Advanced glycation end products induce the expression of interleukin-6 and interleukin-8 by receptor for advanced glycation end product-mediated activation of mitogen-activated protein kinases and nuclear factor-κB in human osteoarthritis chondrocytes - PubMed (original) (raw)

Advanced glycation end products induce the expression of interleukin-6 and interleukin-8 by receptor for advanced glycation end product-mediated activation of mitogen-activated protein kinases and nuclear factor-κB in human osteoarthritis chondrocytes

Zafar Rasheed et al. Rheumatology (Oxford). 2011 May.

Abstract

Objective: To investigate whether advanced glycation end products (AGEs) induce the expression of IL-6 and IL-8 through the receptor for AGEs (RAGE)-activated pathways in human OA chondrocytes.

Methods: OA chondrocytes were stimulated with AGE-modified BSA (AGE-BSA). Gene expression of IL-6 and IL-8 was quantified by TaqMan assays and the production was determined using ELISAs. Immunoblotting was used to analyse the activation of mitogen-activated protein kinases (MAPKs) and the degradation of IκBα. Activation of NF-κB was determined using an ELISA. Pharmacological studies to elucidate the involved pathways were executed using transfection with small interfering RNAs (siRNAs), inhibitors of MAPKs and NF-κB.

Results: AGE-BSA induced the expression of IL-6 and IL-8 in OA chondrocytes, which was inhibited by pre-treatment with soluble RAGE (sRAGE) or RAGE knockdown by siRNAs. Treatment with SB202190 (p38-MAPK inhibitor) or PD98059 (ERK inhibitor) inhibited AGE-BSA-induced IL-6 and IL-8 expression. However, SP600125 (JNK inhibitor) had no effect on AGE-BSA-induced IL-6 expression but inhibited the expression of IL-8. Treatment with NF-κB inhibitors suppressed AGE-BSA-induced IL-6 and IL-8 expression.

Conclusions: This is the first study to demonstrate that AGEs induce the expression of IL-6 and IL-8 in OA chondrocytes. A novel finding of our studies is that in OA chondrocytes, AGE-BSA-induced expression of IL-6, but not of IL-8, was independent of the JNK pathway. Activation of NF-κB was an absolute requirement for both IL-6 and IL-8 expression. These results demonstrate that AGE-BSA-induced expression of IL-6 and IL-8 via RAGE is mediated through different MAPK signalling pathways in OA and possibly in other degenerative diseases.

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Figures

Fig. 1

Expression of IL-6 and IL-8 in AGE-BSA-stimulated primary human OA chondrocytes. Effect of AGE-BSA on the gene expression of IL-6 (a) and IL-8 (b) in primary human OA chondrocytes. Primary human OA chondrocytes were treated with AGE-BSA (5–100 µg/ml) and native BSA (100 µg/ml) for 24 h. Time-dependent effect of AGE-BSA on the gene expression of IL-6 (c) and IL-8 (d) in human OA chondrocytes. Primary chondrocytes were treated with AGE-BSA (100 µg/ml) and native BSA (100 µg/ml) for 0–24 h. Expression of IL-6 or IL-8 mRNA was determined by real-time qRT–PCR using comparative ΔΔ_C_T method. Incubation with native BSA (nBSA) was used as control. Results are representative [mean (

s.e.m

.)] of duplicate experiments with OA chondrocytes obtained from five age- and sex-matched OA donors and differ without a common letter; P < 0.05.

Fig. 2

Enhanced production of IL-6 and IL-8 by AGE-BSA-stimulated primary human OA chondrocytes. Effect of AGE-BSA on the protein production of IL-6 (a) and IL-8 (b) in primary human OA chondrocytes. Primary human OA chondrocytes were treated with AGE-BSA (5–100 µg/ml) and native BSA (100 µg/ml) for 24 h. Kinetics of AGE-BSA-induced production of IL-6 (c) and IL-8 (d) in primary human OA chondrocytes. Primary human OA chondrocytes were treated with AGE-BSA (100 µg/ml) and native BSA (100 µg/ml) for 0–24 h. Production of IL-6 or IL-8 was determined by a sandwich ELISA. Native BSA (nBSA) was used as control. Results are representative [mean (

s.e.m

)] of duplicate experiments with OA chondrocytes obtained from five age- and sex-matched OA donors and differ without a common letter; P < 0.05.

Fig. 3

Effect of sRAGE or RAGE-knockdown on the AGE-BSA or S100A4 protein-induced expression of IL-6 and IL-8 in primary human OA chondrocytes. Primary OA chondrocytes were pre-treated with sRAGE (200 µg/ml) for 2 h and then stimulated with AGE-BSA (50 µg/ml) or S100A4 protein (25 ng/ml) or IL-1β (20 ng/ml) for 24 h. Human OA chondrocytes were transfected with RAGE-siRNA or control siRNA and then stimulated with AGE-BSA for 24 h. Expression of IL-6 (a) and IL-8 (b) mRNA was determined by real-time qPCR and normalized to GAPDH and compared with the levels present in untreated chondrocytes using a comparative ΔΔ_C_T method. Production of IL-6 (c) and IL-8 (d) in the culture medium was quantified by IL-6- or IL-8-specific sandwich ELISA. Results are representative [mean (

s.e.m

)] of duplicate experiments with human OA chondrocytes obtained from two age- and sex-matched OA donors and differ without a common letter; P < 0.05.

Fig. 4

AGE-BSA or S100A4 enhances the production of IL-6 and IL-8 in human OA cartilage explants. Human OA cartilage explants in serum-free DMEM were stimulated with AGE-BSA or S100A4 protein for 24 h at 37°C. Production of IL-6 (a, b) and IL-8 (c, d) in the culture medium was quantified by IL-6- or IL-8-specific sandwich ELISA. Results are representative [mean (

s.e.m

)] of duplicate experiments with human OA chondrocytes obtained from two age- and sex-matched OA donors and differ without a common letter; P < 0.05.

Fig. 5

Activation of RAGE-mediated MAPK signalling by AGE-BSA in primary human OA chondrocytes. Effect of sRAGE (a) and RAGE knockdown (b) on MAPK phosphorylation in AGE-BSA-stimulated OA chondrocytes. After pre-treatment with sRAGE (200 µg/ml) for 2 h at 37°C or RAGE-siRNA (30–100 nM) transfection, human OA chondrocytes (70–80% confluent) were incubated with AGE-BSA (50–100 µg/ml) for 45 min, then the phosphorylation (P) of p38-MAPK, JNK and ERK was determined by western immunoblot analysis. Band images were digitally captured and the band intensities were obtained using UN-San-It software and are expressed in average pixels. Data shown are cumulative of two experiments. Average pixel values presented as mean (

s.e.m

); data without a common letter differ; P < 0.05.

Fig. 6

Different MAPKs regulate AGE-BSA-induced expression of IL-6 and IL-8 in primary human OA chondrocytes. Effect of MAPK inhibition on the gene expression of IL-6 (a) and IL-8 (b) in AGE-BSA-stimulated OA chondrocytes. Primary human OA chondrocytes were pre-treated with inhibitors of p38 (SB202190), JNK (SP600125) and ERK (PD98059) MAPKs for 1 h and then stimulated with AGE-BSA (100 µg/ml) for 24 h. Expression of IL-6 or IL-8 mRNA was determined by real-time qPCR and normalized to GAPDH and compared with the levels present in control using the comparative ΔΔ_C_T method. Effect of MAPK inhibition on the protein production of IL-6 (c) and IL-8 (d) in culture medium of AGE-BSA-stimulated OA chondrocytes. Primary human OA chondrocytes were pre-treated with MAPK inhibitors SB202190, SP600125, PD98059 for 1 h and then stimulated with AGE-BSA (100 µg/ml) for 24 h. Production of IL-6 or IL-8 was quantified by a sandwich ELISA. Primary OA chondrocytes incubated with native BSA (100 µg/ml) were used as control. The concentration of SB202190, SP600125 and PD98059 used in these studies was 100, 10 and 50 µM, respectively. Results are representative [mean (

s.e.m

)] of duplicate experiments with OA chondrocytes obtained from five age- and sex-matched OA donors and differ without a common letter; P < 0.05.

Fig. 7

Activation of RAGE-mediated NF-κB signalling by AGE-BSA in primary human OA chondrocytes. (a) Primary chondrocytes were pre-treated with sRAGE (200 µg/ml) for 2 h prior to AGE-BSA (50 µg/ml) stimulation or (b) RAGE-siRNA-transfected OA chondrocytes were stimulated with AGE-BSA (100 µg/ml) for 45 min and NF-κB p65 was determined in cell extracts (3 µg) by highly sensitive and specific ELISA. TNF-α-treated HeLa cell extract (supplied with kit) was used as a positive control. The assay is developed with a chemiluminescent substrate and the signal is detected using a multimode detector (DTX-880, Beckman Coulter). NF-κB p65 activity was expressed as relative light units (RLUs) and normalized with negative control. Results are representative [mean (

s.e.m

)] of three independent experiments and differ without a common letter; P < 0.05. (c, d) IκBα degradation was analysed by western immunoblotting using antibodies specific for IκBα (Santa Cruz Biotechnology). β-actin was used as protein-loading control. Band images were digitally captured and the band intensities (pixels/band) were obtained using the UN-Scan-It software and are expressed in average pixels. Data shown are cumulative of three experiments and the OD values are mean (

s.e.m

) and differ without a common letter; P < 0.05.

Fig. 8

Activation of NF-κB is required for AGE-BSA-induced expression of IL-6 and IL-8 in primary human OA chondrocytes. Primary human OA chondrocytes were pre-treated with inhibitors specific for IKKα/β (Bay 11-7082), IKKβ (parthenolide), IKKγ (NEMO-BDBP) or proteasome (MG-132) for 1h and then stimulated with AGE-BSA (100 µg/ml) for 24 h. Expression of (a) IL-6 mRNA or (b) IL-8 mRNA was determined as described under Fig. 5. Effect of specific inhibitors of NF-κB on the production of IL-6 (c) and IL-8 (d) in AGE-BSA-stimulated primary human OA chondrocytes. Primary human OA chondrocytes were pre-treated with Bay 11-7082, parthenolide, NEMO-BDBP or MG-132 for 1 h and then stimulated with AGE-BSA (100 µg/ml) for 24 h. Level of IL-6 or IL-8 was quantified by a sandwich ELISA. Primary human OA chondrocytes incubated with native BSA (100 µg/ml) were used as control. Concentration of Bay 11-7082, parthenolide, NEMO-BDBP and MG-132 used was 50, 50, 50 and 100 µM, respectively. Results are representative [mean (

s.e.m

)] of duplicate experiments with OA chondrocytes obtained from age- and sex-matched five OA donors and differ without a common letter P < 0.05.

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Advanced glycation end products induce the expression of interleukin-6 and interleukin-8 by receptor for advanced glycation end product-mediated activation of mitogen-activated protein kinases and nuclear factor-κB in human osteoarthritis chondrocytes - PubMed (original) (raw)