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 gp130(DeltaSTAT/LoxP)) or mice expressing a gp130 allele lacking the essential tyrosine for RAS-MAPK activation (alfpCregp130(Y757F/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 alfpCregp130(Y757F/LoxP) mice, whereas this response was blocked in alfpCre gp130(LoxP/LoxP) and alfpCre gp130(DeltaSTAT/LoxP) mice. In wild-type and alfpCregp130(Y757F/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 (DeltaSTAT/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.

Haptoglobin modifies the hemochromatosis phenotype in mice.

Classic hereditary hemochromatosis (HH) is a common genetic disorder of iron metabolism caused by a mutation in the HFE gene. Whereas the prevalence of the mutation is very high, the clinical penetrance of the disease is low, suggesting that the HFE mutation is a necessary but not sufficient cause of clinical HH. Several candidate modifier genes have been proposed in mice and humans, including haptoglobin. Haptoglobin is the plasma protein with the highest binding affinity for hemoglobin. It delivers free plasma hemoglobin to the reticuloendothelial system, thus reducing loss of hemoglobin through the glomeruli and allowing heme-iron recycling. To gain insight into the role of haptoglobin as a modifier gene in HH, we used Hfe and haptoglobin double-null mice. Here, we show that Hfe and haptoglobin compound mutant mice accumulate significantly less hepatic iron than Hfe-null mice, thus demonstrating that haptoglobin-mediated heme-iron recovery may contribute significantly to iron loading in HH.

Kupffer cells and macrophages are not required for hepatic hepcidin activation during iron overload.

Hepcidin, the iron hormone, is produced by the liver in response to iron and inflammation. Its synthesis during inflammation is triggered by cytokines, but the details of iron activation are obscure. We tested the role of Kupffer cells and macrophages by studying iron-loaded or inflamed mice with selective inactivation of Kupffer cells or the in vitro effect of conditioned human macrophages on hepcidin expression. Hepcidin messenger RNA (mRNA) expression was studied by Northern blot and reverse transcriptase polymerase chain reaction analysis in mice that were treated with 40 mg/kg gadolinium (III) chloride (GdCl(3)) as a Kupffer cell inactivating agent and subjected to inflammatory challenges with either lipopolysaccharide (LPS) and turpentine or iron overload by iron-dextran administration. Similar analyses were performed in human hepatoma cells (HepG2) cultured with medium from LPS- or iron-conditioned macrophages from blood donors or patients with HFE-linked hereditary hemochromatosis (HH). In vivo, LPS and particularly turpentine stimulated hepcidin mRNA expression, and this effect was prevented by the inactivation of Kupffer cells. Also, iron overload markedly upregulated hepatic hepcidin mRNA, but this activity persisted in spite of Kupffer cell blockade. In vitro, the medium of LPS-treated normal or hemocromatotic macrophages turned on hepcidin expression. On the contrary, medium of iron-manipulated macrophages, regardless of their HFE status, did not affect hepcidin mRNA steady-state levels. In conclusion, Kupffer cells are required for the activation of hepcidin synthesis during inflammation, and HH inflamed macrophages are capable of mounting a normal response, eventually leading to hepcidin stimulation. However, both Kupffer cells and human macrophages are dispensable for the regulatory activity exerted by iron on hepatic hepcidin.