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

Abstract 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 , Osteocalcin , and Runx2 . Our results suggest that Vit C-AHK exerts an enhancing effect on osteoblast proliferation and differentiation through activation of Smad1/5/8 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.