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Low-dose γ-radiation inhibits IL-1β-induced dedifferentiation and inflammation of articular chondrocytes via blockage of catenin signaling.

Hong EH, Song JY, Lee SJ, Park IC, Um HD, Park JK, Lee KH, Nam SY, Hwang SG - IUBMB Life (2014)

Bottom Line: Here, we found that LDR, at doses of 0.5-2 centiGray (cGy), inhibited interleukin (IL)-1β-induced chondrocyte destruction without causing side effects, such as cell death and senescence.LDR also inhibited chondrocyte destruction through the catenin pathway induced by epidermal growth factor, phorbol 12-myristate 13-acetate, and retinoic acid.Collectively, these results identify the molecular mechanisms by which LDR suppresses pathophysiological processes and establish LDR as a potentially valuable therapeutic tool for patients with cytokine- or soluble factors-mediated cartilage disorders.

View Article: PubMed Central - PubMed

Affiliation: Division of Radiation Cancer Biology, Korea Institute of Radiological & Medical Sciences, Seoul, Korea; Department of Chemistry, College of Natural Sciences, Hanyang University, Seoul, Korea.

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Effect of LDR on IL-1β-induced expression of catenins and catenin-induced chondrocyte destruction. (A, B): Chondrocytes were treated with different doses of LDR for 48 h in the absence (A) or presence (B) of 10 ng/mL IL-1β. Levels of catenin proteins were determined by Western blotting. (C): Chondrocytes were left untreated or treated with 1 cGy radiation in the absence or presence of 10 ng/mL IL-1β for 48 h. Expression of each catenin was detected by confocal fluorescence microscopy (scale bar: 50 μm). (D, E): Chondrocytes were transfected with 3 μg FLAG-tagged S33A β-catenin or wild-type γ-catenin for 24 h and then exposed to different doses of LDR for an additional 48 h. Levels of differentiation- and inflammation-associated proteins were determined by Western blotting (D). Sox-9 (E, left) or NF-κB transcriptional activity (E, right) was determined by reporter gene assay. Data are expressed as means ± SDs (*P < 0.05, **P < 0.005, ***P < 0.0005 compared with cells treated with IL-1β alone) (E). (F): Cells were left untreated or were pretreated with 5 or 10 μM Bay 11-7082 for 1 h prior to treatment with 10 ng/mL IL-1β. After 48 h, the levels of I-κB and COX-2 proteins were determined by Western blotting. (G): Chondrocytes were transiently transfected with 3 μg of GFP-tagged S83A α-catenin (left), FLAG-tagged S33A β-catenin (middle), or wild-type γ-catenin (right), as indicated, for 24 h and then left untreated or treated with 5 or 10 μM Bay 11-7082 for an additional 24 h. The levels of I-κB and COX-2 proteins were determined by Western blotting.
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fig05: Effect of LDR on IL-1β-induced expression of catenins and catenin-induced chondrocyte destruction. (A, B): Chondrocytes were treated with different doses of LDR for 48 h in the absence (A) or presence (B) of 10 ng/mL IL-1β. Levels of catenin proteins were determined by Western blotting. (C): Chondrocytes were left untreated or treated with 1 cGy radiation in the absence or presence of 10 ng/mL IL-1β for 48 h. Expression of each catenin was detected by confocal fluorescence microscopy (scale bar: 50 μm). (D, E): Chondrocytes were transfected with 3 μg FLAG-tagged S33A β-catenin or wild-type γ-catenin for 24 h and then exposed to different doses of LDR for an additional 48 h. Levels of differentiation- and inflammation-associated proteins were determined by Western blotting (D). Sox-9 (E, left) or NF-κB transcriptional activity (E, right) was determined by reporter gene assay. Data are expressed as means ± SDs (*P < 0.05, **P < 0.005, ***P < 0.0005 compared with cells treated with IL-1β alone) (E). (F): Cells were left untreated or were pretreated with 5 or 10 μM Bay 11-7082 for 1 h prior to treatment with 10 ng/mL IL-1β. After 48 h, the levels of I-κB and COX-2 proteins were determined by Western blotting. (G): Chondrocytes were transiently transfected with 3 μg of GFP-tagged S83A α-catenin (left), FLAG-tagged S33A β-catenin (middle), or wild-type γ-catenin (right), as indicated, for 24 h and then left untreated or treated with 5 or 10 μM Bay 11-7082 for an additional 24 h. The levels of I-κB and COX-2 proteins were determined by Western blotting.

Mentions: We next examined whether LDR, at doses of 0–2 cGy, directly modulates the expression of catenin proteins in chondrocytes. Although LDR alone did not alter the expression of catenin proteins (Fig. 5A), both 0.5 and 1 cGy LDR dramatically attenuated IL-1β-induced expression of all catenin proteins (Fig. 5B). Immunofluorescence analyses showed that treatment of chondrocytes with IL-1β significantly increased the levels of all of the tested catenin proteins in both the cytosol and nuclear regions compared to control cells. At a dose of 1 cGy, LDR reduced catenin expression in IL-1β-treated cells to basal levels (Fig. 5C), indicating that LDR inhibits the IL-1β-dependent post-translational stabilization of catenin. Finally, we examined whether LDR reverses the dedifferentiation and inflammatory response induced by overexpression of the above-mentioned α-, β-, and γ-catenin proteins in chondrocytes. Notably, LDR markedly restored the expression of type II collagen and Sox-9 proteins in catenin-overexpressing chondrocytes (Supporting Information Fig. 1B and Fig. 5D), indicating a reduced commitment to dedifferentiation. LDR also inhibited I-κB degradation and COX-2 expression under the same experimental conditions (Supporting Information Fig. 1B and Fig. 5D). Consistent with this, LDR dramatically increased in Sox-9 activity reduced by catenin proteins and decreased in NF-κB activity induced by catenin proteins in chondrocytes; at a dose of 1 cGy, LDR enhanced Sox-9 activity by approximately 2.2-fold, 2.7-fold, and 2.4-fold compared to that in cells transfected with the above-mentioned α-, β-, and γ-catenin constructs, respectively (Supporting Information Fig. 1C and Fig. 5E, left), and reduced NF-κB activity by approximately 74%, 78%, and 75%, respectively (Supporting Information Fig. 1C and Fig. 5E, right), indicating a reduced commitment to inflammation. To further confirm the role of NF-κB signaling in catenin-mediated induction of COX-2 expression, we treated catenin-overexpressing chondrocytes with the NF-κB inhibitor, BAY. Pretreatment with 5 or 10 μM BAY inhibited I-κB degradation in a dose-dependent manner and consistently reduced COX-2 expression compared to IL-1β-treated chondrocytes (Fig. 5F). Degradation of I-κB and induction of COX-2 in cells transfected with the above-mentioned α-, β-, and γ-catenin constructs were also reversed by BAY treatment (Fig. 5G), indicating that NF-κB signaling is downstream of the catenin pathway.


Low-dose γ-radiation inhibits IL-1β-induced dedifferentiation and inflammation of articular chondrocytes via blockage of catenin signaling.

Hong EH, Song JY, Lee SJ, Park IC, Um HD, Park JK, Lee KH, Nam SY, Hwang SG - IUBMB Life (2014)

Effect of LDR on IL-1β-induced expression of catenins and catenin-induced chondrocyte destruction. (A, B): Chondrocytes were treated with different doses of LDR for 48 h in the absence (A) or presence (B) of 10 ng/mL IL-1β. Levels of catenin proteins were determined by Western blotting. (C): Chondrocytes were left untreated or treated with 1 cGy radiation in the absence or presence of 10 ng/mL IL-1β for 48 h. Expression of each catenin was detected by confocal fluorescence microscopy (scale bar: 50 μm). (D, E): Chondrocytes were transfected with 3 μg FLAG-tagged S33A β-catenin or wild-type γ-catenin for 24 h and then exposed to different doses of LDR for an additional 48 h. Levels of differentiation- and inflammation-associated proteins were determined by Western blotting (D). Sox-9 (E, left) or NF-κB transcriptional activity (E, right) was determined by reporter gene assay. Data are expressed as means ± SDs (*P < 0.05, **P < 0.005, ***P < 0.0005 compared with cells treated with IL-1β alone) (E). (F): Cells were left untreated or were pretreated with 5 or 10 μM Bay 11-7082 for 1 h prior to treatment with 10 ng/mL IL-1β. After 48 h, the levels of I-κB and COX-2 proteins were determined by Western blotting. (G): Chondrocytes were transiently transfected with 3 μg of GFP-tagged S83A α-catenin (left), FLAG-tagged S33A β-catenin (middle), or wild-type γ-catenin (right), as indicated, for 24 h and then left untreated or treated with 5 or 10 μM Bay 11-7082 for an additional 24 h. The levels of I-κB and COX-2 proteins were determined by Western blotting.
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fig05: Effect of LDR on IL-1β-induced expression of catenins and catenin-induced chondrocyte destruction. (A, B): Chondrocytes were treated with different doses of LDR for 48 h in the absence (A) or presence (B) of 10 ng/mL IL-1β. Levels of catenin proteins were determined by Western blotting. (C): Chondrocytes were left untreated or treated with 1 cGy radiation in the absence or presence of 10 ng/mL IL-1β for 48 h. Expression of each catenin was detected by confocal fluorescence microscopy (scale bar: 50 μm). (D, E): Chondrocytes were transfected with 3 μg FLAG-tagged S33A β-catenin or wild-type γ-catenin for 24 h and then exposed to different doses of LDR for an additional 48 h. Levels of differentiation- and inflammation-associated proteins were determined by Western blotting (D). Sox-9 (E, left) or NF-κB transcriptional activity (E, right) was determined by reporter gene assay. Data are expressed as means ± SDs (*P < 0.05, **P < 0.005, ***P < 0.0005 compared with cells treated with IL-1β alone) (E). (F): Cells were left untreated or were pretreated with 5 or 10 μM Bay 11-7082 for 1 h prior to treatment with 10 ng/mL IL-1β. After 48 h, the levels of I-κB and COX-2 proteins were determined by Western blotting. (G): Chondrocytes were transiently transfected with 3 μg of GFP-tagged S83A α-catenin (left), FLAG-tagged S33A β-catenin (middle), or wild-type γ-catenin (right), as indicated, for 24 h and then left untreated or treated with 5 or 10 μM Bay 11-7082 for an additional 24 h. The levels of I-κB and COX-2 proteins were determined by Western blotting.
Mentions: We next examined whether LDR, at doses of 0–2 cGy, directly modulates the expression of catenin proteins in chondrocytes. Although LDR alone did not alter the expression of catenin proteins (Fig. 5A), both 0.5 and 1 cGy LDR dramatically attenuated IL-1β-induced expression of all catenin proteins (Fig. 5B). Immunofluorescence analyses showed that treatment of chondrocytes with IL-1β significantly increased the levels of all of the tested catenin proteins in both the cytosol and nuclear regions compared to control cells. At a dose of 1 cGy, LDR reduced catenin expression in IL-1β-treated cells to basal levels (Fig. 5C), indicating that LDR inhibits the IL-1β-dependent post-translational stabilization of catenin. Finally, we examined whether LDR reverses the dedifferentiation and inflammatory response induced by overexpression of the above-mentioned α-, β-, and γ-catenin proteins in chondrocytes. Notably, LDR markedly restored the expression of type II collagen and Sox-9 proteins in catenin-overexpressing chondrocytes (Supporting Information Fig. 1B and Fig. 5D), indicating a reduced commitment to dedifferentiation. LDR also inhibited I-κB degradation and COX-2 expression under the same experimental conditions (Supporting Information Fig. 1B and Fig. 5D). Consistent with this, LDR dramatically increased in Sox-9 activity reduced by catenin proteins and decreased in NF-κB activity induced by catenin proteins in chondrocytes; at a dose of 1 cGy, LDR enhanced Sox-9 activity by approximately 2.2-fold, 2.7-fold, and 2.4-fold compared to that in cells transfected with the above-mentioned α-, β-, and γ-catenin constructs, respectively (Supporting Information Fig. 1C and Fig. 5E, left), and reduced NF-κB activity by approximately 74%, 78%, and 75%, respectively (Supporting Information Fig. 1C and Fig. 5E, right), indicating a reduced commitment to inflammation. To further confirm the role of NF-κB signaling in catenin-mediated induction of COX-2 expression, we treated catenin-overexpressing chondrocytes with the NF-κB inhibitor, BAY. Pretreatment with 5 or 10 μM BAY inhibited I-κB degradation in a dose-dependent manner and consistently reduced COX-2 expression compared to IL-1β-treated chondrocytes (Fig. 5F). Degradation of I-κB and induction of COX-2 in cells transfected with the above-mentioned α-, β-, and γ-catenin constructs were also reversed by BAY treatment (Fig. 5G), indicating that NF-κB signaling is downstream of the catenin pathway.

Bottom Line: Here, we found that LDR, at doses of 0.5-2 centiGray (cGy), inhibited interleukin (IL)-1β-induced chondrocyte destruction without causing side effects, such as cell death and senescence.LDR also inhibited chondrocyte destruction through the catenin pathway induced by epidermal growth factor, phorbol 12-myristate 13-acetate, and retinoic acid.Collectively, these results identify the molecular mechanisms by which LDR suppresses pathophysiological processes and establish LDR as a potentially valuable therapeutic tool for patients with cytokine- or soluble factors-mediated cartilage disorders.

View Article: PubMed Central - PubMed

Affiliation: Division of Radiation Cancer Biology, Korea Institute of Radiological & Medical Sciences, Seoul, Korea; Department of Chemistry, College of Natural Sciences, Hanyang University, Seoul, Korea.

Show MeSH
Related in: MedlinePlus