Iron-responsive Element-binding Protein Research Articles

Using Biotinylated Iron-Responsive Element to Analyze the Activity of Iron Regulatory Proteins.

Iron regulatory proteins (IRP1 and IRP2) are the master regulators of mammalian iron homeostasis. They bind to the iron-responsive elements (IREs) of the transcripts of iron-related genes to regulate their expression, thereby maintaining cellular iron availability. The primary method to measure the IRE-binding activity of IRPs is the electrophoresis mobility shift assay (EMSA). This method is particularly useful for evaluating IRP1 activity, since IRP1 is a bifunctional enzyme and its protein levels remain similar during conversion between the IRE-binding protein and cytosolic aconitase forms. Here, we exploited a method of using a biotinylated-IRE probe to separate IRE-binding IRPs followed by immunoblotting to analyze the IRE-binding activity. This method allows for the successful measurement of IRP activity in cultured cells and mouse tissues under various iron conditions. By separating IRE-binding IRPs from the rest of the lysates, this method increases the specificity of IRP antibodies and verifies whether a band represents an IRP, thereby revealing some previously unrecognized information about IRPs. With this method, we showed that the S711-phosphorylated IRP1 was found only in the IRE-binding form in PMA-treated Hep3B cells. Second, we found a truncated IRE-binding IRP2 isoform that is generated by proteolytic cleavage on sites in the 73aa insert region of the IRP2 protein. Third, we found that higher levels of SDS, compared to 1-2% SDS in regular loading buffer, could dramatically increase the band intensity of IRPs in immunoblots, especially in HL-60 cells. Fourth, we found that the addition of SDS or LDS to cell lysates activated protein degradation at 37 °C or room temperature, especially in HL-60 cell lysates. As this method is more practical, sensitive, and cost-effective, we believe that its application will enhance future research on iron regulation and metabolism.

Tetramethylpyrazine attenuates renal tubular epithelial cell ferroptosis in contrast-induced nephropathy by inhibiting transferrin receptor and intracellular reactive oxygen species.

Contrast-induced nephropathy (CIN) is a leading cause of hospital-acquired acute kidney injury (AKI). Recently, ferroptosis was reported to be crucial for AKI pathogenesis. Our previous studies indicated antioxidant tetramethylpyrazine (TMP) prevent CIN in vivo. However, whether ferroptosis is involved in TMP nephroprotective mechanism against CIN is unclear. In the present study, we investigated the role of renal tubular epithelial cell ferroptosis in TMP reno-protective effect against CIN and the molecular mechanisms by which TMP regulates ferroptosis. Classical contrast-medium, Iohexol, was used to construct CIN models in rats and HK-2 cells. Results showed that tubular cell injury was accompanied by ferroptosis both in vivo and in vitro, including the typical features of ferroptosis, Fe2+ accumulation, lipid peroxidation and decreased glutathione peroxidase 4 (GPX4). Ferroptosis inhibition by classic inhibitors Fer-1 and DFO promoted cell viability and reduced intracellular ROS production. Additionally, TMP significantly inhibited renal dysfunction, reduced AKI biomarkers, prevented ROS production, inhibited renal Fe2+ accumulation and increased GPX4 expression. Expressions of various proteins associated with iron ion metabolism, including transferrin receptor (TFRC), divalent metal transporter 1, iron-responsive element binding protein 2, ferritin heavy chain 1, ferroportin 1, and heat shock factor binding protein 1, were examined using mechanistic analyses. Among these, TFRC changes were the most significant after TMP pretreatment. Results of siRNA knockdown and plasmid overexpression of TFRC indicated that TFRC is essential for TMP to alleviate ferroptosis and reduce LDH release, Fe2+ accumulation and intracellular ROS. Our findings provide crucial insights about the potential of TMP in treating AKI associated with ferroptosis.

Hyperbaric oxygen improves cerebral ischemia-reperfusion injury in rats via inhibition of ferroptosis

Background: Our previous study found that hyperbaric oxygen (HBO) attenuated cognitive impairment in mice induced by cerebral ischemia-reperfusion injury (CIRI). However, its mechanism of action is not fully understood. In this study, we aimed to establish a rat model of cerebral ischemia-reperfusion, explore the possible role of ferroptosis in the pathogenesis of CIRI, and observe the effect of HBO on ferroptosis-mediated CIRI. Methods: Sprague Dawley (SD) rats were randomly divided into control, model, Ferrostatin-1 (Fer-1), HBO and Fer-1+ HBO groups. Morris water maze, myelin basic protein (MBP) and β-tubulin immunoreactivity were assessed to evaluate the neuroprotective effects of HBO on cerebral ischemia reperfusion injury. Ferroptosis were examined to investigate the mechanism underlying the effects of HBO. Results: Our result showed that Fer-1 and HBO improved learning and memory ability in the navigation trail and probe trail of the Morris water maze and increased MBP and β-tubulin immunoreactivity of the cortex in the model rats. The levels of ferritin, malondialdehyde (MDA) and glutathione (GSH) in the serum were also reversed by Fer-1 and HBO treatment. Mitochondrial cristae dissolution and vacuolization were observed in the model group by transmission electron microscopy and these conditions were improved in the Fer-1 and HBO groups. Furthermore, Fer-1 and HBO treatment reversed Prostaglandin-Endoperoxide Synthase 2 (PTGS2), Iron Responsive Element Binding Protein 2 (IREB2), acyl-CoA synthetase long chain family member 4 (ACSL4) and Solute Carrier Family 7 Member 11 (SLC7A11) mRNA levels and Transferrin Receptor 1 (TFR1), ferritin light chain (FTL), ferritin heavy chain 1 (FTH1), glutathione peroxidase 4 (GPX4), Nuclear factor E2-related factor 2 (Nrf2), lysophosphatidylcholine acyltransferase 3 (LPCAT3), c-Jun N-terminal kinase (JNK), phosphorylated c-Jun N-terminal kinase (P-JNK) phosphorylated Extracellular signal-regulated protein kinase (P-ERK) and mitogen-activated protein kinase/extracellular signal-regulated kinase (MEK) protein levels. The above changes were more pronounced in Fer-1+ HBOGroup. Discussion: The results of the present study indicated that HBO improves cerebral ischemia-reperfusion injury in rats, which may be related to inhibition of ferroptosis. This also means that ferroptosis may become a new target of HBO against CIRI.

Effects of Curcumin on Oxidative Stress and Ferroptosis in Acute Ammonia Stress-Induced Liver Injury in Gibel Carp (Carassius gibelio).

This study investigated the potential role of curcumin (CUR) in preventing oxidative stress and ferroptosis induced by ammonia exposure in gibel carp. Experimental fish (initial weight: 11.22 ± 0.10 g, n = 150) were fed diets supplemented with or without 0.5% CUR for 56 days, followed by a 24 h ammonia (32.5 mg/L) exposure. Liver damages (aspartate aminotransferase (AST), alanine aminotransferase (ALT), adenosine deaminase (ADA), and alkaline phosphatase (ALP)) and oxidative stress enzyme activities (reactive oxygen species (ROS), malondialdehyde (MDA); and the content of antioxidant capacity (T-AOC), superoxide dismutase (SOD), and glutathione peroxidase (GPx)) were induced by ammonia stress. The antioxidant capacity was decreased, as indicated by inhibited gene expression of nuclear factor erythroid 2-related factor 2 (nrf2), heme oxygenase-1 (ho-1), catalase (cat), and sod. Ferroptosis was induced by ammonia stress, as suggested by upregulated mRNA levels of nuclear receptor coactivator 4 (ncoa4), transferrin receptor 1 (tfr1), and iron-responsive element-binding protein 2 (ireb2), and downregulated expression of glutathione peroxidase 4 (gpx4), ferroportin (fpn), and ferritin heavy chain 1 (fth1). In addition, both mRNA and protein levels of ferroptosis markers acyl-CoA synthetase long-chain family member 4 (ACSL4) and prostaglandin-endoperoxide synthase 2 (PTGS2) were upregulated, while cystine/glutamate antiporter (SLC7A11) was downregulated. However, liver injury and ferroptosis in fish induced by ammonia could be attenuated by CUR. Collectively, these findings demonstrate that CUR ameliorates oxidative stress and attenuates ammonia stress-induced ferroptosis. This study provides a new perspective on potential preventive strategies against ammonia stress in gibel carp by dietary CUR.

Optimization of Biotinylated RNA or DNA Pull-Down Assays for Detection of Binding Proteins: Examples of IRP1, IRP2, HuR, AUF1, and Nrf2.

Investigation of RNA- and DNA-binding proteins to a defined regulatory sequence, such as an AU-rich RNA and a DNA enhancer element, is important for understanding gene regulation through their interactions. For in vitro binding studies, an electrophoretic mobility shift assay (EMSA) was widely used in the past. In line with the trend toward using non-radioactive materials in various bioassays, end-labeled biotinylated RNA and DNA oligonucleotides can be more practical probes to study protein-RNA and protein-DNA interactions; thereby, the binding complexes can be pulled down with streptavidin-conjugated resins and identified by Western blotting. However, setting up RNA and DNA pull-down assays with biotinylated probes in optimum protein binding conditions remains challenging. Here, we demonstrate the step-by step optimization of pull-down for IRP (iron-responsive-element-binding protein) with a 5'-biotinylated stem-loop IRE (iron-responsive element) RNA, HuR, and AUF1 with an AU-rich RNA element and Nrf2 binding to an antioxidant-responsive element (ARE) enhancer in the human ferritin H gene. This study was designed to address key technical questions in RNA and DNA pull-down assays: (1) how much RNA and DNA probes we should use; (2) what binding buffer and cell lysis buffer we can use; (3) how to verify the specific interaction; (4) what streptavidin resin (agarose or magnetic beads) works; and (5) what Western blotting results we can expect from varying to optimum conditions. We anticipate that our optimized pull-down conditions can be applicable to other RNA- and DNA-binding proteins along with emerging non-coding small RNA-binding proteins for their in vitro characterization.

KLF14 regulates the growth of hepatocellular carcinoma cells via its modulation of iron homeostasis through the repression of iron-responsive element-binding protein 2

BackgroundHepatocellular carcinoma (HCC) is a multifactor-driven malignant tumor with rapid progression, which causes the difficulty to substantially improve the prognosis of HCC. Limited understanding of the mechanisms in HCC impedes the development of efficacious therapies. Despite Krüpple-Like factors (KLFs) were reported to be participated in HCC pathogenesis, the function of KLF14 in HCC remains largely unexplored.MethodsWe generated KLF14 overexpressed and silenced liver cancer cells, and nude mouse xenograft models for the in vitro and in vivo study. Luciferase reporter assay, ChIP-qPCR, Co-IP, immunofluorescence were performed for mechanism research. The expression of KLF14 in HCC samples was analyzed by quantitative RT-PCR, Western blotting, and immunohistochemistry (IHC) analysis.ResultsKLF14 was significantly downregulated in human HCC tissues, which was highly correlated with poor prognosis. Inhibition of KLF14 promoted liver cancer cells proliferation and overexpression of KLF14 suppressed cells growth. KLF14 exerts its anti-tumor function by inhibiting Iron-responsive element-binding protein 2 (IRP2), which then causes transferrin receptor-1(TfR1) downregulation and ferritin upregulation on the basis of IRP-IREs system. This then leading to cellular iron deficiency and HCC cells growth suppression in vitro and in vivo. Interestingly, KLF14 suppressed the transcription of IRP2 via recruiting SIRT1 to reduce the histone acetylation of the IRP2 promoter, resulting in iron depletion and cell growth suppression. More important, we found fluphenazine is an activator of KLF14, inhibiting HCC cells growth through inducing iron deficiency.ConclusionKLF14 acts as a tumor suppressor which inhibits the proliferation of HCC cells by modulating cellular iron metabolism via the repression of IRP2. We identified Fluphenazine, as an activator of KLF14, could be a potential compound for HCC therapy. Our findings therefore provide an innovative insight into the pathogenesis of HCC and a promising therapeutic target.

Roots of Astragalus propinquus Schischkin Regulate Transmembrane Iron Transport and Ferroptosis to Improve Cerebral Ischemia-Reperfusion Injury.

Background The dried roots of the Astragalus propinquus Schischkin (RAP) plant, as a traditional Chinese medicine, has been widely used to treat stroke, cerebral ischemia, qi deficiency, and hypertension. Buyang Huanwu decoction is traditionally used to treat stroke in China for more than 200 years and has a significant effect on cerebral ischemia, and RAP is monarch medicine of Buyang Huanwu decoction. Therefore, this study was designed to observe the regulatory effect of RAP on transmembrane iron transporters and ferroptosis-related factors in cerebral ischemia-reperfusion injury (CIRI) in rats. Methods Middle cerebral artery occlusion (MCAO) was used to block blood flow in the blood supply area of the middle cerebral artery in seventy male SD rats to induce focal CIRI to establish a rat model of CIRI. RAP was administered to explore the regulatory effect of RAP on iron transmembrane transport under the condition of CIRI. The infarct size was measured using 2,3,5-triphenyl-tetrazolium chloride (TTC) staining, the pathological structure of brain tissue was observed by HE staining, and neuronal injury was evaluated by Nissl staining after treatment. Then, changes in the iron transporters ferritin (Fn), ferritin heavy chain (FHC), ferritin light chain (FLC), transferrin (Tf), transferrin receptor (TfR), divalent metal transporter 1 (DMT1), L-type calcium channel (LTCC), transient receptor potential canonical 6 (TRPC6), and ferroportin 1 (FPN1) were observed by immunohistochemistry staining (IHC) and Western blotting. The expression of key factors of ferroptosis, including the membrane sodium-dependent cystine/glutamate antiporter System Xc− (System Xc−) light chain subunit (XCT) and heavy chain subunit (SLC3A2), glutathione peroxidase 4 (GPX4), nuclear factor erythroid 2-related factor (NRF2), heme oxygenase-1 (HO-1), and iron-responsive element-binding protein 2 (IREB2) in the brain tissues of rats was assessed by Western blotting. RAP decreased the infarct size and neuronal injury after CIRI in rats. Similarly, RAP treatment regulated the expression of iron transporters. As such, RAP was able to reduce the expression of Fn, FHC, FLC, Tf, TfR, DMT1, and TRPC6 and increase the expression of FPN1 through a Tf/TfR-independent pathway after CIRI in rats. Conclusion RAP stimulation inhibited ferroptosis by regulating the expression of the key ferroptosis factors XCT, SLC3A2, GPX4, NRF2, HO-1, and IREB2. In conclusion, RAP regulates transmembrane iron transport and ferroptosis to improve CIRI.

MiR-29a-3p in Exosomes from Heme Oxygenase-1 Modified Bone Marrow Mesenchymal Stem Cells Alleviates Steatotic Liver Ischemia-Reperfusion Injury in Rats by Suppressing Ferroptosis via Iron Responsive Element Binding Protein 2

Hepatic ischemia-reperfusion injury (IRI) is an inevitable result of liver surgery. Steatotic livers are extremely sensitive to IRI and have worse tolerance. Ferroptosis is considered to be one of the main factors of organ IRI. This study is aimed at exploring the role of ferroptosis in the effect of heme oxygenase-1-modified bone marrow mesenchymal stem cells (HO-1/BMMSCs) on steatotic liver IRI and its mechanism. An IRI model of a steatotic liver and a hypoxia reoxygenation (HR) model of steatotic hepatocytes (SHPs) were established. Rat BMMSCs were extracted and transfected with the Ho1 gene to establish HO-1/BMMSCs, and their exosomes were extracted by ultracentrifugation. Ireb2 was knocked down to verify its role in ferroptosis and cell injury in SHP-HR. Public database screening combined with quantitative real-time reverse transcription PCR identified microRNAs (miRNAs) targeting Ireb2 in HO-1/BMMSCs exosomes. miR-29a-3p mimic and inhibitor were used for functional verification experiments. Liver function, histopathology, terminal deoxynulceotidyl transferase nick-end-labeling staining, cell viability, mitochondrial membrane potential, and cell death were measured to evaluate liver tissue and hepatocyte injury. Ferroptosis was assessed by detecting the levels of IREB2, Fe2+, malondialdehyde, glutathione, lipid reactive oxygen species, glutathione peroxidase 4, prostaglandin-endoperoxide synthase 2 mRNA, and mitochondrial morphology. The results revealed that HO-1/BMMSCs improved liver tissue and hepatocyte injury and suppressed ferroptosis in vivo and in vitro. The expression of IREB2 was increased in steatotic liver IRI and SHP-HR. Knocking down Ireb2 reduced the level of Fe2+ and inhibited ferroptosis. HO-1/BMMSC exosomes reduced the expression of IREB2 and inhibited ferroptosis and cell damage. Furthermore, we confirmed high levels of miR-29a-3p in HO-1/BMMSCs exosomes. Overexpression of miR-29a-3p downregulated the expression of Ireb2 and inhibited ferroptosis. Downregulation of miR-29a-3p blocked the protective effect of HO-1/BMMSC exosomes on SHP-HR cell injury. In conclusion, ferroptosis plays an important role in HO-1/BMMSC-mediated alleviation of steatotic liver IRI. HO-1/BMMSCs could suppress ferroptosis by targeting Ireb2 via the exosomal transfer of miR-29a-3p.

MiR-19a suppresses ferroptosis of colorectal cancer cells by targeting IREB2

ABSTRACT Colorectal cancer (CRC) is the most common malignant tumor occurred in digestive system. However, the prognosis of CRC patients is poor. Therefore, it is urgent to illuminate the mechanism suppressing CRC and explore novel targets or therapies for CRC treatment. MicroRNAs (miRNAs) are a class of non-coding RNAs with a length of 20–23 nucleotides encoded by endogenous genes, which are associated with the development of a variety of cancers, including CRC. Studies have shown that miR-19a is identified as oncogenic miRNA and promotes the proliferation, migration and invasion of CRC cells. However, the relationship between miR-19a and ferroptosis in CRC remains unknown. Here, we reported that iron-responsive element-binding protein 2 (IREB2), as an inducer of ferroptosis, was negatively regulated by miR-19a. IREB2 is a direct target of miR-19a. In addition, ferroptosis was suppressed by miR-19a through inhibiting IREB2. Thus, we proposed a novel mechanism of ferroptosis mediated by miR-19a in CRC cells, which could give rise to a new strategy for the therapy of CRC.

Ferrum phosphoricum D12 Treatment Affects J774A.1 Cell Proliferation, Transcription Levels of Iron Metabolism, Antioxidant Defense, and Inflammation-related Genes.

Ferrum phosphoricum (FP) is prescribed as a homeopathic remedy to treat the early stages of fever and inflammation in cases of colds or flu, muscle fatigue and anemia. We aimed to analyze the molecular mechanisms of action of FP D12 on cell proliferation and mRNA expression of iron metabolism, antioxidant defense and inflammation-related genes in mouse J774A.1 macrophages. Cell proliferation was examined using the MTT test. RT-qPCR analyses were performed to estimate gene expression changes. Relative gene expression levels were calculated using the 2-ΔΔCt method. The effect of treatment using FP D12 tablets was compared with that using placebo tablets (PT). FP D12 in low concentrations (0.0125 mg/mL to 0.025 mg/mL) significantly stimulated proliferation of J774A.1 cells by up to 11% (p < 0.01) versus control untreated cells and by up to 40% (p < 0.01) versus PT-treated cells in the respective concentration. FP D12 versus PT induced a significant increase in mRNA expression of ferritin light chain (Ftl1) (by 8-fold, p < 0.01), β-2-microglobulin (B2m) (by 2.5-fold, p < 0.05) and iron-responsive element binding protein 2 (Ireb2) (by 4-fold, p < 0.05), and induced a slight decrease in myosin IE (Myo1e) mRNA expression levels (by 0.4-fold, p < 0.01) in macrophages. A highly significant (r2 = 0.99, p < 0.05) correlation was observed between Ireb2 and B2m transcription levels. Significant stimulation of antioxidant enzyme Gpx-1 (by 1.27-fold, p < 0.01) in cells by 0.025 mg/mL FP D12, but a slight decrease (by 0.12-fold, p < 0.05) in 0.0125 mg/mL-treated cells, was observed. A significant increase in the gene expression of IL-1β (by 3.5-fold, р < 0.05) in macrophages was also detected. Ferrum phosphoricum in D12 dilution potentially exhibits iron retention, antioxidant and immunomodulation activities, possibly by modulating transcription levels of related genes in non-stimulated mouse macrophages.

Curcumin induces ferroptosis in non-small-cell lung cancer via activating autophagy.

BackgroundEmerging studies showed curcumin can inhibit glioblastoma and breast cancer cells via regulating ferroptosis. However, the role of ferroptosis in the inhibitory effect of curcumin on non‐small‐cell lung cancer (NSCLC) remains unclear.MethodsCell counting kit‐8 (CCK‐8) assay was used to measure the viability of A549 and H1299 cells under different conditions. Cell proliferation was examined by Ki67 immunofluorescence. The morphological changes of cells and tumor tissues were observed by optical microscope and hematoxylin and eosin (H&E) staining. Intracellular reactive oxygen species (ROS), malondialdehyde (MDA), superoxide dismutase (SOD), glutathione (GSH), and iron contents were determined by corresponding assay kit. The related protein expression levels were detected by western blot and immunohistochemistry. Transmission electron microscope was used to observe ultrastructure changes of A549 and H1299 cells.ResultsCurcumin inhibited tumor growth and cell proliferation, but promoted cell death. Characteristic changes of ferroptosis were observed in curcumin group, including iron overload, GSH depletion and lipid peroxidation. Meanwhile, the protein level of ACSL4 was higher and the levels of SLC7A11 and GPX4 were lower in curcumin group than that in control group. Incubation of ferroptosis inhibitors ferrostatin‐1 (Fer‐1) or knockdown of iron‐responsive element‐binding protein 2 (IREB2) notably weakened curcumin‐induced anti‐tumor effect and ferroptosis in A549 and H1299 cells. Further investigation suggested that curcumin induced mitochondrial membrane rupture and mitochondrial cristae decrease, increased autolysosome, increased the level of Beclin1 and LC3, and decreased the level of P62. Curcumin‐induced autophagy and subsequent ferroptosis were both alleviated with autophagy inhibitor chloroquine (CQ) or siBeclin1.ConclusionCurcumin induced ferroptosis via activating autophagy in NSCLC, which enhanced the therapeutic effect of NSCLC.

The deubiquitinase OTUD1 enhances iron transport and potentiates host antitumor immunity.

Although iron is required for cell proliferation, iron-dependent programmed cell death serves as a critical barrier to tumor growth and metastasis. Emerging evidence suggests that iron-mediated lipid oxidation also facilitates immune eradication of cancer. However, the regulatory mechanisms of iron metabolism in cancer remain unclear. Here we identify OTUD1 as the deubiquitinase of iron-responsive element-binding protein 2 (IREB2), selectively reduced in colorectal cancer. Clinically, downregulation of OTUD1 is highly correlated with poor outcome of cancer. Mechanistically, OTUD1 promotes transferrin receptor protein 1 (TFRC)-mediated iron transportation through deubiquitinating and stabilizing IREB2, leading to increased ROS generation and ferroptosis. Moreover, the presence of OTUD1 promotes the release of damage-associated molecular patterns (DAMPs), which in turn recruits the leukocytes and strengthens host immune response. Reciprocally, depletion of OTUD1 limits tumor-reactive T-cell accumulation and exacerbates colon cancer progression. Our data demonstrate that OTUD1 plays a stimulatory role in iron transportation and highlight the importance of OTUD1-IREB2-TFRC signaling axis in host antitumor immunity.

Iron chelation rescues hemolytic anemia and skin photosensitivity in congenital erythropoietic porphyria

AbstractCongenital erythropoietic porphyria (CEP) is an inborn error of heme synthesis resulting from uroporphyrinogen III synthase (UROS) deficiency and the accumulation of nonphysiological porphyrin isomer I metabolites. Clinical features are heterogeneous among patients with CEP but usually combine skin photosensitivity and chronic hemolytic anemia, the severity of which is related to porphyrin overload. Therapeutic options include symptomatic strategies only and are unsatisfactory. One promising approach to treating CEP is to reduce the erythroid production of porphyrins through substrate reduction therapy by inhibiting 5-aminolevulinate synthase 2 (ALAS2), the first and rate-limiting enzyme in the heme biosynthetic pathway. We efficiently reduced porphyrin accumulation after RNA interference–mediated downregulation of ALAS2 in human erythroid cellular models of CEP disease. Taking advantage of the physiological iron-dependent posttranscriptional regulation of ALAS2, we evaluated whether iron chelation with deferiprone could decrease ALAS2 expression and subsequent porphyrin production in vitro and in vivo in a CEP murine model. Treatment with deferiprone of UROS-deficient erythroid cell lines and peripheral blood CD34+-derived erythroid cultures from a patient with CEP inhibited iron-dependent protein ALAS2 and iron-responsive element–binding protein 2 expression and reduced porphyrin production. Furthermore, porphyrin accumulation progressively decreased in red blood cells and urine, and skin photosensitivity in CEP mice treated with deferiprone (1 or 3 mg/mL in drinking water) for 26 weeks was reversed. Hemolysis and iron overload improved upon iron chelation with full correction of anemia in CEP mice treated at the highest dose of deferiprone. Our findings highlight, in both mouse and human models, the therapeutic potential of iron restriction to modulate the phenotype in CEP.

Genetic Analysis of EGLN1 C127S Variant in Taiwanese Parkinson's Disease.

Parkinson's disease (PD) is a neurodegenerative disorder related to nigrostriatal dopaminergic neuron degeneration and iron accumulation. As a cellular oxygen sensor, prolyl hydroxylase domain containing protein 2 (PHD2, encoded by egl-9 family hypoxia inducible factor 1, EGLN1) modifies hypoxia-inducible factor alpha (HIF-α) protein for proteasomal destruction under normoxic condition. In addition, 2-oxoglutarate- (OG-) dependent dioxygenase activity of PHD2 is involved in the oxygen and iron regulation of iron-responsive element binding protein 2 (IRP2) stability. Previously increased expression of EGLN1 was found in the substantia nigra of the parkinsonian brain. We investigated the possible role of c.380 G > C (p.C127S) of EGLN1 gene in Taiwanese patients with PD. 479 patients and 435 healthy controls were recruited. Polymerase chain reaction and BsmAI restriction enzyme analysis were applied for analysis. An association between CC genotype and reduced PD risk in the recessive model (CC vs. GG + GC) was found. Our study provides a link between EGLN1 c.380 G > C SNP and the development of PD.

Long Noncoding RNA FENDRR Exhibits Antifibrotic Activity in Pulmonary Fibrosis.

Abnormal activation of lung fibroblasts contributes to the initiation and progression of idiopathic pulmonary fibrosis (IPF). The objective of the present study was to investigate the role of fetal-lethal noncoding developmental regulatory RNA (FENDRR) in the activation of lung fibroblasts. Dysregulated long noncoding RNAs in IPF lungs were identified by next-generation sequencing analysis from the two online datasets. FENDRR expression in lung tissues from patients with IPF and mice with bleomycin-induced pulmonary fibrosis was determined by quantitative real-time PCR. IRP1 (iron-responsive element-binding protein 1), a protein partner of FENDRR, was identified by RNA pulldown-coupled mass spectrometric analysis and confirmed by RNA immunoprecipitation. The interaction region between FENDRR and IRP1 was determined by cross-linking immunoprecipitation. The in vivo role of FENDRR in pulmonary fibrosis was studied using adenovirus-mediated gene transfer in mice. The expression of FENDRR was downregulated in fibrotic human and mouse lungs as well as in primary lung fibroblasts isolated from bleomycin-treated mice. TGF-β1 (transforming growth factor-β1)-SMAD3 signaling inhibited FENDRR expression in lung fibroblasts. FENDRR was preferentially localized in the cytoplasm of adult lung fibroblasts and bound IRP1, suggesting its role in iron metabolism. FENDRR reduced pulmonary fibrosis by inhibiting fibroblast activation by reducing iron concentration and acting as a competing endogenous RNA of the profibrotic microRNA-214. Adenovirus-mediated FENDRR gene transfer in the mouse lung attenuated bleomycin-induced lung fibrosis and improved lung function. Our data suggest that FENDRR is an antifibrotic long noncoding RNA and a potential therapeutic target for pulmonary fibrosis.

MiR-935 Promotes Clear Cell Renal Cell Carcinoma Migration and Invasion by Targeting IREB2.

PurposeClear cell renal cell carcinoma (ccRCC) has the highest rate of metastasis and invasion in RCC and is the third most common adult urinary malignancy. miRNA may serve a critical role in human cancer development and progression, has been confirmed to play a pivotal role in RCC cell invasion and migration. Since miR‑935 had been verified to be an oncogene or tumor suppressor in various cancers, the role of miR‑935 in RCC was unclear.MethodsReal-time quantitative polymerase chain reaction (qRT-PCR) was used to verify miR-935 expression. CCK-8 assay, wound healing assay and transwell assay were used to investigate the cell proliferation, migration and invasion of miR-935. Receiver operating characteristic (ROC) curve analysis was applied to discriminate different clinical classifications. Gain or loss of function approaches were used to investigate the cell proliferation, migration and invasion of miR-935 in vitro. Bioinformatics analysis and dual-luciferase reporter assay were used to identify the target of miR-935.ResultsMiR-935 had a higher expression level in RCC cells and cancer tissues. MiR-935 mimics promoted cell proliferation, migration and invasion, and miR-935 inhibitor inhibited cell inhibit malignancy of cancer cells. Bioinformatics analysis and dual-luciferase reporter assay identified iron-responsive element-binding protein 2 (IREB2) as a direct target of miR-935. qRT-PCR showed IREB2 expression was downregulated in ccRCC cancer tissues and high IREB2 expression had a longer overall survival (OS) and disease-free survival (DFS). Silencing IREB2 could reverse the function of miR-935 inhibitor on cell proliferation and metastasis in renal cancer cells.ConclusionThe study indicated that miR-935 may act as an oncomiRNA and influenced migration and invasion progress of ccRCC by targeting IREB2. Oncogene miR-935 may be a molecular marker and uncover new strategies for ccRCC.

Deferoxamine promotes recovery of traumatic spinal cord injury by inhibiting ferroptosis.

Ferroptosis is an iron-dependent novel cell death pathway. Deferoxamine, a ferroptosis inhibitor, has been reported to promote spinal cord injury repair. It has yet to be clarified whether ferroptosis inhibition represents the mechanism of action of Deferoxamine on spinal cord injury recovery. A rat model of Deferoxamine at thoracic 10 segment was established using a modified Allen's method. Ninety 8-week-old female Wistar rats were used. Rats in the Deferoxamine group were intraperitoneally injected with 100 mg/kg Deferoxamine 30 minutes before injury. Simultaneously, the Sham and Deferoxamine groups served as controls. Drug administration was conducted for 7 consecutive days. The results were as follows: (1) Electron microscopy revealed shrunken mitochondria in the spinal cord injury group. (2) The Basso, Beattie and Bresnahan locomotor rating score showed that recovery of the hindlimb was remarkably better in the Deferoxamine group than in the spinal cord injury group. (3) The iron concentration was lower in the Deferoxamine group than in the spinal cord injury group after injury. (4) Western blot assay revealed that, compared with the spinal cord injury group, GPX4, xCT, and glutathione expression was markedly increased in the Deferoxamine group. (5) Real-time polymerase chain reaction revealed that, compared with the Deferoxamine group, mRNA levels of ferroptosis-related genes Acyl-CoA synthetase family member 2 (ACSF2) and iron-responsive element-binding protein 2 (IREB2) were up-regulated in the Deferoxamine group. (6) Deferoxamine increased survival of neurons and inhibited gliosis. These findings confirm that Deferoxamine can repair spinal cord injury by inhibiting ferroptosis. Targeting ferroptosis is therefore a promising therapeutic approach for spinal cord injury.

Strong correlation of ferrochelatase enzymatic activity with Mitoferrin-1 mRNA in lymphoblasts of patients with protoporphyria

Accumulation of protoporphyrin IX (PPIX) and Zn-PPIX, are the clinical hallmarks of protoporphyria. Phenotypic expression of protoporphyria is due to decreased activity of ferrochelatase (FECH) or to increased activity of aminolevulinic acid synthase (ALAS) in red blood cells. Other genetic defects have been shown to contribute to disease severity including loss of function mutations in the mitochondrial AAA-ATPase, CLPX and mutations in the Iron-responsive element binding protein 2 (IRP2), in mice. It is clear that multiple paths lead to a common phenotype of excess plasma PPIX that causes a phototoxic reaction on sun exposed areas. In this study we examined the association between mitochondrial iron acquisition and utilization with activity of FECH. Our data show that there is a metabolic link between the activity FECH and levels of MFRN1 mRNA. We examined the correlation between FECH activity and MFRN1 mRNA in cell lines established from patients with the classical protoporphyria, porphyria due to defects in ALAS2 mutations. Our data confirm MFRN1 message levels positively correlated with FECH enzymatic activity in all cell types.

Iron-responsive element-binding protein 2 plays an essential role in regulating prostate cancer cell growth.

Iron-responsive element-binding proteins (IRPs) are master regulators of cellular iron homeostasis. Our previous work demonstrated that iron homeostasis is altered in prostate cancer and contributes to prostate cancer progression. Here we report that prostate cancer cells overexpress IRP2 and that overexpression of IRP2 drives the altered iron phenotype of prostate cancer cells. IRP2 knockdown in prostate cancer cell lines reduces intracellular iron and causes cell cycle inhibition and apoptosis. Cell cycle analysis demonstrates that IRP2-depleted prostate cancer cells accumulate in G0/G1 due to induction of p15, p21, and p27. Activation of these pathways is sufficient to significantly reduce the growth of PC3 prostate tumors in vivo. In contrast, IRP1 knockdown does not affect iron homeostasis and only modestly affects cell growth, likely through an iron-independent mechanism. These results demonstrate that upregulation of IRP2 in prostate cancer cells co-opts normal iron regulatory mechanisms to facilitate iron retention and drive enhanced tumor growth.

Ferroptosis: A Novel Anti-tumor Action for Cisplatin.

PurposeFerroptosis is a new mode of regulated cell death, which is completely distinct from other cell death modes based on morphological, biochemical, and genetic criteria. This study evaluated the therapeutic role of ferroptosis in classic chemotherapy drugs, including the underlying mechanism.Materials and MethodsCell viabilitywas detected by using the methylthiazoltetrazlium dye uptake method. RNAiwas used to knockout iron-responsive element binding protein 2, and polymerase chain reaction, western blot was used to evaluate the efficiency. Intracellular reduced glutathione level and glutathione peroxidases activitywere determined by related assay kit. Intracellularreactive oxygen species levelswere determined by flowcytometry. Electron microscopywas used to observe ultrastructure changes in cell.ResultsAmong five chemotherapeutic drugs screened in this study, cisplatin was found to be an inducer for both ferroptosis and apoptosis in A549 and HCT116 cells. The depletion of reduced glutathione caused by cisplatin and the inactivation of glutathione peroxidase played the vital role in the underlying mechanism. Besides, combination therapy of cisplatin and erastin showed significant synergistic effect on their anti-tumor activity.ConclusionFerroptosis had great potential to become a new approach in anti-tumor therapies and make up for some classic drugs, which open up a new way for their utility in clinic.