Limits...
Autophagy mediated CoCrMo particle-induced peri-implant osteolysis by promoting osteoblast apoptosis.

Wang Z, Liu N, Liu K, Zhou G, Gan J, Wang Z, Shi T, He W, Wang L, Guo T, Bao N, Wang R, Huang Z, Chen J, Dong L, Zhao J, Zhang J - Autophagy (2015)

Bottom Line: Both autophagy inhibitor 3-MA (3-methyladenine) and siRNA of Atg5 could dramatically reduce CoPs-induced apoptosis in osteoblasts.Further, inhibition of autophagy with 3-MA ameliorated the severity of osteolysis in PIO animal models.Moreover, 3-MA also prevented osteoblast apoptosis in an antiautophagic way when tested in PIO model.

View Article: PubMed Central - PubMed

Affiliation: a Jinling Hospital; Department of Orthopaedics; State Key Laboratory of Pharmaceutical Biotechnology; Nanjing University ; Nanjing , China.

ABSTRACT
Wear particle-induced osteolysis is the leading cause of aseptic loosening, which is the most common reason for THA (total hip arthroplasty) failure and revision surgery. Although existing studies suggest that osteoblast apoptosis induced by wear debris is involved in aseptic loosening, the underlying mechanism linking wear particles to osteoblast apoptosis remains almost totally unknown. In the present study, we investigated the effect of autophagy on osteoblast apoptosis induced by CoCrMo metal particles (CoPs) in vitro and in a calvarial resorption animal model. Our study demonstrated that CoPs stimulated autophagy in osteoblasts and PIO (particle-induced osteolysis) animal models. Both autophagy inhibitor 3-MA (3-methyladenine) and siRNA of Atg5 could dramatically reduce CoPs-induced apoptosis in osteoblasts. Further, inhibition of autophagy with 3-MA ameliorated the severity of osteolysis in PIO animal models. Moreover, 3-MA also prevented osteoblast apoptosis in an antiautophagic way when tested in PIO model. Collectively, these results suggest that autophagy plays a key role in CoPs-induced osteolysis and that targeting autophagy-related pathways may represent a potential therapeutic approach for treating particle-induced peri-implant osteolysis.

No MeSH data available.


Related in: MedlinePlus

Autophagy mediated the increase in osteoblast apoptosis induced by CoPs. (A) Flow cytometry analysis of ANXA5 and propidium iodide staining of osteoblast cells cultured with various concentrations (0, 10, 50, 100, 200 µg/ml) of CoPs for 24 h. (B) Quantification analysis of apoptotic cells in (A) (both upper- and lower-right quadrants in representative dot plots as shown). Data are represented as means ± S.D. from 3 independent experiments. *, P < 0.05 and **, P < 0.01 vs. the control. (C) Western blots performed after osteoblast cells were cultured with various concentrations (0, 10, 50, 100, 200 µg/ml) of CoPs for 24 h. (D) Flow cytometry analysis of ANXA5 and propidium iodide staining of osteoblast cells cultured with various concentrations (0, 1, 10 mM) of 3-MA, siCtrl and siAtg5 prior to being treated with 50 µg/ml of CoPs for 24 h. (E, G) Quantification analysis of apoptotic cells in (D) (both upper- and lower-right quadrants in representative dot plots as shown). Data are presented as means ± S.D. from 3 independent experiments. *, P < 0.05 and **, P < 0.01 vs. control; ##, P < 0.01 vs. CoPs group. (F) Western blots performed after osteoblast cells were cultured in siCtrl or siAtg5 prior to treatment with CoPs (50 µg/ml) for 24 h. c-CASP3, cleaved CASP3; siCtrl, siControl.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4835204&req=5

f0003: Autophagy mediated the increase in osteoblast apoptosis induced by CoPs. (A) Flow cytometry analysis of ANXA5 and propidium iodide staining of osteoblast cells cultured with various concentrations (0, 10, 50, 100, 200 µg/ml) of CoPs for 24 h. (B) Quantification analysis of apoptotic cells in (A) (both upper- and lower-right quadrants in representative dot plots as shown). Data are represented as means ± S.D. from 3 independent experiments. *, P < 0.05 and **, P < 0.01 vs. the control. (C) Western blots performed after osteoblast cells were cultured with various concentrations (0, 10, 50, 100, 200 µg/ml) of CoPs for 24 h. (D) Flow cytometry analysis of ANXA5 and propidium iodide staining of osteoblast cells cultured with various concentrations (0, 1, 10 mM) of 3-MA, siCtrl and siAtg5 prior to being treated with 50 µg/ml of CoPs for 24 h. (E, G) Quantification analysis of apoptotic cells in (D) (both upper- and lower-right quadrants in representative dot plots as shown). Data are presented as means ± S.D. from 3 independent experiments. *, P < 0.05 and **, P < 0.01 vs. control; ##, P < 0.01 vs. CoPs group. (F) Western blots performed after osteoblast cells were cultured in siCtrl or siAtg5 prior to treatment with CoPs (50 µg/ml) for 24 h. c-CASP3, cleaved CASP3; siCtrl, siControl.

Mentions: The upregulation of osteoblast death reduces bone formation in the periprosthetic region, which is an etiology for wear particles-induced osteolysis.5,6 To study the role of autophagy in CoPs-treated cells, we examined the cell viability of osteoblasts treated with various concentration of CoPs. CoPs treatment sharply decreased the cell viability (Fig. S2A). The autophagy inhibitor 3-MA could rescue the cell viability in a dose-dependent manner (Fig. S2B). Although 3-MA slightly decreased cell viability, there was no significant effect on cell viability at the dose we used (Fig. S2C). As senescence may be involved in aseptic loosening, we measured cell senescence with an SA-GLB1/SA-β-gal (senescence-associated β-galactosidase staining) staining kit.31 The senescence of the cells was not significantly changed among the various CoPs treatment groups (Fig. S3A to D). To determine whether osteoblast apoptosis is involved in the toxicity of CoPs, we examined the effect of CoPs treatment on osteoblast apoptosis. Figures 3A and B illustrated that CoPs could increase the apoptotic rate of cells in a dose-dependent manner. The apoptotic rates in cells treated with 100 μg/ml CoPs and control cells were 57% and 9%, respectively. CASP3/caspase 3, a hallmark of apoptosis, was also assessed by western blot, and a dose-dependent increase in cleaved CASP3 was observed after the exposure of cells to increasing concentrations of CoPs (Fig. 3C). We then investigated the effect of autophagy on CoPs-induced apoptosis. The elevation of the apoptotic rate was significantly ameliorated by treatment with 3-MA (Fig. 3D and E). To further investigate whether upregulation of particle-induced apoptosis was due to autophagy, siRNA was used to knock down the expression of ATG5 (Fig. 3F), a member of the autophagy-related (ATG) protein family that is required for the formation of autophagosomes. The apoptotic rate was significantly decreased in the groups treated with siAtg5 (Fig. 3D and G). Thus, autophagy played a critical role in CoPs-induced osteoblast apoptosis.Figure 3.


Autophagy mediated CoCrMo particle-induced peri-implant osteolysis by promoting osteoblast apoptosis.

Wang Z, Liu N, Liu K, Zhou G, Gan J, Wang Z, Shi T, He W, Wang L, Guo T, Bao N, Wang R, Huang Z, Chen J, Dong L, Zhao J, Zhang J - Autophagy (2015)

Autophagy mediated the increase in osteoblast apoptosis induced by CoPs. (A) Flow cytometry analysis of ANXA5 and propidium iodide staining of osteoblast cells cultured with various concentrations (0, 10, 50, 100, 200 µg/ml) of CoPs for 24 h. (B) Quantification analysis of apoptotic cells in (A) (both upper- and lower-right quadrants in representative dot plots as shown). Data are represented as means ± S.D. from 3 independent experiments. *, P < 0.05 and **, P < 0.01 vs. the control. (C) Western blots performed after osteoblast cells were cultured with various concentrations (0, 10, 50, 100, 200 µg/ml) of CoPs for 24 h. (D) Flow cytometry analysis of ANXA5 and propidium iodide staining of osteoblast cells cultured with various concentrations (0, 1, 10 mM) of 3-MA, siCtrl and siAtg5 prior to being treated with 50 µg/ml of CoPs for 24 h. (E, G) Quantification analysis of apoptotic cells in (D) (both upper- and lower-right quadrants in representative dot plots as shown). Data are presented as means ± S.D. from 3 independent experiments. *, P < 0.05 and **, P < 0.01 vs. control; ##, P < 0.01 vs. CoPs group. (F) Western blots performed after osteoblast cells were cultured in siCtrl or siAtg5 prior to treatment with CoPs (50 µg/ml) for 24 h. c-CASP3, cleaved CASP3; siCtrl, siControl.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4835204&req=5

f0003: Autophagy mediated the increase in osteoblast apoptosis induced by CoPs. (A) Flow cytometry analysis of ANXA5 and propidium iodide staining of osteoblast cells cultured with various concentrations (0, 10, 50, 100, 200 µg/ml) of CoPs for 24 h. (B) Quantification analysis of apoptotic cells in (A) (both upper- and lower-right quadrants in representative dot plots as shown). Data are represented as means ± S.D. from 3 independent experiments. *, P < 0.05 and **, P < 0.01 vs. the control. (C) Western blots performed after osteoblast cells were cultured with various concentrations (0, 10, 50, 100, 200 µg/ml) of CoPs for 24 h. (D) Flow cytometry analysis of ANXA5 and propidium iodide staining of osteoblast cells cultured with various concentrations (0, 1, 10 mM) of 3-MA, siCtrl and siAtg5 prior to being treated with 50 µg/ml of CoPs for 24 h. (E, G) Quantification analysis of apoptotic cells in (D) (both upper- and lower-right quadrants in representative dot plots as shown). Data are presented as means ± S.D. from 3 independent experiments. *, P < 0.05 and **, P < 0.01 vs. control; ##, P < 0.01 vs. CoPs group. (F) Western blots performed after osteoblast cells were cultured in siCtrl or siAtg5 prior to treatment with CoPs (50 µg/ml) for 24 h. c-CASP3, cleaved CASP3; siCtrl, siControl.
Mentions: The upregulation of osteoblast death reduces bone formation in the periprosthetic region, which is an etiology for wear particles-induced osteolysis.5,6 To study the role of autophagy in CoPs-treated cells, we examined the cell viability of osteoblasts treated with various concentration of CoPs. CoPs treatment sharply decreased the cell viability (Fig. S2A). The autophagy inhibitor 3-MA could rescue the cell viability in a dose-dependent manner (Fig. S2B). Although 3-MA slightly decreased cell viability, there was no significant effect on cell viability at the dose we used (Fig. S2C). As senescence may be involved in aseptic loosening, we measured cell senescence with an SA-GLB1/SA-β-gal (senescence-associated β-galactosidase staining) staining kit.31 The senescence of the cells was not significantly changed among the various CoPs treatment groups (Fig. S3A to D). To determine whether osteoblast apoptosis is involved in the toxicity of CoPs, we examined the effect of CoPs treatment on osteoblast apoptosis. Figures 3A and B illustrated that CoPs could increase the apoptotic rate of cells in a dose-dependent manner. The apoptotic rates in cells treated with 100 μg/ml CoPs and control cells were 57% and 9%, respectively. CASP3/caspase 3, a hallmark of apoptosis, was also assessed by western blot, and a dose-dependent increase in cleaved CASP3 was observed after the exposure of cells to increasing concentrations of CoPs (Fig. 3C). We then investigated the effect of autophagy on CoPs-induced apoptosis. The elevation of the apoptotic rate was significantly ameliorated by treatment with 3-MA (Fig. 3D and E). To further investigate whether upregulation of particle-induced apoptosis was due to autophagy, siRNA was used to knock down the expression of ATG5 (Fig. 3F), a member of the autophagy-related (ATG) protein family that is required for the formation of autophagosomes. The apoptotic rate was significantly decreased in the groups treated with siAtg5 (Fig. 3D and G). Thus, autophagy played a critical role in CoPs-induced osteoblast apoptosis.Figure 3.

Bottom Line: Both autophagy inhibitor 3-MA (3-methyladenine) and siRNA of Atg5 could dramatically reduce CoPs-induced apoptosis in osteoblasts.Further, inhibition of autophagy with 3-MA ameliorated the severity of osteolysis in PIO animal models.Moreover, 3-MA also prevented osteoblast apoptosis in an antiautophagic way when tested in PIO model.

View Article: PubMed Central - PubMed

Affiliation: a Jinling Hospital; Department of Orthopaedics; State Key Laboratory of Pharmaceutical Biotechnology; Nanjing University ; Nanjing , China.

ABSTRACT
Wear particle-induced osteolysis is the leading cause of aseptic loosening, which is the most common reason for THA (total hip arthroplasty) failure and revision surgery. Although existing studies suggest that osteoblast apoptosis induced by wear debris is involved in aseptic loosening, the underlying mechanism linking wear particles to osteoblast apoptosis remains almost totally unknown. In the present study, we investigated the effect of autophagy on osteoblast apoptosis induced by CoCrMo metal particles (CoPs) in vitro and in a calvarial resorption animal model. Our study demonstrated that CoPs stimulated autophagy in osteoblasts and PIO (particle-induced osteolysis) animal models. Both autophagy inhibitor 3-MA (3-methyladenine) and siRNA of Atg5 could dramatically reduce CoPs-induced apoptosis in osteoblasts. Further, inhibition of autophagy with 3-MA ameliorated the severity of osteolysis in PIO animal models. Moreover, 3-MA also prevented osteoblast apoptosis in an antiautophagic way when tested in PIO model. Collectively, these results suggest that autophagy plays a key role in CoPs-induced osteolysis and that targeting autophagy-related pathways may represent a potential therapeutic approach for treating particle-induced peri-implant osteolysis.

No MeSH data available.


Related in: MedlinePlus