Al-Jallad Hadil F, Nakano Yukiko, Chen Jeff L Y, McMillan Erin, Lefebvre Céline, Kaartinen Mari T
Division of Oral Biology, Faculty of Dentistry, McGill University, Montreal, QC, Canada.
Matrix Biol. 2006 Apr;25(3):135-48. doi: 10.1016/j.matbio.2005.11.001. Epub 2006 Feb 15.
Transglutaminase (TG) enzymes and protein crosslinking have long been implicated in the formation of mineralized tissues. The aim of this study was to analyze the expression, activity and function of TGs in differentiating osteoblasts to gain further insight into the role of extracellular matrix protein crosslinking in bone formation. MC3T3-E1 (subclone 14) pre-osteoblast cultures were treated with ascorbic acid and beta-glycerophosphate to induce cell differentiation and matrix mineralization. Expression of TG isoforms was analyzed by RT-PCR. TG activity was assessed during osteoblast differentiation by in vitro biochemical assays and by in situ labeling of live cell cultures. We demonstrate that MC3T3-E1/C14 osteoblasts express two TG isoforms--TG2 and FXIIIA. Abundant TG activity was observed during cell differentiation which increased significantly after thrombin treatment, a result confirming the presence of FXIIIA in the cultures. Ascorbic acid treatment, which stimulated collagen secretion and assembly, also stimulated externalization of TG activity, likely from FXIIIA which was externalized upon this treatment as analyzed by immunofluoresence microscopy. Inhibition of TG activity in the cultures by cystamine resulted in complete abrogation of mineralization, attributable to decreased matrix accumulation and an arrested state of osteoblast differentiation as measured by decreased levels of bone sialoprotein, osteocalcin and alkaline phosphatase. Additional functional studies and substrate characterization showed that TG activity was required for the formation of a fibronectin-collagen network during the early stages of matrix formation and assembly. This network, in turn, appeared to be essential for further matrix production and progression of the osteoblast differentiation program, and ultimately for mineralization.
长期以来，转谷氨酰胺酶（TG）和蛋白质交联一直被认为与矿化组织的形成有关。本研究的目的是分析TG在成骨细胞分化过程中的表达、活性和功能，以进一步深入了解细胞外基质蛋白交联在骨形成中的作用。用抗坏血酸和β-甘油磷酸处理MC3T3-E1（亚克隆14）前成骨细胞培养物，以诱导细胞分化和基质矿化。通过RT-PCR分析TG同工型的表达。通过体外生化分析和活细胞培养物的原位标记评估成骨细胞分化过程中的TG活性。我们证明MC3T3-E1/C14成骨细胞表达两种TG同工型——TG2和FXIIIA。在细胞分化过程中观察到丰富的TG活性，凝血酶处理后活性显著增加，这一结果证实培养物中存在FXIIIA。抗坏血酸处理刺激了胶原蛋白的分泌和组装，也刺激了TG活性的外化，通过免疫荧光显微镜分析，这可能来自于经该处理后外化的FXIIIA。胱胺对培养物中TG活性的抑制导致矿化完全消除，这归因于基质积累减少和成骨细胞分化停滞状态，通过骨唾液蛋白、骨钙素和碱性磷酸酶水平降低来衡量。进一步的功能研究和底物表征表明，在基质形成和组装的早期阶段，TG活性是形成纤连蛋白-胶原蛋白网络所必需的。反过来，这个网络似乎对于进一步的基质产生和成骨细胞分化程序的进展至关重要，最终对于矿化也至关重要。
