Division of Infections and Molecular Biology, Department of Health Promotion, Kyushu Dental University, Fukuoka, Japan.
Division of Developmental Stomatognathic Function Science, Department of Health Promotion, Kyushu Dental University, Fukuoka, Japan.
J Cell Physiol. 2019 Feb;234(2):1745-1757. doi: 10.1002/jcp.27045. Epub 2018 Aug 13.
Ameloblastin (Ambn) is an extracellular matrix protein and member of the family of enamel-related gene products. Like amelogenin, Ambn is mainly associated with tooth development, especially biomineralization of enamel. Previous studies have shown reductions in the skeletal dimensions of Ambn-deficient mice, suggesting that the protein also has effects on the differentiation of osteoblasts and/or osteoclasts. However, the specific pathways used by Ambn to influence osteoclast differentiation have yet to be identified. In the present study, two cellular models, one based on bone marrow cells and another on RAW264.7 cells, were used to examine the effects of Ambn on receptor activator of nuclear factor kappa-B ligand (RANKL)-induced osteoclastogenesis. The results showed that Ambn suppresses osteoclast differentiation, cytoskeletal organization, and osteoclast function by the downregulation of the number of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated osteoclasts, actin ring formation, and areas of pit resorption. The expression of the osteoclast-specific genes TRAP, MMP9, cathepsin K, and osteoclast stimulatory transmembrane protein (OC-STAMP) was abolished in the presence of Ambn, while that of nuclear factor of activated T cells cytoplasmic 1 (NFATc1), the master regulatory factor of osteoclastogenesis, was also attenuated by the downregulation of c-Fos expression. In Ambn-induced RAW264.7 cells, phosphorylation of cAMP-response element-binding protein (CREB), c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase (p38 MAPK), but not extracellular signal-regulated kinase 1/2 (ERK1/2), was reduced. Calcium oscillation was also decreased in the presence of Ambn, suggesting its involvement in both RANKL-induced osteoclastogenesis and costimulatory signaling. B-lymphocyte-induced maturation protein-1 (Blimp1), a transcriptional repressor of negative regulators of osteoclastogenesis, was also downregulated by Ambn, resulting in the elevated expression of v-maf musculoaponeurotic fibrosarcoma oncogene family, protein B (MafB), B-cell lymphoma 6 (Bcl6), and interferon regulatory factor-8 (Irf8). Taken together, these findings suggest that Ambn suppresses RANKL-induced osteoclastogenesis by modulating the NFATc1 axis.
成釉蛋白（Ambn）是细胞外基质蛋白，属于釉质相关基因产物家族成员。与釉原蛋白一样，Ambn 主要与牙齿发育有关，特别是釉质的生物矿化。先前的研究表明，Ambn 缺陷型小鼠的骨骼尺寸减小，这表明该蛋白还对成骨细胞和/或破骨细胞的分化有影响。然而，Ambn 影响破骨细胞分化的具体途径尚未确定。在本研究中，使用了两种细胞模型，一种基于骨髓细胞，另一种基于 RAW264.7 细胞，来研究 Ambn 对核因子κB 受体激活剂配体（RANKL）诱导的破骨细胞生成的影响。结果表明，Ambn 通过下调抗酒石酸酸性磷酸酶（TRAP）阳性多核破骨细胞的数量、肌动蛋白环形成和蚀斑吸收面积，抑制破骨细胞分化、细胞骨架组织和破骨细胞功能。在 Ambn 存在的情况下，破骨细胞特异性基因 TRAP、MMP9、组织蛋白酶 K 和破骨细胞刺激跨膜蛋白（OC-STAMP）的表达被消除，而核因子活化 T 细胞细胞质 1（NFATc1），即破骨细胞生成的主要调节因子，其表达也被下调 c-Fos 的表达所减弱。在 Ambn 诱导的 RAW264.7 细胞中，cAMP 反应元件结合蛋白（CREB）、c-Jun N 端激酶（JNK）和 p38 丝裂原活化蛋白激酶（p38 MAPK）的磷酸化减少，但细胞外信号调节激酶 1/2（ERK1/2）的磷酸化没有减少。在 Ambn 存在的情况下，钙振荡也减少，提示其参与了 RANKL 诱导的破骨细胞生成和共刺激信号。B 淋巴细胞诱导成熟蛋白 1（Blimp1）是破骨细胞生成负调节因子的转录抑制因子，也被 Ambn 下调，导致 v-maf 肌肉腱膜纤维肉瘤癌基因家族、蛋白 B（MafB）、B 细胞淋巴瘤 6（Bcl6）和干扰素调节因子 8（Irf8）的表达升高。综上所述，这些发现表明，Ambn 通过调节 NFATc1 轴抑制 RANKL 诱导的破骨细胞生成。
