Interleukin‐6 receptor shedding is enhanced by interleukin‐1β and tumor necrosis factor α and is partially mediated by tumor necrosis factor α–converting enzyme in osteoblast‐like cells

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Abstract
Objective: Interleukin‐6 (IL‐6) and soluble IL‐6 receptor (sIL‐6R) activation of gp130 represents an alternative pathway for osteoclast development in inflammatory conditions. The goal of the present study was to investigate changes in sIL‐6R levels in response to the inflammatory cytokines IL‐1β and tumor necrosis factor α (TNFα) and to determine the role of TNFα‐converting enzyme (TACE) in this process.Methods: Levels of sIL‐6R in the culture media of MG63 and SAOS‐2 osteoblast‐like cell lines after exposure to various agents were determined by immunoassay. TACE protein levels were measured by Western immunoblotting. Cells were transfected with small interfering RNA (siRNA) or with an expression plasmid for IL‐6R and TACE to determine the potential involvement of TACE in IL‐6R shedding.Results: IL‐1β and TNFα increased the levels of sIL‐6R in the culture media of MG63 osteoblast‐like cells. This effect was not influenced by cycloheximide or 5,6‐dichlorobenzimidazole riboside but was markedly inhibited by the calcium chelator EGTA and by the TACE and matrix metalloproteinase inhibitor hydroxamate (Ru36156). IL‐1β and TNFα had no influence on the alternatively spliced form of IL‐6R RNA. Levels of sIL‐6R were reduced when MG63 cells were transiently transfected with TACE siRNA. Transfection of SAOS‐2 cells with expression plasmids for IL‐6R and TACE produced a dose‐dependent increase in sIL‐6R levels.Conclusion: IL‐1β‐ and TNFα‐mediated induction of IL‐6R shedding in osteoblast‐like cells is at least partly dependent on TACE activation.