Heparan Sulfate Regulates Sclerostin Function in Bone Formation

Objectives: Sclerostin functions by binding to LRP5/6 on the osteoblast lineage cells to antagonize canonical Wnt signaling, thus negatively regulating bone formation. Presumably, after sclerostin is secreted by osteocytes, it reaches the target osteoblasts at the bone surfaces by diffusion. However, to date it remains unknown how secreted sclerostin is regulated on the cell surface and in the extracellular matrix. To address this significant gap in sclerostin biology, we focus our study on heparan sulfate (HS)–sclerostin interactions.
Methods: We employ size exclusion chromatography to investigate the oligomerization of sclersotin induced by HS. Digestion assay and SDS-PAGE were used to study the role of HS in the degradation of sclerostin by trypsin. Based on existing NMR structure of sclerostin, we mapped the HS-binding sites of sclerostin by site-directed mutagenesis. This allowed us to further generate a sclerostin knock-in mouse stain (Sost^AA) by introducing point mutations to key residues that are essential for HS binding using CRISPR-Cas9, which renders the sclerostin variant incapable of binding to HS. MicroCT was used to analyze the phenotype of Sost^AA mice.
Results: Our results showed that HS can induce sclerostin to form a stable dimer, and HS is able to protect sclerostin from proteolysis.
By immunostaining we also found abundant HS expression and HS-sclerostin co-localization at the cell surface of both osteoblasts and osteocytes, and the colocalization is especially strong on the dendritic processes of osteocytes. This finding suggests that HS might be involved in the trafficking of sclerostin from osteocytes to osteoblasts. MicroCT analysis revealed Sost^AA mice have substantially increased trabecular bone volume and cortical thickness, and the phenotype become more severe as the mice age.
Conclusions: Our findings provide clear in vivo evidence that the interaction between HS and sclerostin is essential for maintaining normal bone formation, and HS likely plays a multifaceted role in regulating the biological function of sclerostin in bone homeostasis.