Down-regulation Of Transferrin Receptor Research Articles

Hydrogen Attenuates Chronic Intermittent Hypoxia-Induced Cardiac Hypertrophy by Regulating Iron Metabolism.

The present study aimed to investigate the impact of hydrogen (H2) on chronic intermittent hypoxia (CIH)-induced cardiac hypertrophy in mice by modulating iron metabolism. C57BL/6N mice were randomly allocated into four groups: control (Con), CIH, CIH + H2, and H2. The mice were exposed to CIH (21-5% FiO2, 3 min/cycle, 8 h/d), and received inhalation of a hydrogen-oxygen mixture (2 h/d) for 5 weeks. Cardiac and mitochondrial function, levels of reactive oxygen species (ROS), and iron levels were evaluated. The H9C2 cell line was subjected to intermittent hypoxia (IH) and treated with H2. Firstly, we found H2 had a notable impact on cardiac hypertrophy, ameliorated pathological alterations and mitochondrial morphology induced by CIH (p < 0.05). Secondly, H2 exhibited a suppressive effect on oxidative injury by decreasing levels of inducible nitric oxide synthase (i-NOS) (p < 0.05) and 4-hydroxynonenal (4-HNE) (p < 0.01). Thirdly, H2 demonstrated a significant reduction in iron levels within myocardial cells through the upregulation of ferroportin 1 (FPN1) proteins (p < 0.01) and the downregulation of transferrin receptor 1 (TfR1), divalent metal transporter 1 with iron-responsive element (DMT1(+ire)), and ferritin light chain (FTL) mRNA or proteins (p < 0.05). Simultaneously, H2 exhibited the ability to decrease the levels of Fe2+ and ROS in H9C2 cells exposed to IH (p < 0.05). Moreover, H2 mediated the expression of hepcidin, hypoxia-inducible factor-1α (HIF-1α) (p < 0.01), and iron regulatory proteins (IRPs), which might be involved in the regulation of iron-related transporter proteins. These results suggested that H2 may be beneficial in preventing cardiac hypertrophy, a condition associated with reduced iron toxicity.

Cis-monounsaturated fatty acids inhibit ferroptosis through downregulation of transferrin receptor 1

Ferroptosis, a form of cell death mediated by lipid peroxidation, is implicated in various pathological processes. Although monounsaturated fatty acids (MUFAs) can inhibit ferroptotic lipid peroxidation, the underlying structural mechanism of this antagonistic effect remains poorly understood. We hypothesized that MUFAs with different structures (including chain length, conformation, and double bond position) may affect their regulatory effect on ferroptosis. In this study, 11 MUFAs with varying structures were screened to identify those with an inhibitory effect on ferroptosis. Results from 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazoliumbromide assays indicated that only exogenous MUFAs with cis-conformation and centered double bond could inhibit ferroptosis. Meanwhile, it was found that suppressing the expression of SCD1 and SCD5 genes could sensitize cells to ferroptosis indicating the protective role of endogenous MUFA against ferroptosis. Additionally, western blot analysis revealed that cis-MUFAs with centered double bond downregulated the protein levels of transferrin receptor 1. Flow cytometry confirmed that these MUFAs led to decreases in intracellular iron, reactive oxygen species, and lipid peroxides. It was also found that SCD1 inhibitor could enhance ferroptosis inducer-mediated tumor suppression both in vivo and in vitro. Overall, these findings shed light on the particular structural features of MUFAs that contribute to their ferroptosis-resistant properties and suggest the potential therapeutic relevance of natural MUFAs in a range of ferroptosis-related diseases.

Activation of vitamin D receptor inhibits Tau phosphorylation is associated with reduction of iron accumulation in APP/PS1 transgenic mice

Vitamin D deficiency and iron accumulation are prevalent in the brains of Alzheimer's disease (AD) patients, however, whether Vitamin D has a role in the regulations of iron metabolism in the condition of AD remains unknown. Our previous studies revealed that vitamin D deficiency promotes β-amyloid (Aβ) deposition in the APP/PS1 mouse brains, while supplemented with a specific agonist of vitamin D receptor (VDR), paricalcitol (PAL), significantly reduced Aβ production via promoting the lysosomal degradation of β-site APP cleavage enzyme 1 (BACE1). In this study, our data suggested that activation of VDR by PAL significantly reduced the iron accumulation in the cortex and hippocampus of APP/PS1 mice through downregulation of Transferrin receptor (TFR) by reducing iron-regulatory protein 2 (IRP2) expression. Furthermore, activation of VDR effectively reduced the phosphorylations of Tau at Ser396 and Thr181 sites via inhibiting the GSK3β phosphorylation (Tyr216). Taken together, our data suggest that activation of VDR could inhibit the phosphorylations of Tau possibly by repressing the iron accumulation-induced upregulation of GSK3β activity in the brains of APP/PS1 mice. Thus, activation of VDR may be an effective strategy for treating AD.

Characteristics of in Vitro Differentiated Erythrocytes Derived from Human Bmi-1 Extensively Expanded Erythroblasts (E3)

Background and significance: Blood transfusion is an important and sometimes life-saving therapeutic intervention that may not always be available due to donor shortages, alloimmunization, and rare blood groups. Red blood cells (RBCs) produced in vitro may provide an inexhaustible alternative or complimentary source of blood products for those patients without compatible donors, while eliminating the risk of infection and transfusion reactions. Bmi-1 overexpression has been shown to increase the self-renewal potential of mouse erythroblasts. We describe studies employing Bmi-1 overexpression in human erythroblasts to produce cultures of extensively expanded erythroblasts (E3) that can be differentiated into mature erythrocytes, and used for the study and optimization of ex-vivo methods of erythropoiesis with the ultimate goal of mass production of engineered blood red blood cells for transfusion purposes. Methods: Human erythroblasts, derived from peripheral blood of human donors, were transduced with a lentiviral vector expressing Bmi-1 to produce cultures of E3 cells. Following the transfer of proliferating E3 cells to differentiation culture conditions, we characterized changes in cell morphology and expression of erythroid cell surface markers CD49, CD71 and CD235a at various time points. Additionally, we tracked cells numbers, cell viability, and enucleation. At the end of the differentiation process we also analyzed cells for hemoglobinization and blood group expression and compared our results to the original donor cells. Culture conditions were optimized to increase the yield of viable and enucleated cells at the end of the differentiation process. Findings: In vitro maturation and differentiation of Bmi-1-E3 cells is characterized by progressive enucleation (up to 50% of total erythroid cells in some experiments), as well as down-regulation of transferrin receptor (CD71) and α4 integrin (CD49d). As maturation progressed, cells also underwent additional changes including decrease in size, nuclear condensation, and accumulation of hemoglobin in the cytoplasm. We were able to optimize conditions to demonstrate that cells were terminally maturing over time in the culture system. Conclusion(s): The data shows that Bmi-1-E3 cells can be cultured in vitro to produce mature erythroid cells with characteristics desirable for transfusion for those patients who may not otherwise have a suitable source for transfusion. Further work is required in order to increase enucleation and evaluate the survival and functionality of these cells in vivo, as once completely enucleated, cultured red cells do not pose concerns over genotoxicity and should be of adequate quality to be used for transfusion purposes. Simultaneously, our group is developing a mouse model to test the in-vivo performance of these cultured red blood cells. Disclosures Jajosky: Biconcavity Inc.: Other: CEO and partial owner; BioMarin Pharmaceuticals: Current equity holder in publicly-traded company; Magenta Therapeutics: Current equity holder in publicly-traded company.

Ferrous Iron Up-regulation in Fibroblasts of Patients with Beta Propeller Protein-Associated Neurodegeneration (BPAN).

Mutations in WDR45 gene, coding for a beta-propeller protein, have been found in patients affected by Neurodegeneration with Brain Iron Accumulation, NBIA5 (also known as BPAN). BPAN is a movement disorder with Non Transferrin Bound Iron (NTBI) accumulation in the basal ganglia as common hallmark between NBIA classes (Hayflick et al., 2013). WDR45 has been predicted to have a role in autophagy, while the impairment of iron metabolism in the different NBIA subclasses has not currently been clarified. We found the up-regulation of the ferrous iron transporter (-)IRE/Divalent Metal Transporter1 and down-regulation of Transferrin receptor in the fibroblasts of two BPAN affected patients with splicing mutations 235+1G>A (BPAN1) and 517_519ΔVal 173 (BPAN2). The BPAN patients showed a concomitant increase of intracellular ferrous iron after starvation. An altered pattern of iron transporters with iron overload is highlighted in BPAN human fibroblasts, supporting for a role of DMT1 in NBIA. We here present a novel element, about iron accumulation, to the existing knowledge in field of NBIA. Attention is focused to a starvation-dependent iron overload, possibly accounting for iron accumulation in the basal ganglia. Further investigation could clarify iron regulation in BPAN.

A role for transferrin receptor in triggering apoptosis when targeted with gambogic acid

Transferrin receptor (TfR) has been shown to be significantly overexpressed in different types of cancers. We discovered TfR as a target for gambogic acid (GA), used in traditional Chinese medicine and a previously undiscovered link between TfR and the rapid activation of apoptosis. The binding site of GA on TfR is independent of the transferrin binding site, and it appears that GA potentially inhibits TfR internalization. Down-regulation of TfR by RNA interference decreases sensitivity to GA-induced apoptosis, further supporting TfR as the primary GA receptor. In summary, GA binding to TfR induces a unique signal leading to rapid apoptosis of tumor cells. These results suggest that GA may provide an additional approach for targeting the TfR and its use in cancer therapy.

Iron status in manganese alloy production workers.

The aim of this study was to investigate markers of iron status in production workers with current and long-term exposure to manganese (Mn) alloys. A total of 100 Mn-exposed male workers were compared with 100 male controls matched for age in a cross-sectional study. The geometric mean urinary Mn concentration in the exposed workers was 0.9 nmol mmol(-1) creatinine (range = 0.1-126.3), compared with 0.4 nmol mmol(-1) creatinine (range = 0.1-13.1) in the controls. The index group had been exposed to Mn for 20 years on average (range = 2.1-41.0). The geometric mean concentration of soluble transferrin receptor was lower in the exposed subjects than in the controls (2.2 vs 2.6 mg l(-1); P < 0.001) and the concentration was negatively associated with current exposure to "soluble" Mn in the inhalable aerosol fraction and with current smoking habits. An association was found between the concentration of serum soluble transferrin receptor and the concentration of Mn in whole blood (Pearson's r = 0.48; P < 0.001) in the controls. The results suggest that Mn-exposed workers have higher intracellular iron concentration in the erythrocyte precursors than the controls, resulting in a down-regulation of transferrin receptors on the surface of these cells. The concentrations of Mn in the blood of occupationally non-exposed individuals appear to be influenced by iron status, even at physiological iron levels.

Repression of ferritin expression increases the labile iron pool, oxidative stress, and short-term growth of human erythroleukemia cells

AbstractThe role of ferritin expression on the labile iron pool of cells and its implications for the control of cell proliferation were assessed. Antisense oligodeoxynucleotides were used as tools for modulating the expression of heavy and light ferritin subunits of K562 cells. mRNA and protein levels of each subunit were markedly reduced by 2-day treatment with antisense probes against the respective subunit. Although the combined action of antisense probes against both subunits reduced their protein expression, antisense repression of one subunit led to an increased protein expression of the other. Antisense treatment led to a rise in the steady-state labile iron pool, a rise in the production of reactive oxygen species after pro-oxidative challenges and in protein oxidation, and the down-regulation of transferrin receptors. When compared to the repression of individual subunits, co-repression of each subunit evoked a more than additive increase in the labile iron pool and the extent of protein oxidation. These treatments had no detectable effects on the long-term growth of cells. However, repression of ferritin synthesis facilitated the renewal of growth and the proliferation of cells pre-arrested at the G1/S phase. Renewed cell growth was significantly less dependent on external iron supply when ferritin synthesis was repressed and its degradation inhibited by lysosomal antiproteases. This study provides experimental evidence that links the effect of ferritin repression on growth stimulation to the expansion of the labile iron pool.

Relationship between TNF-alpha and iron metabolism in differentiating human monocytic THP-1 cells.

The human monocytic cell line THP-1 differentiates along the macrophage line after phorbol-12-myristate-13-acetate (PMA) supplementation and can be stimulated to secrete tumour necrosis factor alpha (TNF-alpha) by interferon gamma (IFN-gamma) addition. We found that, in the early stage of differentiation (1-48 h), PMA induction elicited an upregulation of intracellular H ferritin and H ferritin binding sites and a downregulation of transferrin receptor. In addition, we found that iron administration to PMA-differentiating cells induced the expression of TNF-alpha mRNA and TNF-alpha secretion to levels even higher than those induced by IFN-gamma alone. The iron chelator desferrioxamine showed the opposite effect and reduced TNF-alpha release. In contrast, preincubation of the cells with iron before PMA induction resulted in a decrease of the TNF-alpha secretion induced by IFN-gamma, whereas the opposite was true after preincubation with desferrioxamine. The data support a co-ordinate interaction between iron and TNF-alpha in monocyte macrophages, with an iron-mediated upregulation of TNF-alpha in the early phase of differentiation and an iron-mediated inhibition at later stages. This complex relationship has to be considered in evaluating the effects of iron on inflammation.

The effect of lead on iron uptake from transferrin in human erythroleukemia (K562) cells.

The effect of lead on cellular iron metabolism has been investigated using human erythroleukemia (K562) cells. When the cells were cultured with 100 microM Pb2+ for 48 h, the rate of cellular iron uptake from transferrin decreased to 46% of that in untreated cells. Scatchard analysis of the binding data revealed that this reduction was the result of a decrease in the number of transferrin receptors rather than an alteration in ligand-receptor affinity. The results of immunoprecipitation of transferrin receptors on the cell surface also confirmed the decreased expression of transferrin receptors by lead-treated cells. The down-regulation of transferrin receptors by treatment with lead did not result from a decrease in the total amount of the receptor, as determined by immunoblotting. Moreover, the biosynthesis of the receptor was unaffected by lead treatment. Thus, the down-regulation of surface transferrin receptors in lead-treated cells might be due to a redistribution of receptors rather than an actual loss of receptors from the cell. Using kinetic analysis, it was shown that redistribution of the receptor did not result from the alteration in the rates of transferrin receptor recycling. A comparison of the amounts of transferrin receptor on the cell surface and in the cycling pool revealed that the sequestration of the receptor from normal flow through the cycle might cause down-regulation of the surface receptor.

Alveolar macrophage activation in experimental legionellosis.

Legionella pneumophila is a facultative intracellular parasite of alveolar macrophages. In vitro studies have shown that lymphokine-activated mononuclear phagocytes inhibit intracellular replication of L. pneumophila. To determine if recovery from legionellosis is associated with activation of alveolar macrophages in vivo to resist L. pneumophila, we studied an animal model of Legionnaires' disease. Rats were exposed to aerosolized L. pneumophila and alveolar macrophages were harvested during the recovery phase of infection. We compared these alveolar exudate macrophages with normal resident alveolar macrophages for the capacity to support or inhibit the intracellular growth of L. pneumophila. We also measured Ia expression as a marker of immunologic activation, and studied binding of bacteria, superoxide release, and the expression of transferrin receptors as potential mechanisms of resistance to L. pneumophila. For perspective on the specificity of these responses, we also studied alveolar exudate cells elicited by inhalation of heat-killed L. pneumophila, live Listeria monocytogenes, and live Escherichia coli. We found that alveolar exudate macrophages elicited by live L. pneumophila, but not heat-killed L. pneumophila, resisted the intracellular growth of L. pneumophila. Exudate macrophages in resolving legionellosis exhibited increased Ia expression, diminished superoxide production, and downregulation of transferrin receptors. Binding of L. pneumophila to exudate macrophages was indistinguishable from that to resident macrophages in the presence of normal serum, and augmented in the presence of immune serum. Alveolar exudate macrophages elicited by E. coli also inhibited growth of L. pneumophila, and exhibited a modest increase in Ia expression without change in transferrin receptors. Exudate cells induced by L. monocytogenes exhibited up-regulation of Ia without diminution of superoxide release. Alveolar cells harvested after inhalation of heat-killed L. pneumophila did not differ from resident alveolar macrophages in the expression of surface markers. These findings suggest that alveolar macrophages are immunologically activated in vivo to serve as effector cells in resolving legionellosis, and that live bacteria are required to induce this expression of immunity. The mechanism of resistance to parasitism by L. pneumophila may entail restriction of the intracellular availability of iron, but does not involve diminished bacterial binding or an augmented respiratory burst.

Transferrin receptor expression and the regulation of placental iron uptake

Placental transferrin receptors, located at the apical side of syncytiotrophoblast, mediate placental iron uptake. Regulation of transferrin receptors on the fetal-maternal exchange area could be a major determinant in the regulation of trans-placental iron transport. Transferrin receptor expression in cultured human term cytotrophoblasts is on a much lower level than in choriocarcinoma cells, with a higher proportion of receptors located on the cell surface. Differentiation of cells, either due to longer culture periods or to 8-bromo-cAMP treatment does not lead to an increase of transferrin receptor expression. In vitro, the level of expression is largely regulated by the cellular density in the culture dishes. Low cellular occupancy of the dish leads to a high level of transferrin receptors. Treatment with iron-sources results in a down regulation of transferrin receptors. Thus, though the level of transferrin receptors in cultured normal trophoblast is at a constant level, unaffected by differentiation, high levels of maternal transferrin-iron availability can lead to a decrease in placental iron uptake. This feed-back mechanism makes placental iron uptake independent of maternal iron stores.

Hemopexin-dependent down-regulation of expression of the human transferrin receptor.

To investigate the regulation mechanism of the uptake of iron and heme iron by the cells and intracellular utilization of iron, we examined the interaction between iron uptake from transferrin and hemopexin-mediated uptake of heme by human leukemic U937 cells or HeLa cells. U937 cells exhibited about 40,000 hemopexin receptors/cell with a dissociation constant (Kd) of 1 nM. Heme bound in hemopexin was taken up by U937 cells or HeLa cells in a receptor-mediated manner. Treatment of both species of cells with hemopexin led to a rapid decrease in iron uptake from transferrin in a hemopexin dose-dependent manner, and the decrease seen in case of treatment with hemin was less than that seen with hemopexin. The decrease of iron uptake by hemopexin contributed to a decrease in cell surface transferrin receptors on hemopexin-treated cells. Immunoblot analysis of the transferrin receptors revealed that the cellular level of receptors in U937 cells did not vary during an 8-h incubation with hemopexin although the number of surface receptors as well as iron uptake decreased within the 2-h incubation. After 4 h of incubation of the cells with hemopexin, a decrease of the synthesis of the receptors occurred. Thus, the down-regulation of transferrin receptors by hemopexin can be attributed to at least two mechanisms. One is a rapid redistribution of the surface receptor into the interior of the cells, and the other is a decrease in the biosynthesis of the receptor. 59Fe from the internalized heme rapidly appeared in non-heme iron (ferritin) coincidently with the induction of heme oxygenase. The results suggest that iron released from heme down-regulates the expression of the transferrin receptors and iron uptake.

Effect of prostaglandin E 2 on gamma-interferon and 1,25(OH) 2D 3 vitamin D 3-induced c- myc reduction during HL-60 cell differentiation

The effect of prostaglandin E 2 (PGE 2) on the reduction of c- myc expression during the differentiation of the human leukemic cell line, HL-60, was examined. PGE 2, a potent inducer of intracellular cyclic AMP (cAMP) in HL-60 cells, augmented monocyte-associated cell surface antigens induced by human gamma-interferon (IFN-γ) or 1α,25-dihydroxyvitamin D 3 (1,25(OH) 2D 3) in these cells. The elevation of intracellular cAMP was induced dose-dependently by PGE 2, but not by IFN-γ or 1,25(OH) 2D 3. Changes were also seen in functional differentiation, such as, the increase of phagocytic capability and superoxide generation. PGE 2 also enhanced the reduction of c- myc expression and the down-regulation of transferrin receptor by IFN-γ or 1,25(OH) 2D 3, whereas PGE 2 alone did not induce these phenotypic changes. These data suggest that IFN-γ and 1,25(OH) 2D 3 reduce c- myc expression of HL-60 cells by a mechanism other than the augmentation of intracellular cAMP.

Analysis of prostaglandin E2 effect on T lymphocyte activation. Abrogation of prostaglandin E2 inhibitory effect by the tumor promotor 12.0 tetradecanoyl phorbol-13 acetate.

We have investigated the inhibitory potential of prostaglandin E2 (PGE2) with respect to intracellular messengers implicated in the signaling system of T-lymphocyte activation pathway. Using the fluorescent indicator Quin 2, it is demonstrated that PGE2 inhibits the increase in cytosolic-free calcium concentration [Ca2+]i. Reconstitution of calcium mobilization in the presence of PGE2 by the calcium ionophore A23187 results in a partial restoration of both interleukin 2 (IL2) production and cell proliferation and has no effect on the inhibition of transferrin receptor expression. In contrast, the treatment of cell cultures with the tumor promotor 12.0 tetra decanoyl phorbol-13-acetate (TPA) abrogates the suppressor activity of PGE2. When T lymphocyte stimulation is provided by the combination of A23187 and TPA, the PGE2 inhibitory effect does not occur. These data also indicate that the down regulation of transferrin receptor by PGE2 is proximal to protein kinase C activation and is not associated with decreased expression of the functional IL2 receptor.

Down regulation of transferrin receptor by prostaglandin E2 : S.C. Chouaib, K. Welte, and B. Dupont; Memorial Sloan-Kettering Cancer Center, New York, NY

Down regulation of transferrin receptor by prostaglandin E2 : S.C. Chouaib, K. Welte, and B. Dupont; Memorial Sloan-Kettering Cancer Center, New York, NY