Friend of GATA suppresses the GATA-induced transcription of hepcidin in hepatocytes through a GATA-regulatory element in the HAMP promoter

Abstract

Hepcidin is an antimicrobial peptide hormone involved in the metabolism of iron, encoded for by the HAMP gene mainly in hepatocytes. It's expressed at lower levels in other cells such as the macrophages. The mechanisms that determine tissue-specific expression of hepcidin remain unclear. GATA-and its co-factor Friend of GATA (FOG) modulate the tissue-specific transcription of other genes involved in the metabolism of iron. GATA proteins are group of evolutionary conserved transcriptional regulators that bind to the consensus motif -WGATAR-in the promoter. We characterized a 1 . 3 kb fragment of the 5 0 -flanking sequence of the HAMP gene in Huh7 cells, which express HAMP. Transfection of 5 0 -deletions of the HAMP promoter in Huh7 cells revealed two regions, K932/K878 and K155/K96, that when deleted decreased promoter activity. Using site-directed mutations in the HAMP promoter region K155/K96 we identified two subregions, K138/K125 and K103/K98, which when mutated suppressed promoter activity by 70 and 90% respectively. Site K103/K98 with a sequence -TTATCT-to which endogenous GATA proteins 4 and 6 bind and transactivate HAMP is a GATA-regulatory element (RE). Mutation of the GATA-RE abrogated binding of GATA proteins 4 and 6 to the promoter and blunted the GATA transactivation of HAMP. FOG proteins 1 and 2 suppressed the endogenous and exogenous GATA activation of the HAMP promoter. We concluded that the GATA-RE, -TTATCT-in the HAMP promoter region K103/K98 is crucial for the GATA-4 and GATA-6 driven transcription of hepcidin in Huh7 cells and that FOG proteins moderate the transcription by suppressing the GATA transactivation of HAMP.

Short-term rapid atrial pacing alters the gene expression profile of rat liver: Cardiohepatic interaction in atrial fibrillation

“…26 A previous study reported that IL-6 stimulation increased the binding of GATA-4 to the HAMP promoter. 27 Our data demonstrated that RAP-induced coagulation factor X production was suppressed by IL-6 neutralizing antibody, suggesting the involvement of IL-6 in the production of factor X, as well as fibrinogen in AF.…”

MyD88 Regulates the Expression of SMAD4 and the Iron Regulatory Hormone Hepcidin

“…Next, we examined the effects of MyD88 on the SMAD4-dependent activation of hepcidin with a luciferase reporter-gene controlled by the human HAMP promoter (HAMP-Luc) ( Bagu and Santos, 2011 ). In line with previous studies ( Bagu and Santos, 2011 ; Besson-Fournier et al, 2012 ), both BMP6 and Activin B, two distinct BMP-signaling activators relevant to hepcidin induction, were able to activate the HAMP-Luc reporter gene ( Figure 2C and Supplementary Figure S1D ). In turn, MyD88 transfection also led to HAMP-Luc activation ( Figure 2C and Supplementary Figures S2C,D ).…”

“…Plasmid pCMV was created by removing MyD88 from pCMV-HA-MyD88 by digestion with restriction enzyme. The wild-type (-1234/+73) HAMP promoter reporter construct HAMP-Luc ( Firefly luciferase) and Renilla luciferase reporter phRL-TK plasmid were used in the present study as previously described ( Bagu and Santos, 2011 ). The QuickChange II site-directed mutagenesis kit (Agilent Technologies, ON, Canada) was used to generate the HAMP-LucΔBMP-RE1 construct, in which the BMP-RE1 site was mutated from GGCGCC to AGAACC ( Verga Falzacappa et al, 2008 ).…”

“…Cells were transiently co-transfected by lipofection using Lipofectamine TM 2000 (Invitrogen). Lipofection included Renilla luciferase (phRL-TK) as the control reporter and the Firefly luciferase under the control of the HAMP promoter (HAMP-Luc) ( Bagu and Santos, 2011 ; Bagu et al, 2013 ) in combination with pCMV-HA-MyD88 (pMyD88) or empty vector pCMV-HA (also referred as pCMV). The total amount of DNA was kept constant.…”

The 17β-Estradiol induced upregulation of the Adhesion G-protein coupled receptor (ADGRG7) is modulated by ESRα and SP1 complex

“…The human hepatocellular carcinoma cell line Huh7 cells was a kind gift from Dr M. Santo and cultured as previously published (Bagu and Santos, 2011). Human breast cancer MCF-7 cells were purchased from American Type Culture Collection (American Type Culture Collection).…”