title: A mouse 3T6 fibroblast cell culture model for the study of normal and protein-engineered collagen synthesis and deposition into the extracellular matrix.
abstract: Mouse 3T6 fibroblasts deposited an organized collagenous extracellular matrix during long-term culture in the presence of ascorbic acid. The matrix produced by the cells had a similar distribution of collagen types as the mouse dermal matrix, comprising predominantly type I with smaller amounts of types III and V collagens. By day 8 of culture more than 70% of the collagen in the 3T6 matrix was involved in covalent crosslinkages and required pepsin digestion for extraction. Incorporation of NaB3H4 into reducible crosslinks and aldehydes directly demonstrated the involvement of the alpha 1 (I)CB6 and alpha 2(I)CB3.5 in crosslinks. The pattern of reducible crosslinks in the in vitro 3T6 matrix was similar to that in mouse skin suggesting a comparable fibril organization. Processing of procollagen to collagen occurred efficiently throughout the culture period and the rate of collagen production was unaltered during 15 days of culture, indicating that the development of a collagenous matrix does not directly play a role in procollagen processing or biosynthetic regulation. The existence of a preformed matrix did however, increase the efficiency with which newly synthesised collagen was incorporated into the pericellular matrix. At day 0, when there was no measurable matrix present, 29% of the collagen synthesised was deposited, while by day 15, 88% of the collagen was laid down in the matrix. The development of this 3T6 culture system, where collagen is efficiently incorporated into an organized extracellular matrix, will facilitate detailed studies on matrix organization and regulation and provide a system in which protein-engineered mutant collagens can be expressed to determine their effects on the production of a functional extracellular matrix.
authors.affiliation: Department of Paediatrics, University of Melbourne, Royal Children's Hospital, Parkville, Victoria, Australia.
authors.forename: S R
authors.initials: SR
authors.lastname: Lamandé
authors.forename: J F
authors.initials: JF
authors.lastname: Bateman
mesh_terms: D000447:Aldehydes; D000818:Animals; D002460:Cell Line; D003094:Collagen / Q000096:biosynthesis* / Q000302:isolation & purification; D003432:Cross-Linking Reagents; D003488:Cyanogen Bromide; D004247:DNA / Q000302:isolation & purification / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D005109:Extracellular Matrix / Q000378:metabolism*; D005347:Fibroblasts / Q000378:metabolism; D051379:Mice; D010446:Peptide Fragments / Q000302:isolation & purification; D010449:Peptide Mapping; D011347:Procollagen / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D011392:Proline / Q000378:metabolism; D015202:Protein Engineering; D011499:Protein Processing, Post-Translational; D014316:Tritium
chemical_list: D000447:Aldehydes; D003432:Cross-Linking Reagents; D010446:Peptide Fragments; D011347:Procollagen; D014316:Tritium; D003094:Collagen; D004247:DNA; D011392:Proline; D003488:Cyanogen Bromide