RESUMO
OBJECTIVE: To determine the effects of interleukin (IL)-6 and IL-1ß stimulation on expression of growth differentiation factor (GDF)-5 and Wnt signaling pathway genes in equine chondrocytes. SAMPLE: Macroscopically normal articular cartilage samples from 6 horses and osteochondral fragments (OCFs) from 3 horses. PROCEDURES: Chondrocyte pellets were prepared and cultured without stimulation or following stimulation with IL-6 or IL-1ß for 1, 2, 12, and 48 hours; expression of GDF-5 was determined with a quantitative real-time PCR assay. Expression of genes in various signaling pathways was determined with microarrays for pellets stimulated for 1 and 2 hours. Immunohistochemical analysis was used to detect GDF-5, glycogen synthase kinase 3ß (GSK-3ß), and ß-catenin proteins in macroscopically normal cartilage samples and OCFs. RESULTS: Chondrocytes stimulated with IL-6 had significantly higher GDF-5 expression within 2 hours versus unstimulated chondrocytes. Microarray analysis of Wnt signaling pathway genes indicated expression of GSK-3ß and coiled-coil domain containing 88C increased after 1 hour and expression of ß-catenin decreased after 2 hours of IL-6 stimulation. Results of immunohistochemical detection of proteins were similar to microarray analysis results. Chondrocytes in macroscopically normal articular cartilage and OCFs had immunostaining for GDF-5. CONCLUSION AND CLINICAL RELEVANCE: Results indicated IL-6 stimulation decreased chondrocyte expression of the canonical Wnt signaling pathway transactivator ß-catenin, induced expression of inhibitors of the Wnt pathway, and increased expression of GDF-5. This suggested IL-6 may inhibit the Wnt signaling pathway with subsequent upregulation of GDF-5 expression. Anabolic extracellular matrix metabolism in OCFs may be attributable to GDF-5 expression. This information could be useful for development of cartilage repair methods.
