Hepcidin mediates transcriptional changes that modulate acute cytokine-induced inflammatory responses in mice
Ivana De Domenico, … , Diane M. Ward, Jerry Kaplan
Ivana De Domenico, … , Diane M. Ward, Jerry Kaplan
Published June 7, 2010
Citation Information: J Clin Invest. 2010;120(7):2395-2405. https://doi.org/10.1172/JCI42011.
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Hepcidin mediates transcriptional changes that modulate acute cytokine-induced inflammatory responses in mice

Abstract

Hepcidin is a peptide hormone that regulates iron homeostasis and acts as an antimicrobial peptide. It is expressed and secreted by a variety of cell types in response to iron loading and inflammation. Hepcidin mediates iron homeostasis by binding to the iron exporter ferroportin, inducing its internalization and degradation via activation of the protein kinase Jak2 and the subsequent phosphorylation of ferroportin. Here we have shown that hepcidin-activated Jak2 also phosphorylates the transcription factor Stat3, resulting in a transcriptional response. Hepcidin treatment of ferroportin-expressing mouse macrophages showed changes in mRNA expression levels of a wide variety of genes. The changes in transcript levels for half of these genes were a direct effect of hepcidin, as shown by cycloheximide insensitivity, and dependent on the presence of Stat3. Hepcidin-mediated transcriptional changes modulated LPS-induced transcription in both cultured macrophages and in vivo mouse models, as demonstrated by suppression of IL-6 and TNF-α transcript and secreted protein. Hepcidin-mediated transcription in mice also suppressed toxicity and morbidity due to single doses of LPS, poly(I:C), and turpentine, which is used to model chronic inflammatory disease. Most notably, we demonstrated that hepcidin pretreatment protected mice from a lethal dose of LPS and that hepcidin-knockout mice could be rescued from LPS toxicity by injection of hepcidin. The results of our study suggest a new function for hepcidin in modulating acute inflammatory responses.

Authors

Ivana De Domenico, Tian Y. Zhang, Curry L. Koening, Ryan W. Branch, Nyall London, Eric Lo, Raymond A. Daynes, James P. Kushner, Dean Li, Diane M. Ward, Jerry Kaplan

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Figure 2

Hepcidin induces a transcriptional response in macrophages.

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Hepcidin induces a transcriptional response in macrophages. (A) Mouse bo...
(A) Mouse bone marrow macrophages were incubated with FAC (10 μM Fe) to induce the expression of Fpn. After 24 hours, cells were incubated in the presence or absence of 1 μg/ml hepcidin for 30 minutes. Cells were placed at 0°C and solubilized and samples immunoprecipitated with rabbit anti-Fpn antibodies. Immunoprecipitated samples were analyzed by Western blot probing for Jak2, Stat3, p-Stat3, or Fpn. (B) Mouse bone marrow macrophages were incubated with FAC (10 μM Fe) for 24 hours, followed by a 4 hour incubation in the absence (–Hep) or presence (+Hep) of hepcidin, Hep20, or protegrin, all at a final concentration of 1 μg/ml. Cells were solubilized, RNA extracted, and Il7, Il17r, and Socs3 mRNAs were analyzed relative to actin mRNA by RT-PCR. (C) Mouse bone marrow macrophages were transfected with either nonspecific (N.S.) or mouse Fpn-, Jak2-, or Stat3-specific siRNA oligonucleotide pools using OligofectAMINE. Cells silenced for mouse Fpn, Jak2, or Stat3 were transfected with siRNA-resistant zebrafish Fpn (Z-Fpn), human Jak2 (H-Jak2), or human Stat3 (H-Stat3), respectively, using Amaxa Nucleofector. Twenty-four hours later, cells were incubated with FAC (10 μM Fe) for 24 hours and then incubated for an additional 4 hours in the absence or presence of hepcidin. Cells were solubilized, RNA was extracted, and Il7 and Il17r mRNA was analyzed relative to actin mRNA by RT-PCR. (D) Mouse bone marrow macrophages were incubated for 24 hours with FAC (10 μM Fe) and then incubated from 0 to 24 hours in the presence of 1 μg/ml hepcidin. Cells were solubilized, RNA was extracted, and Il7, Il17r, and Socs3 mRNA were analyzed relative to actin mRNA by RT-PCR.