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

Abstract

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.

Figure 1

New bone mineralization and maturation is restrained in the nonunion group, accompanied by decreased AMPK activation and autophagic activity. (A) X-ray image showing the healing status of radial defects 16 weeks following surgery. (B) Masson staining (magnification, ×200) detected osteoid (blue) and mineralized bone (red) in calluses obtained from normal bone (control) and fracture ends 4, 8, 12 and 16 weeks post-surgery. (C) Immunohistochemical staining of AMPK, p-AMPK and autophagy-associated markers (LC3B and p62) 4 weeks after surgery (magnification, ×400). AMPK, adenosine monophos-phate-activated protein kinase; LC3B, microtubule-associated proteins 1A/1B light chain 3B; p-AMPK, phosphorylated-AMPK.

Figure 2

AMPK activation promotes the differentiation and mineralization of MC3T3-E1 cells. (A and B) Cells were treated with AICAR (A+, 1 μM; ++, 10 μM) and compound C (C+, 0.1 μM; ++, 1 μM) in differentiation medium for 8 days. (A) ALP, osteocalcin and Runx2 mRNA expression were detected by reverse transcription-quantitative polymerase chain reaction. (B) 5-Bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium staining was used to determine ALP activity. (C) Cells were cultured for 21 days; representative images (magnification, ×100) of mineralized nodules stained by Alizarin red staining are presented. (D) Quantification of Alizarin red staining with cetylpyridinium chloride. Absorbance was measured at 550 nm. ^*P<0.05, ^**P<0.01 compared with the control group; ^#P<0.05, ^##P<0.01 compared with the A++ group. AICAR, 5-aminoimdazole-4-carboxamide-1-β-D-ribofuranoside; ALP, alkaline phosphatase; AMPK, adenosine monophosphate-activated protein kinase; Rnx2, runt-related transcription factor 2.

Figure 3

AMPK activation induces autophagy. (A) Cells were treated with AICAR and (B) AICAR (A++, 10 μM) + compound C (C++, 1 μM) for 24 h, after which western blot analysis was performed. (C and D) Semi-quantitative analysis of western blotting was conducted using ImageJ software and density values were normalized to β-actin, with the control group set as 1. (E) Cells were treated with AICAR (10 μM) or AICAR (10 μM) + compound C (1 μM) for 24 h. Representative images of autophagosomes were captured by transmission electron microscopy (scale bar, 0.2 μm). (F) Relative quantification of autophagosome density; values of the control group were set as 1. ^*P<0.05, ^**P<0.01 compared with the control group; ^#P<0.05, ^##P<0.01 compared with the AICAR(A++) group. AICAR, 5-aminoimdazole-4-carboxamide-1-β-D-ribofuranoside; AMPK, adenosine monophosphate-activated protein kinase; LC3B, microtubule-associated proteins 1A/1B light chain 3B; p-AMPK, phosphorylated-AMPK.

Figure 4

AMPK activation enhances differentiation and mineralization of MC3T3-E1 cells via the induction of autophagy. (A) Cells were treated with AICAR (10 μM) in the presence or absence of 3-MA (5 mM) or CQ (10 μM) for 24 h, after which western blot analysis was conducted. (B) Semi-quantitative results of western blot analysis. (C and D) Cells were cotreated with AICAR (10 μM) and 3-MA (5 mM) or CQ (10 μM) for 8 days. (C) 5-Bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium staining was used to detect ALP activity. (D) ALP, OCN and Runx2 mRNA expression was detected using reverse transcription-polymerase chain reaction. (E) Cells were cultured for 21 days; representative images (magnification, ×100) of Alizarin red staining are presented. (F) Quantification of Alizarin red staining. ^*P<0.05, ^**P<0.01 compared with the control group; ^#P<0.05, ^##P<0.01 compared with the AICAR(A++) group. 3-MA, 3-methyladenine; AICAR, 5-aminoimdazole-4-carboxamide-1-β-D-ribofuranoside; ALP, alkaline phosphatase; AMPK, adenosine monophosphate-activated protein kinase; CQ, chloroquine diphosphatase; OCN, osteocalcin; Runx2, runt-related transcription factor 2.

Figure 5

AMPK activation enhances differentiation of MC3T3-E1 cells via the induction of autophagy. (A) Cells were transfected with the indicated siRNAs, and the mRNA expression levels of BCN1 were measured by RT-qPCR 24, 48 and 72 h post-transfection to assess the silencing effect; siBCN1-II was the most efficient siRNA and was used for further study. (B) Cells were transfected with siBCN1-II and siControl, western blot analyis was performed using an antibody against BCN1 24, 48, 72 and 96 h post-transfection, in order to verify the silencing effect. (C) Semi-quantitative results of western blot analysis. (D and E) A total of 24 h post-transfection with siBCN1-II or siControl, differentiation medium was added and cells were treated with or without AICAR (10 μM) for 8 days. (D) 5-Bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium staining was used to detect ALP activity. (E) ALP, OCN and Runx2 mRNA expression was detected by RT-PCR. siControl cells were treated with AICAR. ^*P<0.05, ^**P<0.01 compared with the control group; ^#P<0.05, ^##P<0.01 compared with the siControl group. AICAR, 5-aminoimdazole-4-carboxamide-1-β-D-ribofuranoside; ALP, alkaline phosphatase; AMPK, adenosine monophosphate-activated protein kinase; BCN1, Beclin 1; OCN, osteocalcin; Runx2, runt-related transcription factor 2; si/siRNA, small interfering RNA.