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.

谷氨酰胺转移酶（Transglutaminase）活性可能源自谷氨酰胺转移酶2（Transglutaminase 2, TG2）与凝血因子XIIIA（Factor XIIIA, FXIIIA），该活性与成骨细胞（osteoblast）分化密切相关，且对I型胶原（type I collagen）与纤连蛋白（fibronectin）的基质沉积不可或缺。本研究采用不可逆谷氨酰胺转移酶抑制剂开展“阻断-示踪”实验，靶向定位成骨细胞分化过程中的目标酶类。实验结果显示，该不可逆谷氨酰胺转移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量。通过蛋白质印迹（Western blotting）与免疫荧光显微镜（immunofluorescence microscopy）对丹酰基探针进行示踪分析，结果表明该抑制剂靶向结合质膜相关的FXIIIA。TG2似乎并未参与成骨细胞表面的交联活性。使用NC9抑制FXIIIA会导致分泌囊泡向质膜的递送出现缺陷，这一现象源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）的水平，证实NC9抑制FXIIIA会导致微管稳定性下降。此外，NC9可阻断Glu-tubulin向150千道尔顿（kDa）的高分子量Glu-tubulin形式的修饰，该修饰产物特异性定位于质膜。FXIIIA酶与其交联活性均与质膜相关的微管蛋白（tubulin）存在共定位现象，由此可见，FXIIIA的交联活性可定向稳定微管与质膜之间的相互作用。本研究首次揭示了谷氨酰胺转移酶活性影响成骨细胞中蛋白质分泌与基质沉积的潜在机制，并提出了质膜FXIIIA在微管动力学中发挥的全新功能。