肥胖对小鼠十二指肠 DMT1和 FPN1表达的影响

目的：观察肥胖对小鼠十二指肠二价金属离子转运体（divalent metal transporter 1,DMT1）mRNA、膜铁转运蛋白（ferroportin1,FPN1）mRNA及蛋白表达的变化,探讨肥胖影响铁吸收的机制。方法 C57BL／6J小鼠随机分为正常对照组和肥胖模型组,每组6只,通过喂养高脂饲料喂养建立肥胖模型,对照组采用普通饲料饲养,实验干预期14周。建模完成后,采用实时荧光定量PCR方法检测小鼠十二指肠DMT1、FPN1 mRNA 的表达,用Western blot检测小鼠十二指肠FPN1蛋白表达。结果与对照组小鼠相比,肥胖模型组小鼠十二指肠DMT1、FPN1 mRNA表达以及FPN1蛋白表达水平降低,差异具有统计学意义（ P ＜0.05）。结论肥胖会下调机体十二指肠DMT1、FPN1的表达,导致铁吸收不良,为进一步研究肥胖引起铁缺乏机制提供理论和实验依据。     

Decreased DMT1 and increased ferroportin 1 expression is the mechanisms of reduced iron retention in macrophages by erythropoietin in rats

Recycled iron from reticuloendothelial macrophages to erythroid precursors is important to maintain the iron homeostasis. However, the molecular mechanisms underlying iron homeostasis in macrophages are poorly understood. In this study, male Sprague-Dawley rats were treated with recombinant human erythropoietin (rHuEpo, 500 IU/day, s.c.) for 3 days. At the fifth day, peritoneal exudate macrophages were harvested, and then (55)Fe uptake and release were measured by liquid scintillation counting method. The expression of divalent metal transporter 1 (DMT1) and ferroportin 1 (FPN1) in peritoneal exudate macrophages was detected by RT-PCR and Western blot. In order to exclude the direct effect of rHuEpo on macrophages, the parallel experiments were performed with incubation normal peritoneal exudate macrophages with rHuEpo (2 IU/ml). Our results showed rHuEpo injection reduced the peritoneal exudate macrophages iron retention. The uptake of Fe(II) was decreased via the suppression of DMT1 (+IRE) expression and the release of Fe(II) was increased with increasing the expression of FPN1 in macrophages. Moreover, the expression of HAMP mRNA was four times lower in rHuEpo-treated liver of rats than control group (CG). HAMP mRNA expression was increased; the synthesis of DMT1 had no significant change, whereas the FPN1 was decreased in normal peritoneal exudate macrophages after treatment with rHuEpo in vitro. We conclude that hepcidin may play a major, causative role in the change of FPN1 synthesis and that decreased the iron retention in macrophages of rHuEpo-treated rats.     

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.     

Iron uptake and Nramp2/DMT1/DCT1 in human bronchial epithelial cells

The capacity of natural resistance-associated macrophage protein-2 [Nramp2; also called divalent metal transporter-1 (DMT1) and divalent cation transporter-1 (DCT1)] to transport iron and its ubiquitous expression make it a likely candidate for transferrin-independent uptake of iron in peripheral tissues. We tested the hypothesis that non-transferrin-bound iron uptake by airway epithelial cells is associated with Nramp2/DMT1/DCT1 and that exposure to iron can increase Nramp2/DMT1/DCT1 mRNA and protein expression and transport of this metal. Exposure of BEAS-2B cells to ferric ammonium citrate (FAC) resulted in a decrease in Fe(3+) concentration in the supernatant that was dependent on time and initial iron concentration. In the presence of internalized calcein, FAC quenched the fluorescent signal, indicating intracellular transport of the metal. The Nramp2/DMT1/DCT1 mRNA isoform without an iron-response element (IRE) increased with exposure of BEAS-2B cells to FAC. RT-PCR demonstrated no change in the mRNA for the isoform with an IRE. Similarly, Western blot analysis for the isoform without an IRE confirmed an increased expression of this protein after FAC exposure, whereas the isoform with an IRE exhibited no change. Finally, immunohistochemistry revealed an increase in the isoform without an IRE in the rat lung epithelium after instillation of FAC. Comparable to mRNA and protein increases, iron transport was elevated after pretreatment of BEAS-2B cells with iron-containing compounds. We conclude that airway epithelial cells increase mRNA and expression of the Nramp2/DMT1/DCT1 without an IRE after exposure to iron. The increase results in an elevated transport of iron and its probable detoxification by these cells.     

6-Hydroxydopamine promotes iron traffic in primary cultured astrocytes

It is well known that disrupted brain iron homeostasis was involved in Parkinson’s disease. We previously reported 6-hydroxydopamine (6-OHDA) could enhance iron influx and attenuate iron efflux process, thus promote iron accumulation in neurons. Astrocytes, the major glial cell type in the central nervous system, are largely responsible for iron distribution in the brain. However, how iron metabolism changes in astrocytes with 6-OHDA treatment are not fully elucidated. In the present study, we first observed that both iron influx and efflux were enhanced with 10 μM 6-OHDA treatment for 24 h in primary cultured astrocytes. In accordance with these iron traffic modulations, both mRNA and protein levels of iron importer divalent metal transporter 1 with iron responsive element (DMT1+IRE) and exporter ferroportin 1 (FPN1) were up-regulated in these cells. L-ferritin mRNA levels were increased. Iron regulatory protein 1 (IRP1) showed a dynamic regulation with 6-OHDA treatment, as indicated by a moderate up-regulation at 12 h, however, down-regulation at 24 h. We further demonstrated that 6-OHDA treatment could induce activation of nuclear factor-kappaB (NF-κB) p65. IκBα activation inhibitor BAY11-7082 fully blocked 6-OHDA induced NF-κB p65 phosphorylation and DMT1 + IRE up-regulation. These results suggest that 6-OHDA might promote iron transport rate in astrocytes by regulating iron transporters, IRP1 expression and NF-κB p65 activation, indicating a different response between neurons and astrocytes.     

TNF, IFN-gamma, and endotoxin increase expression of DMT1 in bronchial epithelial cells

Regulation of the metal transport protein divalent metal transporter-1 (DMT1) may contribute to the uptake and detoxification of iron by cells resident in the respiratory tract. Inflammation has been associated with an increased availability of this metal resulting in an oxidative stress. Because proinflammatory cytokines and LPS have been demonstrated to affect an elevated expression of DMT1 in a macrophage cell line, we tested the hypothesis that tumor necrosis factor (TNF)-alpha, interferon (IFN)-gamma, and LPS increase DMT1 expression in airway epithelial cells. We used RT-PCR to detect mRNA for both -IRE DMT1 and +IRE DMT1 in BEAS-2B cells. Treatment with TNF-alpha, IFN-gamma, or LPS increased both forms. Western blot analysis also demonstrated an increase in the expression of both isoforms of DMT1 after these treatments. Twenty-four hours after exposure of an animal model to TNF-alpha, IFN-gamma, or LPS, a significant increase in pulmonary expression of -IRE DMT1 was seen by immunohistochemistry; the level of +IRE DMT1 was too low in the lung to be visualized using this methodology. Finally, iron transport into BEAS-2B cells was increased after inclusion of TNF-alpha, IFN-gamma, or LPS in the media. We conclude that proinflammatory cytokines and LPS increase mRNA and protein expression of DMT1 in airway cells in vitro and in vivo. Furthermore, both -IRE and +IRE isoforms are elevated after exposures. Increased expression of this protein appears to be included in a coordinated response of the cell and tissue where the function might be to diminish availability of metal.     

Inflammation alters the expression of DMT1, FPN1 and hepcidin,and it causes iron accumulation in central nervous system cells

Inflammation and iron accumulation are present in a variety of neurodegenerative diseases that include Alzheimer's disease and Parkinson's disease. The study of the putative association between inflammation and iron accumulation in central nervous system cells is relevant to understand the contribution of these processes to the progression of neuronal death. In this study, we analyzed the effects of the inflammatory cytokines tumor necrosis factor alpha (TNF‐α) and interleukin 6 (IL‐6) and of lipopolysaccharide on total cell iron content and on the expression and abundance of the iron transporters divalent metal transporter 1 (DMT1) and Ferroportin 1 (FPN1) in neurons, astrocytes and microglia obtained from rat brain. Considering previous reports indicating that inflammatory stimuli induce the systemic synthesis of the master iron regulator hepcidin, we identified brain cells that produce hepcidin in response to inflammatory stimuli, as well as hepcidin‐target cells. We found that inflammatory stimuli increased the expression of DMT1 in neurons, astrocytes, and microglia. Inflammatory stimuli also induced the expression of hepcidin in astrocytes and microglia, but not in neurons. Incubation with hepcidin decreased the expression of FPN1 in the three cell types. The net result of these changes was increased iron accumulation in neurons and microglia but not in astrocytes. The data presented here establish for the first time a causal association between inflammation and iron accumulation in brain cells, probably promoted by changes in DMT1 and FPN1 expression and mediated in part by hepcidin. This connection may potentially contribute to the progression of neurodegenerative diseases by enhancing iron‐induced oxidative damage.     

Molecular analysis of increased iron status in moderately exercised rats

Although iron plays a critical role in exercise, the regulatory mechanism of iron metabolism remains poorly understood. The aims of the present study were to investigate the effects of different intensity exercise on body iron status and the regulatory mechanism of duodenal iron absorption. Thirty female Sprague-Dawley rats (90–100 g) were randomly divided into three groups: a control group (remained sedentary, CG), a moderately exercised group (swam 1.5 h/day, MG) and a strenuously exercised group (swam with different load, SG). Serum iron status, serum ferritin and Hct were examined after 10 weeks of swimming. Western blot was performed to detect the expression of iron transport proteins: divalent metal transporter1 (DMT1) and ferroportin 1 (FPN1) in duodenal epithelium. The expression of hepcidin mRNA in liver was examined by RT-PCR. The results showed: (1) the body iron status in MG was kept at a high level compared to that of CG and SG, (2) Western blot showed DMT1 with iron responsive element (IRE) and FPN1 in duodenal epithelium which were higher in MG than that of CG and (3) the expression of hepatic hepcidin mRNA was down regulated in MG (p < 0.05). The data suggested that moderate exercise improved iron status and that was likely regulated by increased DMT1 with IRE and FPN1 expression. Hepcidin signaling pathway may involve in the regulation of duodenal iron absorption proteins. Xiang Lin Duan and Yan Zhong Chang share Senior Authorship     

Divalent Metal Transporter 1 Expression and Regulation in Human Placenta

Divalent metal transporter 1 (DMT1) is likely responsible for the release of iron from endosomes to the cytoplasm in placental syncytiotrophoblasts (STB). To determine the localization and the regulation of DMT1 expression by iron directly in placenta, the expression of DMT1 in human term placental tissues and BeWo cells (human placental choriocarcinoma cell line) was detected and the change in expression in response to different iron treatments on BeWo cells was observed. DMT1 was shown to be most prominent near the maternal side in human term placenta and predominantly in the cytoplasm of BeWo cells. BeWo cells were treated with desferrioxamine (DFO) and human holotransferrin (hTf-2Fe) and it was found that both DMT1 mRNA and protein increased significantly with DFO treatment and decreased with hTf-2Fe treatment. Further, DMT1 mRNA responded more significantly to treatments if it possessed an iron-responsive element than mRNA without this element. This study indicated that DMT1 is likely involved in endosomal iron transport in placental STB and placental DMT1 + IRE expression was primarily regulated by the IRE/IRP mechanism.     

Sub-lethal levels of amyloid β-peptide oligomers decrease non-transferrin-bound iron uptake and do not potentiate iron toxicity in primary hippocampal neurons

Two major lesions are pathological hallmarks in Alzheimer's disease (AD): the presence of neurofibrillary tangles formed by intracellular aggregates of the hyperphosphorylated form of the cytoskeletal tau protein, and of senile plaques composed of extracellular aggregates of amyloid beta (Aβ) peptide. Current hypotheses regard soluble amyloid beta oligomers (AβOs) as pathological causative agents in AD. These aggregates cause significant calcium deregulation and mediate neurotoxicity by disrupting synaptic activity. Additionally, the presence of high concentrations of metal ions such as copper, zinc, aluminum and iron in neurofibrillary tangles and senile plaques, plus the fact that they accelerate the rate of formation of Aβ fibrils and AβOs in vitro, suggests that accumulation of these metals in the brain is relevant to AD pathology. A common cellular response to AβOs and transition metals such as copper and iron is the generation of oxidative stress, with the ensuing damage to cellular components. Using hippocampal neurons in primary culture, we report here the effects of treatment with AβOs on the (+)IRE and (-)IRE mRNA levels of the divalent metal transporter DMT1. We found that non-lethal AβOs concentrations decreased DMT1 (-)IRE without affecting DMT1 (+)IRE mRNA levels, and inhibited non-transferrin bound iron uptake. In addition, since both iron and AβOs induce oxidative damage, we studied whether their neurotoxic effects are synergistic. In the range of concentrations and times used in this study, AβOs did not potentiate iron-induced cell death while iron chelation did not decrease AβOs-induced cell death. The lack of synergism between iron and AβOs suggests that these two neurotoxic agents converge in a common target, which initiates signaling processes that promote neurodegeneration.     

铁代谢蛋白在肾脏的表达与功能

最近的研究证实,肾小管细胞具有能力表达包括转铁蛋白受体1(transferrin receptor-1,TfR1)、二价金属离子转运蛋白1(divalent metal transporter-1,DMT1)、膜铁转运蛋白1(ferroportin-1,FPN1)、铁调节蛋白(iron regulatory protein,IRP)和铁调素(hepcidin,Hepc)在内的几乎所有铁代谢蛋白.这些蛋白质的存在以及相关研究显示肾脏可能具有排出多余铁的功能,因此对体铁平衡起有十分重要的作用.     

1B/(-)IRE DMT1 expression during brain ischemia contributes to cell death mediated by NF-κB/RelA acetylation at Lys310

The molecular mechanisms responsible for increasing iron and neurodegeneration in brain ischemia are an interesting area of research which could open new therapeutic approaches. Previous evidence has shown that activation of nuclear factor kappa B (NF-κB) through RelA acetylation on Lys310 is the prerequisite for p50/RelA-mediated apoptosis in cellular and animal models of brain ischemia. We hypothesized that the increase of iron through a NF-κB-regulated 1B isoform of the divalent metal transporter-1 (1B/DMT1) might contribute to post-ischemic neuronal damage. Both in mice subjected to transient middle cerebral artery occlusion (MCAO) and in neuronally differentiated SK-N-SH cells exposed to oxygen-glucose-deprivation (OGD), 1A/DMT1 was only barely expressed while the 1B/DMT1 without iron-response-element (-IRE) protein and mRNA were early up-regulated. Either OGD or over-expression of 1B/(-)IRE DMT1 isoform significantly increased iron uptake, as detected by total reflection X-ray fluorescence, and iron-dependent cell death. Iron chelation by deferoxamine treatment or (-)IRE DMT1 RNA silencing displayed significant neuroprotection against OGD which concomitantly decreased intracellular iron levels. We found evidence that 1B/(-)IRE DMT1 was a target gene for RelA activation and acetylation on Lys310 residue during ischemia. Chromatin immunoprecipitation analysis of the 1B/DMT1 promoter showed there was increased interaction with RelA and acetylation of H3 histone during OGD exposure of cortical neurons. Over-expression of wild-type RelA increased 1B/DMT1 promoter-luciferase activity, the (-)IRE DMT1 protein, as well as neuronal death. Expression of the acetylation-resistant RelA-K310R construct, which carried a mutation from lysine 310 to arginine, but not the acetyl-mimic mutant RelA-K310Q, down-regulated the 1B/DMT1 promoter, consequently offering neuroprotection. Our data showed that 1B/(-)IRE DMT1 expression and intracellular iron influx are early downstream responses to NF-κB/RelA activation and acetylation during brain ischemia and contribute to the pathogenesis of stroke-induced neuronal damage.     

Protoporphyrin IX regulates peripheral benzodiazepine receptor associated protein 7 (PAP7) and divalent metal transporter 1 (DMT1) in K562 cells

BackgroundProtoporphyrin IX (PP IX), the immediate precursor to heme, combines with ferrous iron to make this product. The effects of exogenous PP IX on iron metabolism remain to be elucidated. Peripheral-type benzodiazepine receptor (PBR) is implicated in the transport of coproporphyrinogen into the mitochondria for conversion to PP IX. We have demonstrated that PBR-Associated Protein 7 (PAP7) bound to the Iron Responsive Element (IRE) isoform of divalent metal transporter 1 (DMT1). PP IX and PAP7 are ligands for PBR, thus, we hypothesized that PAP7 interact with PP IX via PBR.MethodsWe have examined in K562 cells, which can be induced to undergo erythroid differentiation by PP IX and hemin, the effects of PP IX on the expression of PAP7 and other proteins involved in cellular iron metabolism, transferrin receptor 1 (TfR1), DMT1, ferritin heavy chain (FTH), c-Myc and C/EBPα by western blot and quantitative real time PCR analyses.ResultsPP IX significantly decreased mRNA levels of DMT1 (IRE) and (non-IRE) from 4 h. PP IX markedly decreased protein levels of C/EBPα, PAP7 and DMT1. In contrast, hemin, which like PP IX also induces K562 cell differentiation, had no effect on PAP7 or DMT1 expression.ConclusionWe hypothesize that PP IX binds to PBR displacing PAP7 protein, which is then degraded, decreasing the interaction of PAP7 with DMT1 (IRE) and resulting in increased turnover of DMT1.General significanceThese results suggest that exogenous PP IX disrupts iron metabolism by decreasing the protein expression levels of PAP7, DMT1 and C/EBPα.     

Correlation between the expression of divalent metal transporter 1 and the content of hypoxia-inducible factor-1 in hypoxic HepG2 cells

Transferrin and transferrin receptor are two key proteins of iron metabolism that have been identified to be hypoxia-inducible genes. Divalent metal transporter 1 (DMT1) is also a key transporter of iron under physiological conditions. In addition, in the 5' regulatory region of human DMT1 (between -412 and -570), there are two motifs (CCAAAGTGCTGGG) that are similar to hypoxia-inducible factor-1 (HIF-1) binding sites. It was therefore speculated that DMT1 might also be a hypoxia-inducible gene. We investigated the effects of hypoxia and hypoxia/re-oxygenation on the expression of DMT1 and the content of HIF-1alpha in HepG2 cells. As we expected, a very similar tendency in the responses of the expression of HIF-1alpha, DMT1+IRE (iron response element) and DMT1-IRE proteins to chemical (CoCl(2)) or physical hypoxia was observed. A highly significant correlation was found between the expression of DMT1 proteins and the contents of HIF-1 in hypoxic cells. After the cells were exposed to hypoxia and subsequent normoxia, no HIF-1alpha could be detected and a significant decrease in DMT1+IRE expression (P<0.05), but not in DMT1-IRE protein (versus the hypoxia group), was observed. The findings implied that the HIF-1 pathway might have a role in the regulation of DMT1+IRE expression during hypoxia.     

Hepcidin expression and iron transport in alveolar macrophages

Alveolar macrophages express many proteins important in iron homeostasis, including the iron importer divalent metal transport 1 (DMT1) and the iron exporter ferroportin 1 (FPN1) that likely participate in lung defense. We found the iron regulatory hormone hepcidin (HAMP) is also produced by alveolar macrophages. In mouse alveolar macrophages, HAMP mRNA was detected at a low level when not stimulated but at a high level when exposed to lipopolysaccharide (LPS). LPS also affected the mRNA levels of the iron transporters, with DMT1 being upregulated and FPN1 downregulated. However, iron had no effect on HAMP expression but was able to upregulate both DMT1 and FPN1 in alveolar macrophages. IL-1 and IL-6, which are important in HAMP augmentation in hepatocytes, also did not affect HAMP expression in alveolar macrophages. In fact, the LPS-induced alterations in the expression of HAMP as well as DMT1 and FPN1 were preserved in the alveolar macrophages isolated from IL-1 receptor or IL-6-deficient mice. When alveolar macrophages were loaded with transferrin-bound (55)Fe, the subsequent release of (55)Fe was inhibited significantly by LPS. In addition, treatment of these cells with either LPS or HAMP caused the diminishment of the surface FPN1. These findings are consistent with the current model that HAMP production leads to a decreased iron efflux. Our studies suggest that iron mobilization by alveolar macrophages can be affected by iron and LPS via several pathways, including HAMP-mediated degradation of FPN1, and that these cells may use unique regulatory mechanisms to cope with iron imbalance in the lung.     

Brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor inhibit ferrous iron influx via divalent metal transporter 1 and iron regulatory protein 1 regulation in ventral mesencephalic neurons

Iron accumulation is observed in the substantia nigra of patients with Parkinson's disease. However, it is unknown whether neurotrophic factors, brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF) participate in the modulation of neuronal iron metabolism. Here, we investigated the effects and underlying mechanisms of BDNF and GDNF on the iron influx process in primary cultured ventral mesencephalic neurons. 6-hydroxydopamine-induced enhanced ferrous iron influx via improper up-regulation of divalent metal transporter 1 with iron responsive element (DMT1 + IRE) was consistently relieved by BDNF and GDNF. Both the mRNA and protein levels of DMT1 + IRE were down-regulated by BDNF or GDNF treatment alone. We further demonstrated the involvement of iron regulatory protein 1 (IRP1) in BDNF- and GDNF-induced DMT1 + IRE expression. Extracellular-regulated kinase 1/2 (ERK1/2) and Akt were activated and participated in these processes. Inhibition of ERK1/2 and Akt phosphorylation abolished the down-regulation of IRP1 and DMT1 + IRE induced by BDNF and GDNF. Taken together, these results show that BDNF and GDNF ameliorate iron accumulation via the ERK/Akt pathway, followed by inhibition of IRP1 and DMT1 + IRE expression, which may provide new targets for the neuroprotective effects of these neurotrophic factors.