AMP-activated protein kinase stimulates osteoblast differentiation and mineralization through autophagy induction

Previous studies have reported that adenosine monophosphate‑activated protein kinase (AMPK) activation can enhance osteoblast differentiation and mineralization; however, the underlying mechanism is not fully understood. Autophagy also serves an important role in osteoblast mineralization and bone homeostasis. The present study aimed to explore whether activation of AMPK could enhance osteoblast differentiation and mineralization via the induction of Autophagy. The fracture healing and nonunion animal models were established and verified by X-ray imaging. Bone maturation was measured by Masson staining and the expression of AMPK, p-AMPK, microtubule-associated proteins 1A/1B light chain 3B II, and p62 in the fracture ends were detected by immunohistochemical staining. The mRNA expression levels of Alkaline Phosphatase (ALP), osteocalcin ,runt-related transcription factor 2 and BCN1 were determined by reverse transcription-quantitative polymerase chain reaction. 5-Bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium staining was used to determine ALP activity and alizarin red staining was adopted to examine mineralization. Western blot analysis was performed to detect protein expression. Autophagosome was observed by Transmission electron microscopy. Small interfering (si)RNA was used to knock down the expression of target gene. In vivo experiments demonstrated that new bone mineralization and maturation was markedly restrained in the nonunion group, alongside decreased AMPK activation and autophagic activity, compared with in the fracture healing group. The results of an in vitro study indicated that AMPK activation stimulated the osteogenic differentiation of MC3T3‑E1 cells, with increases in ALP activity, mineralization, and the mRNA expression levels of ALP, osteocalcin and runt-related transcription factor 2. Furthermore, AMPK activation induced Autophagy, as determined by upregulation of microtubule‑associated proteins 1A/1B light chain 3B, increased autophagosome density and downregulation of p62. In addition, inhibition of Autophagy reversed the effects of AMPK activation on osteoblast differentiation. These results suggested that AMPK activation may stimulate osteoblast differentiation and mineralization via the induction of Autophagy, and provides evidence to suggest that enhancing AMPK activation and autophagic activity may be a potential novel approach to promote fracture healing.