Posttranslational processing of hepcidin in human hepatocytes is mediated by the prohormone convertase furin

Abstract

Hepcidin is encoded as an 84 amino acid prepropeptide containing a typical N-terminal 24 amino acid endoplasmic reticulum targeting signal sequence, and a 35 amino acid proregion (pro) with a consensus furin cleavage site immediately followed by the C-terminal 25 amino acid bioactive iron-regulatory hormone (mature peptide). We performed pulse-chase studies of posttranslational processing of hepcidin in human hepatoma HepG2 cells and in primary human hepatocytes induced with bone morphogenic protein (BMP-9). In some experiments, the cells were treated with the furin protease inhibitor decanoyl-Arg-Val-Lys-Arg-chloromethylketone (CMK) or furin siRNA. In the absence of furin inhibitor, hepcidin was found to be processed in less than 1 h and secreted as a 3 kDa form reactive with anti-mature but not anti-pro antibody. In the presence of furin inhibitors or furin siRNA, a 6 kDa form reactive with both anti-pro and anti-mature antibody was rapidly secreted into the medium. Processing was not affected by inhibitors of the hypoxia inducible factor (HIF) pathway, or by treatment with 30 microM holo- or apo-transferrin. In conclusion, the hepatic prohormone convertase furin mediates the posttranslational processing of hepcidin. The proteolytic cleavage of prohepcidin to hepcidin is not regulated by iron-transferrin or the HIF pathway.

Figure 1. Amino acid sequence of hepcidin precursor

Underlined residues were radiolabeled with ³⁵-S labeled amino acids. The boxed region denotes the furin consensus cleavage site. The molecular weight of the hepcidin precursor is shown.

Figure 2. Pulse-chase study of hepcidin processing in Hep-G2 cells treated with and without furin proproteinase inhibitor

Cells were labeled with ³⁵S-met-cys for 1 hr then subjected to cold chase for the times in minutes indicated above the lane. Lane EQ was labeled for 3 hours without cold chase prior to processing to incorporate radioactivity into all peptide forms. Cell lysates (top panel) or the corresponding culture media (lower panel) were immunoprecipitated with either anti-pro (propiece) or anti-mature (hepcidin) antibody and analyzed on SDS-tricine PAGE. The autoradiograms of the gels are shown with the molecular weight standards indicated on the right side of the figure. Three forms of hepcidin are seen as marked by arrows. A cleaved form of the propiece is indicated by the small arrow (p) and the asterisk (panels B and D). In the right panel, cells were treated with furin inhibitor (decanoyl-RVKR-CMK) during the amino acid depletion step (1 hr) and during the radiolabeling procedure.

Figure 3. Pulse-chase study of hepcidin processing in HepG2 cells transfected with furin siRNA

Cells were transfected twice over a 48 hr period with either blank vector (panels A and B) or vector containing furin siRNA (panels C and D). Cells were labeled with ³⁵S-met-cys for 1 hr then subjected to cold chase for the times indicated. Lane EQ was labeled for 3 hr. Cell lysates (top panel) or the corresponding culture media (lower panel) were immunoprecipitated with either anti-pro (propiece) or anti-mature (hepcidin) antibody and analyzed on SDS-tricine PAGE. The autoradiogram of the gels are shown with the corresponding time of cold chase indicated above the lane (minutes). Molecular weight standards are indicated on the right panels. Three forms of hepcidin are seen as indicated by the arrows. Cleaved forms of the propiece are indicated by the small arrows (p) and the asterisks (panels B and D).

Figure 4. Hepcidin processing in HepG2 cells treated with apo- and holo-transferrin and with prolyl-hydroxylase inhibitors

Cells were labeled with ³⁵S-met-cys for 3 hr. Cell lysates or the corresponding culture media were immunoprecipitated with anti-mature (hepcidin) antibody and analyzed on SDS-tricine PAGE. The autoradiograms of the gels are shown with the molecular weight standards indicated on the right side of the figure. In the upper panel, cells were treated with solvent control (C), or 30 μM apo-transferrin (A) or holo-transferrin (H); in the lower panel, cells were treated with solvent control (C) or 500 μM DMOG or 10 μM 2,4-DPD. All treatments were for 18 hr prior to and during radiolabeling.

Figure 5. Pulse-chase study of hepcidin processing in primary human hepatocytes

Cells were treated for 18 hr with 10 ng/ml BMP-9 prior to, and during the depletion and radiolabeling steps to increase hepcidin synthesis. Cells were labeled with ³⁵S-met-cys and ¹⁴C-amino acids for 1 hr then subjected to cold chase for the times indicated above the lanes, in minutes. Cell lysates and conditioned media in lane EQ are from hepatocytes labeled for 4 hr prior to processing. Cell lysates (top panel) or the corresponding media (lower panel) were immunoprecipitated with anti-mature hepcidin antibody (panel A) or anti-pro-peptide (panel B) and analyzed on SDS-tricine PAGE. Autoradiograms of the gels are shown with the molecular weight standards indicated. In the right half of the autoradiogram, cells were treated with furin inhibitor (decanoyl-RVKR-CMK) during the amino acid depletion step (1 hr) prior to and during the radiolabeling procedure.