Frara Nagat, Abdelmagid Samir M, Sondag Gregory R, Moussa Fouad M, Yingling Vanessa R, Owen Thomas A, Popoff Steven N, Barbe Mary F, Safadi Fayez F
Department of Anatomy and Cell Biology, Temple University School of Medicine, Philadelphia, Pennsylvania.
Department of Stem Cell Biology and Regenerative Medicine, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio.
J Cell Physiol. 2016 Jan;231(1):72-83. doi: 10.1002/jcp.25020.
Initial identification of osteoactivin (OA)/glycoprotein non-melanoma clone B (gpnmb) was demonstrated in an osteopetrotic rat model, where OA expression was increased threefold in mutant bones, compared to normal. OA mRNA and protein expression increase during active bone regeneration post-fracture, and primary rat osteoblasts show increased OA expression during differentiation in vitro. To further examine OA/gpnmb as an osteoinductive agent, we characterized the skeletal phenotype of transgenic mouse overexpressing OA/gpnmb under the CMV-promoter (OA-Tg). Western blot analysis showed increased OA/gpnmb in OA-Tg osteoblasts, compared to wild-type (WT). In OA-Tg mouse femurs versus WT littermates, micro-CT analysis showed increased trabecular bone volume and thickness, and cortical bone thickness; histomorphometry showed increased osteoblast numbers, bone formation and mineral apposition rates in OA-Tg mice; and biomechanical testing showed higher peak moment and stiffness. Given that OA/gpnmb is also over-expressed in osteoclasts in OA-Tg mice, we evaluated bone resorption by ELISA and histomorphometry, and observed decreased serum CTX-1 and RANK-L, and decreased osteoclast numbers in OA-Tg, compared to WT mice, indicating decreased bone remodeling in OA-Tg mice. The proliferation rate of OA-Tg osteoblasts in vitro was higher, compared to WT, as was alkaline phosphatase staining and activity, the latter indicating enhanced differentiation of OA-Tg osteoprogenitors. Quantitative RT-PCR analysis showed increased TGF-β1 and TGF-β receptors I and II expression in OA-Tg osteoblasts, compared to WT. Together, these data suggest that OA overexpression has an osteoinductive effect on bone mass in vivo and stimulates osteoprogenitor differentiation ex vivo.
骨激活素（OA）/非黑色素瘤糖蛋白B克隆（gpnmb）最初是在骨石化大鼠模型中被鉴定出来的，与正常情况相比，OA在突变骨中的表达增加了两倍。在骨折后的活跃骨再生过程中，OA的mRNA和蛋白表达增加，并且原代大鼠成骨细胞在体外分化过程中OA表达也增加。为了进一步研究OA/gpnmb作为一种骨诱导剂，我们对在巨细胞病毒启动子（CMV）驱动下过表达OA/gpnmb的转基因小鼠（OA-Tg）的骨骼表型进行了特征分析。蛋白质免疫印迹分析显示，与野生型（WT）相比，OA-Tg成骨细胞中OA/gpnmb增加。与WT同窝仔鼠相比，OA-Tg小鼠股骨的显微CT分析显示骨小梁体积和厚度增加，皮质骨厚度增加；组织形态计量学显示OA-Tg小鼠的成骨细胞数量、骨形成和矿物质沉积率增加；生物力学测试显示峰值力矩和刚度更高。鉴于OA/gpnmb在OA-Tg小鼠的破骨细胞中也过表达，我们通过酶联免疫吸附测定法（ELISA）和组织形态计量学评估了骨吸收情况，并且观察到与WT小鼠相比，OA-Tg小鼠血清I型胶原C端肽（CTX-1）和核因子κB受体活化因子配体（RANK-L）降低，破骨细胞数量减少，这表明OA-Tg小鼠的骨重塑减少。与WT相比，OA-Tg成骨细胞在体外的增殖率更高，碱性磷酸酶染色和活性也是如此，后者表明OA-Tg成骨祖细胞的分化增强。定量逆转录聚合酶链反应（RT-PCR）分析显示与WT相比，OA-Tg成骨细胞中转化生长因子-β1（TGF-β1）及其I型和II型受体的表达增加。总之，这些数据表明OA过表达在体内对骨量具有骨诱导作用，并在体外刺激成骨祖细胞分化。
