DMT1 Protein Research Articles

Cell Cycle-Related Centromere Protein F Deficiency Suppresses Ovarian Cancer Cell Growth by Inducing Ferroptosis

Objectives: This study aimed to investigate the involvement of the cell cycle-related protein centromere protein F (CENPF) in the development of ovarian cancer (OC) and explored its relationship with ferroptosis. Design: The databases were analysed to identify differential expression of cell cycle-related proteins between individuals with OC and normal individuals. Immunohistochemistry and statistical analysis were conducted on ovarian tissues obtained from 40 patients with epithelial OC and 20 normal individuals. In vitro experiments were performed using SKOV3 and HEY epithelial OC cell lines. Participants/Materials, Setting, Methods: The mRNA microarray dataset, consisting of GSE14001, GSE54388, GSE40595, and GSE14407, was downloaded from the Gene Expression Omnibus (GEO) database to investigate the genes associated with cell cycle regulation in OC cells. CENPF was selected as the subject of study through differential analysis.Assessed the expression of CENPF in both OC patients and normal ovarian tissues using immunohistochemistry. Lentivirus infection was employed to downregulate CENPF expression, and subsequent experiments including Cell Counting Kit-8 assay, cell cycle analysis, transwell assay, and wound-healing assay were conducted to investigate the effects of CENPF on proliferation, invasion, migration, and cell cycle regulation in OC cells. The reactive oxygen species (ROS) and the malondialdehyde (MDA) assays were performed to assess the involvement of CENPF in cellular redox reactions. Western blot analysis was conducted to examine the expression levels of ferroptosis-related proteins (GPX4, SLC7A11, DMT1, and protein 53 [p53]). Results: By querying and integrating cell cycle-related genes from the GEO database, in silico analyses using The Cancer Genome Atlas database combined with immunohistochemical studies, we discovered that CENPF is upregulated in OC tissues and is related to survival. Downregulation of CENPF inhibited biological function of OC cells, increased intracellular ROS and MDA levels, and downregulated the GPX4 protein and the SLC7A11/xCT protein, but upregulated the DMT1 protein and the tumour p53 expression to induce ferroptosis. Limitations: This study did not investigate ferroptosis-related studies following CENPF overexpression, and the findings have not been validated in animal studies. Conclusions: Our findings demonstrated that the deficiency of CENPF played a crucial anti-oncogenic role in the progression of OC through the mechanism of ferroptosis.

Myeloid Ptpn2 Deficiency in Mice Causes Functional Iron Deficiency

Background: Anemia is the most prevalent extraintestinal complication of inflammatory bowel disease (IBD) and is associated with the manifestation of Crohn’s disease. Anemia significantly reduces the quality of life, increases hospitalization visits, and increases medical care expenses of IBD patients. Iron deficiency is the leading cause of anemia in IBD; however, the physiological mechanisms involved are still poorly understood. Macrophages significantly contribute to iron homeostasis by mechanisms including iron recycling by phagocytosing senescent erythrocytes and damaged cells, and nurturing erythropoiesis in the bone marrow. In this study, we investigated the role of the IBD candidate gene, protein tyrosine phosphatase non-receptor type 2 ( PTPN2), in regulating macrophage iron homeostasis. Methods: Myeloid cell-specific knockout mice ( Ptpn2LysMCre) and control littermates ( Ptpn2fl/fl) were analyzed for serum iron content and liver and spleen non-heme iron. Duodenal epithelial cell (DEC) proteins were assayed by Western blotting. Localization of the brush border ferrous iron transporter, DMT1, and the coupled ferrous iron absorption driver, NHE3, in duodenal tissue was determined by immunohistochemistry (IHC). Gene expression profiles of macrophages were analyzed by Nanostring. Results: Ptpn2LysMCre mice had reduced serum iron ( p<0.0001; n=12) and reduced spleen non-heme iron (p=0.0017; n=6), whereas liver non-heme iron was unaltered (n=6), indicating iron deficiency in select tissues. DMT1 (n=8) and NHE3 (n=5) protein expression in DECs was unaltered and IHC confirmed the unaltered localization of DMT1 and NHE3 (n=6) in the duodenal epithelium of Ptpn2LysMCre mice, suggesting the reduction in serum iron was not due to an impairment in intestinal non-heme iron uptake. Nanostring analyses revealed that the gene expression of the iron transporter Dmt1, iron exporter Fpn, and iron importer Tfr1 were significantly downregulated (p<0.05; n=5) in macrophages from Ptpn2LysMCre mice, indicating an iron sequestering phenotype. Conclusions: Loss of myeloid Ptpn2 in mice causes functional iron deficiency. These findings indicate a major role of myeloid PTPN2 in regulating systemic iron homeostasis. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Constitutive Loss of Ptpn2 in Mice Causes Features of Anemia Including Iron Deficiency

Background: Anemia is the most frequent extraintestinal manifestation of inflammatory bowel disease (IBD) and is associated with the risk of Crohn’s disease. Anemia significantly impairs quality of life, increases hospitalization rates, and increases medical care costs of IBD patients. Iron deficiency is the most frequent cause of anemia in IBD; however, the mechanisms involved are still poorly understood. Here, we investigated the role of the IBD risk gene, protein tyrosine phosphatase non-receptor type 2 ( PTPN2), in regulating iron homeostasis. Methods: Constitutive Ptpn2 wild type (WT), heterozygous (Het), and knockout (KO) 3-week-old mice were analyzed for serum iron content, complete blood count parameters, and intestinal and extraintestinal non-heme iron. Protein and RNA from duodenal epithelial cells (DECs) were assayed by western blotting and qPCR. Localization of the brush border ferrous iron transporter, DMT1, and the ferrous iron absorption driver, NHE3, in duodenal tissue was determined by immunohistochemistry (IHC). Results: Ptpn2-KO mice had reduced i) serum iron ( p=0.0007; n=10); ii) serum iron carrier transferrin saturation ( p=0.0077; n=8); iii) hemoglobin ( p=0.0002; n=5); iv) hematocrit ( p=0.0023; n=5); v) intestinal non-heme iron: duodenum ( p=0.0128; n=10) and jejunum ( p=0.0006; n=8); and vi) extraintestinal tissue non-heme iron: liver ( p=0.0014; n=10), spleen ( p=0.04; n=10), kidney ( p=0.0004; n=7), and gastrocnemius muscle ( p=0.0436; n=9) vs. Ptpn2-WT and Het mice. This indicated that loss of Ptpn2 causes characteristics of anemia including decreased serum iron and tissue iron storage and impaired intestinal iron homeostasis. DEC gene expression of the intracellular iron storage molecule ferritin ( Fth1) was reduced ( p=0.048; n=4) while the basolateral cellular iron importer transferrin receptor 1 ( Tfrc1) was significantly increased ( p=0.0048; n=5) in Ptpn2-KO mice. Reduced FTH1 protein expression ( p=0.007; n=12) and increased TFRC1 protein expression ( p=0.0021; n=8) in DECs of Ptpn2-KO mice was confirmed by western blot, indicating cellular iron deficiency. DMT1 (n=6) and NHE3 (n=8) protein expression in DECs was unaltered, whereas IHC showed reduced apical membrane DMT1 and NHE3 in the duodenal epithelium of Ptpn2-KO mice (n=6), suggesting a possible mechanism of impaired intestinal non-heme iron uptake. Conclusions: Loss of Ptpn2 in mice causes features of anemia including iron deficiency associated with mislocalization of DMT1 and reduced apical membrane expression of NHE3. These findings identify a critical role for PTPN2 in regulating systemic iron homeostasis. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The relation among myocardial iron load and the severity of heart failure

Abstract Heart failure (HF) symptoms are related to reduced circulatory perfusion, peripheral congestion, also impaired peripheral muscle strength & oxygen utilization due to iron deficiency. Despite many premises, the correlation between myocardial iron load and failing heart function was not proved. The purpose of this study was to assess iron itself and iron-related proteins in human failing (FH) vs non-failing (NFH) myocardium. The study group consisted of 58 explanted FHs vs the control consisted of 31 NFHs unsuitable for transplantation. HF study group presented with LV dilatation (LVDD 67±8 mm; LVSD 59±13 mm), reduced contraction (LVEF 20±7.5%), RV enlargement (RVD 42±6.5 mm), pulmonary hypertension (2.21±0.93 Wu) and elevated NTproBNP (3955.5±2250.5pg/ml). We assessed iron serum markers: ferritin (FR), transferrin/saturation (TSAT), transferrin receptor (soluble sTfR), sTFR/log FR; myocardial iron homeostasis assessment: M-Iron (Instrumental Neutron Activation Analysis, μg/g) and iron gathering proteins (by ELISA): divalent metal transporter (DMT-1); L-type calcium channel (L-CH), TfR-1/TfR-2; ferritins: heavy (FT-H), light (FT-L) chain, mitochondrial (FT-MT); ferroportin (FPN) and oxidative stress marker: 4-hydroxynonenal (4-HNE). In FH vs NFH, M-Iron was reduced (164.6±25.2 vs 182.3±23.9; p=0.0358) however, did not apply to the entire group. Therefore we divvied the FH group into two subgroups (based on M-Iron concentration in NFH controls). Average iron concertation in non-iron-deficient (NIDM) subgroup was 172.5±19.1 (n=35) and in iron-deficient (IDM) subgroup wad 127.0±16.5; (n=23, p=0.0001). Regardless M-Iron load in FH vs NFH myocardial proteins expressions were reduced: L-CH (NIDM vs IDM vs NFH: 49.2±9.2; 42.5±4.2; 67.3±23.0, p=0.0022); FT-L (16.4±7.3; 11.6±4.8; 26.3±7.5, p= <0.0001, respectively); FT-MT (41.2±7.6; 38.7±4.1; 59.3±10.4 p≤0,0001, respectively). With regard to M-Iron load, only in DMT-1 expression was changed (NIDM vs IDM vs NFH 4.8±0.7; 4.0±0.5; 5.8±0.8; p≤0.0001). 4-HNE levels in FH vs NFH were increased, regardless M-Iron load (NIDM-Iron vs IDM-Iron vs NFH; 55.7±29.2; 57.2±47.8; 9.9±8.1 p≤0.0001). Interestingly, the iron load (NIDM-Iron vs IDM-Iron) did not modify the assessed clinical parameters. With regard to commonly used systemic iron markers only sTFR/log FR proved M-Iron load difference (NIDM-Iron vs IDM-Iron 0.6±0.2; 0.7±0.3; p=0.047910) In FH, we observed, the subgroup with the reduced M-Iron load. The observed reduction of iron gathering proteins were independent on M-Iron load, except DMT-1 the main iron transportation protein into the cell. The results showed that the HF degree and concertation of iron proteins are unlikely to be affected by the M-Iron load, except DMT-1 protein. The oxidative stress was also regardless from M-Iron load, and increased in the whole FH group. Commonly used systemic iron markers except sTFR/log FR did not define M-Iron load. Funding Acknowledgement Type of funding sources: Public Institution(s). Main funding source(s): The Cardinal Stefan Wyszyński National Institute of Cardiology

IRE-dependent Regulation of Intestinal Dmt1 Prevails During Chronic Dietary Iron Deficiency but is Dispensable in Conditions of Acute Erythropoietic Stress.

IRE-dependent Regulation of Intestinal Dmt1 Prevails During Chronic Dietary Iron Deficiency but is Dispensable in Conditions of Acute Erythropoietic Stress.

The Nramp2/DMT1 iron transporter is induced in the duodenum of microcytic anemia mk mice but is not properly targeted to the intestinal brush border

AbstractMicrocytic anemia (mk) mice and Belgrade (b) rats are severely iron deficient because of impaired intestinal iron absorption and defective iron metabolism in peripheral tissues. Both animals carry a glycine to arginine substitution at position 185 in the iron transporter known as Nramp2/DMT1 (divalent metal transporter 1). DMT1 messenger RNA (mRNA) and protein expression has been examined in the gastrointestinal tract ofmk mice. Northern blot analysis indicates that, by comparison to mk/+ heterozygotes, mk/mkhomozygotes show a dramatic increase in the level of DMT1 mRNA in the duodenum. This increase in RNA expression is paralleled by a concomitant increase of the 100-kd DMT1 isoform I protein expression in the duodenum. Immunohistochemical analyses show that, as for normal mice on a low-iron diet, DMT1 expression in enterocytes ofmk/mk mice is restricted to the duodenum. However, and in contrast to normal enterocytes, little if any expression of DMT1 is seen at the apical membrane in mk/mk mice. These results suggest that the G185R mutation, which was shown to impair the transport properties of DMT1, also affects the membrane targeting of the protein in mk/mk enterocytes. This loss of function of DMT1 is paralleled by a dramatic increase in expression of the defective protein in mk/mk mice. This is consistent with a feedback regulation of DMT1 expression by iron stores.

Transmembrane protein western blotting: Impact of sample preparation on detection of SLC11A2 (DMT1) and SLC40A1 (ferroportin).

Western blotting has been widely used for investigation of protein expression, posttranslational modifications, and interactions. Because western blotting usually involves heat-denaturation of samples prior to gel loading, clarification of detailed procedures for sample preparation have been omitted or neglected in many publications. We show here the case that even excellent primary antibodies failed to detect a specific protein of interest due to a routine heating practice of protein samples. We performed western blotting for transmembrane iron transporter proteins; SLC11A2 (divalent metal transporter 1, DMT1), SLC40A1 (ferroportin 1, Fpn1), and transferrin receptor-1 (TfR1), along with cytoplasmic iron storage protein ferritin H. Our results in 12 human culture cell lysates indicated that only unheated samples prior to gel loading gave rise to clear resolution of DMT1 protein, while heated samples (95°C, 5min) caused the loss of resolution due to DMT1 protein aggregates. Unheated samples also resulted in better resolution for Fpn1 and TfR1 western blots. Conversely, only heated samples allowed to detect ferritin H, otherwise ferritin polymers failed to get into the gel. Neither different lysis/sample loading buffers nor sonication improved the resolution of DMT1 and Fpn1 western blots. Thus, heating samples most critically affected the outcome of western blotting, suggesting the similar cases for thousands of other transmembrane and heat-sensitive proteins.

Zinc induces iron egress from intestinal Caco-2 cells via induction of Hephaestin: A role for PI3K in intestinal iron absorption

In previous studies we demonstrated that zinc stimulates iron uptake in intestinal Caco-2 cells via Zinc-PI3K-IRP2-DMT1 axis. In the current study we investigated the effect of zinc on basolateral iron release and characterized the associated mechanisms. In Caco-2 cells grown on permeable supports, zinc induced iron transport and expression of DMT1, HEPH mRNA and protein, but not that of FPN1. LY294002, an inhibitor of PI3K, inhibited the zinc-induced iron transport, DMT1, HEPH mRNA and protein expression. In addition, LY294002 also inhibited the basal expression of HEPH and FPN1 resulting in blockade of iron egress from cells. In addition, siRNA-silencing of HEPH led to inhibition of both zinc-induced and basal iron transport. Conversely, TPEN, a chelator of zinc, inhibited iron uptake, DMT1, HEPH and FPN1 mRNA and protein expression. These results suggest that intestinal cell zinc status is a critical determinant of iron absorption and effects are mediated via activation of PI3K. Further, PI3K pathway appears to selectively modulate the expression of iron transporters and iron absorption, therefore this might serve as a therapeutic target in iron overload disorders.

Metabolic response to dietary supplementation with iron and folic acid in the rat

AbstractFolic acid (FA) and iron absorption is performed by transporters encoded by the Slc46a1 and Slc11a2 genes, respectively. The aim of this study was to determine how FA and iron deficiency in rats, and subsequent supplementation of the animals’ diet with these nutrients, affects Slc11a2 and Slc46a1 gene expression and the metabolic biomarkers of FA and iron status.150 female Wistar rats were assigned to a group fed a diet deficient in iron and FA (D, n = 120) or to a group fed a control diet (C, n = 30, AIN-93M) for 28 days. After this period, the rats were randomized to a group fed for 2, 10, or 21 days with a diet deficient in FA and supplemented with iron (DFE), a diet deficient in iron and supplemented with FA (DFOL), a diet supplemented with iron and FA (FEFOL), or a diet deficient in iron and folic (D).Transcript and protein levels were determined in the duodenum using the real-time PCR and western blot methods, respectively. FA concentrations in serum were assessed using the electrochemiluminescence method, and homocysteine concentrations were determined with an enzymatic assay. Iron metabolism was evaluated by measuring the total and unsaturated iron-binding capacity (TIBC and UIBC) in serum, and iron concentrations were determined using a photometric method. Hepcidin concentrations were measured in plasma with an immunoenzymatic method. Morphological parameters of the blood were determined with a Sysmex XT-4000 analyzer.The D group had higher Slc11a2 transcript levels than the DFE group at every time-point (p < 0.01). There were higher Slc46a1 mRNA levels in the DFE group than in the FEFOL group at the third time-point (p < 0.05). We did not observe any differences in PCFT or DMT1 protein abundance. The DFOL, FEFOL, and C groups had higher serum FA concentrations at the second and third time-points (p < 0.001), as well as the lowest homocysteine concentrations (p < 0.001). TIBC concentrations were lowest in the DFE and the FEFOL groups at the final time-point (p < 0.001). We did not observe any differences in hepcidin concentrations. Simultaneous supplementation with FA and iron resulted in significantly higher hemoglobin concentrations at the third time-point (p < 0.01).This study shows that dietary FA and iron deficiency, and subsequent supplementation with moderate amounts of these nutrients, may affect the transcription but not the protein abundance of FA and iron transporters in the rat duodenum.

Prenatal Ethanol Exposure Misregulates Genes Involved in Iron Homeostasis Promoting a Maladaptation of Iron Dependent Hippocampal Synaptic Transmission and Plasticity.

Prenatal ethanol exposure (PAE) induces behavioral maladptations in offspring, including a deficit in memory formation which is part of the umbrella sign of fetal alcohol spectrum disorder. Clinical and preclinical studies have shown that iron depletion exacerbates cognitive problems in offspring exposed to ethanol in utero and that PAE promotes dysregulation in brain iron homeostasis. However, the mechanisms underlying brain iron dysregulation and neuronal activity defects in adolescent offspring of PAE are unclear and poorly understand. Here, we used a PAE rat model to analyze messenger RNA (mRNA) and protein expression of iron homeostasis genes such as transferrin receptor (TfR), divalent metal transporter (DMT1), ferroportin (FPN1), and ferritin (FT) in brain areas associated with memory formation such as the prefrontal cortex (PFC), ventral tegmental area, and hippocampus. Interestingly, we found that 21 day old PAE rats have higher mRNA expression of DMT1 in the PFC, and TfR in the hippocampus, compared to control animals. In contrast FPN has lower mRNA expression in the PFC, and FT and FPN1 have lower expression in the hippocampus. In agreement with these results, we found a 1.5–2 fold increase of TfR and DMT1 protein levels both in the hippocampus and the PFC. Additionally, using an electrophysiological approach, we found that in hippocampal slices from PAE rats, iron treatment decreased long-term potentiation (LTP), but not AMPAR basal transmission (AMPAR fEPSP). In contrast, in control slices Fe-NTA did not affect LTP but decreased significantly the AMPAR fEPSP. Meanwhile, iron chelation with deferiprone decreased AMPAR transmission in PAE and control slices and decreased LTP only in controls slices. These results suggest that PAE affects iron homeostasis of specific brain areas—PFC and hippocampus—which could be involved in maladaptive cognition observed in this animal model.

Ceruloplasmin Plays a Neuroprotective Role in Cerebral Ischemia.

Ceruloplasmin (Cp) is a ferroxidase that also plays a role in iron efflux from cells. It can thus help to regulate cellular iron homeostasis. In the CNS, Cp is expressed as a membrane-anchored form by astrocytes. Here, we assessed the role of Cp in permanent middle cerebral artery occlusion (pMCAO) comparing wildtype and Cp null mice. Our studies show that the lesion size is larger and functional recovery impaired in Cp null mice compared to wildtype mice. Expression of Cp increased ninefold at 72 h after pMCAO and remained elevated about twofold at day 14. We also assessed changes in mRNA and protein expression of molecules involved in iron homeostasis. As expected there was a reduction in ferroportin in Cp null mice at 72 h. There was also a remarkable increase in DMT1 protein in both genotypes at 72 h, being much higher in wildtype mice (19.5-fold), that then remained elevated about twofold at 14 days. No difference was seen in transferrin receptor 1 (TfR1) expression, except a small reduction in wildtype mice at 72 h, suggesting that the increase in DMT1 may underlie iron uptake independent of TfR1-endosomal uptake. There was also an increase of ferritin light chain in both genotypes. Iron histochemistry showed increased iron accumulation after pMCAO, initially along the lesion border and later throughout the lesion. Immunofluorescence labeling for ferritin (a surrogate marker for iron) and GFAP or CD11b showed increased ferritin in GFAP+ astrocytes along the lesion border in Cp null mice, while CD11b+ macrophages expressed ferritin equally in both genotypes. Increased lipid peroxidation assessed by 4HNE staining was increased threefold in Cp null mice at 72 h after pMCAO; and 3-nitrotyrosine labeling showed a similar trend. Three key pro-inflammatory cytokines (IL-1β, TNFα, and IL-6) were markedly increased at 24 h after pMCAO equally in both genotypes, and remained elevated at lower levels later, indicating that the lack of Cp does not alter key inflammatory cytokine expression after pMCAO. These data indicate that Cp expression is rapidly upregulated after pMCAO, and loss of Cp results in dysregulation of iron homeostasis, increased oxidative damage, greater lesion size and impaired recovery of function.

Increased DMT-1 expression in placentas of women living in high-Cd-contaminated areas of Thailand.

Cadmium (Cd) is a toxic heavy metal and contamination was reported in soil and rice in several areas of Thailand. Humans are normally exposed to environmental Cd, leading to gradual Cd accumulation in their bodies, including the placenta. DMT-1 is a divalent metal transporter which is found in placental tissue and plays a vital role in the transportation of Fe2+ and Cd2+. This study investigated DMT-1 protein and mRNA expressions in full term human placentas comparing those from high-Cd-contaminated areas (high-Cd group) and low-Cd-contaminated areas (low-Cd group), n = 6 per group. The maternal blood Cd (B-Cd) and placental Cd (P-Cd) of the high-Cd group was significantly raised in comparison with those in the low-Cd group. DMT-1 in the fetal portion of the placentas was localized in the apical and basal portions of the cytoplasm of the syncytiotrophoblastic cells, the endothelium of fetal capillaries which is functional structure of the placental barrier, and was also found in the cytoplasm of Hofbauer cells. Moreover, DMT-1 localization in the maternal portion was also detected in most decidual cells. In addition, the DMT-1 protein and mRNA expressions in the high-Cd group were significantly higher than those in the low-Cd group. Therefore, we suggest that pregnant women, who are exposed to environmental Cd, show an increased level of Cd in their maternal blood and this Cd can accumulate in the placenta. Intracellular Cd may induce DMT-1 mRNA transcription which further translates into DMT-1 protein, which can then function as a reciprocal Cd transporter in placental tissue.

Hephaestin and ceruloplasmin facilitate iron metabolism in the mouse kidney.

Multicopper ferroxidases (MCFs) play an important role in cellular iron homeostasis. However, the role of MCFs in renal metabolism remains unclear. We used Hephaestin (Heph) and Ceruloplasmin (Cp) single or double (Heph/Cp) knockout (KO) mice to study the roles of MCFs in the kidney. Renal iron levels and the expression of iron metabolism genes were examined. The non-heme iron content both in the renal cortex and medulla of Heph/Cp KO mice was significantly increased. Perls’ Prussian blue staining showed iron accumulation on the apical side of renal tubular cells in Heph/Cp KO mice. A significant increase in ferritin protein expression was also observed in the renal medulla and cortex of Heph/Cp KO mice. Both DMT1 and TfR1 protein expression were significantly decreased in the renal medulla of Heph/Cp KO mice, while the expression of DMT1 protein was significantly increased in the renal cortex of these animals. Significant increase in proteinuria and total urinary iron was observed in the double knockout mice, and this was associated with compromised structural integrity. These results suggest that KO of both the HEPH and CP genes leads to kidney iron deposition and toxicity, MCFs could protect kidney against a damage from iron excess.

Ndfip2 is a potential regulator of the iron transporter DMT1 in the liver.

The regulation of divalent metal ion transporter DMT1, the primary non-heme iron importer in mammals, is critical for maintaining iron homeostasis. Previously we identified ubiquitin-dependent regulation of DMT1 involving the Nedd4 family of ubiquitin ligases and the Ndfip1 and Ndfip2 adaptors. We also established the in vivo function of Ndfip1 in the regulation of DMT1 in the duodenum of mice. Here we have studied the function of Ndfip2 using Ndfip2-deficient mice. The DMT1 protein levels in the duodenum were comparable in wild type and Ndfip2−/− mice, as was the transport activity of isolated enterocytes. A complete blood examination showed no significant differences between wild type and Ndfip2−/− mice in any of the hematological parameters measured. However, when fed a low iron diet, female Ndfip2−/− mice showed a decrease in liver iron content, although they maintained normal serum iron levels and transferrin saturation, compared to wild type female mice that showed a reduction in serum iron and transferrin saturation. Ndfip2−/− female mice also showed an increase in DMT1 expression in the liver, with no change in male mice. We suggest that Ndfip2 controls DMT1 in the liver with female mice showing a greater response to altered dietary iron than the male mice.

Influence of Iron Supplementation on DMT1 (IRE)-induced Transport of Lead by Brain Barrier Systems in vivo

ObjectiveTo investigate the potential involvement of DMT1 (IRE) protein in the brain vascular system in vivo during Pb exposure. MethodsThree groups of male Sprague-Dawley rats were exposed to Pb in drinking water, among which two groups were concurrently administered by oral gavage once every other day as the low and high Fe treatment group, respectively, for 6 weeks. At the same time, the group only supplied with high Fe was also set as a reference. The animals were decapitated, then brain capillary-rich fraction was isolate from cerebral cortex. Western blot method was used to identify protein expression, and RT-PCR to detect the change of the mRNA. ResultsPb exposure significantly increased Pb concentrations in cerebral cortex. Low Fe dose significantly reduced the cortex Pb levels, However, high Fe dose increased the cortex Pb levels. Interestingly, changes of DMT1 (IRE) protein in brain capillary-rich fraction were highly related to the Pb level, but those of DMT1 (IRE) mRNA were not significantly different. Moreover, the consistent changes in the levels of p-ERK1/2 or IRP1 with the changes in the levels of DMT1 (IRE). ConclusionThese results suggest that Pb is transported into the brain through DMT1 (IRE), and the ERK MAPK pathway is involved in DMT1 (IRE)-mediated transport regulation in brain vascular system in vivo.

DHA supplementation: A nutritional strategy to improve prenatal Fe homeostasis and prevent birth outcomes related with Fe-deficiency

The effect of maternal DHA supplementation on expression of key genes and proteins involved in Fe metabolism and mineral placenta content was evaluated for the first time. One hundred and ten pregnant women were randomly assigned to one of the groups: control (n = 54, 400 ml/day of the control dairy drink; DHA-supplemented (n = 56, 400 ml/day of the DHA-enriched dairy drink). Placenta DMT1, FPN1, TfR1 and Hamp1 mRNA and protein expressions were analysed. Hepcidin concentration in the mother and umbilical cord and placenta mineral content were assessed. DMT1, FPN1, TfR1 and Hamp1 gene expressions together with serum hepcidin increased in umbilical cord and artery of the DHA-supplemented group. In this group, Fe concentration increased in plasma, umbilical cord vein and artery. Ca concentrations raised in umbilical cord vein and artery, Cu, V and Mn levels. Docosahexaenoic acid (DHA) supplementation improved transplacental Fe homeostasis, enhancing Fe transfer and boosting neonates Fe stores at delivery.

Differentiation‐dependent Regulation of Intestinal Iron Absorption: Cell and Molecular Mechanisms

Differentiation of intestinal epithelial cells is associated with up‐ and down‐regulation of a variety of genes, but little is known about the differentiation‐dependent regulation of genes encoding proteins involved in intestinal iron absorption. We have addressed this issue using human‐derived intestinal epithelial Caco‐2 cells and crypt/villus epithelial cells isolated from wild‐type mice as models. We examined expression of divalent metal‐ion transporter‐1 (DMT‐1; brush border iron importer), ferroportin (FPN; basolateral iron exporter) and hephaestin (HEPH; basolateral iron oxidase). Differentiation of Caco‐2 cells was associated with a significant up‐regulation of iron uptake. This up‐regulation was associated with a significant increase in the expression of DMT‐1 and HEPH mRNA and protein, as well as increased expression of FPN mRNA, but not FPN protein. Similarly, iron uptake and the expression of DMT‐1, HEPH, and FPN mRNAs was higher in mouse villous cells than crypt cells. Promoter‐reporter constructs were used to show that the activity of the promoters of DMT‐1 and HEPH increased with differentiation of Caco‐2 cells. These studies demonstrate that differentiation of intestinal epithelial cells is associated with up‐regulation of the iron uptake process and increased expression of DMT‐1, HEPH, and FPN. The expression of all these proteins of iron transport can be mediated at the post‐transcriptional level according to intracellular iron availability, but our results indicate that up‐regulation of DMT‐1 and HEPH during differentiation appears to be mediated at least in part via transcriptional regulatory mechanisms. (Supported by funds from the Department of Veterans Affairs.)

The changes of gene expression of iron transporters for duodenal iron uptake and export in diet-induced obese mice

This study aims to determine the gene expression changes of iron transporters-divalent metal transporter 1 (DMT1) and ferroportin 1 (Fpn1) in the duodenal tissue of diet-induced obese mice. C57BL/6J mice were randomly divided into normal control (NC) and obesity model (OM) group, 6 in each, and fed on conventional and high-fat diet respectively for 14 weeks by table of random number. Then the DMT1 and Fpn1 mRNA contents in duodenal tissues of the animals were measured by Real-time PCR method, and the protein expression levels were analyzed by Western blot test. The Real-time PCR detection results showed that, compared with the NC group for which the mRNA expression level was defined as 1.0, the Fpn1 mRNA expression in OM group (0.58±0.11) was reduced significantly (t = 6.71, P = 0.014), whereas the relative expression level of DMT1 mRNA in OM group (0.89±0.26) showed no obvious alteration (t = 2.01, P = 0.122). Western blot results showed that the relative protein expression levels of Fpn1 in OM and NC group were 0.32±0.06 and 0.65±0.19, respectively, and the difference was statistically significant (t = 5.37, P = 0.026). The DMT1 protein relative abundance was 0.88±0.21 in OM group and 0.92±0.17 in NC group, and the difference has no statistical significance (t = 1.84, P = 0.185). Fpn1 gene expression is inhibited in the duodenum of diet-induced obesity mouse while DMT1 expression keeps unchanged, and this implies that decreased iron export from enterocytes into circulation might be responsible for the impaired iron absorption in obesity.

DMT1 iron uptake in the PNS: bridging the gap between injury and regeneration.

Previous studies by our group demonstrated the key role of iron in Schwann cell maturation through an increase in cAMP, PKA activation and CREB phosphorylation. These studies opened the door to further research on non-transferrin-bound iron uptake, which revealed the presence of DMT1 mRNA all along SC progeny, hinting at a constitutive role of DMT1 in ensuring the provision of iron in the PNS. In light of these previous results, the present work evaluates the participation of DMT1 in the remyelination process following a demyelinating lesion promoted by sciatic nerve crush--a reversible model of Wallerian degeneration. DMT1 was observed to colocalize with a SC marker S100β at all survival times analyzed. In turn, the assessment of DMT1 mRNA expression exhibited an increase 7 days post-injury, while DMT1 protein levels showed an increase 14 days after crush at the lesion site and distal stump; finally, an increase in iron levels became evident as from 14 days post-injury, in parallel with DMT1 values. To sum up, the present work unveils the role of DMT1 in mediating the neuroregenerative action of iron.

Effects of different sources of copper on Ctr1, ATP7A, ATP7B, MT and DMT1 protein and gene expression in Caco-2 cells

Copper sulfate (CuSO4), micron copper oxide (micron CuO) and nano copper oxide (nano CuO) at different concentrations were, respectively, added to culture media containing Caco-2 cells and their effects on Ctr1, ATP7A/7B, MT and DMT1 gene expression and protein expression were investigated and compared. The results showed that nano CuO promoted mRNA expression of Ctr1 in Caco-2 cells, and the difference was significant compared with micron CuO and CuSO4. Nano CuO was more effective in promoting the expression of Ctr1 protein than CuSO4 and micron CuO at the same concentration. Nano CuO at a concentration of 62.5μM increased the mRNA expression levels of ATP7A and ATP7B, and the difference was significant compared with CuSO4. The addition of CuSO4 and nano CuO to the culture media promoted the expression of ATP7B proteins. CuSO4 at a concentration of 125μM increased the mRNA expression level of MT in Caco-2 cells, and the difference was significant compared with nano CuO and micron CuO. Nano CuO at a concentration of 62.5μM inhibited the mRNA expression of DMT1, and the difference was significant compared with CuSO4 and micron CuO. Thus, the effects of CuSO4, micron CuO and nano CuO on the expression of copper transport proteins and the genes encoding these proteins differed considerably. Nano CuO has a different uptake and transport mechanism in Caco-2 cells to those of CuSO4 and micron CuO.