Pathomechanism of LYPD6 enhancing abnormal hyperostosis via Wnt/β-catenin signaling pathway in diffuse idiopathic skeletal hyperostosis.

Diffuse idiopathic skeletal hyperostosis (DISH) is a noninflammatory metabolic bone disorder marked by progressive ectopic ossification at spinal and peripheral entheses. Its molecular etiology is poorly understood. We investigated whether Ly6/uPAR domain-containing 6 (LYPD6) regulates osteoblast differentiation and drives pathological hyperostosis in DISH. Primary osteoblasts from DISH patients and matched controls were assessed for ALP activity, calcium deposition, and expression of osteogenic markers. RNA sequencing identified LYPD6 as a candidate; its expression was validated by qRT-PCR, Western blot, and immunofluorescence. siRNA-mediated knockdown and lentiviral overexpression of LYPD6 were performed in patient osteoblasts and MC3T3-E1 cells. A global LYPD6 knockout mouse model underwent micro-CT, femoral fracture healing assays, and biomechanical testing. Mechanistic studies evaluated LRP6 phosphorylation, β-catenin stabilization and nuclear translocation, and in silico docking of LYPD6 to LRP6. DISH osteoblasts displayed increased ALP activity, mineralization, and upregulated Runx2, OPN, Col1a1, BSP II, and Osterix. LYPD6 was among the top upregulated genes and confirmed at mRNA and protein levels. LYPD6 knockdown impaired mineralization and osteogenic marker expression, whereas overexpression enhanced them. LYPD6-/- mice exhibited reduced trabecular and cortical bone mass, delayed fracture healing, and weaker biomechanical properties. At the molecular level, LYPD6 promoted LRP6 phosphorylation, stabilized β-catenin, and increased its nuclear localization; docking data predict direct LYPD6-LRP6 binding. Aberrant upregulation of LYPD6 drives pathological hyperostosis in DISH via activation of the canonical Wnt/β-catenin pathway. LYPD6 may serve as both a biomarker for DISH and a pharmacological target. Modulating its activity could inform future therapies: transient inhibition of LYPD6 might prevent or attenuate ectopic ossification in DISH and related hyperostotic disorders, whereas short-term activation could enhance bone repair in osteoporotic or nonunion fracture settings.