S100A4: A Novel Regulator of Biomineralization

S100A4：新型生物矿化调节剂

• 批准号: 6626081
• 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 2005-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6626081
• 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，并有助于开发矿化组织疾病的治疗方法。S100 A4是一种钙结合蛋白，与癌症转移有关，然而，最近的证据表明，S100 A4也在成骨细胞等正常细胞中表达。然而，在矿化基质形成开始之前，S100 A4表达水平显著降低。在MC 3 T3 E1衍生的成骨细胞克隆中通过反义方法抑制S100 A4蛋白合成显著加速并增加体外矿化结节的形成。此外，这些克隆表达骨唾液酸蛋白蛋白质骨钙素，标志物与基质矿化和成骨细胞表型，在他们从控制细胞缺席的阶段。在此，我们假设S100 A4可能通过调节成骨细胞分化而作为矿化的负调节因子发挥作用。为了解决这一假设，提出了以下具体目标：1。确定表达低水平S100 A4（受S100 A4反义抑制）的MC 3 T3 E1衍生细胞克隆的细胞分化早期、中期和晚期的Cbfa 1和成骨细胞表型标志物的表达水平。2. 在MC 3 TE 1前成骨细胞中过表达S100 A4。3.评价过表达S100 A4的MC 3 T3 E1衍生细胞克隆体外形成矿化结节的能力，并测定细胞分化早期、中期和晚期的Cbfa和成骨细胞表型标志物的表达。4. 将低表达和高表达S100 A4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S1004 A4在成骨细胞分化和生物矿化中的潜在调节作用提供见解。