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Objective Hedgehog (HH) signaling has recently been associated with cartilage degradation in osteoarthritis (OA). Because interleukin-1β (IL-1β) has been implicated as a principal instigator of OA, we sought to determine whether IL-1β induces the expression of sonic HH (SHH) and its regulation by microRNAs (miRNAs) in human chondrocytes. Methods Expression of SHH protein in human OA cartilage and in an animal model of OA was determined by immunohistochemical analysis and immunofluorescence analysis, respectively. Gene and protein expression in IL-1β- or SHH-stimulated OA chondrocytes was determined by TaqMan assays and Western blotting, respectively. The effect of overexpression of miRNA-602 (miR-602) and miR-608 or their antagomirs on SHH expression was evaluated by transient transfection of human chondrocytes and HEK 293 cells. The role of signaling pathways was evaluated using small molecule inhibitors. Binding of miRNAs with the putative seed sequence in SHH messenger RNA (mRNA) was validated using a luciferase reporter assay. Results Expression of SHH, patched 1, Gli-1, HH-interacting protein, matrix metalloproteinase 13 (MMP-13), and Colα1(X) was high in damaged OA cartilage. In damaged cartilage and in IL-1β-stimulated OA chondrocytes, expression of SHH was inversely correlated with expression of miR-608. Cotransfection of OA chondrocytes with miR-608 or miR-602 mimic inhibited reporter activity, and mutation of the miRNA seed sequences abolished the repression of reporter activity. Overexpression of miR-602 or miR-608 inhibited the expression of SHH mRNA and protein, and this was abrogated by antagomirs. Stimulation with recombinant human SHH protein up-regulated MMP-13 expression, and inhibition of HH signaling blocked MMP-13 expression in OA chondrocytes. Conclusion MiR-602 and miR-608 are important posttranscription regulators of SHH expression in OA chondrocytes, and their suppression by IL-1β may contribute to the enhanced expression of SHH and MMP-13 in OA. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM PUBLISHER]