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Involvement of ayu NOD2 in NF-?B and MAPK signaling pathways: Insights into functional conservation of NOD2 in antibacterial innate immunity.

ABSTRACT: Nucleotide oligomerization domain 2 (NOD2) is a major cytoplasmic sensor for pathogens and is critical for the clearance of cytosolic bacteria in mammals. However, studies regarding NOD2, especially the initiated signaling pathways, are scarce in teleost species. In this study, we identified a NOD2 molecule (PaNOD2) from ayu (Plecoglossus altivelis). Bioinformatics analysis showed the structure of NOD2 to be highly conserved during vertebrate evolution. Dual-luciferase reporter assays examined the activation of NF-?B signaling and Western blotting analysis detected the phosphorylation of three MAP kinases (p-38, Erk1/2, and JNK1/2). Functional study revealed that, like its mammalian counterparts, PaNOD2 was the receptor of the bacterial cell wall component muramyl dipeptide (MDP), and the leucine-rich repeat motif was responsible for the recognition and binding of PaNOD2 with the ligand. Overexpression of PaNOD2 activated the NF-?B signaling pathway, leading to the upregulation of inflammatory cytokines, including TNF-? and IL-1? in HEK293T cells and ayu head kidney-derived monocytes/macrophages (MO/M?). Particularly, we found that PaNOD2 activated the MAPK signaling pathways, as indicated by the increased phosphorylation of p-38, Erk1/2, and JNK1/2, which have not been characterized in any teleost species previously. Our findings proved that the NOD2 molecule and initiated pathways are conserved between mammals and ayu. Therefore, ayu could be used as an animal model to investigate NOD2-based diseases and therapeutic applications.

SUBMITTER: Ren Y

PROVIDER: S-EPMC6378557 | biostudies-literature | 2019 Mar

REPOSITORIES: biostudies-literature

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Involvement of ayu NOD2 in NF-κB and MAPK signaling pathways: Insights into functional conservation of NOD2 in antibacterial innate immunity.

Ren Yi Y Liu Shui-Fang SF Nie Li L Cai Shi-Yu SY Chen Jiong J

Zoological research 20180525 2

Nucleotide oligomerization domain 2 (NOD2) is a major cytoplasmic sensor for pathogens and is critical for the clearance of cytosolic bacteria in mammals. However, studies regarding NOD2, especially the initiated signaling pathways, are scarce in teleost species. In this study, we identified a NOD2 molecule (PaNOD2) from ayu (Plecoglossus altivelis). Bioinformatics analysis showed the structure of NOD2 to be highly conserved during vertebrate evolution. Dual-luciferase reporter assays examined t ...[more]

PMID: 29872030

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Project description:The overall objective of this study was to investigate the mechanism of inflammation on chondrocyte injury and the protective effect of catalpol on chondrocytes in an inflammatory environment. Chondrocytes were isolated and cultured from the knee joints of three-day-old newborn mice. Alcian Blue staining and the immunocytochemistry staining of type II collagen were used to identify the purity of chondrocytes. Primary chondrocytes were stimulated by IL-1β (10 ng/mL) and subjected to transcriptome analysis. Differentially expressed genes (DEGs) were further analyzed based on Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses. In this experimental study, we performed the viability assay to determine the effects of different concentrations of catalpol on the cell viability of chondrocytes. Chondrocytes were seeded in six-well plates and exposed to 10 μM catalpol 2 h prior to treatment with IL-1β (10 ng/mL). Quantitative real-time (qPCR) and Western blotting were performed to evaluate the RNA and protein expression, respectively. Based on the results of transcriptomics analysis, we found the NOD2 signaling pathway, the NF-kappa B signaling pathway, and the MAPK signaling pathway showed significant changes in chondrocyte damage caused by inflammation. Catalpol (10 μM and 100 μM) could significantly reduce NO, IL-6, IL-1β, and TNF-α in supernatant of chondrocytes. Catalpol significantly inhibited the mRNA expression of IL-1, IL-6, and IL-12 in chondrocytes induced by IL-1β. Catalpol markedly inhibited MMP3, MMP13 mRNA, and protein levels. Catalpol could significantly reduce TNF-α mRNA levels in inflammatory chondrocytes. Inflammation causes significant increases in mRNA levels and protein levels of NOD2, mRNA levels, and protein levels were markedly suppressed by catalpol. In addition, catalpol could significantly increase IKBα protein levels and significantly lower intranuclear P65 levels. Catalpol significantly lowered the phosphorylation protein levels of ERK, p38, and JNK. Our transcriptomic analysis demonstrated that the activation of NOD2 and its downstream pathways, NF-κB and MAPK, is an important cause of the inflammatory injury to chondrocytes induced by IL-1β. Catalpol inhibited the activation of the NOD2 signaling pathway, which reduced the phosphorylation of ERK, p38, and JNK, inhibited the degradation of IκBα, inhibited p65 translocation into the nucleus, reduced the release of inflammatory cytokines, and attenuated the inflammatory damage to chondrocytes.

Dataset Information

Involvement of ayu NOD2 in NF-?B and MAPK signaling pathways: Insights into functional conservation of NOD2 in antibacterial innate immunity.

Publications

Involvement of ayu NOD2 in NF-κB and MAPK signaling pathways: Insights into functional conservation of NOD2 in antibacterial innate immunity.

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