Kajita Tomonari, Ariyoshi Wataru, Okinaga Toshinori, Mitsugi Sho, Tominaga Kazuhiro, Nishihara Tatsuji
Division of Infections and Molecular Biology, Department of Health Promotion, Kyushu Dental University, Kitakyushu, Fukuoka, Japan.
Division of Oral and Maxillofacial Surgery, Department of Science and Physical Functions, Kyushu Dental University, Kitakyushu, Fukuoka, Japan.
J Cell Biochem. 2018 Aug;119(8):6974-6985. doi: 10.1002/jcb.26906. Epub 2018 May 8.
Several growth factors in bone tissues are reported to be associated with osteoclastogenesis. Activin-A, a member of the transforming growth factor-β (TGF-β) family is known to be present in bone tissues and an important regulator in osteoclastogenesis with SMAD-mediated signaling being crucial for inducing osteoclast differentiation. In the present study, we examined the effect and underlying mechanisms of activin-A on osteoclast formation in vitro culture systems. Activin-A enhanced osteoclast formation in both mouse bone marrow cells and monocyte/macrophage cell line RAW 264.7 cells induced by receptor activator of nuclear factor kappa B (NF-κB) ligand (RANKL) and/or macrophage stimulating factor (M-CSF). We also found that activin-A stimulated bone resorption and actin ring formation induced by RANKL and/or M-CSF. Furthermore, activin-A enhanced RANKL-induced expression of nuclear factor of activated T cell cytoplasmic 1 (NFATc1), a key regulator of osteoclastogenesis, thereby increasing osteoclastogenesis-related marker gene expression, including tartrate-resistant acid phosphatase, osteoclast stimulatory transmembrane protein, and cathepsin K. Blockage of receptor binding by follistatin, an activing-binding protein suppressed the activin-A-mediated stimulation of NFATc1. In addition, activin-A increased RANKL-induced c-fos expression without significantly affecting the NF-κB and mitogen-activated protein kinase (MAPK) signaling pathway. Pre-treatment of the cells with a specific inhibitor of SMAD2/3 attenuated the activin-A-induced expression of NFATc1 and co-immunoprecipitation assay revealed that treatment with activin-A increased physical interaction of phosphorylated-c-fos and phosphorylated-SMAD2 protein induced by RANKL. These results suggest that activin-A enhances RANKL-induced osteoclast formation mediated by interaction of c-fos and smad2/3.
据报道，骨组织中的几种生长因子与破骨细胞生成有关。激活素-A是转化生长因子-β（TGF-β）家族的成员，已知存在于骨组织中，是破骨细胞生成的重要调节因子，SMAD介导的信号传导对于诱导破骨细胞分化至关重要。在本研究中，我们研究了激活素-A在体外培养系统中对破骨细胞形成的影响及其潜在机制。激活素-A增强了由核因子κB（NF-κB）配体（RANKL）和/或巨噬细胞刺激因子（M-CSF）诱导的小鼠骨髓细胞和单核细胞/巨噬细胞系RAW 264.7细胞中的破骨细胞形成。我们还发现激活素-A刺激了由RANKL和/或M-CSF诱导的骨吸收和肌动蛋白环形成。此外，激活素-A增强了RANKL诱导的活化T细胞核因子胞质1（NFATc1）的表达，NFATc1是破骨细胞生成的关键调节因子，从而增加了破骨细胞生成相关标记基因的表达，包括抗酒石酸酸性磷酸酶、破骨细胞刺激跨膜蛋白和组织蛋白酶K。卵泡抑素（一种激活素结合蛋白）阻断受体结合可抑制激活素-A介导的NFATc1刺激。此外，激活素-A增加了RANKL诱导的c-fos表达，而对NF-κB和丝裂原活化蛋白激酶（MAPK）信号通路没有显著影响。用SMAD2/3特异性抑制剂预处理细胞可减弱激活素-A诱导的NFATc1表达，免疫共沉淀试验表明，激活素-A处理增加了RANKL诱导的磷酸化-c-fos和磷酸化-SMAD2蛋白的物理相互作用。这些结果表明，激活素-A通过c-fos和smad2/3的相互作用增强了RANKL诱导的破骨细胞形成。
