Experimental Studies Of The Effects Of HEPC1 And HEPC2 On Iron Store And Release In Mouse Macrophages

Hepcidin(HEPC or HAMP), the liver-specific expressing antimicrobial peptide, is cysteine-rich and has wide spectrum of antimicrobial activities. Homologous hepcidin was found in species from fish to human. Hepcidin mature peptide contains only about twenty amino acids. In contrast to the human genome, which contains only one copy of the gene, the mouse genome contains 2 highly similar hepcidin genes, hepc1 and hepc2. Soon after it was discovered, hepcidin was found involving in iron homeostasis, and now is regarded as a key iron-regulatory hormone. In vivo hepcidin reduced intestines iron absorption, placenta iron transport and the release of iron from reticuloendothelial system (RES) macrophages, but the mechanisms are still unknown. It has been reported that in vitro hepcidin reduced the iron absorption of the intestine isolated from rats, and it also decreased iron uptake of the human intestinal epithelial cell line Caco-2 cells, indicating that hepcidin could act on enterocytes directly and reduce their iron absorption although the mechanism is still unclear. To research the mechanism by which hepcidin increased RES iron retention, we have first investigated whether in vitro hepcidin affects macrophages iron retention. The brief protocol and major results were described as follow. Objective 1. To construct eukaryotic expression vectors of HEPC1 and HEPC2 genes. 2. To investigate the effects of HEPC1 and HEPC2 on iron store and release in mouse macrophage lines Ana-1 and RAW264.7. Methods 1. Construction of the eukaryotic expression vectors of HEPC1 and HEPC2 genes Total RNA was isolated from liver of mouse, and the cDNAs of HEPC1 and HEPC2 were synthesized by RT-PCR. The specific amplification products were respectively cloned into pcDNA3, and then identified by restriction enzymes digestion and sequencing analysis. 2. The effects of HEPC1 and HEPC2 on iron store and release in Ana-1 cells NIH3T3 cells were transfected with pcDNA3, pcDNA3/HEPC1 or pcDNA3/HEPC2 using lipofectin reagent. The positive clones of the transfected cells were obtained by G418 screening, and then identified by RT-PCR and dot-blot. The culture supernatants were collected after the NIH3T3 cells stably transfected with pcDNA3, pcDNA3/HEPC1 and pcDNA3/HEPC2 respectively were cultured in IMDM with 10% FCS at 37°C, 5% CO2 for 24 hours. To evaluate the effects of HEPC1 and HEPC2 on iron store in Ana-1 cells, Ana-1 cells continued to be cultured for 16 hours after the collected culture supernatants and citrate-iron were added, and then the intracellular iron concentrations were determined by atomic absorption spectroscopy (AAS). To evaluate the effects of HEPC1 and HEPC2 on iron release in Ana-1 cells, Ana-1 cells were cultured in IMDM with high concentration citrate-iron for 24 hours, then were washed and continued to be cultured for 16 hours in collected culture supernatants. Ana-1 cells were collected and their intracellular iron concentrations were determined by AAS. 3. The effects of HEPC1 and HEPC2 on iron store and release in RAW264.7 cells RAW264.7 cells were transfected with pcDNA3, pcDNA3/HEPC1 or pcDNA3/HEPC2 using lipofectin reagent. The positive clones of the transfected cells were obtained by G418 screening, and then identified by RT-PCR. RAW264.7 cells respectively stably transfected with pcDNA3, pcDNA3/HEPC1 and pcDNA3/HEPC2 were cultured in IMDM with 10% FCS at 37°C, 5% CO2, and were treated with 25μmol/L FeCl3 and 1μCi59FeCl3 for 24 hours. To evaluate the effects of HEPC1 and HEPC2 on iron store in RAW264.7 cells, all the RAW264.7 cells were washed and counted for radioactivity in a gamma counter. To evaluate the effects of HEPC1 and HEPC2 on iron release in RAW264.7 cells, all the treated RAW264.7 cells were washed and continued to be cultured in IMDM with 10% FCS for 8 hours. Cells and their supernatants were collected and counted for radioactivity in a gamma counter. The percentage of iron release was calculated as [(cpm in supernatant) / (cpm in supernatant +cpm cells)] ×100. Results 1. Construction of the eukaryotic expression vectors of HEPC1 and HEPC2 genes The cDNAs of HEPC1 and HEPC2 were obtained from the tissue of mouse liver by RT-PCR. DNA sequencing analysis of the positive recombinants suggested the eukaryoticexpression vectors of HEPC1 and HEPC2 were successfully constructed. 2. The effects of HEPC1 and HEPC2 on iron store and release in Ana-1 cells RT-PCR and dot-blot analysises showed that the NIH3T3 cells stably transfected respectively with pcDNA3, pcDNA3/HEPC1 and pcDNA3/HEPC2 were screened successfully, and HEPC1 and HEPC2 proteins were secreted into the culture supernatants. The culture supernatants of NIH3T3 cells stably transfected with pcDNA3/HEPC1 or pcDNA3/HEPC2 increased iron store of Ana-1 cells and reduced iron release from Ana-1 cells compared with the culture supernatants of NIH3T3 cells stably transfected with pcDNA3. 3. The effects of HEPC1 and HEPC2 on iron store and release in RAW264.7 cells RT-PCR analysis showed RAW264.7 cells stably transfected respectively with pcDNA3, pcDNA3/HEPC1 and pcDNA3/HEPC2 were gained successfully. RAW264.7 cells stably transfected with pcDNA3/HEPC1 and pcDNA3/HEPC2 respectively increased iron store by 1.65 fold and 1.71 fold, and reduced iron release by 67.6% and 57.9% respectively, comparing with RAW264.7 cells stably transfected with pcDNA3 using iron isotopic analysis. Conclusion 1. The cDNAs of HEPC1 and HEPC2 were obtained from the tissue of mouse liver by RT-PCR, and the eukaryotic expression vectors of HEPC1 and HEPC2 genes were successfully constructed. 2. HEPC1 and HEPC2 can increase mouse macrophage lines Ana-1 and RAW264.7 iron store and reduce their iron release,indicating that hepcidin can increase macrophages iron retention directly in vitro. So the mechanism by which hepcidin increased RES iron retention in vivo may be the direct act of hepcidin on macrophages.