Transferrin Receptor Gene Expression Research Articles

Biomineralized ceria nanoparticles target the heart to improve diabetic cardiac remodeling by regulating mitochondrial oxidative stress and decreasing excessive mitophagy

Abstract Background An altered state of cardiomyocytes due to high glucose concentrations results in cell hypertrophy, leading to diabetic cardiomyopathy (DCM).The molecular regulatory process of mitochondrial dyshomeostasis and mitophagy, oxidative stress, and reactive oxygen species (ROS) production in diabetes is still not fully understood. Mitochondrial dyshomeostasis and mitophagy could be critical factors in the heart remodeling that occurs in diabetes. Purpose Our transcriptome sequencing of heart tissue revealed upregulation of the gene encoding the transferrin receptor (TfR) in the diabetic rat (Figure 1A). We then validated in vitro that high glucose increased TfR protein expression in the HL-1 cell line (Figure 1B). Herein, we developed a novel therapeutic ceria nanoparticle via biomineralization using transferrin (Tf) protein as a temple and assessed its targeted therapy for DM-induced cardiac remodeling via Tf-TfR mediated endocytosis. Method We synthesized Tf protein-based ceria NPs (CeO2@Tf) via biomineralization. Then the physicochemical and biological properties of NPs were characterized. Cellular uptake, cytotoxicity test, intracellularmitochondrial membrane potential (MMP), and ROS were verified with mouse cardiomyocytes HL-1 cells line. Afterward, the male C57BL/6N mice were randomly divided into control, DM, DM+Metformin (Met), and DM+NPs groups with eight weeks of treatment. Heart structure and function were measured by echocardiographic. The heart tissues were stained with hematoxylin, eosin (H&E), and Masson's trichrome stain to observe morphological changes and fibrosis. Western blot was used to determine mitochondrial homeostasis regulation-related proteins and mitophagy pathway. Results TEM and X-ray photoelectron spectroscopy characterized the properties successfully. MTT assay shows that HL-1 cell viabilities were not influenced by CeO2@Tf at 200 µM.High-glucose treatment led to increased ROS and decreased intracellular MMP in HL-1 cells. DM mice cardiac transcriptome sequencing results showed upregulation of transferrin receptor gene expression. Echocardiographic showed increased LAD and abnormal diastolic and systolic functions. All these abnormal changes were improved in the DM+ Met and DM+NPs groups. Our investigations revealed proteins linked to the PINK1-Parkin signaling pathway (Figure 2A-B) and found that the level of mitophagy in the diabetic group was overly high, which was improved after the application of CeO2@Tf. We observed the mitochondrial autophagosomes through transmission electron microscopy (Figure 2C). The autophagosomes in the diabetic group increased significantly and improved after treatment with CeO2@Tf. Conclusions These consistent biochemical and histopathological results suggest that the biomimetic mineralization of CeO2@Tf could inhibit oxidative stress and improve mitochondrial function in vitro induced by high-glucose and in vivo caused by DM, alleviating cardiac remodeling in DM mice.

Antiproliferative activity of marine-derived oligopeptides combined with androgen receptor inhibition in preclinical prostate cancer models.

e15186 Background: Recent cancer statistics reveal a sustained increase in the incidence of prostate cancer (PC) of 3% per year. More than half of this increase is attributable to advanced disease. Advanced PC remains largely incurable, owing to inevitable progression. Escalation of therapy can restore tumor control, but significant sequelae may result. Anticancer peptides of marine origin appear intrinsically target specific and may constitute a novel approach to improve the therapeutic sensitivity of resistant cancers. Recently published in vitro work showed that a marine soluble protein hydrolysate (SPH) potentiated the anticancer effect of androgen-receptor inhibitors (ARis) in PC cells. SPH also induced transcriptional upregulation of ferritin heavy chain 1 (FTH1) in LNCaP and PC3 cell lines. Given the growing recognition that iron dysmetabolism plays a key role in PC, we applied the findings to this current study. Methods: Eight candidate FTH1-modulatory peptides (FT-001 to -008, all 7- or 8-mer peptides) were identified in the SPH peptide mix and subsequently sequenced and synthesized. Antiproliferative effects of individual FT peptides were assessed in combination with ARis (bicalutamide/BIC or enzalutamide/ENZ) in phenotypically distinct PC cell lines (LNCaP, PC3, VCaP, ENZR-VCaP) and compared to the inhibitory effect of SPH-ARi combination or ARi monotherapy. Results: FT-002 or -005 (10 μM concentration), showed significantly greater inhibition of LNCaP and PC3 cell viability when co-administered with BIC IC50 (0.4 μM & 10 μM respectively), than SPH (160 μM) with BIC or BIC monotherapy (p < 0.001). Similarly, either peptide combined with ENZ IC50 (1.4 μM) exhibited significant antiproliferative effects on androgen-sensitive VCaP cells when compared to SPH (160 μM) & ENZ co-treatment (p < 0.01) and ENZ monotreatment (p < 0.001). Both peptides (10 μM) produced a similar pattern of results in ENZ-resistant VCaP cells when treated with ENZ at its corresponding IC50 (47 μM) (p < 0.001). Differential gene expression profiling of all tested cells showed a consistent and significant upregulation of FTH1 and concomitant downregulation of transferrin receptor (TFRC) gene expression of greater than 2-fold for FT/ARi therapy. Conclusions: FT-002 and -005 synergistically combine with ARis to significantly attenuate PC tumor growth in vitro. Gene expression profiling implicates peptide-mediated FTH1 and TFRC modulation in the mode of action to interfere with tumor cell iron metabolism. Novel research perspectives implicate ferritin and FTH1 expression levels in prostate carcinogenesis and tumor progression. Further research in xenograft models is warranted, in order to validate the current findings and further investigate the cellular pathways responsible for the observed anticancer activity seen with FT-002 and FT-005.

COVID-19 compromises iron homeostasis: Transferrin as a target of investigation

ImportanceSince the beginning of the COVID-19 pandemic, numerous metabolic alterations have been observed in individuals with this disease. It is known that SARS-CoV-2 can mimic the action of hepcidin, altering intracellular iron metabolism, but gaps remain in the understanding of possible outcomes in other pathways involved in the iron cycle. ObjectiveTo profile iron, ferritin and hepcidin levels and transferrin receptor gene expression in patients diagnosed with COVID-19 between June 2020 and September 2020. Design, setting and participantsCross-sectional study that evaluated iron metabolism markers in 427 participants, 218 with COVID-19 and 209 without the disease. ExposuresThe primary exposure was positive diagnose to COVID-19 in general population of Santo André and São Bernardo cities. The positive and negative diagnose were determinate through RT-qPCR. Main outcomes and measuresDevido a evidências de alterações do ciclo do ferro em pacientes diagnosticados com COVID-19 e devido a corregulação entre hepcidina e receptor de transferrina, uma análise da expressão gênica deste último, poderia trazer insights sobre o estado de ferro celular. A hipótese foi confirmada, mostrando aumento da expressão de receptor de transferrina concomitante com redução do nível de hepcidina circulante. ResultsSerum iron presented lower values in individuals diagnosed with COVID-19, whereas serum ferritin presented much higher values in infected patients. Elderly subjects had lower serum iron levels and higher ferritin levels, and men with COVID-19 had higher ferritin values than women. Serum hepcidin was lower in the COVID-19 patient group and transferrin receptor gene expression was higher in the infected patient group compared to controls. Conclusions and relevanceCOVID-19 causes changes in several iron cycle pathways, with iron and ferritin levels being markers that reflect the state and evolution of infection, as well as the prognosis of the disease. The increased expression of the transferrin receptor gene suggests increased iron internalization and the mimicry of hepcidin action by SARS-CoV-2, reduces iron export via ferroportin, which would explain the low circulating levels of iron by intracellular trapping.

Influence of supplementation of probiotic bacteria Lactobacillus plantarum and Lactobacillus curvatus on selected parameters of liver iron metabolism in rats on high-fat iron-deficient diet

• Lactobacillus curvatus and L. plantarum increase serum ferroportin. • L. curvatus enhances the expression of the Tfrc gene in the liver. • Increased expression of Tfrc and SLC11A2 genes stimulates hepatic iron accumulation. • Fecal Lactobacillus content shows negative correlation with HJV gene expression. Iron deficiency constitutes the most frequent diet deficit. A high-fat diet also leads to disturbances in iron metabolism. There is evidence that probiotics favorably influence iron turnover. To compare the influence of supplementation with probiotic Lactobacillus plantarum and Lactobacillus curvatus with and without iron supply on liver iron metabolism in rats on high-fat iron-deficient diet. In the first stage (8 weeks), rats were fed a control diet (group C, n = 8), high-fat diet (group HF, n = 8), or high-fat iron-deficient diet (group HFDEF, n = 48). In the second stage (8 weeks), the rats in the C and HF groups were kept on the same diets, while the rats in the HFDEF group were divided into 6 groups (n = 8 each) and fed as follows: group HFDEF- high-fat iron-deficient diet, group HFDEFFe- high-fat iron-deficient diet with iron supplementation, groups HFDEFLp and HFDEFLc- high-fat iron-deficient diet with L. plantarum or L. curvatus supplementation, respectively, and groups HFDEFFeLp and HFDEFFeLc- high-fat iron-deficient diet supplemented with iron and L. plantarum or L. curvatus , respectively. The rats were subsequently sacrificed, and their serum and liver samples were collected for analysis. Serum ferroportin level was higher in the HFDEFFe, HFDEFLp, HFDEFLc, and HFDEFFeLp groups than in the C and HFDEF groups. The hepatic expression of type 1 transferrin receptor gene ( Tfrc ) was higher in the HFDEFFeLc group than in the C group. No differences in serum iron, hepcidin, hemojuvelin, transferrin, and ferritin levels and in hepatic type 2 transferrin receptor gene expression were observed between the groups. In rats on high-fat iron-deficient diet, the supplementation of L. curvatus and L. plantarum increased serum ferroportin concentration, and L. curvatus enhanced liver expression of the Tfrc gene.

Role of TFRC as a Novel Prognostic Biomarker and in Immunotherapy for Pancreatic Carcinoma.

Objective: To explore the expression of the transferrin receptor (TFRC) gene in pancreatic cancer and to analyze the pathogenesis and immunotherapy of TFRC in patients using bioinformatics methods. Methods: We used public data from the cancer genome atlas (TCGA) and gene expression omnibus databases to explore the expression level of the TFRC gene in pancreatic cancer patients. At the same time, we analyzed the correlation between the TFRC gene expression and patient survival, and further analyzed the correlation between TFRC and survival time of patients with different clinicopathological characteristics. Co-expressed genes and pathway enrichment analyses were used to analyze the mechanism of the TFRC in the occurrence and development of pancreatic cancer. Ultimately, we used the R software to examine the relationship between TFRC and immune phenotypes and immune cell infiltration using the TCGA database. Results: The results of the study showed that TFRC is highly expressed in pancreatic cancer tissue. The upregulated expression of TFRC was negatively correlated with the survival in patients with pancreatic cancer. The bioinformatics analysis showed that TFRC plays a role in the occurrence and development of pancreatic cancer mainly through signaling pathways (including cell adhesion molecule binding, condensed chromosomes, chromosome segregation, and cell cycle checkpoints). Finally, TFRC is associated with immune phenotypes and immune cell infiltration, which may influence immunotherapy. Conclusion: TFRC is significantly increased in pancreatic cancer and is associated with a poor prognosis. Moreover, research on TFRC may generate new ideas for the immunotherapy of pancreatic cancer.

Importance of Potential New Biomarkers in Patient with Serouse Ovarian Cancer

Ovarian cancer remains the gynecological cancer with the highest mortality rate. In our study, we compare a number of proteins from different effector pathways to assess their usefulness in the diagnosis of ovarian cancer. The tissue expression of the tested proteins was assessed by two methods: qRT-PCR and an immunohistochemical analysis. A significantly higher level of mRNA expression was found in the ovarian cancer group for YAP and TEAD4 (p = 0.004 and p = 0.003, respectively). There was no statistical significance in the expression of mRNA for SMAD3, and there was borderline statistical significance for SMAD2 between the groups of ovarian cancer patients and other subgroups of patients with simple cysts and healthy ovarian tissue (p = 0.726 and p = 0.046, respectively). Significantly higher levels of transferrin receptor (CD71), H2A.X, and ADH1A gene expression were found in the ovarian cancer group compared to the control group for YAP, and TEAD4 showed strong nuclear and cytoplasmic staining in ovarian carcinoma and weak staining in non-carcinoma ovarian samples, ADH1A1 showed strong staining in the cytoplasm of carcinoma sections and a weak positive reaction in the non-carcinoma section, H2A.X showed strong positive nuclear staining in carcinoma sections and moderate positive staining in non-carcinoma samples, and CD71 showed moderate positive staining in carcinoma and non-carcinoma samples. YAP, TEAD4, and ADH1A proteins appear to be promising biomarkers in the diagnosis of ovarian cancer.

Phlebotomy-induced anemia alters hippocampal neurochemistry in neonatal mice.

Phlebotomy-induced anemia (PIA) is common in preterm infants. The hippocampus undergoes rapid differentiation during late fetal/early neonatal life and relies on adequate oxygen and iron to support oxidative metabolism necessary for development. Anemia shortchanges these two critical substrates, potentially altering hippocampal development and function. PIA (hematocrit <25%) was induced in neonatal mice pups from postnatal day (P)3 to P14. Neurochemical concentrations in the hippocampus were determined using in vivo (1)H NMR spectroscopy at 9.4T and compared with control animals at P14. Gene expression was assessed using quantitative real-time polymerase chain reaction (qRT-PCR). PIA decreased brain iron concentration, increased hippocampal lactate and creatine concentrations, and decreased phosphoethanolamine (PE) concentration and the phosphocreatine/creatine ratio. Hippocampal transferrin receptor (Tfrc) gene expression was increased, while the expression of calcium/calmodulin-dependent protein kinase type IIα (CamKIIα) was decreased in PIA mice. This clinically relevant model of neonatal anemia alters hippocampal energy and phospholipid metabolism and gene expression during a critical developmental period. Low target hematocrits for preterm neonates in the neonatal intensive care unit (NICU) may have potential adverse neural implications.

The Contact with MDS Endothelial Cells Alters the Pattern of Lineage-Specific Gene Expression During Normal Hematopoietic Differentiation

AbstractAbstract 1718In the embryo, hematopoiesis and angiogenesis occur concurrently, since endothelial and hematopoietic cells derive from a common cell precursor, the hemangioblast. In the post natal life, the bone marrow (BM) vascular niche has an important role in supporting hematopoietic stem cells (HSC) proliferation and differentiation. Moreover, during the processes of homing and mobilization, BM vascular niche regulates HSC migration. In BM failure, these functions are promoted by vascular niches located outside of the BM, such as in the spleen, thus confirming that endothelial cells are relevant to the normal hematopoietic differentiation. The dominant feature of myelodyspslatic syndromes (MDS) is the impaired capacity of HSC to give rise to a terminally differentiated normal cell population in one or more lineages. We hypothesized that in MDS patients the vascular niches within and eventually outside of the BM might be altered and that they do not support adequately the physiologic hematopoietic differentiation, thus contributing to the pathogenesis of the disease. To investigate this hypothesis, we isolated and expanded endothelial colony forming cells (ECFCs) (Ingram et al. Blood. 2004;104: 2752) from the peripheral blood of patients with MDS (RCMD) and we used ECFCs as a surrogate of the vascular niche to differentiate hematopoietic cells. Normal CD34+ were seeded over a layer of endothelial cells and cultured for 8 days in different cytokine- containing media able to induce selective granulocytic-macrophage, erythroid or megakaryocyte differentiation. At baseline and after 5 and 8 days of co-culture, hematopoietic cells were recovered by gentle repeated washings, and then they were analyzed with RT-PCR for the expression of lineage specific genes. For each lineage, two genes, mainly involved either in the early or in the late phase of the differentiation, respectively, were analyzed. Basically, we found that co-culturing CD34+ cells in contact with a normal endothelial layer the gene expression observed in the corresponding no-layer cultures was amplified. This effect was strictly dependent on the physical contact between hematopoietic and endothelial cells, since it was abolished when cells were grown separated from a porous membrane. We observed that in co-cultures with normal hematopoietic cells and MDS ECFCs, the modulatory effect exerted by the endothelial layer was partially or completely lost. For example, CD34+ cells undergoing granulocyte differentiation showed a progressive decline of PU.1 gene expression, followed by the rise of the expression of MPO gene; these effects were much more evident if cells were cultured over normal ECFCs. In contrast, this pattern of gene expression was deeply perturbed when normal CD34+ cells were cultured with MDS ECFCs: actually, at day 7, the PU.1 RNA level was six folds higher and the MPO RNA level was three folds lower than that observed in co-cultures with normal ECFCs. Similar figures were observed for RUNX1 and GP1b gene expression in cells undergoing megakaryocyte differentiation. Moreover, CD34+ cells undergoing erythroid differentiation on normal ECFCs exhibited the progressive up-regulation of FOG-1 and of transferrin receptor (TFR) gene expression: interestingly, the co-culture with MDS ECFCs resulted in a lower expression of FOG -1 gene but also in a surprisingly higher expression of the TFR gene. Overall, these data suggest that in MDS does exist an impairment of talking between endothelial and hematopoietic cells. To understand which molecular pathways are involved could represent the basis for a novel therapy approach. Disclosures:No relevant conflicts of interest to declare.

Transferrin receptor gene and protein expression and localization in human IUGR and normal term placentas

Iron (Fe) deficiency in pregnancy is associated to low birth weight and premature delivery while in adults it can result in increased blood pressure and cardiovascular disease. Cellular Fe uptake is mediated by the Transferrin Receptor 1 (TFRC), located in the trophoblast membranes. Here, we measured TFRC mRNA expression (Real Time PCR) and TFRC protein expression and localization (Western Blotting and immunohistochemistry) in IUGR compared to control placentas. A total of 50 IUGR and 56 control placentas were studied at the time of elective cesarean section. IUGR was defined by ultrasound in utero, and confirmed by birth weight <10th percentile. Three different severity groups were identified depending on the umbilical artery pulsatility index and fetal heart rate. TFRC mRNA expression was significantly lower in IUGR placentas compared to controls (p < 0.05), and this was confirmed for TFRC protein levels. In both experiments the most severe IUGR group presented lower expression compared to the other groups, and this was also related to umbilical venous oxygen levels. TFRC protein localization in the villous trophoblast did not differ in the groups, and was predominantly present in the syncytiotrophoblast. In conclusion, these are the first observations about TFRC expression in human IUGR placentas, demonstrating its significant decrease in IUGR vs controls. Thus, Fe transport could be limited in IUGR placentas. Further studies are needed to study components of the placental Fe transport system and to clarify the regulation mechanisms involved in TFRC expression, possibly altered in IUGR placentas.

In Vitro Evaluation of Magnetic Resonance Imaging Contrast Agents for Labeling Human Liver Cells: Implications for Clinical Translation

Magnetic resonance imaging (MRI) is a promising approach for non-invasive monitoring after liver cell transplantation. We compared in vitro labeling of human liver cells with nano-sized (SPIO) and micron-sized iron oxide particles (MPIO). The cellular iron load was quantified and phantom studies were performed using 3.0-T MRI. Transferrin receptor and ferritin gene expression, reactive oxygen species (ROS) formation, transaminase leakage, and urea synthesis were investigated over 6 days. Incubation with MPIO produced stronger signal extinctions in MRI at similar iron loads within shorter labeling time. MPIO had no negative effects on the cellular iron homeostasis or cell performance, whereas SPIO caused temporary ROS formation and non-physiologic activation of the iron metabolic pathway. Our findings suggest that MPIO are suited for clinical translation of strategies for cellular imaging with MRI. Attention should be paid to iron release and oxidative stress caused by biodegradable contrast agents.

Iron overload alters iron-regulatory genes and proteins, down-regulates osteoblastic phenotype, and is associated with apoptosis in fetal rat calvaria cultures

Iron overload has been implicated in decreased bone mineral density. However, the effect of iron overload on osteoblast lineage cells remains poorly understood. The purpose of this study was to examine osteoblast differentiation, function, and apoptosis in iron-loaded cells from fetal rat calvaria. Cells were incubated with media supplemented with 0–10 μM ferrous sulfate (FeSO 4) during differentiation (days 6–20). Intracellular iron status was assessed by measuring iron content in cell layers and changes in transferrin receptor (TrfR) and ferritin gene and protein expression. Osteoblast differentiation and function were evaluated by measuring osteoblast phenotypic gene markers and capacity of cultures to form mineralized bone nodules. Apoptotic hallmarks were evaluated by microscopy. A 2.3-fold increase in media iron concentration resulted in saturable accumulation of iron in the cell layer 20-fold higher than control ( p < 0.05) by mid-differentiation (day 15, D15). Iron accumulation resulted in rapid and sustained down-regulation of TrfR gene and protein levels (within 24 h) and up-regulation of light and heavy chain ferritin protein levels at late differentiation (day 20, D20). Concurrently, osteoblast phenotype gene markers were suppressed by D15 and a decreased number of mineralized nodules at D20 were observed. Apoptotic events were observed within 24 h of iron loading. These results provide evidence that iron overload alters iron metabolism and suppresses differentiation and function of cells in the osteoblast lineage associated with increased apoptosis.

Changes in hepatic gene expression in dogs with experimentally induced nutritional iron deficiency

We investigated hepatic gene expression in dogs with experimentally induced nutritional iron deficiency (ID). Our hypothesis was that ID would result in decreased hepcidin gene expression, and possibly in altered expression of other genes associated with iron metabolism. Liver biopsies were collected from each of 3 dogs before induction of ID, at the point of maximal ID, and after resolution of ID. Using Affymetrix microarray technology and analytical tools specifically designed for microarray data, we identified genes that had at least a 2-fold change in expression in response to ID. Four genes were selected for further analysis by reverse transcriptase PCR (RT-PCR). Dogs with ID had markedly decreased expression of the hepcidin gene (mean decrease of 40-fold for one probe and >100-fold for another probe) and increased expression of the transferrin receptor gene (mean increase of >7-fold). There was also mildly decreased expression of the "similar to calreticulin" gene and a gene of unknown function. Results of RT-PCR analysis were consistent with microarray findings. Changes in hepcidin and transferrin receptor gene expression were consistent with the known biology of iron metabolism. The decrease in expression of a gene identified as "similar to calreticulin," while not statistically significant, was consistent with the findings of other investigators that suggest iron plays a role in calreticulin expression.

Hematopoietic-specific Stat5-null mice display microcytic hypochromic anemia associated with reduced transferrin receptor gene expression

AbstractIron is essential for all cells but is toxic in excess, so iron absorption and distribution are tightly regulated. Serum iron is bound to transferrin and enters erythroid cells primarily via receptor-mediated endocytosis of the transferrin receptor (Tfr1). Tfr1 is essential for developing erythrocytes and reduced Tfr1 expression is associated with anemia. The transcription factors STAT5A/B are activated by many cytokines, including erythropoietin. Stat5a/b−/− mice are severely anemic and die perinatally, but no link has been made to iron homeostasis. To study the function of STAT5A/B in vivo, we deleted the floxed Stat5a/b locus in hematopoietic cells with a Tie2-Cre transgene. These mice exhibited microcytic, hypochromic anemia, as did lethally irradiated mice that received a transplant of Stat5a/b−/− fetal liver cells. Flow cytometry and RNA analyses of erythroid cells from mutant mice revealed a 50% reduction in Tfr1 mRNA and protein. We detected STAT5A/B binding sites in the first intron of the Tfr1 gene and found that expression of constitutively active STAT5A in an erythroid cell line increased Tfr1 levels. Chromatin immunoprecipitation experiments confirmed the binding of STAT5A/B to these sites. We conclude that STAT5A/B is an important regulator of iron update in erythroid progenitor cells via its control of Tfr1 transcription.

Clone and expression of human transferrin receptor gene: A marker gene for magnetic resonance imaging

Objective To clone human transferrin receptor(hTfR)gene and construct expression vector producing recombination protein.Methods Human transferrin receptor gene cDNA was amplified by RT-PCR from human embryonic liver and lung tissue.Recombinant pcDNA3-hTfR and pEGFP-Cl-hTfR plasmids were constructed and confirmed by DNA sequencing.These plasmids were stably transfected into the HEK293 cells.The protein expression in vitro was confirmed by Western Blot.The efficiency of expression and the location of hTfR were also investigated by fluorescence microscopy and confocal fluorescence microscopy.Results The full length cDNA of hTfR gene(2332 bp)was cloned and sequenced.The hTfR(190 000)was overexpressed in transfeeted HEK293 cells by Western blot analysis. Fluorescence micrographs displayed that the hTfR was expressed at high level and located predominantly in the cell surface.Conclusions Human transferrin receptor(hTfR)gene has been successfully cloned and obtained high-level expression in HEK293 cells,and the recombination protein of hTfR distributed predominantly in the cell membrane.

Gene targeting using a promoterless gene trap vector (“targeted trapping”) is an efficient method to mutate a large fraction of genes

A powerful tool for postgenomic analysis of mammalian gene function is gene targeting in mouse ES cells. We report that homologous recombination using a promoterless gene trap vector ("targeting trapping") yields targeting frequencies averaging above 50%, a significant increase compared with current approaches. These high frequencies appear to be due to the stringency of selection with promoterless constructs, because most random insertions are silent and eliminated by drug selection. The promoterless design requires that the targeted gene be expressed in ES cells at levels exceeding a certain threshold (which we estimate to be approximately 1% of the transferrin receptor gene expression level, for the secretory trap vector used here). Analysis of 127 genes that had been trapped by random (nontargeted) gene trapping with the same vector shows that virtually all are expressed in ES cells above this threshold, suggesting that targeted and random trapping share similar requirements for expression levels. In a random sampling of 130 genes encoding secretory proteins, about half were expressed above threshold, suggesting that about half of all secretory genes are accessible by either targeted or random gene trapping. The simplicity and high efficiency of the method facilitate systematic targeting of a large fraction of the genome by individual investigators and large-scale consortia alike.

House keeping genes and gene expression analysis in transplant recipients: a note of caution

Background: House keeping genes are often used as a means of standardising results obtained in gene expression investigations. This study was performed to investigate whether β-actin, β2-microglobulin (two genes frequently quoted as house keeping genes) and/or transferrin receptor would be suitable house keeping genes for use in gene expression analysis of renal transplant recipients. Methods: Sequential expression of all three genes was measured in the peripheral blood mononuclear cells of 13 living donors and 45 renal transplant recipients, pre-operatively and then daily for up to 2 weeks. Fifteen of the recipients experienced an episode of biopsy proven acute rejection. Gene expression measurement was performed using quantitative real time ‘TaqMan’ PCR technology. Results: Gene expression of all three genes was unchanged in the living donor cohort. However, in the transplant recipients there were significant increases in expression following transplantation in the non-rejectors, and preceding the diagnosis of acute rejection. In the latter group, levels returned to pre-transplant values after the commencement of anti-rejection therapy. Conclusions: β-actin, β2-microglobulin and transferrin receptor gene expression, although not influenced by surgery, is influenced by transplantation, acute rejection and anti-rejection therapy making these genes unsuitable as house keeping genes following renal transplantation. These findings may cast doubt on the results of some studies that used these genes for the purposes of standardisation when looking at cDNA measurement. We suggest that any group wishing to use a house keeping gene ensure that its expression is independent of study parameters prior to the start of the study.

Diallyl Disulfide Increases Rat H-Ferritin, L-Ferritin and Transferrin Receptor Genes In Vitro in Hepatic Cells and In Vivo in Liver

Of the oil-soluble organosulfur compounds derived from garlic, diallyl disulfide (DADS) is one of the most abundant. We examined the effect of DADS on gene expression in rat liver. By suppressive subtractive hybridization, we identified the heavy (H)-ferritin gene as a DADS-stimulated gene in the rat liver epithelial (REL) cells. DADS stimulation of H- and L (light)-ferritin mRNA was analyzed in REL cells and in rat liver. Incubation of the REL cells in 10 micro mol/L DADS for 4 h increased H-ferritin 1.9 +/- 0.2-fold, n = 3) and light(L)-ferritin mRNA 1.5 +/- 0.2-fold, n = 3). Stimulation did not occur in the presence of an inhibitor of transcription, actinomycin D. Stimulation of ferritin at the RNA and protein levels was also found in rats administered a DADS-enriched oil solution intragastrically. There was a 3 +/- 1.1-fold increase in H- and 3 +/- 0.14-fold increase for L-ferritin mRNA 24 h after the end of the infusion in the presence of DADS, (n = 3). The expression of the transferrin receptor, an iron transporter, was also enhanced by DADS in rat liver. In conclusion, our data suggest that DADS could modify iron homeostasis through the modulation of ferritin and transferrin receptor gene expression.

Role of Ets-1 in transcriptional regulation of transferrin receptor and erythroid differentiation.

High expression of transferrin receptor (TfR) on the membrane of erythroid cells accounts for the high level of iron required to sustain heme synthesis. Several studies indicate that during erythroid differentiation TfR expression is highly dependent on transcriptional regulation. In this study we characterized the minimal region able to confer transcriptional regulation during erythroid differentiation in Friend leukemia cells (FLC). This region of 120 bp, upstream the transcription start site, contains an overlapping consensus recognition sequence for AP1/CREB/ATF transcription factors and for proteins of the Ets family and a GC rich region. Here, we report that both the Ets and the Ap1/CRE like sites are essential for promoter activity during erythroid differentiation. We showed that Ets-1 binds to the EBS-TfR and its binding activity decreases in FLC induced to differentiate and during normal erythroid differentiation. Consistent with this, FLC constitutively expressing Ets-1 show a decrease in TfR gene expression, globin mRNA and hemoglobin synthesis. We conclude that Ets-1 binding activity is modulated during erythroid maturation and that a deregulated expression of this transcription factor interferes with terminal erythroid differentiation.

Transferrin receptor gene expression and transferrin-bound iron uptake are increased during postischemic rat liver reperfusion

Iron-catalyzed production of reactive oxygen species is a cause of liver injury after ischemia/reperfusion (I/R). The aim of the present study was to address the regulation of transferrin receptor (TfR), which mediates cellular iron uptake, during I/R. The molecular mechanisms controlling TfR gene expression in vivo during I/R of rat liver were investigated by molecular biology procedures. We also analyzed transferrin-bound iron uptake into surviving liver slices. Increased amounts of TfR protein and messenger RNA (mRNA) were found 2 to 6 hours after reestablishment of blood supply. RNA bandshift analysis showed that iron regulatory protein (IRP) activity was decreased in the first hours of reperfusion, thus indicating that IRP-mediated mRNA stabilization was not involved in early TfR upregulation. On the contrary, increased transcription of the TfR gene in isolated nuclei was observed during reperfusion; during the ischemic phase this was preceded by enhanced binding of hypoxia inducible factor (HIF-1) to a DNA sequence derived from the TfR promoter. TfR2 mRNA levels were also enhanced after reperfusion. The increased expression of TfR at the cell surface resulted in increased uptake of transferrin-bound-iron into surviving liver slices; however, iron was not incorporated into ferritin. In conclusion, HIF-1 mediated activation of TfR gene transcription and IRP-mediated increase of TfR mRNA stability ensure a steady induction of TfR, and hence higher iron uptake in reperfused rat liver. TfR-mediated entry of the metal into liver cells may represent a source of catalitically active iron, which may play a role in reperfusion damage. (H EPATOLOGY 2002;36:103-111.)

Validation of endogenous controls for gene expression studies in human adipocytes and preadipocytes.

Quantitative gene expression protocols require adequate controls to monitor intersample variation. Quantitative approaches to describe relative changes in gene expression use endogenous controls--"housekeeping" genes. Given the low amounts of mRNA in fat cells, RT-PCR is the method of choice, and housekeeping genes are widely used as endogenous controls. However, literature reports suggest changes in gene expression of typical housekeeping genes (e. g. GAPDH, beta-actin, 18S rRNA) upon hormonal stimulation or during adipogenic differentiation. Thus, we tested the influence of 6 hormones and adipogenic differentiation on gene expression levels of 11 commonly used housekeeping genes in primary cultured mature human adipocytes and preadipocytes. Using the TaqMan RT-PCR technique and "Human Endogenous Control Assays" (PE Biosystems), we found several housekeeping genes with at least twice the difference in expression levels between stimulated and unstimulated cells (such as acidic ribosomal protein, beta-actin, beta(2)-microglobulin and beta-glucuronidase). Only GAPDH and transferrin receptor gene expression levels did not change under any of the stimuli tested, thus appeared best suited for gene expression studies in human adipose cells across a wide range of experimental settings.