Vitamin K2 stimulates MC3T3-E1 osteoblast differentiation and mineralization through autophagy induction

Vitamin K2 likely exerts its protective effects during osteoporosis by promoting osteoblast differentiation and mineralization. However, the precise mechanism remains to be fully elucidated. Autophagy maintains cell homeostasis by breaking down and eliminating damaged proteins and organelles. Increasing evidence in recent years has implicated autophagy in the development of osteoporosis. The aim of the present study was to verify whether vitamin K2 (VK2) can induce autophagy during the differentiation and mineralization of osteoblasts. In the present study, MC3T3-E1 osteoblasts were treated with various doses of VK2 (10(-8)-10(-3) M) for 1-5 days. The results revealed no cytotoxicity at concentrations below 10(-5) M, but cell viability was reduced in a dose-dependent manner at concentrations above 10(-5) M. Furthermore, MC3T3-E1 osteoblasts were seeded in 6-well plates in complete medium supplemented with dexamethasone, -glycerophosphate and vitamin C (VC) for osteogenic differentiation. MC3T3-E1 osteoblasts treated with different concentrations (10(-5), 10(-6) and 10(-7) M) of VK2 for 24 h on days 1, 3, 5 and 7 of the differentiation protocol. It was confirmed that VK2 promoted osteoblast differentiation and mineralization by using alkaline phosphatase (ALP) and alizarin red staining. Using western blotting, immunofluorescence, monodansylcadaverine staining and reverse transcription-quantitative polymerase chain reaction, it was observed that VK2 induced autophagy in osteoblasts. The results revealed that VK2 (1 mu M) significantly increased ALP activity and the conversion of microtubule associated protein 1 light chain 3- (LC3)II to LC3I in MC3T3-E1 osteoblasts (P<0.05) at every time point. The number of fluorescent bodies and the intensity increased with VK2, and decreased following treatment with 3-MA+VK2. There was an increase in the mRNA expression levels of ALP, osteocalcin (OCN) and Runt-related transcription factor 2 in VK2-treated cells (P<0.01). The present study further confirmed the association between autophagy and osteoblast differentiation and mineralization through treatment with an autophagy inhibitor [3-methyladenine (3-MA)]. Osteoblasts treated with 3-MA exhibited significant inhibition of ALP activity and osteogenic differentiation (both P<0.05). In addition, ALP activity and osteogenesis in the VK2+3-MA group was lower compared with VK2-treated cells (P<0.05 for both). The present study confirmed that VK2 stimulated autophagy in MC3T3 cells to promote differentiation and mineralization, which may be a potential therapeutic target for osteoporosis.