Synthesis of collagen by bovine chondrocytes cultured in alginate; posttranslational modifications and cell-matrix interaction.
The extracellular matrix synthesized by articular chondrocytes cultured in alginate beads was investigated. Collagen levels increased sigmoidally with time and remained constant after 2 weeks of culture. The presence of cartilage-specific type II collagen was confirmed immunohistochemically. Predominantly type II collagen was present in the alginate bead, as reflected by the unique extent of lysyl hydroxylation, glycosylation, and pyridinoline crosslink formation measured. Collagen crosslinks, predominantly hydroxylysylpyridinoline (> 93%), were observed after 7 to 11 days of culture and their formation was effectively blocked by beta-aminopropionitrile (BAPN). Unexpectedly, BAPN treatment resulted in a 100% increase of collagen levels, without influencing cell proliferation and proteoglycan levels. In control cultures 90% of the synthesized collagen was retained in the cell-associated matrix, while in BAPN-treated cultures half of the collagen was found in the interterritorial matrix compartment further removed from the cells. This suggests that impaired crosslinking of collagen interferes with pericellular collagen deposition, causing upregulation of collagen synthesis by impaired cell-matrix interactions. Integrins are likely to be involved in this feedback inhibition by extracellular collagen since the cyclic RGD-containing peptide CGRGDSPC downregulated collagen synthesis by 37%.
Beekman
B
B
Department of Vascular and Connective Tissue Research, TNO Prevention and Health, Leiden, The Netherlands.
Verzijl
N
N
Bank
R A
RA
von der Mark
K
K
TeKoppele
J M
JM
D000464:Alginates; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000629:Aminopropionitrile / Q000494:pharmacology; D000818:Animals; D002358:Cartilage, Articular / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D002417:Cattle; D002455:Cell Division; D002478:Cells, Cultured; D003094:Collagen / Q000096:biosynthesis*; D004247:DNA / Q000032:analysis; D005109:Extracellular Matrix / Q000502:physiology; D020723:Glucuronic Acid; D006031:Glycosylation; D006603:Hexuronic Acids; D007700:Kinetics; D009842:Oligopeptides / Q000494:pharmacology; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D011509:Proteoglycans / Q000032:analysis; D013997:Time Factors
D000464:Alginates; D000596:Amino Acids; D006603:Hexuronic Acids; D009842:Oligopeptides; D011509:Proteoglycans; D000629:Aminopropionitrile; C015484:pyridinoline; C047981:arginyl-glycyl-aspartic acid; D020723:Glucuronic Acid; D003094:Collagen; D004247:DNA; C058161:glycyl-arginyl-glycyl-aspartyl-seryl-proline