S100A4: A Novel Regulator of Biomineralization

S100A4：新型生物矿化调节剂

• 批准号: 6486312
• 负责人: WAGNER R DUARTE
• 金额: $ 7.28万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-04-01 至 2004-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6486312
• 关键词: biomarker; calcium binding protein; cell differentiation; cell growth regulation; cell population study; developmental genetics; disease /disorder model; gene expression; genetic regulation; histology; inborn immunodeficiency; laboratory mouse; normal ossification; northern blottings; osteoblasts; osteogenesis; protein structure function; stem cell transplantation; transcription factor; transfection; western blottings

The long-term objective of the present research is to elucidate the roles of certain factors that regulate mineralized tissue formation and to contribute to the development of therapies for mineralized tissue disorders. S100A4 is a calcium-binding protein that has been implicated in cancer metastasis, however, recent evidence has shown that S100A4 is also expressed by normal cells such as osteoblastic cells. However, prior to the initiation of mineralized matrix formation, the level of S100A4 expression is markedly diminished. Inhibition of S100A4 protein synthesis by an antisense approach in MC3T3E1-derived osteoblastic cell clones significantly accelerated and increased the formation of mineralized nodules in vitro. Furthermore those clones expressed bone sialoprotein protein osteocalcin, markers associated with matrix mineralization and osteoblastic phenotype, at the stage when they were absent from the control cells. Here we hypothesize that S100A4 functions as a negative regulator of mineralization likely by modulating osteoblast differentiation. To address this hypothesis, the following specific aims are proposed: 1. To determine the expression levels of Cbfa1 and osteoblastic phenotypic markers for early, intermediate, and late stages of cell differentiation of MC3T3E1-derived cell clones expressing low levels of S100A4 (inhibited by S100A4 antisense). 2. To over-express S100A4 in MC3TE1 pre-osteoblastic cells. 3. To evaluate the ability of MC3T3E1-derived cell clones over- expressing S100A4 to form mineralized nodules in vitro and determine the expression of Cbfa and osteoblastic phenotypic markers for early, intermediate, and late stages of cell differentiation. 4. To transplant the cell clones expressing low levels of S100A4 and those over-expressing S100A4 into immunodeficient mice and evaluate their capabilities to form mineralized matrices in vivo. The combination of this in vitro/in vivo approach would provide insights into the potential regulatory roles of S1004A4 in osteoblast differentiation and biomineralization.

本研究的长期目标是阐明某些调节矿化组织形成的因素的作用，并为矿化组织疾病的治疗方法的发展做出贡献。S100A4是一种钙结合蛋白，与肿瘤转移有关，但最近的研究表明，S100A4在正常细胞如成骨细胞中也有表达。然而，在矿化基质形成开始之前，S100A4的表达水平明显降低。在MC3T3E1来源的成骨细胞克隆中，通过反义方法抑制S100A4蛋白的合成显著加速和增加体外矿化结节的形成。此外，这些克隆在对照细胞中没有表达与基质矿化和成骨细胞表型相关的标记物--骨涎蛋白-骨钙素。这里我们假设S100A4可能通过调节成骨细胞分化而发挥矿化的负调节作用。为了解决这一假设，提出了以下具体目标：1.确定低水平表达S100A4(被S100A4反义抑制)的MC3T3E1来源细胞克隆分化早、中、晚期Cbfa1和成骨表型标志物的表达水平。2.在MC3TE1前成骨细胞中过表达S100A4。3.检测高表达S100A4的MC3T3E1来源细胞克隆在体外形成矿化结节的能力，并检测CBFA和成骨细胞表型标志物在细胞分化早、中、晚期的表达。4.将低表达S100A4和高表达S100A4的细胞克隆移植到免疫缺陷小鼠体内，评价其体内形成矿化基质的能力。这种体外/体内方法的结合将为S1004A4在成骨细胞分化和生物矿化中的潜在调控作用提供深入的见解。