To define, for the C5.18 chondrocyte-restricted rat cell line, (1) the capacities for recognition of alternatively spliced segments of the adhesion protein fibronectin (FN), (2) the integrin subunits required for such recognition, and (3) differences in such FN recognition vs the multipotential chondroprogenitor line, RCJ 3.1.
C5.18 and RCJ 3.1 cells were tested for their capacities to adhere to recombinant alternatively spliced segments of rat FN, presented on plastic surfaces either in isolation or in partial FNs spanning the 7th through 15th type III repeats (III7-15 FNs). The effects on such adhesion of cations and integrin subunit-specific antibodies were tested.
Despite significant augmentation in chondrocyte-specific gene expression in C5.18 relative to the RCJ 3.1 cells, the two lines exhibited similar recognition of FN spliced segments and partial isoforms. Specifically, both lines adhered to the extra type III repeat A (EIIIA) and V, but not extra type III repeat B (EIIIB), segments. There were different cation and integrin subunit requirements for adhesion to EIIIA vs V segments, and only the V segment was recognized in the context of a III7-15 FN. Such recognition was mediated via a "second" arginine-glycine-aspartic acid (RGD) sequence that is present in the V95 subsegment in rat, but not human, FN.
The chondrocyte lineage-committed C5.18 cell line, similar to its multipotential chondroprogenitor, RCJ 3.1, recognizes the "cartilage-restricted" EIIIA and V segments of FN with cation, integrin, and molecular context requirements that are specific to each of these segments.