REGULATION OF COLLAGENASE GENE EXPRESSION

胶原酶基因表达的调控

• 批准号: 2078581
• 负责人: CONSTANCE E BRINCKERHOFF
• 金额: $ 24.53万
• 依托单位: DARTMOUTH COLLEGE
• 依托单位国家: 美国
• 财政年份: 1980
• 资助国家: 美国
• 起止时间: 1980-03-01 至 1999-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2078581
• 关键词: DNA footprinting; chemical binding; collagenase; gel mobility shift assay; gene expression; genetic promoter element; genetic regulation; genetic transcription; messenger RNA; metalloenzyme; nuclear runoff assay; nucleic acid sequence; phorbols; retinoid binding proteins; site directed mutagenesis; tissue /cell culture; transfection

This proposal seeks to understand the molecular mechanisms controlling expression of the gene for collagenase (matrix metalloproteinase-1; MMP- 1), the only enzyme active at neutral pH that can degrade the interstitial collagens (types I, II and III). Collagenase can be induced by phorbol esters (PMA) and by cytokines such as interleukin-1 (IL-1), and suppressed by vitamin A analogues (retinoids). Collagenase is over- expressed in the joints of patients with rheumatoid arthritis, and this over-expression contributes significantly to the pathophysiology of this disease. To study mechanisms of transcriptional activation and suppression of the collagenase gene, we made chimeric constructs containing fragments of the collagenase promoter linked to a reporter gene, and we transfected these chimeric constructs into fibroblasts, cells that normally express the collagenase gene endogenously. We found that although the AP-1 (activator protein) motif, 5'-TGAGTCAC-3', which binds the transcription factors Fos and Jun is important, additional sequences are essential for both activation and suppression. We also found that upstream sequences compensate for mutations in the AP-1 site, and that repression by retinoic acid involves differential activity of several Retinoic Acid Receptors (RARs and RXRs), at multiple sites along the promoter in a RAR type-specific manner. Furthermore, the mechanisms mediating IL-1 induction of collagenase mRNA may not be identical to those mediating induction by PMA. Different cis-acting elements in the promoter may respond transcriptionally, and post-transcriptional mechanisms, i.e. mRNA stability, may also contribute. Since the 3' untranslated region of collagenase mRNA contains 3 copies of the sequence AUUUA, which is associated with mRNA instability, these sequences may be important in regulating IL-1 induced collagenase gene expression. Thus, the specific aims of this proposal are to: (1) Continue our studies to (a) identify cis-acting regulatory elements in the collagenase promoter involved in basal/constitutive transcription and in the response to PMA (b) determine their dependency on a functional AP-1 site, and (c) characterize the protein-DNA interactions involved; (2) Identify retinoic acid responsive elements within the collagenase promoter and characterize the specific role of RARs (alpha, Beta and gamma) and RXR alpha in modulating basal and phorbol induced transcription; and (3) Characterize the transcriptional and post-transcriptional mechanisms (i.e., mRNA stability and the role of the AUUUA sequences) that regulate collagenase gene expression in response to IL-1. Knowledge of mechanisms regulating expression of the collagenase gene will enhance our understanding of the pathophysiology of arthritic disease and may form the basis for new and novel therapies designed to reduce joint destruction.

该提案旨在了解控制的分子机制 胶原酶基因的表达（基质金属蛋白酶-1；MMP- 1），唯一在中性pH下具有活性并能降解 间质胶原蛋白（I、II 和 III 型）。 可以诱导胶原酶 通过佛波酯 (PMA) 和细胞因子，例如白介素-1 (IL-1)， 并被维生素 A 类似物（类维生素A）抑制。 胶原酶过多 在类风湿性关节炎患者的关节中表达， 过度表达对这种疾病的病理生理学有显着贡献 疾病。 研究转录激活机制 抑制胶原酶基因，我们制作了嵌合结构 含有与报告基因连接的胶原酶启动子片段 基因，我们将这些嵌合构建体转染到成纤维细胞中， 通常内源表达胶原酶基因的细胞。 我们发现 虽然 AP-1（激活蛋白）基序 5'-TGAGTCAC-3'， 结合转录因子 Fos 和 Jun 很重要，另外 序列对于激活和抑制都是必需的。 我们也 发现上游序列补偿了 AP-1 位点的突变， 视黄酸的抑制涉及不同的活性 多个视黄酸受体（RAR 和 RXR），位于沿线的多个位点 以 RAR 类型特异性方式启动子。 此外，机制 介导 IL-1 诱导胶原酶 mRNA 可能与 那些通过 PMA 介导诱导的。 不同的顺式作用元件 启动子可以在转录上做出反应，并且在转录后 机制，即 mRNA 稳定性，也可能有所贡献。 自从 3' 胶原酶 mRNA 的非翻译区包含 3 个序列拷贝 AUUUA，与 mRNA 不稳定相关，这些序列可能是 在调节 IL-1 诱导的胶原酶基因表达中发挥重要作用。 因此， 该提案的具体目标是： (1) 继续我们的研究 (a) 鉴定胶原酶启动子中的顺式作用调控元件 参与基础/组成转录和对 PMA 的反应 (b) 确定它们对功能性 AP-1 站点的依赖性，以及 (c) 描述所涉及的蛋白质-DNA 相互作用的特征； (2) 识别 胶原酶启动子内的视黄酸反应元件和 描述 RAR（α、Beta 和 gamma）和 RXR 的具体作用 α 调节基础和佛波醇诱导的转录；和（3） 表征转录和转录后机制 （即 mRNA 稳定性和 AUUUA 序列的作用） 胶原酶基因表达响应 IL-1。 机制知识 调节胶原酶基因的表达将增强我们的 了解关节炎疾病的病理生理学并可能形成 旨在减少关节损伤的新疗法的基础 破坏。