IL-17A and TNF synergistically drive expression of proinflammatory mediators in synovial fibroblasts via IκBζ-dependent induction of ELF3.

OBJECTIVE: IL-17A and TNF act in synergy to induce proinflammatory mediators in synovial fibroblasts thus contributing to diseases associated with chronic arthritis. Many of these factors are regulated by transcription factor E74-like factor-3 (ELF3). Therefore, we sought to investigate ELF3 as a downstream target of IL-17A and TNF signalling and to characterize its role in the molecular mechanism of synergy between IL-17A and TNF. METHODS: Regulation of ELF3 expression by IL-17A and TNF was studied in synovial fibroblasts of RA and OA patients and RA synovial explants. Signalling leading to ELF3 mRNA induction and the impact of ELF3 on the response to IL-17A and TNF were studied using siRNA, transient overexpression and signalling inhibitors in synovial fibroblasts and HEK293 cells. RESULTS: ELF3 was marginally affected by IL-17A or TNF alone, but their combination resulted in high and sustained expression. ELF3 expression was regulated by the nuclear factor-κB (NF-κB) pathway and CCAAT/enhancer-binding protein ß (C/EBPß), but its induction required synthesis of the NF-κB co-factor IκB (inhibitor of NF-κB) ζ. siRNA-mediated depletion of ELF3 attenuated the induction of cytokines and matrix metalloproteinases by the combination of IL-17A and TNF. Overexpression of ELF3 or IκBζ showed synergistic effect with TNF in upregulating expression of chemokine (C-C motif) ligand 8 (CCL8), and depletion of ELF3 abrogated CCL8 mRNA induction by the combination of IκBζ overexpression and TNF. CONCLUSION: Altogether, our results establish ELF3 as an important mediator of the synergistic effect of IL-17A and TNF in synovial fibroblasts. The findings provide novel information of the pathogenic mechanisms of IL-17A in chronic arthritis and implicate ELF3 as a potential therapeutic target.

Resveratrol inhibits MUC5AC expression by regulating SPDEF in lung cancer cells.

BACKGROUND: MUC5AC was recently identified to play important roles in the proliferation and metastasis of malignant mucinous lung tumor cells. Resveratrol (Res), a natural compound with anticancer effects in lung cancer cells, has been reported to inhibit mucin production in airway epithelial cells. This study aimed to investigate the inhibitory effect of Res on MUC5AC expression in lung mucinous adenocarcinoma cells and the potential mechanisms. METHODS: Mucus-producing A549 human lung carcinoma cells were used to test the effects of Res on SPDEF and MUC5AC expression. Gene and protein expression was assessed by real-time quantitative PCR (qPCR), immunofluorescence and western blotting assays. SPDEF lentivirus was used to upregulate SPDEF expression levels in mucus-producing A549 human lung carcinoma cells. Cell proliferation was assessed by Cell Counting Kit-8 (CCK-8) assay. RESULTS: Res decreased MUC5AC expression in an SPDEF-dependent manner in mucus-producing A549 human lung carcinoma cells, and this change was accompanied by decreased ERK expression and AKT pathway activation. Moreover, SPDEF was found to be overexpressed in lung adenocarcinoma (LUAD), especially in mucinous adenocarcinoma. In-vitro functional assays showed that overexpression of SPDEF reduced the chemosensitivity of A549 cells to cisplatin (DDP). In addition, Res treatment increased A549 cell chemosensitivity to DDP by inhibiting the SPDEF-MUC5AC axis. CONCLUSION: Our results indicate that the SPDEF-MUC5AC axis is associated with DDP sensitivity, and that Res decreases SPDEF and MUC5AC expression by inhibiting ERK and AKT signaling in A549 cells, which provides a potential pharmacotherapy for the prevention and therapeutic management of mucinous adenocarcinoma.

Transcriptomic profile of Pea3 family members reveal regulatory codes for axon outgrowth and neuronal connection specificity.

PEA3 transcription factor subfamily is present in a variety of tissues with branching morphogenesis, and play a particularly significant role in neural circuit formation and specificity. Many target genes in axon guidance and cell-cell adhesion pathways have been identified for Pea3 transcription factor (but not for Erm or Er81); however it was not so far clear whether all Pea3 subfamily members regulate same target genes, or whether there are unique targets for each subfamily member that help explain the exclusivity and specificity of these proteins in neuronal circuit formation. In this study, using transcriptomics and qPCR analyses in SH-SY5Y neuroblastoma cells, hypothalamic and hippocampal cell line, we have identified cell type-specific and subfamily member-specific targets for PEA3 transcription factor subfamily. While Pea3 upregulates transcription of Sema3D and represses Sema5B, for example, Erm and Er81 upregulate Sema5A and Er81 regulates Unc5C and Sema4G while repressing EFNB3 in SH-SY5Y neuroblastoma cells. We furthermore present a molecular model of how unique sites within the ETS domain of each family member can help recognize specific target motifs. Such cell-context and member-specific combinatorial expression profiles help identify cell-cell and cell-extracellular matrix communication networks and how they establish specific connections.

Novel ETV6-RUNX1 Mouse Model to Study the Role of ELF-MF in Childhood B-Acute Lymphoblastic Leukemia: a Pilot Study.

Exposure to extremely low-frequency magnetic fields (ELF-MFs) has been classified by the International Agency for Research on Cancer (IARC) as "possibly carcinogenic to humans," based on limited scientific evidence concerning childhood leukemia. This assessment emphasized the lack of appropriate animal models recapitulating the natural history of this disease. Childhood B-cell acute lymphoblastic leukemia (B-ALL) is the result of complex interactions between genetic susceptibility and exposure to exogenous agents. The most common chromosomal alteration is the ETV6-RUNX1 fusion gene, which confers a low risk of developing the malignancy by originating a preleukemic clone requiring secondary hits for full-blown disease to appear. To develop potential prophylactic interventions, we need to identify the environmental triggers of the second hit. Recently, we generated a B-ALL mouse model of the human ETV6-RUNX1+ preleukemic state. Here, we present the results from the ARIMMORA pilot study, obtained by exposing 34 Sca1-ETV6-RUNX1 mice (vs. 27 unexposed) to a 50 Hz magnetic field of 1.5 mT with both fundamental and harmonic content, with an on/off cycle of 10 min/5 min, for 20 h/day, from conception until 3 months of age. Mice were monitored until 2 years of age and peripheral blood was periodically analyzed by flow cytometry. One of the exposed mice developed B-ALL while none of the non-exposed did. Although the results are statistically non-significant due to the limited number of mice used in this pilot experiment, overall, the results show that the newly developed Sca1-ETV6-RUNX1 mouse can be successfully used for ELF-MF exposure studies about the etiology of childhood B-ALL. Bioelectromagnetics. 2019;40:343-353. © 2019 Bioelectromagnetics Society.

XRP44X, an Inhibitor of Ras/Erk Activation of the Transcription Factor Elk3, Inhibits Tumour Growth and Metastasis in Mice.

Transcription factors have an important role in cancer but are difficult targets for the development of tumour therapies. These factors include the Ets family, and in this study Elk3 that is activated by Ras oncogene /Erk signalling, and is involved in angiogenesis, malignant progression and epithelial-mesenchymal type processes. We previously described the identification and in-vitro characterisation of an inhibitor of Ras / Erk activation of Elk3 that also affects microtubules, XRP44X. We now report an initial characterisation of the effects of XRP44X in-vivo on tumour growth and metastasis in three preclinical models mouse models, subcutaneous xenografts, intra-cardiac injection-bone metastasis and the TRAMP transgenic mouse model of prostate cancer progression. XRP44X inhibits tumour growth and metastasis, with limited toxicity. Tumours from XRP44X-treated animals have decreased expression of genes containing Elk3-like binding motifs in their promoters, Elk3 protein and phosphorylated Elk3, suggesting that perhaps XRP44X acts in part by inhibiting the activity of Elk3. Further studies are now warranted to develop XRP44X for tumour therapy.

Characterization of the 5'-flanking region of the mouse asparagine-linked glycosylation 12 homolog gene.

Recently, we characterized multiple roles of the endoplasmic reticulum stress responsive element (ERSE) in the promotion of a unique head-to-head gene pair: mammalian asparagine-linked glycosylation 12 homolog (ALG12) and cysteine-rich with EGF-like domains 2 (CRELD2). This bidirectional promoter, which consists of fewer than 400 base pairs, separates the two genes. It has been demonstrated that the ALG12 promoter shows less transcriptional activity through ERSE, but its basic regulatory mechanism has not been characterized. In this study, we focused on well-conserved binding elements for the transcription factors for ATF6, NF-Y and YY1 and the Sp1 and Ets families in the 5'-flanking region of the mouse ALG12 gene. We characterized their dominant roles in regulating ALG12 promoter activities using several deletion and mutation luciferase reporter constructs. The ALG12 gene is expressed in three distinct cell lines: Neuro2a, C6 glioma and HeLa cells. The reporter activity in each cell line decreased similarly with serial deletions of the mouse ALG12 promoter. Mutations in the ERSE and adjacent NF-Y-binding element slightly affected reporter activity. Each of the mutations in the GC-rich sequence and YY1-binding element reduced ALG12 promoter activity, and the combination of these mutations additively decreased reporter activity. Each mutation in the tandem-arranged Ets-family consensus sequences partially attenuated ALG12 promoter activity, and mutations of all three Ets-binding elements decreased promoter activity by approximately 40%. Mutation of the three conserved regulatory elements (GC-rich, YY1 and Ets) in the ALG12 promoter decreased reporter activity by more than 90%. Our results suggest that the promoter activity of the mouse ALG12 gene is regulated in a similar manner in the three cell lines tested in this study. The well-conserved consensus sequences in the promoter of this gene synergistically contribute to maintaining basal gene expression.