Recent discoveries have indicated that the hormone hepcidin plays a major role in the control of iron homeostasis. Hepcidin regulates the iron level in the blood through the interaction with ferroportin, an iron exporter molecule, causing its internalization and degradation. As a result, hepcidin increases cellular iron sequestration, and decreases the iron concentration in the plasma. Only mature hepcidin (result of the cleavage of prohepcidin by furin proteases) has biological activity; however, prohepcidin, the prohormone form, is also present in the plasma. In this study, we aimed to identify new protein–protein interactions of preprohepcidin, prohepcidin and hepcidin using the BacterioMatch two‐hybrid system. Screening assays were carried out on a human liver cDNA library. Preprohepcidin screening gave the following results: α‐1 antitrypsin, transthyretin and α‐1‐acid glycoprotein showed strong interactions with preprohepcidin. We further confirmed and examined the α‐1 antitrypsin binding in vitro (glutathione S‐transferase, pull down, coimmunoprecipitation, MALDI‐TOF) and in vivo (ELISA, cross‐linking assay). Our results demonstrated that the serine protease inhibitor α‐1 antitrypsin binds preprohepcidin within the cell during maturation. Furthermore, α‐1 antitrypsin binds prohepcidin significantly in the plasma. This observation may explain the presence of prohormone in the circulation, as well as the post‐translational regulation of the mature hormone level in the blood. In addition, the lack of cleavage protection in patients with α‐1 antitrypsin deficiency may be the reason for the disturbance in their iron homeostasis.

Hepcidin is the major regulatory peptide hormone of iron metabolism, encoded by the HAMP (hepcidin antimicrobial peptide) gene. Hepcidin is expressed mainly in hepatocytes, but is also found in the blood in both a mature and prohormone form. Although, the function of mature hepcidin and the regulation of the HAMP gene have been extensively studied, the intracellular localization and the fate of prohepcidin remains controversial. In the present study, we propose a novel role for prohepcidin in the regulation of its own transcription. Using indirect immunofluorescence and mCherry tagging, a portion of prohepcidin was detected in the nucleus of hepatocytes. Prohepcidin was found to specifically bind to the STAT3 (signal transducer and activator of transcription 3) site in the promoter of HAMP. Overexpression of prohepcidin in WRL68 cells decreased HAMP promoter activity, whereas decreasing the amount of prohepcidin caused increased promoter activity measured by a luciferase reporter-gene assay. Moreover, overexpression of the known prohepcidin-binding partner, α-1 antitrypsin caused increased HAMP promoter activity, suggesting that only the non-α-1 antitrypsin-bound prohepcidin affects the expression of its own gene. The results of the present study indicate that prohepcidin can bind to and transcriptionally regulate the expression of HAMP, suggesting a novel autoregulatory pathway of hepcidin gene expression in hepatocytes.