Investigation of the zinc-ZIP8-MTF1 signalling pathway in human OA chondrocytes

Abstract

Osteoarthritis (OA) is a common musculoskeletal disease characterised by articular cartilage loss resulting in severe joint pain, chronic morbidity, and premature mortality. There are no licensed OA drugs and current treatments involve pain relief with analgesics and joint replacement surgery. In murine cartilage chondrocytes the zinc-ZIP8-MTF1 signalling pathway causes cartilage breakdown. Pathway activation by interleukin-1β (IL-1β) stimulates upregulation of zinc transporter gene Slc39a8 (coding for ZIP8), increased intracellular zinc levels, nuclear translocation of the zinc-dependent MTF1 transcription factor and upregulation of genes encoding cartilage matrix-degrading proteins including Mmp13 and Adam-ts5. This thesis aimed to investigate if the IL-1-mediated zinc-ZIP8-MTF1 pathway is active in human chondrocytes and if this pathway contributes to OA pathogenesis. Analysis of OA-related transcriptomic datasets revealed that SLC39A14 but not SLC39A8, was dysregulated in human OA cartilage and mouse OA models. Human articular chondrocytes (HACs), and the chondrocyte cell lines SW1353 and T/C-28a2, were cultured in monolayer and treated with IL-1α and/or ZnCl2 or IL-1β. Genes involved in inflammation, zinc homeostasis and cartilage degradation were measured by quantitative PCR. MTF1 activity and subcellular localisation were evaluated using luciferase reporter assays, immunocytochemistry (ICC) and western blots. SLC39A8 and SLC39A14 were upregulated in IL-1α-treated HACs 5.5 and 2.9-fold respectively but were selectively elevated in SW1353 (SLC39A8, p <0.0001) and T/C-28a2 cells (SLC39A14, p = 0.0001). IL-1 raised intracellular zinc ion levels in HACs (p=0.0004) but not in the cell lines. IL-1 upregulated MMP13 in T/C-28a2 lines and SW1353 cells, while both MMP13 and ADAM-TS5 were upregulated in T/C-28a2 cells. MMP13 and ADAM-TS5 expression was non-significant after IL-1 treatment in HACs. IL-1 repressed MTF1 transactivation activity in SW1353 cells (14.5%, p=0.0114), even when MTF1 itself was overexpressed (17.2% p<0.0001). Western blotting and ICC results were non-correlative. Zinc-ZIP8-MTF1-mediated cartilage matrix-degrading gene upregulation induced through IL-1α as a contributing factor to human OA pathogenesis was inconclusive.