Plasma Membrane Factor XIIIA Transglutaminase Activity Regulates Osteoblast Matrix Secretion and Deposition by Affecting Microtubule Dynamics

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.

潜在源自转谷氨酰胺酶2（Transglutaminase 2，TG2）与凝血因子XIII A亚基（Factor XIIIA，FXIIIA）的转谷氨酰胺酶活性，已被证实与成骨细胞分化相关，且该活性是I型胶原与纤连蛋白基质沉积的必要条件。本研究采用不可逆转谷氨酰胺酶抑制剂开展“阻断-追踪”实验，靶向解析成骨细胞分化过程中的靶标酶活性。研究结果表明，该不可逆转谷氨酰胺酶抑制剂可强效抑制成骨细胞分化与矿化过程，并降低纤连蛋白与I型胶原的分泌量及其从细胞表面的释放水平。通过蛋白质免疫印迹（Western blotting）与免疫荧光显微镜对丹酰探针的追踪实验证实，该抑制剂靶向结合质膜结合型FXIIIA。TG2似乎并未参与成骨细胞表面的交联活性调控。使用NC9抑制FXIIIA后，分泌囊泡向质膜的递送出现功能缺陷，该缺陷可归因于微管网络紊乱以及微管与质膜的结合能力下降。通过检测细胞内谷氨酰化微管蛋白（Glu-tubulin）水平可知，NC9对FXIIIA的抑制会导致微管不稳定。此外，NC9可阻断谷氨酰化微管蛋白转化为150 kDa的高分子量谷氨酰化微管蛋白亚型，该亚型特异性定位于质膜。FXIIIA酶与其交联活性与质膜结合的微管蛋白共定位，因此推测FXIIIA的交联活性可定向稳定微管与质膜之间的相互作用。本研究首次揭示了转谷氨酰胺酶活性影响成骨细胞内蛋白质分泌与基质沉积的潜在机制，并提出质膜结合型FXIIIA在微管动态调控中具有全新功能的观点。