Galectin-1 induces inflammation and cartilage degeneration in osteoarthritis through NF-κB-signaling

Abstract

Purpose: Cartilage consists of numerous glycoproteins and proteoglycans, which are of significance for its functionality. In osteoarthritis (OA), we previously observed an altered glycophenotype of cartilage and chondrocytes. We also examined the presence of galectins and found that Galectin-1 (Gal-1) predominantly accumulated in regions of severely degenerated cartilage. The interaction of glycans and galectins is crucial in many physiological or pathological processes. While a protective role of Gal-1 is described in rheumatoid arthritis (RA), little is known about the role of galectins in onset and progression of OA. Therefore, the present study investigates in detail the presence and biological activity of Gal-1 in OA cartilage and chondrocytes. Methods: OA cartilage was obtained from patients who underwent total knee replacement surgery. The degeneration status of cartilage from 29 patients was assessed in a total of 231 regions of interest using the Mankin score (MS). Gal-1 presence was detected by immunohistochemical staining and the percentages of positive chondrocytes were assessed. OA chondrocytes were isolated and secretion of Gal-1 to the cell culture medium was determined by ELISA. Binding of Gal-1-FITC to the surface of chondrocytes was analyzed in cell suspension. Expression of cartilage matrix components and matrix metalloproteinases (MMPs) in Gal-1-treated chondrocytes was analyzed with RT-qPCR. Cell culture supernatants were subjected to ELISA for determination of MMP levels. For microarray analyses, RNA was isolated from chondrocytes cultured in absence or presence of Gal-1 (n=5). Bioinformatic analyses were performed using Gene Set Enrichment Analyses (GSEA) and subsequent NF-κB analyses were carried out using quantitative Western blot and RT-qPCR. Results: Immunohistochemical staining revealed increasing amounts of Gal-1-positive chondrocytes in regions of degenerated cartilage. Correlation analysis indicated a significant positive correlation between Gal-1 presence (% chondrocyte positivity) and cartilage degeneration (p<0.0001, Wilcoxon‘s signed rank test). In vitro, Gal-1 was secreted by chondrocytes, whereby this secretion was not inducible by pro-inflammatory cytokines. Binding of Gal-1 to chondrocyte surfaces and resulting effects were inhibitable by cognate sugar (lactose). Gal-1 treatment of chondrocytes increased the expression of matrix degrading enzymes (ADAMTS4, MMP1, MMP3, MMP13) and pro-inflammatory cytokines (IL1B, TNFA), while the expression of matrix components (AGC1, COL2A1) was decreased. In agreement, elevated secretion levels of pro-MMP-1, MMP-3 and pro-MMP-13 were detected. Analyses of the microarray data revealed an overexpression of chemokines and cytokines in Gal-1-treated chondrocytes. GSEA showed an overrepresentation of NF-κB DNA binding motifs in the promoters of most significantly induced genes prompting further investigation of the NF-κB signaling cascade. Western blot analyses showed that Gal-1 induced phosphorylation of IκBα and p65, and RT-qPCR experiments revealed an impairment of Gal-1-mediated IL1B transcription by inhibition of specific NF-κB pathway components. Conclusions: This study is first to suggest a functional link between Gal-1 and OA manifestation. In situ, Gal-1 presence correlates with cartilage degeneration, which points towards its qualification as novel tissue biomarker. In vitro, Gal-1 induces the production of pro-inflammatory cytokines and matrix degrading enzymes. Importantly, Gal-1 secretion is not induced by pro-inflammatory conditions, indicating its position as an upstream mediator acting as a trigger of inflammation and matrix breakdown in OA. The deleterious activity of Gal-1 in OA, which is mediated by the NF-κB pathway, stands in contrast to its protective role in RA. In summary, our results highlight the role of Gal-1 in onset and progression of OA and suggest that targeting Gal-1 signaling may be a promising therapeutic strategy.