Division of Biomedical Sciences, Faculty of Dentistry, McGill University, Montreal, Quebec, Canada.
Division of Experimental Medicine, Department of Medicine, Faculty of Medicine, McGill University, Montreal, Quebec, Canada.
J Cell Physiol. 2018 Sep;233(9):7497-7513. doi: 10.1002/jcp.26603. Epub 2018 Apr 16.
Osteoclasts, bone resorbing cells, derive from monocyte/macrophage cell lineage. Increased osteoclast activity is responsible for bone destruction in diseases such as osteoporosis, periodontitis and rheumatoid arthritis. Transglutaminases (TGs), protein crosslinking enzymes, were recently found involved in osteoclastogenesis in vivo, however their mechanisms of action have remained unknown. In this study, we have investigated the role of TG activity in osteoclastogenesis in vitro using four TG inhibitors, NC9, Z006, T101, and monodansyl cadaverine. Our results showed that all TG inhibitors were capable of blocking the entire osteoclastogenesis process. The most potent of the inhibitors, NC9 when added to cultures at different phases of osteoclastogenesis, inhibited differentiation, migration, and fusion of pre-osteoclasts as well as resorption activity of mature osteoclasts. Further investigation into the mechanisms revealed that NC9 increased RhoA levels and blocked podosome belt formation suggesting that TG activity regulates actin dynamics in pre-osteoclasts. The inhibitory effect of NC9 on osteoclastogenesis as well as podosome belt formation was completely reversed with a Rho-family inhibitor Exoenzyme C3. Microtubule architecture, acetylation, and detyrosination of α-tubulin were not affected. Finally, we demonstrated that macrophages and osteoclasts expressed mRNA of three TGs:TG1, TG2, and Factor XIII-A which were all differentially regulated in these cells during differentiation. Immunofluoresence microscopic analysis showed that all three enzymes co-localized to podosomes in osteoclasts. Taken together, our data suggests that TG activity regulates differentiation, migration and fusion of osteoclasts via affecting actin dynamics and that this may involve contribution from all three TG enzymes.
破骨细胞是一种骨吸收细胞，来源于单核/巨噬细胞细胞系。破骨细胞活性的增加是导致骨质疏松症、牙周炎和类风湿性关节炎等疾病骨破坏的原因。转谷氨酰胺酶（TGs）是一种蛋白质交联酶，最近被发现参与体内破骨细胞的生成，但它们的作用机制仍不清楚。在这项研究中，我们使用四种 TG 抑制剂 NC9、Z006、T101 和单丹磺酰尸胺，研究了 TG 活性在体外破骨细胞生成中的作用。我们的结果表明，所有 TG 抑制剂都能够阻断整个破骨细胞生成过程。抑制剂中最有效的 NC9，在添加到破骨细胞生成的不同阶段的培养物中时，能够抑制前破骨细胞的分化、迁移和融合，以及成熟破骨细胞的吸收活性。进一步的机制研究表明，NC9 增加了 RhoA 水平并阻断了破骨细胞伪足带的形成，表明 TG 活性调节前破骨细胞中的肌动蛋白动力学。NC9 对破骨细胞生成和伪足带形成的抑制作用，可被 Rho 家族抑制剂 Exoenzyme C3 完全逆转。微管结构、α-微管蛋白的乙酰化和去酪氨酸化没有受到影响。最后，我们证明巨噬细胞和破骨细胞表达了三种 TG：TG1、TG2 和因子 XIII-A 的 mRNA，这些基因在细胞分化过程中均有差异表达。免疫荧光显微镜分析显示，这三种酶都在破骨细胞的伪足中共同定位。总之，我们的数据表明，TG 活性通过影响肌动蛋白动力学来调节破骨细胞的分化、迁移和融合，而这可能涉及到三种 TG 酶的共同作用。
