Syndecan 4, a heparan sulfate proteoglycan, has been associated with osteoarthritis. The present study was undertaken to analyze the functional role of syndecan 4 in endochondral ossification of mouse embryos and in adult fracture repair, which, like osteoarthritis, involves an inflammatory component.
Sdc4 promoter activity was analyzed in Sdc4−/− lacZ-knockin mice, using β-galactosidase staining. Endochondral ossification in embryos from embryonic day 16.5 was assessed by histologic and immunohistologic staining. Bone fracture repair was analyzed in femora of adult mice on days 7 and 14 postfracture. To evaluate Sdc2 and Sdc4 gene expression with and without tumor necrosis factor α (TNFα) and Wnt-3a stimulation, quantitative real-time polymerase chain reaction was performed.
In Sdc4−/− lacZ-knockin animals, syndecan 4 promoter activity was detectable at all stages of chondrocyte differentiation, and Sdc4 deficiency inhibited chondrocyte proliferation. Aggrecan turnover in the uncalcified cartilage of the epiphysis was decreased transiently in vivo, but this did not lead to a growth phenotype at birth. In contrast, among adult mice, fracture healing was markedly delayed in Sdc4−/− animals and was accompanied by increased callus formation. Blocking of inflammation via anti-TNFα treatment during fracture healing reduced these changes in Sdc4−/− mice to levels observed in wild-type controls. We analyzed the differences between the mild embryonic and the severe adult phenotype, and found a compensatory up-regulation of syndecan 2 in the developing cartilage of Sdc4−/− mice that was absent in adult tissue. Stimulation of chondrocytes with Wnt-3a in vitro led to increased expression of syndecan 2, while stimulation with TNFα resulted in up-regulation of syndecan 4 but decreased expression of syndecan 2. TNFα stimulation reduced syndecan 2 expression and increased syndecan 4 expression even in the presence of Wnt-3a, suggesting that inflammation has a strong effect on the regulation of syndecan expression.
Our results demonstrate that syndecan 4 is functionally involved in endochondral ossification and that its loss impairs fracture healing, due to inhibition of compensatory mechanisms under inflammatory conditions.