Transferrin Receptor Activity Research Articles

Neutrophil-like cell membrane-coated siRNA of lncRNA AABR07017145.1 therapy for cardiac hypertrophy via inhibiting ferroptosis of CMECs

Cardiac microvascular dysfunction is associated with cardiac hypertrophy and can eventually lead to heart failure. Dysregulation of long non-coding RNAs (lncRNAs) has recently been recognized as one of the key mechanisms involved in cardiac hypertrophy. However, the potential roles and underlying mechanisms of lncRNAs in cardiac microvascular dysfunction have not been explicitly delineated. Our results confirmed that cardiac microvascular dysfunction was related to cardiac hypertrophy and ferroptosis of cardiac microvascular endothelial cells (CMECs) occurred during cardiac hypertrophy. Using a combination of in vivo and in vitro studies, we identified a lncRNA AABR07017145.1, named as lncRNA AAB for short, and revealed that lncRNA AAB was upregulated in the hearts of cardiac hypertrophy rats as well as in the Ang II-induced CMECs. Importantly, we found that lncRNA AAB sponged and sequestered miR-30b-5p to induce the imbalance of MMP9/TIMP1, which enhanced the activation of transferrin receptor 1 (TFR-1) and then eventually led to the ferroptosis of CMECs. Moreover, we have developed a delivery system based on neutrophil membrane (NM)-camouflaged mesoporous silica nanocomplex (MSN) for inhibition of cardiac hypertrophy, indicating the potential role of silenced lncRNA AAB (si-AAB) and overexpressed miR-30b-5p as the novel therapy for cardiac hypertrophy.

A Syndrome of IgA-Related Polycythemia

Polycythemia is characterized by the risk of complications such as thrombosis. Whereas red blood cell intrinsic genetic abnormalities, such as JAK2 mutation account for most of the cases of polycythemia, pathophysiologic mechanisms involved in other etiologies of polycythemia remains poorly understood. We previously reported that pIgA1 controls erythropoiesis through activation of transferrin receptor (Coulon et al, Nat Med 2011), but data in patients are still lacking to involve pIgA1 in normal or pathologic erythropoiesis. IgA nephropathy (IgAN) is associated with elevated levelsof polymeric IgA (pIgA1) and circulating IgA1 complexes. In patients with IgAN and unexplained polycythemia, we hypothesized that pIgA1 could also be involved in the increased red blood cell production.METHODSSera from patients with IgAN and unexplained polycythemia (IgAN-Pcy, persistent hematocrit (Ht) >54%) were collected after written consent. Human progenitor CD34+ cells with addition of IgAN-Pcy or control serum were plated in semi-solid methycellulose medium and quantified, and IgA1 depletion was performed as previously described (Coulon et al, Nat Med 2011).We performed a multivariate linear regression to analyze Hb levels among various groups of a CKD cohort (696 patients with glomerulonephritis but no ESRD, IgAN n=171), and in a post-transplant cohort (2600 patients, IgAN n=271).RESULTSWe report 6 patients with IgAN-Pcy who developed polycythemia (median Ht55%). No JAK2 mutations were found. All patients had normal blood oxygen level, as well as EPO levels (median 7.6 mUI/L, range 5.8-9.9) and abdominal ultrasound. In contrast, in vitro at low dose of Epo, we found that the number of erythroid burst forming unit (BFU-E)-derived colonies was increased in the presence of IgAN-Pcy serum compared to control (73 vs 52, p<0.05). Removal of IgA1 from IgAN-Pcy patients normalized the number of colonies while add-back of these IgA1 increased it to initial values. Emphasizing our finding, among glomerulonephritis, Hb level was significantly higher in patients with IgAN by multivariate analysis (13.1g/dL vs 12.2 g/dL, p=0.01). In the post-transplant cohort, the mean elevation of Hb at M3 and M12 compared to M0 was higher in IgAN compared to other patients (Dhb M0-M3 0.87 and 0.22g/dL; M0-M12 1.36 and 0.71g/dL respectively), suggesting that Hb recovery is faster after acute anemia following renal transplantation.CONCLUSIONThese data suggest a role of pIgA1 in erythropoiesis regulation and describe a new etiology of polycythemia. DisclosuresNo relevant conflicts of interest to declare.

Abstracts of the 17th International Symposium on Bioluminescence and Chemiluminescence ‐ (ISBC 2012)

WARNING : The light-emitting molecular structures responsible for the chemiluminescence and fluorescence phenomena are not necessarily the same!

A guide to diagnosis of iron deficiency and iron deficiency anemia in digestive diseases

Iron deficiency (ID), with or without anemia, is often caused by digestive diseases and should always be investigated, except in very specific situations, as its causes could be serious diseases, such as cancer. Diagnosis of ID is not always easy. Low serum levels of ferritin or transferrin saturation, imply a situation of absolute or functional ID. It is sometimes difficult to differentiate ID anemia from anemia of chronic diseases, which can coexist. In this case, other parameters, such as soluble transferrin receptor activity can be very useful. After an initial evaluation by clinical history, urine analysis, and serological tests for celiac disease, gastroscopy and colonoscopy are the key diagnostic tools for investigating the origin of ID, and will detect the most important and prevalent diseases. If both tests are normal and anemia is not severe, treatment with oral iron can be indicated, along with stopping any treatment with non-steroidal anti-inflammatory drugs. In the absence of response to oral iron, or if the anemia is severe or clinical suspicion of important disease persists, we must insist on diagnostic evaluation. Repeat endoscopic studies should be considered in many cases and if both still show normal results, investigating the small bowel must be considered. The main techniques in this case are capsule endoscopy, followed by enteroscopy.

New liver cell mutants defective in the endocytic pathway

To isolate mutant liver cells defective in the endocytic pathway, a selection strategy using toxic ligands for two distinct membrane receptors was utilized. Rare survivors termed trafficking mutants (Trf2–Trf7) were stable and more resistant than the parental HuH-7 cells to both toxin conjugates. They differed from the previously isolated Trf1 HuH-7 mutant as they expressed casein kinase 2 α″ (CK2α″) which is missing from Trf1 cells and which corrects the Trf1 trafficking phenotype. Binding of 125I-asialoorosomucoid (ASOR) and cell surface expression of asialoglycoprotein receptor (ASGPR) were reduced approximately 20%–60% in Trf2–Trf7 cells compared to parental HuH-7, without a reduction in total cellular ASGPR. Based on 125I-transferrin binding, cell surface transferrin receptor activity was reduced between 13% and 88% in the various mutant cell lines. Distinctive phenotypic traits were identified in the differential resistance of Trf2–Trf7 to a panel of lectins and toxins and to UV light-induced cell death. By following the endocytic uptake and trafficking of Alexa488-ASOR, significant differences in endosomal fusion between parental HuH-7 and the Trf mutants became apparent. Unlike parental HuH-7 cells in which the fusion of endosomes into larger vesicles was evident as early as 20 min, ASOR endocytosed into the Trf mutants remained within small vesicles for up to 60 min. Identifying the biochemical and genetic mechanisms underlying these phenotypes should uncover novel and unpredicted protein–protein or protein–lipid interactions that orchestrate specific steps in membrane protein trafficking.

Activation of Transferrin Receptor 1 by c-Myc Enhances Cellular Proliferation and Tumorigenesis

Overexpression of transferrin receptor 1 (TFRC1), a major mediator of iron uptake in mammalian cells, is a common feature of human malignancies. Therapeutic strategies designed to interfere with tumor iron metabolism have targeted TFRC1. The c-Myc oncogenic transcription factor stimulates proliferation and growth by activating thousands of target genes. Here we demonstrate that TFRC1 is a critical downstream target of c-Myc. Using in vitro and in vivo models of B-cell lymphoma, we show that TFRC1 expression is activated by c-Myc. Chromatin immunoprecipitation experiments reveal that c-Myc directly binds a conserved region of TFRC1. In light of these findings, we sought to determine whether TFRC1 is required for c-Myc-mediated cellular proliferation and cell size control. TFRC1 inhibition decreases cellular proliferation and results in G1 arrest without affecting cell size. Consistent with these findings, expression profiling reveals that TFRC1 depletion alters expression of genes that regulate the cell cycle. Furthermore, enforced TFRC1 expression confers a growth advantage to cells and significantly enhances the rate of c-Myc-mediated tumor formation in vivo. These findings provide a molecular basis for increased TFRC1 expression in human tumors, illuminate the role of TFRC1 in the c-Myc target gene network, and support strategies that target TFRC1 for cancer therapy.

Extracellular H2O2 and not superoxide determines the compartment-specific activation of transferrin receptor by iron regulatory protein 1

Iron regulatory protein 1 (IRP1) functions as translational regulator that plays a central role in coordinating the cellular iron metabolism by binding to the mRNA of target genes such as the transferrin receptor (TfR)—the major iron uptake protein. Reactive oxygen species such as H2O2 and that are both co-released by inflammatory cells modulate IRP1 in opposing directions. While H2O2—similar to iron depletion—strongly induces IRP1 via a signalling cascade, inactivates the mRNA binding activity by a direct chemical attack. These findings have raised the question of whether compartmentalization may be an important mechanism for isolating these biological reactants when released from inflammatory cells during the oxygen burst cascade. To address this question, we studied cytosolic IRP1 and its downstream target TfR in conjunction with a tightly controlled biochemical modulation of extracellular and H2O2 levels mimicking the oxygen burst cascade of inflammatory cells. We here demonstrate that IRP1 activity and expression of TfR are solely dependent on H2O2 when co-released with from xanthine oxidase. Our findings confirm that extracellular H2O2 determines the functionality of the IRP1 cluster and its downstream targets while the reactivity of is limited to its compartment of origin.

Transferrin receptor activity and localisation in the rat duodenum.

It is not known how the efficiency of intestinal iron absorption is regulated. One hypothesis suggests that an interaction between the transferrin receptor (TfR) and the haemochromatosis protein (HFE) regulates the level of iron loading in crypt cells. The hypothesis goes on to suggest that this determines the amount of transport protein, expressed in villus enterocytes, that is involved in iron absorption. Mice with haploinsufficiency for TfR are iron deficient and this is thought to be caused by reduced iron absorption. This suggests that TfR may play a role in the regulation and/or mechanism of iron absorption. We investigated TfR function and distribution by measuring iron uptake from plasma transferrin and by immunohistochemistry. The uptake of transferrin-bound iron (Tf-Fe2) into mucosal cells subsequently separated along the crypt-villus axis was compared to the presence of TfR, determined by immunohistochemistry using frozen and wax sections. Frozen sections showed TfR staining in crypt and villus epithelial cells. In wax sections TfR was only identified in a supranuclear region commencing in enterocytes at the crypt-villus junction and attaining greatest levels at the villus tip. This indicates that the processing of wax tissue exposes a TfR epitope that otherwise remains undetectable when studied in frozen sections. This appearance in paraffin sections was inversely related to the uptake of Tf-Fe2. Supranuclear TfR was not associated with lysosomes, since there was no difference in the uptake of normal Tf-Fe2 and that of the non-digestible cellobiose Tf-Fe2, and Western blot analysis revealed similar amounts of TfR in crypt and villus cells. Also the uptake of Tf-Fe2 increased linearly with time, albeit less in villus than crypt cells, suggesting that maturation of an efflux system in villus cells is not responsible for this difference. We hypothesise that TfR in the supranuclear region of villus enterocytes may play a role in iron absorption.

Transferrin receptor activity and localisation in the rat duodenum

Transferrin receptor activity and localisation in the rat duodenum

Iron proteins of duodenal enterocytes isolated from mice with genetically and experimentally altered iron metabolism.

The molecular basis for the control of iron absorption by the duodenum remains unknown: however, ferritin (Ft) and the iron status of enterocytes have been suggested as regulatory factors. We determined the iron and Ft status of duodenal enterocytes from mice with hypotransferrinaemia, a genetic defect leading to greatly enhanced iron absorption, and for comparison we also investigated mice with experimentally-altered iron absorption. Duodenal enterocytes were isolated and analysed for Ft and non-haem iron content and for transferrin binding (as a measure of transferrin receptor activity). RNA was extracted from the duodenal mucosa and examined for transferrin receptor and H- and L-Ft mRNA levels by Northern hybridization analysis. Ft levels were elevated in enterocytes of hypotransferrinaemic mice, similar to that seen in iron dextran-injected mice of the CD1-strain. Enterocyte Ft levels were reduced in mice fed a diet diminished in iron, but unchanged in hypoxic mice enterocytes. Enterocytes of hypotransferrinaemic mice had normal non-haem iron levels and transferrin binding; however, enterocytes from CD-1 mice fed a low iron diet had increased transferrin binding and a decreased non-haem iron content. Duodenal mRNA levels for transferrin receptor and H-Ft were unchanged in hypotransferrinaemic mice, whereas L-Ft was increased. We conclude from the Ft and non-haem iron contents and transferrin binding that duodenal enterocytes from hypotransferrinaemic mice are not simply iron deficient, leading to increased expression of iron carriers proteins. Duodenal iron absorption can be enhanced in mice even when enterocyte Ft levels are raised or unchanged, suggesting that iron absorption is regulated by developmentally programmed expression of iron transporters by enterocytes.

Characterization of isolated duodenal epithelial cells along a crypt-villus axis in rats fed diets with different iron content.

The intestinal mucosa is characterized by cell proliferation, commitment, differentiation, digestion and absorption. These processes occur at specified locations along the crypt to villus axis. A technique is reported for the isolation of cells along this axis which allows the study of any one of these processes in an enriched population of cells. As an example, the uptake of transferrin-bound iron by enterocytes was studied. Rats were fed diets normal, high (30% carbonyl iron) or low in iron for 12 days. Cells from either the duodenum or ileum were isolated by incubating in a Ca(2+)-, Mg2+-free, cation chelating solution for varying periods. The incorporation of thymidine into DNA was measured in these cells as a marker of the crypt region, while alkaline phosphatase and sucrase activities marked mature enterocytes. The in vivo uptake of transferrin-bound 59Fe was measured in cells isolated either 2 or 4 h after intravenous injection. This procedure resulted in the isolation of 10 fractions of viable cells. Earlier fractions were enriched at least 10-fold in villus cells and the last fractions in crypt cells. Cells in intermediate fractions were at various stages of development. Uptake of transferrin-bound iron into enterocytes was highest with feeding an iron-loaded diet compared with control or iron-deficient diets. However, with all diets uptake was highest in crypt cells and this fell at the crypt-villus junction to be only 25%, as high at the villus tip as the crypt. A technique for the reproducible isolation of viable enterocytes along a crypt-villus axis is described. Transferrin receptor activity changes with maturation of the enterocyte.

Ferritin gene expression and transferrin receptor activity in intestine of rats with varying iron stores.

Expression of transferrin receptor and ferritin genes has been shown previously to be under transcriptional and posttranscriptional regulation, the latter being reciprocally regulated according to cellular iron levels. This study examined transferrin receptor function and ferritin gene expression along the crypt-villus axis of the intestinal tract in rats with varying iron stores. Altered iron stores were produced by feeding a control diet and diets low or high in iron (2% carbonyl iron) for 8-10 wk. Expression and activity of the ferritin genes were assessed by in situ hybridization and immunohistochemical localization, respectively. Transferrin receptor activity was determined by the uptake of intravenously injected transferrin-bound iron and was shown to increase with the level of iron loading. In all iron status groups, ferritin mRNA was seen at highest levels in the epithelial cells of the crypt and macrophages within the lamina propria and at lower levels in villus epithelial cells. In all groups, ferritin protein was not seen in the crypt region but was seen with increasing staining in the apical two-thirds of the villus cells of control and iron-loaded, but not iron-deficient, rats. Ferritin staining increased with iron loading. We conclude that in undifferentiated crypt cells ferritin genes are transcribed, but the message is not translated. After differentiation, these genes appear to be controlled posttranscriptionally by cellular iron stores.

Transferrin Receptor Activity as a Marker in Transitional Cell Carcinoma of the Bladder

An immunohistochemical staining method was used to study transferrin receptors (TFR) in bladder tissue from 10 patients with benign conditions and 33 with bladder cancer. All patients with bladder cancer were followed up by cystoscopy at intervals of 3 months (mean follow-up period 11 months). The relationship between TFR activity and recurrence rates was investigated. It was not possible to demonstrate TFR in normal bladder mucosa except for the proliferating cells of the basal layer. TFR activity in malignant tissue correlated well with the histological grade and pathological stage of the tumour. Patients with low grade superficial tumours showing TFR activity had a higher recurrence rate than those with no TFR activity. It was concluded that TFR activity in low grade superficial bladder tumours is a useful marker for predicting the recurrence rate.

Modulation of transferrin-receptor activity and recycling after induced differentiation of BeWo choriocarcinoma cells

BeWo human choriocarcinoma cells normally grow as cytotrophoblast cells. However, in the presence of 100 microM-forskolin or 5 mM-theophylline, these cells form syncytia similar to morphologically well differentiated syncytiotrophoblasts. We have examined the effect of syncytia formation on transferrin-receptor activity and recycling. Although cellular proliferation stops upon growth in the presence of forskolin or theophylline, the number of cell-surface transferrin-receptors unexpectedly increased 2-fold, whereas the total cellular number increased at most 15%. The rate of biosynthesis of the transferrin receptor as well as class I MHC glycoprotein did not change measurably during syncytium formation. The biosynthesis of human chorionic gonadotropin increased 35-fold after 30 h of growth in the presence of theophylline. The redistribution of the transferrin receptor in syncytia is maintained by a decreased rate constant of endocytosis (0.141 min-1 compared with 0.231 min-1 for control cells) and an increased rate constant of externalization (0.122 min-1 compared with 0.060 min-1 for control cells). These altered rates of endocytosis and externalization resulted in an increased rate of iron accumulation in the syncytia. Furthermore, the recycling time of the transferrin receptor decreased in cells grown in the presence of theophylline (14.6 min compared with 21.2 min in control cells).

Loss of transferrin receptor activity in Neisseria meningitidis correlates with inability to use transferrin as an iron source.

Although Neisseria meningitidis does not produce siderophores, it is able to obtain iron from human transferrin. We observed saturable specific binding of 125I-labeled human transferrin to meningococci. Human lactoferrin and mouse transferrin did not compete with human transferrin for binding, whereas human apotransferrin and 100% iron-saturated transferrin competed equally well. Meningococci thus have a specific receptor for human transferrin. Scatchard analysis yielded a relatively low Kd of 0.7 microM and an apparent copy number of 2,900 receptors per CFU. Receptor activity was iron-regulated. A meningococcal transformant specifically unable to utilize transferrin as an iron source had decreased transferrin receptor activity. These data are consistent with the hypothesis that receptor-mediated binding of transferrin is a rate-limiting step in meningococcal iron uptake from transferrin.

Purification and characterization of an estrogen-inducible membrane glycoprotein. Evidence that it is a transferrin receptor.

A number of N-linked membrane glycoproteins are induced during chick oviduct differentiation. We have purified a major estrogen-inducible glycoprotein (Mr = 91,000) to homogeneity by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Comparison of partial NH2-terminal sequence data with membrane glycoproteins having similar Mr showed a limited homology with human and murine transferrin receptors. We observed that oviduct membranes contain estrogen-inducible transferrin receptor activity (Kd = 2-8 x 10(-8) M). Analytical purification of the putative receptor on an ovotransferrin-Affi-Gel affinity column and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis reveals a protein of Mr, 180,000, which contains two disulfide-linked subunits of Mr 91,000. The receptor reacts very strongly with antibodies prepared against the 91-kDa glycoprotein on Western blots. Western blot analysis confirms that the 91-kDa glycoprotein is induced by estrogen. The protein has 2% total carbohydrate with Man, GlcNAc, Gal, GalNAc, and NeuAc in a molar ratio of 6:4:2:1:1. The protein contains at least one O-linked moiety. Analysis of the O-linked moiety by glycosidase digestions and gel filtration indicates there are sialo tetra- and trisaccharides and a neutral disaccharide(s). Labeled N-linked glycopeptides were prepared by pronase digestion, beta-elimination, and 3H-acetylation. The N-linked oligosaccharides include high mannose and complex neutral nonbisected biantennary types in an approximate ratio of 3:1 as determined by serial lectin affinity chromatography.

Loss of transferrin receptor activity in Neisseria meningitidis correlates with inability to use transferrin as an iron source.

Loss of transferrin receptor activity in Neisseria meningitidis correlates with inability to use transferrin as an iron source.

Determination of transferrin receptors on frozen sections of malignant B-cell lymphomas by immunofluorescence with a monoclonal antibody.

A series of 34 B-cell lymphomas was studied for the presence of transferrin receptors by immunofluorescence on frozen tissue sections with a monoclonal antibody. These lymphomas were classified by light microscopic study and furthermore investigated by immunohistochemical and morphometric methods. There were diffuse centrocytic, diffuse centroblastic, and follicular and diffuse centrocytic/centroblastic lymphomas. Significant differences in log mean nuclear area in these four histologic groups were found, as well as a significant correlation between log nuclear area and transferrin receptors as measured semiquantitatively by fluorescence intensity of the cell membranes. In addition, it was found that there is a strong correlation between the transferrin receptor activity and malignancy grading on histologic and morphometric basis. Therefore, the transferrin receptor determination on frozen sections appears to be a good method of malignancy grading.

The 1985 Walter Hubert lecture. Malignant cell differentiation as a potential therapeutic approach.

Most drugs available for cancer chemotherapy exert their effects through cytodestruction. Although significant advances have been attained with these cytotoxic agents in several malignant diseases, response is often accompanied by significant morbidity and many common malignant tumours respond poorly to existing cytotoxic therapy. Development of chemotherapeutic agents with non-cytodestructive actions appears desirable. Considerable evidence exists which indicates that (a) the malignant state is not irreversible and represents a disease of altered maturation, and (b) some experimental tumour systems can be induced by chemical agents to differentiate to mature end-stage cells with no proliferative potential. Thus, it is conceivable that therapeutic agents can be developed which convert cancer cells to benign forms. To study the phenomenon of blocked maturation, squamous carcinoma SqCC/Y1 cells were employed in culture. Using this system it was possible to demonstrate that physiological levels of retinoic acid and epidermal growth factor were capable of preventing the differentiation of these malignant keratinocytes into a mature tissue-like structure. The terminal differentiation caused by certain antineoplastic agents was investigated in HL-60 promyelocytic leukaemia cells to provide information on the mechanism by which chemotherapeutic agents induce cells to by-pass a maturation block. The anthracyclines aclacinomycin A and marcellomycin were potent inhibitors of N-glycosidically linked glycoprotein biosynthesis and transferrin receptor activity, and active inducers of maturation; temporal studies suggested that the biochemical effects were associated with the differentiation process. 6-Thioguanine produced cytotoxicity in parental cells by forming analog nucleotide. In hypoxanthine-guanine phosphoribosyltransferase negative HL-60 cells the 6-thiopurine initiated maturation; this action was due to the free base (and possibly the deoxyribonucleoside), a finding which separated termination of proliferation due to cytotoxicity from that caused by maturation.

Dissociation of protein kinase C activation from phorbol ester-induced maturation of HL-60 leukemia cells.

The role of C-kinase in the induction of maturation of HL-60 promyelocytic leukemia cells was examined using two activators of this kinase, 12-O-tetradecanoyl phorbol 13-acetate (TPA) and 1-oleoyl-2-acetylglycerol (OAG). At 10(-8) M, a concentration that induced maturation, TPA effectively stimulated C-kinase activity in cell-free preparations by increasing the affinity of the enzyme for Ca2+. Similar activation was observed with 20 micrograms/ml of OAG. At these concentrations, addition of either compound to intact cells stimulated the phosphorylation of cellular proteins. Treatment with TPA resulted in an increased phosphorylation of 14 proteins, 9 of which also changed in response to OAG. In addition to the effects on protein phosphorylation, TPA and OAG both affected choline lipid metabolism. TPA at 10(-8) M stimulated the incorporation of [methyl-3H]choline into phosphatidylcholine, sphingomyelin, and lysophosphatidylcholine. OAG at 20 micrograms/ml had quantitatively similar effects on the labeling of the former two lipids, but did not affect incorporation of choline into lysophosphatidylcholine. Despite the similar biochemical effects of TPA and OAG, the diglyceride was unable to induce HL-60 cell maturation as measured by inhibition of cell growth, development of nonspecific esterase activity, phagocytosis, adherence of cells to plastic, and loss of transferrin receptor activity. The lack of effect is not due to metabolism of OAG; maturation could not be induced by treating cells with fresh OAG every 2 h for a period of 12 h. These results suggest a dissociation of the activation of C-kinase and the induction of HL-60 cell maturation by TPA.