Characterization of IL-6 Responsive Elements in Hepcidin Promoters at the Molecular Level: Lessons from the Two Closely Related Promoter Sequences, Murine Hepc1 and Hepc2.

Hepcidin is an antimicrobial peptide that plays an important role in regulation of iron metabolism. There is one hepcidin peptide in humans and two hepcidin peptides in mice, hepcidin 1 and hepcidin 2, the murine mature peptides sharing 68% homology at the amino-acid level. The promoter region of these two genes is highly conserved (92% homology of 500 bp promoter sequence from the start of translation). However, regulation of transcription of these two genes is different. Human hepcidin and murine hepcidin 1 mRNA levels become elevated in response to iron excess and inflammation, while murine hepcidin 2 responds only to iron. Wrighting and Andrews (Blood Ahead of Print July 2006) demonstrated that the inflammatory pathway is mediated by STAT3 and that the binding site includes nucleotides −145 to −143 from start of translation in the human hepcidin promoter. Moreover, mutation of these nucleotides led to loss of IL-6 responsiveness. Alignment of the nucleotide sequences of hepcidin promoters revealed that there are two major differences between the murine hepcidin 2 (mHepc2) and the human hepcidin (hHEPC) and the murine hepcidin 1 (mHepc1) sequence in the proximal 140 bp promoter region. There is a substitution of T for A at −136 in the STAT3/AP1 site and an inversion of ApG to GpA at position −127–8 in the AP1 site. We cloned mHepc1 and mHepc2 promoters into a pGL3 basic luciferase reporter vector and mutated the two different sites in order to determine if creating the mHepc2 sequence in the mHepc1 promoter eliminated IL-6 responsiveness and if creating the mHepc1 sequence in the mHepc2 promoter restored IL-6 responsiveness of that promoter. We also created the equivalent A→T mutation in the STAT3/AP1 site in the hHEPC construct and the TpTpC→GpGpA mutation at −143–5, the same mutant as made by Wrighting and Andrews. Plasmids were transfected into human hepatoma HepG2 cells and human kidney HEK293T cells. As expected, the wildtype hHEPC reporter construct was induced 10 fold or more by IL-6. The T→A and TpTpC→GpGpA hHEPC mutants both displayed significantly reduced, but not absent, IL-6 responsiveness. The basal level of the native mHepc1-driven reporter was at least 10-fold higher than that of the native mHepc2. Furthermore, the mHepc1 promoter was similarly responsive to IL-6 as the hHEPC while the response of mHepc2 promoter was negligible. Interestingly, although the mHepc1 promoter-driven reporter manifests baseline expression even in cells of non-hepatic origin (kidney HEK293T cells), the ability to respond to IL-6 is restricted to cells of hepatic origin. We observed a significantly lower basal level of luciferase reporter activity in the mHepc1 promoter mutated to mHepc2 (A→ T and GpA→ApG), however, it was still IL-6 responsive. In contrast, complementary mutagenesis of mHepc2 to mHepc1 (T→ A and ApG→GpA) increased the basal level of reporter expression and significantly increased the mHepc2 promoter responsiveness to IL-6 although not to the extent seen with mHepc1 promoter. We conclude that the promoter region at nt −126 to −145 from the start of translation is important for the basal level of expression of hepcidin and is important for IL-6 responsiveness in the hHEPC promoter but there are probably additional regions responsible for IL-6 responsiveness in the mHepc1 promoter.