Reduced Iron Uptake Research Articles

Lif-deficiency promote systemic Iron metabolism disorders and increases the susceptibility of osteoblasts to ferroptosis

Leukemia inhibitory factor (LIF) is a multifunctional cytokine that plays a crucial role in various biological processes. However, LIF involvement in iron metabolism remains almost unexplored. This study aimed to explore the impact of LIF on systemic iron transportation and its potential role in ferroptosis in osteoblasts. We observed that the Lif-deficient (Lif−/−) mice is characterized by a reduction in bone mass and a decrease in bone mineral density compared with wild-type (WT) mice. Energy-dispersive X-ray spectroscopy revealed a marked increase in iron content on the surface of femurs from Lif−/− mice. Meanwhile, iron stores test lower iron levels in the spleens and higher levels in the femurs of Lif−/− mice. Besides, Lif−/− mice display increased levels of serum iron, total iron-binding capacity, unsaturated iron-binding capacity, and transferrin saturation and serum ferritin relative to WT mice. Hepcidin mRNA expression reduction in the liver of Lif−/− mice. It also holds true in the AML-12 hepatocyte cell line after Lif-knockdown. Immunohistochemistry and RT-PCR revealed elevated ferroportin (FPN) in duodenal cells of Lif−/− mice. Lif-deficiency decreases SLC7A11 levels in osteoblasts. In addition, overexpression of LIF downregulates CD71, DCYTB, and DMT1, thereby reducing iron uptake in iron-overloaded cells. Femur immunohistochemistry (IHC) revealed increased ACSL4 and decreased GPX4 and SLC7A11, indicating an increase in ferroptosis of osteoblasts in Lif−/− mice. Whole-transcriptome sequencing showed gene expression changes after Lif-knockdown, exhibiting a negative correlation with genes involved in long-chain fatty acid transport, mitochondrial organization, and the p38 MAPK signaling pathway. These results demonstrate that Lif-deficiency alter systemic iron metabolism and increases the susceptibility of osteoblasts to ferroptosis.

Scanning iron response regulator binding sites using Dap-seq in the Brucella genome.

Iron is an essential element required for all organisms. Iron response regulator (Irr) is a crucial transcriptional regulator and can affect the growth and iron uptake of Brucella. The growth rate of Brucella melitensis M5-90 irr mutant was significantly lower than that of B. melitensis M5-90 under normal or iron-sufficient conditions, however, the growth rate of the B. melitensis M5-90 irr mutant was significantly higher than that of B. melitensis M5-90 under iron-limited conditions. In addition, irr mutation significantly reduced iron uptake under iron-limited conditions. Previous studies suggested that the Irr protein has multiple target genes in the Brucella genome that are involved in iron metabolism. Therefore, in the present study, a Dap-seq approach was used to investigate the other iron metabolism genes that are also regulated by the Irr protein in Brucella. A total of seven genes were identified as target genes for Irr in this study and the expression levels of these seven genes was identified using qRT-PCR. The electrophoretic mobility shift assay confirmed that six out of the seven genes, namely rirA (BME_RS13665), membrane protein (BME_RS01725), hypothetical protein (BME_RS09560), ftrA (BME_RS14525), cation-transporting P-type ATPase (zntA) (BME_RS10660), and 2Fe-2S binding protein (BME_RS13655), interact with the Irr protein. Furthermore, the iron utilization and growth assay experiments confirmed that rirA was involve in iron metabolism and growth of Brucella. In summary, our results identified six genes regulated by the Irr protein that may participate in iron metabolism, and the rirA was identified as a regulon of Irr and it also plays a role in iron metabolism of Brucella. Collectively, these results provide valuable insights for the exploration of Brucella iron metabolism.

MicroRNA let-7d attenuates hypertrophic scar fibrosis through modulation of iron metabolism by reducing DMT1 expression.

Hypertrophic scar is an unavoidable result of wound healing following burns and trauma, which remains a challenging problem for clinicians. Previously, we demonstrated that exosomal microRNAs (miRs) of human amniotic epithelial cells accelerated wound healing and inhibited scar formation. However, the underlying mechanism is still unclear. In this particular study, we found that miR-let-7d reduced collagen deposition, and this was accompanied by decreased level of iron content in myofibroblasts. Importantly, inhibition of miR-let-7d in myofibroblasts accelerated collagen deposition and promoted cell proliferation. In addition, bioinformatics prediction combined with classical dual-luciferase reporter gene assay demonstrated that the cellular iron importer divalent metal transporter 1 (DMT1) was a target gene of miR-let-7d, and the miR-let-7d mimics inhibited the expression of DMT1 in myofibroblasts. Moreover, silencing of DMT1 with small interfering RNA (siRNA) reduced the deposition of extracellular matrix. Consistent with the results in vitro, the miR-let-7d mimics effectively ameliorated hypertrophic scar fibrosis in a rabbit ear hypertrophic scar model. Taken together, our results indicated for the first time that miR-let-7d attenuated hypertrophic scar fibrosis through modulation of iron metabolism by reducing iron uptake through DMT1, which may provide a novel therapeutic strategy for hypertrophic scar.

Resveratrol ameliorates iron overload induced liver fibrosis in mice by regulating iron homeostasis.

This study is intended to explore the protective effects of resveratrol (RES) on iron overload-induced liver fibrosis and its mechanism. Iron dextran (50 mg/kg) was injected intraperitoneally in all groups except the control group. Mice in the L-RES, M-RES and H-RES groups were gavaged with RES solution at 25, 50 mg/kg and 100 mg/kg, respectively, 4 h before injection of iron dextran every day; mice in the deferoxamine (DFO) group were injected with DFO intraperitoneally (100 mg/kg); mice in the control group received isovolumetric saline. After seven weeks of RES administration, serum alanine aminotransferase (ALT), aspartate aminotransferase (AST) activities and liver hydroxyproline (Hyp) levels were reduced; the malondialdehyde (MDA) activities decreased and the levels of superoxide dismutase (SOD) and glutathione (GSH) were raised. Hematoxylin and eosin (H&E), Prussian, and Masson staining indicated that RES treatment could improve cell damage and reduce hepatic iron deposition and collagen deposition in iron-overload mice. The expression of Bcl-2 was increased, the expression levels of Bax and caspase-3 were decreased under RES treatment. Moreover, RES reduced the expression of hepcidin, ferritin (Ft), divalent metal transporter-1 (DMT-1), transferrin receptor-2 (TFR-2), and raised the expression of ferroprotein-1 (FPN-1). In conclusion, RES could ameliorate iron overload-induced liver fibrosis, and the potential mechanisms may be related to antioxidant, anti-inflammatory, anti-apoptotic, and more importantly, regulation of iron homeostasis by reducing iron uptake and increasing iron export.

Transferrin receptor‐mediated iron uptake is essential for colon tissue homeostasis

Background and aimsTransferrin receptor (TFRC) is the major mediator for iron entry into a cell. Under excessive iron conditions, TFRC is expected to be downregulated via the iron regulatory protein and the iron‐responsive element machinery to reduce iron uptake and toxicity. However, the mechanism by which the expression of TFRC is maintained high in iron‐enriched cancer cells and its contribution to cancer development are enigmatic.MethodsThe regulation and function of TFRC‐mediated iron uptake in colon were tested in mouse models with colon‐specific TFRC disruption and colon‐derived cell lines. Transcriptome analysis of patient‐derived tumor colonoids was performed to identify molecular targets of iron.ResultsOur work shows TFRC is induced by adenomatous polyposis coli gene loss‐driven β‐catenin activation in colorectal cancer, whereas TFRC‐mediated intratumoral iron accumulation potentiates β‐catenin signaling via directly enhancing the activity of tankyrase. TFRC‐mediated iron import is at the center of this novel feed‐forward loop to facilitate colonic epithelial cell survival. Mechanistically, disruption of TFRC led to a reduction of colonic iron levels and iron‐dependent tankyrase activity, which caused stabilization of Axin2 and subsequent repression of the b‐catenin/c‐Myc/E2F1/DNA polymerase delta1 (POLD1) axis. POLD1 knockdown, iron chelation and TFRC disruption increased DNA replication stress, DNA damage response, apoptosis and reduced colon tumor growth. Strikingly, a combination of iron chelators and DNA damaging agents caused a synergistic effect in inducing DNA damage response and reducing colon tumor cell growth.ConclusionTogether, the TFRC/iron/tankyrase/Axin2/b‐catenin/c‐Myc/E2F1/POLD1 axis is essential for colon homeostasis and may provide potential novel strategies for colorectal cancer therapy.

Iron-induced NCOA4 condensation regulates ferritin fate and iron homeostasis.

Iron is not only essential but also a toxic trace element. Under iron repletion, ferritin maintains cellular iron homeostasis by storing iron to avoid iron toxicity. Under iron depletion, the ferritin-specific autophagy adaptor NCOA4 delivers ferritin to lysosomes via macroautophagy to enable cells to use stored iron. Here, we show that NCOA4 also plays crucial roles in the regulation of ferritin fate under iron repletion. NCOA4 forms insoluble condensates via multivalent interactions generated by the binding of iron to its intrinsically disordered region. This sequesters NCOA4 away from ferritin and allows ferritin accumulation in the early phase of iron repletion. Under prolonged iron repletion, NCOA4 condensates can deliver ferritin to lysosomes via a TAX1BP1-dependent non-canonical autophagy pathway, thereby preventing relative iron deficiency due to excessive iron storage and reduced iron uptake. Together, these observations suggest that the NCOA4-ferritin axis modulates intracellular iron homeostasis in accordance with cellular iron availability.

Effect of enhanced CaCl2, MgSO4, and KH2PO4 on improved in vitro growth of potato.

Potato (Solanum tuberosum L.) is a major global food crop. Contemporary potato production largely utilizes micropropagation to produce healthy seed potatoes. The micropropagation of potatoes is widely achieved through nodal explants using the conventional Murashige and Skoog (MS) medium. Currently, effective culture media that can facilitate rapid propagation are increasingly required for new cultivars that have been developed to possess improved traits. In this study, we evaluated the effect of enhanced meso nutrients (CaCl2.2H2O, MgSO4, and KH2PO4) in MS medium on the growth of S. tuberosum. The cultivars used in this study were representative of Japanese, European, and Peruvian lines. Enhanced meso nutrients improved the overall quality of all cultivars, as indicated by longer shoots and larger leaves with dark color, compared with MS medium only. Shoots grown on enhanced mesos were approximately 1.5 times longer than on MS medium. Quantitative ion analysis revealed that plantlets with improved shoot length and leaf quality in most cultivars had increased calcium, magnesium, potassium, and phosphorus uptake than plantlets on MS medium. The results suggest that the reduced iron uptake on 3.0×MS, compared with 2.0× or 2.5×MS mesos, reduced plant growth. This study revealed for the first time that mesos concentrations higher than MS medium concentrations, complemented by enhanced calcium, magnesium, potassium, phosphorus, and iron uptake, play a significant role in improving the in vitro growth of potato.

Molecular mechanism of zero valent iron-enhanced microbial azo reduction

Zero valent iron (ZVI)–microbe technology has an increasing application on the removal of organic pollution, yet the molecular mechanism of microbe respond to ZVI is still a mystery. Here, we established a successive ZVI-enhanced microbial system to remove azo dye (a typical organic pollutant) by Shewanella decolorationis S12 (S. decolorationis S12, an effective azo dye degradation bacterium) and examined the gene expression time course (10, 30, 60, and 120 min) by whole genome transcriptional analysis. The addition of ZVI to the microbial degradation system increases the rate of azo reduction from ~60% to over 99% in 16 h reaction, suggesting the synergistic effect of ZVI and S12 on azo dye degradation. Comparing with the treatment without ZVI, less filamentous cells were observed in ZVI treated system, and approximately 8% genes affiliated with 10 different gene expression profiles in S. decolorationis S12 were significantly changed in 120 min during the ZVI-enhanced azo reduction. Intriguingly, MarR transcriptional factor might play a vital role in regulating ZVI-enhanced azo reduction in the aspect of energy production, iron homeostasis, and detoxification. Further investigation showed that the induced [Ni–Fe] H2ase genes (hyaABCDEF) and azoreductase genes (mtrABC-omcA) contributed to ZVI-enhanced energy production, while the reduced iron uptake (hmuVCB and feoAB), induced sulfate assimilation (cysPTWA) and cysteine biosynthesis (cysM) related genes were essential to iron homeostasis and detoxification. This study disentangles underlying mechanisms of ZVI-enhanced organic pollution biotreatment in S. decolorationis S12.

Managing Genetic Hemochromatosis: An Overview of Dietary Measures, Which May Reduce Intestinal Iron Absorption in Persons With Iron Overload.

Genetic hemochromatosis causes iron overload by excess absorption of dietary iron, due to a decreased expression of hepcidin. The objective was to elaborate dietary recommendations that can reduce intestinal iron absorption in hemochromatosis patients, based on our present knowledge of the iron contained in nutrients and the mechanisms of iron uptake. This is a narrative review. Literature search in PubMed and Google Scholar of papers dealing with iron absorption from the diet was conducted. Most important proposed dietary recommendations are: 1) Choose a varied vegetarian, semi-vegetarian or flexitarian diet. A “veggie-lacto-ovo-poultry-pescetarian” diet seems optimal. Avoid iron enriched foods and iron supplements. 2) Eat many vegetables and fruits, at least 600 g per day. Choose protein rich pulses and legumes (e.g., kidney- and soya beans). Fresh fruits should be eaten between meals. 3) Abstain from red meat from mammals and choose the lean, white meat from poultry. Avoid processed meat, offal and blood containing foods. Eat no more than 200 g meat from poultry per week. Choose fish, eggs, vegetables and protein rich legumes the other days. Eat fish two to four times a week as main course, 350 - 500 g fish per week, of which half should be fat fish. 4) Choose whole grain products in cereals and bread. Avoid iron enriched grains. Choose non-sourdough, yeast-fermented bread with at least 50% whole grain. 5) Choose vegetable oils, and low-fat dairy products. 6) Eat less sugar and salt. Choose whole foods and foods with minimal processing and none or little added sugar or salt. 7) Quench your thirst in water. Drink green- or black tea, coffee, or low-fat milk with the meals, alternatively water or non-alcoholic beer. Fruit juices must be consumed between meals. Abstain from alcoholic beverages. Drink soft drinks, non-alcoholic beer, or non-alcoholic wine instead. These advices are close to the official Danish dietary recommendations in 2021. In the management of hemochromatosis, dietary modifications that lower iron intake and decrease iron bioavailability may provide additional measures to reduce iron uptake from the foods and reduce the number of phlebotomies. However, there is a need for large, prospective, randomized studies that specifically evaluate the effect of dietary interventions.

Efficacy and Safety of Amlodipine in Preventing Myocardial Iron Overload in Patients with Transfusion-Dependent Thalassemia Major : A Systematic Review and Meta-Analysis

Background: Patients diagnosed with thalassemia major who are transfusion dependant, have iron accumulation leading to iron toxicity and severe impairment in organs like heart, liver and endocrine organs which are highly sensitive to iron toxicity. This makes iron chelation therapy imperative for these patients. Half of the deaths resulting from iron toxicity related complications are attributed to cardiac complications. Iron chelation therapies have not been completely successful to prevent iron toxicity related complications like arrhythmia, cardiomyopathy and heart failure. Higher doses of iron chelation therapies have been associated with various side effects. Studies have shown L-type calcium channel blocker might be able to reduce iron uptake by myocardium. The aim of this meta-analysis is to assess the efficacy and safety of amlodipine to reduce myocardial iron concentration (MIC). Methods: We used PICO framework to do a systematic literature search using four database PubMed, Cochrane, Embase, and Web of Science using keywords, "Thalassemia" AND "Amlodipine" from the inception till July 2020. The initial search showed 90 articles out of which, six randomized clinical trials (RCT) (N= 226) were selected after exclusion of case reports, case series, preclinical trials, review articles, meta-analysis, and trials not providing any information about preventing iron overload in patients with transfusion dependent thalassemia. We extracted the data for myocardial iron concentration (MIC), myocardial T2, ferritin, hepatic iron/liver iron concentration (LIC), liver T2, left ventricular ejection fraction, response rate and adverse effects. DerSimonian-Laird random effects model was used to derive mean differences along with their 95% confidence interval (CI) using comprehensive meta-analysis version 3.0. Results: In six RCT, 96 patients were tested in experimental group and 97 in control group. In five RCT total number of male participants were 45 in experimental group and 54 in control group. 33 patients had splenectomy in experimental group and 41 in control group. The age range was 8 years to 31 years. The myocardial T2 score increased in amlodipine group compared to standard chelation group with significant mean difference estimated to be -0.62 (95% CI: -0.95-0.29, p-value: <0.001) in favor of amlodipine in meta-analysis of the four trials (Fig 1.). Statistically significant reduction in myocardial iron was seen in two trials on adding amlodipine to standard chelation therapy (N=55) (Table 1.) (Khaled et al and Fernandes et al). Significant difference was reported in liver T2 score and LIC at the end of six months between amlodipine and control group by Khaled et al. But, there was no statistically significant mean difference in serum ferritin and in liver MRI T2 between amlodipine group and control group with mean difference of -1143 (95% CI: -2410 to 124, p-value = 0.07) and -0.06 (95% CI: -0.463 to 0.338, p-value = 0.76) in meta-analysis of four and two trials respectively (Fig 2. And Fig 3.). El-Haggar et al compared amlodipine with spirulina and statistically significant improvement in myocardial T2 and NT-proBNP level was seen in both groups. Spirulina group also showed significant reduction in serum ferritin, which showed spirulina could also help reduce iron overload. Only mild adverse effects were reported by trials (Table 2.). No cases of severe hypotension, palpitation or any other serious adverse effects were seen in the amlodipine group. Conclusion: This systematic review and meta-analysis suggests that addition of amlodipine 2.5-5 mg/day to standard chelation therapy with monitoring for potential adverse effects, could benefit patients with thalassemia major by reducing cardiac iron overload and thus improve survival and quality of life. Future studies are required to study the role of amlodipine in reducing iron overload in endocrine organs that also absorb iron through voltage-gated channels, particularly considering the close association of cardiac siderosis with endocrine complications and the correlation of pancreas and MICs. Disclosures Anwer: Incyte, Seattle Genetics, Acetylon Pharmaceuticals, AbbVie Pharma, Astellas Pharma, Celegene, Millennium Pharmaceuticals.: Honoraria, Research Funding, Speakers Bureau.

A Review of Nutrients and Compounds, Which Promote or Inhibit Intestinal Iron Absorption: Making a Platform for Dietary Measures That Can Reduce Iron Uptake in Patients with Genetic Haemochromatosis.

Objective To provide an overview of nutrients and compounds, which influence human intestinal iron absorption, thereby making a platform for elaboration of dietary recommendations that can reduce iron uptake in patients with genetic haemochromatosis. Design Review. Setting. A literature search in PubMed and Google Scholar of papers dealing with iron absorption. Results The most important promoters of iron absorption in foods are ascorbic acid, lactic acid (produced by fermentation), meat factors in animal meat, the presence of heme iron, and alcohol which stimulate iron uptake by inhibition of hepcidin expression. The most important inhibitors of iron uptake are phytic acid/phytates, polyphenols/tannins, proteins from soya beans, milk, eggs, and calcium. Oxalic acid/oxalate does not seem to influence iron uptake. Turmeric/curcumin may stimulate iron uptake through a decrease in hepcidin expression and inhibit uptake by complex formation with iron, but the net effect has not been clarified. Conclusions In haemochromatosis, iron absorption is enhanced due to a decreased expression of hepcidin. Dietary modifications that lower iron intake and decrease iron bioavailability may provide additional measures to reduce iron uptake from the foods. This could stimulate the patients' active cooperation in the treatment of their disorder and reduce the number of phlebotomies.

Functional Iron Indices and Highly Sensitive CRP Post Single Dose Irradiation with Cobalt60 and the Ameliorating Effects of Single and Combined Doses of Aqueous Extracts of Parquetina nigrescens, Camellia sinensis and Telfairia occidentalis in Guinea Pigs

Background: The relationship between iron, hypoxia, inflammation, and erythropoietin in cellular homeostasis is well documented. Patients on radiotherapy are known with active immune/inflammatory disorders often accompanied with reduced iron uptake or unavailability of circulatory iron and hence, must be adequately evaluated. The present study hypothesized “aqueous extracts of Camellia sinensis, Telfairia occidentalis and Parquetina nigrescens have chemical properties of ameliorating and restoring to normal, functional iron deficiency sequel to Cobalt 60 irradiation effect”.
 Materials and Methods: Fifty-Five young male guinea-pigs approximately 450 gram in weight were recruited and thirty were randomly assigned to 3 groups (A, B and C) for the study. Groups A and B were further divided into 4 (A1-4 and B1-4) with 3 animals (n=3) per group. Three guinea-pigs were also assigned to group C. Groups A and B belonged to Pre and post-irradiation groups while groups C served as control. Each animal was given 400r (4.0 Gy) whole-body gamma-irradiation under general anaesthesia, using a Co60 therapy unit as a source. Groups A1, A2, A3 and A4 had 1,400 mg/kg C. sinensis, 4000 mg/kg P. nigrescens, 3,500 mg/kg T. occidentalis and Combined dose (1,400 mg/kg C. sinensis + 400 mg/kg P. nigrescens + 3,500 mg/kg T. occidentalis) respectively twice daily 72 hours prior to irradiation and continued throughout the 14 days of the study. Groups B1, B2, B3 and B4 had similar treatment but commenced 24 hours after exposure to radiation and likewise continued throughout the 14 days of the study. Group C were not given any treatment but also had irradiation.
 Results: Total Iron Binding Capacity, Ferritin, Serum Transferrin receptor and Iron were all increased significantly for all the extracts pre and post irradiation. However, C-reactive protein decreased significantly.
 Conclusion: Aqueous extracts of Camellia sinensis, Parquetina nigrescens and Telfairia occidentalis leaves have good ameliorating effect on irradiation-induced injuries.

Iron deficiency in worsening heart failure is associated with reduced estimated protein intake, fluid retention, inflammation, and antiplatelet use.

AimsIron deficiency (ID) is common in heart failure (HF) patients and negatively impacts symptoms and prognosis. The aetiology of ID in HF is largely unknown. We studied determinants and the biomarker profile of ID in a large international HF cohort.Methods and resultsWe studied 2357 worsening HF patients from the BIOSTAT-CHF cohort. ID was defined as transferrin saturation <20%. Univariable and multivariable logistic regression models were constructed to identify determinants for ID. We measured 92 cardiovascular markers (Olink Cardiovascular III) to establish a biomarker profile of ID. The primary endpoint was the composite of all-cause mortality and first HF rehospitalization. Mean age (±standard deviation) of all patients was 69 ± 12.0 years, 26.1% were female and median N-terminal pro B-type natriuretic peptide levels (+interquartile range) were 4305 (2360–8329) ng/L. Iron deficiency was present in 1453 patients (61.6%), with highest prevalence in females (71.1% vs. 58.3%; P < 0.001). Independent determinants of ID were female sex, lower estimated protein intake, higher heart rate, presence of peripheral oedema and orthopnoea, chronic kidney disease, lower haemoglobin, higher C-reactive protein levels, lower serum albumin levels, and P2Y12 inhibitor use (all P < 0.05). None of these determinants were sex-specific. The biomarker profile of ID largely consisted of pro-inflammatory markers, including paraoxonase 3 (PON3) and tartrate-resistant acid phosphatase type 5. In multivariable Cox proportional hazard regression analyses, ID was associated to worse outcome, independently of predictors of ID (hazard ratio 1.25, 95% confidence interval 1.06–1.46; P = 0.007).ConclusionOur data suggest that the aetiology of ID in worsening HF is complex, multifactorial and seems to consist of a combination of reduced iron uptake (malnutrition, fluid overload), impaired iron storage (inflammation, chronic kidney disease), and iron loss (antiplatelets).

Pea Ferritin Stability under Gastric pH Conditions Determines the Mechanism of Iron Uptake in Caco-2 Cells

BackgroundIron deficiency is an enduring global health problem that requires new remedial approaches. Iron absorption from soybean-derived ferritin, an ∼550-kDa iron storage protein, is comparable to bioavailable ferrous sulfate (FeSO4). However, the absorption of ferritin is reported to involve an endocytic mechanism, independent of divalent metal ion transporter 1 (DMT-1), the transporter for nonheme iron.ObjectiveOur overall aim was to examine the potential of purified ferritin from peas (Pisum sativum) as a food supplement by measuring its stability under gastric pH treatment and the mechanisms of iron uptake into Caco-2 cells.MethodsCaco-2 cells were treated with native or gastric pH–treated pea ferritin in combination with dietary modulators of nonheme iron uptake, small interfering RNA targeting DMT-1, or chemical inhibitors of endocytosis. Cellular ferritin formation, a surrogate measure of iron uptake, and internalization of pea ferritin with the use of specific antibodies were measured. The production of reactive oxygen species (ROS) in response to equimolar concentrations of native pea ferritin and FeSO4 was also compared.ResultsPea ferritin exposed to gastric pH treatment was degraded, and the released iron was transported into Caco-2 cells by DMT-1. Inhibitors of DMT-1 and nonheme iron absorption reduced iron uptake by 26–40%. Conversely, in the absence of gastric pH treatment, the iron uptake of native pea ferritin was unaffected by inhibitors of nonheme iron absorption, and the protein was observed to be internalized in Caco-2 cells. Chlorpromazine (clathrin-mediated endocytosis inhibitor) reduced the native pea ferritin content within cells by ∼30%, which confirmed that the native pea ferritin was transported into cells via a clathrin-mediated endocytic pathway. In addition, 60% less ROS production resulted from native pea ferritin in comparison to FeSO4.ConclusionWith consideration that nonheme dietary inhibitors display no effect on iron uptake and the low oxidative potential relative to FeSO4, intact pea ferritin appears to be a promising iron supplement.

The Protective Role of Mitochondrial Ferritin on Erastin-Induced Ferroptosis.

Ferroptosis, a newly identified form of regulated cell death, is characterized by overwhelming iron-dependent accumulation of lethal lipid reactive oxygen species (ROS). Preventing cellular iron overload by reducing iron uptake and increasing iron storage may contribute to inhibit ferroptosis. Mitochondrial ferritin (FtMt) is an iron-storage protein that is located in the mitochondria, which has a significant role in modulating cellular iron metabolism. Recent studies showed that FtMt played inhibitory effects on oxidative stress-dependent neuronal cell damage. However, the potential role of FtMt in the progress of ferroptosis in neuronal cells has not been studied. To explore this, we established ferroptosis models of cell and drosophila by erastin treatment. We found that overexpression of FtMt in neuroblastoma SH-SY5Y cells significantly inhibited erastin-induced ferroptosis, which very likely was achieved by regulation of iron homeostasis. Upon erastin treatment, significant increases of cellular labile iron pool (LIP) and cytosolic ROS were observed in wild-type SH-SY5Y cells, but not in the FtMt-overexpressed cells. Consistent with that, the alterations of iron-related proteins in FtMt-overexpressed cells were different from that of the control cells. We further investigated the role of FtMt in erastin-induced ferroptosis in transgenic drosophila. We found that the wild-type drosophilas fed an erastin-containing diet didn't survive more than 3 weeks. In contrast, the FtMt overexpressing drosophilas fed the same diet were survival very well. These results indicated that FtMt played a protective role in erastin-induced ferroptosis.

Altered levels of AtHSCB disrupts iron translocation from roots to shoots.

Plants overexpressing AtHSCB and hscb knockdown mutants showed altered iron homeostasis. The overexpression of AtHSCB led to activation of the iron uptake system and iron accumulation in roots without concomitant transport to shoots, resulting in reduced iron content in the aerial parts of plants. By contrast, hscb knockdown mutants presented the opposite phenotype, with iron accumulation in shoots despite the reduced levels of iron uptake in roots. AtHSCB play a key role in iron metabolism, probably taking part in the control of iron translocation from roots to shoots. Many aspects of plant iron metabolism remain obscure. The most known and studied homeostatic mechanism is the control of iron uptake in the roots by shoots. Nevertheless, this mechanism likely involves various unknown sensors and unidentified signals sent from one tissue to another which need to be identified. Here, we characterized Arabidopsis thaliana plants overexpressing AtHSCB, encoding a mitochondrial cochaperone involved in [Fe-S] cluster biosynthesis, and hscb knockdown mutants, which exhibit altered shoot/root Fe partitioning. Overexpression of AtHSCB induced an increase in root iron uptake and content along with iron deficiency in shoots. Conversely, hscb knockdown mutants exhibited increased iron accumulation in shoots and reduced iron uptake in roots. Different experiments, including foliar iron application, citrate supplementation and iron deficiency treatment, indicate that the shoot-directed control of iron uptake in roots functions properly in these lines, implying that [Fe-S] clusters are not involved in this regulatory mechanism. The most likely explanation is that both lines have altered Fe transport from roots to shoots. This could be consistent with a defect in a homeostatic mechanism operating at the root-to-shoot translocation level, which would be independent of the shoot control over root iron deficiency responses. In summary, the phenotypes of these plants indicate that AtHSCB plays a role in iron metabolism.

Deciphering the Substrate Specificity of SbnA, the Enzyme Catalyzing the First Step in Staphyloferrin B Biosynthesis.

Staphylococcus aureus assembles the siderophore, staphyloferrin B, from l-2,3-diaminopropionic acid (l-Dap), α-ketoglutarate, and citrate. Recently, SbnA and SbnB were shown to produce l-Dap and α-ketoglutarate from O-phospho-l-serine (OPS) and l-glutamate. SbnA is a pyridoxal 5′-phosphate (PLP)-dependent enzyme with homology to O-acetyl-l-serine sulfhydrylases; however, SbnA utilizes OPS instead of O-acetyl-l-serine (OAS), and l-glutamate serves as a nitrogen donor instead of a sulfide. In this work, we examined how SbnA dictates substrate specificity for OPS and l-glutamate using a combination of X-ray crystallography, enzyme kinetics, and site-directed mutagenesis. Analysis of SbnA crystals incubated with OPS revealed the structure of the PLP-α-aminoacrylate intermediate. Formation of the intermediate induced closure of the active site pocket by narrowing the channel leading to the active site and forming a second substrate binding pocket that likely binds l-glutamate. Three active site residues were identified: Arg132, Tyr152, Ser185 that were essential for OPS recognition and turnover. The Y152F/S185G SbnA double mutant was completely inactive, and its crystal structure revealed that the mutations induced a closed form of the enzyme in the absence of the α-aminoacrylate intermediate. Lastly, l-cysteine was shown to be a competitive inhibitor of SbnA by forming a nonproductive external aldimine with the PLP cofactor. These results suggest a regulatory link between siderophore and l-cysteine biosynthesis, revealing a potential mechanism to reduce iron uptake under oxidative stress.

ZIP8 Is an Iron and Zinc Transporter Whose Cell-surface Expression Is Up-regulated by Cellular Iron Loading

ZIP8 (SLC39A8) belongs to the ZIP family of metal-ion transporters. Among the ZIP proteins, ZIP8 is most closely related to ZIP14, which can transport iron, zinc, manganese, and cadmium. Here we investigated the iron transport ability of ZIP8, its subcellular localization, pH dependence, and regulation by iron. Transfection of HEK 293T cells with ZIP8 cDNA enhanced the uptake of (59)Fe and (65)Zn by 200 and 40%, respectively, compared with controls. Excess iron inhibited the uptake of zinc and vice versa. In RNA-injected Xenopus oocytes, ZIP8-mediated (55)Fe(2+) transport was saturable (K(0.5) of ∼0.7 μm) and inhibited by zinc. ZIP8 also mediated the uptake of (109)Cd(2+), (57)Co(2+), (65)Zn(2+) > (54)Mn(2+), but not (64)Cu (I or II). By using immunofluorescence analysis, we found that ZIP8 expressed in HEK 293T cells localized to the plasma membrane and partially in early endosomes. Iron loading increased total and cell-surface levels of ZIP8 in H4IIE rat hepatoma cells. We also determined by using site-directed mutagenesis that asparagine residues 40, 88, and 96 of rat ZIP8 are glycosylated and that N-glycosylation is not required for iron or zinc transport. Analysis of 20 different human tissues revealed abundant ZIP8 expression in lung and placenta and showed that its expression profile differs markedly from ZIP14, suggesting nonredundant functions. Suppression of endogenous ZIP8 expression in BeWo cells, a placental cell line, reduced iron uptake by ∼40%, suggesting that ZIP8 participates in placental iron transport. Collectively, these data identify ZIP8 as an iron transport protein that may function in iron metabolism.

A defect in iron uptake enhances the susceptibility of Cryptococcus neoformans to azole antifungal drugs

The high-affinity reductive iron uptake system that includes a ferroxidase (Cfo1) and an iron permease (Cft1) is critical for the pathogenesis of Cryptococcus neoformans. In addition, a mutant lacking CFO1 or CFT1 not only has reduced iron uptake but also displays a markedly increased susceptibility to azole antifungal drugs. Altered antifungal susceptibility of the mutants was of particular interest because the iron uptake system has been proposed as an alternative target for antifungal treatment. In this study, we used transcriptome analysis to begin exploring the molecular mechanisms of altered antifungal susceptibility in a cfo1 mutant. The wild-type strain and the cfo1 mutant were cultured with or without the azole antifungal drug fluconazole and their transcriptomes were compared following sequencing with Illumina Genome Analyzer IIx (GAIIx) technology. As expected, treatment of both strains with fluconazole caused elevated expression of genes in the ergosterol biosynthetic pathway that includes the target enzyme Erg11. Additionally, genes differentially expressed in the cfo1 mutant were involved in iron uptake and homeostasis, mitochondrial functions and respiration. The cfo1 mutant also displayed phenotypes consistent with these changes including a reduced ratio of NAD+/NADH and down-regulation of Fe–S cluster synthesis. Moreover, combination treatment of the wild-type strain with fluconazole and the respiration inhibitor diphenyleneiodonium dramatically increased susceptibility to fluconazole. This result supports the hypothesis that down-regulation of genes required for respiration contributed to the altered fluconazole susceptibility of the cfo1 mutant. Overall, our data suggest that iron uptake and homeostasis play a key role in antifungal susceptibility and could be used as novel targets for combination treatment of cryptococcosis. Indeed, we found that iron chelation in combination with fluconazole treatment synergistically inhibited the growth of C. neoformans.

Hepcidin directly inhibits transferrin receptor 1 expression in astrocytes via a cyclic AMP‐protein kinase a pathway

Hepcidin, an iron-regulatory hormone, plays a central role in iron homeostasis in peripheral tissues. The widespread distribution of hepcidin in the brain implies that the hormone may be essential for brain iron homeostasis. Here, we investigated the effects of hepcidin on the expression of iron uptake proteins, including transferrin receptor 1 (TfR1) and divalent metal transporter1 (DMT1) and the release protein ferroportin1 (Fpn1) in the cultured astrocytes. The effects of hepcidin on iron uptake, including transferrin-bound iron (Tf-Fe) and non-transferrin-bound iron (NTBI), and iron release were also studied. Our results demonstrated that astrocytes, when treated with hepcidin peptide or infected with hepcidin expression adenovirus (ad-hepcidin), showed a significant ability in reducing iron uptake (both Tf-Fe and NTBI), and iron release, which were accompanied by decreased expressions of TfR1, DMT1, and Fpn1. Moreover, we found that the effect of hepcidin in reducing TfR1 expression, which is dependent on the cyclic AMP-protein kinase A pathway, was the primary and dominant event. In conclusion, our results demonstrated that hepcidin controlled iron uptake and release by regulating expression of iron transport proteins. The findings also implied the existence of a novel hepcidin-receptor on the membrane of astrocytes.