REGULATION OF OSTEOBLAST GENE EXPRESSION BY CYTOKINES

细胞因子对成骨细胞基因表达的调节

• 批准号: 3161103
• 负责人: JOHN R HARRISON
• 金额: $ 13.37万
• 依托单位: UNIVERSITY OF CONNECTICUT SCH OF MED/DNT
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-09-15 至 1995-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3161103
• 关键词: DNA footprinting; DNA methylation; collagen; gel mobility shift assay; gene deletion mutation; gene expression; genetic mapping; genetic promoter element; interleukin 1; molecular cloning; northern blottings; nucleic acid sequence; osteoblasts; phorbols; prostaglandin endoperoxide synthase; site directed mutagenesis; tissue /cell culture; transcription factor; tumor necrosis factor alpha

The clonal osteoblastic cell line MC3T3-El has been used as a model to study the mechanisms of interleukin-1 (IL-1) and tumor necrosis factor-alpha (TNF) action on type I collagen and prostaglandin H synthase transcription in bone cells. Using MC3T3-El cells permanently transfected with ColCAT 3.6, a chimeric gene consisting of 3.6 kb of alpha1(I) flanking sequence directing transcription of the structural thloramphenicol acetyltransferase (CAT) gene, we found that cytokines regulate alpha1(I) promoter activity in a differentiation-dependent manner. In cells plated at high density, which exhibit an osteoblastic phenotype and express high levels of CAT activity, IL-1 and TNF caused repression of alpha1(I) transcription. On the other hand, in cells plated at low density, which are less differentiated, a stimulatory transcriptional response to cytokines was observed. Phorbol 12-myristate 13-acetate (PMA), which mimics IL-1 and TNF actions in some systems, repressed ColCAT 3.6 activity regardless of the differentiation state. However, a dramatic stimulatory effect of PMA, but not cytokines, was unmasked in differentiated cells harboring a 5'-deletion construct which lacks the upstream basal enhancer region of the alpha1(I) promoter. The location of cytokine and PMA response elements in the alpha1(I) promoter will be mapped using a functional CAT assay. Initially, a series of 5' deletion mutants will be tested for cytokine and PMA responsiveness. ColCAT 3.6 mutants containing internal deletions will be prepared to assess the role of downstream sequences in the presence of the intact upstream enhancer. Putative response elements will be tested for their ability to confer responsiveness to a heterologous thymidine kinase promoter. Regions responsive to IL-1, TNF or PMA will be examined for constitutive or inducible DNA-protein interactions by mobility shift, DNase protection and methylation interference analysis. The function of putative response elements identified by these methods will be assessed by site-directed mutagenesis. The 5' flanking region of the PGH synthase gene, which encodes the rate-limiting enzyme in prostaglandin biosynthesis, will be cloned and sequenced. Regulation of PGH synthase transcription by cytokines will be analyzed in a manner similar to that described for the alpha1(I) promoter. These studies should contribute to understanding the mechanisms by which cytokines alter type I collagen and prostaglandin synthesis in bone. Thus, these studies could have important implications for the pathogenesis of osteoporosis and bone loss associated with chronic inflammatory disease.

克隆成骨细胞系MC3T3-EL已被用作模型 研究白介素-1（IL-1）和肿瘤坏死的机制 因子-Alpha（TNF）对I型胶原蛋白和前列腺素H合酶的作用 骨细胞中的转录。 永久使用MC3T3-EL细胞 用Colcat 3.6转染，一个由3.6 kb组成的嵌合基因 alpha1（i）指导结构转录的侧翼序列 硫甲烯醇乙酰基转移酶（CAT）基因，我们发现细胞因子 调节α1（i）启动子活性在分化依赖性中 方式。 在以高密度镀以的细胞中，表现出成骨细胞 表型和表达高水平的CAT活性，IL-1和TNF引起 抑制alpha1（i）转录。 另一方面，在细胞中 以较小的分化为低密度的镀板，刺激性 观察到对细胞因子的转录反应。 Phorbol 12-杂种 在某些系统中模仿IL-1和TNF动作的13-乙酸盐（PMA）， 抑制Colcat 3.6活性不管分化状态如何。 但是，PMA但不是细胞因子的戏剧性刺激作用是 在具有5'脱落构建体的分化细胞中揭露，该细胞 缺少α1（i）启动子的上游基础增强子区域。 这 α1（i）启动子中细胞因子和PMA响应元件的位置 将使用功能性猫测定法映射。 最初，一系列5' 缺失突变体将测试细胞因子和PMA反应性。 Colcat 3.6包含内部缺失的突变体将准备好 评估在完整存在下下游序列的作用 上游增强剂。 假定的响应元素将进行测试 能够赋予异源胸苷激酶的响应能力 发起人。 将检查对IL-1，TNF或PMA响应的区域的检查 迁移率转移的组成型或诱导DNA蛋白相互作用， DNase保护和甲基化干扰分析。 功能 这些方法确定的推定响应元素将被评估 通过定点诱变。 PGH合酶的5'侧翼区域 基因，该基因编码前列腺素中的限速酶 生物合成将被克隆和测序。 PGH合酶的调节 细胞因子的转录将以类似的方式进行分析 描述为alpha1（i）启动子。 这些研究应有助于 了解细胞因子改变I型胶原蛋白的机制和 骨骼中的前列腺素合成。 因此，这些研究可能有 对骨质疏松和骨质流失的发病机理的重要意义 与慢性炎症性疾病有关。