Inhibition of HIF-1α restrains fracture healing via regulation of autophagy in a rat model

Abstract

It has been demonstrated that bone fracture is associated with the activation of autophagy, and upregulation of autophagy could promote fracture healing. Previous study by our group demonstrated that activating the HIF-1α pathway via administration of cobalt (II) chloride (CoCl₂) could promote fracture healing in vivo. However, the role of hypoxia-inducible factor-1α (HIF-1α) in autophagy remains unknown. In the current study, rats were divided into two groups following tibial fracture and treated with echinomycin or dimethyl sulfoxide (DMSO). Rats were sacrificed at 7, 14, 28 and 42 days after fracture. The evaluation of fracture healing was performed by micro-computed tomography. In addition, the effects of echinomycin on microtubule-associated protein 1 light chain 3 (LC3 II), runt-related transcription factor 2 (Runx2), alkaline phosphatase (ALP), Unc-51-like autophagy activating kinase 1 (ULK1) and P62 were detected at the mRNA and protein levels by reverse transcription-quantitative polymerase chain reaction, western blotting and immunohistochemistry. The results demonstrated that the expression of LC3 II was markedly decreased following systemic administration of echinomycin (0.05 mg/kg every other day for 42 days, intraperitoneally). Furthermore, the levels of Runx2, ALP and ULK1 were decreased, while those of P62 were increased, at the mRNA and protein levels in rats treated with echinomycin in vivo. In summary, the current study suggested that HIF-1α may serve an important role in fracture healing via the downregulation of autophagy.

Keywords: autophagy; echinomycin; fracture; hypoxia-inducible factor-1α.

Figure 1.

Micro-computed tomography images of the fracture callus at days 28 and 42 after fracture. Representative images of each group at (A) day 28 and (B) day 42 are presented. DMSO, dimethyl sulfoxide.

Figure 2.

mRNA levels of (A) ALP, (B) Runx2, (C) LC3 II, (D) ULK1, (E) P62 and (F) HIF-1α at the indicated time points. *P<0.05, **P<0.01 vs. DMSO-treated group. ALP, alkaline phosphatase; Runx2, runt-related transcription factor 2; LC3 II, microtubule-associated protein 1 light chain 3; ULK1, Unc-51 like autophagy activating kinase 1; HIF-1α, hypoxia-inducible factor-1α; DMSO, dimethyl sulfoxide.

Figure 3.

Representative images of immunohistochemical staining of Runx2, ALP and LC3 II in bone tissues at day 42 after fracture (magnification, ×400). ALP, alkaline phosphatase; Runx2, runt-related transcription factor 2; LC3 II, microtubule-associated protein 1 light chain 3; DMSO, dimethyl sulfoxide.

Figure 4.

Western blot analysis of HIF-1α, ULK1, P62, LC3 II, Runx2 and ALP at the indicated time points after fracture. HIF-1α, hypoxia-inducible factor-1α; ULK1, Unc-51-like autophagy activating kinase 1; LC3 II, microtubule-associated protein 1 light chain 3; Runx2, runt-related transcription factor 2; ALP, alkaline phosphatase; DMSO, dimethyl sulfoxide.

Figure 5.

Quantitative analysis of HIF-1α, ULK1, P62, LC3 II, Runx2 and ALP protein expression at the indicated time points after fracture. *P<0.01 vs. DMSO-treated group. HIF-1α, hypoxia-inducible factor-1α; ULK1, Unc-51-like autophagy activating kinase 1; LC3 II, microtubule-associated protein 1 light chain 3; Runx2, runt-related transcription factor 2; ALP, alkaline phosphatase; DMSO, dimethyl sulfoxide.