Koga Ayaka, Nagai-Yoshioka Yoshie, Yamasaki Ryota, Adachi Yoshiyuki, Fujii Wataru, Ariyoshi Wataru
Department of Health Sciences, Kyushu Dental University, Kitakyushu, Fukuoka, Japan.
Division of Infections and Molecular Biology, Department of Health Promotion, Kyushu Dental University, Kitakyushu, Fukuoka, Japan.
J Cell Biochem. 2025 Jan;126(1):e30682. doi: 10.1002/jcb.30682. Epub 2024 Nov 28.
Osteoclasts derived from hematopoietic stem cells express immunoreceptors on their cell surface. Previously, we showed that the β-glucan curdlan suppressed osteoclastogenesis via binding to dectin-1, a pattern recognition receptor. Curdlan negatively regulates osteoclast differentiation and bone resorption capacity by suppressing the expression of nuclear factor of activated T cells 1 (NFATc1), a master factor for osteoclast differentiation, in a dectin-1-dependent manner; however, the mechanism involved in this process has not yet been fully elucidated. In this study, we aimed to elucidate the molecular mechanism involved in the suppression of RANKL-induced osteoclast differentiation by curdlan. Real-time RT-qPCR results showed that curdlan suppressed the expression of NFATc1 in cells of the osteoclast progenitor cell line RAW264.7 overexpressing dectin-1 (d-RAW cells), without altering the expression of negative regulators. Therefore, we examined the effect of curdlan on the NF-κB pathway, which is important for the induction of NFATc1 expression. Western blot analysis results showed that curdlan addition suppressed RANKL-induced NF-κB activation in the vector control line (c-RAW) cells with low expression of dectin-1, in d-RAW cells, and the parental RAW264.7 (RAW) cells. The results of tartrate-resistant alkaline phosphatase staining and real-time RT-qPCR showed that curdlan addition suppressed osteoclast differentiation in RAW cells, suggesting the presence of a dectin-1-independent modification system. Finally, we focused on the complement receptor 3 (CR3), which binds β-glucan, and revealed that blocking the binding of β-glucan to the CD11b molecule, a component of CR3, by neutralizing antibody, recovered the suppression of IκBα degradation by curdlan. These results suggest that the suppression of osteoclast differentiation by curdlan involves not only the dectin-1-dependent pathway but also the negative regulation of NFATc1 via modification of the NF-κB pathway via CR3 recognition. The results of this study may aid to establish treatment methods for metabolic bone diseases and inflammatory bone destruction and to clarify their pathogenesis.
源自造血干细胞的破骨细胞在其细胞表面表达免疫受体。此前，我们发现β-葡聚糖可德兰通过与模式识别受体dectin-1结合来抑制破骨细胞生成。可德兰以dectin-1依赖的方式，通过抑制破骨细胞分化的主要因子活化T细胞核因子1（NFATc1）的表达，对破骨细胞分化和骨吸收能力进行负调控；然而，这一过程所涉及的机制尚未完全阐明。在本研究中，我们旨在阐明可德兰抑制RANKL诱导的破骨细胞分化所涉及的分子机制。实时RT-qPCR结果显示，可德兰可抑制过表达dectin-1的破骨细胞祖细胞系RAW264.7（d-RAW细胞）中NFATc1的表达，而不改变负调控因子的表达。因此，我们研究了可德兰对NF-κB通路的影响，该通路对NFATc1表达的诱导很重要。蛋白质免疫印迹分析结果显示，添加可德兰可抑制RANKL诱导的dectin-1低表达的载体对照系（c-RAW）细胞、d-RAW细胞和亲本RAW264.7（RAW）细胞中的NF-κB活化。抗酒石酸酸性磷酸酶染色和实时RT-qPCR结果显示，添加可德兰可抑制RAW细胞中的破骨细胞分化，提示存在dectin-1非依赖的修饰系统。最后，我们聚焦于与β-葡聚糖结合的补体受体3（CR3），并发现用中和抗体阻断β-葡聚糖与CR3的组成成分CD11b分子的结合，可恢复可德兰对IκBα降解的抑制作用。这些结果表明，可德兰对破骨细胞分化的抑制不仅涉及dectin-1依赖的通路，还涉及通过CR3识别对NF-κB通路进行修饰从而对NFATc1进行负调控。本研究结果可能有助于建立代谢性骨病和炎性骨破坏的治疗方法，并阐明其发病机制。
