophages, in particular for immune responses, IL production, and hypoxia pathways. Mice deficient for c-Jun in macrophages show an amelioration of inflammation and bone destruction in the serum-induced arthritis model. In vivo and in vitro gene profiling, together with chromatin immunoprecipitation analysis of macrophages, revealed direct activation of the proinflammatory factor cyclooxygenase-2 and indirect inhibition of the anti-inflammatory factor arginase-1 by c-Jun. Thus, c-Jun regulates the activation state of macrophages and promotes arthritis via differentially regulating cyclooxygenase-2 and arginase-1 levels.

Octacosanol Attenuates Inflammation in Both RAW264.7 Macrophages and a Mouse Model of Colitis.

Octacosanol has multiple biological functions. In this study, the anti-inflammatory effect and molecular mechanism of octacosanol were evaluated by using dextran sulfate sodium (DSS)-induced ulcerative colitis model in mice and lipopolysaccharide (LPS)-stimulated mouse macrophage RAW264.7 cells. The colitis mouse model was induced by 3.0% DSS in 8-week ICR mice and octacosanol orally administered with 100 mg/kg/day. The results showed that octacosanol significantly improved the health status of mice and reduced DSS-induced pathological damage in the colonic tissues. Octacosanol obviously inhibited the mRNA and protein expression levels of pro-inflammatory factors of colonic tissues. In vitro, octacosanol administration significantly reduced the expression of mRNA or protein of pro-inflammatory cytokines and the phosphorylation of c-Jun N-terminal kinase and p38, and it also partly prevented LPS-induced translocations of NF-κB and AP-1. Octacosanol has anti-inflammatory effect, and its molecular mechanism may be involved in downregulating the expression of inflammatory factors and blocking of MAPK/NF-κB/AP-1 signaling pathway.

3-Methoxy-catalposide inhibits inflammatory effects in lipopolysaccharide-stimulated RAW264.7 macrophages.

Pseudolysimachion rotundum var. subintegrum is utilized as a traditional herbal remedy to treat cough, bronchitis, and asthma in Korea, Russia, China, and Europe. Here, we show that 3-methoxy-catalposide, a novel iridoide glycoside isolated from P. rotundum var. subintegrum has the anti-inflammatory activity in lipopolysaccharide (LPS)-stimulated macrophages. The chemical structure of 3-methoxy-catalposide was determined by NMR, optical rotation and HRESIMS. In in vitro experiment, RAW264.7 cells were treated with 3-methoxy-catalposide for 2h before exposure to LPS for different times. Inflammatory gene and protein expressions were assayed using RT-PCR and ELISA. Activities of signal proteins were examined using western analysis. Our results demonstrated that 3-methoxy-catalposide significantly inhibits the expression of cyclooxygenase (COX)-2 and inducible nitric oxide synthase (iNOS) in RAW264.7 cells stimulated by LPS, thereby suppressing the release of prostaglandin E2 (PGE2) and nitric oxide (NO). Moreover, 3-methoxy-catalposide markedly reduced the LPS-induced expression of pro-inflammatory genes, such as interleukin (IL)-6, IL-1ß, and TNF-α. Further, 3-methoxy-catalposide inhibited both LPS-induced activation of three MAP kinases (ERK 1/2, JNK, and p38) and the nuclear translocation of NF-κB and AP-1. These results support that 3-methoxy-catalposide may be a promising candidate for inflammation treatment.

The regulation of TIM-3 transcription in T cells involves c-Jun binding but not CpG methylation at the TIM-3 promoter.

Tim-3 is an immunomodulatory protein that is expressed constitutively on monocytes but is induced in activated T cells. The mechanisms involved in the regulation of TIM-3 transcription are poorly understood. In the present study, we investigated whether methylation of the TIM-3 promoter is involved in regulatingTIM-3 transcription in T cells, and identified a transcription factor that regulates TIM-3 transcription by associating with the TIM-3 minimal promoter region. Pyrosequencing of the TIM-3 promoter up to -1440bp revealed 11 hypermethylated CpG sites and 4 hypomethylated CpG sites in human CD4(+) T cells as well as in CD11b(+) cells. Dimethylation of histone H3 lysine 4 (H3K4), a mark of transcriptional activation, was predominantly found in the proximal TIM-3 promoter -954 to -34bp region, whereas trimethylation of H3K9 and H3K27, which are markers of transcriptional suppression, were mostly observed in the distal promoter -1549 to -1048bp region in human CD4(+) T cells and CD11b(+) cells. However, no change in the methylation status of CpG sites and the histone H3 in the TIM-3 promoter was found during induction of TIM-3 transcription in T cells. Finally, AP-1 involvement in TIM-3 transcription was shown in relation with the TIM-3 minimal promoter -146 to +144bp region. The present study defines the minimal TIM-3 promoter region and demonstrates its interaction with c-Jun during TIM-3 transcription in CD4(+) T cells.