Expression of ABCG2 (BCRP) in mouse models with enhanced erythropoiesis

Haem is a structural component of numerous cellular proteins which contributes significantly to iron metabolic processes in mammals but its toxicity demands that cellular levels must be tightly regulated. Breast Cancer Resistance Protein (BCRP/ABCG2), an ATP Binding Cassette G-member protein has been shown to possess porhyrin/haem efflux function. The current study evaluated the expression and regulation of Abcg2 mRNA and protein levels in mouse tissues involved in erythropoiesis. Abcg2 mRNA expression was enhanced in bone marrow hemopoietic progenitor cells from mice that were treated with phenylhydrazine (PHZ). Abcg2 mRNA expression was increased particularly in the extramedullary haematopoietic tissues from all the mice models with enhanced erythropoiesis. Haem oxygenase (ho1) levels tended to increase in the liver of mice with enhanced erythropoiesis and gene expression patterns differed from those observed in the spleen. Efflux of haem biosynthetic metabolites might be dependent on the relative abundance of Abcg2 or ho1 during erythropoiesis. Abcg2 appears to act principally as a safety valve regulating porphyrin levels during the early stages of erythropoiesis and its role in systemic haem metabolism and erythrophagocytosis, in particular, awaits further clarification

The transcription factor ATOH8 is regulated by erythropoietic activity and regulates HAMP transcription and cellular pSMAD1,5,8 levels

ATOH8 has previously been shown to be an iron-regulated transcription factor, however its role in iron metabolism is not known. ATOH8 expression in HEK293 cells resulted in increased endogenous HAMP mRNA levels as well as HAMP promoter activity. Mutation of the E-box or SMAD response elements within the HAMP promoter significantly reduced the effects of ATOH8, indicating that ATOH8 activates HAMP transcription directly as well as through bone morphogenic protein (BMP) signalling. In support of the former, Chromatin immunoprecipitation assays provided evidence that ATOH8 binds to E-box regions within the HAMP promoter while the latter was supported by the finding that ATOH8 expression in HEK293 cells led to increased phosphorylated SMAD1,5,8 levels. Liver Atoh8 levels were reduced in mice under conditions associated with increased erythropoietic activity such as hypoxia, haemolytic anaemia, hypotransferrinaemia and erythropoietin treatment and increased by inhibitors of erythropoiesis. Hepatic Atoh8mRNA levels increased in mice treated with holo transferrin, suggesting that Atoh8 responds to changes in plasma iron. ATOH8 is therefore a novel transcriptional regulator of HAMP, which is responsive to changes in plasma iron and erythroid activity and could explain how changes in erythroid activity lead to regulation of HAMP

Iron absorption in hepcidin1 knockout mice

Hepcidin, the Fe-regulatory peptide, has been shown to inhibit Fe absorption and reticuloendothelial Fe recycling. The present study was conducted to explore the mechanism of in vivo Fe regulation through genetic disruption of hepcidin1 and acute effects of hepcidin treatment in hepcidin1 knockout (Hepc1− / − ) and heterozygous mice. Hepcidin1 disruption resulted in significantly increased intestinal Fe uptake. Hepcidin injection inhibited Fe absorption in both genotypes, but the effects were more evident in the knockout mice. Hepcidin administration was also associated with decreased membrane localisation of ferroportin in the duodenum, liver and, most significantly, in the spleen of Hepc1− / −  mice. Hypoferraemia was induced in heterozygous mice by hepcidin treatment, but not in Hepc1− / −  mice, 4 h after injection. Interestingly, Fe absorption and serum Fe levels in Hepc1− / −  and heterozygous mice fed a low-Fe diet were not affected by hepcidin injection. The present study demonstrates that hepcidin deficiency causes increased Fe absorption. The effects of hepcidin were abolished by dietary Fe deficiency, indicating that the response to hepcidin may be influenced by dietary Fe level or Fe status.</jats:p

BMPER Protein Is a Negative Regulator of Hepcidin and Is Up-regulated in Hypotransferrinemic Mice

The BMP/SMAD4 pathway has major effects on liver hepcidin levels. Bone morphogenetic protein-binding endothelial cell precursor-derived regulator (Bmper), a known regulator of BMP signaling, was found to be overexpressed at the mRNA and protein levels in liver of genetically hypotransferrinemic mice (Trfhpx/hpx). Soluble BMPER peptide inhibited BMP2- and BMP6-dependent hepcidin promoter activity in both HepG2 and HuH7 cells. These effects correlated with reduced cellular levels of pSMAD1/5/8. Addition of BMPER peptide to primary human hepatocytes abolished the BMP2-dependent increase in hepcidin mRNA, whereas injection of Bmper peptide into mice resulted in reduced liver hepcidin and increased serum iron levels. Thus Bmper may play an important role in suppressing hepcidin production in hypotransferrinemic mice

Effects of ME on spatial memory in mice.

<p>(<b>A</b>) Effects of ME on escape latency during 3 test trials of day1, 2 and 3 of the treatment using Morris water maze test. ME (50 or 100 mg/kg) or water (control) was orally administered to the mice 45 min before the trials (n=6). The escape latency (sec) was expressed as Mean ± SEM. * <i>P</i> < 0.05 compared to control in the same test trial on the same test day; # <i>P</i> <0.05 compared to the trial 1 of the same test condition in the same test day. (<b>B</b>) Effects of ME on time in right quadrant (probe trial) on day 1-3 and 14-16 of the treatment using Morris water maze test. The mice were orally administered with water (control) or ME (50 and 100 mg/kg) at 45 minutes before the test. The escape latency (sec) was expressed as Mean ± SEM (n=6). * <i>P</i> < 0.05 compared to control on the same test day; # <i>P</i> < 0.05 compared to 50 mg/kg ME-treated group on the same test day.</p

Cytotoxicity screening of mangosteen extract in SK-N-SH cells.

<p>SK-N-SH cells were treated with mangosteen extract at the concentrations of 25-800 µg/ml. After 24-hour incubation, cytotoxicity was determined by MTT assay. Data were presented as mean ± SEM (n=8 for each group). ***<i>P</i> < 0.001 compared to control.</p