Bei Yin, Tianqian Hui, Fanyuan Yu, Haiyun Luo, Xueyang Liao, Jing Yang, Chenglin Wang, Ling Ye
State Key Laboratory of Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu, China.
State Key Laboratory of Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu, China.
J Endod. 2017 Feb;43(2):306-314.e2. doi: 10.1016/j.joen.2016.10.020. Epub 2016 Dec 29.
Pulpitis 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.
Human 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 of ASH1L and trimethylation of lysine 4 histone 3 (H3K4me3). The presence of ASH1L in HDPCs that had been generated by TNF-α stimulation was analyzed by Western blot procedures and cellular immunofluorescence. Once detected, ASH1L was silenced through the use of specific small interfering RNA. The effects of ASH1L on 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 whether ASH1L and 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 of ASH1L. We also examined the specific pathway involved in ASH1L regulation of the MMPs.
After stimulating HDPCs with TNF-α, ASH1L emerged as 1 of the most strongly induced epigenetic mediators. We found that TNF-α treatment induced the expression of ASH1L through the nuclear factor kappa B and MAPK signal pathways. ASH1L was found in both the nucleus and the cytoplasm. TNF-α treatment was particularly active in inducing the accumulation of ASH1L in cellular cytoplasm. As is also consistent with in vitro results, ASH1L was found in increased quantities in experimental dental pulpitis tissue. ASH1L knockdown 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. ASH1L knockdown activated the MAPK signal pathway in TNF-α-triggered HDPCs, the inhibition of which reversed the induction of MMPs.
Our research identifies a mechanism by which ASH1L suppresses the occurrence and operation of MMPs during pulpitis. It does this through the MAPK pathway.
牙髓炎是牙髓对外部刺激产生的炎症反应。该反应涉及大量细胞和分子活动。遗传和表观遗传调节因子均参与牙髓炎的发生。然而，表观遗传机制仍知之甚少。在本研究中，我们探讨了类无小同源框（ASH1L）基因在牙髓炎过程中的作用。
用促炎细胞因子肿瘤坏死因子α（TNF-α）刺激人牙髓细胞（HDPCs）。进行基因表达谱分析以评估表观遗传调节因子的发生情况。对大鼠实验性牙髓炎的牙髓组织进行免疫荧光检测，以检测ASH1L的存在及组蛋白H3第4位赖氨酸三甲基化（H3K4me3）情况。通过蛋白质免疫印迹法和细胞免疫荧光分析TNF-α刺激产生的HDPCs中ASH1L的存在情况。一旦检测到ASH1L，便通过使用特异性小干扰RNA将其沉默。然后通过定量聚合酶链反应分析、蛋白质免疫印迹法和酶谱分析来检测ASH1L对基质金属蛋白酶（MMPs）发生及活性的影响。进行染色质免疫沉淀以检测MMPs启动子区域是否存在ASH1L和H3K4me3。然后我们使用蛋白质免疫印迹法检测核因子κB和丝裂原活化蛋白激酶（MAPK）对ASH1L沉默的反应。我们还研究了ASH1L调节MMPs的具体途径。
用TNF-α刺激HDPCs后，ASH1L成为诱导最强的表观遗传介质之一。我们发现TNF-α处理通过核因子κB和MAPK信号通路诱导ASH1L表达。ASH1L在细胞核和细胞质中均有发现。TNF-α处理在诱导ASH1L在细胞质中积累方面特别活跃。与体外实验结果一致，在实验性牙髓炎组织中发现ASH1L含量增加。ASH1L敲低显著上调MMP-1、MMP-2和MMP-13的表达。它还对TNF-α刺激的HDPCs中MMP-2的酶活性产生影响。ASH1L敲低激活了TNF-α触发的HDPCs中的MAPK信号通路，抑制该通路可逆转MMPs的诱导。
我们的研究确定了一种机制，即ASH1L在牙髓炎期间通过MAPK途径抑制MMPs的发生及活性。
