Parisuthiman Duenpim, Mochida Yoshiyuki, Duarte Wagner R, Yamauchi Mitsuo
Dental Research Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.
J Bone Miner Res. 2005 Oct;20(10):1878-86. doi: 10.1359/JBMR.050612. Epub 2005 Jun 27.
MC3T3-E1 cell-derived clones expressing higher (S) or lower (AS) levels of biglycan were generated and characterized. The processes of cell differentiation and matrix mineralization were accelerated in S but delayed in AS, indicating that BGN modulates osteoblastic cell differentiation.
Biglycan (BGN), a member of the small leucine-rich proteoglycan family, is one of the major proteoglycans found in bone and has been implicated in bone formation. In this study, the effects of over- or underexpression of BGN on osteoblastic cell phenotypes and matrix mineralization were studied.
MC3T3-E1 cells were transfected with vectors containing the BGN cDNA in a sense or antisense orientation to generate clones expressing higher (S clones) or lower (AS clones) levels of BGN. MC3T3-E1 cells and those transfected with an empty vector (EV) were used as controls. The levels of BGN synthesized by these clones were evaluated by Western blot analysis. Cell growth was analyzed by cell counting and cell differentiation by the gene expression patterns of several osteoblastic markers using quantitative real-time PCR. The abilities of these clones to form mineralized matrices were evaluated by in vitro and in vivo mineralization assays. Furthermore, the clones were treated with BMP-4 and their responsiveness was assessed.
The cell growth in these clones was unaffected; however, osteoblast differentiation was significantly accelerated in S clones and suppressed in AS clones. The in vitro matrix mineralization in S clones was significantly enhanced but severely impaired in AS clones. When transplanted into immunodeficient mice, S clone transplants exhibited larger areas of lamellar bonelike matrices, whereas only minute amounts of woven bone-type structure was found in AS transplants. The response to BMP-4 was higher in S clones but poorer in AS clones compared with that of controls.
BGN modulates osteoblast differentiation, possibly by regulating BMP signaling, and consequently matrix mineralization.
生成并鉴定了表达高水平（S）或低水平（AS）双糖链蛋白聚糖的MC3T3-E1细胞衍生克隆。细胞分化和基质矿化过程在S克隆中加速而在AS克隆中延迟，表明双糖链蛋白聚糖（BGN）调节成骨细胞分化。
双糖链蛋白聚糖（BGN）是富含亮氨酸的小分子蛋白聚糖家族成员之一，是骨骼中发现的主要蛋白聚糖之一，并与骨形成有关。在本研究中，研究了BGN过表达或低表达对成骨细胞表型和基质矿化的影响。
用含有正义或反义方向BGN cDNA的载体转染MC3T3-E1细胞，以生成表达高水平BGN（S克隆）或低水平BGN（AS克隆）的克隆。将MC3T3-E1细胞和用空载体（EV）转染的细胞用作对照。通过蛋白质印迹分析评估这些克隆合成的BGN水平。通过细胞计数分析细胞生长，并使用定量实时PCR通过几种成骨细胞标志物的基因表达模式分析细胞分化。通过体外和体内矿化试验评估这些克隆形成矿化基质的能力。此外，用骨形态发生蛋白-4（BMP-4）处理这些克隆并评估其反应性。
这些克隆中的细胞生长未受影响；然而，成骨细胞分化在S克隆中显著加速而在AS克隆中受到抑制。S克隆中的体外基质矿化显著增强，而AS克隆中则严重受损。当移植到免疫缺陷小鼠中时，S克隆移植表现出更大面积的板层骨样基质，而在AS移植中仅发现少量编织骨型结构。与对照相比，S克隆对BMP-4的反应更高，而AS克隆则较差。
BGN可能通过调节BMP信号传导从而调节基质矿化来调节成骨细胞分化。
