AP-1 Pathways Research Articles

Priming Tumor Cells by Immunogenic Modulation to Enhance IL‐27 Immunetherapy Efficacy

Immunogenic modulation is an emerging strategy for treating bone‐metastatic prostate tumors, which present an altered balance between bone resorption and bone formation, with enhanced tumor growth and severe pain and fractures. There remains a critical lack of therapies that can significantly reduce tumor burden while simultaneously restoring the bone. Emerging immune therapies include Interleukin‐27 (IL‐27), a multifunctional cytokine that drives antitumor activity through STAT1/T‐bet/IFNg pathway immune‐activation and anti‐angiogenic mechanisms. IL‐27 also is a pro‐osteogenic factor, as it inhibits osteoclast formation and increases the pool of mature functional osteoblasts. Our goal for this work was to examine whether IL‐27 immunetherapy efficacy still could be improved by the addition of ‘priming’ strategies such as chemotherapy‐induced immunogenic modulation in order to more efficiently promote tumor cell elimination by the immune system. Immunogenic modulation (IMO) develops following tumor cell exposure to chemotherapy, with alterations in tumor cell phenotype that prime cells for elimination by the immune system. Our results suggest that chemotherapy can reduce tumor cell viability, however, aggressive prostate tumor cells still are relatively resistant even at high doses. We have identified a potential cytokine combination that can sensitize aggressive prostate cancer cells (PC3) to low or sublethal doses of recently FDA‐approved chemotherapy agents. Gene expression analyses using quantitative real time PCR arrays suggest that single chemotherapuetic agents promote a fairly low modulation and magnitude of apoptotic transcripts. Immune therapy alone (IL27 plus OP1 or 27OP1) upregulated several apoptosis genes, however, the combination of 27OP1 synergized with either cabozantinib or cabazitaxel, especially in augmenting expression of proapoptotic genes BNIP2 (>52‐fold, p<0.05), and CARD8 (>24‐fold) and immune modulatory gene CD70 (>124‐fold). Preliminary pathway analyses with the gene expression data (Pathgen) generated hypotheses of possible mechanisms involving gene regulation by AP1 or SMAD related pathways. Functional assays using luciferase reporter assays showed that AP1 activity was significantly upregulated by 27OP1 (2‐fold), Cabozantinib (2‐fold), Cabazitaxel+27OP1 (3‐fold), and Cabozantinib+27OP1 (5‐fold). SMAD1/5/8 activity was significantly upregulated by OP1 (3.5‐fold), 27OP1 (6‐fold), Cabozantinib (2‐fold), Cabazitaxel+27OP1 (7‐fold), and Cabozantinib+27OP1 (12‐fold). These results suggest that 27OP1 synergizes with cabozantinib or cabazitaxel at least through AP1 and SMAD‐regulatory mechanisms. Future studies will examine the effectiveness of these combinations in vivo, and preclinical studies are planned to examine whether low‐dose chemotherapy can augment 27OP1 immunotherapy efficacy. Ultimately, our goal is to identify the optimal therapeutic niche for immunotherapy in combination with current immunomodulatory chemotherapies. We anticipate that osteo‐immune therapies will have a significant impact in the treatment of prostate and other bone‐trophic cancer, since this modality may be efficient in reducing tumor burden while restoring bone, which will be an exciting prospect for cancer therapy.Support or Funding InformationWe acknowledge R21CA179699, R01CA196947 and start up funds from the BMS department

RHBDD1 upregulates EGFR via the AP-1 pathway in colorectal cancer.

Our previous study showed that RHBDD1 can activate the EGFR signaling pathway to promote colorectal cancer growth. In the present study, EGFR was decreased when RHBDD1 was knocked down or inactivated. Further analysis found that c-Jun and EGFR protein expression was decreased in RHBDD1 knockdown and inactivated cells. c-Jun overexpression in RHBDD1-inactivated cells rescued EGFR expression in a dose-dependent manner. RHBDD1 overexpression in RHBDD1-inactivated cells restored EGFR expression, but this effect was counteracted by c-Jun knockdown. Furthermore, EGFR and c-Jun were attenuated in the RHBDD1 knockdown and inactivated groups in animal tumor models. Tissue microarray assays demonstrated a correlation between RHBDD1 and EGFR in colorectal cancer patients. Therefore, our findings indicate that RHBDD1 stimulates EGFR expression by promoting the AP-1 pathway.

Galectin-1 knockdown improves drug sensitivity of breast cancer by reducing P-glycoprotein expression through inhibiting the Raf-1/AP-1 signaling pathway.

Galectin-1 (Gal-1), a member of the galectin family of carbohydrate binding proteins, plays a pivotal role in various cellular processes of tumorigenesis. The regulatory effect of Gal-1 on multidrug resistance (MDR) breast cancer cells is still unclear. qRT-PCR and western blot showed that Gal-1 and MDR gene 1 (MDR1) were both highly expressed in breast tumor tissues and cell lines. MTT assay and flow cytometry revealed that Gal-1 knockdown improved sensitivity to paclitaxel (PTX) and adriamycin (ADR) in MCF-7/PTX and MCF-7/ADR cells via inhibition of cell viability and promotion of cell apoptosis, while MDR1 overexpression weakened the sensitivity to PTX and ADR induced by Gal-1 knockdown. Furthermore, the negative effects of Gal-1 knockdown on sensitivity to PTX and ADR in MCF-7/PTX and MCF-7/ADR cells were revealed to be mediated via the suppression of Raf-1/AP-1 pathway. In conclusion, Gal-1 knockdown dramatically improved drug sensitivity of breast cancer by reducing P-glycoprotein (P-gp) expression via inhibiting the Raf-1/AP-1 pathway, providing a novel therapeutic target to overcome MDR in breast cancer.

Rhein suppresses matrix metalloproteinase production by regulating the Rac1/ROS/MAPK/AP-1 pathway in human ovarian carcinoma cells.

Matrix metalloproteinases (MMPs) are a family of calcium-dependent zinc-containing endopeptidases, which play an integral role in migration and invasion of ovarian cancer. Rac1 proteins might mostly influence cell migration and invasion by generating endogenous reactive oxygen species. Therefore, inhibiting MMPs and regulating the Rac1/ROS/MAPK/AP-1 pathway may be a new therapeutic strategy for ovarian cancer. In this study, we found that rhein could suppress the invasion and migration of SKOV3-PM4 cells with characteristics of directional highly lymphatic metastasis. Phorbol 12-myristate 13-acetate (PMA), which is a Rac1 activator, significantly enhanced the expression levels of MMP-2, -3, -9 and -19 proteins, whereas the results of rhein and Rac1 inhibitor NSC23766 were just the opposite. The inhibitory effects of rhein were associated with the upregulation of tissue inhibitors of metalloproteinase (TIMP)-1, TIMP-2 and NM23-H1. Subsequent mechanism studies revealed that rhein reduce the production of reactive oxygen species (ROS) and lower NADPH oxidase activity. Furthermore, rhein significantly inhibited JNK, AP-1 phosphorylation in the cells treated with PMA. The results obtained from the cells treated with NSC23766 alone or NSC23766 combined with rhein, were consistent with rhein treatment alone. Taken together, these results indicate that rhein may be a potential inhibitor of Rac1 and can inhibit the migration and invasion of SKOV3-PM4 cells through modulating matrix metalloproteinases and RAC1/ROS/MAPK/AP-1 signaling pathway-associated proteins.

JS-III-49, a hydroquinone derivative, exerts anti-inflammatory activity by targeting Akt and p38.

Since previous studies have reported that hydroquinone (HQ) exerted immunosuppressive and anti-inflammatory activity, various HQ derivatives have been synthesized and their biological activities investigated. In this study, we explored the anti-inflammatory activity of JS-III-49, a novel HQ derivative, in macrophage-mediated inflammatory responses. JS-III-49 suppressed the production of the inflammatory mediators nitric oxide (NO) and prostaglandin E2 (PGE2) and down-regulated the mRNA expression of the inflammatory enzymes cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) as well as the expression of the pro-inflammatory cytokines interleukin-6 (IL-6) and IL-1b without cytotoxicity in LPS-stimulated RAW264.7 cells. JS-III-49 inhibited nuclear translocation of the NF-kB transcription factors p65 and p50 by directly targeting Akt, an upstream kinase of the NF-kB pathway, in LPS-stimulated RAW264.7 cells. However, JS-III-49 did not directly inhibit the kinase activities of Src and Syk, which are upstream kinases of Akt, in LPS-stimulated RAW264.7 cells. Moreover, JS-III-49 suppressed the nuclear translocation of c-Fos, one of the components of AP-1, by specifically targeting p38, an upstream mitogen-activated protein kinase (MAPK) in the AP-1 pathway in LPS-stimulated RAW264.7 cells. These results suggest that JS-III-49 plays an anti-inflammatory role in LPS-stimulated macrophages by targeting Akt and p38 in the NF-kB and AP-1 pathways, respectively.

T Cell Transcriptomes from Paroxysmal Nocturnal Hemoglobinuria Patients Reveal Novel Signaling Pathways

Background. Paroxysmal nocturnal hemoglobinuria (PNH) is a rare acquired blood disease, characterized by hemolytic anemia, bone marrow (BM) failure, and venous thrombosis. The etiology of PNH is a somatic mutation in the phosphatidylinositol glycan class A gene (PIG-A) on the X chromosome, which causes deficiency in glycosyl phosphatidylinositol-anchored proteins (GPI-APs). The involvement of T cells in PNH is strongly supported by clinical overlap between PNH and aplastic anemia (AA); the presence of GPI-AP deficient cells in AA associated with favorable response to immunosuppressive therapy; and an oligoclonal T cell repertoire in PNH patients. However, the molecular mechanisms responsible for the aberrant immune responses in PNH patients are not well understood. To identify aberrant molecular mechanisms involved in immune targeting of hematopoietic stem cells in BM, RNA sequencing (RNA-seq) was applied to examine the transcriptome of T cell subsets from PNH patients and healthy controls.Method. Blood samples were obtained after informed consent from 15 PNH patients and 15 age-matched healthy controls. For RNA extraction, freshly isolated peripheral blood mononuclear cells were sorted on the same day of blood draw to obtain four different T cell (CD3+ CD14- CD19- ViViD-) populations [CD4+ naïve (CD45RA+ CD45RO-), CD4+ memory (CD45RA- CD45RO+), CD8+ naïve (CD45RA+ CD45RO-), and CD8+ memory (CD45RA- CD45RO+) T cells] by fluorescence-activated cell sorter . RNA-Seq analysis from three PNH and three healthy controls was performed using the Illumina HiSeq™ 2000 platform. The Ingenuity® Pathway Analysis and Gene set enrichment analysis (GSEA) were employed to elucidate transcriptional pathways. RNA-seq data were validated by flow cytometry and quantitative real-time RT-PCR (RT-qPCR).Results and Discussion. Differentially expressed gene analysis of four T cell subsets showed distinct gene expression signatures in individual T cell subsets. In CD4+ naïve T cells, 11 gene expression levels were significantly different: five upregulated (including SRRM2 and TNFSF8) and six downregulated genes (including GIMAP6) (> 2 fold change, false discovery rate [FDR] < 0.05). In CD4+ memory T cells, 25 gene expression levels were significantly different: 15 upregulated (including JUND and TOB1) and 10 downregulated genes (including GIMAP4). In CD8+ naive T cells, only two gene expression levels were significantly different: upregulated CTSW and downregulated RPL9. In CD8+ memory T cells, seven gene expression levels were significantly different: two upregulated (CTSW and DPP4) and five downregulated genes (including SLC12A7). Further, differentially expressed gene analysis was performed by combining CD4+ naïve, CD4+ memory, CD8+ naïve, and CD8+ memory T cells from PNH or healthy controls, respectively. Out of 55 gene expression levels that were significantly different, 41 were upregulated (including TNFAIP3, JUN, JUND, TOB1, TNFSF8, and CD69) and 14 downregulated (including GIMAP4). By canonical pathways analysis, putative gene network interactions of differentially expressed genes were significantly enriched for canonical pathways of TNFR1, TNFR2, IL-17A, and CD27 signaling. By GSEA, the most significantly upregulated gene sets in CD4+ naïve, CD4+ memory, CD8+ naïve, and CD8+ memory T cells from PNH patients displayed gene signatures related to the "IGF1 pathway", "Pre-NOTCH expression and processing", "AP-1 pathway", and "ATF2 pathway", respectively. For validation of the RNA-seq data, we chose seven genes (TNFAIP3, JUN, JUND, TOB1, TNFSF8, CD69, and CTSW) because these are important mediators involved in regulation for T cells and dysregulation of these genes is associated with autoimmune diseases. Differential expression levels of TNFAIP3, JUN, and TOB1 were validated by RT-qPCR. By flow cytometry, higher expression of CD69 and TNFSF8 was confirmed in CD4+ and CD8+T cells from PNH compared to healthy controls.Conclusion. Using RNA-seq, we identified novel molecular mechanisms and pathways which may underlie the aberrant T cell immune status in PNH. Specific dysregulation of T cell intracellular signaling may contribute to BM failure and the inflammatory environment in PNH. Understanding these pathways may provide new therapeutic strategies to modulate T cell immune responses in BM failure. DisclosuresHosokawa:Aplastic Anemia and MDS International Foundation: Research Funding. Rios:GSK/Novartis: Research Funding. Weinstein:GSK/Novartis: Research Funding. Townsley:GSK/Novartis: Research Funding.

Cap Analysis of Gene Expression (CAGE) Sequencing Reveals Alterations of the Transcriptional Signatures of FLT3-ITD with Secondary D835 TKD Mutations in Acute Myeloid Leukemia

Mutations in the gene encoding Fms-like tyrosine kinase-3 (FLT3) are found in approximately 30% of acute myeloid leukemia (AML) patients, with two distinct groups of mutations; the internal tandem duplication mutations in juxtamembrane domain (FLT3-ITD) and the point mutations within the tyrosine kinase domains (FLT3-TKD).The second-generation tyrosine kinase inhibitor (TKI) quizartinib which primarily targets an inactive (type II) kinase conformation, has demonstrated responses in relapsed/refractory FLT3-ITD AML patients. However, duration of response to quizartinib is limited largely by secondary mutations in FLT3-TKD, most commonly at D835 activation loop, emerging in at least 20% of patients.To elucidate the alterations of transcript signatures of quizartinib-resistant FLT3-ITD/D835 mutants in AML, we performed cap analysis of gene expression (CAGE) sequencing utilizing 11 FLT3-ITD and 6 FLT3-ITD/D835 primary AML cells with wild type NPM1. CAGE identifies and quantifies the 5’ ends of capped mRNA transcripts, which enables the identification of transcription start sites (TSS) and allows investigating promoter structures necessary for gene expression. The TSS of genes altered in AML cells with FLT3-ITD/D835 compared with FLT3-ITD were mapped, and CAGE detected upregulation of 124 genes and downregulation of 25 genes in FLT3-ITD/D835 cells (FDR < 0.05). Specifically, several genes closely linked with activation of MAPK signaling (MAPK6, TCF4), AKT/mTOR signaling (NBN) and both (TNFAIP6) were upregulated in the D835 acquired cells. Expression of inflammatory response cytokine genes IL1B and IL8 were also increased in FLT3-ITD/D835 cells.The FLT related functional pathway analysis by KEGG ontology indicated that Ras pathway with its downstream Raf/MAPK and PI3K/AKT/mTOR signaling including transcription factor NFKB are the most significantly activated pathways in FLT3-ITD/D835 cells compared to FLT3-ITD cells. No additional activation of STAT5 pathway, a direct downstream signaling of mutated FLT3, was found in FLT3-ITD/D835 cells.To assess the underlying molecular basis of transcription signature, we performed an unbiased search for enriched sequence motifs using HOMER software. HOMER revealed upregulation of 80 motifs and downregulation of 51 motifs in AML cells with FLT3-ITD/D835 compared with FLT3-ITD (q < 0.05). Motifs of AP-1, JUN, CEBP, ATF and NFKB were substantially enriched in FLT3-ITD/D835 compared to FLT3-ITD cells. ETS, SPI-1 and RUNX1 were the most common binding motifs altered (both increased and decreased) by D835 mutation acquisition. Of note, AP-1 modulates inflammatory response and is tightly regulated by MAPK.To validate the transcriptional changes and the highlighted gene ontology results of AML primary samples, we utilized paired isogenic FLT3-ITD or FLT3-ITD/D835Y transfected Ba/F3 cell lines. As predicted, type II kinase inhibitor quizartinib was active against FLT3-ITD Ba/F3 cells (IC50; 0.6nM) but not FLT3-ITD/D835Y Ba/F3 cells (IC50; > 5000nM). Type I kinase inhibitor crenolanib was highly active against both FLT3-ITD (IC50; 2.1nM) and FLT3-ITD/D835Y cells (IC50; 16.4nM). Focusing on FLT related Ras pathway, we first investigated MAPK activation in these cells. Immunoblot analysis demonstrated substantial reduction of phospho- (p-) ERK after quizartinib (5nM) or crenolanib (50nM) treatment both in FLT3-ITD and FLT3-ITD/D835Y cells, irrespective of their susceptibility. We next examined p-S6, the target of PI3K/AKT/mTOR and MAPK signaling, and observed that crenolanib but not quizartinib successfully diminished p-S6 expression in FLT3-ITD/D835Y cells. FLT3-ITD/D835 cells showed higher baseline expression of NFkB regulator p-IkB compared to FLT3-ITD cells, which was not repressed by crenolanib.In conclusion, our study showed that the acquired D835 mutation in FLT3-ITD AML cells facilitates transcriptional network activation to induce proinflammatory genes through Ras-MAPK-NF-κB/AP-1 pathway and Ras-PI3K-AKT-mTOR signaling. This transcriptional modulation promotes oncogenic pro-survival and proliferative signaling in AML cells and defines differential sensitivity against Type I and Type II kinase inhibitors. DisclosuresShah:ARIAD: Research Funding; BMS: Research Funding; Daiichi-Sankyo: Research Funding; Pfizer: Research Funding; Plexxikon: Research Funding. Konopleva:Reata Pharmaceuticals: Equity Ownership; Abbvie: Consultancy, Research Funding; Genentech: Consultancy, Research Funding; Stemline: Consultancy, Research Funding; Eli Lilly: Research Funding; Cellectis: Research Funding; Calithera: Research Funding.

Anti-Inflammatory Effects of Chloranthalactone B in LPS-Stimulated RAW264.7 Cells.

Chloranthalactone B (CTB), a lindenane-type sesquiterpenoid, was obtained from the Chinese medicinal herb Sarcandra glabra, which is frequently used as a remedy for inflammatory diseases. However, the anti-inflammatory mechanisms of CTB have not been fully elucidated. In this study, we investigated the molecular mechanisms underlying these effects in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. CTB strongly inhibited the production of nitric oxide and pro-inflammatory mediators such as prostaglandin E2, tumor necrosis factor α (TNF-α), interleukin-1β (IL-1β), and IL-6 in RAW264.7 cells stimulated with LPS. A reverse-transcription polymerase chain reaction assay and Western blot further confirmed that CTB inhibited the expression of inducible nitric oxide synthase, cyclooxygenase-2, TNF-α, and IL-1β at the transcriptional level, and decreased the luciferase activities of activator protein (AP)-1 reporter promoters. These data suggest that inhibition occurred at the transcriptional level. In addition, CTB blocked the activation of p38 mitogen-activated protein kinase (MAPK) but not c-Jun N-terminal kinase or extracellular signal-regulated kinase 1/2. Furthermore, CTB suppressed the phosphorylation of MKK3/6 by targeting the binding sites via formation of hydrogen bonds. Our findings clearly show that CTB inhibits the production of inflammatory mediators by inhibiting the AP-1 and p38 MAPK pathways. Therefore, CTB could potentially be used as an anti-inflammatory agent.

Differential inflammasome activation signatures following intracellular infection of human macrophages with Mycobacterium bovis BCG or Trypanosoma cruzi

Pathogens frequently exploit or evade inflammasome activation in order to survive and proliferate. Alternatively, inadequate inflammasome activation by attenuated microorganisms or adjuvanted subunit vaccines may contribute to poor longevity of protection. To further understand these pathways, we determined the differential inflammasome transcriptome of human THP monocyte-derived macrophages in response to Mycobacterium bovis BCG, as compared to LPS or Trypanosoma cruzi. The results identify the highly specific innate recognition programs associated with inflammasome activation by human macrophages exposed to these microbial stimuli. BCG, T. cruzi, and LPS strongly induced expression of both unique and overlapping genes downstream of TLR signaling pathways including cytokines and chemokines that mediate inflammation and regulate cell death pathways. Compared to LPS, BCG failed to directly activate anti-apoptotic molecules and multiple NLR and inflammasome complex components including caspase-1, and actively repressed important signaling intermediates in AP-1 and NFκB transcription factor pathways. Both BCG and T. cruzi repressed expression of TXNIP, an anti-oxidant inhibitor that recruits caspase-1 to the NLRP3 inflammasome, while T. cruzi infection uniquely failed to activate TNF-α. These results identify unique pathogen specific strategies to activate inflammation and modulate cell death that may drive inflammatory outcomes and suggest avenues of investigation to optimize host immunity.

Molecular Weight-Dependent Immunostimulative Activity of Low Molecular Weight Chitosan via Regulating NF-κB and AP-1 Signaling Pathways in RAW264.7 Macrophages

Chitosan and its derivatives such as low molecular weight chitosans (LMWCs) have been found to possess many important biological properties, such as antioxidant and antitumor effects. In our previous study, LMWCs were found to elicit a strong immunomodulatory response in macrophages dependent on molecular weight. Herein we further investigated the molecular weight-dependent immunostimulative activity of LMWCs and elucidated its mechanism of action on RAW264.7 macrophages. LMWCs (3 kDa and 50 kDa of molecular weight) could significantly enhance the mRNA expression levels of COX-2, IL-10 and MCP-1 in a molecular weight and concentration-dependent manner. The results suggested that LMWCs elicited a significant immunomodulatory response, which was dependent on the dose and the molecular weight. Regarding the possible molecular mechanism of action, LMWCs promoted the expression of the genes of key molecules in NF-κB and AP-1 pathways, including IKKβ, TRAF6 and JNK1, and induced the phosphorylation of protein IKBα in RAW264.7 macrophage. Moreover, LMWCs increased nuclear translocation of p65 and activation of activator protein-1 (AP-1, C-Jun and C-Fos) in a molecular weight-dependent manner. Taken together, our findings suggested that LMWCs exert immunostimulative activity via activation of NF-κB and AP-1 pathways in RAW264.7 macrophages in a molecular weight-dependent manner and that 3 kDa LMWC shows great potential as a novel agent for the treatment of immune suppression diseases and in future vaccines.

Cerbera manghas methanol extract exerts anti-inflammatory activity by targeting c-Jun N-terminal kinase in the AP-1 pathway

Ethnopharmacological relevanceCerbera manghas L. (Apocynaceae) is a medicinal plant traditionally used to ameliorate the clinical signs of inflammatory diseases and hypertension. Aim of studyAlthough C. manghas L. has long been used as a traditional remedy for various diseases, the underlying molecular and cellular mechanisms are poorly understood. A detailed investigation of these mechanisms is necessary to demonstrate the ethnopharmaceutical utility of this plant. Materials and MethodsThe effects of C. manghas methanol extract (Cm-ME) on the production of inflammatory mediators and the expression of proinflammatory cytokines and identification of molecular targets were investigated using lipopolysaccharide (LPS)-treated macrophages in vitro. In addition, the inhibitory effects of Cm-ME orally administered were tested by LPS/D-galactosamine (D-GalN)-induced hepatitis and LPS-induced peritonitis mouse models in vivo. ResultsCm-ME downregulated the production of prostaglandin (PG)E2 and the mRNA expression of cyclooxygenase (COX)-2, tumor necrosis factor (TNF)-α, and interleukin (IL)-1β in LPS-stimulated RAW264.7 cells under non-toxic concentration of Cm-ME. This extract inhibited the nuclear translocation of c-Jun and p-ATF2, the phosphorylation of JNK and p38, and AP-1 activity. Western blot analysis and in vitro kinase assay confirmed that JNK is a direct pharmacological target of Cm-ME action. In addition, Cm-ME significantly ameliorated the clinical signs of LPS/D-GalN-induced hepatitis and lowered the production of nitric oxide (NO) and the phosphorylation of JNK in LPS-induced peritonitis conditions. ConclusionCm-ME exerts anti-inflammatory actions on LPS-stimulated macrophages and in mouse models of acute inflammatory disease. These actions are predominantly mediated by targeting JNK in the AP-1 signaling pathway.

Global DNA methylation and PTEN hypermethylation alterations in lung tissues from human silicosis.

Silicosis is a respiratory disease caused by long-term silica dust exposure. Our previous study has demonstrated that silica mediates the activation of phosphatidylinositol 3-kinase (PI3K)/phosphatase and tensin homolog deleted on chromosome 10 (PTEN)/serine or threonine kinase (AKT)/mitogen-activated protein kinases (MAPK)/AP-1 pathway in human embryo lung fibroblasts (HELFs). The purpose of this study is to identify genome-wide aberrant DNA methylation profiling in lung tissues from silicosis patients. We performed Illumina Human Methylation 450K Beadchip arrays to investigate the methylation alteration in formalin-fixed, paraffin-embedded (FFPE) lung specimens, immunohistochemistry to detect the level of c-Jun and PTEN proteins; methylation specific PCR (MS-PCR) to identify PTEN and c-Jun promoter methylation in HELFs. We found 86,770 CpG sites and 79,660 CpG sites significantly differed in methylation status in early-stage and advanced-stage compared with GEO normal lung methylation data. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed the methylated status of MAPK signaling pathway was considered changed. The number of PTEN and c-Jun CpG promoter methylated-sites were increased in advanced-stage. Early-stage showed the positive expression of c-Jun and PTEN protein and negative or mild expression in advanced-stage. PTEN promoter was no differentially methylated and c-Jun promoter differed at 12 and 24 h in HELFs. Abnormal DNA methylation on genome-scale was implicated in silicosis, and PTEN promoter hypermethylation might be associated with decrease of PTEN protein.

Andrographolide ameliorates diabetic nephropathy by attenuating hyperglycemia-mediated renal oxidative stress and inflammation via Akt/NF-κB pathway

Diabetic nephropathy (DN) is characterized by proliferation of mesangial cells, mesangial hypertrophy and extracellular matrix (ECM) accumulation. Our recent study found that andrographolide inhibited high glucose-induced mesangial cell proliferation and fibronectin expression through inhibition of AP-1 pathway. However, whether andrographolide has reno-protective roles in DN has not been fully elucidated. Here, we studied the pharmacological effects of andrographolide against the progression of DN and high glucose-induced mesangial dysfunction. Diabetes was induced in C57BL/6 mice by intraperitoneal injection of streptozotocin (STZ). After 1 weeks after STZ injection, normal diet was substituted with a high-fat diet (HFD). Diabetic mice were intraperitoneal injected with andrographolide (2 mg/kg, twice a week). After 8 weeks, functional and histological analyses were carried out. Parallel experiments uncovering the molecular mechanism by which andrographolide prevents from DN was performed in mesangial cells. Andrographolide inhibited the increases in fasting blood glucose, triglyceride, kidney/body weight ratio, blood urea nitrogen, serum creatinine and 24-h albuminuria in diabetic mice. Andrographolide also prevented renal hypertrophy and ECM accumulation. Furthermore, andrographolide markedly attenuated NOX1 expression, ROS production and pro-inflammatory cytokines as well. Additionally, andrographolide inhibited Akt/NF-κB signaling pathway. These results demonstrate that andrographolide is protective against the progression of experimental DN by inhibiting renal oxidative stress, inflammation and fibrosis.

Overexpression of HDAC6 induces pro-inflammatory responses by regulating ROS-MAPK-NF-κB/AP-1 signaling pathways in macrophages

Although histone deacetylase 6 (HDAC6) has been implicated in inflammatory diseases, direct involvement and its action mechanism of HDAC6 in the transcriptional regulation of pro-inflammatory genes have been unclear. In this study, we investigated the possible role of HDAC6 in the expression of pro-inflammatory mediators, indicator of macrophage activation, in RAW 264.7 cells and primary mouse macrophages. HDAC6 overexpression significantly enhanced expression of pro-inflammatory cytokines, such as TNF-α, IL-1β, and IL-6, with concomitant reduction in acetylated α-tubulin. HDAC6 overexpression significantly induced ROS generation via upregulation of NADPH oxidase expression and activity. Inhibition of ROS generation by N-acetyl cysteine, diphenyl iodonium and apocynin suppressed HDAC6-induced pro-inflammatory cytokines. An HDAC6 enzymatic inhibitor significantly inhibited ROS generation and expression of HDAC6-induced pro-inflammatory mediators, indicating the requirement of HDAC6 enzymatic activity for induction of pro-inflammatory cytokines. In addition, HDAC6 overexpression increased activation of MAPK species including ERK, JNK, and p38. Furthermore, HDAC6 overexpression resulted in activation of the NF-κB and AP-1 signaling pathways. Overall, our results provide the first evidence that HDAC6 is capable of inducing expression of pro-inflammatory genes by regulating the ROS-MAPK-NF-κB/AP-1 pathways and serves as a molecular target for inflammation.

Src/Syk/IRAK1-targeted anti-inflammatory action of Torreya nucifera butanol fraction in lipopolysaccharide-activated RAW264.7 cells

Ethnopharmacological relevanceSeed of Torreya nucifera (L.) Siebold & Zucc is used to treat several diseases in Asia. Reports document that T. nucifera has anti-cancer, anti-inflammatory, anti-oxidative activities. In spite of numerous findings on its pharmacological effects, the understanding of the molecular inhibitory mechanisms of the plant remains to be studied. Therefore, we aimed to explore in vitro anti-inflammatory mechanisms of ethyl acetate fraction (Tn-EE-BF) prepared from the seed of T. nucifera in LPS-stimulated macrophage inflammatory responses. Materials and methodsFor this purpose, we measured nitric oxide (NO) and prostaglandin E2 (PGE2) in LPS-stimulated macrophages. Additionally, using RT-PCR, luciferase reporter gene assay, immunoblotting analysis, and kinase assay, the levels of inflammatory genes, transcription factors, and inflammatory signal-regulatory proteins were investigated. Finally, the constituent of Tn-EE-BF was identified using HPLC. ResultsTn-EE-BF inhibits NO and PGE2 production and also blocks mRNA levels of inducible NO synthase (iNOS), tumor necrosis factor (TNF)-α, and cyclooxygenase (COX)-2 in a dose dependent manner. Tn-EE-BF reduces nuclear levels of the transcriptional factors NF-κB (p65) and AP-1 (c-Jun and FRA-1). Surprisingly, we found that Tn-EE-BF inhibits phosphorylation levels of Src and Syk in the NF-κB pathway, as well as, IRAK1 at the protein level, part of the AP-1 pathway. By kinase assay, we confirmed that Src, Syk, and IRAK1 are suppressed directly. HPLC analysis indicates that arctigenin, amentoflavone, and quercetin may be active components with anti-inflammatory activities. ConclusionTn-EE-BF exhibits anti-inflammatory activities by direct inhibition of Src/Syk/NF-κB and IRAK1/AP-1.

A cold water extract of Fucus vesiculosus inhibits lipopolysaccharide (LPS) induced pro-inflammatory responses in the porcine colon ex-vivo model

Abstract The objectives of this experiment were to compare the in-vitro anti-inflammatory activity of crude extracts (cold water (CWE), hot water and 80% ethanol extract) of F. vesiculosus and to predict the key molecular targets of the extract with most anti-inflammatory activity in-vitro . In TNF-α challenged Caco-2 cells, of the three crude extracts, the CWE exhibited maximum inhibition of IL-8 production. In the ex-vivo challenged porcine colonic tissue, CWE inhibited the expression (> 2 fold) of inflammatory mediators ( PTGS2 , C5 , LYZ ), cytokines ( IL17A , IL8 ), chemokines ( CCL2 , CXCL2 , CXCL10 , CXCL11 ), cell adhesion molecules ( ICAM1 , VCAM1 ), toll like receptors ( TLR4 , TLR7 ) and components of NF-κB ( NFKB1 , RELB ), MAPK ( MAP3K8 ) and AP-1 ( CJUN ) pathways. The gene expression analysis suggest that the CWE does contain immunomodulatory bioactive compound/s that mediated through the interferon regulatory system 2, TNF-α inducing protein 3 and TNF-α receptor 2 and thus has potential application for human and animal health.

Trim69 regulates zebrafish brain development by ap-1 pathway.

Proteins belonging to the TRIM family have been implicated in a variety of cellular processes such as apoptosis, differentiation, neurogenesis, muscular physiology and innate immune responses. Trim69, previously identified as a novel gene cloned from a human testis cDNA library, has a homologous gene in zebrafish and this study focused on investigating the function of trim69 in zebrafish neurogenesis. Trim69 was found to be expressed in zebrafish embryo brain at the early stages. Knockdown of trim69 led to deformed brain development, obvious signs of apoptosis present in the head, and decreased expression of neuronal differentiation and stem cell markers. This phenotype was rescued upon co-injection of human mRNA together along with the trim69 knockdown. Results of this study also showed an interaction between TRIM69 and c-Jun in human cells, and upon TRIM69 knock down c-Jun expression subsequently increased, whereas the over-expression of TRIM69 led to the down-regulation of c-Jun. Additionally, knockdown both c-Jun and trim69 can rescue the deformed brain, evident cellular apoptosis in the head and decreased expression of neuronal differentiation and stem cell markers. Overall, our results support a role for trim69 in the development of the zebrafish brain through ap-1 pathway.

Mutant KRAS Conversion of Conventional T Cells into Regulatory T Cells.

Constitutive activation of the KRAS oncogene in human malignancies is associated with aggressive tumor growth and poor prognosis. Similar to other oncogenes, KRAS acts in a cell-intrinsic manner to affect tumor growth or survival. However, we describe here a different, cell-extrinsic mechanism through which mutant KRAS contributes to tumor development. Tumor cells carrying mutated KRAS induced highly suppressive T cells, and silencing KRAS reversed this effect. Overexpression of the mutant KRAS(G12V)gene in wild-type KRAS tumor cells led to regulatory T-cell (Treg) induction. We also demonstrate that mutant KRAS induces the secretion of IL10 and transforming growth factor-β1 (both required for Treg induction) by tumor cells through the activation of the MEK-ERK-AP1 pathway. Finally, we report that inhibition of KRAS reduces the infiltration of Tregs in KRAS-driven lung tumorigenesis even before tumor formation. This cell-extrinsic mechanism allows tumor cells harboring a mutant KRAS oncogene to escape immune recognition. Thus, an oncogene can promote tumor progression independent of its transforming activity by increasing the number and function of Tregs. This has a significant clinical potential, in which targeting KRAS and its downstream signaling pathways could be used as powerful immune modulators in cancer immunotherapy.

TNF-alpha promotes lymphangiogenesis and lymphatic metastasis of gallbladder cancer through the ERK1/2/AP-1/VEGF-D pathway.

BackgroundTumor necrosis factor-alpha (TNF-α), a key player in cancer-related inflammation, was recently demonstrated to be involved in the lymphatic metastasis of gallbladder cancer (GBC). Vascular endothelial growth factor D (VEGF-D) is a key lymphangiogenic factor that is associated with lymphangiogenesis and lymph node metastasis in GBC. However, whether VEGF-D is involved in TNF-α-induced lymphatic metastasis of GBC remains undetermined.MethodsThe expression of VEGF-D in patient specimens was detected by immunohistochemistry and the relationship between VEGF-D in the tissue and TNF-α in the bile of the matching patients was analyzed. The VEGF-D mRNA and protein levels after treatment with exogenous TNF-α in NOZ, GBC-SD and SGC-996 cell lines were measured by real-time PCR and ELISA. The promoter activity and transcriptional regulation of VEGF-D were analyzed with the relative luciferase reporter assay, mutant constructs, electrophoretic mobility shift assay (EMSA), chromatin immunoprecipitation (ChIP) assay, RNA interference and Western blotting. Inhibitors of JNK, p38 MAPK and ERK1/2 were used to explore the upstream signaling effector of AP-1. We used lentiviral vector expressing a VEGF-D shRNA construct to knockdown VEGF-D gene in NOZ and GBC-SD cells. The role of the TNF-α-VEGF-D axis in the tube formation of human dermal lymphatic endothelial cells (HDLECs) was determined using a three-dimensional coculture system. The role of the TNF-α - VEGF-D axis in lymphangiogenesis and lymph node metastasis was studied via animal experiment.ResultsTNF-α levels in the bile of GBC patients were positively correlated with VEGF-D expression in the clinical specimens. TNF-α can upregulate the protein expression and promoter activity of VEGF-D through the ERK1/2 - AP-1 pathway. Moreover, TNF-α can promote tube formation of HDLECs, lymphangiogenesis and lymph node metastasis of GBC by upregulation of VEGF-D in vitro and in vivo.ConclusionTaken together, our data suggest that TNF-α can promote lymphangiogenesis and lymphatic metastasis of GBC through the ERK1/2/AP-1/VEGF-D pathway.

Genome-Wide Chromatin Landscape Transitions Identify Novel Pathways in Early Commitment to Osteoblast Differentiation.

Bone continuously undergoes remodeling by a tightly regulated process that involves osteoblast differentiation from Mesenchymal Stem Cells (MSC). However, commitment of MSC to osteoblastic lineage is a poorly understood process. Chromatin organization functions as a molecular gatekeeper of DNA functions. Detection of sites that are hypersensitive to Dnase I has been used for detailed examination of changes in response to hormones and differentiation cues. To investigate the early steps in commitment of MSC to osteoblasts, we used a model human temperature-sensitive cell line, hFOB. When shifted to non-permissive temperature, these cells undergo "spontaneous" differentiation that takes several weeks, a process that is greatly accelerated by osteogenic induction media. We performed Dnase I hypersensitivity assays combined with deep sequencing to identify genome-wide potential regulatory events in cells undergoing early steps of commitment to osteoblasts. Massive reorganization of chromatin occurred within hours of differentiation. Whereas ~30% of unique DHS sites were located in the promoters, the majority was outside of the promoters, designated as enhancers. Many of them were at novel genomic sites and need to be confirmed experimentally. We developed a novel method for identification of cellular networks based solely on DHS enhancers signature correlated to gene expression. The analysis of enhancers that were unique to differentiating cells led to identification of bone developmental program encompassing 147 genes that directly or indirectly participate in osteogenesis. Identification of these pathways provided an unprecedented view of genomic regulation during early steps of differentiation and changes related to WNT, AP-1 and other pathways may have therapeutic implications.