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. 2009 Jun;94(6):765-72.
doi: 10.3324/haematol.2008.003541. Epub 2009 May 19.

Cross-talk between the mitogen activated protein kinase and bone morphogenetic protein/hemojuvelin pathways is required for the induction of hepcidin by holotransferrin in primary mouse hepatocytes

Guillemette Ramey  1 , Jean-Christophe Deschemin, Sophie Vaulont
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Cross-talk between the mitogen activated protein kinase and bone morphogenetic protein/hemojuvelin pathways is required for the induction of hepcidin by holotransferrin in primary mouse hepatocytes (VSports app下载)

Guillemette Ramey et al. Haematologica. 2009 Jun.

"V体育官网" Abstract

Background: The circulating hormone hepcidin plays a central role in iron homeostasis. Our goal was to establish an ex vivo iron-sensing model and to characterize the molecular mechanisms linking iron to hepcidin. VSports手机版.

Design and methods: Murine hepatocytes were isolated by the collagenase method, either from wild type or HFE knockout mice, and cultured 42 h without serum before treatments. V体育安卓版.

Results: After 42 h of serum-free culture, hepcidin gene expression was undetectable in the hepatocytes. Hepcidin gene expression could, however, be re-activated by an additional 24 h of incubation with 10% serum V体育ios版. Interestingly, addition of 30 microM holotransferrin consistently increased serum-dependent hepcidin levels 3- to 5-fold. The effects of serum and serum+holotransferrin were direct, transcriptional, independent of de novo protein synthesis and required the presence of bone morphogenetic protein. Transferrin receptor-2 activation by its ligand holotransferrin led to extracellular signal regulated kinase (ERK)/mitogen activated protein kinase pathway stimulation and the ERK specific inhibitor U0-126 blunted holotransferrin-mediated induction of hepcidin. ERK activation by holotransferrin provoked increased levels of phospho-Smad1/5/8 highlighting cross-talk between the bone morphogenetic protein/hemojuvelin and ERK1/2 pathways. Finally, we demonstrated, using hepatocytes isolated from Hfe(-/-) mice, that HFE was not critical for the hepcidin response to holotransferrin but important for basal hepcidin expression. .

Conclusions: We demonstrate that hepatocytes are liver iron-sensor cells and that transferrin receptor-2, by signaling through the ERK1/2 pathway, and bone morphogenetic protein/hemojuvelin, by signaling through the Smad pathways, coordinately regulate the iron-sensing machinery linking holotransferrin to hepcidin VSports最新版本. .

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Figures

Figure 1.
Figure 1.
Hepcidin mRNA levels in hepatocytes in primary culture treated with serum and serum plus human holotransferrin [(holoTf (h)] Hepcidin mRNA levels in primary hepatocytes was determined by northern blot analysis. Hepatocytes were treated, after a 42 h period of serum-free culture, with either 0% serum (control cells), 10% serum, 30 μM human holotransferrin, or both, for 24 h. Total liver RNA (20 μg) was subjected to electrophoresis, blotted, and hybridized with hepcidin and 18S-labeled probes. The experiment was performed at least three times and a representative result is shown.
Figure 2.
Figure 2.
Effects of noggin and neutralizing BMP2/4 antibodies on hepcidin response to serum and serum+holotransferrin and kinetic study of BMP2 mRNA levels. Relative changes in hepcidin mRNA levels were quantified by real-time quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) performed on cDNA synthesized from 2 μg total RNA (β-actin normalized, arbitrary units) (A) Hepatocytes were treated, after a 42 h period of serum-free culture, with either 0% serum (control cells), 10% serum, or 10% serum + 30 μM human holotransferrin [holoTf(h)], in the presence of either 1 μg/mL noggin or 20 μg/mL BMP2/4 antibodies (Ab BMP2/4), for 24 h. Results are expressed relative to hepatocytes cultured for 66 h without serum. Mean±SD for three independent samples. The experiment was performed three times and a representative result is shown. Statistical analysis was performed using Student’s t test (unpaired, two-tailed). ***p<0.001 as compared to untreated hepatocytes. (B) Hepatocytes were treated, after a 42 h period of serum-free culture, with either 0% serum (control cells), 10% serum or 10% serum in the presence of 30 μM human holoTf for 2, 4, 6, 8, 12 or 24 h. Relative changes in BMP2 mRNA levels were quantified by real-time qRT-PCR performed on cDNA synthesized from 2 μg total RNA (β-actin normalized, arbitrary units). Results are expressed relative to hepatocytes cultured for 66 h without serum. Mean±SD for three independent samples. The experiment was performed three times and a representative result is shown. Statistical analysis was performed using Student’s t test (unpaired, two tailed).
Figure 3.
Figure 3.
Effect of holotransferrin treatment on ERK1/2 activation and of a specific ERK1/2 inhibitor on hepcidin mRNA induction by holotransferrin. (A) After a 42 h period of serum-free culture, hepatocytes were treated either with 30 μM holotransferrin (holoTf) alone (A), 10% serum alone (B), or both (C) for 5, 10, 15, 30, or 120 min. Activation of the ERK1/2 pathway was assessed by immunoblotting 80 μg hepatocyte extracts with the phospho-ERK1/2 antibody. The relative amount of ERK1/2 was ascertained by immunoblot analysis using ERK1/2 antibody. Hepatocytes were treated for 15 min with 50 ng/mL EGF as a positive control for the activation of the ERK1/2 pathway. Molecular weight markers (kDa) are indicated on the left. (D) Relative changes in hepcidin mRNA levels as quantified by real-time qRT-PCR performed on cDNA synthesized from 2 μg total RNA (β-actin normalized, arbitrary units). Hepatocytes were treated for 24 h, after a 42 h period of serum-free culture, with either 0% serum (control cells) or 10% serum with or without 30 μM holoTf, in the presence or absence of 10 μM U0-126 inhibitor added 1 h prior treatment. Results are expressed relative to hepatocytes cultured for 66 h without serum. Mean±SD for four independent samples. The experiment was performed at least three times and a representative result is shown Statistical analysis was performed using Student’s t test (unpaired, two tailed). NS: non significant.
Figure 4.
Figure 4.
Effect of holotransferrin (holoTf) treatment on Smad1/5/8 activation. After a 42 h period of serum-free culture, hepatocytes were treated with either 10% serum alone (S), 30 μM holotransferrin alone (H), serum + holoTf (SH), or serum + holoTf in the presence of 10 μM U0-126 (SHI) for 5, 10, 15, 30, or 120 min. Activation of the Smad pathway was assessed by immunoblotting 120 μg of hepatocyte extracts with the phospho-Smad1/5/8 antibody. Lane 9 is a positive control with hepatocytes treated for 24 h with 20 ng/mL BMP2. Molecular weight markers (kDa) are indicated on the left. Blot densitometry of two independent experiments is shown.
Figure 5.
Figure 5.
Effects of HFE deficiency on hepcidin response to serum and serum+holotransferrin. Relative changes in hepcidin mRNA levels were quantified by real-time qRT-PCR performed on cDNA synthesized from 2 μg total RNA (β-actin normalized, arbitrary units). (A) Hepatocytes isolated from either Hfe+/+ mice or Hfe−/−mice were treated, after a 42 h period of serum-free culture, with either 0% serum (control cells), 10% serum or 10% serum in the presence of 30 μM human holotransferrin [holoTf (h)] for 24 h. Results are expressed relative to hepatocytes cultured for 66 h without serum. Mean±SD for three independent samples. The experiment was performed twice and a representative result is shown. Statistical analysis was performed using Student’s t test (unpaired, two tailed).
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References

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