ASH1L Suppresses Matrix Metalloproteinase through Mitogen‐activated Protein Kinase Signaling Pathway in Pulpitis
ASH1L Suppresses Matrix Metalloproteinase through Mitogen‐activated Protein Kinase Signaling Pathway in Pulpitis
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DOI：
10.1016/j.joen.2016.10.020
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发表时间：
2017-02
影响因子：
4.2
通讯作者：
Yin Bei;T. Hui;F. Yu;Haiyun Luo;Xueyang Liao;Yang Jing;Chenglin Wang;Ye Ling
中科院分区：
文献类型：
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作者：
Yin Bei;T. Hui;F. Yu;Haiyun Luo;Xueyang Liao;Yang Jing;Chenglin Wang;Ye Ling
牙髓炎是牙髓对外界刺激产生的炎症反应。这种反应需要大量的细胞和分子活动。牙髓炎的发生与遗传和表观遗传调节因子有关。然而，表观遗传机制仍然知之甚少。本研究以人牙髓细胞（HDPCs）为研究对象，探讨ASH 1 L基因在牙髓炎发生、发展过程中的作用。进行基因表达谱分析以评估表观遗传调节剂的发生。用免疫荧光法检测大鼠实验性牙髓炎牙髓组织中ASH 1和组蛋白3（H3 K4 me 3）的发生。通过Western印迹和细胞免疫荧光分析TNF-α刺激产生的ASH 1 Lin HDPCs的存在。一旦检测到，ASH 1 L通过使用特定的小干扰RNA沉默。然后通过定量聚合酶链反应、蛋白质印迹和酶谱分析来检测ASH 1 L对基质金属蛋白酶（MMPs）的发生和运作的影响。染色质免疫沉淀法检测MMPs启动子区是否存在ASH 1和H3 K4 me 3。然后，我们使用蛋白质印迹法检测核因子κ B和丝裂原活化蛋白激酶（MAPK）对ASH 1 L沉默的反应。结果TNF-α刺激HDPCs后，ASH 1 L成为诱导作用最强的表观遗传介质之一。结果表明，TNF-α通过核因子κ B和MAPK信号通路诱导ASH 1 L的表达，ASH 1 L在细胞核和细胞质中均有表达。TNF-α处理在诱导ASH 1 Lin细胞质积累方面特别活跃。与体外结果一致的是，在实验性牙髓炎组织中发现ASH 1 L的量增加，ASH 1 L敲低显著上调MMP-1、MMP-2和MMP-13的发生。ASH 1 L对TNF-α刺激的HDPCs中MMP-2的活性也有影响。ASH 1 L的敲低激活了TNF-α刺激的HDPCs中的MAPK信号通路，抑制该通路可逆转MMP-2的诱导。它是通过MAPK途径实现的。
IntroductionPulpitis is an inflammation of dental pulp produced by a response to external stimuli. The response entails substantial cellular and molecular activities. Both genetic and epigenetic regulators contribute to the occurrence of pulpitis. However, the epigenetic mechanisms are still poorly understood. In this research, we studied the role of the absent, small, or homeotic-like (ASH1L) gene in the process of pulpitis.MethodsHuman dental pulp cells (HDPCs) were stimulated with proinflammatory cytokine tumor necrosis factor alpha (TNF-α). Gene expression profiling was performed to assess the occurrence of epigenetic regulators. Pulp tissue from rat experimental pulpitis was subjected to immunofluorescence to detect the occurrence ofASH1Land trimethylation of lysine 4 histone 3 (H3K4me3). The presence ofASH1Lin HDPCs that had been generated by TNF-α stimulation was analyzed by Western blot procedures and cellular immunofluorescence. Once detected,ASH1Lwas silenced through the use of specific small interfering RNA. The effects ofASH1Lon the occurrence and operation of matrix metalloproteinases (MMPs) were then tested by analysis of quantitative polymerase chain reactions, Western blotting, and zymography. Chromatin immunoprecipitation was performed to detect whetherASH1Land H3K4me3 were present in the promoter regions of MMPs. We then used Western blot procedures to examine the nuclear factor kappa B and the mitogen-activated protein kinase (MAPK) responses to the silencing ofASH1L. We also examined the specific pathway involved inASH1Lregulation of the MMPs.ResultsAfter stimulating HDPCs with TNF-α,ASH1Lemerged as 1 of the most strongly induced epigenetic mediators. We found that TNF-α treatment induced the expression ofASH1Lthrough the nuclear factor kappa B and MAPK signal pathways.ASH1Lwas found in both the nucleus and the cytoplasm. TNF-α treatment was particularly active in inducing the accumulation ofASH1Lin cellular cytoplasm. As is also consistent within vitroresults,ASH1Lwas found in increased quantities in experimental dental pulpitis tissue.ASH1Lknockdown markedly up-regulated the occurrence of MMP-1, MMP-2, and MMP-13. It also exercised an impact on the enzymatic activity of MMP-2 in HDPCs that had been stimulated with TNF-α.ASH1Lknockdown activated the MAPK signal pathway in TNF-α–triggered HDPCs, the inhibition of which reversed the induction of MMPs.ConclusionsOur research identifies a mechanism by whichASH1Lsuppresses the occurrence and operation of MMPs during pulpitis. It does this through the MAPK pathway.