Vitamin C-linker-conjugated tripeptide AHK stimulates BMP-2-induced osteogenic differentiation of mouse myoblast C2C12 cells

Vitamin C-linker-conjugated Ala-His-Lys tripeptide (Vit C-AHK) is a derivative of Vitamin C-conjugated tripeptides, which were originally developed as a component of a product for collagen synthesis enhancement or human dermal fibroblast growth. Here, we investigated the effect of Vit C-AHK on bone morphogenetic protein (BMP)-2-induced osteoblast differentiation in a cell culture model. Vit C-AHK enhanced proliferation of C2C12 cells and induction of BMP-2-induced alkaline phosphatase, a typical marker of osteoblast differentiation. Vit C-AHK also stimulated the phosphorylation and translocation of Smad1/5/8 to the nucleus and phosphorylation of mitogen-activated protein kinases (MAPKs) including ERK1/2 and p38. In addition, Vit C-AHK enhanced the BMP-2-induced mRNA expression of osteoblast differentiation-related genes such as ALP, BMP-2, Osteocakin, and Runx2. Our results suggest that Vit C-AHK exerts an enhancing effect on osteoblast proliferation and differentiation through activation of Smadl/5/8 and MAPK ERK1/2 and p38 signaling and without significant cytotoxicity. These results provide important data for the development of peptide-based bone-regenerative agents and treatment of bone-related disorders.