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: 6317115
• 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/6317115
• 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，是成年人牙齿脱落的主要原因。细胞因子，转化生长因子-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物的合成，在牙周组织和骨重建中发挥作用。成骨细胞不仅合成和激活潜在的复合体转化生长因子-β，还在许多功能上对转化生长因子-β作出反应，包括增加骨基质蛋白的产生。转化生长因子-β在成骨细胞和破骨细胞偶联中也发挥重要作用。本实验室在正常人成骨细胞中发现了一个转化生长因子-β诱导早期基因(TIEG)，并将其鉴定为对转化生长因子-β的即时反应基因。TIEG编码一个72 kDa的3锌指转录因子样的磷蛋白。该实验室和其他实验室的早期研究已经将TIEG的表达水平与包括HOB细胞在内的几种不同类型的细胞中的转化生长因子-β反应相关联，包括对细胞增殖和凋亡的抑制。我们最近报道了TIEG在MG-63细胞中的过表达，通过抑制细胞增殖和促进骨基质蛋白基因的表达，模拟了转化生长因子-β对MG-63细胞的影响，并呈剂量依赖性。现在我们有证据表明，TIEG的表达通过在其启动子水平下调抑制Smad 7基因的表达来增强Smad结合元件-报告基因的活性。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质；以及4)通过随机序列寡核苷酸方法确定TIEG的DNA共识结合元件。我们将把这个元件与上面在Smad7启动子-报告基因实验中确定的序列进行比较。这些研究的结果应该会为转化生长因子-β作用的机制以及TIEG在人类成骨细胞和牙周炎等骨骼疾病中的作用提供新的见解。