IL1 AND GINGIVAL COLLAGENASE GENE REGULATION

IL1 和牙龈胶原酶基因调控

• 批准号: 2684006
• 负责人: DINESH S TEWARI
• 金额: $ 11.04万
• 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/2684006
• 关键词: DNA footprinting; antisense nucleic acid; chloramphenicol acetyltransferase; collagenase; complementary DNA; enhancer binding protein; 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) 刺激牙龈成纤维细胞的产生 金属蛋白酶（例如胶原酶、溶基质素（蛋白聚糖酶））和 因此与牙龈软组织破坏有关 牙周炎。连接 IL-1 牙龈的细胞内生化事件 成纤维细胞受体激活到金属蛋白酶基因激活是 不太了解。很可能有许多蛋白质（例如 转录前/转录后调节因子、酶 参与信号转导）在这些调节中发挥关键作用 金属蛋白酶。滑膜和牙龈成纤维细胞的最新研究 文化中的相关分子事件提供了新的见解 随着细胞金属蛋白酶的 IL-1 诱导， 有关这些金属蛋白酶诱导的可用信息 牙龈成纤维细胞。 基于此，本提案的目标 涉及一系列旨在开始识别六种牙龈的实验 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 具体目标是： (l) 获得四种 IL-的全长 cDNA 序列 1 个从人类牙龈中获得的诱导基因（之前未鉴定） 成纤维细胞，表征它们并表达它们的蛋白质，(2) 确定 if/how 蛋白质产物源自这三个 IL-1 诱导基因和两个 其他已知基因（C/EBP 和 NFkB）被 IL-1 上调，功能性结合 至启动子区域或调节金属蛋白酶转录和（3） 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。这些目标将通过使用以下方式来实现： (a) 牙龈成纤维细胞系和原代培养物 从正常人和牙周炎患者获得的牙龈组织； (二) 全长 cDNA 克隆分离、表征和测序； (三) 计算机序列分析； (d) 体外蛋白质翻译； (e) 共同 转染、过度表达和表达不足（反义）研究；和 (f) 凝胶位移结合和足迹分析。这些建议的体外 人类牙龈成纤维细胞的研究提供了一个模型系统 IL-1 诱导的基因激活的基本分子信息，以及 从而识别可能参与发病机制的事件 牙周炎。这些基因的鉴定有望带来更好的结果 定义结缔组织破坏的分子基础 牙周炎。因此，重要的治疗意义 目前的建议是，这些 IL-1 诱导的生化事件的阻断剂 在临床上可能对牙周炎的未来治疗有用。