Division of Biomedical Sciences, Faculty of Dentistry, McGill University, Montreal, Quebec, Canada.
PLoS One. 2011 Jan 20;6(1):e15893. doi: 10.1371/journal.pone.0015893.
Transglutaminase activity, arising potentially from transglutaminase 2 (TG2) and Factor XIIIA (FXIIIA), has been linked to osteoblast differentiation where it is required for type I collagen and fibronectin matrix deposition. In this study we have used an irreversible TG-inhibitor to 'block -and-track' enzyme(s) targeted during osteoblast differentiation. We show that the irreversible TG-inhibitor is highly potent in inhibiting osteoblast differentiation and mineralization and reduces secretion of both fibronectin and type I collagen and their release from the cell surface. Tracking of the dansyl probe by Western blotting and immunofluorescence microscopy demonstrated that the inhibitor targets plasma membrane-associated FXIIIA. TG2 appears not to contribute to crosslinking activity on the osteoblast surface. Inhibition of FXIIIA with NC9 resulted in defective secretory vesicle delivery to the plasma membrane which was attributable to a disorganized microtubule network and decreased microtubule association with the plasma membrane. NC9 inhibition of FXIIIA resulted in destabilization of microtubules as assessed by cellular Glu-tubulin levels. Furthermore, NC9 blocked modification of Glu-tubulin into 150 kDa high-molecular weight Glu-tubulin form which was specifically localized to the plasma membrane. FXIIIA enzyme and its crosslinking activity were colocalized with plasma membrane-associated tubulin, and thus, it appears that FXIIIA crosslinking activity is directed towards stabilizing the interaction of microtubules with the plasma membrane. Our work provides the first mechanistic cues as to how transglutaminase activity could affect protein secretion and matrix deposition in osteoblasts and suggests a novel function for plasma membrane FXIIIA in microtubule dynamics.
转谷氨酰胺酶活性，可能来自转谷氨酰胺酶 2（TG2）和因子 XIIIa（FXIIIA），与成骨细胞分化有关，在那里它需要 I 型胶原和纤维连接蛋白基质的沉积。在这项研究中，我们使用不可逆的 TG 抑制剂来“阻断和跟踪”成骨细胞分化过程中靶向的酶。我们表明，不可逆的 TG 抑制剂在抑制成骨细胞分化和矿化方面非常有效，并减少纤维连接蛋白和 I 型胶原的分泌及其从细胞表面的释放。通过 Western blot 和免疫荧光显微镜追踪丹磺酰探针表明，抑制剂靶向质膜相关的 FXIIIA。TG2 似乎不会对成骨细胞表面的交联活性做出贡献。NC9 抑制 FXIIIA 导致分泌囊泡向质膜的传递缺陷，这归因于微管网络的紊乱和微管与质膜的结合减少。NC9 抑制 FXIIIA 导致微管的不稳定性，如细胞 Glu-微管蛋白水平所示。此外，NC9 阻断 Glu-微管蛋白修饰成 150 kDa 高分子量 Glu-微管蛋白形式，该形式特异性定位于质膜。FXIIIA 酶及其交联活性与质膜相关的微管蛋白共定位，因此，似乎 FXIIIA 交联活性的作用是稳定微管与质膜的相互作用。我们的工作提供了第一个关于转谷氨酰胺酶活性如何影响成骨细胞中蛋白质分泌和基质沉积的机制线索，并表明质膜 FXIIIA 在微管动力学中的一个新功能。
