STAT3 Is Required for IL-6-gp130–Dependent Activation of Hepcidin In Vivo

Background & 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). We studied gp130-dependent pathways and hepcidin mRNA expression by Western blot, reverse-transcription polymerase chain reaction, and Northern blot in vivo and ex vivo. Results: IL-6 stimulated phospho STAT3, serum amyloid A (SAA), and suppressor of cytokine signaling 3 (SOCS3) expression in livers of wild-type and alfpCregp130Y757F/LoxP mice, whereas this response was blocked in alfpCre gp130LoxP/LoxP and alfpCre gp130ΔSTAT/LoxP mice. In wild-type and alfpCregp130Y757F/LoxP animals, significantly higher hepcidin mRNA expression was found 3 to 6 hours after IL-6 stimulation. In contrast, no IL-6-dependent regulation of hepcidin mRNA expression was found in alfpgp130 ΔSTAT/LoxP and AlfpCre gp130 LoxP/LoxP animals. In primary hepatocytes, higher hepcidin mRNA expression after IL-6 stimulation was only observed when gp130-STAT3-dependent signaling was intact. Conclusions: We have demonstrated that both in vivo and in vitro STAT3 is the key transcription factor responsible for IL-6 activation of hepcidin gene expression in the liver. Background & 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). We studied gp130-dependent pathways and hepcidin mRNA expression by Western blot, reverse-transcription polymerase chain reaction, and Northern blot in vivo and ex vivo. Results: IL-6 stimulated phospho STAT3, serum amyloid A (SAA), and suppressor of cytokine signaling 3 (SOCS3) expression in livers of wild-type and alfpCregp130Y757F/LoxP mice, whereas this response was blocked in alfpCre gp130LoxP/LoxP and alfpCre gp130ΔSTAT/LoxP mice. In wild-type and alfpCregp130Y757F/LoxP animals, significantly higher hepcidin mRNA expression was found 3 to 6 hours after IL-6 stimulation. In contrast, no IL-6-dependent regulation of hepcidin mRNA expression was found in alfpgp130 ΔSTAT/LoxP and AlfpCre gp130 LoxP/LoxP animals. In primary hepatocytes, higher hepcidin mRNA expression after IL-6 stimulation was only observed when gp130-STAT3-dependent signaling was intact. Conclusions: We have demonstrated that both in vivo and in vitro STAT3 is the key transcription factor responsible for IL-6 activation of hepcidin gene expression in the liver. See editorial on page 447. See editorial on page 447. Hepcidin is an antimicrobial peptide produced by the liver in response to inflammatory stimuli and iron.1Krause A. Neitz S. Magert H.J. et al.LEAP-1, a novel highly disulfide-bonded human peptide, exhibits antimicrobial activity.FEBS Lett. 2000; 480: 147-150Abstract Full Text Full Text PDF PubMed Scopus (1109) Google Scholar, 2Park C.H. Valore E.V. Waring A.J. Ganz T. Hepcidin, a urinary antimicrobial peptide synthesized in the liver.J Biol Chem. 2001; 276: 7806-7810Crossref PubMed Scopus (1806) Google Scholar, 3Pigeon C. Ilyin G. Courselaud B. et al.A new mouse liver-specific gene, encoding a protein homologous to human antimicrobial peptide hepcidin, is overexpressed during iron overload.J Biol Chem. 2001; 276: 7811-7819Crossref PubMed Scopus (1448) Google Scholar It is the master regulator of iron homeostasis and is involved in the pathogenesis of anemia of inflammation and hemochromatosis.4Ganz T. Hepcidin in iron metabolism.Curr Opin Hematol. 2004; 11: 251-254Crossref PubMed Scopus (151) Google Scholar, 5Pietrangelo A. Trautwein C. Mechanisms of disease: the role of hepcidin in iron homeostasis—implications for hemochromatosis and other disorders.Nat Clin Pract Gastroenterol Hepatol. 2004; 1: 3945Crossref Scopus (57) Google Scholar Evidence from transgenic mouse models indicates that hepcidin is the principal down-regulator of the transport of iron across the small intestine and the placenta and its release from macrophages. Mice with genetic disruption of either the upstream stimulatory factor 2 (USF2), which fail to express either copy of the duplicated hepcidin genes immediately downstream of USF2,6Nicolas G. Bennoun M. Devaux I. et al.Lack of hepcidin gene expression and severe tissue iron overload in upstream stimulatory factor 2 (USF2) knockout mice.Proc Natl Acad Sci U S A. 2001; 98: 8780-8785Crossref PubMed Scopus (1097) Google Scholar or C-EBPa,7Courselaud B. Pigeon C. Inoue Y. et al.C/EBPalpha regulates hepatic transcription of hepcidin, an antimicrobial peptide and regulator of iron metabolism Cross-talk between C/EBP pathway and iron metabolism.J Biol Chem. 2002; 277: 41163-41170Crossref PubMed Scopus (229) Google Scholar required for hepcidin transcriptional control, develop iron overload resembling hemochromatosis. Transgenic animals over expressing hepcidin die perinatally due to severe iron-deficiency anemia occurring in the context of reticuloendothelial cell iron overload.8Nicolas G. Bennoun M. Porteu A. et al.Severe iron deficiency anemia in transgenic mice expressing liver hepcidin.Proc Natl Acad Sci U S A. 2002; 99: 4596-4601Crossref PubMed Scopus (773) Google Scholar In vivo injection of hepcidin into mice significantly reduced mucosal iron uptake and transfer to the carcass, independently on iron status or presence of HFE, the hemochromatosis protein,9Laftah A.H. Ramesh B. Simpson R.J. et al.Effect of hepcidin on intestinal iron absorption in mice.Blood. 2004; 103: 3940-3944Crossref PubMed Scopus (188) Google Scholar or induces hypoferremia in humans.10Rivera S. Nemeth E. Gabayan V. Lopez M.A. Farshidi D. Ganz T. Synthetic hepcidin causes rapid dose-dependent hypoferremia and is concentrated in ferroportin-containing organs.Blood. 2005; 106: 2196-2199Crossref PubMed Scopus (277) Google Scholar The present view is that hepcidin down-regulates iron efflux from intestine and macrophages by interacting with the main iron export protein in mammals, ferroportin (FPN). In fact, it has been recently shown that hepcidin binds to FPN in cultured cells stably expressing FPN, and, following complex internalization, leads to FPN degradation.11Nemeth E. Tuttle M.S. Powelson J. et al.Hepcidin regulates cellular iron efflux by binding to ferroportin and inducing its internalization.Science. 2004; 306: 2090-2093Crossref PubMed Scopus (3777) Google Scholar Moreover, hepcidin is highly concentrated in organs expressing FPN.10Rivera S. Nemeth E. Gabayan V. Lopez M.A. Farshidi D. Ganz T. Synthetic hepcidin causes rapid dose-dependent hypoferremia and is concentrated in ferroportin-containing organs.Blood. 2005; 106: 2196-2199Crossref PubMed Scopus (277) Google Scholar This implies decreased FPN expression and reduced iron egress from cells such as enterocytes and macrophages whenever circulating hepcidin levels are high, namely, inflammation3Pigeon C. Ilyin G. Courselaud B. et al.A new mouse liver-specific gene, encoding a protein homologous to human antimicrobial peptide hepcidin, is overexpressed during iron overload.J Biol Chem. 2001; 276: 7811-7819Crossref PubMed Scopus (1448) Google Scholar, 12Nicolas G. Chauvet C. Viatte L. et al.The gene encoding the iron regulatory peptide hepcidin is regulated by anemia, hypoxia, and inflammation.J Clin Invest. 2002; 110: 1037-1044Crossref PubMed Scopus (1380) Google Scholar and iron overload. 3Pigeon C. Ilyin G. Courselaud B. et al.A new mouse liver-specific gene, encoding a protein homologous to human antimicrobial peptide hepcidin, is overexpressed during iron overload.J Biol Chem. 2001; 276: 7811-7819Crossref PubMed Scopus (1448) Google Scholar, 13Bridle K.R. Frazer D.M. Wilkins S.J. et al.Disrupted hepcidin regulation in HFE-associated haemochromatosis and the liver as a regulator of body iron homoeostasis.Lancet. 2003; 361: 669-673Abstract Full Text Full Text PDF PubMed Scopus (554) Google Scholar, 14Gehrke S.G. Kulaksiz H. Herrmann T. et al.Expression of hepcidin in hereditary hemochromatosis: evidence for a regulation in response to serum transferrin saturation and non-transferrin-bound iron.Blood. 2003; 102: 371-376Crossref PubMed Scopus (221) Google Scholar, 15Nemeth E. Valore E.V. Territo M. Schiller G. Lichtenstein A. Ganz T. Hepcidin, a putative mediator of anemia of inflammation, is a type II acute-phase protein.Blood. 2003; 101: 2461-2463Crossref PubMed Scopus (1213) Google Scholar Hepcidin is readily stimulated by lipopolysaccharide (LPS)3Pigeon C. Ilyin G. Courselaud B. et al.A new mouse liver-specific gene, encoding a protein homologous to human antimicrobial peptide hepcidin, is overexpressed during iron overload.J Biol Chem. 2001; 276: 7811-7819Crossref PubMed Scopus (1448) Google Scholar or turpentine12Nicolas G. Chauvet C. Viatte L. et al.The gene encoding the iron regulatory peptide hepcidin is regulated by anemia, hypoxia, and inflammation.J Clin Invest. 2002; 110: 1037-1044Crossref PubMed Scopus (1380) Google Scholar both in vivo and in vitro. This stimulation is indirect and appears to be mainly mediated by the inflammatory cytokine interleukin-6 (IL-6).15Nemeth E. Valore E.V. Territo M. Schiller G. Lichtenstein A. Ganz T. Hepcidin, a putative mediator of anemia of inflammation, is a type II acute-phase protein.Blood. 2003; 101: 2461-2463Crossref PubMed Scopus (1213) Google Scholar In fact, neutralizing antibodies against IL-6 block hepcidin induction in vitro, and IL-6 knockout (KO) mice fail to up-regulate hepcidin in response to turpentine.16Nemeth E. Rivera S. Gabayan V. et al.IL-6 mediates hypoferremia of inflammation by inducing the synthesis of the iron regulatory hormone hepcidin.J Clin Invest. 2004; 113: 1271-1276Crossref PubMed Scopus (2073) Google Scholar, 17Lee P. Peng H. Gelbart T. Beutler E. The IL-6- and lipopolysaccharide-induced transcription of hepcidin in HFE-, transferrin receptor 2-, and beta2-microglobulin-deficient hepatocytes.Proc Natl Acad Sci U S A. 2004; 101: 9263-9265Crossref PubMed Scopus (186) Google Scholar However, it must be noted that the stimulatory effect of LPS may be also accounted for, at least to some extent, by the activity of other cytokines, such as IL-1, as recently shown.18Lee 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 (459) Google Scholar For signal transduction, IL-6 binds the membrane-bound IL-6 receptor (gp80). The IL-6-gp80 dimer interacts with gp130, the signal transducing unit in this complex. Formation of gp130-containing complexes results in activation of intracellular JAKs, which phosphorylate tyrosine residues in the cytoplasmic domain of gp130. The phosphorylation of gp130 leads to dimerization of gp130 and activation of STAT1 and STAT3, as well as the SHP2-RAS-MAPK pathway (where SHP2 is Src homology 2 domain-containing protein tyrosine phosphatase).19Stahl N. Farruggella T.J. Boulton T.G. Zhong Z. Darnell Jr., J.E. Yancopoulos G.D. Choice of STATs and other substrates specified by modular tyrosine-based motifs in cytokine receptors.Science. 1995; 267: 1349-1353Crossref PubMed Scopus (869) Google Scholar, 20Nakajima K. Yamanaka Y. Nakae K. et al.A central role for Stat3 in IL-6-induced regulation of growth and differentiation in M1 leukemia cells.Embo J. 1996; 15: 3651-3658Crossref PubMed Scopus (526) Google Scholar, 21Fukada T. Hibi M. Yamanaka Y. et al.Two signals are necessary for cell proliferation induced by a cytokine receptor gp130: involvement of STAT3 in anti-apoptosis.Immunity. 1996; 5: 449-460Abstract Full Text Full Text PDF PubMed Scopus (585) Google Scholar The use of distinct downstream signaling pathways from different phosphorylation sites of the gp130 intracellular domain enables ligand- and tissue-specific activation of specific target genes. For these reasons, IL-6 signaling allows a complex plasticity.22Ernst M. Jenkins B.J. Acquiring signalling specificity from the cytokine receptor gp130.Trends Genet. 2004; 20: 23-32Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar, 23Rose-John S. Coordination of interleukin-6 biology by membrane bound and soluble receptors.Adv Exp Med Biol. 2001; 495: 145-151Crossref PubMed Scopus (43) Google Scholar The aim of the present study is to identify the IL-6-gp130–dependent pathway that leads to hepcidin activation in hepatocytes. Cre gp130LoxP/LoxP hepatocyte-specific gp130–/– mice were generated by breeding alfpCre mice with mice expressing LoxP-flanked gp130 alleles. Genomic DNA was isolated and analyzed by polymerase chain reaction (PCR) as described previously.23Rose-John S. Coordination of interleukin-6 biology by membrane bound and soluble receptors.Adv Exp Med Biol. 2001; 495: 145-151Crossref PubMed Scopus (43) Google Scholar Gp130LoxP/LoxP mice without Cre expression were used as control (wild-type [WT]) animals. AlfpCre gp130ΔSTAT/LoxP hepatocyte-specific gp130ΔSTAT/LoxP animals were generated by breeding alfpCre gp130LoxP/LoxP with gp130ΔSTAT/ΔSTAT knockin (KI) mice. Gp130ΔSTAT/ΔSTAT expresses a truncated gp130 KI allele that lacks the essential region for the activation of STAT1 and -3 signaling.25Ernst M. Inglese M. Waring P. et al.Defective gp130-mediated signal transducer and activator of transcription (STAT) signaling results in degenerative joint disease, gastrointestinal ulceration, and failure of uterine implantation.J Exp Med. 2001; 194: 189-203Crossref PubMed Scopus (209) Google Scholar The genotype was detected by PCR analysis for alfpCre, gp130LoxP, and gp130ΔSTAT allele as described previously.25Ernst M. Inglese M. Waring P. et al.Defective gp130-mediated signal transducer and activator of transcription (STAT) signaling results in degenerative joint disease, gastrointestinal ulceration, and failure of uterine implantation.J Exp Med. 2001; 194: 189-203Crossref PubMed Scopus (209) Google Scholar, 26Tebbutt N.C. Giraud A.S. Inglese M. et al.Reciprocal regulation of gastrointestinal homeostasis by SHP2 and STAT-mediated trefoil gene activation in gp130 mutant mice.Nat Med. 2002; 8: 1089-1097Crossref PubMed Scopus (417) Google Scholar AlfpCre gp130Y757F/LoxP hepatocyte-specific gp130Y757F/LoxP animals were generated by crossing alfpCre gp130LoxP/LoxP with gp130Y757F/Y757F KI mice. The gp130Y757F/Y757F mice express a gp130 allele with a point mutation at Y757. The Y757 is the essential tyrosine for the recruiting of SHP2 and the activation of downstream signals of the RAS-MAPK cascade.26Tebbutt N.C. Giraud A.S. Inglese M. et al.Reciprocal regulation of gastrointestinal homeostasis by SHP2 and STAT-mediated trefoil gene activation in gp130 mutant mice.Nat Med. 2002; 8: 1089-1097Crossref PubMed Scopus (417) Google Scholar The genotype was analyzed by PCR analysis for alfpCre, gp130LoxP, and gp130Y757F allele as described previously.24Streetz K.L. Wustefeld T. Klein C. et al.Lack of gp130 expression in hepatocytes promotes liver injury.Gastroenterology. 2003; 125: 532-543Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, 26Tebbutt N.C. Giraud A.S. Inglese M. et al.Reciprocal regulation of gastrointestinal homeostasis by SHP2 and STAT-mediated trefoil gene activation in gp130 mutant mice.Nat Med. 2002; 8: 1089-1097Crossref PubMed Scopus (417) Google Scholar Hepatocytes from the AlfpCre gp130LoxP/LoxP mice are KO for the gp130 allele, whereas the nonhepatocyte cell populations are homozygous for the WT floxed allele. The hepatocytes from the AlfpCre gp130Y757F/LoxP mice and from the AlfpCre gp130ΔSTAT/LoxP mice are hemizygous for the mutant allele, whereas the nonhepatocyte cell populations of the mice are heterozygous for the mutant allele. All cell populations from the gp130LoxP/LoxP are homozygous for the WT gp130 allele. These latter mice serve as control (WT) mice. Control and gp130-modified KO-KI mice received 1 IP injection of 250 μg/kg recombinant IL-6. Recombinant IL-6 was produced and provided by P. Heinrich (Aachen, Germany) and S. Rose-John (Kiel, Germany) as described before.2Park C.H. Valore E.V. Waring A.J. Ganz T. Hepcidin, a urinary antimicrobial peptide synthesized in the liver.J Biol Chem. 2001; 276: 7806-7810Crossref PubMed Scopus (1806) Google Scholar Primary mouse hepatocytes were isolated by collagenase perfusion.28Gjessing R. Seglen P.O. Adsorption, simple binding and complex binding of rat hepatocytes to various in vitro substrata.Exp Cell Res. 1980; 129: 239-249Crossref PubMed Scopus (40) Google Scholar Mouse liver was perfused with HEPES buffer and then with collagenase solution in a retrograde fashion. Isolated hepatocytes were washed with PBS. 8 × 105 cells were plated on 6-cm dishes in Dulbecco’s modified Eagle medium containing 10% fetal calf serum and a combination of penicillin and streptomycin. The medium was changed after 4 hours. The cells were cultured under serum-free conditions overnight. The next day, cells were stimulated with 200 ng/mL recombinant IL-6. Cells were harvested at the indicated time points, and whole cell proteins, RNA, and nuclear proteins were isolated. After liver tissue collection, samples were immediately placed in RNAlater (Ambion, Austin, TX) solution for storage before processing. Hepatocyte RNA was prepared using TRIzol reagent according to the manufacturer’s instructions (Invitrogen, Carlsbad, CA). Total RNA (15 μg per lane) was separated on a 1% agarose formaldehyde gel, transferred to nylon membrane (Amersham Pharmacia Biotech, UK), and ultraviolet (UV) light–crosslinked. Hepcidin probe (317–base pair [bp]) was generated by PCR from hepatic mouse RNA with the following primers: forward, 5’ACCATGGCACTCAGCACTCG3’ and reverse, 5’GCGGCTCTAGGCTATGTTTTG3’ and cloned in pcDNA3.1/V5/His-TOPO. Hybridization was performed at 42°C with random-primed 32P-labeled cDNA probes for hepcidin and β-actin.29Cleveland D.W. Lopata M.A. MacDonald R.J. Cowan N.J. Rutter W.J. Kirschner M.W. Number and evolutionary conservation of alpha- and beta-tubulin and cytoplasmic beta- and gamma-actin genes using specific cloned cDNA probes.Cell. 1980; 20: 95-105Abstract Full Text PDF PubMed Scopus (1312) Google Scholar The cDNA was generated by reverse transcription of 5 μg of total mouse liver RNA or isolated hepatocyte RNA, with 100 ng random hexamer (Roche, Mannheim, GmbH-Germany), 250 μM dNTPs (Promega Corp., Madison, WI), and 200 U M-MLV Reverse Transcriptase (Promega Corp., Madison, WI) in 1X reverse transcriptase buffer for 1 hour at 42°C. Expression of mouse hepcidin and GAPDH were analyzed using Platinum SYBR Green SuperMix (Invitrogen, Carlsbad, CA). The primers were as follows: SOCS3, forward 5′-CCC GCG GGC ACC TTT CTT AT–3′ and reverse 5′-CAC TGG ATG CGT AGG TTC TTG GTC–3′; SAA, forward 5′-TCT CTG GGG CAA CAT AGT ATA CCT CTC AT –3′ and reverse 5′-TTT ATT ACC CTC TCC TCC TCA AGC AGT TAC–3′; hepcidin, forward 5′-GCCTGTCTCCTGCTTCTCCT-3′ and reverse 5′-GCTCTGTAGTCTGTCTCATCTGTT-3′; GAPDH, forward 5′-CAATGTGTCCGTCGTGGATCT-3′ and reverse 5′-GTCCTCAGTGTAGCCCAAGATG-3′. Amplification was performed at 60°C to 64°C for 45 cycles in iCycler Thermal Cycler (Bio-Rad Hercules, CA), and data were analyzed using iCycler iQ Optical System Software. The relative expression in each sample was calculated by a mathematical method based on the real-time PCR efficiencies using as references GAPDH mRNA.30Pfaffl M.W. A new mathematical model for relative quantification in real-time RT-PCR.Nucleic Acids Res. 2001; 29: e45Crossref PubMed Scopus (26444) Google Scholar All samples were assayed in triplicate. After 45 amplification cycles, threshold cycle values were automatically calculated, and femtograms of starting cDNA were calculated from a standard curve covering a range of 4 orders of magnitude. Both hepcidin and GAPDH standard curves ranged from 1 to 1000 fg per 25-μL reaction. Ratios of hepcidin to GAPDH starting quantity were calculated. Statistical differences of results between various treatment groups and relevant control groups were analyzed using the Student t test. Livers were removed, pooled, rinsed in ice-cold PBS, and were used to prepare liver nuclear extracts.31Wuestefeld T. Klein C. Streetz K.L. Betz U. Lauber J. Buer J. Manns M.P. Muller W. Trautwein C. Interleukin-6/glycoprotein 130-dependent pathways are protective during liver regeneration.J Biol Chem. 2003; 278: 11281-11288Crossref PubMed Scopus (152) Google Scholar All the steps were performed at 4°C. Nuclear protein extracts were separated on a 10% SDS-polyacrylamide gel and blotted onto a nitrocellulose membrane (Whatman, Dassel, Germany) in 1% SDS, 20% methanol, 400 mmol/L glycine, 50 mmol/L Tris-HCl, pH 8.3 at 4°C for 2 hours at 200 mA. Western blot was performed as described previously.32Luedde T. Assmus U. Wustefeld T. Meyer zu Vilsendorf A. Roskams T. Schmidt-Supprian M. Rajewsky K. Brenner D.A. Manns M.P. Pasparakis M. Trautwein C. Deletion of IKK2 in hepatocytes does not sensitize these cells to TNF-induced apoptosis but protects from ischemia/reperfusion injury.J Clin Invest. 2005; 115: 849-859Crossref PubMed Scopus (160) Google Scholar For primary antibody incubation, membranes were probed with anti-phospho-STAT3 specific for pTyr705 (Cell Signaling, Danvers, MA, #9131L) or anti-GAPDH antibody (Biogenesis, Kingston, NH, 4699-9555). As a secondary antibody, anti-rabbit IgG (Jackson Immuno Research Laboratories, Inc., Jackson, West Grove, PA, #711-035-152) or anti-mouse IgG (Chemicon International, Temecula, CA, #AQ 127P) were used. The antigen-antibody complexes were visualized using the ECL-chemiluminescence kit (Amersham, Arlington Heights, IL). IL-6 signaling is dependent on gp130. In order to characterize IL-6–dependent regulation of hepcidin in hepatocytes in vivo, we used genetically modified animals that combine conditional KO and KI technology to modify gp130-dependent pathways in hepatocytes in vivo.27Klein C. Wustefeld T. Assmus U. et al.The IL-6-gp130-STAT3 pathway in hepatocytes triggers liver protection in T cell-mediated liver injury.J Clin Invest. 2005; 115: 860-869Crossref PubMed Scopus (175) Google Scholar Four different animal strains were used: alfpgp130LoxP/LoxP, alfpCre gp130ΔSTAT/LoxP, alfpCre gp130Y757F/LoxP, and WT controls. In order to characterize the modification of gp130-dependent signaling in vivo in hepatocytes, these 4 strains were stimulated with 250 μg/kg recombinant IL-6, and livers were harvested at different time points. IL-6–induced activation of P-STAT3 was monitored by Western blot analysis. In WT animals, P-STAT3 was first found after 3 hours and was most prominent 6 hours after IL-6 stimulation. No P-STAT3 signal was found in alfpgp130LoxP/LoxP and alfpCre gp130ΔSTAT/LoxP mice, whereas stronger P-STAT3 activation was found in gp130Y757F/LoxP compared to WT animals 6 hours after IL-6 stimulation (Figure 1A). In all animals, no P-Stat1 activation could be detected after IL-6 stimulation as reported previously24Streetz K.L. Wustefeld T. Klein C. et al.Lack of gp130 expression in hepatocytes promotes liver injury.Gastroenterology. 2003; 125: 532-543Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar (data not shown). As gp130-dependent marker genes, we next determined the suppressor of cytokine signaling 3 (SOCS3) and serum amyloid A (SAA) mRNA expression in these animals using quantitative real-time PCR. A maximum in SOCS3 expression was found in WT and gp130Y757F/LoxP animals 3 hours after stimulation, whereas no induction of SOCS3 mRNA could be detected in alfpgp130LoxP/LoxP and alfpCre gp130ΔSTAT/LoxP mice (Figure 1B). SOCS3 mRNA expression was induced 50-fold in alfpCre gp130Y757/LoxP mice and was thus 4-fold higher compared to the level found in WT animals. SAA expression was only induced in WT and gp130Y757F/LoxP mice. Although 3 and 6 hours after IL-6 stimulation, SAA mRNA expression was comparable between the 2 animal strains, its expression level was significantly higher in WT animals at the 24-hour time point (Figure 1C). In order to dissect the molecular pathway underlying hepcidin activation in response to IL-6 in vivo, we now use the hepatocyte-specific gp130 genetically modified animals to determine hepcidin mRNA expression at different time points after IL-6 stimulation by qRT-PCR. In the WT animals, significantly higher hepcidin mRNA expression (P = .001) was found 3 and 6 hours after IL-6 stimulation, and baseline levels were found after 24 hours (Figure 2A). A similar kinetic in hepcidin mRNA expression after IL-6 expression was found in alfpCregp130Y757F/LoxP mice showing the maximum of induction after 3 hours. However, 24 hours after stimulation in alfpCregp130Y757F/LoxP mice, hepcidin mRNA expression significantly decreased compared with the baseline level (P = .009) (Figure 2A). In contrast to these results, no IL-6–dependent regulation of hepcidin mRNA expression was found in alfpgp130 ΔSTAT/LoxP and AlfpCre gp130 LoxP/LoxP animals. In the first 6 hours, hepcidin mRNA expression was significantly different compared to the baseline level. These results indicated that IL-6–dependent hepcidin mRNA expression is linked to gp130-dependent STAT3 activation. To confirm the results of these experiments, we also performed Northern blot analysis with a specific hepcidin cDNA probe. Figure 2B, in agreement with the RT-PCR data, shows that hepcidin mRNA is stimulated at 3 hours after IL-6 injection in WT mice and alfpCre gp130Y757F/LoxP (see lane 2 as compared to 1 and 10 as compared to 9, respectively), whereas mice with the disrupted gp130 or STAT3 pathways fail to up-regulate hepcidin mRNA (see lane 6 as compared to 5 and 14 as compared to 13, respectively). For further confirmation, we studied hepcidin mRNA expression in primary hepatocytes derived from these animals. Twenty-four hours after seeding, hepatocytes were stimulated with 200 ng/mL of IL-6. Quantitative RT-PCR was performed at different time points after stimulation (Figure 2C). As observed in the whole animal, higher hepcidin mRNA expression after IL-6 stimulation was only observed when gp130-STAT3–dependent signaling was not altered. Hence, higher hepcidin mRNA levels were found in WT and alfpCregp130Y757F/LoxP mice, whereas no up-regulation was found in alfpCre gp130ΔSTAT/LoxP and alfpCre gp130LoxP/LoxP animals (Figure 2C). In contrast to the in vivo situation, hepcidin mRNA levels did not return to the baseline levels 24 hours after stimulation. Hepcidin is a main player in the disturbances of iron homeostasis during inflammatory states. Anemia of inflammation, or anemia of chronic disease, is an acquired disorder commonly encountered in patients with chronic infections, malignancy, trauma, and inflammatory bowel diseases.33Cartwright G.E. The anemia of chronic disorders.Semin Hematol. 1966; 3: 351-375PubMed Google Scholar It is usually mild or moderate, but it can be severe enough to require transfusions. The hallmarks of the disorder are normocytic anemia (although it can be microcytic at later stages), low serum iron, and normal or high serum ferritin level. The central pathogenic abnormality is that iron is not efficiently recycled from reticuloendothelial macrophages (RE) to erythroid precursors (leading to iron sequestration in RE cells), and intestinal iron absorption is interrupted, causing circulatory hypoferremia.34Roy C.N. Weinstein D.A. Andrews N.C. 2002 E. Mead Johnson Award for Research in Pediatrics Lecture: the molecular biology of the anemia of chronic disease: a hypothesis.Pediatr Res. 2003; 53: 507-512Crossref PubMed Scopus (62) Google Scholar Most of these abnormalities had been traditionally ascribed to the effect of inflammatory cytokines on iron metabolism. Recently, Weinstein et al35Weinstein D.A. Roy C.N. Fleming M.D. Loda M.F. Wolfsdorf J.I. Andrews N.C. Inappropriate expression of hepcidin is associated with iron refractory anemia: implications for the anemia of chronic disease.Blood. 2002; 100: 3776-3781Crossref PubMed Scopus (552) Google Scholar reported that patients with type 1a glycogen-storage disease and refractory chronic anemia had large hepatic adenomas that synthesize inappropriately high levels of hepcidin. When the adenomas were removed, the anemia resolved spontaneously.35Weinstein D.A. Roy C.N. Fleming M.D. Loda M.F. Wolfsdorf J.I. Andrews N.C. Inappropriate expression of hepcidin is associated with iron refractory anemia: implications for the anemia of chronic disease.Blood. 2002; 100: 3776-3781Crossref PubMed Scopus (552) Google Scholar Studies in mice have shown that injections of turpentine, a standard inflammatory stimulus, into mice induce hepcidin mRNA 4-fold and lead to a 2-fold decrease in serum iron.12Nicolas G. Chauvet C. Viatte L. et al.The gene encoding the iron regulatory peptide hepcidin is regulated by anemia, hypoxia, and inflammation.J Clin Invest. 2002; 110: 1037-1044Crossref PubMed Scopus (1380) Google Scholar The hypoferremic response to turpentine-induced inflammation is absent in the USF2/hepcidin-deficient mice12Nicolas G. Chauvet C. Viatte L. et al.The gene encoding the iron regulatory peptide hepcidin is regulated by anemia, hypoxia, and inflammation.J Clin Invest. 2002; 110: 1037-1044Crossref PubMed Scopus (1380) Google Scholar and in IL-6 KO mice,16Nemeth E. Rivera S. Gabayan V. et al.IL-6 mediates hypoferremia of inflammation by inducing the synthesis of the iron regulatory hormone hepcidin.J Clin Invest. 2004; 113: 1271-1276Crossref PubMed Scopus (2073) Google Scholar indicating that this response is dependent on an intact IL-6-gp130-hepcidin pathway. In patients with anemia of inflammation due to chronic infections or severe inflammatory diseases, a 100-fold increase in urinary hepcidin excretion has been reported, and IL-6 infusion in human volunteers stimulates urinary hepcidin excretion within 2 hours and induces hypoferremia.16Nemeth E. Rivera S. Gabayan V. et al.IL-6 mediates hypoferremia of inflammation by inducing the synthesis of the iron regulatory hormone hepcidin.J Clin Invest. 2004; 113: 1271-1276Crossref PubMed Scopus (2073) Google Scholar Overall, these data clearly indicate that the (IL-6–mediated) up-regulation of hepcidin by inflammation plays a major role in the derangement of iron homeostasis observed in acute or chronic inflammatory diseases. In this study, we have taken advantage of different mice strains in which the main IL-6 signaling pathways have been genetically disrupted to dissect the signals involved in hepcidin activation by IL-6. These animals were first stimulated in vivo by IL-6, and the gp130-dependent signaling cascades were analyzed. IL-6 is unable to activate STAT1 in vivo in hepatocytes.24Streetz K.L. Wustefeld T. Klein C. et al.Lack of gp130 expression in hepatocytes promotes liver injury.Gastroenterology. 2003; 125: 532-543Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, 27Klein C. Wustefeld T. Assmus U. et al.The IL-6-gp130-STAT3 pathway in hepatocytes triggers liver protection in T cell-mediated liver injury.J Clin Invest. 2005; 115: 860-869Crossref PubMed Scopus (175) Google Scholar In WT animals, IL-6 resulted in STAT3 activation as evidenced by phospho-STAT3 expression. STAT3 activation triggered target gene transcription of SOCS3 and SAA. This response was blunted in the 2 animals—AlfpCre gp130LoxP/LoxP and AlfpCre gp130ΔSTAT/LoxP—where no gp130-dependent STAT docking sites were expressed in hepatocytes. SOCS3 is involved in inhibiting gp130-dependent STAT3 activation via the RAS/MAP docking site at gp130 tyrosine 757.36Heinrich 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 (2579) Google Scholar Thus, this explains stronger STAT3, SAA, and SOCS3 activation in AlfpCre gp130Y757F/LoxP mice, as in these animals, this negative feedback loop is blocked. Our in vivo and in vitro data point to STAT3 as the key transcription factors responsible for IL-6 activation of hepcidin gene transcription. The fact that in cultured hepatocytes with a disrupted RAS/MAPK pathway, the response of hepcidin mRNA to IL-6 was lower than in WT cells, particularly at 3 hours and 24 hours (Figure 2C), may suggest that RAS, at least to some extent, is also involved in the IL-6–dependent regulation of hepcidin. At present, we cannot rule out this possibility. However, this phenomenon may be somehow related to the in vitro settings, as it was not found in our in vivo experiments. Even in vitro, it must be noted that, throughout the study, hepcidin response to IL-6 was consistently higher in hepatocytes with the disrupted RAS pathway as compared to those lacking a functional STAT3 pathway. Moreover, the central role of STAT3 is also supported by the results of a recent in vitro study showing that in a transfected hepatoma cell line, STAT3 was clearly required for response of a 0.6-kilobase hepcidin promoter fragment to IL-6.37Wrighting D.M. Andrews N.C. Interleukin-6 induces hepcidin expression through STAT3.Blood. 2006; 108: 3204-3209Crossref PubMed Scopus (733) Google Scholar Some transcription factors might be important for hepcidin expression: C/EBPalpha, SMADs, and STATs.7Courselaud B. Pigeon C. Inoue Y. et al.C/EBPalpha regulates hepatic transcription of hepcidin, an antimicrobial peptide and regulator of iron metabolism Cross-talk between C/EBP pathway and iron metabolism.J Biol Chem. 2002; 277: 41163-41170Crossref PubMed Scopus (229) Google Scholar, 38Wang R.H. Li C. Xu X. et al.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 (515) Google Scholar, 39Babitt J.L. Huang F.W. Wrighting D.M. et al.Bone morphogenetic protein signaling by hemojuvelin regulates hepcidin expression.Nat Genet. 2006; 38: 531-539Crossref PubMed Scopus (853) Google Scholar The C/EBPα KO animal develops liver iron overload suggesting a key role of this factor in hepcidin control.7Courselaud B. Pigeon C. Inoue Y. et al.C/EBPalpha regulates hepatic transcription of hepcidin, an antimicrobial peptide and regulator of iron metabolism Cross-talk between C/EBP pathway and iron metabolism.J Biol Chem. 2002; 277: 41163-41170Crossref PubMed Scopus (229) Google Scholar More recently, a Cre-loxP-mediated liver-specific disruption of SMAD4 involved in TGFβ superfamily signaling resulted in markedly decreased hepcidin expression and accumulation of iron in many organs, particularly in the liver, kidney, and pancreas.38Wang R.H. Li C. Xu X. et al.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 (515) Google Scholar Interestingly, in this study, SMAD4 KO animals failed to respond to IL-6 stimulation. However, as indicated by the author, the interpretation is not straightforward, as other IL-6 target genes could be stimulated by IL-6, and the possibility exists that SMAD4 might simply facilitate opening of the chromatin on the hepcidin promoter. In fact, mice lacking hemojuvelin, a bone morphogenic protein coreceptor involved in hepcidin transcriptional control upstream of SMAD439Babitt J.L. Huang F.W. Wrighting D.M. et al.Bone morphogenetic protein signaling by hemojuvelin regulates hepcidin expression.Nat Genet. 2006; 38: 531-539Crossref PubMed Scopus (853) Google Scholar up-regulate hepcidin in response to IL-6.40Niederkofler V. Salie R. Arber S. Hemojuvelin is essential for dietary iron sensing, and its mutation leads to severe iron overload.J Clin Invest. 2005; 115: 2180-2186Crossref PubMed Scopus (328) Google Scholar In addition, also in view of our results, it cannot be ruled out as possible cross-talk between SMAD4 and STAT3 on hepcidin promoter as shown to occur for other IL-6 target genes.41Zauberman A. Lapter S. Zipori D. Smad proteins suppress CCAAT/enhancer-binding protein (C/EBP) beta- and STAT3-mediated transcriptional activation of the haptoglobin promoter.J Biol Chem. 2001; 276: 24719-24725Crossref PubMed Scopus (57) Google Scholar In conclusion, although the complex interplay of different transcription factors and signaling molecules on hepcidin activation during IL-6 stimulus has yet to be clarified, our data show that STAT3 plays a key role in the IL-6-gp130-hepcidin axis in vivo. Hepcidin Activation During Inflammation: Make It STATGastroenterologyVol. 132Issue 1PreviewThe central role for the peptide hormone hepcidin in the regulation of iron metabolism is increasingly apparent.1 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. Full-Text PDF CorrectionGastroenterologyVol. 132Issue 3PreviewPietrangelo A, Dierssen U, Valli L, Garuti C, Rump A, Corradini E, Ernst M, Klein C, Trautwein C. STAT3 Is Required for IL-6-gp130–Dependent Activation of Hepcidin In Vivo. Gastroenterology 2007;132:294–300. Full-Text PDF