The hepcidin circuits act: Balancing iron and inflammation

Authors


  • Potential conflict of interest: Nothing to report.

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. (HEPATOLOGY 2011 )

De Domenico I, Zhang TY, Koening CL, Branch RW, London N, Lo E, et al. Hepcidin mediates transcriptional changes that modulate acute cytokine-induced inflammatory responses in mice. J Clin Invest 2010; 120:2395-2405. (Reprinted with permission.)

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.

Comment

Hepcidin is a peptide hormone primarily known as the key regulator of iron homeostasis. This peptide, which binds the only known cellular iron exporter, ferroportin (Fpn), leads to its internalization and degradation in hepatocytes, enterocytes, and macrophages, prevents iron transport to plasma, and causes cellular retention of iron.1 Hepcidin is also an amphipathic peptide with antimicrobial activity similar to that of the defensin family of proteins.2 Hepcidin expression is up-regulated in response to iron stores, inflammation, and endoplasmic reticulum stress and is inhibited by anemia, erythropoiesis, hypoxia, and oxidative stress.3 Other factors that have been proposed to regulate hepcidin expression include leptin,4 p53,5 estradiol,6 and circadian rhythms.6

Hepcidin regulation due to iron stores is mediated via the bone morphogenetic protein (BMP)/Sma- and Mad-related protein (SMAD) pathway and the hemochromatosis/transferrin receptor 1/transferrin receptor 2 complex on hepatocytes in response to plasma transferrin levels.3 In the proposed mechanism, soluble BMPs (most notably BMP6) bind to bone morphogenetic protein receptors (BMPRs) and the BMP coreceptor hemojuvelin (HJV) in response to cellular iron levels; this initiates the phosphorylation of SMAD1, SMAD5, and SMAD8 and subsequent interactions with SMAD4.7, 8 This complex is then translocated to the nucleus, at which it binds to bone morphogenetic protein–responsive elements (BMP-REs) within the hepcidin promoter up-regulating hepcidin expression. Recently, two new negative regulators of this pathway have been identified: SMAD7, which directly binds the hepcidin promoter to repress transcription,9 and transmembrane protease serine 6 (TMPRSS6), which acts by cleaving HJV at the cell membrane to inhibit BMP signaling.10 These negative regulators may be important in limiting hepcidin production to prevent iron deficiency; mutations in TMPRSS6 are responsible for iron-refractory iron deficiency anemia.11

Hepcidin expression is also induced during infection and inflammation through the activation of the transcription factor signal transducer and activator of transcription 3 (STAT3) by the inflammatory cytokine interleukin-6 (IL-6).12 The binding of this cytokine to its cellular receptor leads to the recruitment of Janus kinase 2 (JAK2), which phosphorylates STAT3; STAT3 is then translocated into the nucleus and binds to the STAT3 binding motif at −64/−72 in the hepcidin promoter region, which induces hepcidin transcription.12, 13

Previously, De Domenico et al.14 reported that JAK2 activation and phosphorylation of Fpn are key steps in the hepcidin-mediated internalization of Fpn. In this study,15 they identified over 400 differentially expressed genes by messenger RNA microarray analysis in Fpn-expressing bone marrow–derived macrophages treated with hepcidin. Using cycloheximide to prevent de novo protein synthesis, the authors showed that the expression of approximately half of these genes was a direct result of hepcidin treatment and was not due to downstream gene activation. These data also suggest a novel signal transduction role for hepcidin in mediating the transcription of a large number of genes. Next, by treating cells with hepcidin-20, a hepcidin derivative incapable of binding Fpn, or protegrin, an antimicrobial defensin family peptide, De Domenico et al. confirmed that these results were specifically due to the binding of hepcidin by Fpn. The results of treatment with these analogues showed no effects on several genes previously up-regulated by hepcidin treatment. The role of STAT3 in this hepcidin-mediated transcriptional response was confirmed by coimmunoprecipitation of JAK2 and STAT3 with anti-Fpn antibodies but only in the presence of hepcidin. The silencing of Fpn, JAK2, and STAT3 with small interfering RNA (siRNA) pools showed that many representative genes of high and moderate abundance were affected by Fpn and JAK2 silencing, but not all of these were also affected by STAT3 silencing. Three prominent examples were prostate transmembrane protein, androgen-induced 1, and matrix metallopeptidase 9. Changes in the expression of these genes were also resistant to the addition of cycloheximide, and this suggests that other transcription factors in addition to STAT3 are responsive to this hepcidin/Fpn/JAK2 response.

A novel finding of the current study by De Domenico et al.15 is the fact that hepcidin is involved in an Fpn/JAK2/STAT3-dependent anti-inflammatory negative feedback loop; hepcidin incubation of Fpn-expressing macrophages after lipopolysaccharide (LPS)-induced cytokine release resulted in down-regulation of IL-6 and tumor necrosis factor α (TNF-α) expression (Fig. 1). This effect was abolished when siRNAs were added to silence the effect of suppressor of cytokine signaling 3 (SOCS3), a known negative regulator of the JAK/STAT signaling pathway. The authors suggested that this feedback mechanism may have arisen to limit the inflammatory response to bacterial infections.

Figure 1.

Hepcidin and STAT3: balancing iron and inflammation. Hepcidin gene expression is up-regulated by inflammatory cytokines and iron through the JAK/STAT and BMP/SMAD pathways, respectively. It is down-regulated by EPO, hypoxia, and ROS through their modulation of C/EBPα expression and activation of TMPRSS6, which cleaves HJV. Activation of JAK2 after hepcidin binding of Fpn results in Fpn phosphorylation; this is targeted for internalization and degradation. Hepcidin-mediated JAK2 activation also induces STAT3 phosphorylation, which initiates the regulation of a large number of STAT3-responsive genes; these genes include SOCS3, which suppresses the expression of IL-6 and TNF-α. This further reduces the induction of HAMP expression by these inflammatory cytokines and completes a negative feedback loop. Abbreviations: EPO, erythropoietin; HAMP, hepcidin antimicrobial peptide; IL-6R, interleukin-6 receptor; ROS, reactive oxygen species; STAT3-RE, signal transducer and activator of transcription 3–responsive element; TLR, toll-like receptor.

De Domenico et al.15 also demonstrated a novel therapeutic potential for hepcidin; in vivo testing of a hepcidin pretreatment for sublethal doses of LPS, turpentine, and polyinosinic:polycytidylic acid [poly(I:C)] showed reduced toxicity and morbidity. Compared with mice treated with LPS alone, hepcidin-pretreated mice have lower serum protein and hepatic mRNA levels of both IL-6 and TNF-α, higher temperatures, more energy, and better coordination. Hepcidin pretreatments also protected mice from lethal injections of LPS, and the mice regained normal function within 48 hours. However, a long-term inflammatory model of cecal ligation and puncture demonstrated that the effects of hepcidin are more likely to be effective in regulation of acute rather than chronic inflammation. Although serum IL-6 levels were reduced at early time points, these levels rose beyond those of controls later in the study.

This interesting report by De Domenico et al.15 raises a number of additional questions. First, this study was performed with bone marrow–derived macrophages, and its findings were confirmed in peritoneal macrophages; additional studies are warranted to define the nature of the hepcidin-mediated transcriptional response in other Fpn-expressing cell types. The authors noted that this response may be specific to the cell type, and this effect potentially has broad implications for iron regulation and inflammatory signaling. As mentioned previously, TNF-α and IL-6 were down-regulated by the expression of SOCS3 through a hepcidin/Fpn/STAT3-mediated pathway. Unlike IL-6, TNFα does not have a direct role in the aforementioned STAT3-mediated hepcidin up-regulation, but its level is often elevated in many chronic inflammatory disorders. TNF-α is known to down-regulate intestinal iron absorption and induce ferritin synthesis,16 but a link to the STAT3 or SMAD4 pathway is a necessary finding for understanding hepcidin-mediated inflammatory regulation. Interferon-γ, an important regulator of macrophage iron homeostasis and immune function, may also provide a meaningful link, especially with respect to the immune response to iron.17 Lipocalin 2 was highly up-regulated in this study and was previously shown to be up-regulated by a deficiency in the hemochromatosis gene, HFE.18 C/EBPα and other key factors of hepcidin expression such as upstream stimulatory factors (USF1 and USF2) and erythropoeisis (e.g., growth differentiation factor 15 and twisted gastrulation homolog 1) are also not discussed in this article. Further elaboration of these genes and others in this study could help us to further the understanding of iron and inflammatory balance by STAT3.

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