Distinctive pro-inflammatory gene signatures induced in articular chondrocytes by oncostatin M and IL-6 are regulated by Suppressor of Cytokine Signaling-3

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Distinctive pro-inflammatory gene signatures induced in articular chondrocytes by oncostatin M and IL-6 are regulated by Suppressor of Cytokine Signaling-3 X. Liu, R. Liu, B.A. Croker, K.E. Lawlor, G.K. Smyth, I.P. Wicks Osteoarthritis and Cartilage Volume 23, Issue 10, Pages 1743-1754 (October 2015) DOI: 10.1016/j.joca.2015.05.011 Copyright © 2015 Osteoarthritis Research Society International Terms and Conditions

Fig. 1 Microarray analysis of wild-type (WT) chondrocytes stimulated with gp130 cytokines. (A) Venn diagram showing the number of differentially expressed genes (false discovery rate < 0.05) in primary epiphyseal chondrocytes stimulated with OSM, IL-6 in conjunction with soluble IL-6R (IL-6/sIL-6R), IL-11, and leukemia inhibitory factor (LIF). The table summarizes the total number and the number of unique differentially expressed genes for each cytokine. (B) Log2-fold changes and P-values for the 13 genes responding to all four gp130 cytokines. (C) Scatter plots of log2-fold expression changes induced by OSM compared to the changes induced by other gp130 cytokines. All expressed genes are shown without any filtering for statistical significance. Three independent experiments were conducted. Each biological replicate utilized epiphyseal chondrocytes from 15 mice. Osteoarthritis and Cartilage 2015 23, 1743-1754DOI: (10.1016/j.joca.2015.05.011) Copyright © 2015 Osteoarthritis Research Society International Terms and Conditions

Fig. 2 Validation of induced gene expression in wild-type (WT) chondrocytes stimulated with OSM or IL-6/sIL-6R. Quantitative-polymerase chain reaction was performed to analyze (A) Il19 (B) Ccl7 (C) Saa1 (D) Lrg1 (E) Bcl3 and (F) Sphk1 mRNA expression in mouse epiphyseal chondrocytes left unstimulated or stimulated with OSM or IL-6/sIL-6R for 1, 2, 4 and 8 h. Results show sets of experimental data from three independent biological experiments. Each experiment utilized epiphyseal chondrocytes from ten mice. * = P < 0.05, ** = P < 0.01 and *** = P < 0.001. Osteoarthritis and Cartilage 2015 23, 1743-1754DOI: (10.1016/j.joca.2015.05.011) Copyright © 2015 Osteoarthritis Research Society International Terms and Conditions

Fig. 3 Differences between OSM and IL-6/sIL-6R stimulation for genes associated with gene ontology (GO) terms for joint inflammation, cartilage degradation and enzyme inhibitor activity. For each of the GO terms (A) “Cytokine activity” (B) “Metallopeptidase activity” and (C) Inhibitors of Enzyme Activity”, genes are shown that were differentially expressed (FDR < 0.05) after OSM relative to IL-6/sIL-6R stimulation in wild-type chondrocytes. Black bars show genes that were more highly up-regulated after OSM stimulation and white bars show genes that were more up-regulated after IL-6/sIL-6R stimulation. The horizontal axis shows the log2 expression difference between cells stimulated by that cytokine relative to those stimulated by the other. Osteoarthritis and Cartilage 2015 23, 1743-1754DOI: (10.1016/j.joca.2015.05.011) Copyright © 2015 Osteoarthritis Research Society International Terms and Conditions

Fig. 4 Microarray analysis of SOCS-3–deficient chondrocytes stimulated with gp130 cytokines. (A) Venn diagram showing the number of differentially expressed genes (FDR < 0.05) in Socs3Δ/Δcol2 chondrocytes stimulated with OSM, IL-6/sIL-6R, IL-11 and LIF. The table summarizes the total number and the number of unique differentially expressed genes for each cytokine. Genes showing SOCS-3-dependency in their expression changes after (B) OSM or (C) IL-6/sIL-6R-stimulation. Genes are shown that produced significantly different (FDR < 0.05) cytokine-induced expression changes in Socs3Δ/Δcol2 chondrocytes compared to wild-type chondrocytes. Log FC values are delta log2-fold changes between the two genotypes. For OSM, the 10 largest positive and negative delta log2-fold changes are shown. For IL-6/sIL-6R, all 15 significant genes are shown. Gene lists were cross-referenced with the Interferon Regulated Gene (IRG) database. Osteoarthritis and Cartilage 2015 23, 1743-1754DOI: (10.1016/j.joca.2015.05.011) Copyright © 2015 Osteoarthritis Research Society International Terms and Conditions