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.
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.


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.


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. 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.
Ramey
Guillemette
G
Institut Cochin, Faculté de Médecine Cochin Port Royal, Paris, France.
Deschemin
Jean-Christophe
JC
Vaulont
Sophie
S
D000818:Animals; D023181:Antimicrobial Cationic Peptides / Q000235:genetics*; D015152:Blotting, Northern; D019485:Bone Morphogenetic Proteins / Q000235:genetics* / Q000378:metabolism; D002478:Cells, Cultured; D016895:Culture Media, Serum-Free / Q000494:pharmacology; D005260:Female; D058851:GPI-Linked Proteins; D005786:Gene Expression Regulation / Q000187:drug effects; D000071020:Hemochromatosis Protein; D022781:Hepatocytes / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D064451:Hepcidins; D006801:Humans; D008297:Male; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D008815:Mice, Inbred Strains; D018345:Mice, Knockout; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011990:Receptors, Transferrin / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D013997:Time Factors; D014168:Transferrin / Q000494:pharmacology*
D023181:Antimicrobial Cationic Peptides; D019485:Bone Morphogenetic Proteins; D016895:Culture Media, Serum-Free; D058851:GPI-Linked Proteins; C578702:HAMP protein, human; C504414:HJV protein, mouse; C579599:Hamp protein, mouse; D000071020:Hemochromatosis Protein; D064451:Hepcidins; D008565:Membrane Proteins; D012333:RNA, Messenger; D011990:Receptors, Transferrin; C503641:TFR2 protein, mouse; D014168:Transferrin; C110714:holotransferrin; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases