Franceschi R T, Iyer B S
Department of Biological Chemistry, University of Texas Health Sciences Center, Houston.
J Bone Miner Res. 1992 Feb;7(2):235-46. doi: 10.1002/jbmr.5650070216.
The MC3T3-E1 mouse calvaria-derived cell line has been used to study the role of collagen synthesis in osteoblast differentiation. MC3T3-E1 cells, like several previously characterized osteoblast culture systems, expressed osteoblast markers and formed a mineralized extracellular matrix only after exposure to ascorbic acid. Mineralization was stimulated further by beta-glycerol phosphate. Ultrastructural observations indicated that the extracellular matrix produced by ascorbic acid-treated cells was highly organized and contained well-banded collagen fibrils. Expression of osteoblast markers followed a clear temporal sequence. The earliest effects of ascorbic acid were to stimulate type I procollagen mRNA and collagen synthesis (24 h after ascorbate addition), followed by induction of alkaline phosphatase (48-72 h) and osteocalcin (96-144 h) mRNAs. Procollagen mRNA, which was expressed constitutively in the absence of ascorbate, increased only twofold after vitamin C addition. In contrast, alkaline phosphatase and osteocalcin mRNAs were undetectable in untreated cultures. Actions of ascorbic acid on osteoblast marker gene expression are mediated by increases in collagen synthesis and/or accumulation because (1) parallel dose-response relationships were obtained for ascorbic acid stimulation of collagen accumulation and alkaline phosphatase activity, and (2) the specific collagen synthesis inhibitors, 3,4-dehydroproline and cis-4-hydroxyproline, reversibly blocked ascorbic acid-dependent collagen synthesis and osteoblast marker gene expression.
MC3T3-E1小鼠颅盖骨来源的细胞系已被用于研究胶原蛋白合成在成骨细胞分化中的作用。MC3T3-E1细胞与几种先前已表征的成骨细胞培养系统一样，表达成骨细胞标志物，并且仅在暴露于抗坏血酸后才形成矿化的细胞外基质。β-甘油磷酸进一步刺激矿化。超微结构观察表明，抗坏血酸处理的细胞产生的细胞外基质高度有序，并且含有具有良好条纹的胶原纤维。成骨细胞标志物的表达遵循明确的时间顺序。抗坏血酸的最早作用是刺激I型前胶原mRNA和胶原蛋白合成（添加抗坏血酸后24小时），随后诱导碱性磷酸酶（48 - 72小时）和骨钙素（96 - 144小时）mRNA。在没有抗坏血酸的情况下组成性表达的前胶原mRNA，在添加维生素C后仅增加两倍。相比之下，在未处理的培养物中未检测到碱性磷酸酶和骨钙素mRNA。抗坏血酸对成骨细胞标志物基因表达的作用是由胶原蛋白合成和/或积累的增加介导的，因为（1）抗坏血酸刺激胶原蛋白积累和碱性磷酸酶活性获得了平行的剂量反应关系，并且（2）特异性胶原蛋白合成抑制剂3,4-脱氢脯氨酸和顺式-4-羟脯氨酸可逆地阻断了抗坏血酸依赖性胶原蛋白合成和成骨细胞标志物基因表达。
