Cp is an acute phase reactant protein that also acts as a ferroxidase, and thus indirectly decreases the production of the reactive oxygen species hydroxyl radical. Ceruloplasmin (Cp) expression is induced by a variety of central nervous system injuries, but the mechanism by which this occurs is unclear. Based on the fact that peripheral nerve injury induces interleukin-6 (IL-6) expression and that there are three IL-6 response elements in the upstream region of the Cp gene, we studied their role in transcriptional regulation of Cp in astrocytic C6 glioma cells, using transfection of a rat Cp-luciferase construct, followed by sequential and simultaneous mutation of the IL-6 response elements. We found that 0.8 kb of sequence upstream to the rat ceruloplasmin start site was sufficient to drive luciferase expression in C6 glioma cells. Cells transfected with Cp-luc and treated with 100 ng/ml rat IL-6 induced 216.8% +/- 4.6% of control activity. Mutagenesis of the IL-6 response elements decreased luciferase activity, with the maximal decline (9.7 +/- 0.7% of wild-type) after mutation of the second site. Mutagenesis of multiple sites decreased activity beyond mutagenesis of single sites with mutation of all three sites decreasing activity to 5.3 +/- 0.4% of wild-type. Gel shift and supershift assays indicated that activation of Cp in these cells was not via STAT-3. These results are consistent with a signaling process via IL-6 response elements for Cp upregulation.