Faculty of Medicine and Health Sciences (Division of Experimental Medicine), McGill University, Montreal, QC H3A 0C7, Canada.
Faculty of Dental Medicine and Oral Health Sciences (Biomedical Sciences), McGill University, Montreal, QC H3A 0C7, Canada.
Cells. 2023 Aug 24;12(17):2139. doi: 10.3390/cells12172139.
Osteoclasts are multinucleated, bone-resorbing giant cells derived from monocyte-macrophage cell lines. Increased bone resorption results in loss of bone mass and osteoporosis. Osteoclast and bone marrow macrophages have been shown to express three TG enzymes (TG2, Factor XIII-A, and TG1) and TG activity to regulate osteoclast differentiation from bone marrow macrophages in vitro. In vivo and in vitro studies have demonstrated that the deletion of TG2 causes increased osteoclastogenesis and a significant loss of bone mass in mice (-/- mice). Here, we confirm that TG2 deficiency results in increased osteoclastogenesis in vitro and show that this increase can be reversed by a TG inhibitor, NC9, suggesting that other TGs are responsible for driving osteoclastogenesis in the absence of TG2. An assessment of total TG activity with 5-(biotinamido)-pentylamine, as well as TG1 and FXIII-A activities using TG-specific Hitomi peptides (bK5 and bF11) in -/- bone marrow flushes, bone marrow macrophages, and osteoclasts, showed a significant increase in total TG activity and TG1 activity. Factor XIII-A activity was unchanged. Aspartate proteases, such as cathepsins, are involved in the degradation of organic bone matrix and can be produced by osteoclasts. Moreover, Cathepsin D was shown in previous work to be increased in TG2-null cells and is known to activate TG1. We show that Pepstatin A, an aspartate protease inhibitor, blocks osteoclastogenesis in wild-type and Tgm2-/- cells and decreases TG1 activity in Tgm2-/- osteoclasts. Cathepsin D protein levels were unaltered in Tgm2-/-cells and its activity moderately but significantly increased. -/- and +/+ bone marrow macrophages and osteoclasts also expressed Cathepsin E, and Renin of the aspartate protease family, suggesting their potential involvement in this process. Our study brings further support to the observation that TGs are significant regulators of osteoclastogenesis and that the absence of TG2 can cause increased activity of other TGs, such as TG1.
破骨细胞是源自单核细胞-巨噬细胞细胞系的多核、骨吸收巨细胞。骨吸收增加导致骨量丢失和骨质疏松症。已经表明破骨细胞和骨髓巨噬细胞表达三种 TG 酶（TG2、因子 XIII-A 和 TG1）和 TG 活性，以调节体外骨髓巨噬细胞向破骨细胞的分化。体内和体外研究表明，TG2 的缺失导致破骨细胞生成增加和小鼠骨量显著丢失（-/- 小鼠）。在这里，我们证实 TG2 缺乏导致体外破骨细胞生成增加，并表明这种增加可以被 TG 抑制剂 NC9 逆转，表明在没有 TG2 的情况下，其他 TG 负责驱动破骨细胞生成。使用针对 TG 的 Hitomi 肽（bK5 和 bF11）评估 5-（生物素氨基）戊基胺的总 TG 活性以及 TG1 和 FXIII-A 活性，在-/-骨髓冲洗液、骨髓巨噬细胞和破骨细胞中，总 TG 活性和 TG1 活性均显著增加。FXIII-A 活性不变。天冬氨酸蛋白酶，如组织蛋白酶，参与有机骨基质的降解，并且可以由破骨细胞产生。此外，在以前的工作中表明组织蛋白酶 D 在 TG2 缺失细胞中增加，并且已知其激活 TG1。我们表明天冬氨酸蛋白酶抑制剂 Pepstatin A 阻断野生型和 Tgm2-/-细胞的破骨细胞生成，并降低 Tgm2-/-破骨细胞中的 TG1 活性。Tgm2-/-细胞中的组织蛋白酶 D 蛋白水平不变，其活性适度但显著增加。-/-和 +/+骨髓巨噬细胞和破骨细胞也表达天冬氨酸蛋白酶家族的组织蛋白酶 E 和肾素，表明它们可能参与该过程。我们的研究进一步支持这样一种观察结果，即 TG 是破骨细胞生成的重要调节剂，并且 TG2 的缺失会导致其他 TG（如 TG1）的活性增加。
