Sulfation of chondroitin sulfate proteoglycans is necessary for proper Indian hedgehog signaling in the developing growth plate

Abstract

In contrast to the functional role of heparan sulfate proteoglycans (HSPGs), the importance of chondroitin sulfate proteoglycans (CSPGs) in modulating signaling pathways involving hedgehog proteins, wingless-related proteins and fibroblast growth factors remains unclear. To elucidate the importance of sulfated CSPGs in signaling paradigms required for endochondral bone formation, the brachymorphic (bm) mouse was used as a model for undersulfated CSPGs. The bm mouse exhibits a postnatal chondrodysplasia caused by a mutation in the phosphoadenosine phosphosulfate (PAPS) synthetase (Papss2) gene, leading to reduced levels of PAPS and undersulfated proteoglycans. Biochemical analysis of the glycosaminoglycan (GAG) content in bm cartilage via sulfate labeling and fluorophore-assisted carbohydrate electrophoresis revealed preferential undersulfation of chondroitin chains (CS) and normal sulfation of heparan sulfate chains. In situ hybridization and immunohistochemical analysis of bm limb growth plates showed diminished Indian hedgehog (Ihh) signaling and abnormal Ihh protein distribution in the extracellular matrix. Consistent with the decrease in hedgehog signaling, BrdU incorporation exhibited a significant reduction in chondrocyte proliferation. Direct measurements of Ihh binding to defined GAG chains demonstrated that Ihh interacts with CS, particularly chondroitin-4-sulfate. Furthermore, co-immunoprecipitation experiments showed that Ihh binds to the major cartilage CSPG aggrecan via its CS chains. Overall, this study demonstrates an important function for CSPGs in modulating Ihh signaling in the developing growth plate, and highlights the importance of carbohydrate sulfation in regulating growth factor signaling.