Advanced glycation end products induce peroxisome proliferator-activated receptor γ down-regulation-related inflammatory signals in human chondrocytes via Toll-like receptor-4 and receptor for advanced glycation end products

Abstract

Accumulation of advanced glycation end products (AGEs) in joints is important in the development of cartilage destruction and damage in age-related osteoarthritis (OA). The aim of this study was to investigate the roles of peroxisome proliferator-activated receptor γ (PPARγ), toll-like receptor 4 (TLR4), and receptor for AGEs (RAGE) in AGEs-induced inflammatory signalings in human OA chondrocytes. Human articular chondrocytes were isolated and cultured. The productions of metalloproteinase-13 and interleukin-6 were quantified using the specific ELISA kits. The expressions of related signaling proteins were determined by Western blotting. Our results showed that AGEs enhanced the productions of interleukin-6 and metalloproteinase-13 and the expressions of cyclooxygenase-2 and high-mobility group protein B1 and resulted in the reduction of collagen II expression in human OA chondrocytes. AGEs could also activate nuclear factor (NF)-κB activation. Stimulation of human OA chondrocytes with AGEs significantly induced the up-regulation of TLR4 and RAGE expressions and the down-regulation of PPARγ expression in a time- and concentration-dependent manner. Neutralizing antibodies of TLR4 and RAGE effectively reversed the AGEs-induced inflammatory signalings and PPARγ down-regulation. PPARγ agonist pioglitazone could also reverse the AGEs-increased inflammatory signalings. Specific inhibitors for p38 mitogen-activated protein kinases, c-Jun N-terminal kinase and NF-κB suppressed AGEs-induced PPARγ down-regulation and reduction of collagen II expression. Taken together, these findings suggest that AGEs induce PPARγ down-regulation-mediated inflammatory signalings and reduction of collagen II expression in human OA chondrocytes via TLR4 and RAGE, which may play a crucial role in the development of osteoarthritis pathogenesis induced by AGEs accumulation.

Figure 1. AGEs induce inflammatory signalings in human OA chondrocytes.

Human OA chondrocytes (1×10⁶/ml) were incubated with AGEs (5–100 µg/ml) for 24 hours and cytotoxic effect was determined by MTT assay (A). Productions of MMP-13 (B) and IL-6 (C) were quantified by the ELISA kits. Protein expressions of collagen II (D) were determined by Western blotting. Densitometric analysis for collagen levels corrected to β-actin is shown. All data are presented as mean ± SEM for three independent experiments. *: P<0.05 versus control.

Figure 2. AGEs induce inflammatory signalings in human OA chondrocytes.

Human OA chondrocytes (1×10⁶/ml) were incubated with AGEs (5–100 µg/ml) for 24 hours (A, C) or 0.5–24 hours (B, D). Protein expressions of COX-2 (A, B) and HMGB1 (C, D) were determined by Western blotting. Densitometric analysis for COX-2 and HMGB1 levels corrected to α-tubulin is shown. All data are presented as mean ± SEM for three independent experiments. *: P<0.05 versus control.

Figure 3. AGEs activate NF-κB signaling in human OA chondrocytes.

Human OA chondrocytes (1×10⁶/ml) were incubated with AGEs (50 µg/ml) for indicated time intervals. The phosphorylations of IKKα/β (A), IκBα (B) and p65 (C) and the degradation of IκBα (B) were determined by Western blotting. In D, Chondrocytes were pretreated with PDTC (20 µM) for 1 hour followed by treatment with AGEs for 2 hours. Protein expression of collagen II was determined by Western blotting. Densitometric analysis for p-IKKα/β, p-IκBα, IκBα, p-p65, and collagen II levels corrected to α-tubulin is shown. All data are presented as mean ± SEM for three independent experiments. *: P<0.05 versus control. #: P<0.05 versus AGEs alone.

Figure 4. AGEs activate NF-κB activity in human OA chondrocytes, which can be reversed by pioglitazone.

Human OA chondrocytes (1×10⁶/ml) were incubated with AGEs (50 µg/ml) for indicated time intervals. The expressions of nuclear p65 (A) and cytosol IκBα degradation (B) were determined by Western blotting. In C, chondrocytes were pretreated with PDTC (20 µM) for 1 hour followed by treatment with AGEs for 2 hours. Protein expression of PPARγ was determined by Western blotting. Densitometric analysis for nuclear p65, cytosolic IκBα, and PPARγ levels corrected to Histone H1, α-tubulin, and β-actin, respectively, is shown. In D, chondrocytes (1×10⁶/ml) were pretreated with pioglitazone (10 and 50 µg/ml) for 1 hour followed by stimulating with AGEs (50 µg/ml) for 24 hours. NF-κB activity was measured using NF-κB (p65) Transcription Assay kit and quantified with a spectrophotometric plate reader at wavelengths of 450 nm. All data are presented as mean ± SEM for three independent experiments. *: P<0.05 versus control. #: P<0.05 versus AGEs alone.

Figure 5. Involvement of TLR4 and RAGE in AGEs-induced COX-2 and HMGB1 protein expressions in human OA chondrocytes.

In A-D, human OA chondrocytes (1×10⁶/ml) were treated with AGEs (5–100 µg/ml) for 24 hours (A, C) or 0.5–24 hours (B, D). Protein expressions of TLR4 (A, B) and RAGE (C, D) were measured by Western blotting. In E–H, human OA chondrocytes (1×10⁶/ml) were pretreated with neutralizing antibodies of TLR4 (20 µg/ml) and RAGE (10 µg/ml) for 1 hour followed by treatment with AGEs (50 µg/ml) for 24 hours. Protein expressions of COX-2 (E, F) and HMGB1 (G, H) were determined by Western blotting. Results shown are representative of at least three independent experiments.

Figure 6. Densitometric analysis for TLR4, RAGE, COX-2, and HMGB1 levels.

Values are corrected to α-tubulin or β-actin levels. All data are presented as mean ± SEM for three independent experiments. *: P<0.05 versus control. #: P<0.05 versus AGEs alone.

Figure 7. AGEs down-regulate PPARγ protein expression in human OA chondrocytes.

Human OA chondrocytes (1×10⁶/ml) were treated with AGEs (5–100 µg/ml) for 24 hours (A) or 0.5–24 hours (B). In C and D, human OA chondrocytes were pretreated with neutralizing antibodies of RAGE (10 µg/ml; C) and TLR4 (20 µg/ml; D) for 1 hour and then stimulated with AGEs (50 µg/ml) for 24 hours. PPARγ protein expression was measured by Western blotting. Densitometric analysis for PPARγ level corrected to β-actin is shown. All data are presented as mean ± SEM for three independent experiments. *: P<0.05 versus control. #: P<0.05 versus AGEs alone.

Figure 8. Effects of pioglitazone on inflammatory signalings in human OA chondrocytes.

Human OA chondrocytes (1×10⁶/ml) were pretreated with pioglitazone (10 and 50 µg/ml) for 1 hour followed by stimulating with AGEs (50 µg/ml) for 24 hours. Productions of MMP-13 (A) and IL-6 (B) were quantified by specific ELISA kits. Protein expressions of COX-2, HMGB1, and collagen II were determined by Western blotting (C). Densitometric analysis for COX-2, HMGB1, and collagen II levels corrected to β-actin is shown. All data are presented as mean ± SEM for three independent experiments. *: P<0.05 versus control. #: P<0.05 versus AGEs alone.

Figure 9. Involvement of MAPK signaling in AGEs-induced PPARγ down-regulation and reduction of collagen II expression.

Human OA chondrocytes (1×10⁶/ml) were incubated with AGEs (50 µg/ml) for 0.5–24 hours (A, B) or 24 hours (C). The phosphorylations of JNK (A) and p38MAPK (B) were determined by Western blotting. In C, chondrocytes were pretreated with SP600125 (10 and 20 µM) or SB203580 (1 and 10 µM) for 1 hour followed by treatment with AGEs for 24 hours. Protein expressions of PPARγ and collagen II was determined by Western blotting. Densitometric analysis for p-JNK, p-p38MAPK, PPARγ, and collagen II levels corrected to JNK, p38MAPK, β-actin, and β-actin, respectively, is shown. All data are presented as mean ± SEM for three independent experiments. *: P<0.05 versus control. #: P<0.05 versus AGEs alone.

Figure 10. ERK signaling is not involved in the effects of AGEs on chondrocytes.

Human OA chondrocytes (1 ×10⁶/ml) were incubated with AGEs (50 µg/ml) for 0.5–24 hours (A) or 24 hours (B,). The phosphorylation of ERK (A) were determined by Western blotting. In B and C, chondrocytes were pretreated with PD98059 (10 and 20 µM) for 1 hour followed by treatment with AGEs for 24 hours. Protein expressions of PPARγ and collagen II was determined by Western blotting. Densitometric analysis for p-ERK, PPARγ and collagen II levels corrected to ERK, β-actin, and β-actin, respectively, is shown. All data are presented as mean ± SEM for three independent experiments. *: P<0.05 versus control.

Figure 11. The proposed schematic representation of AGEs-induced inflammatory signalings and resulted reduction of collagen II expression mediated by the down-regulation of PPARγ via TLR4 and RAGE in human OA chondrocytes is shown.