title: Relationship between collagen synthesis and expression of the osteoblast phenotype in MC3T3-E1 cells.
abstract: 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.
authors.affiliation: Department of Biological Chemistry, University of Texas Health Sciences Center, Houston.
authors.forename: R T
authors.initials: RT
authors.lastname: Franceschi
authors.forename: B S
authors.initials: BS
authors.lastname: Iyer
mesh_terms: D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D001205:Ascorbic Acid / Q000502:physiology*; D015415:Biomarkers / Q000737:chemistry; D002454:Cell Differentiation / Q000502:physiology; D002460:Cell Line; D003094:Collagen / Q000032:analysis / Q000096:biosynthesis*; D005109:Extracellular Matrix / Q000502:physiology*; D051379:Mice; D008854:Microscopy, Electron; D008903:Minerals / Q000378:metabolism; D010006:Osteoblasts / Q000378:metabolism*; D010641:Phenotype; D012313:RNA / Q000302:isolation & purification; D012333:RNA, Messenger / Q000096:biosynthesis; D014961:X-Ray Diffraction
chemical_list: D015415:Biomarkers; D008903:Minerals; D012333:RNA, Messenger; D012313:RNA; D003094:Collagen; D000469:Alkaline Phosphatase; D001205:Ascorbic Acid