Direct Upregulation of Iron Regulatory Hormone Hepcidin by Interferon Alpha

Interferon (IFN) activates various immune systems in vivo and is administered for the patients with various diseases such as viral hepatitis B,C and malignant tumors. During the treatment with IFN, iron metabolism has been reported to be important. For instances, iron overload has reported to contribute to the poor response rate of IFN treatment and poor prognosis in the patients with hepatitis C. However, it has been unclear whether IFN itself effects on the iron metabolism. Therefore, we aimed to determine the effect of IFN itself on iron metabolism.

The mice were subcutaneously administered the mouse IFN alpha (10⁴ and 10⁵ IU/mouse/day) and sacrificed after 3, 5 and 7 days. Then the serum, the bone marrow, the liver, the spleen, and the duodenum tissues were collected. To analyze serum hepcidin levels, liquid chromatography-tandem mass spectrometry was performed. The changes of mRNA and protein expressions involved in iron metabolism such as transferrin receptor 1, hepcidin, and ferroportin, were also analyzed by quantitative real time RT-PCR and western blotting. Some parts of tissues were processed for the formalin-fixed paraffin embedding blocks, and Hematoxylin-Eosin and immunofluorescence staining for ferroportin. To evaluate the direct effects of IFN alpha on the expression of hepcidin by hepatocytes, primary cultured hepatocytes obtained from the mouse liver and human hepatoma-derived cultured cells were treated with human IFN alpha, and then RNA and the protein were extracted after 24 hours of treatment, followed by the analysis of hepcidin mRNA and transcription factors, such as STAT3.

Among the analyzed genes and proteins that are involved in iron metabolism, the hepcidin expression was highly modulated by IFN alpha. Both the serum hepcidin level and hepcidin mRNA expression in the liver were increased in IFN alpha-treated mice. And the expression of ferroportin, target molecule of hepcidin, was decreased in the duodenum of IFN alpha-treated mice. The analysis in vitro showed the upregulation of hepcidin mRNA by the IFNa treatment both in primary hepatocytes of the mouse liver and human hepatoma-derived cells. Interestingly, the upregulation of activated STAT3 was also observed in those cells, and that was cancelled by the addition of neutralizing antibody (IFNAR1) for IFN receptors.

Our results showed that IFN alpha directly upregulated the hepcidin expression by the liver. Concerning the mechanism of the upregulation of hepcidin, the upregulation of activated STAT3 by IFN should be involved. Commonly, IFN alpha accelerated the heterodimer formation of transcription of STAT1 and STAT2 via the activation of JAK by the binding of IFN alpha with IFN receptor on cell surface. The upstream lesion of the hepcidin gene does not have the binding site for STAT1/STAT2 heterodimer, but have the binding site for STAT3 homodimer. In addition, IFNa has been reported to activate STAT3 in human primary hepatocytes (Radaeva, S. et al. Gastroenterology 2002; 122:1020–34). Combined these, presumably IFN alpha directly effected on hepatocytes to enhance the transcription of hepcidin gene via activated STAT3 homodimer. In addition to the upregulation of hepcidin, our results showed the decreased duodenum expression of ferroportin in IFN alpha-treated mice. Ferroportin is expressed on the basolateral cell membrane of enterocytes and has function of iron transport from the enterocytes to blood. Ferroportin has also been reported to internalize and be degraded when hepcidin binds to ferroportin on the cell surface, resulting in the decrease of iron absorption from the gastrointestinal tract. Therefore, the upregulation of hepcidin by IFN alpha treatment observed in the present study should be physiologically functional, and this indicated that iron absorption should be decreased during IFN treatment. This effect is considered to be favorable because this would inhibit iron overload during IFN treatment and may increase the effect of IFN.

Sasaki:Novartis Pharma K.K.: Research Funding. Kohgo:Novartis: Research Funding, Speakers Bureau; Chugai Roche: Research Funding; Asahikasei Kurare Medical: Research Funding; Sapporo Brewery: Research Funding; Kyorin Pharma: Research Funding.