Characterization of Type II and Type XI Collagen Synthesis by an Immortalized Rat Chondrocyte Cell Line (IRC) Having a Low Level of Type II Collagen mRNA Expression

Abstract

The biosynthesis of type XI and type II collagens was examined using a stable rat chondrocyte cell line established by W. E. Horton et al. (1988, Exp. Cell Res. 178, 457-468.). These cells (IRC; immortalized rat chondrocytes) were created by transformation with a murine retrovirus carrying the v-myc and v-raf oncogenes. They grow in suspension culture as multicellular aggregates and synthesize typical cartilage proteins, aggrecan and link protein. Type II collagen is absent or synthesized at severely reduced levels, as shown by Northern analysis of mRNA. Thus, this cell type represents a unique model in which to study cartilage matrix protein interactions in the absence of type II collagen. A more detailed look at the proteins secreted into the medium by metabolically labeled IRC cell revealed the presence of collagenase-sensitive bands when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The bands were identified as the α1, α2, and α3 chains of heterotimeric type XI collagen by electrophoretic migration after pepsin digestion, by CNBr peptide mapping, and by immunoprecipitation with antibodies to rat α1(XI). mRNA for all three chains was detected by Northern blot analysis. The data indicate that the low level of α1(II) mRNA previously detected in these cells is translated into proα3(XI) polypeptide chains which are incorporated into molecules of type XI. Under normal culture conditions, homotrimers of type II collagen were not detected. The carboxyl propeptide domain of the fibrillar collagens directs chain selection and molecular assembly of the trimeric molecules. The sequence of the carboxyl propeptide domain from proα3(XI) of IRC cells was found to be identical to this domain from proα1(II) of swarm rat chondrosarcoma, supporting previous evidence that proα3(XI) and proα1(II) have the same primary structure. When cultured in the presence of 50 mM arginine, IRC cells could be induced to synthesize proα1(II) chains in excess over proα1(XI) and proα2(XI). Only under these conditions were type II collagen molecules detected, suggesting a preferential association of proα1(II) with the proα1 and/or proα2 chains of type XI collagen.