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: 6754457
• 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/6754457
• 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.

牙周炎是一种慢性炎症性疾病，与结缔组织破坏和牙槽骨丢失有关，是成年人牙齿丢失的主要原因。 细胞因子TGF-β在炎症部位释放，并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的TGF-β复合物，它们还在许多功能中对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起重要作用。 该实验室在正常人成骨细胞（hOB）中发现了TGF-β诱导型早期基因（TIEG），并将其表征为TGF-β的即时反应基因。TIEG编码一个72 kDa的3-锌指转录因子样磷蛋白。 本实验室和其他实验室的早期研究已经将TIEG表达水平与几种不同细胞类型（包括hOB细胞）中的TGF-β反应（包括细胞增殖和凋亡的抑制）相关联。我们最近报道，TIEG在MG-63细胞中的过表达通过抑制细胞增殖和以TIEG剂量依赖性模式增强骨基质蛋白基因表达来模拟TGF-β对这些细胞的作用。 现在，我们显示的证据表明，TIEG表达增强Smad结合元件报告基因的活性，下调抑制性Smad 7基因的表达在其启动子的水平。 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敲除小鼠在胚胎发育和成年骨骼中的作用; 3）利用酵母双杂交系统鉴定与TIEG蛋白相互作用的蛋白;（4）利用随机序列寡核苷酸方法，获得TIEG的DNA共有结合元件。 我们将比较该元件与上文在Smad 7启动子-报告基因实验中鉴定的序列。 这些研究的结果应该提供新的见解TGF-β的作用机制和TIEG在人类成骨细胞和骨疾病，如牙周炎的作用。