ROLE OF A TGF-BETA REGULATED GENE IN HUMAN OSTEOBLASTS

TGF-β 调控基因在人类成骨细胞中的作用

• 批准号: 6516651
• 负责人: THOMAS C SPELSBERG
• 金额: $ 28.96万
• 依托单位: MAYO CLINIC ROCHESTER
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-09-01 至 2006-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6516651
• 关键词: DNA binding protein; DNA footprinting; affinity chromatography; binding sites; biological signal transduction; gel mobility shift assay; gene expression; gene induction /repression; gene targeting; genetic promoter element; genetically modified animals; laboratory mouse; methylation; osteoblasts; pathologic process; periodontitis; phosphoproteins; physiologic bone resorption; polymerase chain reaction; protein metabolism; protein structure function; regulatory gene; tissue /cell culture; transforming growth factors; yeast two hybrid system

Periodontitis is a chronic inflammatory disease associated with destruction of connective tissue and alveolar bone loss and is the primary cause of tooth loss in adults. The cytokine, TGF-beta, is released at inflammatory sites and plays a role in periodontal tissue and bone remodeling by regulating the synthesis of matrix proteins and matrix metalloprotease inhibitors. Not only do bone forming osteoblasts synthesize and activate latent complex TGF-beta, they also respond to TGF-beta in many functions, including increased bone matrix protein production. TGF-beta also plays an important role in osteoblast and osteoclast coupling. This laboratory discovered a TGF-beta inducible early gene (TIEG) in normal human osteoblasts (hOB) and characterized it as an immediate response gene for TGF-beta. TIEG encodes a 72kDa, 3-zinc finger, transcription factor-like, phosphoprotein. Early studies in this and other laboratories have correlated the levels of TIEG expression with TGF-beta responses, including the inhibition of cell proliferation and apoptosis, in several different cell types, including hOB cells. We have recently reported that TIEG overexpression in MG-63 cells mimics the TGF-beta effects on these cells by inhibiting cell proliferation and enhancing bone matrix protein gene expression in a TIEG dose-dependent pattern. Now we show evidence that TIEG expression enhances Smad binding element-reporter gene activity by down regulating the inhibitory Smad 7 gene expression at the level of its promoter. To elucidate the biological role of TIEG in human osteoblasts, we plan to determine: 1) the mechanism by which TIEG down-regulates Smad 7 promoter; 2) the effects of TIEG knockout mice in both embryo development and adult skeleton; and identify 3) proteins that interact with TIEG protein using the yeast-two hybrid systems; and 4) the DNA consensus binding element for TIEG by using random sequence oligonucleotide approach. We will compare this element to the sequences identified above in the Smad 7 promoter-reporter gene experiments. The outcome of these studies should provide new insights into the mechanisms of TGF-beta action and the role of TIEG in human osteoblast cells and bone disease such as periodontitis.

牙周炎是一种与结缔组织破坏和牙槽骨丢失相关的慢性炎症性疾病，是成人牙齿脱落的主要原因。 细胞因子 TGF-β 在炎症部位释放，通过调节基质蛋白和基质金属蛋白酶抑制剂的合成，在牙周组织和骨重塑中发挥作用。 成骨细胞不仅合成并激活潜在复合物 TGF-β，而且还在许多功能上对 TGF-β 做出反应，包括增加骨基质蛋白的产生。 TGF-β还在成骨细胞和破骨细胞偶联中发挥重要作用。 该实验室在正常人成骨细胞（hOB）中发现了 TGF-β 诱导早期基因（TIEG），并将其鉴定为 TGF-β 的立即反应基因。 TIEG 编码 72kDa、3-锌指、转录因子样磷蛋白。 该实验室和其他实验室的早期研究已将 TIEG 表达水平与 TGF-β 反应相关联，包括几种不同细胞类型（包括 hOB 细胞）中细胞增殖和凋亡的抑制。我们最近报道，MG-63 细胞中的 TIEG 过表达模拟了 TGF-β 对这些细胞的影响，通过抑制细胞增殖并以 TIEG 剂量依赖性模式增强骨基质蛋白基因表达。 现在我们证明，TIEG 表达通过在启动子水平下调抑制性 Smad 7 基因表达来增强 Smad 结合元件报告基因活性。 为了阐明 TIEG 在人成骨细胞中的生物学作用，我们计划确定：1）TIEG 下调 Smad 7 启动子的机制； 2）TIEG基因敲除小鼠对胚胎发育和成年骨骼的影响；并鉴定 3) 使用酵母-两种杂交系统与 TIEG 蛋白相互作用的蛋白； 4)使用随机序列寡核苷酸方法获得TIEG的DNA共有结合元件。 我们将将此元件与上面在 Smad 7 启动子-报告基因实验中鉴定的序列进行比较。 这些研究的结果应该为 TGF-β 作用机制以及 TIEG 在人类成骨细胞和牙周炎等骨疾病中的作用提供新的见解。