Chondrocytes respond to an altered heparan sulfate composition with distinct changes of heparan sulfate structure and increased levels of chondroitin sulfate

Heparan sulfate (HS) regulates the activity of many signaling molecules critical for the development of endochondral bones. Even so, mice with a genetically altered HS metabolism display a relatively mild skeletal phenotype compared to the defects observed in other tissues and organs pointing to a reduced HS dependency of growth-factor signaling in chondrocytes. To understand this difference, we have investigated the glycosaminoglycan (GAG) composition in two mouse lines that produce either reduced levels of HS (Ext1^gt/gt mice) or HS lacking 2-O-sulfation (Hs2st1^-/- mice). Analysis by RPIP-HPLC revealed an increased level of sulfated disaccarides not affected by the mutation in both mouse lines indicating that chondrocytes attempt to restore a critical level of sulfation. In addition, in both mutant lines we also detected significantly elevated levels of CS. Size exclusion chromatography further demonstrated that Ext1^gt/gt mutants produce more but shorter CS chains, while the CS chains produced by (Hs2st1^-/- mice) mutants are of similar length to that of wild type littermates indicating that chondrocytes produce more rather than longer CS chains. Expression analysis revealed an upregulation of aggrecan, which likely carries most of the additionally produced CS. Together the results of this study demonstrate for the first time that not only a reduced HS synthesis but also an altered HS structure leads to increased levels of CS in mammalian tissues. Furthermore, as chondrocytes produce 100-fold more CS than HS the increased CS levels point to an active, precursor-independent mechanism that senses the quality of HS in a vast excess of CS. Interestingly, reducing the level of cell surface CS by chondroitinase treatment leads to reduced Bmp2 induced Smad1/5/9 phosphorylation. In addition, Erk phosphorylation is increased independent of Fgf18 treatment indicating that both, HS and CS, affect growth factor signaling in chondrocytes in distinct manners.