Protein Kinase C Involvement in Interleukin‐6 Production by Parathyroid Hormone and Tumor Necrosis Factor‐α in UMR‐106 Osteoblastic Cells

The cytokine interleukin‐6 (IL‐6) is increased in bone and bone cells by several resorptive stimuli, including parathyroid hormone (PTH), IL‐1β, and tumor necrosis factor‐α (TNF‐α). The current studies were designed to determine the contribution of the protein kinase C (PKC) signaling pathway to the effects of these three agents to increase IL‐6 in UMR‐106 rat osteoblastic cells. Cells were pretreated with vehicle (dimethylsulf‐oxide [DMSO]) or the phorbol ester, phorbol 12,13‐dibutyrate (PDB; 300 nM) for 48 h to down‐regulate phorbol‐sensitive PKC isozymes. Either PTH (0.1–10 nM), IL‐1β (0.1–10 nM), or TNF‐α (5 nM and 10 nM) was then added for 24 h in the continued presence of vehicle or PDB. PKC isozymes were visualized by Western immunoblotting and IL‐6 was determined by bioassay. PDB pretreatment caused a partial down‐regulation of the conventional α‐PKC and β I ‐PKC isozymes and complete down‐regulation of the novel δ‐isoenzyme and ϵ‐isozymes but it had no effect on the atypical Ξ‐PKC isozyme. PDB pretreatment reduced IL‐6 responses to 5 nM and 10 nM PTH by 61% and 33%, respectively, reduced IL‐6 responses to 5nM and 10 nM TNF‐α by 54% and 42%, respectively, and failed to inhibit the IL‐6 responses to 0.1–10 nM IL‐1β. The PDB pretreatment protocol significantly enhanced PTH‐stimulated cyclic adenosine monophosphate (cAMP) production. The PKC inhibitor calphostin C also decreased IL‐6 responses to PTH. Thus, in this osteoblast cell line, the PKC pathway is an important component of the signaling pathway for the IL‐6 production stimulated by PTH and TNF‐α but not that from IL‐1β. (J Bone Miner Res 2000;15:885–893)