Department of Orthopaedic Surgery, The First Affiliated Hospital of Soochow University, Suzhou, Jiangsu, China.
Department of Orthopaedic Surgery, The First People's Hospital of Wenling, Wenling, Zhejiang, China.
J Cell Physiol. 2020 Mar;235(3):2220-2231. doi: 10.1002/jcp.29131. Epub 2019 Sep 5.
Osteonectin binds strongly to type I collagen and hydroxyapatite and plays a crucial role in extracellular matrix mineralization. Previous studies have also shown that p38 signaling pathway is an important regulator for osteoblast mineralization. This study focused on the role of osteonectin in regulating extracellular matrix mineralization via the p38 signaling pathway. Osteoblasts were isolated and cultured from parietal bones of neonatal Sprague-Dawley rats. The gene and protein expressions of noncollagen proteins (BSP, bone sialoprotein; OCN, osteocalcin; OPN, osteopontin), p38 mitogen-activated protein kinase, and SIBLINGs (Small Integrin-Binding LIgand N-linked Glycoproteins) members (DMP1, dentine matrix protein 1, DSPP, dentin sialophosphoprotein, and MEPE, matrix extracellular phosphoglycoprotein) were detected by reverse-transcription quantitative polymerase chain reaction and western blot analysis. Alizarin red staining, intracellular calcium assay, and transmission electron microscopy were used to detect mineralization. Initially, by adding osteonectin at different concentrations in osteoblasts and detecting the above mineralization indexes, 1 µg/ml was determined to be the optima osteonectin concentration, which significantly increased gene expressions of BSP, OPN, OCN, DMP1, MEPE, DSPP, and p38 in osteoblasts, p38 and p-p38 protein expressions were also significantly increased, mineralized nodules were significantly enhanced; when added with SB203580 (a specific inhibitor for p38) these effects were inhibited. Furthermore, osteoblasts transfected with Ad-p38 also significantly upregulated the protein and gene expressions of noncollagens and SIBLINGs members, whereas transfection of p38-rhRNA showed the opposite effect. Our data suggest that osteonectin regulates the extracellular matrix mineralization of osteoblasts through the P38 signaling pathway.
骨连接蛋白与 I 型胶原和羟磷灰石结合能力强，在细胞外基质矿化中发挥着关键作用。先前的研究还表明，p38 信号通路是成骨细胞矿化的重要调节剂。本研究重点研究骨连接蛋白通过 p38 信号通路在调节细胞外基质矿化中的作用。从新生 Sprague-Dawley 大鼠顶骨中分离和培养成骨细胞。通过逆转录定量聚合酶链反应和 Western blot 分析检测非胶原蛋白（BSP、骨唾液蛋白；OCN、骨钙素；OPN、骨桥蛋白）、p38 有丝分裂原激活蛋白激酶和 SIBLINGs（小整合素结合配体 N-连接糖蛋白）成员（DMP1、牙本质基质蛋白 1、DSPP、牙本质涎磷蛋白和 MEPE、基质细胞外磷糖蛋白）的基因和蛋白表达。茜素红染色、细胞内钙测定和透射电镜用于检测矿化。首先，通过向成骨细胞中添加不同浓度的骨连接蛋白并检测上述矿化指标，确定 1μg/ml 为最佳骨连接蛋白浓度，该浓度显著增加成骨细胞中 BSP、OPN、OCN、DMP1、MEPE、DSPP 和 p38 的基因表达，p38 和 p-p38 蛋白表达也显著增加，矿化结节明显增强；加入 SB203580（p38 的特异性抑制剂）后，这些作用被抑制。此外，转染 Ad-p38 的成骨细胞也显著上调非胶原和 SIBLINGs 成员的蛋白和基因表达，而转染 p38-rhRNA 则表现出相反的效果。我们的数据表明，骨连接蛋白通过 P38 信号通路调节成骨细胞细胞外基质矿化。
