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. 2019 Aug;186(4):e108-e112.
doi: 10.1111/bjh.15939. Epub 2019 May 2.

Role of MFHAS1 in regulating hepcidin expression via the BMP/SMAD and MAPK/ERK1/2 signalling pathways

Chutima Kumkhaek  1 , Christian LaChance  2 , Wulin Aerbajinai  1 , Jianqiong Zhu  1 , Griffin P Rodgers  1
Affiliations

Role of MFHAS1 in regulating hepcidin expression via the BMP/SMAD and MAPK/ERK1/2 signalling pathways

Chutima Kumkhaek et al. Br J Haematol. 2019 Aug.
No abstract available

Keywords: BMP/SMAD; Hsp70; MAPK/ERK1/2; MFHAS1; hepcidin.

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Figures

Fig 1.
Fig 1.. MFHAS1 decreases BMP6-stimulated hepcidin upregulation, SMAD1/5/9 phosphorylation, and nuclear translocation of p-SMAD1/5/9, but increases ERK1/2 phosphorylation, in MFHAS1-overexpressing human hepatocytes.
(A) Human hepatocytes were transfected with empty vector (Control pCMV6) or Myc-DDK-tagged MFHAS1-pCMV6-Entry plasmid (MFHAS1 pCMV6) for 48 h and then treated with or without 50 ng/ml BMP6 for 24 h. Human hepatocytes without transfection were used as an internal control and indicated as non-transfected. Semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) analysis of MFHAS1 gene expression was then conducted, with GAPDH used as an internal control. (B) Mean relative hepcidin (HAMP) gene expression levels in human hepatocytes treated as described in A, shown as fold induction compared with levels in non-transfected cells without BMP6 treatment by quantitative PCR. Values were normalized to the expression level of the housekeeping gene GAPDH. Error bars represent the standard deviation from three individual experiments, *p < 0.05 compared with non-transfected- or control pCMV6-transfected BMP6-treated human hepatocytes. (C) Human hepatocytes were transfected with Control pCMV6 or MFHAS1 pCMV6 plasmid for 48 h and then treated with or without 50 ng/ml BMP6 for 1 h. Whole-cell lysates were analysed by Western blotting using antibodies targeting MFHAS1, phosphorylated SMAD1/5/9 (p-Smad1/5/9), Smad1, phosphorylated ERK1/2 (p-Erk1/2) and ERK1/2. β-actin was used as an internal control. Blots are representative images of three independent experiments showing similar results. (D) Representative images of immunofluorescence staining for p-SMAD1/5/9 (green), Myc-MFHAS1 (red), and DAPI (blue) in human hepatocytes treated as described in C.
Fig 2.
Fig 2.. Hsp70 is a binding partner for MFHAS1.
(A) HuH-7 cells were transiently transfected with Myc-DDK-tagged MFHAS1-pCMV6-Entry plasmid for 48 h, then treated with 50 ng/ml BMP6 for 1 h. Cell lysates were subjected to immunoprecipitation with anti-mouse IgG (IgG) or anti-Myc coupled to Dynabeads™ Protein G (Myc), and the eluted bound proteins run on NuPAGE™ 4–12% Bis-Tris gels and then stained with SimplyBlue™ SafeStain (Thermo Fisher Scientific, Waltham, MA, USA).. Protein bands not seen in the control IgG lane were selected for analysis. MFHAS1, TfR1, Hsp90, Hsp70, Hsp60 and MAPK1 were identified by performing mass-spectrometry analysis. (B) Cell lysates from HUDEP-2 cells at day 12 of erythroid differentiation were subjected to immunoprecipitation (IP) and subsequent Western blot analysis (IB) using the indicated antibodies. Lysate (2%) was used as input. (C) Representative images of immunofluorescence staining for MFHAS1 (green), Hsp70 (red), and DAPI (blue) in HUDEP-2 cells at day 12 of erythroid differentiation. WW: molecular weight.
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