Miao Di, Godovikova Valentina, Qian Xu, Seshadrinathan Suchithra, Kapila Yvonne L, Fenno J Christopher
Department of Periodontics and Oral Medicine, School of Dentistry, University of Michigan, Ann Arbor, MI, United States.
Department of Biologic and Materials Sciences, School of Dentistry, University of Michigan, Ann Arbor, MI, United States.
Arch Oral Biol. 2014 Oct;59(10):1056-64. doi: 10.1016/j.archoralbio.2014.06.003. Epub 2014 Jun 14.
Periodontal pathogens initiate chronic dysregulation of inflammation and tissue homeostasis that characterize periodontal disease. To better understand oral microbe-host tissue interactions, we investigated expression and activation of MMP-2 in periodontal ligament cells following Treponema denticola challenge.
Cultured PDL cells were challenged with T. denticola, and bacterial adherence, internalization and survival were assayed by immunofluorescence microscopy and antibiotic protection assays, respectively. MMP-2 activation was detected by zymography. MMP-2, MT1/MMP and TIMP-2 expression following T. denticola challenge was determined by qRT-PCR. Promoter methylation of MMP-2 and MT1/MMP was screened by methylation-sensitive restriction analysis and by bisulfite DNA sequencing.
T. denticola adhered to and was internalized by PDL cells but did not survive intracellularly beyond 24h. Importantly, while dentilisin activity in PDL culture supernatants gradually decreased following T. denticola challenge, MMP-2 activation persisted for up to 5 days, suggesting involvement of other regulatory mechanisms. Transcription and expression of MT1/MMP and TIMP-2 increased in response to T. denticola challenge. However, consistent with previously reported constitutive pro-MMP-2 expression in PDL cells, the MMP-2 promoter was hypomethylated, independent of T. denticola challenge.
MMP-2 promoter hypomethylation is consistent with constitutive pro-MMP-2 expression in PDL cells. This, coupled with T. denticola-mediated upregulation of MMP-2-related genes and chronic activation of pro-MMP-2, mimics key in vivo mechanisms of periodontal disease chronicity, in particular MMP-2-dependent matrix degradation and bone resorption. Adherence and/or internalization of T. denticola may contribute to these processes by one or more regulatory mechanisms, including contact-dependent signal transduction or other epigenetic mechanisms.
牙周病原体引发炎症和组织稳态的慢性失调，这是牙周病的特征。为了更好地理解口腔微生物与宿主组织的相互作用，我们研究了牙龈卟啉单胞菌攻击后牙周膜细胞中MMP-2的表达和激活情况。
用牙龈卟啉单胞菌攻击培养的牙周膜细胞，分别通过免疫荧光显微镜和抗生素保护试验检测细菌的黏附、内化和存活情况。用酶谱法检测MMP-2的激活情况。通过qRT-PCR测定牙龈卟啉单胞菌攻击后MMP-2、MT1/MMP和TIMP-2的表达。通过甲基化敏感限制性分析和亚硫酸氢盐DNA测序筛选MMP-2和MT1/MMP的启动子甲基化情况。
牙龈卟啉单胞菌黏附并被牙周膜细胞内化，但在细胞内24小时后无法存活。重要的是，虽然牙龈卟啉单胞菌攻击后牙周膜培养上清液中的牙本质素活性逐渐降低，但MMP-2的激活持续长达5天，提示存在其他调节机制。MT1/MMP和TIMP-2的转录和表达在牙龈卟啉单胞菌攻击后增加。然而，与先前报道的牙周膜细胞中组成型前MMP-2表达一致，MMP-2启动子低甲基化，与牙龈卟啉单胞菌攻击无关。
MMP-2启动子低甲基化与牙周膜细胞中组成型前MMP-2表达一致。这与牙龈卟啉单胞菌介导的MMP-2相关基因上调和前MMP-2的慢性激活一起，模拟了牙周病慢性化的关键体内机制，特别是MMP-2依赖性基质降解和骨吸收。牙龈卟啉单胞菌的黏附和/或内化可能通过一种或多种调节机制，包括接触依赖性信号转导或其他表观遗传机制，促成这些过程。
