Matsuguchi Tetsuya, Chiba Norika, Bandow Kenjiro, Kakimoto Kyoko, Masuda Akio, Ohnishi Tomokazu
Department of Oral Biochemistry, Field of Developmental Medicine, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima, Japan.
J Bone Miner Res. 2009 Mar;24(3):398-410. doi: 10.1359/jbmr.081107.
Osteoblasts differentiate from mesodermal progenitors and play a pivotal role in bone formation and mineralization. Several transcription factors including runt-related transcription factor 2 (RUNX2), Osterix (OSX), and activating transcription factor4 (ATF4) are known to be crucial for the process, whereas the upstream signal transduction controlling the osteoblast differentiation sequence is largely unknown. Here, we explored the role of c-jun N-terminal kinase (JNK) in osteoblast differentiation using in vitro differentiation models of primary osteoblasts and MC3T3-E1 cells with ascorbic acid/beta-glycerophosphate treatment. Terminal osteoblast differentiation, represented by matrix mineralization, was significantly inhibited by the inactivation of JNK with its specific inhibitor and exogenous overexpression of MKP-M (MAP kinase phosphatase isolated from macrophages), which preferentially inactivates JNK. Conversely, enhanced mineral deposition was observed by inducible overexpression of p54(JNK2), whereas it was not observed by the overexpression of p46(JNK1) or p46(JNK2), indicating a distinct enhancing role of p54(JNK2) in osteoblast differentiation. Inactivation of JNK significantly inhibited late-stage molecular events of osteoblast differentiation, including gene expression of osteocalcin (Ocn) and bone sialoprotein (Bsp). In contrast, earlier differentiation events including alkaline phosphatase (ALP) activation and osteopontin (Opn) expression were not inhibited by JNK inactivation. Although the expression levels of two transcription factor genes, Runx2 and Osx, were not significantly affected by JNK inactivation, induction of Atf4 mRNA during osteoblast differentiation was significantly inhibited. Taken together, these data indicate that JNK activity is specifically required for the late-stage differentiation events of osteoblasts.
成骨细胞从中胚层祖细胞分化而来，在骨形成和矿化过程中起关键作用。已知包括 runt 相关转录因子 2（RUNX2）、osterix（OSX）和激活转录因子 4（ATF4）在内的几种转录因子对该过程至关重要，而控制成骨细胞分化序列的上游信号转导在很大程度上尚不清楚。在这里，我们使用原代成骨细胞和经抗坏血酸/β-甘油磷酸处理的 MC3T3-E1 细胞的体外分化模型，探讨了 c-jun N 末端激酶（JNK）在成骨细胞分化中的作用。以基质矿化为代表的终末成骨细胞分化，通过用其特异性抑制剂使 JNK 失活以及外源性过表达 MKP-M（从巨噬细胞中分离的丝裂原活化蛋白激酶磷酸酶）而受到显著抑制，MKP-M 优先使 JNK 失活。相反，通过诱导过表达 p54（JNK2）观察到矿质沉积增强，而通过过表达 p46（JNK1）或 p46（JNK2）未观察到这种增强，这表明 p54（JNK2）在成骨细胞分化中具有独特的增强作用。JNK 的失活显著抑制了成骨细胞分化的晚期分子事件，包括骨钙素（Ocn）和骨唾液蛋白（Bsp）的基因表达。相比之下，早期分化事件，包括碱性磷酸酶（ALP）激活和骨桥蛋白（Opn）表达，不受 JNK 失活的抑制。尽管 Runx2 和 Osx 这两个转录因子基因的表达水平不受 JNK 失活的显著影响，但在成骨细胞分化过程中 Atf4 mRNA 的诱导受到显著抑制。综上所述，这些数据表明 JNK 活性是成骨细胞晚期分化事件所特需的。
