Interaction of heparan sulfate and integrin signaling in maintaining the articular cartilage

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Heparan sulfate (HS) carrying proteoglycans are important components of the articular cartilage (AC) matrix. HS chains bind to many secreted proteins, such as signaling factors, cytokines and proteases, thereby regulating their distribution, activity and receptor binding. The specificity of these interactions is determined by the degree of HS sulfation and its pattern.During the first funding period, we could show that mice carrying clones of HS-deficient cells in the AC and mice with altered Ndst1-dependent sulfation show reduced OA progression during aging and after anterior crucial ligament transection (ACLT). We demonstrated reduced Aggrecan degradation by AdamTS and Mmp proteases and found a decreased gelatinase activity of Mmp2 in Ndst1 mutants. Biochemical analysis of the relative composition of HS and chondroitin sulfate (CS) in the ECM of chondrocytes with reduced HS levels (Extgt/gt) or lacking 2-O sulfation (Hs2st1-/-) revealed that an altered HS structure is compensated by increased CS levels resulting in a decreased cartilage stiffness in the growth plate cartilage of Extgt/gt mice (with SP1). To decipher how distinct alterations of the HS structure affect OA, we surgically destabilized the medial meniscus (DMM) in Hs2st1 mutants and in mice lacking HS epimerization (Glce-/-). We did not detect an acceleration of OA, but due to the mild OA induced by DMM we could not assess a possible protective function.We now plan to induce OA by the harsher ACLT method to identify a potential protective effect against OA in Hs2st1 mutants (WP1). To receive comprehensive insight into the relevance of the HS structure on AC homeostasis, we will complete the ongoing systematic analysis of GAG composition (RPIP-HPLC), cartilage stiffness (AFM; SP1) and subchondral bone structure (nCT; SP5) of Ext1, Ndst1 and Hs2st1 mutants (WP4). Furthermore, the initiated transcriptome analyses of the impact of HS on mechanical stress responses (Extgt/gt) will be completed (with SP3; WP3). To address the potential role of HS as a therapeutic target, we plan to inhibit HS function by the small molecule inhibitor Surfen in cartilage explants and in mice after ACLT surgery (WP2). We will analyze cartilage degradation, OA progression, osteocyte numbers (with SP4) and synovial inflammation (with SP6). To further investigate the role of integrin signaling in sensing the altered HS structure, we will treat cells with Surfen and corroborate preliminary findings of altered Actin cytoskeleton, focal adhesions and cell migration (WP5). Epistasis will be evaluated by co-activation or inhibition of integrin signaling, in vitro, and the results will be confirmed in primary HS-deficient cells (Ext1fl/fl;R26RmT/mG). To detect mechanisms that translate the information on ECM composition into gene expression changes, we will investigate signaling pathways affected by an altered cytoskeleton (YAP) or by HS deficiency (Bmp/Smad, Ihh, Wnt).