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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

Activation of autophagy following exposure to CoPs in murine calvaria resorption models and osteoblast cells. (A) Immunofluorescence was performed to examine the expression of LC3 in osteoblasts. Sections of murine calvaria are presented for animals from each group. Scale bar: 50 µm. Red, LC3; green, osteoblasts (BGLAP); blue, DAPI nuclear staining. (B) Immunofluorescence was performed to examine the expression of LC3 in osteoblast cells. Cells were treated with 50 µg/ml CoPs for 12 h. Scale bar: 10 µm. Red, LC3; blue, DAPI nuclear staining. (C) Quantification of the percentage of cells with LC3 puncta shown in (B). Data are presented as the means ± S.E.M. from 3 independent experiments. **, P < 0.01 vs. control. (D) Western blots performed after osteoblast cells were treated with various concentrations (0, 10, 50, 100 µg/ml) of CoPs for 12 h. Rap, rapamycin. (E) Western blots performed after osteoblast cells were cultured in CQ for 3 h prior to being treated with 50 µg/ml CoPs for 12 h. (F) Transmission electron microscopy of osteoblasts cultured with or without 10 mM 3-MA prior to being treated with 50 µg/ml CoPs for 12 h. The double-membrane autophagosome (AP) containing CoPs clusters and the rough endoplasmic reticulum are shown in the middle and right images of the upper panel. High-magnification view of an autolysosome containing CoPs clusters, partially degraded ribosomes and rough endoplasmic reticulum in the middle image of the lower panel. AVd, degradative autophagic vacuole.
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f0001: Activation of autophagy following exposure to CoPs in murine calvaria resorption models and osteoblast cells. (A) Immunofluorescence was performed to examine the expression of LC3 in osteoblasts. Sections of murine calvaria are presented for animals from each group. Scale bar: 50 µm. Red, LC3; green, osteoblasts (BGLAP); blue, DAPI nuclear staining. (B) Immunofluorescence was performed to examine the expression of LC3 in osteoblast cells. Cells were treated with 50 µg/ml CoPs for 12 h. Scale bar: 10 µm. Red, LC3; blue, DAPI nuclear staining. (C) Quantification of the percentage of cells with LC3 puncta shown in (B). Data are presented as the means ± S.E.M. from 3 independent experiments. **, P < 0.01 vs. control. (D) Western blots performed after osteoblast cells were treated with various concentrations (0, 10, 50, 100 µg/ml) of CoPs for 12 h. Rap, rapamycin. (E) Western blots performed after osteoblast cells were cultured in CQ for 3 h prior to being treated with 50 µg/ml CoPs for 12 h. (F) Transmission electron microscopy of osteoblasts cultured with or without 10 mM 3-MA prior to being treated with 50 µg/ml CoPs for 12 h. The double-membrane autophagosome (AP) containing CoPs clusters and the rough endoplasmic reticulum are shown in the middle and right images of the upper panel. High-magnification view of an autolysosome containing CoPs clusters, partially degraded ribosomes and rough endoplasmic reticulum in the middle image of the lower panel. AVd, degradative autophagic vacuole.

Mentions: Whether autophagy is involved in wear particle-induced osteolysis is unknown. In the present study, we performed immunofluorescence analysis of MAP1LC3/LC3 (microtubule-associated protein 1 light chain 3), a marker of autophagy,23-25 in sections of CoPs-stimulated calvarias obtained from a commonly used murine calvaria resorption model. As shown in Figure 1A, the level of LC3 was significantly increased in osteoblasts compared with that of the control. We further examined the rise of autophagy in osteoblasts in vitro. Confocal microscopy revealed that CoPs can induce the accumulation of LC3-positive puncta (Fig. 1B and C). For western blot analysis, we used LC3-II/GAPDH ratios as indicators of autophagy instead of LC3-II/LC3-I or LC3-II/ (LC3-I+ LC3-II) because LC3-II tends to be more sensitive than LC3-I in immunoblotting.23 We observed a marked increase in LC3-II induced by CoPs when compared with the control (Fig. 1D). To further interpret the autophagy induction by CoPs, we treated cells with CQ (chloroquine), a lysosomal degradation inhibitor, and observed that CoPs and chloroquine cotreatment resulted in significantly elevated levels of LC3-II relative to treatment with either CoPs or chloroquine alone (Fig. 1E). The accumulation of autophagosomes was also confirmed using electron microscopy. As presented in Figure 1F, under normal conditions, autophagosomes were rarely detected, whereas their levels were elevated upon CoPs treatment. The autophagy inhibitor 3-MA decreased the accumulation of autophagosomes. These results suggested that osteoblasts underwent autophagy in response to CoPs.Figure 1.


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)

Activation of autophagy following exposure to CoPs in murine calvaria resorption models and osteoblast cells. (A) Immunofluorescence was performed to examine the expression of LC3 in osteoblasts. Sections of murine calvaria are presented for animals from each group. Scale bar: 50 µm. Red, LC3; green, osteoblasts (BGLAP); blue, DAPI nuclear staining. (B) Immunofluorescence was performed to examine the expression of LC3 in osteoblast cells. Cells were treated with 50 µg/ml CoPs for 12 h. Scale bar: 10 µm. Red, LC3; blue, DAPI nuclear staining. (C) Quantification of the percentage of cells with LC3 puncta shown in (B). Data are presented as the means ± S.E.M. from 3 independent experiments. **, P < 0.01 vs. control. (D) Western blots performed after osteoblast cells were treated with various concentrations (0, 10, 50, 100 µg/ml) of CoPs for 12 h. Rap, rapamycin. (E) Western blots performed after osteoblast cells were cultured in CQ for 3 h prior to being treated with 50 µg/ml CoPs for 12 h. (F) Transmission electron microscopy of osteoblasts cultured with or without 10 mM 3-MA prior to being treated with 50 µg/ml CoPs for 12 h. The double-membrane autophagosome (AP) containing CoPs clusters and the rough endoplasmic reticulum are shown in the middle and right images of the upper panel. High-magnification view of an autolysosome containing CoPs clusters, partially degraded ribosomes and rough endoplasmic reticulum in the middle image of the lower panel. AVd, degradative autophagic vacuole.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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f0001: Activation of autophagy following exposure to CoPs in murine calvaria resorption models and osteoblast cells. (A) Immunofluorescence was performed to examine the expression of LC3 in osteoblasts. Sections of murine calvaria are presented for animals from each group. Scale bar: 50 µm. Red, LC3; green, osteoblasts (BGLAP); blue, DAPI nuclear staining. (B) Immunofluorescence was performed to examine the expression of LC3 in osteoblast cells. Cells were treated with 50 µg/ml CoPs for 12 h. Scale bar: 10 µm. Red, LC3; blue, DAPI nuclear staining. (C) Quantification of the percentage of cells with LC3 puncta shown in (B). Data are presented as the means ± S.E.M. from 3 independent experiments. **, P < 0.01 vs. control. (D) Western blots performed after osteoblast cells were treated with various concentrations (0, 10, 50, 100 µg/ml) of CoPs for 12 h. Rap, rapamycin. (E) Western blots performed after osteoblast cells were cultured in CQ for 3 h prior to being treated with 50 µg/ml CoPs for 12 h. (F) Transmission electron microscopy of osteoblasts cultured with or without 10 mM 3-MA prior to being treated with 50 µg/ml CoPs for 12 h. The double-membrane autophagosome (AP) containing CoPs clusters and the rough endoplasmic reticulum are shown in the middle and right images of the upper panel. High-magnification view of an autolysosome containing CoPs clusters, partially degraded ribosomes and rough endoplasmic reticulum in the middle image of the lower panel. AVd, degradative autophagic vacuole.
Mentions: Whether autophagy is involved in wear particle-induced osteolysis is unknown. In the present study, we performed immunofluorescence analysis of MAP1LC3/LC3 (microtubule-associated protein 1 light chain 3), a marker of autophagy,23-25 in sections of CoPs-stimulated calvarias obtained from a commonly used murine calvaria resorption model. As shown in Figure 1A, the level of LC3 was significantly increased in osteoblasts compared with that of the control. We further examined the rise of autophagy in osteoblasts in vitro. Confocal microscopy revealed that CoPs can induce the accumulation of LC3-positive puncta (Fig. 1B and C). For western blot analysis, we used LC3-II/GAPDH ratios as indicators of autophagy instead of LC3-II/LC3-I or LC3-II/ (LC3-I+ LC3-II) because LC3-II tends to be more sensitive than LC3-I in immunoblotting.23 We observed a marked increase in LC3-II induced by CoPs when compared with the control (Fig. 1D). To further interpret the autophagy induction by CoPs, we treated cells with CQ (chloroquine), a lysosomal degradation inhibitor, and observed that CoPs and chloroquine cotreatment resulted in significantly elevated levels of LC3-II relative to treatment with either CoPs or chloroquine alone (Fig. 1E). The accumulation of autophagosomes was also confirmed using electron microscopy. As presented in Figure 1F, under normal conditions, autophagosomes were rarely detected, whereas their levels were elevated upon CoPs treatment. The autophagy inhibitor 3-MA decreased the accumulation of autophagosomes. These results suggested that osteoblasts underwent autophagy in response to CoPs.Figure 1.

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