Abalone-derived dermatopontin and its RFK-containing peptide promote osteogenesis via TGF-β/Smad signaling pathway

Bone regeneration depends on the coordinated interplay of cellular differentiation and extracellular matrix (ECM) organization. Dermatopontin (DPT), a non-collagenous ECM protein, is essential for collagen fibrillogenesis and cell-matrix interactions, particularly during wound healing. Bioinformatic analysis of DPT from the marine mollusk Haliotis diversicolor reveals a conserved RFK motif known to activate TGF-β signaling, suggesting that it may also be involved in osteogenesis, a process that requires collagen deposition in the ECM. This study examined the osteogenic potential of recombinant DPT (rDPT) and a synthetic short peptide containing the RFK motif (1RFK-DPT) in MC3T3-E1 pre-osteoblasts. Both rDPT and 1RFK-DPT demonstrated non-cytotoxicity in MC3T3-E1 cells and upregulated the expression of key osteogenic marker genes, including RUNX2, OSX, ALP, OPN, COL1A1, and OCN. Translationally, rDPT increased COL1A1 protein expression and its deposition, as demonstrated by Western blotting and immunofluorescence analysis, as well as enhanced ECM mineralization, as shown by Alizarin Red S staining. In silico molecular docking supported a potential interaction between the RFK motif and the latent TGF-β complex. Additionally, rDPT rapidly induced Smad2 phosphorylation within 15 min of treatment, which was effectively blocked by SB-431542, a specific inhibitor of the TGF-β type I receptor (ALK5), indicating that DPT acts through the canonical TGF-β/Smad signaling pathway. In summary, both full-length rDPT and 1RFK-DPT peptides enhance osteogenesis by promoting osteoblast differentiation and matrix mineralization via TGF-β/Smad signaling activation. These results expand the list of marine-derived bioactive compounds with a potential for bone regenerative medicine.