Selim Abdulhafez A, Abdelmagid Samir M, Kanaan Reem A, Smock Steven L, Owen Thomas A, Popoff Steven N, Safadi Fayez F
Department of Anatomy and Cell Biology, Temple University, School of Medicine, Philadelphia, PA 19140, USA.
Crit Rev Eukaryot Gene Expr. 2003;13(2-4):265-75. doi: 10.1615/critreveukaryotgeneexpr.v13.i24.180.
Osteoactivin (OA) is a novel protein identified by mRNA differential display using bone from osteopetrotic versus normal rats. Bioinformatic analysis showed that OA cDNA has an open reading frame of 1716 bp encoding a protein of 572 aa, the first 21 aa constitute a signal peptide. OA sequence analysis also demonstrated 13 putative N-glycosylation sites suggestive of a heavily glycosylated protein. In this study, we localized OA protein in primary osteoblast culture by immunofluorescent staining and Western blot analysis. Primary osteoblast cultures pass through three stages: proliferation from day 1 to 7, matrix formation from day 7 to 14, and matrix mineralization from day 14 to 21. OA protein was detected at all stages examined, with maximal expression at 3 weeks when osteoblasts are terminally differentiated. Using the Chariot transfection reagent as a vehicle to deliver anti-OA antibody into the cells, we demonstrated that anti-OA antibody significantly inhibited osteoblast differentiation markers, including alkaline phosphatase activity, nodule formation, osteocalcin production, and calcium deposition, without affecting cell proliferation or viability. These data suggest that OA is an osteoblast-related protein that plays an important role in the regulation of osteoblast differentiation and function.
骨激活素（OA）是一种通过对骨石化大鼠与正常大鼠的骨骼进行mRNA差异显示鉴定出的新型蛋白质。生物信息学分析表明，OA cDNA具有一个1716 bp的开放阅读框，编码一个572个氨基酸的蛋白质，前21个氨基酸构成一个信号肽。OA序列分析还显示有13个推定的N-糖基化位点，提示该蛋白高度糖基化。在本研究中，我们通过免疫荧光染色和蛋白质印迹分析将OA蛋白定位在原代成骨细胞培养物中。原代成骨细胞培养经历三个阶段：第1天至第7天的增殖期、第7天至第14天的基质形成期以及第14天至第21天的基质矿化期。在所检测的所有阶段均检测到OA蛋白，在成骨细胞终末分化的3周时表达最高。使用Chariot转染试剂作为载体将抗OA抗体导入细胞，我们证明抗OA抗体显著抑制成骨细胞分化标志物，包括碱性磷酸酶活性、结节形成、骨钙素产生和钙沉积，而不影响细胞增殖或活力。这些数据表明，OA是一种与成骨细胞相关的蛋白质，在成骨细胞分化和功能的调节中起重要作用。
