Relationship between collagen synthesis and expression of the osteoblast phenotype in MC3T3‐E1 cells

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 β‐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.

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