We have previously shown that SV40 large T oncogene is able to induce mouse chondrocyte proliferation without loss of expression of types II, IX, and XI
collagen, as well as cartilage
aggrecan and
link protein. The cell line obtained (termed MC 615) also expressed some
type I collagen in monolayer and we have investigated if anchorage-independent conditions could inhibit
type I collagen synthesis and promote
hypertrophy and
type X collagen synthesis. The MC 615 cells were grown in
agarose in the presence of serum, and GAG accumulation,
DNA content, and matrix synthesis rates were monitored after incubation with [35S]
sulfate and [3H]- or [14C]
proline. SDS-PAGE analysis of
pepsin-extracted samples showed that
type I collagen was still synthesized by the MC 615 cells, from the beginning of the culture and at low or high density.
Type II collagen synthesis was demonstrated by immunoblotting, but
type X collagen synthesis was not detected, indicating that the MC 615 chondrocytes immortalized by large T were still blocked in their maturation pathway. The cells were also grown over
agarose and electron microscopy (E. M.) analysis of the cell aggregates showed an extracellular matrix rich in
proteoglycans and in type II-containing
collagen fibrils. To gain insight into the role of
type IX collagen in
cartilage collagen assembly and/or matrix organization, we also immortalized embryonic chondrocytes isolated from mice lacking alpha 1 (IX)
collagen and obtained a clone termed 4KO 91. As found for the MC 615 cells, the 4KO 91 cells synthesized
type II collagen as demonstrated by Western blotting and some
type I collagen identified by the presence of alpha 2(I) chains after electrophoretic analysis of
pepsin-digested
collagen chains. E. M. analysis of the extracellular matrices synthesized by the two cell lines revealed differences in
collagen structure and organization. In the absence of alpha 1 (IX)
collagen chains, the
collagen fibrils seemed to fuse laterally, suggesting that
collagen IX acts as a "spacer" between fibrils, to keep them apart.