We previously demonstrated that parathyroid hormone (PTH) induced interleukin-6 (IL-6) expression in osteoblasts primarily via the cAMP-PKA signaling pathway. In this study, we explored the molecular mechanism(s) underlying this action. We used a fragment of the human IL-6 proximal promoter containing two previously identified, juxtaposed CRE and C/EBP sites at -165/-158 bp (CRE165) and -157/-148 bp (C/EBP157) to drive the expression of a luciferase reporter (IL6-Luc225). Osteoblastic MC3T3-E1 cells were stably and transiently transfected with IL6-Luc225 constructs carrying mutations or deletions of the CRE and C/EBP sites. We demonstrated that agonists that stimulated the cAMP-PKA pathway, also induced IL6-Luc225 activity. The proximal CRE sequence (CRE165) was important in mediating cAMP induction of IL-6 promoter activity, whereas the C/EBP157 did not play a role in this. We also identified a distal CRE-like site (CRE209) that may involve in mediating cAMP-PKA induction of IL-6 promoter activity. Electrophoretic mobility shift assays showed the formation of DNA/protein complexes on the proximal CRE/C/EBP site. We used antibody supershift assays to show the presence of CREM/CREB, phosporylated CREB, C/EBPβ, and C/EBPδ” in the complexes. Our data suggest that PTH-mediated induction of IL-6 promoter activity is regulated primarily by the cAMP-PKA pathway and CRE165 may play a key role in mediating this response.
