Regulation of transferrin receptor-1 mRNA by the interplay between IRE-binding proteins and miR-7/miR-141 in the 3'-IRE stem-loops.

Intracellular iron is tightly regulated by coordinated expression of iron transport and storage genes, such as transferrin receptor-1 (TfR1) and ferritin. They are primarily regulated by iron through iron-induced dissociation of iron-regulatory proteins (IRPs) from iron-responsive elements (IREs) in the 3'-UTR (untranslated region) of TfR1 or 5'-UTR of ferritin mRNA, resulting in destabilization of TfR1 mRNA and release of ferritin translation block. Thus high iron decreases iron transport via TfR1 mRNA degradation and increases iron storage via ferritin translational up-regulation. However, the molecular mechanism of TfR1 mRNA destabilization in response to iron remains elusive. Here, we demonstrate that miR-7-5p and miR-141-3p target 3'-TfR1 IREs and down-regulate TfR1 mRNA and protein expression. Conversely, miR-7-5p and miR-141-3p antagomiRs partially but significantly blocked iron- or IRP knockdown-induced down-regulation of TfR1 mRNA, suggesting the interplay between these microRNAs and IRPs along with involvement of another uncharacterized mechanism in TfR1 mRNA degradation. Luciferase reporter assays using 3'-UTR TfR1 IRE mutants suggested that the IREs C and E are targets of miR-7-5p and miR-141-3p, respectively. Furthermore, miR-7 expression was inversely correlated with TfR1 mRNA in human pancreatic adenocarcinoma patient samples. These results suggest a role of microRNAs in the TfR1 regulation in the IRP-IRE system.

MRI reporter gene MagA suppresses transferrin receptor and maps Fe2+ dependent lung cancer.

As a magnetic resonance imaging (MRI) reporter gene, MagA has become a powerful tool to monitor dynamic gene expression and allowed concomitant high resolution anatomical and functional imaging of subcellular genetic information. Here we establish a stably expressed MagA method for lung cancer MRI. The results show that MagA can not only enhance both in vitro and in vivo MRI contrast by specifically alternating the transverse relaxation rate of water, but also inhibit the malignant growth of lung tumor. In addition, MagA can regulate magnetic nanoparticle production in grafted tissues and also suppress transferrin receptor expression by acting as an iron transporter, and meanwhile can permit iron biomineralization in the presence of mammalian iron homeostasis. This work provides experimental evidence for the safe preclinical applications of MagA as both a potential inhibitor and an MRI-based tracing tool for iron ion-dependent lung cancer.

Fasudil alleviates pressure overload-induced heart failure by activating Nrf2-mediated antioxidant responses.

The RhoA/Rho-kinase cascade plays an important role in many aspects of cardiovascular function. This study aims to investigate the protective effects of fasudil, a Rho-kinase inhibitor, on pressure overload induced heart failure in rats. Pressure overload induced heart failure was induced in SD rats by banding the abdominal aorta for 8 weeks. The rats were divided into four groups: Sham, TAC, TAC plus low dose of fasudil, and TAC plus high dose of fasudil group. Low dose and high dose fasudil were 5 and 10 mg/kg/day, respectively. Rats in the Sham and TAC groups were treated with vehicle. Fasudil effectively inhibited TAC-induced heart failure, as evaluated by echocardiography and transmission electron microscopy. Fasudil could significantly promote superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) activity and significantly decrease malondialdehyde (MDA) content in a dose-dependent maner in TAC rats. Consistently, fasudil evoked significant nuclear translocation of Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) with increased DNA/promoter binding and transactivation of Nrf2 targets. In addition, fasudil increased the content of iron as well as transferrin receptor 1 (TfR1) in TAC rats. A mild oxidative stress induced by iron may activate the antioxidant enzymes by feedback response. Taken together, these results indicate that the protective effect of fasudil may be due to its strong antioxidative activities which related with the activated Nrf2 and its down-regulated genes. These findings provide a new treatment concept and support the benefit of fasudil treatment in heart failure.

Dietary Iron Fortification Normalizes Fetal Hematology, Hepcidin, and Iron Distribution in a Rat Model of Prenatal Alcohol Exposure.

BACKGROUND: Prenatal alcohol exposure (PAE) causes neurodevelopmental disability. Clinical and animal studies show gestational iron deficiency (ID) exacerbates PAE's behavioral and growth deficits. In rat, PAE manifests an inability to establish iron homeostasis, increasing hepcidin (maternal and fetal), and fetal liver iron while decreasing brain iron and promoting anemia. Here, we hypothesize dietary iron fortification during pregnancy may mitigate alcohol's disruption of fetal iron homeostasis. METHODS: Pregnant Long-Evans rats, fed iron-sufficient (100 ppm iron) or iron-fortified (IF; 500 ppm iron) diets, received either 5 g/kg alcohol (PAE) or isocaloric maltodextrin daily on gestational days (GD) 13.5 through 19.5. Maternal and fetal outcomes were evaluated on GD20.5. RESULTS: PAE reduced mean fetal weight (p < 0.001) regardless of maternal iron status, suggesting iron fortification did not improve fetal growth. Both PAE (p < 0.01) and IF (p = 0.035) increased fetal liver iron. In fetal brain, PAE (p = 0.015) affected total (p < 0.001) and nonheme iron (p < 0.001) such that iron fortification normalized (p = 0.99) the alcohol-mediated reductions in brain iron and nonheme iron. Iron fortification also improved fetal hematologic indices in PAE including hemoglobin, hematocrit, and mean cell volume (ps<0.001). Iron fortification also normalized hepcidin expression in alcohol-exposed maternal and fetal liver. Neither diet nor PAE affected transferrin (Tf) and ferritin (FTN) content in fetal liver, nor Tf or transferrin receptor in fetal brain. However, IF-PAE fetal brains trended to less FTN content (p = 0.074), suggesting greater availability of nonstorage iron. In PAE, hepcidin levels were linearly related to increased liver iron stores and decreased red blood cell count and brain iron. CONCLUSIONS: Maternal oral iron fortification mitigated PAE's disruption of fetal iron homeostasis and improved brain iron content, hematologic indices, and hepcidin production in this rat PAE model. Clinical studies show maternal ID substantially enhances fetal vulnerability to PAE, and our work supports increased maternal dietary iron intake may improve fetal iron status in alcohol-exposed pregnancies.

Unique high and homogenous surface expression of the transferrin receptor CD71 on murine plasmacytoid dendritic cells in different tissues.

Plasmacytoid dendritic cells (pDC) are of increasing interest in cancer vaccine development, but many functions of these highly specialized, multifaceted cells are poorly understood. The transferrin receptor CD71 has also been suggested to function as an antigen uptake receptor on professional antigen-presenting cells. In this study, we employed multiparameter flow cytometry to investigate CD71 expression on various leukocyte subsets, including DC subsets, granulocytes, macrophages, T and B lymphocytes, γδ T cells, and natural killer cells. Cells from various lymphoid and non-lymphoid murine tissues were analyzed using fluorochrome-conjugated monoclonal antibodies. High CD71 expression (90-100%) was observed, uniquely on pDC amongst the leukocyte populations examined, in both lymphoid and non-lymphoid tissues, including other DC subsets. In contrast, CD71 expression on non-tissue pDC, in the bone marrow and peripheral blood, was reduced. The cause and function of this high tissue pDC-selective CD71 expression remain to be examined.

Upregulation of transferrin receptor-1 induces cholangiocarcinoma progression via induction of labile iron pool.

Labile iron pool is a cellular source of ions available for Fenton reactions resulting in oxidative stress. Living organisms avoid an excess of free irons by a tight control of iron homeostasis. We investigated the altered expression of iron regulatory proteins and iron discrimination in the development of liver fluke-associated cholangiocarcinoma. Additionally, the levels of labile iron pool and the functions of transferrin receptor-1 on cholangiocarcinoma development were also identified. Iron deposition was determined using the Prussian blue staining method in human cholangiocarcinoma tissues. We investigated the alteration of iron regulatory proteins including transferrin, transferrin receptor-1, ferritin, ferroportin, hepcidin, and divalent metal transporter-1 in cholangiocarcinoma tissues using immunohistochemistry. The clinicopathological data of cholangiocarcinoma patients and the expressions of proteins were analyzed. Moreover, the level of intracellular labile iron pool in cholangiocarcinoma cell lines was identified by the RhoNox-1 staining method. We further demonstrated transferrin receptor-1 functions on cell proliferation and migration upon small interfering RNA for human transferrin receptor 1 transfection. Results show that Iron was strongly stained in tumor tissues, whereas negative staining was observed in normal bile ducts of healthy donors. Interestingly, high iron accumulation was significantly correlated with poor prognosis of cholangiocarcinoma patients. The expressions of iron regulatory proteins in human cholangiocarcinoma tissues and normal liver from cadaveric donors revealed that transferrin receptor-1 expression was increased in the cancer cells of cholangiocarcinoma tissues when compared with the adjacent normal bile ducts and was significantly correlated with cholangiocarcinoma metastasis. Labile iron pool level and transferrin receptor-1 expression were significantly increased in KKU-214 and KKU-213 when compared with cholangiocyte cells (MMNK1). Additionally, the suppression of transferrin receptor-1 expression significantly decreased intracellular labile iron pool, cholangiocarcinoma migration, and cell proliferation when compared with control media and control small interfering RNA. In Conclusion, high expression of transferrin receptor-1 resulting in iron uptake contributes to increase in the labile iron pool which plays roles in cholangiocarcinoma progression with aggressive clinical outcomes.

Real-Time Specific Light-Up Sensing of Transferrin Receptor: Image-Guided Photodynamic Ablation of Cancer Cells through Controlled Cytomembrane Disintegration.

Transferrin receptor (TfR) represents a unique target for specific imaging of cancer cells and targeted delivery of therapeutic reagents. Detection and qualification of TfR is thus of great importance for cancer diagnosis and therapy. In this contribution, a light-up probe TPETH-2T7 was developed by conjugating a red-emissive photosensitizer with aggregation-induced emission (AIE) characteristics to a TfR-targeting peptide T7. The probe is almost nonemissive by itself, but it gives turn-on fluorescence in the presence of TfR with a detection limit of 0.45 µg/mL. Cellular experiments show that the probe specifically binds to TfR-overexpressed cancer cells. Real-time imaging results reveal that the probe stains the MDA-MB-231 cell membrane in 30 min, which is followed by probe internalization. Experiments on image-guided photodynamic cancer ablation show that the therapeutic performance is better when TPETH-2T7 is localized on the cell membrane as compared to that being internalized into cells. Confocal laser scanning microscopy (CLSM) study reveals that cytomembrane disintegration allows quick ablation of MDA-MB-231 cells.

Release of Vesicular Stomatitis Virus Spike Protein G-Pseudotyped Lentivirus from the Host Cell Is Impaired upon Low-Density Lipoprotein Receptor Overexpression.

Production of a vesicular stomatitis virus spike protein G (VSVG)-pseudotyped lentiviral expression vector in HEK293 cells decreased on overexpression of low-density lipoprotein receptor (LDLR) but not that of ICAM1 or TfR1. Reverse transcription-quantitative PCR (RT-qPCR) revealed a reduction in vector RNA as a function of LDLR expression. Decreased syncytium formation suggested diminished surface expression of VSVG. Intracellular VSVG granules colocalized with LDLR, ER-Golgi intermediate compartment protein 53 (ERGIC53), LAMP2, and vimentin but not with GM130 or calnexin, suggesting that VSVG interacts with LDLR within the ERGIC, resulting in rerouting into the aggresome/autophagosome pathway.

Effect of testosterone on hepcidin, ferroportin, ferritin and iron binding capacity in patients with hypogonadotropic hypogonadism and type 2 diabetes.

CONTEXT: As the syndrome of hypogonadotropic hypogonadism (HH) is associated with anaemia and the administration of testosterone restores haematocrit to normal, we investigated the potential underlying mechanisms. DESIGN: Randomized, double-blind, placebo-controlled trial. METHODS: We measured basal serum concentrations of erythropoietin, iron, iron binding capacity, transferrin (saturated and unsaturated), ferritin and hepcidin and the expression of ferroportin and transferrin receptor (TR) in peripheral blood mononuclear cells (MNC) of 94 men with type 2 diabetes. Forty-four men had HH (defined as subnormal free testosterone along with low or normal LH concentrations) while 50 were eugonadal. Men with HH were randomized to testosterone or placebo treatment every 2 weeks for 15 weeks. Blood samples were collected at baseline, 3 and 15 weeks after starting treatment. Twenty men in testosterone group and 14 men in placebo group completed the study. RESULTS: Haematocrit levels were lower in men with HH (41·1 ± 3·9% vs 43·8 ± 3·4%, P = 0·001). There were no differences in plasma concentrations of hepcidin, ferritin, erythropoietin, transferrin or iron, or in the expression of ferroportin or TR in MNC among HH and eugonadal men. Haematocrit increased to 45·3 ± 4·5%, hepcidin decreased by 28 ± 7% and erythropoietin increased by 21 ± 7% after testosterone therapy (P < 0·05). There was no significant change in ferritin concentrations, but transferrin concentration increased while transferrin saturation and iron concentrations decreased (P < 0·05). Ferroportin and TR mRNA expression in MNC increased by 70 ± 13% and 43 ± 10%, respectively (P < 0·01), after testosterone therapy. CONCLUSIONS: The increase in haematocrit following testosterone therapy is associated with an increase in erythropoietin, the suppression of hepcidin, and an increase in the expression of ferroportin and TR.

AMD-like retinopathy associated with intravenous iron.

Iron accumulation in the retina is associated with the development of age-related macular degeneration (AMD). IV iron is a common method to treat iron deficiency anemia in adults, and its retinal manifestations have not hitherto been identified. To assess whether IV iron formulations can be retina-toxic, we generated a mouse model for iron-induced retinal damage. Male C57BL/6J mice were randomized into groups receiving IV iron-sucrose (+Fe) or 30% sucrose (-Fe). Iron levels in neurosensory retina (NSR), retinal pigment epithelium (RPE), and choroid were assessed using immunofluorescence, quantitative PCR, and the Perls' iron stain. Iron levels were most increased in the RPE and choroid while levels in the NSR did not differ significantly in +Fe mice compared to controls. Eyes from +Fe mice shared histological features with AMD, including Bruch's membrane (BrM) thickening with complement C3 deposition, as well as RPE hypertrophy and vacuolization. This focal degeneration correlated with areas of high choroidal iron levels. Ultrastructural analysis provided further detail of the RPE/photoreceptor outer segment vacuolization and Bruch's membrane thickening. Findings were correlated with a clinical case of a 43-year-old patient who developed numerous retinal drusen, the hallmark of AMD, within 11 months of IV iron therapy. Our results suggest that IV iron therapy may have the potential to induce or exacerbate a form of retinal degeneration. This retinal degeneration shares features with AMD, indicating the need for further study of AMD risk in patients receiving IV iron treatment.

Clinical value of high expression level of CD71 in acute myeloid leukemia.

CD71 (transferrin receptor 1, TfR-1) is a type II membrane glycoprotein and associated closely with tumors. It was recognized as an indication for diagnosing acute erythroid leukemia (AEL). High expression level of CD71 has been identified as a negative prognostic marker for many solid tumors. However, whether CD71 should be identified as an adverse marker in acute myeloid leukemia (AML) remained conflicting. We studied 214 AML patients for analysis of clinical and laboratory data. Taking the CD71 expression level of 60% as a standard, we divided our patients into two groups. We discovered that AML with high expression level of CD71 was prone to linked with severe anemia (P=0.004), thrombocytopenia (P<0.001) and complex karyotype (P=0.024) and had increasing expression level of CD117 (P=0.001). No statistically significant correlations in age, gender, WBC counting, molecular markers between the two groups. And moreover, high expression level of CD71 did not alter the pattern of survival time.

A convenient method for hTfR1 inclusion body purification.

Human transferrin receptor, referred as hTfR1, is ubiquitously expressed at low levels in most normal human tissues; however, the expression level of hTfR1 at the blood-brain barrier (BBB) and in tumor tissues is relatively higher. hTfR1 is a type II homodimeric transmembrane protein. The extracellular domain of hTfR1 consists of three domains: helical domain, apical, and protease-like domain. In order to prepare hTfR1 antibody, which can be utilized to deliver drugs across BBB through receptor-mediated endocytosis, we began to express the nonligand binding domain of hTfR1 in Escherichia coli BL21 Transetta (DE3). The TfR1 gene was first obtained from HepG2 cells by reverse-transcription polymerase chain reaction (RT-PCR) and then inserted into pET 32a(c+) vector. The protein was expressed in the form of inclusion body with extremely high purity by the E. coli BL21 Transetta (DE3), and the purity was further improved by size-exclusion chromatography. The Western blot test indicated that the recombinant protein was TfR1 as expected. Above all, this report provided a convenient protocol that could be fulfilled in order to prepare hTfR1 inclusion body, which failed to be purified by an Ni(2+) affinity column.

Gene delivery in malignant B cells using the combination of lentiviruses conjugated to anti-transferrin receptor antibodies and an immunoglobulin promoter.

BACKGROUND: We previously developed an antibody-avidin fusion protein (ch128.1Av) specific for the human transferrin receptor 1 (TfR1; CD71) to be used as a delivery vector for cancer therapy and showed that ch128.1Av delivers the biotinylated plant toxin saporin-6 into malignant B cells. However, as a result of widespread expression of TfR1, delivery of the toxin to normal cells is a concern. Therefore, we explored the potential of a dual targeted lentiviral-mediated gene therapy strategy to restrict gene expression to malignant B cells. Targeting occurs through the use of ch128.1Av or its parental antibody without avidin (ch128.1) and through transcriptional regulation using an immunoglobulin promoter. METHODS: Flow cytometry was used to detect the expression of enhanced green fluorescent protein (EGFP) in a panel of cell lines. Cell viability after specific delivery of the therapeutic gene FCU1, a chimeric enzyme consisting of cytosine deaminase genetically fused to uracil phosphoribosyltransferse that converts the 5-fluorocytosine (5-FC) prodrug into toxic metabolites, was monitored using the MTS or WST-1 viability assay. RESULTS: We found that EGFP was specifically expressed in a panel of human malignant B-cell lines, but not in human malignant T-cell lines. EGFP expression was observed in all cell lines when a ubiquitous promoter was used. Furthermore, we show the decrease of cell viability in malignant plasma cells in the presence of 5-FC and the FCU1 gene. CONCLUSIONS: The present study demonstrates that gene expression can be restricted to malignant B cells and suggests that this dual targeted gene therapy strategy may help to circumvent the potential side effects of certain TfR1-targeted protein delivery approaches.

Iron availability is increased in individual human ovarian follicles in close proximity to an endometrioma compared with distal ones.

STUDY QUESTION: Does the iron content of an endometrioma represent a potential source of toxicity for the adjacent follicles? SUMMARY ANSWER: The presence of an endometrioma increases iron and H/L ferritin levels, and transferrin receptor (TfR1) mRNA in individual follicles proximal to the endometrioma and is accompanied by reduced oocyte retrieval. WHAT IS KNOWN ALREADY: Levels of free iron in endometriotic ovarian cysts are much higher than those in normal serum or in non-endometriotic ovarian cysts. The presence of an endometrioma exerts a detrimental effect on the surrounding healthy ovarian tissue as reflected by a reduced number of developing follicles and oocytes retrieved in IVF cycles. STUDY DESIGN, SIZE, DURATION: This is a research study with prospective collection and evaluation of individual follicles (follicular fluid and luteinized granulosa cells) from the affected and the healthy ovaries of 13 women with unilateral endometrioma. PARTICIPANTS/MATERIALS, SETTING, METHODS: Individual follicular samples (145) were obtained from 13 women with endometriosis-related infertility undergoing IVF-ICSI procedures from May 2012 to March 2013. All women had unilateral endometrioma not previously treated with surgery; the contralateral ovary was free of endometriomas and previous surgery. The average ± SEM age was 35.36 ± 2.5 years with anti-Mullerian hormone levels of 2.03 ± 0.55 ng/ml. Follicles were classified as: (i) proximal follicles, in physical contact with the endometrioma; (ii) distal follicles, present in the affected ovary but not in close contact with the endometrioma and (iii) contralateral follicles, in the contralateral healthy ovary. Iron content was measured by the FerroZine method. H/L ferritin subunits were evaluated by specific enzyme-linked immunosorbant assays. Expression of H ferritin and TfR1 was examined by semi-quantitative RT-PCR. Oocyte retrieval rates and Day 3 embryo quality were analyzed. MAIN RESULTS AND THE ROLE OF CHANCE: Total iron levels were higher in endometrioma-proximal follicles compared with endometrioma-distal ones (P = 0.009) and to follicles in the healthy ovary (P = 0.02). L ferritin was higher in proximal versus distal follicles (P = 0.044) or follicles from the healthy ovary (P = 0.027). H ferritin was higher in the proximal and distal follicles compared with follicles in the healthy ovary (P = 0.042 and P = 0.0067, respectively). H ferritin transcript levels in granulosa cells were higher in proximal follicles versus follicles from healthy ovary (P = 0.02). TfR1 transcript levels were higher in proximal versus distal follicles (P = 0.03) and versus follicles from the healthy ovary (P = 0.04). The oocyte retrieval rate was lower in proximal and distal follicles than in follicles from the healthy ovary (P = 0.001 and P = 0.04, respectively). LIMITATIONS, REASONS FOR CAUTION: This is a study on a relatively small sample size and confirmation in a larger group of patients may be required. The method used to purify luteinized granulosa cells offers the best combination of purity, viability and total number of cells recovered. However, a minor contamination by CD45(+) cells (<5%) cannot be excluded. WIDER IMPLICATIONS OF THE FINDINGS: This study represents a further in-depth analysis of the toxic influence of the endometrioma content on the surrounding follicles. We demonstrate the presence of iron-related compounds that are potentially toxic to developing ovarian follicles adjacent to the endometrioma during IVF. Our findings provide novel information that suggests that when surgical removal of the endometrioma is not the option, follicle aspiration at sites distant from the endometrioma might increase the probability of retrieving oocytes. STUDY FUNDING/COMPETING INTEREST(S): This project was supported by Fondazione Giorgio Pardi, Milan, Italy. The authors have no competing financial interests in relation to the content of this research paper. TRIAL REGISTRATION NUMBER: NA.

Angiotensin II inhibits iron uptake and release in cultured neurons.

Based on the well-confirmed roles of angiotensin II (ANGII) in iron transport of peripheral organs and cells, the causative link of excess brain iron with and the involvement of ANGII in neurodegenerative disorders, we speculated that ANGII might also have an effect on expression of iron transport proteins in the brain. In the present study, we investigated effects of ANGII on iron uptake and release using the radio-isotope methods as well as expression of cell iron transport proteins by Western blot analysis in cultured neurons. Our findings demonstrated for the first time that ANGII significantly reduced transferrin-bound iron and non-transferrin bound iron uptake and iron release as well as expression of two major iron uptake proteins transferrin receptor 1 and divalent metal transporter 1 and the key iron exporter ferroportin 1 in cultured neurons. The findings suggested that endogenous ANGII might have a physiological significance in brain iron metabolism.

Immunohistochemical demonstration of transferrin receptor 1 and 2 in human hepatocellular carcinoma tissue.

BACKGROUND/AIMS: Recent studies have confirmed that iron overload is involved not only in liver carcinogenesis, but in its progression. Results in studies using liver cancer cell lines have suggested a relationship between transferrin receptor (TfR) expression and liver carcinogenesis, but TfR expression has not yet been analyzed in human hepatocellular carcinoma (HCC) tissues. METHODOLOGY: We immunohistochemically assessed the expression of TfR1 and TfR2 in tumor tissues and adjacent non-tumorous liver tissues from 41 HCC patients who underwent partial hepatectomy. We evaluated uptake of iron in hepatocytes and HCC cells using iron staining. RESULTS: The expression TfR was significantly higher in HCC samples than in adjacent non-tumor tissue (p < 0.001). TfR expression was significantly related to serum alpha-fetoprotein (p < 0.05) and des-gamma carboxy prothrombin (p < 0.05) concentrations. We also found iron deposition in non-tumor tissue from 25 patients, but in only two HCC samples, consistent with findings that hepatocellular iron uptake decreases with liver carcinogenesis. CONCLUSIONS: We investigated the expression of TfR1 and TfR2 in human HCC tissues by immunohistochemistry, the first report demonstrating TfR2 expression immunohistochemically in human HCC. These results suggest that TfR is expressed in response to iron deficiency during liver carcinogenesis.

[The process of heme synthesis in bone marrow mesenchymal stem cells cultured under fibroblast growth factor bFGF and hypoxic conditions].

It was demonstrated that fibroblast growth factor bFGF influences the process of heme synthesis, the proliferation activity and viability of bone marrow mesenchymal stem cells in culture under hypoxic conditions. The addition of fibroblast growth factor bFGF (7 ng/ml) to the medium under above conditions led to the accumulation of aminolevulinic acid--an early porphyrin and heme precursor, an increase in CD 71 expression--a transferrin receptor, and also a decrease in porphyrin pigments and heme contents--a late precursor and end products of heme synthesis, respectively. It was found that cultivation of the cells under hypoxic conditions and bFGF is an optimum to maintain high viability and proliferation capacity of the mesenchymal stem cells.

Micromanaging Iron Homeostasis: hypoxia-inducible micro-RNA-210 suppresses iron homeostasis-related proteins.

Iron is fundamental for sustaining life for living organisms, and the iron metabolism is finely regulated at different levels. In cancer cells, deregulation of the iron metabolism induces oxidative stress and drives tumor progression and metastasis; however, the molecular mechanisms of iron homeostasis are not fully understood. Here we found that iron deficiency as well as hypoxia promoted microRNA-210 (miR-210) expression. A central mediator of miR-210 transcriptional activation is the hypoxia-inducible factor (HIF)-1α, and the hypoxia-response element in the miR-210 promoter is confirmed experimentally. This is in agreement with the data from in vivo studies that have demonstrated the presence of miR-210-expressing cells at the chronic hypoxic regions of xenografted tumors. Furthermore we found two essential molecules for iron homeostasis, iron-sulfur cluster scaffold protein (ISCU) and transferrin receptor 1 (TfR), are a direct target of miR-210. Transfection of miR-210 decreases the uptake of transferrin by inhibiting the expression of TfR. In addition, inhibition of miR-210 by anti-miR-210 up-regulates ISCU expression. These findings suggest that miR-210 works as an iron sensor and is involved in the maintenance of iron homeostasis by sustaining the TfR expression level to stimulate cell proliferation and promote cell survival in the hypoxic region within tumors.

DMT1 (IRE) expression in intestinal and erythroid cells is regulated by peripheral benzodiazepine receptor-associated protein 7.

The divalent metal transporter 1 (DMT1) is essential for cellular uptake of iron, mediating iron absorption across the duodenal brush border membrane. We have previously shown that with iron feeding DMT1 in the brush border membrane undergoes endocytosis into the subapical compartment of enterocytes. To understand the mechanisms of iron-induced endocytosis of DMT1, we used the yeast two-hybrid system to find proteins that interact with DMT1 and isolated from a rat duodenal cDNA library a protein that interacts specifically with the IRE containing isoform of DMT1 {DMT1 [iron-responsive element (IRE)]}. The protein (Genbank AY336075) is 97.5% identical with peripheral benzodiazepine receptor-associated protein 7 (PAP7), a protein that interacts with the peripheral benzodiazepine receptor. PAP7 is ubiquitously expressed in the rat and in multiple cell lines with consensus sequences including a nuclear localization signal and a Golgi dynamic domain. PAP7, expressed on the brush border of rat duodenum, copurified with DMT1 in brush border membrane vesicles, and following iron feeding, was internalized in parallel with the internalization of DMT1. To determine if PAP7 plays a role in cellular iron metabolism, we downregulated PAP7 expression in K562 cells with small interfering RNA. Following the decrease in PAP7 protein, DMT1 (IRE) protein but not mRNA was significantly downregulated but without effect on DMT1 (non-IRE), transferin (Tf)R1, or ferritin expression. Lowered levels of PAP7 resulted also in decreased cell proliferation and G(1) cell cycle arrest. These data are consistent with PAP7 interacting with DMT1 (IRE) and regulating DMT1 (IRE) expression in K562 cells by modulating expression of DMT1 (IRE) protein.