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Effects of interleukin-6 and interleukin-1β on expression of growth differentiation factor-5 and Wnt signaling pathway genes in equine chondrocytes

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  • 1 Department of Biomedical Sciences and Veterinary Public Health, Division of Pathology, Pharmacology and Toxicology, Swedish University of Agricultural Sciences, 750 07 Uppsala, Sweden.
  • | 2 BIOMATCELL, VINN Excellence Center of Biomaterials and Cell Therapy at the Department of Biomaterials, Institute of Clinical Sciences.
  • | 3 Department of Biomedical Sciences and Veterinary Public Health, Division of Pathology, Pharmacology and Toxicology, Swedish University of Agricultural Sciences, 750 07 Uppsala, Sweden.
  • | 4 Department of Clinical Chemistry and Transfusion Medicine, Institute of Biomedicine, Sahlgrenska University Hospital.
  • | 5 University of Gothenburg, 411 37 Gothenburg, Sweden; and System Biology Research Centre, School of Humanities and Informatics, University of Skövde, 541 28 Skövde, Sweden.
  • | 6 Department of Clinical Chemistry and Transfusion Medicine, Institute of Biomedicine, Sahlgrenska University Hospital.
  • | 7 Department of Biomedical Sciences and Veterinary Public Health, Division of Pathology, Pharmacology and Toxicology, Swedish University of Agricultural Sciences, 750 07 Uppsala, Sweden.
  • | 8 Department of Biomedical Sciences and Veterinary Public Health, Division of Pathology, Pharmacology and Toxicology, Swedish University of Agricultural Sciences, 750 07 Uppsala, Sweden.

Abstract

Objective—To determine the effects of interleukin (IL)-6 and IL-1β 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-1β 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 3β (GSK-3β), and β-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-3β and coiled-coil domain containing 88C increased after 1 hour and expression of β-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 β-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.

Contributor Notes

Drs. Svala and Skiöldebrand's present address is Department of Clinical Chemistry and Transfusion Medicine, Institute of Biomedicine, Sahlgrenska University Hospital, University of Gothenburg, 411 37 Gothenburg, Sweden. Dr. Henriksson's present address is Department of Orthopaedics, Institute of Clinical Sciences, Sahlgrenska University Hospital, University of Gothenburg, 411 37 Gothenburg, Sweden.

Supported by grants from the Swedish-Norwegian Foundation for Equine Research (H0947014) and the Swedish Research Council for Environment, Agricultural Sciences and Spatial Planning (Formas 2006–1947).

The authors declare that they have no conflicts of interest.

Address correspondence to Dr. Svala (emilia.svala@slu.se).