Oxygen-dependent secretion of a bioactive hepcidin-GFP chimera

• Published in: Biochemical and biophysical research communications 2013-06, Vol.435 (4), p.540-545
• Main Authors: Chachami, Georgia, Lyberopoulou, Aggeliki, Kalousi, Alkmini, Paraskeva, Efrosyni, Pantopoulos, Kostas, Simos, George
• Format: Article
• Language: English
• Subjects: Animals; Antimicrobial Cationic Peptides - metabolism; Carcinoma, Hepatocellular - metabolism; Cell Line, Tumor; Chimera - genetics; Chimera - metabolism; GFP; Green Fluorescent Proteins - genetics; Green Fluorescent Proteins - metabolism; Hepatoma; Hepcidin; Hepcidins; Humans; Hypoxia; Iron; Liver; Liver Neoplasms - metabolism; Macrophages; Mice; Oxygen - metabolism; Recombinant Proteins - metabolism
• ISSN: 0006-291X; 1090-2104
• DOI: 10.1016/j.bbrc.2013.04.085

•GFP was fused to hepcidin C-terminus and the chimera was expressed in Huh7 cells.•Stable low expression led to secretion of a properly matured hepcidin-GFP.•Secreted hepcidin-GFP was active in terms of iron homeostasis.•Treatment with hypoxia altered the cellular distribution of pro-hepcidin-GFP.•Hypoxia reduced the secretion of mature hepcidin-GFP. Hepcidin, a hepatic hormone, regulates serum iron levels by controlling both intestinal iron absorption and iron release from macrophages. Although transcription of hepcidin is controlled by diverse stimuli, it remains elusive if post-transcriptional steps of its production are also regulated. To address this issue, GFP was fused to the C-terminus of hepcidin and the chimeric hepcidin-GFP protein was expressed in hepatoma Huh7 cells. Expression and secretion of hepcidin-GFP were analyzed by fluorescence microscopy or western blotting and its activity was assessed by in vitro biological assays. Transient over-expression of hepcidin-GFP resulted in production and secretion of premature forms. On the other hand, stable low-level expression led to synthesis and secretion of a properly matured hepcidin-GFP. This form was biologically active since it affected appropriately the levels of IRP2 and ferritin in human THP1 monocytes and targeted ferroportin in mouse J774 macrophages. Treatment of hepcidin-GFP expressing cells with hypoxia (0.1% O2) altered the subcellular distribution of pro-hepcidin-GFP and significantly reduced the secretion of mature hepcidin-GFP. Our hepcidin-GFP expression system allows the investigation of post-transcriptional processing of hepcidin and implicates hypoxia in its secretion control.