IL1 AND GINGIVAL COLLAGENASE GENE REGULATION

IL1 和牙龈胶原酶基因调控

• 批准号: 2133098
• 负责人: DINESH S TEWARI
• 金额: $ 10.04万
• 依托单位: TEMPLE UNIV OF THE COMMONWEALTH
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-04-01 至 2000-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2133098
• 关键词: DNA binding protein; DNA footprinting; antisense nucleic acid; chloramphenicol acetyltransferase; collagenase; complementary DNA; enzyme induction /repression; fibroblasts; gel mobility shift assay; genetic promoter element; genetic regulation; gingiva; human subject; interleukin 1; metalloenzyme; northern blottings; nuclear runoff assay; nucleic acid sequence; oligonucleotides; periodontitis; stromelysin; tissue /cell culture; transcription factor; transfection /expression vector; western blottings

Interleukin-1 (IL-1) stimulates production of gingival fibroblast metalloproteinases (e . g. collagenase, stromelysin (proteoglycanase)) and thereby has been linked to gingival soft tissue destruction associated with periodontitis. The intracellular biochemical events that link IL-1 gingival fibroblast receptor activation to metalloproteinase gene activation are poorly understood. It is likely that a number of proteins (e.g. pretranscriptional/post-transcriptional regulatory factors, enzymes involved in signal transduction) play key roles in the modulation of these metalloproteinases. Recent studies with synovial and gingival fibroblasts in culture have provided new insights on the molecular events associated with the IL-1 induction of cellular metalloproteinases, there is much less information available on the induction of these metalloproteinases in gingival fibroblasts. Based on this, the objective of the present proposal involves a series of experiments designed to begin to identify six gingival fibroblast genes and products induced by IL-1 that could be involved in the modulation of metalloproteinases likely associated with periodontitis. The specific aims are to: (l) To obtain full length cDNA sequences for four IL- 1 induced genes (previously unidentified) obtained from human gingival fibroblasts, characterize them and express their proteins, (2) To determine if/how protein products derived from these three IL-1 induced genes and two other known genes (C/EBP and NFkB) upregulated by IL-1, functionally bind to the promoter region or modulate metalloproteinase transcription and (3) To determine if these selected genes functionally participate in the IL-1 induction of metalloproteinases. These aims will be accomplished by using: (a) gingival fibroblast cell lines and primary cultures derived from gingival tissue obtained from normal and patients with periodontitis; (b) full length cDNA clone isolation, characterization and sequencing; (c) computerized sequence analysis; (d) in vitro protein translation; (e) co- transfection, overexpression and underexpression (anti sense) studies; and (f) gel shift binding and foot printing assays. These proposed in-vitro studies with human gingival fibroblasts offer a model system to provide fundamental molecular information on gene activation induced by IL-1, and thereby identifying events which are likely involved in the pathogenesis of periodontitis. Identification of such genes holds promise for better defining the molecular basis of connective tissue destruction in periodontitis. Consequently, an important therapeutic implication of the present proposal is that blockers of these IL-1 induced biochemical events could be useful clinically for the future treatment of periodontitis.

白细胞介素1(IL-1)刺激牙周成纤维细胞产生 金属蛋白酶(E.胶原酶、基质分解酶(蛋白葡聚糖酶))和 从而与牙周软组织破坏有关 牙周炎。与IL-1牙龈相关的细胞内生化事件 成纤维细胞受体激活对金属蛋白酶基因激活的影响 人们对此知之甚少。很可能有一些蛋白质(例如 转录前/转录后调节因子、酶 参与信号转导)在这些信号的调节中起着关键作用 金属蛋白酶。滑膜和牙龈成纤维细胞的研究近况 为相关的分子事件提供了新的见解 随着IL-1诱导的细胞金属蛋白酶，有更少的 有关这些金属蛋白酶在体内诱导作用的信息 牙龈成纤维细胞。基于此，本提案的目标是 涉及一系列实验，旨在开始识别六种牙龈 IL-1诱导的成纤维细胞基因和产物可能参与 金属蛋白酶的调节可能与牙周炎有关。这个 具体目的是：(L)获得四种IL-4的全长c DNA序列 1个从人牙龈中获得的诱导基因(以前未鉴定) 成纤维细胞，鉴定它们并表达它们的蛋白，(2)确定 如果/如何从这三个IL-1诱导基因和两个基因获得蛋白质产物 其他已知基因(C/EBP和NFkB)由IL-1上调，功能结合 到启动子区域或调控金属蛋白酶转录和(3) 以确定这些选定的基因是否在功能上参与IL-1 金属蛋白酶的诱导。这些目标将通过以下方式实现： (A)牙龈成纤维细胞系和来源于 牙龈组织取自正常人和牙周炎患者； 全长cdna克隆分离、鉴定及测序； 计算机序列分析；(D)体外蛋白质翻译；(E)共- 转基因、过表达和低表达(反义)研究；以及 (F)凝胶移位绑定和足印分析。这些建议是体外培养的 对人牙龈成纤维细胞的研究提供了一个模型系统，以提供 IL-1诱导基因激活的基本分子信息，以及 从而识别可能与发病机制有关的事件 牙周炎。这些基因的识别为更好的研究提供了希望 确定结缔组织破坏的分子基础 牙周炎。因此，一项重要的治疗意义在于 目前的建议是，这些IL-1的阻滞剂诱导生化事件 对今后牙周炎的治疗有一定的临床应用价值。