Gastroenterology

Gastroenterology

Volume 132, Issue 1, January 2007, Pages 294-300
Gastroenterology

Basic–liver, pancreas, and biliary tract
STAT3 Is Required for IL-6-gp130–Dependent Activation of Hepcidin In Vivo

https://doi.org/10.1053/j.gastro.2006.10.018Get rights and content

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.

Section snippets

Generation and genotyping of gp130-mutated animals

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.23 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. Gp130

RNA Isolation and Analysis

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).

Northern-Blot Analysis

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.

Quantitative Reverse-Transcription PCR

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

Characterization and Validation of Hepatocyte-Specific alfpgp130LoxP/LoxP,alfpgp130ΔSTAT/LoxP, and gp130Y757F/LoxP Mice

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.27 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

Discussion

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.33 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

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Supported by a Telethon grant No. 0644015372 and the SFB 542, Teilprojekt C14.

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