Expression of the (V+C)− fibronectin isoform is tightly linked to the presence of a cartilaginous matrix

Fibronectin is encoded by a single gene, but heterogeneity is introduced by alternative splicing of the pre-mRNA. An unique splice variant, designated (V+C)-, which deletes nucleotides encoding the V, III-15 and I-10 segments, has been identified in articular cartilage. In this study, a ribonuclease protection assay was used to quantitate expression of the (V+C)- isoform in eight canine cartilaginous tissues and in chondrocytes cultured as monolayers or in alginate beads. The (V+C)- fibronectin isoform was detected in all cartilaginous tissues examined, ranging from a low of 11% of steady-state fibronectin mRNA in the nucleus pulposus to 71% in the rib. An age dependent increase, from 18% in the epiphyseal cartilage of a newborn to 54% in the articular cartilage of dogs over 10 months of age, was observed. The ubiquitous presence of this isoform in cartilaginous tissues and its absence in all non-cartilaginous tissues examined to date is consistent with a very strong association of the (V+C)- fibronectin isoform with the cartilaginous phenotype. Results from a ribonuclease protection assay using a probe extending into the V region from III-14 were combined with the quantitative information about (V+C)- fibronection expression to develop an over-all profile of splicing within the V region in cartilage. Monolayer culture of articular chondrocytes altered fibronectin splicing patterns. The (V+C)- isoform was rapidly lost and ED-A(+) fibronectin was induced. Three-dimensional culture in alginate beads prevented induction of ED-A(+) fibronection, but failed to sustain expression of the (V+C)- isoform. Thus, some matrix component or structure, lost in cell culture, may be essential to maintain expression of the (V+C)- isoform. The possible relationship of changing patterns of fibronectin isoforms in cultured chondrocytes to maintenance of the differentiated phenotype is discussed.