title: Characterisation of hepcidin response to holotransferrin treatment in CHO TRVb-1 cells.
abstract: Iron overload coupled with low hepcidin levels are characteristics of hereditary haemochromatosis. To understand the role of transferrin receptor (TFR) and intracellular iron in hepcidin secretion, Chinese hamster ovary transferrin receptor variant (CHO TRVb-1) cells were used that express iron-response-element-depleted human TFRC mRNA (TFRC∆IRE). Results showed that CHO TRVb-1 cells expressed higher basal levels of cell-surface TFR1 than HepG2 cells (2.2-fold; p < 0.01) and following 5 g/L holotransferrin treatment maintained constitutive over-expression at 24h and 48 h, contrasting the HepG2 cells where the receptor levels significantly declined. Despite this, the intracellular iron content was neither higher than HepG2 cells nor increased over time under basal or holotransferrin-treated conditions. Interestingly, hepcidin secretion in CHO TRVb-1 cells exceeded basal levels at all time-points (p < 0.02) and matched levels in HepG2 cells following treatment. While TFRC mRNA expression showed expected elevation (2h, p < 0.03; 4h; p < 0.05), slc40a1 mRNA expression was also elevated (2 h, p < 0.05; 4 h, p < 0.03), unlike the HepG2 cells. In conclusion, the CHO TRVb-1 cells prevented cellular iron-overload by elevating slc40a1 expression, thereby highlighting its significance in the absence of iron-regulated TFRC mRNA. Furthermore, hepcidin response to holotransferrin treatment was similar to HepG2 cells and resembled the human physiological response.
authors.affiliation: Department of Biomedical Sciences, University of Westminster, London W1W 6UW, UK.
authors.forename: Kosha
authors.initials: K
authors.lastname: Mehta
authors.affiliation: Department of Biomedical Sciences, University of Westminster, London W1W 6UW, UK.
authors.forename: Pamela
authors.initials: P
authors.lastname: Greenwell
authors.affiliation: Faculty of Health & Life Sciences, Coventry University, Coventry, CV1 5FB, UK.
authors.forename: Derek
authors.initials: D
authors.lastname: Renshaw
authors.affiliation: Department of Clinical Biochemistry, Charing Cross Hospital, Imperial College Healthcare NHS Trust, London W6 8RF, UK.
authors.forename: Mark
authors.initials: M
authors.lastname: Busbridge
authors.affiliation: Randall Division of Cell and Molecular Biophysics, King's College London SE1 1UL, UK.
authors.forename: Mitla
authors.initials: M
authors.lastname: Garcia
authors.affiliation: Department of Life Sciences, University of Bedfordshire, Luton, LU1 3JU, UK.
authors.forename: Sebastien
authors.initials: S
authors.lastname: Farnaud
authors.affiliation: Department of Biomedical Sciences, University of Westminster, London W1W 6UW, UK. Electronic address: v.b.patel@westminster.ac.uk.
authors.forename: Vinood B
authors.initials: VB
authors.lastname: Patel
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D027682:Cation Transport Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D017124:Conserved Sequence; D006224:Cricetinae; D003412:Cricetulus; D015870:Gene Expression; D056945:Hep G2 Cells; D064451:Hepcidins / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D042541:Intracellular Space / Q000378:metabolism; D007501:Iron / Q000378:metabolism; D008928:Mitochondria / Q000378:metabolism; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000235:genetics; D011990:Receptors, Transferrin / Q000235:genetics* / Q000378:metabolism*; D016415:Sequence Alignment; D014168:Transferrin / Q000494:pharmacology*; D000098742:Ferroportin
chemical_list: D027682:Cation Transport Proteins; D064451:Hepcidins; D012333:RNA, Messenger; D011990:Receptors, Transferrin; D014168:Transferrin; C110714:holotransferrin; D000098742:Ferroportin; D007501:Iron
keywords: Hepcidin; Iron regulation; Iron response element; Transferrin receptor