Dual role of insulin in transcriptional regulation of the acute phase reactant ceruloplasmin.

Insulin is a potent negative regulator of the response of hepatic cells to pro-inflammatory cytokines, particularly, interleukin (IL)-6. The action of insulin is target-selective because it inhibits transcription of most but not all acute phase genes. We here show that ceruloplasmin (Cp), an acute phase reactant with important functions in iron homeostasis, is subject to a unique dual regulation by insulin. IL-6 increased Cp mRNA expression in HepG2 cells by approximately 5-fold. Simultaneous treatment with insulin reduced this stimulation by half. Surprisingly, insulin by itself caused a 2-4-fold induction in Cp mRNA expression. The mechanism of induction by insulin was studied by transfecting into HepG2 cells chimeric constructs of the Cp 5'-flanking region driving luciferase. The activity of a 4800-bp segment of the Cp 5'-flanking region was increased 3-fold by insulin. Deletion and mutation analyses showed the requirement for a single hypoxia-responsive element in a 96-bp segment approximately 3600 bp upstream of the initiation site. The domains required for the two activities of insulin were distinct: The distal, hypoxia-responsive element-containing site was sufficient for Cp transactivation by insulin; in contrast, an 848-bp region adjacent to the initiation site was sufficient for IL-6 transactivation of Cp and for the inhibitory activity of insulin. The role of hypoxia-inducible factor-1 in the induction of Cp by insulin was shown by electrophoretic mobility shift assays and by the absence of insulin-stimulated Cp promoter activation in mouse Hepa c4 cells deficient in hypoxia-inducible factor-1 activity. Taken together these results show that insulin functions as a bidirectional, condition-dependent regulator of hepatic cell Cp expression. The unique regulation of Cp may reflect its dual roles in inflammation and iron homeostasis.