Transglutaminase activity regulates osteoblast differentiation and matrix mineralization in MOT3-E1 osteoblast cultures
Transglutaminase activity regulates osteoblast differentiation and matrix mineralization in MOT3-E1 osteoblast cultures
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DOI：
10.1016/j.matbio.2005.11.001
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发表时间：
2006-04-01
期刊：
影响因子：
6.9
通讯作者：
Kaartinen, MT
中科院分区：
文献类型：
--
作者：
Al-Jallad, HF;Nakano, Y;Kaartinen, MT
转氨酶（TG）酶和蛋白质交联长期以来一直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-甘油磷酸盐处理MC 3 T3-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同种型的表达。通过体外生物化学测定和活细胞培养物原位标记，在成骨细胞分化过程中评估TG活性。我们证明MC 3 T3 E1/C14成骨细胞表达两种TG亚型-TG 2和FXIIIA。在细胞分化过程中观察到丰富的TG活性，其在凝血酶处理后显著增加，结果证实了培养物中FXIIIA的存在。抗坏血酸处理，刺激胶原蛋白的分泌和组装，也刺激TG活性的外化，可能来自FXIIIA，其通过免疫荧光显微镜分析在该处理后外化。胱胺对培养物中TG活性的抑制导致矿化的完全废除，这归因于基质积累减少和成骨细胞分化的停滞状态，如通过骨唾液蛋白、骨钙素和碱性磷酸酶水平降低所测量的。额外的功能研究和基板表征表明，TG活性所需的纤维连接蛋白-胶原蛋白网络的形成在早期阶段的基质形成和组装。反过来，这个网络似乎是必要的进一步基质生产和进展的成骨细胞分化程序，并最终矿化。(c)2005年Elsevier B. V./国际矩阵生物学学会。All rights reserved.
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 MC3T3E1/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. (c) 2005 Elsevier B.V./International Society of Matrix Biology. All rights reserved.