Thumpd1 preserves osteogenic TGF-β signaling by promoting USP4-dependent deubiquitination and stabilization of TGF-βRII.

Impaired osteogenic signaling contributes to defective bone formation, but the mechanisms that preserve receptor stability during skeletal development remain incompletely understood. We investigated the role of Thumpd1 in skeletal development and osteogenic differentiation and defined the downstream signaling mechanism. Thumpd1-deficient zebrafish were evaluated for embryonic development, craniofacial cartilage, skeletal mineralization, and bone microarchitecture, while bone marrow mesenchymal stem cells (BMSCs) were used to assess cell-intrinsic osteogenesis. Thumpd1 deficiency caused developmental delay, abnormal craniofacial cartilage morphology, reduced cartilage extracellular-matrix components expression, impaired ossification, and decreased bone mineral density and bone volume. In contrast, osteoclast differentiation and resorptive activity were not substantially increased. Thumpd1-deficient BMSCs showed reduced alkaline phosphatase activity, matrix mineralization, and osteogenic marker expression. Transcriptomic profiling identified ubiquitin-specific protease 4 (USP4) as a prominently downregulated factor. Mechanistically, Thumpd1 loss reduced USP4 expression, enhanced ubiquitination, and destabilized transforming growth factor-β receptor type II (TGF-βRII). TGF-βRII re-expression restored canonical SMAD2 signaling and osteogenic differentiation after USP4 depletion, while tgfb1a rescue partially reversed skeletal defects in vivo. These findings identify Thumpd1 as a regulator of skeletal development and define a Thumpd1-USP4-TGF-βRII proteostatic pathway contributing to impaired osteogenesis.