Characterization of hepcidin response to holotransferrin in novel recombinant TfR1 HepG2 cells.
Hepcidin is the key regulator of systemic iron homeostasis. The iron-sensing mechanisms and the role of intracellular iron in modulating hepatic hepcidin secretion are unclear. Therefore, we created a novel cell line, recombinant-TfR1 HepG2, expressing iron-response-element-independent TFRC mRNA to promote cellular iron-overload and examined the effect of excess holotransferrin (5g/L) on cell-surface TfR1, iron content, hepcidin secretion and mRNA expressions of TFRC, HAMP, SLC40A1, HFE and TFR2. Results showed that the recombinant cells exceeded levels of cell-surface TfR1 in wild-type cells under basal (2.8-fold; p<0.03) and holotransferrin-supplemented conditions for 24h and 48h (4.4- and 7.5-fold, respectively; p<0.01). Also, these cells showed higher intracellular iron content than wild-type cells under basal (3-fold; p<0.03) and holotransferrin-supplemented conditions (6.6-fold at 4h; p<0.01). However, hepcidin secretion was not higher than wild-type cells. Moreover, holotransferrin treatment to recombinant cells did not elevate HAMP responses compared to untreated or wild-type cells. In conclusion, increased intracellular iron content in recombinant cells did not increase hepcidin responses compared to wild-type cells, resembling hemochromatosis. Furthermore, TFR2 expression altered within 4h of treatment, while HFE expression altered later at 24h and 48h, suggesting that TFR2 may function prior to HFE in HAMP regulation.
Department of Biomedical Sciences, University of Westminster, London, UK.
Kosha
K
Mehta
Department of Clinical Biochemistry, Charing Cross Hospital, Imperial College Healthcare NHS Trust, London, UK.
Mark
M
Busbridge
Faculty of Health & Life Sciences, Coventry University, Coventry, UK.
Derek
D
Renshaw
School of Engineering and Design, Brunel University, London, UK.
Robert W
RW
Evans
Department of Life Sciences, University of Bedfordshire, Luton, UK.
Sebastien
S
Farnaud
Department of Biomedical Sciences, University of Westminster, London, UK. Electronic address: v.b.patel@westminster.ac.uk.
Vinood B
VB
Patel
D015703:Antigens, CD / Q000187:drug effects / Q000235:genetics; D000071020:Hemochromatosis Protein / Q000097:blood / Q000187:drug effects; D056945:Hep G2 Cells; D064451:Hepcidins / Q000097:blood* / Q000187:drug effects; D006801:Humans; D007501:Iron / Q000097:blood; D019190:Iron Overload; D012333:RNA, Messenger / Q000097:blood; D011990:Receptors, Transferrin / Q000187:drug effects / Q000235:genetics; D011994:Recombinant Proteins; D035341:Telomeric Repeat Binding Protein 2 / Q000097:blood / Q000187:drug effects; D013997:Time Factors; D014168:Transferrin / Q000494:pharmacology*
D015703:Antigens, CD; C068126:CD71 antigen; C103673:HFE protein, human; D000071020:Hemochromatosis Protein; D064451:Hepcidins; D012333:RNA, Messenger; D011990:Receptors, Transferrin; D011994:Recombinant Proteins; C504753:TERF2 protein, human; D035341:Telomeric Repeat Binding Protein 2; D014168:Transferrin; C110714:holotransferrin; D007501:Iron
HepG2; Hepatocyte; Hepcidin; Iron; Transferrin