Interleukin-17 plays a role in pulp inflammation partly by WNT5A protein induction.

OBJECTIVE: Our study aimed to investigate the role of interleukin (IL)-17 in dental pulp inflammation and the relationship between WNT5A and IL-17. METHODS: Immunohistochemical staining was used to detect the expression of tumor necrosis factor-α (TNF-α), WNT5A and IL-17 in pulp tissues. Anti-IL-17 neutralizing antibody was used in rat pulpitis model and to study the role of IL-17 in pulpitis. TNF-α, WNT5A or IL-17 recombinant protein were used to treat human dental pulp cells. RT-PCR, Western blot, and Enzyme linked immunosorbent assay were used to detect the expression of mRNA and protein. Transwell assay was used to measure the migration of THP-1 cells, which is a human monocytic cell line. RESULTS: IL-17 and WNT5A are co-expressed in TNF-α high-expressed region in human and rat pulpitis tissue. IL-17 mainly contributes to its positive regulatory role in inflammation through up regulate cytokines and mediated macrophages migration. Anti-IL-17 neutralizing antibody can suppress the inflammatory cell infiltration and TNF-α expression in dental pulpitis. TNF-α promotes the expression of IL-17 partly through WNT5A and WNT5A regulates IL-17 expression by mitogen-activated protein kinase (MAPK)-(P38 and ERK) pathway. CONCLUSIONS: IL-17 acts as an inflammatory mediator in dental pulp inflammation. The expression of IL-17 can be partially regulated by WNT5A.

EZH2 regulates dental pulp inflammation by direct effect on inflammatory factors.

OBJECTIVE: Pulpitis is a multi-factorial disease that could be caused by complex interactions between genetics, epigenetics and environmental factors. We aimed to evaluate the role of Enhancer of Zeste Homolog 2 (EZH2) in the inflammatory response of human dental pulp cells (HDPCs) and dental pulp tissues. METHODS: The expressions of inflammatory cytokines in HDPCs treated by EZH2 complex or EZH2 siRNA with or without rhTNF-α were examined by quantitative real-time polymerase chain reaction (q-PCR). The levels of secreted inflammatory cytokines including IL-6, IL-8, IL-15, CCL2 and CXCL12 in culture supernatants were measured by Luminex assay. In rat pulpitis model, the effects of EZH2 on dental pulp tissues were verified by histology. We invested the mechanisms of the effect of EZH2 on the inflammatory factors by ChIP assay. RESULTS: EZH2 down-regulation inhibited the expression of inflammatory factors, including IL-6, IL-8, IL-15, CCL2 and CXCL12 in HDPCs. EZH2 complex promoted the expression and secretion of these inflammatory factors in HDPCs, while EZH2 silencing could attenuate the promotion of inflammatory factors that were induced by rhTNF-α. In pulpitis models of rats, EZH2 down-regulation inhibited the inflammatory process of dental pulp while EZH2 complex showed no significant facilitation of pulpal inflammation. In addition, EZH2 could bind on the promoters of IL-6, IL-8 and CCL2, but not IL-15 and CXCL12, to affect the transcription of these proinflammatory cytokines. CONCLUSIONS: In HDPCs, EZH2 could induce inflammation, while EZH2 down-regulation could attenuate the inflammatory responses. EZH2 plays an important role in this inflammatory process of dental pulp.

Inhibition of SOX9 Promotes Inflammatory and Immune Responses of Dental Pulp.

INTRODUCTION: The process of pulpitis is characterized by extracellular matrix imbalance and inflammatory cell infiltration. As an essential transcription factor, sex-determining region Y-box 9 (SOX9) is significantly inhibited by tumor necrosis factor alpha in inflammatory joint diseases. The aim of this study was to explore the role of SOX9 in extracellular matrix balance, cytokine expression, and the immune response in dental pulp. METHODS: The expression of SOX9 in normal and inflamed pulp tissue/human dental pulp cells (HDPCs) was detected by immunohistochemistry, Western blot, and quantitative polymerase chain reaction (qPCR). SOX9 small interfering RNA was used to knock down SOX9 expression of dental cells in vitro; extracellular matrix imbalance was analyzed by qPCR, Western blot, and gelatin/collagen zymography, and the secretion of cytokines was scanned by antibody arrays. The immune response of THP-1 was investigated by cell migration assay, cell attachment assay, phagocytosis assay, and enzyme-linked immunosorbent assay. The interaction of SOX9 with target genes was explored by chromatin immunoprecipitation (ChIP). RESULTS: SOX9 was strongly expressed in normal dental pulp tissue and HDPCs and reduced in inflamed pulp. SOX9 knockdown could inhibit the production of type I collagen, stimulate the enzymatic activities of MMP2 and MMP13, and regulate the production of interleukin (IL) 8 of HDPCs. SOX9 knockdown also effectively suppressed the differentiation and functional activities of THP-1. ChIP showed that the binding of the SOX9 protein with matrix metalloproteinase (MMP)-1, MMP-13, and IL-8 gene promoters was reduced after being treated with recombinant human tumor necrosis factor alpha. CONCLUSIONS: SOX9 was inhibited in inflamed dental pulp and may participate in the regulation of extracellular matrix balance, the inflammatory process, and the immune response.

EZH2 promotes DNA replication by stabilizing interaction of POLδ and PCNA via methylation-mediated PCNA trimerization.

BACKGROUND: Proliferating cell nuclear antigen (PCNA), a ring-shaped homotrimer complex, promotes DNA replication via binding to DNA polymerase. Trimerized PCNA is critical for DNA replication. Enhancer of zeste homologue 2 (EZH2), which primarily acts as a histone methyltransferase, is essential for proliferation. However, how EZH2 promotes proliferation by controlling DNA replication through PCNA remains elusive. RESULTS: Here, we showed that low EZH2 levels repressed the proliferation of human dental pulp cells (hDPCs). The EZH2 protein level was dramatically upregulated in hDPCs at S phase in the absence of H3K27 trimethylation. Molecularly, EZH2 interacted with PCNA via the PIP box and dimethylated PCNA at lysine 110. Dimethylation of PCNA is essential for stabilization of the PCNA trimer and the binding of DNA polymerase δ to PCNA. CONCLUSIONS: Our data reveal the direct interaction between PCNA and EZH2 and a novel mechanism by which EZH2 orchestrates genome duplication.

EZH2, a potential regulator of dental pulp inflammation and regeneration.

INTRODUCTION: Dental pulp has limited capability to regenerate, which happens in the early stage of pulpitis. An ambiguous relationship exists; inflammation may impair or support pulp regeneration. Epigenetics, which is involved in cell proliferation and inflammation, could regulate human dental pulp cell (HDPCs) regeneration. The aim of this study was to determine the role of the epigenetic mark, enhancer of zeste homolog 2 (EZH2), in the inflammation, proliferation, and regeneration of dental pulp. We used trimethylated histone H3 lysine 27(H3K27me3) and its lysine demethylase 6B (KDM6B) to monitor functional effects of altered EZH2 levels. METHODS: We detected epigenetic marks (EZH2, H3K27me3, and KDM6B) in pulp tissue by immunohistochemistry and immunofluorescence. EZH2 levels in HDPCs in inflammatory responses or differentiation were analyzed by quantitative polymerase chain reaction and Western blot. Quantitative polymerase chain reaction was used to assess the effects of EZH2 inhibition on interleukins in HDPCs upon tumor necrosis factor alpha stimulation. Cell proliferation was tested by cell counting kit-8, cell cycle, and apoptosis analysis. HDPC differentiation was investigated by quantitative polymerase chain reaction, alkaline phosphatase activity, and oil red O staining. RESULTS: EZH2 and H3K27me3 were decreased, whereas KDM6B was increased in infected pulp tissue and cells, which were similar to HDPC differentiation. EZH2 inhibition suppressed IL-1b, IL-6, and IL-8 messenger RNA (mRNA) in HDPCs upon inflammatory stimuli and impeded HDPC proliferation by decreasing cell number, arresting cell cycle, and increasing apoptosis. Suppressed EZH2 impaired adipogenesis, peroxisome proliferator-activated receptor r (PPAR-r), and CCAAT-enhancer binding protein a (CEBP/a) mRNA in adipogenic induction while enhancing alkaline phosphatase activity, Osx, and bone sialoprotein (BSP) mRNA in mineralization induction of HDPCs. CONCLUSIONS: EZH2 inhibited HDPC osteogenic differentiation while enhancing inflammatory response and proliferation, suggesting its role in pulp inflammation, proliferation, and regeneration.

[Amaxa Nucleofector(TM) nuclear transfection apparatus transfers L1210 cell line].

Mouse L1210 leukemia cell line is widely used as a model in the study of tumorigenesis, as well as the efficacy of chemotherapeutic drugs; however, like other suspension cell lines, the mouse L1210 cell line has lowest transfection efficiency, that many barriers exist to study about the structure, function, as well as metabolism in leukemia cells. This study was aimed to obtain higher transfection efficiency of L1210 cell line to facilitate scientific research. The transfection efficiencies of nucleofector and liposome in L1210 leukemia cells were detected by converted fluorescence microscopy and flow cytometry using EGFP (enhance green fluorescent protein); cell viability was observed by trypan blue exclusion test. The results showed that the transfection efficiency of nucleofector primarily through reporter gene pEGFP by Amaxa Nucleofector(TM) nuclear transfer apparatus was significantly higher than lipofectamine 2000 transfection, furthermore, in the same cell density (2 × 10(6)/ml) and plasmid content (10 µg), the transfection efficiency of nuclear transfer apparatus default mode A-20 was higher than that of other modes (S-18, T-20). Its survival rate was up to 50.5% after 24 hours. Cell viability of liposome transfection reached to 88% after 24 hours, but the transfection efficiency was lower (< 1%). It is concluded that the nuclear transfer apparatus A-20 transfected L1210 can reach higher transfection efficiency up to 61.6%, which is significantly higher than that of lipofectamine transfection. The survival rate is up to 50.5% well meeting the needs of scientific research. Higher transfection efficiency is helpful for in-depth research about the morphology, functions and pathogenesis in leukemia model L1210, and provides more searching space for the treatment of leukemia diseases.