Effect of Recombinant Human Erythroferrone Protein on Hepcidin Gene (Hamp1) Expression in HepG2 and HuH7 Cells

Abstract

Iron is essential for all living organisms. It is strictly controlled by iron transporters, transferrin receptors, ferroportin and hepcidin. Erythroferrone (ERFE) is an iron-regulatory hormone which is highly expressed in erythroblasts by erythropoietin (EPO) stimulation and osteoblasts independently of EPO by sequestering bone morphogenetic proteins and inhibiting hepatic hepcidin expression. Although the hepcidin suppressive function of ERFE is known, its receptors still require investigation. Here, we aim to identify ERFE receptors on the HepG2 and Huh7 cells responsible for ERFE. Recombinant ERFE (rERFE) was first produced in HEK293 cells transfected with pcDNA3.1 + ERFE, then purified and detected by Western blot. The liver cells were treated with an rERFE-rich medium of transfected HEK293 cells and a purified rERFE-supplemented medium at various time points, and hepcidin gene (Hamp1) expression was determined using qRT-PCR. The results show that 37-kD rERFE was expressed in HEK293 cells. Hamp1 was suppressed at 3 h and 6 h in Huh7 cells after rERFE treatments (p < 0.05), then restored to the original levels. Hamp1 was activated after treatment with purified rERFE for 24 h and 48 h. Together, these results reveal that ERFE suppressed Hamp1 expression in liver cells, possibly acting on membrane ERFE receptor, which in Huh7 cells was more sensitive to the ERFE concentrate.

Keywords: erythroferrone; hepcidin; iron; receptor; recombinant protein.

Figure 1

Western blot analysis of ERFE–FLAG protein expression in culture medium and pellet of HEK293 cells transfected with pcDNA3.1 + ERFE–FLAG plasmid. HEK293T cells were transfected with pcDNA3.1 + ERFE–FLAG plasmid or standard pcDNA3.1 + (mock) and cultured in 10% FBS-supplemented DMEM for 24 h. The expressed ERFE–FLAG protein was analyzed in the medium and the cells using Western blot technique. Abbreviations: DMEM = Dulbecco’s Modified Eagle Medium, ERFE = erythroferrone, FBS = fetal bovine serum, HEK293 = human embryonic kidney 293.

Figure 2

Western blot analysis of expressed ERFE–FLAG protein purified from the medium of HEK293 cells transfected with pcDNA3.1 + ERFE–FLAG plasmid. HEK293T cells were transfected with pcDNA3.1 + ERFE–FLAG plasmid or standard pcDNA3.1 + (mock) and cultured in 10% FBS-supplemented DMEM for 24 h. The expressed ERFE–FLAG protein was purified using immuno-precipitation technique and analyzed using Western blot technique. Abbreviations: DMEM = Dulbecco’s Modified Eagle Medium, ERFE = erythroferrone, FBS = fetal bovine serum, HEK293 = human embryonic kidney 293.

Figure 3

Relative hepcidin mRNA levels in HepG2 cells at various time points after ERFE treatment. HepG2 cells were cultured in 50% (v/v) DMEM obtained from HEK293 cells transfected with pcDNA3.1 + ERFE (ERFE–FLAG protein) or pcDNA3.1+ (MOCK) (A) or in 3 μg/mL purified ERFE-supplemented DMEM (B) for 1–48 h and determined mRNA levels using qRT-PCR. Data which were obtained from four repetitions and normalized with constitutive RPL19 are expressed as mean ± SD of fold-change hepcidin mRNA expression using −delta Ct (Rpl19-Hamp). * p < 0.05 when compared with MOCK. Abbreviations: Ct = control, DMEM = Dulbecco’s Modified Eagle Medium, ERFE = erythroferrone, HEK293 = human embryonic kidney 293 cells, qRT-PCR = quantitative real-time polymerase chain reaction.

Figure 4

Relative hepcidin mRNA levels in Huh7 cells at various time points after ERFE treatment. Huh7 cells were cultured in 50% (v/v) DMEM obtained from HEK293 cells transfected with pcDNA3.1 + ERFE (ERFE–FLAG protein) or pcDNA3.1+ (MOCK) (A) or in 3 μg/mL purified ERFE-supplemented DMEM (B) for 1–48 h and determined mRNA levels using qRT-PCR. Data which were obtained from four repetitions and normalized with constitutive RPL19 are expressed as mean ± SD of fold-change hepcidin mRNA expression using −delta Ct (Rpl19-Hamp). * p < 0.05 when compared with MOCK. Abbreviations: Ct = control, DMEM = Dulbecco’s Modified Eagle Medium, ERFE = erythroferrone, HEK293 = human embryonic kidney 293 cells, qRT-PCR = quantitative real-time polymerase chain reaction.

Figure 5

Relative hepcidin mRNA levels in HepG2 cells after 24 h of ERFE treatment. HepG2 cells were cultured in ERFE–FLAG-rich DMEM of the transfected HEK293 cells (A) or purified ERFE-supplemented DMEM (B) and mRNA levels determined using qRT-PCR. Data on fold changes in hepcidin mRNA expression using −delta Ct (Rpl19-Hamp) were obtained from four repetitions and normalized with constitutive RPL19 are expressed as fold changes and as mean ± SD. * p < 0.05 when compared to no treatment. Abbreviations: Ct = control, DMEM = Dulbecco’s Modified Eagle Medium, ERFE = erythroferrone, FBS = fetal bovine serum, HEK293 = human embryonic kidney 293 cells, qRT-PCR = quantitative real-time polymerase chain reaction, RPL19 = ribosomal protein L19.

Figure 6

Relative hepcidin mRNA levels in Huh7 cells after 24 h of ERFE treatment. Huh7 cells were cultured in the ERFE–FLAG-rich DMEM of transfected HEK293 cells (A) or purified ERFE-supplemented DMEM (B) and mRNA levels determined using qRT-PCR. Data were obtained from four repetitions and normalized with constitutive RPL19, and are expressed as mean ± SD of fold-change hepcidin mRNA expression using −delta Ct (Rpl19-Hamp). p < 0.05 when compared to no treatment. Abbreviations: Ct = control, DMEM = Dulbecco’s Modified Eagle Medium, ERFE = erythroferrone, FBS = fetal bovine serum, HEK293 = human embryonic kidney 293 cells, qRT-PCR = quantitative real-time polymerase chain reaction.