Diferric Transferrin-Sensed Hepcidin Upregulation in Human Hepatoma-Derived Cell Line Is Differentially Controlled in Each Isoform 20, 22, 25; Confirmation by a Novel Simultaneous Quantification by Liquid Chromatography/ Tandem Mass Spectrometry.

Hepcidin is a key molecule of body iron metabolism, and the expression at mRNA level is thought to be upregulated by iron loading. As the mature processed form of human hepcidin is known to have 3 isoforms, hepcidin -20, -22, and -25, and hepcidin -25 is thought to be the major isoform active in iron metabolism. However, the physiological roles of other isoforms are poorly understood. Concerning the study on the regulatory mechanism on hepcidin expression, most studies have been only performed at the transcriptional level because of the difficulty of quantification of hepcidin in cell culture media; therefore, the experiments in vitro would be valuable. We therefore developed a sensitive new method for measuring hepcidin that can simultaneously measure the isoforms in culture media, and studied the expression patterns of isoforms at mature protein level in various human hepatoma-derived cell lines with and without diferric transferrin.

Quantification of human hepcidin -20, -22, -25 was performed using liquid chromatography (LC) - tandem mass spectrometry (MS) which we newly developed. Selected reaction monitoring (SRM) transitions and the collision energies were settled for each isoform respectively. Quantification of hepcidin isoforms in culture medium of 13 strains of hepatoma-derived cell lines was performed. Various stimulants for hepcidin expression, such as interleukin-6, diferric transferrin and etc, were also used for investigating the response patterns of hepcidin isoforms.

Upon optimization of SRM conditions, the most intense precursor ions were selected in each mass spectrum to detect hepcidin isoforms. Product ions were selected to maximize sensitivity and selectivity. Despite using culture media including 10% FBS as matrix, isoform peaks were not interfered with by a blank matrix, indicating the method has good selectivity. Calibration curves were constructed over the range 2-1,000 ng/mL, and linearity of the calibration curves by weighted (1/x²) linear regression was excellent (correlation coefficient: r=0.9974 for hepcidin-20, r=0.9937 for hepcidin-22, r=0.9950 for hepcidin-25). Accuracies for back-corrected concentrations were 99.7-122.1% for hepcidin-20, 102.6-132.5% for hepcidin-22, and 99.1-141.2% for hepcidin-25. These results indicate that the method is adequate for quantifying hepcidin isoforms in culture media. We also found that substantial difference of hepcidin isoforms' expression patterns among human hepatoma-derived cell lines, and the patterns were divided into 5 groups. Response patterns for various stimulants were also different among those groups. Especially, human diferric transferrin upregulates hepcidin-20 and -22 in WRL68 cells, and hepcidin-22 in Hep3B, HuH-2, HuH-4, and HuH-6 cells; this should be the first report that human diferric transferrin upregulates hepcidin isoforms other than hepcidin-25 in human hepatocyte-derived cells.

We have devised a novel method for simultaneous quantification of hepcidin isoforms in culture media. Although most previous studies only observe the changes of hepcidin expression at mRNA level, our method revealed heterogeneous expressions of hepcidin isoforms and hepcidin upregulation by human diferric transferrin in human hepatocyte-derived cells at the peptide level. The fact of hepcidin isoforms' upregulation by human diferric transferrin in human hepatocyte-derived cells might be the clue to elucidate the mechanism for iron sensor in human body. We believe that this novel quantification method can contribute to further progress, especially in vitro research on the regulation of hepcidin expression.

No relevant conflicts of interest to declare.