S100A4: A Novel Regulator of Biomineralization

本研究的长期目标是阐明调节矿化组织形成的某些因素的作用，并有助于矿化组织疾病治疗的发展。S100A4是一种与癌症转移有关的钙结合蛋白，然而，最近的证据表明，S100A4也在正常细胞（如成骨细胞）中表达。然而，在矿化基质形成开始之前，S100A4的表达水平明显降低。通过反义途径抑制mc3t3e1来源的成骨细胞克隆中S100A4蛋白的合成可显著加速并增加矿化结节的形成。此外，这些克隆表达骨涎蛋白和骨钙蛋白，以及与基质矿化和成骨细胞表型相关的标志物，而这些标志物在对照细胞中缺失。在这里，我们假设S100A4可能通过调节成骨细胞分化而起到矿化的负调节作用。为了解决这一假设，提出以下具体目标：1。测定表达低水平S100A4（受S100A4反义抑制）的mc3t3e1衍生细胞克隆在细胞分化早期、中期和晚期的Cbfa1和成骨表型标志物的表达水平。2. 目的：在MC3TE1成骨前细胞中过表达S100A4。3. 评估过表达S100A4的mc3t3e1衍生细胞克隆在体外形成矿化结节的能力，并确定Cbfa和成骨细胞表型标志物在细胞分化早期、中期和晚期的表达。4. 将低表达S100A4和过表达S100A4的细胞克隆移植到免疫缺陷小鼠体内，评估其在体内形成矿化基质的能力。这种体外/体内方法的结合将有助于深入了解S1004A4在成骨细胞分化和生物矿化中的潜在调节作用。

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.