Hepcidin Activation During Inflammation: Make It STAT
2007; Elsevier BV; Volume: 132; Issue: 1 Linguagem: Inglês
10.1053/j.gastro.2006.11.049
ISSN1528-0012
Autores Tópico(s)MicroRNA in disease regulation
ResumoSee “STAT3 is required for IL-6–gp130–dependent activation of hepcidin in vivo” by Pietrangelo A, Dierssen U, Valli L, Garuti C, Rump A, Corradini E, Ernst M, Klein C, and Trautwein C on page 294.The central role for the peptide hormone hepcidin in the regulation of iron metabolism is increasingly apparent.1Ganz T. Hepcidin—a peptide hormone at the interface of innate immunity and iron metabolism.Curr Top Microbiol Immunol. 2006; 306: 183-198Crossref PubMed Scopus (118) Google Scholar By serving as a negative regulator of iron efflux from absorptive enterocytes and from reticuloendothelial macrophages, hepcidin influences both dietary iron absorption and tissue distribution. In iron deficiency, hepatocellular production decreases, thereby maximizing dietary iron absorption and mobilization of iron stores for use in erythropoiesis. By contrast, hepcidin production increases during inflammation, bringing about a decrease in dietary iron absorption and sequestration of iron in reticuloendothelial macrophages. These latter changes characterize the hypoferremia of inflammation and contribute to the hematologic changes observed in the anemia of chronic diseases. The hypoferremia in this setting is thought to disadvantage the proliferation of certain infectious organisms in the bloodstream. As such, hepcidin can be considered an acute phase plasma protein, which contributes to host defense. Several studies have demonstrated that interleukin (IL)-6 is an important activator of hepcidin expression during inflammation. The signal transduction pathway through which hepcidin is regulated by IL-6 has been the focus of several recent publications.2Wrighting D.M. Andrews N.C. Interleukin-6 induces hepcidin expression through STAT3.Blood. 2006; 108: 3204-3209Crossref PubMed Scopus (686) Google Scholar, 3Verga Falzacappa M.V. Vujic Spasic M. Kessler R. Stolte J. Hentze M.W. Muckenthaler M.U. STAT-3 mediates hepatic hepcidin expression and its inflammatory stimulation.Blood. 2006; (Aug 31); [Epub ahead of print]PubMed Google Scholar, 4Pietrangelo A. Dierrsen U. Valli L. Garuti C. Rump A. Corradini E. Klein C. Trautwein C. STAT3 is required for IL-6-GP130-dependent activation of hepcidin in vivo.Gastroenterology. 2007; 132: 294-300Abstract Full Text Full Text PDF PubMed Scopus (243) Google ScholarAlthough IL-6 signal transduction has a number of complexities,5Heinrich P.C. Behrmann I. Haan S. Hermanns H.M. Muller-Newen G. Schaper F. Principles of interleukin (IL)-6-type cytokine signalling and its regulation.Biochem J. 2003; 374: 1-20Crossref PubMed Scopus (2462) Google Scholar the prototypic pathway can be summarized as follows. Released primarily by Kupffer cells, IL-6 interacts with receptors on hepatocytes to affect the rate of transcription of target genes. The IL-6 receptor complex consists of 2 major subunits, an 80-kDa α subunit (IL-6-R) with ligand specificity and a 130-kDa β subunit (gp130) with signal transduction capability (Figure 1). Binding of IL-6 to the IL-6-R subunit effects homodimerization of gp130. This dimerization recruits cytoplasmic JAK (Janus kinase) proteins to phosphorylate gp130.5Heinrich P.C. Behrmann I. Haan S. Hermanns H.M. Muller-Newen G. Schaper F. Principles of interleukin (IL)-6-type cytokine signalling and its regulation.Biochem J. 2003; 374: 1-20Crossref PubMed Scopus (2462) Google Scholar The phosphorylation of gp130 then activates 2 distinct downstream pathways: (1) the MAPK (mitogen-activated protein kinase) pathway and (2) the STAT (signal transducer and activator of transcription) pathway. Activation of the MAPK pathway requires interaction of SHP2 with a specific domain on gp130, which includes a critical tyrosine residue (Y757). Downstream events in the MAPK pathway culminate in the nuclear translocation of ERK (extracellular signal-regulated kinase) proteins, which in turn influence the activities of specific transcription factors. The second or STAT pathway does not require Y757 on gp130. Upon phosphorylation of gp130 by JAK, certain STAT proteins (STAT1 or STAT3) bind to a distinct gp130 domain, and are themselves phosphorylated. They are then released from gp130, dimerize, and translocate to the nucleus. In the nucleus the STAT dimers bind to specific cis-acting genomic elements to influence the transcription of genes participating in the acute phase response.In this issue of Gastroenterology, Pietrangelo et al4Pietrangelo A. Dierrsen U. Valli L. Garuti C. Rump A. Corradini E. Klein C. Trautwein C. STAT3 is required for IL-6-GP130-dependent activation of hepcidin in vivo.Gastroenterology. 2007; 132: 294-300Abstract Full Text Full Text PDF PubMed Scopus (243) Google Scholar make innovative use of murine model systems to dissect the contribution of gp130 to the regulation of hepcidin expression by IL-6. Three transgenic mouse models were used, each with a different hepatocellular-specific alteration in gp130 expression: (1) gp130 knockout; (2) mutation of the tyrosine (Y757) residue essential for activation of the MAPK (but not STAT) signaling pathway; and (3) truncation of gp130 to remove the STAT (but not the MAPK) signaling domain.As expected, wild-type mice demonstrated an increase in liver hepcidin expression in response to IL-6. However, mice with the hepatocellular knockout of gp130 did not. The investigators found that mice with the hepatocellular Y757 gp130 mutation retained the IL-6 mediated activation of hepcidin expression, suggesting that activation of the MAPK pathway was not essential. However, mice with the defect in the gp130 STAT pathway had no change in hepcidin expression in response to IL-6. Based on the additional finding that IL-6 induces hepatic expression of phospho-STAT3, but not phospho-STAT1, the authors conclude that STAT3 is the key transcription factor responsible for IL-6–induced activation of hepcidin gene expression. This report represents the first in vivo demonstration of the role of the gp130/STAT3 pathway in the regulation of hepcidin.The investigators performed additional studies using cultured hepatocytes from the same mouse model systems. The cell culture results confirmed the participation of the gp130/STAT pathway in the activation of hepcidin by IL-6. However, unlike the situation in vivo, the cultured hepatocytes carrying the gp130 Y757 mutation showed an enhanced hepcidin response to IL-6. Although this suggests a possible role for the MAPK pathway in the activation of hepcidin by IL-6, the authors propose another explanation. They speculate that the enhanced response is due to the requirement of gp130 Y757 for not only MAPK activation, but also for binding of SOCS3 (suppressor of cytokine signaling). SOCS3 expression is increased by IL-6 to provide a negative feedback regulation of gp130 signaling. Supporting the concept that loss of the negative feedback hyperactivates the STAT pathway is the observation that mice carrying the Y757 mutation have increased phophoSTAT3 in response to IL-6 compared with wild-type mice. It is unclear why hyperactivation of the STAT pathway increased hepcidin expression in cell culture but not in vivo. Regardless of residual questions regarding the potential role of the MAPK pathway, the authors effectively demonstrate the role of gp130-mediated activation of STAT3 in the regulation of hepcidin expression by IL-6.Two recently published studies on the regulation of hepcidin by IL-6 have dissected events downstream of gp130. Wrighting and Andrews2Wrighting D.M. Andrews N.C. Interleukin-6 induces hepcidin expression through STAT3.Blood. 2006; 108: 3204-3209Crossref PubMed Scopus (686) Google Scholar used the human hepatoma cell line HepG2 to study the potential involvement of STAT3 on hepcidin gene transcription. Using promoter-reporter gene constructs they demonstrated that activation of hepcidin by IL-6 depended on a region containing a putative STAT3 binding element. STAT3 was shown moreover by chromatin immunoprecipitation to interact directly with this region of the hepcidin promoter. The participation of STAT3 was functionally confirmed by using RNAi, as well as dominant-negative constructs. Verga Falzacappa et al3Verga Falzacappa M.V. Vujic Spasic M. Kessler R. Stolte J. Hentze M.W. Muckenthaler M.U. STAT-3 mediates hepatic hepcidin expression and its inflammatory stimulation.Blood. 2006; (Aug 31); [Epub ahead of print]PubMed Google Scholar performed similar studies in the human liver cell line Huh7, and narrowed the STAT3 element to the sequence TTCTTGGAA located 64 bp upstream of the hepcidin promoter. Furthermore, they reported that the response to conditioned medium from a monocyte/macrophage cell line was dependent on this element. Interestingly, both studies demonstrated that STAT3 is required not only for the inflammatory response, but also for steady state hepcidin mRNA expression under control culture conditions.The effect of inflammation on hepcidin may not be entirely mediated by IL-6. For example, IL-6 knockout mice maintain some hepcidin responsiveness to LPS.6Lee P. Peng H. Gelbart T. Wang L. Beutler E. Regulation of hepcidin transcription by interleukin-1 and interleukin-6.Proc Natl Acad Sci U S A. 2005; 102: 1906-1910Crossref PubMed Scopus (436) Google Scholar Moreover, the combination of anti–IL-6 and anti–IL-1 neutralizing antibodies were necessary to abrogate completely the stimulatory effect of conditioned macrophage media on hepcidin in cultured mouse hepatocytes. Although IL-1 can induce IL-6 production in hepatocytes, studies indicate that IL-1 has a direct action in stimulating hepcidin expression in both mouse hepatocytes and human HuH7 cells.6Lee P. Peng H. Gelbart T. Wang L. Beutler E. Regulation of hepcidin transcription by interleukin-1 and interleukin-6.Proc Natl Acad Sci U S A. 2005; 102: 1906-1910Crossref PubMed Scopus (436) Google Scholar, 7Inamura J. Ikuta K. Jimbo J. Shindo M. Sato K. Torimoto Y. Kohgo Y. Upregulation of hepcidin by interleukin-1β in human hepatoma cell lines.Hepatol Res. 2005; 33: 198-205Crossref PubMed Scopus (37) Google Scholar The signal-transduction pathway responsible for the effect of IL-1 on hepcidin expression remains to be elucidated.In addition to the gp130/STAT3 pathway, another signal transduction pathway to hepcidin is the transforming growth factor-β/bone morphogenetic protein (TGF-β/BMP) superfamily. The role for this pathway was evidenced by the loss of hepcidin expression in mice with a hepatocellular-specific knockout of SMAD4.8Wang R.H. Li C. Xu X. Zheng Y. Xiao C. Zerfas P. Cooperman S. Eckhaus M. Rouault T. Mishra L. Deng C.X. A role of SMAD4 in iron metabolism through the positive regulation of hepcidin expression.Cell Metab. 2005; 2: 399-409Abstract Full Text Full Text PDF PubMed Scopus (496) Google Scholar SMAD4 is the common mediator for nuclear translocation in the signal transduction from the TGF-β/BMP superfamily (Figure 1). Further evidence was suggested by the observation that hemojuvelin, the protein mutated in the most common form of juvenile hemochromatosis, has homology to certain BMP co-receptors. Several studies have clearly demonstrated that hepcidin expression is regulated by BMPs in cell culture8Wang R.H. Li C. Xu X. Zheng Y. Xiao C. Zerfas P. Cooperman S. Eckhaus M. Rouault T. Mishra L. Deng C.X. A role of SMAD4 in iron metabolism through the positive regulation of hepcidin expression.Cell Metab. 2005; 2: 399-409Abstract Full Text Full Text PDF PubMed Scopus (496) Google Scholar, 9Babitt J.L. Huang F.W. Wrighting D.M. Xia Y. Sidis Y. Samad T.A. Campagna J.A. Chung R.T. Schneyer A.L. Woolf C.J. Andrews N.C. Lin H.Y. Bone morphogenetic protein signaling by hemojuvelin regulates hepcidin expression.Nat Genet. 2006; 38: 531-539Crossref PubMed Scopus (824) Google Scholar, 10Truksa J. Peng H. Lee P. Beutler E. Bone morphogenetic proteins 2, 4, and 9 stimulate murine hepcidin 1 expression independently of Hfe, transferrin receptor 2 (Tfr2), and IL-6.Proc Natl Acad Sci U S A. 2006; 103: 10289-10293Crossref PubMed Scopus (266) Google Scholar and that this regulation is strongly potentiated by hemojuvelin.9Babitt J.L. Huang F.W. Wrighting D.M. Xia Y. Sidis Y. Samad T.A. Campagna J.A. Chung R.T. Schneyer A.L. Woolf C.J. Andrews N.C. Lin H.Y. Bone morphogenetic protein signaling by hemojuvelin regulates hepcidin expression.Nat Genet. 2006; 38: 531-539Crossref PubMed Scopus (824) Google Scholar It is possible that the STAT3 and SMAD pathways leading to hepcidin regulation are not completely separate. Indeed, the effect of IL-6 on hepcidin expression depends on SMAD4, as evidenced by the failure of IL-6 to increase hepcidin expression in mice with hepatocellular knockout of SMAD4.8Wang R.H. Li C. Xu X. Zheng Y. Xiao C. Zerfas P. Cooperman S. Eckhaus M. Rouault T. Mishra L. Deng C.X. A role of SMAD4 in iron metabolism through the positive regulation of hepcidin expression.Cell Metab. 2005; 2: 399-409Abstract Full Text Full Text PDF PubMed Scopus (496) Google Scholar Responsiveness of other acute-phase genes to IL-6 was, however, intact in these mice. The authors suggested that effect of SMAD4 was essentially permissive, namely, that it facilitated accessibility of the hepcidin promoter to transcription factors specifically involved in IL-6 signal transduction to hepcidin. However, there is precedent for crosstalk between participants of the STAT3 and SMAD pathways in the regulation of other genes.11Jenkins B.J. Grail D. Nheu T. Najdovska M. Wang B. Waring P. Inglese M. McLoughlin R.M. Jones S.A. Topley N. Baumann H. Judd L.M. Giraud A.S. Boussioutas A. Zhu H.J. Ernst M. Hyperactivation of Stat3 in gp130 mutant mice promotes gastric hyperproliferation and desensitizes TGF-β signaling.Nat Med. 2005; 11: 845-852Crossref PubMed Scopus (253) Google Scholar, 12Zhang X.L. Topley N. Ito T. Phillips A. Interleukin-6 regulation of transforming growth factor (TGF)-β receptor compartmentalization and turnover enhances TGF-β1 signaling.J Biol Chem. 2005; 280: 12239-12245Crossref PubMed Scopus (158) Google Scholar, 13Long J. Wang G. Matsuura I. He D. Liu F. Activation of Smad transcriptional activity by protein inhibitor of activated STAT3 (PIAS3).Proc Natl Acad Sci U S A. 2004; 101: 99-104Crossref PubMed Scopus (83) Google ScholarThere has been considerable investigation into the regulation of hepcidin expression in situations other than inflammation.1Ganz T. Hepcidin—a peptide hormone at the interface of innate immunity and iron metabolism.Curr Top Microbiol Immunol. 2006; 306: 183-198Crossref PubMed Scopus (118) Google Scholar Hepcidin is up-regulated in response to dietary iron and down-regulated in response to iron deficiency, anemia, hypoxemia, or erythropoietic activity. Moreover, hepcidin expression is abnormally low in most forms of hereditary hemochromatosis. The signal transduction pathways regulating hepcidin expression in these settings are not as well delineated. The transcription factor C/EBPα is clearly involved in regulating hepcidin transcription.3Verga Falzacappa M.V. Vujic Spasic M. Kessler R. Stolte J. Hentze M.W. Muckenthaler M.U. STAT-3 mediates hepatic hepcidin expression and its inflammatory stimulation.Blood. 2006; (Aug 31); [Epub ahead of print]PubMed Google Scholar, 14Courselaud B. Pigeon C. Inoue Y. Inoue J. Gonzalez F.J. Leroyer P. Gilot D. Boudjema K. Guguen-Guillouzo C. Brissot P. Loreal O. Ilyin G. C/EBPα regulates hepatic transcription of hepcidin, an antimicrobial peptide and regulator of iron metabolism.J Biol Chem. 2002; 277: 41163-41170Crossref PubMed Scopus (225) Google Scholar C/EBPα knockout mice demonstrate decreased hepcidin expression and iron overload.14Courselaud B. Pigeon C. Inoue Y. Inoue J. Gonzalez F.J. Leroyer P. Gilot D. Boudjema K. Guguen-Guillouzo C. Brissot P. Loreal O. Ilyin G. C/EBPα regulates hepatic transcription of hepcidin, an antimicrobial peptide and regulator of iron metabolism.J Biol Chem. 2002; 277: 41163-41170Crossref PubMed Scopus (225) Google Scholar Moreover, mice with dietary iron loading demonstrate increased hepatic expression of C/EBPα protein, potentially contributing to the iron-induced increase in hepcidin expression.14Courselaud B. Pigeon C. Inoue Y. Inoue J. Gonzalez F.J. Leroyer P. Gilot D. Boudjema K. Guguen-Guillouzo C. Brissot P. Loreal O. Ilyin G. C/EBPα regulates hepatic transcription of hepcidin, an antimicrobial peptide and regulator of iron metabolism.J Biol Chem. 2002; 277: 41163-41170Crossref PubMed Scopus (225) Google Scholar Other key molecules involved in hepcidin regulation include HFE and transferrin receptor 2 (TfR2). Mutations in each of these genes results in decreased hepcidin expression and consequently hemochromatosis. Cultured hepatocytes from HFE knockout and TfR2 mutant mice retain the ability to respond to BMPs or to IL-6 with an increase in hepcidin expression.6Lee P. Peng H. Gelbart T. Wang L. Beutler E. Regulation of hepcidin transcription by interleukin-1 and interleukin-6.Proc Natl Acad Sci U S A. 2005; 102: 1906-1910Crossref PubMed Scopus (436) Google Scholar, 10Truksa J. Peng H. Lee P. Beutler E. Bone morphogenetic proteins 2, 4, and 9 stimulate murine hepcidin 1 expression independently of Hfe, transferrin receptor 2 (Tfr2), and IL-6.Proc Natl Acad Sci U S A. 2006; 103: 10289-10293Crossref PubMed Scopus (266) Google Scholar Although this result suggests their participation in a separate pathway, it remains a possibility that HFE and TfR2 facilitate basal hepcidin signaling through the TGFβ/BMP-SMAD or the gp130/STAT pathway.In summary, the major signal transduction pathway responsible for the regulation of hepcidin by IL-6 has now been defined. The current study by Pietrangelo et al,4Pietrangelo A. Dierrsen U. Valli L. Garuti C. Rump A. Corradini E. Klein C. Trautwein C. STAT3 is required for IL-6-GP130-dependent activation of hepcidin in vivo.Gastroenterology. 2007; 132: 294-300Abstract Full Text Full Text PDF PubMed Scopus (243) Google Scholar taken together with other recent reports,2Wrighting D.M. Andrews N.C. Interleukin-6 induces hepcidin expression through STAT3.Blood. 2006; 108: 3204-3209Crossref PubMed Scopus (686) Google Scholar, 3Verga Falzacappa M.V. Vujic Spasic M. Kessler R. Stolte J. Hentze M.W. Muckenthaler M.U. STAT-3 mediates hepatic hepcidin expression and its inflammatory stimulation.Blood. 2006; (Aug 31); [Epub ahead of print]PubMed Google Scholar have convincingly demonstrated the central role of the gp130/STAT3 signaling pathway in the activation of hepcidin during inflammation. Much exciting work remains in defining the role of this and other signaling pathways in the regulation of hepcidin by physiologic stimuli and in the pathogenesis of hereditary hemochromatosis. See “STAT3 is required for IL-6–gp130–dependent activation of hepcidin in vivo” by Pietrangelo A, Dierssen U, Valli L, Garuti C, Rump A, Corradini E, Ernst M, Klein C, and Trautwein C on page 294. See “STAT3 is required for IL-6–gp130–dependent activation of hepcidin in vivo” by Pietrangelo A, Dierssen U, Valli L, Garuti C, Rump A, Corradini E, Ernst M, Klein C, and Trautwein C on page 294. See “STAT3 is required for IL-6–gp130–dependent activation of hepcidin in vivo” by Pietrangelo A, Dierssen U, Valli L, Garuti C, Rump A, Corradini E, Ernst M, Klein C, and Trautwein C on page 294. The central role for the peptide hormone hepcidin in the regulation of iron metabolism is increasingly apparent.1Ganz T. Hepcidin—a peptide hormone at the interface of innate immunity and iron metabolism.Curr Top Microbiol Immunol. 2006; 306: 183-198Crossref PubMed Scopus (118) Google Scholar By serving as a negative regulator of iron efflux from absorptive enterocytes and from reticuloendothelial macrophages, hepcidin influences both dietary iron absorption and tissue distribution. In iron deficiency, hepatocellular production decreases, thereby maximizing dietary iron absorption and mobilization of iron stores for use in erythropoiesis. By contrast, hepcidin production increases during inflammation, bringing about a decrease in dietary iron absorption and sequestration of iron in reticuloendothelial macrophages. These latter changes characterize the hypoferremia of inflammation and contribute to the hematologic changes observed in the anemia of chronic diseases. The hypoferremia in this setting is thought to disadvantage the proliferation of certain infectious organisms in the bloodstream. As such, hepcidin can be considered an acute phase plasma protein, which contributes to host defense. Several studies have demonstrated that interleukin (IL)-6 is an important activator of hepcidin expression during inflammation. The signal transduction pathway through which hepcidin is regulated by IL-6 has been the focus of several recent publications.2Wrighting D.M. Andrews N.C. Interleukin-6 induces hepcidin expression through STAT3.Blood. 2006; 108: 3204-3209Crossref PubMed Scopus (686) Google Scholar, 3Verga Falzacappa M.V. Vujic Spasic M. Kessler R. Stolte J. Hentze M.W. Muckenthaler M.U. STAT-3 mediates hepatic hepcidin expression and its inflammatory stimulation.Blood. 2006; (Aug 31); [Epub ahead of print]PubMed Google Scholar, 4Pietrangelo A. Dierrsen U. Valli L. Garuti C. Rump A. Corradini E. Klein C. Trautwein C. STAT3 is required for IL-6-GP130-dependent activation of hepcidin in vivo.Gastroenterology. 2007; 132: 294-300Abstract Full Text Full Text PDF PubMed Scopus (243) Google Scholar Although IL-6 signal transduction has a number of complexities,5Heinrich P.C. Behrmann I. Haan S. Hermanns H.M. Muller-Newen G. Schaper F. Principles of interleukin (IL)-6-type cytokine signalling and its regulation.Biochem J. 2003; 374: 1-20Crossref PubMed Scopus (2462) Google Scholar the prototypic pathway can be summarized as follows. Released primarily by Kupffer cells, IL-6 interacts with receptors on hepatocytes to affect the rate of transcription of target genes. The IL-6 receptor complex consists of 2 major subunits, an 80-kDa α subunit (IL-6-R) with ligand specificity and a 130-kDa β subunit (gp130) with signal transduction capability (Figure 1). Binding of IL-6 to the IL-6-R subunit effects homodimerization of gp130. This dimerization recruits cytoplasmic JAK (Janus kinase) proteins to phosphorylate gp130.5Heinrich P.C. Behrmann I. Haan S. Hermanns H.M. Muller-Newen G. Schaper F. Principles of interleukin (IL)-6-type cytokine signalling and its regulation.Biochem J. 2003; 374: 1-20Crossref PubMed Scopus (2462) Google Scholar The phosphorylation of gp130 then activates 2 distinct downstream pathways: (1) the MAPK (mitogen-activated protein kinase) pathway and (2) the STAT (signal transducer and activator of transcription) pathway. Activation of the MAPK pathway requires interaction of SHP2 with a specific domain on gp130, which includes a critical tyrosine residue (Y757). Downstream events in the MAPK pathway culminate in the nuclear translocation of ERK (extracellular signal-regulated kinase) proteins, which in turn influence the activities of specific transcription factors. The second or STAT pathway does not require Y757 on gp130. Upon phosphorylation of gp130 by JAK, certain STAT proteins (STAT1 or STAT3) bind to a distinct gp130 domain, and are themselves phosphorylated. They are then released from gp130, dimerize, and translocate to the nucleus. In the nucleus the STAT dimers bind to specific cis-acting genomic elements to influence the transcription of genes participating in the acute phase response. In this issue of Gastroenterology, Pietrangelo et al4Pietrangelo A. Dierrsen U. Valli L. Garuti C. Rump A. Corradini E. Klein C. Trautwein C. STAT3 is required for IL-6-GP130-dependent activation of hepcidin in vivo.Gastroenterology. 2007; 132: 294-300Abstract Full Text Full Text PDF PubMed Scopus (243) Google Scholar make innovative use of murine model systems to dissect the contribution of gp130 to the regulation of hepcidin expression by IL-6. Three transgenic mouse models were used, each with a different hepatocellular-specific alteration in gp130 expression: (1) gp130 knockout; (2) mutation of the tyrosine (Y757) residue essential for activation of the MAPK (but not STAT) signaling pathway; and (3) truncation of gp130 to remove the STAT (but not the MAPK) signaling domain. As expected, wild-type mice demonstrated an increase in liver hepcidin expression in response to IL-6. However, mice with the hepatocellular knockout of gp130 did not. The investigators found that mice with the hepatocellular Y757 gp130 mutation retained the IL-6 mediated activation of hepcidin expression, suggesting that activation of the MAPK pathway was not essential. However, mice with the defect in the gp130 STAT pathway had no change in hepcidin expression in response to IL-6. Based on the additional finding that IL-6 induces hepatic expression of phospho-STAT3, but not phospho-STAT1, the authors conclude that STAT3 is the key transcription factor responsible for IL-6–induced activation of hepcidin gene expression. This report represents the first in vivo demonstration of the role of the gp130/STAT3 pathway in the regulation of hepcidin. The investigators performed additional studies using cultured hepatocytes from the same mouse model systems. The cell culture results confirmed the participation of the gp130/STAT pathway in the activation of hepcidin by IL-6. However, unlike the situation in vivo, the cultured hepatocytes carrying the gp130 Y757 mutation showed an enhanced hepcidin response to IL-6. Although this suggests a possible role for the MAPK pathway in the activation of hepcidin by IL-6, the authors propose another explanation. They speculate that the enhanced response is due to the requirement of gp130 Y757 for not only MAPK activation, but also for binding of SOCS3 (suppressor of cytokine signaling). SOCS3 expression is increased by IL-6 to provide a negative feedback regulation of gp130 signaling. Supporting the concept that loss of the negative feedback hyperactivates the STAT pathway is the observation that mice carrying the Y757 mutation have increased phophoSTAT3 in response to IL-6 compared with wild-type mice. It is unclear why hyperactivation of the STAT pathway increased hepcidin expression in cell culture but not in vivo. Regardless of residual questions regarding the potential role of the MAPK pathway, the authors effectively demonstrate the role of gp130-mediated activation of STAT3 in the regulation of hepcidin expression by IL-6. Two recently published studies on the regulation of hepcidin by IL-6 have dissected events downstream of gp130. Wrighting and Andrews2Wrighting D.M. Andrews N.C. Interleukin-6 induces hepcidin expression through STAT3.Blood. 2006; 108: 3204-3209Crossref PubMed Scopus (686) Google Scholar used the human hepatoma cell line HepG2 to study the potential involvement of STAT3 on hepcidin gene transcription. Using promoter-reporter gene constructs they demonstrated that activation of hepcidin by IL-6 depended on a region containing a putative STAT3 binding element. STAT3 was shown moreover by chromatin immunoprecipitation to interact directly with this region of the hepcidin promoter. The participation of STAT3 was functionally confirmed by using RNAi, as well as dominant-negative constructs. Verga Falzacappa et al3Verga Falzacappa M.V. Vujic Spasic M. Kessler R. Stolte J. Hentze M.W. Muckenthaler M.U. STAT-3 mediates hepatic hepcidin expression and its inflammatory stimulation.Blood. 2006; (Aug 31); [Epub ahead of print]PubMed Google Scholar performed similar studies in the human liver cell line Huh7, and narrowed the STAT3 element to the sequence TTCTTGGAA located 64 bp upstream of the hepcidin promoter. Furthermore, they reported that the response to conditioned medium from a monocyte/macrophage cell line was dependent on this element. Interestingly, both studies demonstrated that STAT3 is required not only for the inflammatory response, but also for steady state hepcidin mRNA expression under control culture conditions. The effect of inflammation on hepcidin may not be entirely mediated by IL-6. For example, IL-6 knockout mice maintain some hepcidin responsiveness to LPS.6Lee P. Peng H. Gelbart T. Wang L. Beutler E. Regulation of hepcidin transcription by interleukin-1 and interleukin-6.Proc Natl Acad Sci U S A. 2005; 102: 1906-1910Crossref PubMed Scopus (436) Google Scholar Moreover, the combination of anti–IL-6 and anti–IL-1 neutralizing antibodies were necessary to abrogate completely the stimulatory effect of conditioned macrophage media on hepcidin in cultured mouse hepatocytes. Although IL-1 can induce IL-6 production in hepatocytes, studies indicate that IL-1 has a direct action in stimulating hepcidin expression in both mouse hepatocytes and human HuH7 cells.6Lee P. Peng H. Gelbart T. Wang L. Beutler E. Regulation of hepcidin transcription by interleukin-1 and interleukin-6.Proc Natl Acad Sci U S A. 2005; 102: 1906-1910Crossref PubMed Scopus (436) Google Scholar, 7Inamura J. Ikuta K. Jimbo J. Shindo M. Sato K. Torimoto Y. Kohgo Y. Upregulation of hepcidin by interleukin-1β in human hepatoma cell lines.Hepatol Res. 2005; 33: 198-205Crossref PubMed Scopus (37) Google Scholar The signal-transduction pathway responsible for the effect of IL-1 on hepcidin expression remains to be elucidated. In addition to the gp130/STAT3 pathway, another signal transduction pathway to hepcidin is the transforming growth factor-β/bone morphogenetic protein (TGF-β/BMP) superfamily. The role for this pathway was evidenced by the loss of hepcidin expression in mice with a hepatocellular-specific knockout of SMAD4.8Wang R.H. Li C. Xu X. Zheng Y. Xiao C. Zerfas P. Cooperman S. Eckhaus M. Rouault T. Mishra L. Deng C.X. A role of SMAD4 in iron metabolism through the positive regulation of hepcidin expression.Cell Metab. 2005; 2: 399-409Abstract Full Text Full Text PDF PubMed Scopus (496) Google Scholar SMAD4 is the common mediator for nuclear translocation in the signal transduction from the TGF-β/BMP superfamily (Figure 1). Further evidence was suggested by the observation that hemojuvelin, the protein mutated in the most common form of juvenile hemochromatosis, has homology to certain BMP co-receptors. Several studies have clearly demonstrated that hepcidin expression is regulated by BMPs in cell culture8Wang R.H. Li C. Xu X. Zheng Y. Xiao C. Zerfas P. Cooperman S. Eckhaus M. Rouault T. Mishra L. Deng C.X. A role of SMAD4 in iron metabolism through the positive regulation of hepcidin expression.Cell Metab. 2005; 2: 399-409Abstract Full Text Full Text PDF PubMed Scopus (496) Google Scholar, 9Babitt J.L. Huang F.W. Wrighting D.M. Xia Y. Sidis Y. Samad T.A. Campagna J.A. Chung R.T. Schneyer A.L. Woolf C.J. Andrews N.C. Lin H.Y. Bone morphogenetic protein signaling by hemojuvelin regulates hepcidin expression.Nat Genet. 2006; 38: 531-539Crossref PubMed Scopus (824) Google Scholar, 10Truksa J. Peng H. Lee P. Beutler E. Bone morphogenetic proteins 2, 4, and 9 stimulate murine hepcidin 1 expression independently of Hfe, transferrin receptor 2 (Tfr2), and IL-6.Proc Natl Acad Sci U S A. 2006; 103: 10289-10293Crossref PubMed Scopus (266) Google Scholar and that this regulation is strongly potentiated by hemojuvelin.9Babitt J.L. Huang F.W. Wrighting D.M. Xia Y. Sidis Y. Samad T.A. Campagna J.A. Chung R.T. Schneyer A.L. Woolf C.J. Andrews N.C. Lin H.Y. Bone morphogenetic protein signaling by hemojuvelin regulates hepcidin expression.Nat Genet. 2006; 38: 531-539Crossref PubMed Scopus (824) Google Scholar It is possible that the STAT3 and SMAD pathways leading to hepcidin regulation are not completely separate. Indeed, the effect of IL-6 on hepcidin expression depends on SMAD4, as evidenced by the failure of IL-6 to increase hepcidin expression in mice with hepatocellular knockout of SMAD4.8Wang R.H. Li C. Xu X. Zheng Y. Xiao C. Zerfas P. Cooperman S. Eckhaus M. Rouault T. Mishra L. Deng C.X. A role of SMAD4 in iron metabolism through the positive regulation of hepcidin expression.Cell Metab. 2005; 2: 399-409Abstract Full Text Full Text PDF PubMed Scopus (496) Google Scholar Responsiveness of other acute-phase genes to IL-6 was, however, intact in these mice. The authors suggested that effect of SMAD4 was essentially permissive, namely, that it facilitated accessibility of the hepcidin promoter to transcription factors specifically involved in IL-6 signal transduction to hepcidin. However, there is precedent for crosstalk between participants of the STAT3 and SMAD pathways in the regulation of other genes.11Jenkins B.J. Grail D. Nheu T. Najdovska M. Wang B. Waring P. Inglese M. McLoughlin R.M. Jones S.A. Topley N. Baumann H. Judd L.M. Giraud A.S. Boussioutas A. Zhu H.J. Ernst M. Hyperactivation of Stat3 in gp130 mutant mice promotes gastric hyperproliferation and desensitizes TGF-β signaling.Nat Med. 2005; 11: 845-852Crossref PubMed Scopus (253) Google Scholar, 12Zhang X.L. Topley N. Ito T. Phillips A. Interleukin-6 regulation of transforming growth factor (TGF)-β receptor compartmentalization and turnover enhances TGF-β1 signaling.J Biol Chem. 2005; 280: 12239-12245Crossref PubMed Scopus (158) Google Scholar, 13Long J. Wang G. Matsuura I. He D. Liu F. Activation of Smad transcriptional activity by protein inhibitor of activated STAT3 (PIAS3).Proc Natl Acad Sci U S A. 2004; 101: 99-104Crossref PubMed Scopus (83) Google Scholar There has been considerable investigation into the regulation of hepcidin expression in situations other than inflammation.1Ganz T. Hepcidin—a peptide hormone at the interface of innate immunity and iron metabolism.Curr Top Microbiol Immunol. 2006; 306: 183-198Crossref PubMed Scopus (118) Google Scholar Hepcidin is up-regulated in response to dietary iron and down-regulated in response to iron deficiency, anemia, hypoxemia, or erythropoietic activity. Moreover, hepcidin expression is abnormally low in most forms of hereditary hemochromatosis. The signal transduction pathways regulating hepcidin expression in these settings are not as well delineated. The transcription factor C/EBPα is clearly involved in regulating hepcidin transcription.3Verga Falzacappa M.V. Vujic Spasic M. Kessler R. Stolte J. Hentze M.W. Muckenthaler M.U. STAT-3 mediates hepatic hepcidin expression and its inflammatory stimulation.Blood. 2006; (Aug 31); [Epub ahead of print]PubMed Google Scholar, 14Courselaud B. Pigeon C. Inoue Y. Inoue J. Gonzalez F.J. Leroyer P. Gilot D. Boudjema K. Guguen-Guillouzo C. Brissot P. Loreal O. Ilyin G. C/EBPα regulates hepatic transcription of hepcidin, an antimicrobial peptide and regulator of iron metabolism.J Biol Chem. 2002; 277: 41163-41170Crossref PubMed Scopus (225) Google Scholar C/EBPα knockout mice demonstrate decreased hepcidin expression and iron overload.14Courselaud B. Pigeon C. Inoue Y. Inoue J. Gonzalez F.J. Leroyer P. Gilot D. Boudjema K. Guguen-Guillouzo C. Brissot P. Loreal O. Ilyin G. C/EBPα regulates hepatic transcription of hepcidin, an antimicrobial peptide and regulator of iron metabolism.J Biol Chem. 2002; 277: 41163-41170Crossref PubMed Scopus (225) Google Scholar Moreover, mice with dietary iron loading demonstrate increased hepatic expression of C/EBPα protein, potentially contributing to the iron-induced increase in hepcidin expression.14Courselaud B. Pigeon C. Inoue Y. Inoue J. Gonzalez F.J. Leroyer P. Gilot D. Boudjema K. Guguen-Guillouzo C. Brissot P. Loreal O. Ilyin G. C/EBPα regulates hepatic transcription of hepcidin, an antimicrobial peptide and regulator of iron metabolism.J Biol Chem. 2002; 277: 41163-41170Crossref PubMed Scopus (225) Google Scholar Other key molecules involved in hepcidin regulation include HFE and transferrin receptor 2 (TfR2). Mutations in each of these genes results in decreased hepcidin expression and consequently hemochromatosis. Cultured hepatocytes from HFE knockout and TfR2 mutant mice retain the ability to respond to BMPs or to IL-6 with an increase in hepcidin expression.6Lee P. Peng H. Gelbart T. Wang L. Beutler E. Regulation of hepcidin transcription by interleukin-1 and interleukin-6.Proc Natl Acad Sci U S A. 2005; 102: 1906-1910Crossref PubMed Scopus (436) Google Scholar, 10Truksa J. Peng H. Lee P. Beutler E. Bone morphogenetic proteins 2, 4, and 9 stimulate murine hepcidin 1 expression independently of Hfe, transferrin receptor 2 (Tfr2), and IL-6.Proc Natl Acad Sci U S A. 2006; 103: 10289-10293Crossref PubMed Scopus (266) Google Scholar Although this result suggests their participation in a separate pathway, it remains a possibility that HFE and TfR2 facilitate basal hepcidin signaling through the TGFβ/BMP-SMAD or the gp130/STAT pathway. In summary, the major signal transduction pathway responsible for the regulation of hepcidin by IL-6 has now been defined. The current study by Pietrangelo et al,4Pietrangelo A. Dierrsen U. Valli L. Garuti C. Rump A. Corradini E. Klein C. Trautwein C. STAT3 is required for IL-6-GP130-dependent activation of hepcidin in vivo.Gastroenterology. 2007; 132: 294-300Abstract Full Text Full Text PDF PubMed Scopus (243) Google Scholar taken together with other recent reports,2Wrighting D.M. Andrews N.C. Interleukin-6 induces hepcidin expression through STAT3.Blood. 2006; 108: 3204-3209Crossref PubMed Scopus (686) Google Scholar, 3Verga Falzacappa M.V. Vujic Spasic M. Kessler R. Stolte J. Hentze M.W. Muckenthaler M.U. STAT-3 mediates hepatic hepcidin expression and its inflammatory stimulation.Blood. 2006; (Aug 31); [Epub ahead of print]PubMed Google Scholar have convincingly demonstrated the central role of the gp130/STAT3 signaling pathway in the activation of hepcidin during inflammation. Much exciting work remains in defining the role of this and other signaling pathways in the regulation of hepcidin by physiologic stimuli and in the pathogenesis of hereditary hemochromatosis. STAT3 Is Required for IL-6-gp130–Dependent Activation of Hepcidin In VivoGastroenterologyVol. 132Issue 1PreviewBackground & Aims: Hepcidin is a peptide hormone that is central to the regulation of iron homeostasis. In response to interleukin 6 (IL-6), hepatocytes produce hepcidin that decreases iron release/transfer from enterocytes and macrophages and causes hypoferremia. To clarify the molecular pathways involved in hepcidin activation by IL-6, we used different mice strains in which the main IL-6/gp130 signaling pathways have been genetically disrupted. Methods: We generated mice with hepatocyte-specific deletion of the IL-6 signal-transducing gp130 receptor (alfpgp130 LoxP/LoxP), with a gp130 receptor lacking the essential region for STAT1 and -3 activation (alfpCre gp130ΔSTAT/LoxP) or mice expressing a gp130 allele lacking the essential tyrosine for RAS-MAPK activation (alfpCregp130Y757F/LoxP). Full-Text PDF
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