Targeting DKK1 prevents development of alcohol-induced osteonecrosis of the femoral head in rats

Abstract

Osteonecrosis of the femoral head (ONFH) is a devastating bone disease characterized by avascular or aseptic necrosis of the femoral head, and alcohol consumption is reported one of the leading risks to this disease. Previous studies have linked Dickkopf-1 (DKK1) to the occurrence of ONFH, but the role of DKK1 in alcohol-induced ONFH (AONFH) has not been fully discussed. In this study, we found that the expression level of DKK1 was dramatically increased in serum and bone samples from AONFH patients, experimental AONFH rats, and cultured bone marrow mesenchymal stem cells (BMMSCs) with ethanol stimulation. Elevated DKK1 inhibited Wnt/β-catenin signaling in vivo and in vitro, while knockdown of DKK1 enhanced the nuclear translocation of β-catenin and promoted osteogenesis and inhibited adipogenesis in the BMMSC cell line C3H10T1/2. Local injection of DKK1 knockout lentivirus into the femoral head of rats alleviated the progression of AONFH, with activated Wnt/β-catenin signaling, increased bone formation, reduced number of empty adipose lacunae and restored blood supply. In conclusion, our findings confirmed the important role of DKK1 and canonical Wnt/β-catenin pathway in AONFH. We propose that DKK1 may be a prognostic marker of AONFH and targeting DKK1 to activate the canonical Wnt/β-catenin pathway and restore osteogenic potential could be a promising therapeutic strategy to prevent AONFH.

Keywords: Alcohol-induced ONFH; BMMSCs; Dickkopf-1; Wnt/β-catenin signaling; rats.

Figure 1

DKK-1 expression is increased in serum and bone samples of patients with AONFH. A. ELISA results of DKK-1 expression in serum of patients with AONFH (n = 35) and normal controls (n = 30). B. Representative photographs and quantification of DKK1 expression in bone specimens from patients with AONFH and normal controls; n = 5 for each group, scale bar = 50 μm, **P < 0.01, ***P < 0.001 by Student’s t test. All data are shown as means ± SD.

Figure 2

DKK-1 accumulates in osteoblasts of bone samples from an AONFH rat model. A. Micro-CT scanning images of femoral heads from AONFH and control rats; n = 5 for each group. B. Quantification of the value of BV/TV and BMD in both groups; n = 5 for each group. C. H&E staining of the femoral head in AONFH and control rats; White arrows indicate necrotic bone marrow cells and black arrows indicate empty lacunas. n = 5 for each group, scale bar = 50 μm. D. Images and quantification of TUNEL staining in the femoral heads from both groups. n = 5 for each group. E. IHC staining of DKK-1 and quantification of DKK-1-positive cells in the femoral heads from both groups. n = 5 for each group. F. Co-staining of OCN (green) and DKK-1 (red) by IF and quantification of positive cells in the femoral head from both groups; n = 5 for each group, scale bar = 50 μm. **P < 0.01, ***P < 0.001 by Student’s t test. All data are shown as means ± SD.

Figure 3

Ethanol treatment elevates DKK-1 expression and promotes adipogenesis in bone marrow mesenchymal stem cells. A. Images of Oil red O staining of cultured BMMSCs stimulated with different concentrations of ethanol (0, 10, 50, 100 mmol/L) for 3 days. B. Western blot results of DKK-1, β-catenin, PPARγ and CEBPα in BMMSCs stimulated with 50 mmol/L ethanol for different time periods (0, 1, 3, 5, and 7 days). β-actin was used as an internal control. The experiment was repeated three different times.

Figure 4

Ethanol treatment increases DKK-1 expression and inhibits Wnt/β-catenin signaling in vitro and in vivo. A. Co-staining of DKK-1 (red) and β-catenin (green) by IF in BMMSCs stimulated with 50 mmol/L ethanol or control (saline). Expression of β-catenin in the nuclei was quantified. The experiment was repeated three different times. Scale bar = 20 μm. B. Co-staining of DKK-1 (red) and β-catenin (green) by IF in the femoral head from AONFH and control rats; n = 5 for each group, scale bar = 50 μm. *P <0.05, ***P < 0.001 by Student’s t test. All data are shown as means ± SD.

Figure 5

Knockdown of DKK-1 promotes osteogenesis and inhibits adipogenesis in bone marrow mesenchymal stem cells via activation of Wnt/β-catenin signaling. A. Western blot results of DKK-1, β-catenin, OCN, Runx2 and Osterix in C3H10T1/2 cells transfected with negative control or DKK-1 siRNAs, and cultured in osteogenic induction medium for 3 days. B. Alizarin red staining of C3H10T1/2 cells transfected with negative control or DKK-1 siRNA, and cultured in osteogenic induction medium for 14 days. C. Western blot results of DKK-1, PPARγ, CEBPα and aP2 in C3H10T1/2 cells transfected with negative control or DKK-1 siRNAs, and cultured in adipogenic induction medium for 3 days. D. Oil red O staining of C3H10T1/2 cells transfected with negative control or DKK-1 siRNAs, and cultured in adipogenic induction medium for 14 days. All experiments were repeated three different times.

Figure 6

Knockout of DKK-1 with lentivirus injection alleviates AONFH in rats. A. Micro-CT scans of the femoral heads from AONFH and AONFH with DKK-1-KO rats; n = 5 for each group. B. Quantification of the value of bone volume/tissue volume (BV/TV) and bone mineral density (BMD) in both groups; n = 5 for each group. C. H&E staining of the femoral head in AONFH and AONFH with DKK-1-KO rats; n = 5 for each group, scale bar = 50 μm. D. Images and quantification of TUNEL staining in the femoral heads from both groups. n = 5 for each group. E. Representative IF staining photographs and quantification of DKK-1 and β-catenin in femoral heads from AONFH and AONFH with DKK-1-KO rats; n = 5 for each group. F. Representative IF staining photographs and quantification of OCN and CD31 in femoral heads from AONFH and AONFH with DKK-1-KO rats; n = 5 for each group, scale bar = 50 μm. *P < 0.05, **P < 0.01, ***P < 0.001 by Student’s t test. All data are shown as means ± SD.