Divalent Metal Transporter Research Articles

Hypoxia-inducible factor-prolyl hydroxylase inhibitor Roxadustat (FG-4592) reduces renal fibrosis in Dahl salt-sensitive rats.

Although hypoxia-inducible factor-prolyl hydroxylase (HIF-PH) inhibitors have been developed for the treatment of renal anemia, their effects on cardiac and renal dysfunction remain unknown. We previously reported on Dahl salt-sensitive rats, in a rat model of salt-sensitive hypertension, that exhibited anemia and impaired expression of duodenal iron transporters after the development of hypertensive cardiac and renal dysfunction. Therefore, we investigated the effects of Roxadustat (FG-4592), an HIF-PH inhibitor, on anemia, iron regulation, and cardiac and renal dysfunction in Dahl salt-sensitive rats. Six-week-old male Dahl salt-sensitive rats were fed a normal or high-salt diet for 8 weeks. A further subset of Dahl salt-sensitive rats, that were fed a high-salt diet, was administered Roxadustat for 8 weeks. Dahl salt-sensitive rats fed a high-salt diet developed hypertension, cardiac and renal dysfunction, and anemia after 8 weeks of feeding. Roxadustat increased hemoglobin and serum erythropoietin levels in Dahl salt-sensitive rats fed a high-salt diet. With regard to the iron-regulating system, Roxadustat lowered hepatic hepcidin gene expression and increased the gene expression of duodenal iron transporters, such as cytochrome b and divalent metal transporter 1 , in Dahl salt-sensitive rats fed a high-salt diet. Roxadustat did not affect the development of hypertension and cardiac hypertrophy in Dahl salt-sensitive rats with a high-salt diet; however, Roxadustat treatment attenuated renal fibrosis in these rats. Roxadustat ameliorated anemia with affecting the gene expression of the iron-regulating system, and did not affect cardiac hypertrophy but attenuated renal fibrosis in Dahl salt-sensitive rats fed a high-salt diet.

The Iron Chaperone Poly C Binding Protein 1 Regulates Iron Efflux through Intestinal Ferroportin in Mice

Iron is an essential nutrient required by all cells but used primarily for red blood cell production. Because humans have no effective mechanism for ridding the body of excess iron, the absorption of dietary iron must be precisely regulated. The critical site of regulation is the transfer of iron from the absorptive enterocyte to the portal circulation via the sole iron efflux transporter, ferroportin (FPN). Here we report that poly(rC)-binding protein 1 (PCBP1), the major cytosolic iron chaperone, is necessary for the regulation of iron flux through FPN in the intestine of mice. PCBP1 binds iron-glutathione complexes in the cytosol of cells and delivers the iron to ferritin, the iron storage protein, to iron centers in enzymes, and to iron-sulfur cluster chaperones. PCBP1 also limits the chemical reactivity of iron, thereby limiting iron-catalyzed oxidative damage in cells. To explore the role of PCBP1 in dietary iron absorption, we developed mice with an inducible, enterocyte-specific deletion of PCBP1. Mice lacking PCBP1 in the intestinal epithelium exhibit low levels of enterocyte iron, poor retention of dietary iron in enterocyte ferritin, and excess efflux of iron through FPN. Kinetic studies of iron transport using a stable isotope ( 57Fe) coupled with a mini-hepcidin that blocks FPN activity demonstrated that PCBP1 deletion had no effect of intestinal iron uptake but caused high rates of iron efflux through FPN. Surprisingly, excess iron efflux occurred despite lower levels of FPN protein in enterocytes and upregulation of the iron regulatory hormone hepcidin. PCBP1 deletion and the resulting unregulated dietary iron absorption led to poor growth, severe anemia on a low iron diet and liver oxidative stress with iron loading on a high iron diet. To investigate the roles of PCBP1 more precisely in enterocytes, we developed 3-D organoid cultures from intestinal epithelium which functionally expressed the major iron trafficking proteins: divalent metal transporter 1, PCBP1, ferritin, transferrin receptor 1 and FPN. Ex vivo culture of wild-type and PCBP1-depleted enteroids demonstrated normal patterns of FPN expression and hepcidin-mediated turnover. However, fluorescence measurements of kinetically labile iron pools in enteroids competent or blocked for FPN iron efflux indicated that PCBP1 dramatically altered efflux activity. PCBP1 functioned as a cytosolic buffer for iron, binding and retaining iron and thereby keeping “free” iron at very low levels. This low concentration of free iron limited its availability for FPN-mediated efflux and allowed hepcidin-mediated changes in FPN levels to regulate the amount of iron exported from the enterocyte. Thus, by binding enterocyte iron, PCBP1 reduces the concentration of unchaperoned, “free” iron to a low level. This low level of cytosolic free iron is necessary for hepcidin-mediated regulation of FPN activity.

Investigation of the activity of a novel tropolone in osteosarcoma.

Osteosarcoma (OS) is a primary malignant bone tumor characterized by frequent metastasis, rapid disease progression, and a high rate of mortality. Treatment options for OS have remained largely unchanged for decades, consisting primarily of cytotoxic chemotherapy and surgery, thus necessitating the urgent need for novel therapies. Tropolones are naturally occurring seven-membered non-benzenoid aromatic compounds that possess antiproliferative effects in a wide array of cancer cell types. MO-OH-Nap is an α-substituted tropolone that has activity as an iron chelator. Here, we demonstrate that MO-OH-Nap activates all three arms of the unfolded protein response (UPR) pathway and induces apoptosis in a panel of human OS cell lines. Co-incubation with ferric chloride or ammonium ferrous sulfate completely prevents the induction of apoptotic and UPR markers in MO-OH-Nap-treated OS cells. MO-OH-Nap upregulates transferrin receptor 1 (TFR1) protein levels, as well as TFR1, divalent metal transporter 1 (DMT1), iron-regulatory proteins (IRP1, IRP2), ferroportin (FPN), and zinc transporter 14 (ZIP14) transcript levels, demonstrating the impact of MO-OH-Nap on iron-homeostasis pathways in OS cells. Furthermore, MO-OH-Nap treatment restricts the migration and invasion of OS cells in vitro. Lastly, metabolomic profiling of MO-OH-Nap-treated OS cells revealed distinct changes in purine and pyrimidine metabolism. Collectively, we demonstrate that MO-OH-Nap-induced cytotoxic effects in OS cells are dependent on the tropolone's ability to alter cellular iron availability and that this agent exploits key metabolic pathways. These studies support further evaluation of MO-OH-Nap as a novel treatment for OS.

373 Effects of Different Trace Mineral Supplementation Strategies on Liver Mineral Status and Gene Expression of At-Risk Mineral Deficient Feedlot Cattle

Abstract Grazing lands in the western US are deficient in several trace minerals known to be critical for livestock health and performance. Mineral supplementation is often not possible since animals graze rough terrain, making mineral delivery logistically difficult. Therefore, trace minerals are often supplemented during the growing and feedlot period to maximize cattle health and production. However, morbidity and mortality rates are often greater in these mineral deficient cattle, at least in part due to their mineral deficiencies, and producers welcome input on best practices for mineral supplementation. This study aimed to evaluate how different mineral supplementation strategies affect liver mineral concentrations and abundance of mRNA involved in mineral transport in mineral deficient cattle receiving different mineral supplementation strategies. Sixty mineral deficient feedlot steers were obtained and initially stratified by weight into one of four treatment groups (n = 15): no mineral supplementation (CON); dietary mineral supplementations at industry recommended levels (DM); multimin injection at labeled dose (MM); or a combination of the MM and DM treatments (MM+DM). Treatments were given for the first 40 days of the trial and steers were fed ad libitum in pens equipped with GrowSafe bunks. Liver biopsies were conducted on days 0, 5, 10, 20, 30 and 40 to assess liver mineral concentrations of copper (Cu), manganese (Mn), selenium (Se), and zinc (Zn). Total mRNA was isolated from liver biopsies on days 0, 20, and 40 and mRNA abundance of genes involved in trace mineral transport was measured. The liver mineral data was analyzed using the PROC MIXED procedure of SAS with day as a repeated measure to evaluate the fixed effect of treatment. A series of contrasts were also conducted to determine whether mRNA abundance differed between the mineral treatments and the CON. There was a treatmentxday interaction (P < 0.05) on the Cu concentration where the MM treatment peaked at day 20 and then started to decrease towards day 40. All mineral treatments increased (P < 0.05) the liver Se concentration and mRNA abundance of zinc transporter (ZNT) 7, ZNT9, and Mn superoxide dismutase (MnSOD) compared with the CON. Steers receiving the DM treatment increased (P < 0.05) the mRNA abundance of Zrt- and Irt-like proteins (ZIP) 1, ZNT1, ZNT3, and ZNT4 compared with the CON. When compared with the CON treatment, the DM and MM+DM had a tendency (P = 0.07) to increase the abundance of divalent metal transporter 1 (DMT1) and ZNT1, as well as the liver Mn concentration. Overall, both the MM+DM and DM treatments improved mineral status by increasing liver mineral concentrations. However, the DM treatment was more effective at increasing the mRNA abundance of mineral transporters in the liver. Further research is needed to determine which pathways were influenced by an improved trace mineral status.

Iron Dys-Homeostasis Contributed to Liver Gvhd after Allogeneic Hematopoietic Stem Cell Transplantation

Graft-versus-host disease (GVHD), which results from immunological attack to recipient organs or tissues (such as the skin, liver and gut) by donor allogeneic T cells, limits the success of allogeneic hematopoietic stem cell transplantation (allo-HSCT). A correlation of high serum ferritin levels with increased mortality after allo-HSCT has been suggested by several analyses. While the role of iron dys-homeostasis in the risk of GVHD remains unclear. Whether iron dys-homeostasis contributes to the development of GVHD or iron dys-homeostasis is only a result of severe GVHD remains unknown. To assess the status of iron metabolism during GVHD following allo-HSCT, we established aGVHD mouse model by transplantation of T cell-depleted bone marrow (TCD BM) with or without splenic T cells from C57BL/6 (B6) mice to BALB/c mice after being lethally irradiated. By 28 days post-transplantation, when aGVHD stable developed, the serum iron level was significantly higher in aGVHD group compared with control. Meanwhile, ferrous-ion load in aGVHD-involved liver was also increased, while the total iron load did not differ significantly in aGVHD-involved liver compared with control. The ferrous-ion load as well as the total iron load in intestine, lung and spleen were comparable between aGVHD group and control group. To further determine the dysregulation of iron metabolism during liver aGVHD, we detected the expression levels of pivotal iron regulatory genes in liver and duodenum. The expression levels of transferrin receptor 1 ( Tfr1) and divalent metal transporter 1 ( Dmt1) were significantly upregulated in aGVHD-involved liver, which contributed to ferrous-ion overload in the liver aGVHD. Besides, Ferroportin 1 was down-regulated in aGVHD-involved duodenum, which is the result of the upregulated hepcidin ( Hamp1) in liver. Our data suggested that the increased serum iron in aGVHD was not related to iron absorption by duodenum, but may be associated with ineffectively use of iron, which was caused by the impaired marrow function after myeloablative conditioning. To explore whether ferrous-ion overload in liver would lead to induction of reactive oxygen species (ROS) via the Fenton reaction, and eventually results in liver damage during aGVHD, we detected the activity of superoxide dismutase (SOD) and the load of thiol and lipid peroxidation product, malondialdehyde (MDA). The activity of SOD and thiol load both decreased significantly in aGVHD-involved liver, while the serum MDA level increased in aGVHD group, indicating that iron dys-homeostasis may aggravate liver GVHD damage by induction of lipid peroxidation injury. Finally, to explore how iron overload affect GVHD, we fed recipient mice with high-iron diet and low-iron diet since three weeks before transplantation, respectively. The survival rates and GVHD score were significantly worse in high-iron diet group compared with normal-iron diet group. Interestingly, low-iron diet didn't show survival benefit compared with the normal-iron diet. This suggests that excessive iron deficiency is not beneficial for preventing and treating GVHD. Thus, we applied apo-transferrin (apo-Tf) administration to receipt mice, which can render the poorly soluble non-transferrin bonding iron (NTBI) into a soluble transferrin-bound form, and prevent iron from being rapidly uptake by the liver. The administration of apo-Tf alleviated the organ damage during aGVHD, and reduced the infiltration of CD4 +IFN-γ +T cells, CD4 +IL17 +T cells and CD8 +IL17 +T cells in target organs such as intestine, liver and lung. These data suggested that ferrous-ion overload leads to the increase of NTBI, which aggravate GVHD tissue damage. Our findings reveal a novel mechanism of iron dys-homeostasis during the development of liver aGVHD, which may reveal promising therapeutic targeting in the prevention and treatment of liver aGVHD.

SPI1 exacerbates iron accumulation and promotes osteoclast formation through inhibiting the expression of Hepcidin

BackgroundOsteoporosis (OP) can be caused by an overactive osteoclastic function. Anti-osteoporosis considerable therapeutic effects in tissue repair and regeneration because bone resorption is a unique osteoclast function. In this study, we mainly explored the underlying mechanisms of osteoclasts' effects on osteoporosis. MethodsRAW264.7 cells were used and induced toward osteoclast and iron accumulation by M-CSF and RANKL administration. We investigated Hepcidin and divalent metal transporter 1 (DMT1) on iron accumulation and osteoclast formation in an ovariectomy (OVX)-induced osteoporosis. Osteoporosis was induced in mice by OVX, and treated with Hepcidin (10, 20, 40, 80 mg/kg, respectively) and overexpression of DMT1 by tail vein injection. Hepcidin, SPI1, and DMT1 were detected by immunohistochemical staining, western blot and RT-PCR. The bioinformatics assays, luciferase assays, and Chromatin Immunoprecipitation (ChIP) verified that Hepcidin was a direct SPI1 transcriptional target. Iron accumulation was detected by laser scanning confocal microscopy, Perl's iron staining and iron content assay. The formation of osteoclasts was assessed using tartrate-resistant acid phosphatase (TRAP) staining. ResultsWe found that RAW264.7 cells differentiated into osteoclasts when exposed to M-CSF and RANKL, which increased the protein levels of osteoclastogenesis-related genes, including c-Fos, MMP9, and Acp5. We also observed higher concentration of iron accumulation when M-CSF and RANKL were administered. However, Hepcidin inhibited the osteoclast differentiation cells and decreased intracellular iron concentration primary osteoclasts derived from RAW264.7. Spi-1 proto-oncogene (SPI1) transcriptionally repressed the expression of Hepcidin, increased DMT1, facilitated the differentiation and iron accumulation of mouse osteoclasts. Overexpression of SPI1 significantly declined luciferase activity of HAMP promoter and increased the enrichment of HAMP promoter. Furthermore, our results showed that Hepcidin inhibited osteoclast differentiation and iron accumulation in mouse osteoclasts and OVX mice. ConclusionTherefore, the study revealed that SPI1 could inhibit Hepcidin expression contribute to iron accumulation and osteoclast formation via DMT1 signaling activation in mouse with OVX.

The efficiency and safety evaluation of hemoglobin hydrolysate as a non-heme iron fortifier

Hemoglobin hydrolysate is derived from the enzymatic degradation of hemoglobin. This work aimed to evaluate whether hemoglobin hydrolysate promotes the absorption of non-heme iron and the safety of absorbed iron in mice by analyzing the iron binding content, iron circulation, and liver homeostasis. We found that hemoglobin hydrolysate promoted the absorption of non-heme iron with high efficiency in duodenum by spontaneously binding non-heme iron during digestion, and increased hepatic iron content by up-regulating divalent metal transporter 1, zinc transporter 14, but hepatic iron content only increased at 3 weeks. Duodenal iron entered the blood through ferroportin without restriction at 3 weeks, and excessive iron entered the liver and then affected the hepatocyte membranes permeability and lipid synthesis through oxidative stress. With the prolongation of dietary intervention, the up-regulated hepcidin acted on the ferroportin to restrict excess iron from entering the blood, and then the hepatic homeostasis recovered. In addition, hemoglobin hydrolysate enhanced the hepatic antioxidant capacity. Taken together, hemoglobin hydrolysate has a strong ability to promote the absorption of non-heme iron in vivo, and the absorbed iron is relatively safe due to the regulation of hepcidin.

HMGB1 Mediates Inflammation-Induced DMT1 Increase and Dopaminergic Neurodegeneration in the Early Stage of Parkinsonism.

Both neuroinflammation and iron accumulation play roles in the pathogenesis of Parkinson's disease (PD). However, whether inflammation induces iron dyshomeostasis in dopaminergic neurons at an early stage of PD, at which no quantifiable dopaminergic neuron loss can be observed, is still unknown. As for the inflammation mediators, although several cytokines have been reported to increase in PD, the functions of these cytokines in the SN are double-edged and controversial. In this study, whether inflammation could induce iron dyshomeostasis in dopaminergic neurons through high mobility group protein B1 (HMGB1) in the early stage of PD is explored. Lipopolysaccharide (LPS), a toxin that primarily activates glia cells, and 6-hydroxydopamine (6-OHDA), the neurotoxin that firstly impacts dopaminergic neurons, were utilized to mimic PD in rats. We found a common and exceedingly early over-production of HMGB1, followed by an increase of divalent metal transporter 1 with iron responsive element (DMT1+) in the dopaminergic neurons before quantifiable neuronal loss. HMGB1 neutralizing antibody suppressed inflammation in the SN, DMT1+ elevation in dopaminergic neurons, and dopaminergic neuronal loss in both LPS and 6-OHDA administration-induced PD models. On the contrary, interleukin-1β inhibitor diacerein failed to suppress these outcomes induced by 6-OHDA. Our findings not only demonstrate that inflammation could be one of the causes of DMT1+ increase in dopaminergic neurons, but also highlight HMGB1 as a pivotal early mediator of inflammation-induced iron increase and subsequent neurodegeneration, thereby HMGB1could serve as a potential target for early-stage PD treatment.

Heme oxygenase-1 increases intracellular iron storage and suppresses inflammatory response of macrophages by inhibiting M1 polarization.

Heme oxygenase-1 (HO-1) catalyzes the first and rate-limiting enzymatic step of heme degradation, producing carbon monoxide, biliverdin, and free iron. Most iron is derived from aged erythrocytes by the decomposition of heme, which happened mainly in macrophages. However, the role of HO-1 on iron metabolism and function of macrophage is unclear. The present study investigated the effect of HO-1 on iron metabolism in macrophages, and explored the role of HO-1 on inflammatory response, polarization, and migration of macrophages. HO-1 inducer Hemin or HO-1 inhibitor zinc protoporphyrin was intravenously injected to C57BL/6J mice every 4 d for 28 d. We found that HO-1 was mainly located in the cytoplasm of splenic macrophages of mice. Activation of HO-1 by Hemin significantly increased iron deposition in the spleen, up-regulated the gene expression of ferritin and ferroportin, and down-regulated gene expression of divalent metal transporter 1 and hepcidin. Induced HO-1 by Hemin treatment increased intracellular iron levels of macrophages, slowed down the absorption of extracellular iron, and accelerated the excretion of intracellular iron. In addition, activation of HO-1 significantly decreased the expression of pro-inflammatory cytokines including interleukin (IL)-6, IL-1β, and inducible nitric oxide synthase, but increased the expression of anti-inflammatory cytokines such as IL-10. Furthermore, activation of HO-1 inhibited macrophages to M1-type polarization, and increased the migration rate of macrophages. This study demonstrated that HO-1 was able to regulate iron metabolism, exert anti-inflammatory effects, and inhibit macrophages polarization to M1 type.

Anemia in female adolescents at Karanganyar regency: A cross-sectional study associated with polymorphism of duodenal cytochrome B gene and daily consumptions of fruits and vegetables.

The prevalence of anemia in female adolescents increases every year. A duodenal Cytochrome B (CYBRD1) enzyme is involved in the regulation of iron metabolism. G797A gene polymorphism of the CYBRD1 reduces nonheme iron transport into the enterocytes, which was mediated by the divalent metal transporter 1 protein. Daily consumption of fruits and vegetables has been recommended for the prevention of non- communicable diseases, including anemia. This study aimed to analyze the association of CYBRD1 polymorphism and daily consumption of fruits and vegetables with anemia in female adolescents in the Karanganyar regency. This cross-sectional study recruited 233 female students in 6 senior and vocational high schools in Karanganyar regency, which were selected using purposive sampling. Data on fruit and vegetable consumption were collected using Semi-Quantitative Food Frequency Questionnaire (SQ-FFQ). Polymorphism of the G797A CYBRD1 gene was determined using the amplification refractory mutation system polymerase chain reaction. All collected data were analyzed using chi-square and multiple logistic regression tests with p < 0.05. Anemia was found in 13.73% of female students. Inadequate intakes of fruits and vegetables were found in 63.09 and 51.07% of female students, respectively. The AA genotype (OR = 5.779; 95%CI: 0.974-34.289; p = 0.053), inadequate fruit consumption (OR = 1.497; 95%CI: 0.603-3.718; p = 0.133), and inadequate vegetable consumption (OR = 11.99; 95%CI: 3.457-41.586; p < 0.001) increased higher risk of anemia, compared with their counterparts. G797A CYBRD1 gene polymorphism and daily consumption of vegetables increase the risk of anemia but not for daily consumption of fruits in female adolescents in the Karanganyar regency.

Ebselen, Iron Uptake Inhibitor, Alleviates Iron Overload-Induced Senescence-Like Neuronal Cells SH-SY5Y via Suppressing the mTORC1 Signaling Pathway.

Increasing evidence highlights that excessive iron accumulation in the brain plays a vital role in neuronal senescence and is implicated in the pathogenesis of age-related neurodegenerative diseases, including Alzheimer's disease (AD) and Parkinson's disease (PD). Therefore, the chemical compounds that eliminate an iron overload may provide better protection against oxidative stress conditions that cause the accumulation of senescent cells during brain aging. Ebselen has been identified as a strongly useful compound in the research on redox biology mechanisms. We hypothesized that ebselen could alleviate an iron overload-induced oxidative stress and consequently reverses the senescence-like phenotypes in the neuronal cells. In the present study, SH-SY5Y cells were treated with ferric ammonium citrate (FAC) before ebselen, and the evaluation of the cellular iron homeostasis, the indicators of oxidative stress, and the onset of senescence phenotypes and mechanisms were carried out accordingly. Our findings showed that ebselen ameliorated the FAC-mediated iron overload by decreasing the expression of divalent metal transporter 1 (DMT1) and ferritin light chain (FT-L) proteins. In contrast, it increased the expression of ferroportin 1 (FPN1) protein and its correlation led to a decrease in the expression of the cytosolic labile iron pool (LIP). Furthermore, ebselen significantly reduced reactive oxygen species (ROS) and rescued the mitochondrial membrane potential (ΔΨm). Notably, ebselen restored the biomarkers of cellular senescence by reducing the number of senescence-associated β-galactosidase (SA-β-gal) positive cells and senescence-associated secretory phenotypes (SASP). This also suppressed the expression of p53 protein targeting DNA damage response (DDR)/p21 cyclin-dependent kinase (CDK) inhibitor through a mTORC1 signaling pathway. Potentially, ebselen could be a therapeutic agent for treating brain aging and AD by mitigating iron accumulation and restoring senescence in SH-SY5Y cells.

Iron-responsive element of Divalent metal transporter 1 (Dmt1) controls Notch-mediated cell fates.

Notch receptor activation is regulated by the intramembrane protease γ-secretase, which cleaves and liberates the Notch intracellular domain (Nicd) that regulates gene transcription. While γ-secretase cleavage is necessary, we demonstrate it is insufficient for Notch activation and requires vesicular trafficking. Here, we report Divalent metal transporter 1 (Dmt1, Slc11A2) as a novel and essential regulator of Notch signalling. Dmt1-deficient cells are defective in Notch signalling and have perturbed endolysosomal trafficking and function. Dmt1 encodes for two isoforms, with and without an iron response element (ire). We show that isoform-specific silencing of Dmt1-ire and Dmt1+ire has opposite consequences on Notch-dependent cell fates in cell lines and intestinal organoids. Loss of Dmt1-ire suppresses Notch activation and promotes differentiation, whereas loss of Dmt1+ire causes Notch activation and maintains stem-progenitor cell fates. Dmt1 isoform expression correlates with Notch and Wnt signalling in Apc-deficient intestinal organoids and human colorectal cancers. Consistently, Dmt1-ire silencing induces Notch-dependent differentiation in colorectal cancer cells. These data identify Dmt1 isoforms as binary switches controlling Notch cell fate decisions in normal and tumour cells.

The Endolysosomal Transporter DMT1 is Required for Morphine Regulation of Neuronal Ferritin Heavy Chain

NeuroHIV and other neurologic disorders present with altered iron metabolism in central nervous system neurons. Many people with HIV also use opioids, which can worsen neuroHIV symptoms by further dysregulating neuronal iron metabolism. Our previous work demonstrated that the μ-opioid agonist morphine causes neuronal endolysosomes to release their iron stores, and neurons respond by upregulating ferritin heavy chain (FHC), an iron storage protein associated with cognitive impairment in neuroHIV. Here, we investigated if this process required divalent metal transporter 1 (DMT1), a well-known iron transporter expressed on endolysosomes. We first optimized conditions to detect DMT1 isoforms (DMT1 1B ± iron responsive element) using fluorescently labeled rat DMT1 constructs expressed in HEK-293 cells. We also expressed these constructs in primary rat cortical neurons to compare their expression and subcellular distribution with endogenous DMT1 isoforms. We found endogenous DMT1 isoforms in the cytoplasm that colocalized with lysosomal-associated protein 1 (LAMP1), a marker of endolysosomes. Next, we blocked endogenous DMT1 isoforms using ebselen, a potent pharmacological inhibitor of DMT1 iron transport. Ebselen pre-treatment blocked morphine’s ability to upregulate FHC protein, suggesting this pathway requires DMT1 iron transport from endolysosomes. This was further validated using viral-mediated genetic silencing of DMT1±IRE in cortical neurons, which also blocked FHC upregulation in the presence of morphine. Overall, our work demonstrates that the μ-opioid agonist morphine utilizes the endolysosomal iron transporter DMT1 to modulate neuronal cellular iron metabolism, upregulate FHC protein, and contribute to cognitive decline in neuroHIV.Graphical Morphine requires DMT1 to upregulate neuronal FHC. Cortical neurons treated with morphine release their endolysosomal iron stores to the cytoplasm and upregulate FHC, an iron storage protein associated with dendritic spine deficits and cognitive impairment in neuroHIV. This pathway requires the endolysosomal iron transporter DMT1, as pharmacological and genetic inhibitors of the transporter completely block morphine’s ability to upregulate FHC. Created with BioRender.com.

Cytochrome b561 regulates iron metabolism by activating the Akt/mTOR pathway to promote Breast Cancer Cells proliferation

Breast cancer (BC) is the leading cause of cancer-related mortality in women, necessitating the development of novel therapeutic targets. While cytochrome b561 (CYB561) expression is associated with poor prognosis in BC, the precise role of CYB561 in BC and its potential mechanisms remain unclear. In the present study, we found that CYB561 plays an essential role in BC growth. CYB561 expression was up-regulated in surgically resected cancerous tissues and in six BC cell lines. Lentivirus-mediated CYB561 knockdown in BC cells significantly reduced their proliferation, migration, and invasiveness. CYB561 participates in the regulation of iron metabolism in BC. CYB561 knockdown reduced total iron content, increased ferrous iron content, and down-regulated the expression of proteins associated with iron metabolism (transferrin receptor 1, divalent metal transporter 1, and ferritin heavy chain 1). Conversely, up-regulation of CYB561 through co-incubation with exogenous iron (ferric ammonium citrate) produced contrary outcomes. Additionally, CYB561 activated the protein kinase B/mammalian target of rapamycin (Akt/mTOR) signaling pathway in BC cells. Down-regulation of CYB561 expression inhibited the Akt/mTOR signaling pathway activity. The application of an mTOR agonist (MHY1485) rescued this negative effect, as well as the inhibitory effect of CYB561 knockdown on cell proliferation. Importantly, the dual mTOR inhibitor MLN0128 (50 nM, 48 h) down-regulated CYB561 expression and the iron metabolism-related proteins transferrin receptor, divalent metal transporter 1, and ferritin heavy chain 1, whereas the mTOR agonist MHY1485 rescued the down-regulation of CYB561 knockdown on iron metabolism-related proteins. We conclude that CYB561 promotes the proliferation of BC cells by regulating iron metabolism through the activation of the Akt/mTOR signaling pathway.

Blood Levels of Environmental Heavy Metals are Associated with Poorer Iron Status in Ugandan Children: A Cross-Sectional Study

BackgroundIron deficiency (ID) and environmental exposure to metals frequently co-occur among Ugandan children, but little is known about their associations, although iron and other divalent metals share the same intestinal absorption transporter, divalent metal transporter 1 (DMT1). ObjectivesWe examined associations between iron status and blood concentrations of lead, manganese (Mn), cobalt (Co), and cadmium, both singly and as a mixture. MethodsWe used data on sociodemographic status, iron biomarkers, and blood concentrations of heavy metals collected from a cross-sectional survey of 100 children aged 6–59 mo in Kampala, Uganda. We compared blood concentrations of metals in ID with iron-sufficient children. We examined associations between a metal mixture and iron biomarkers using multiple linear regression and weighted quintile sum regression. ResultsThe median (interquartile range) blood Mn (μg/L) was higher in ID children defined by soluble transferrin receptor (sTfR) and ferritin (ID compared with iron-sufficient children): (sTfR [21.3 {15.1, 28.8}, 11.2 {8.6, 18.5}], ferritin [19.5 {15.0, 27.2}, 11.2 {8.8, 19.6}]; P < 0.001 for both). Similarly, the median (interquartile range) blood Co (μg/L) was higher in ID children by ferritin ([0.5 {0.4, 0.9}, 0.4 {0.3, 0.5}], P = 0.05). Based on the multiple linear regression results, higher blood Co and Mn were associated with poorer iron status (defined by all 4 iron indicators for Co and by sTfR for Mn). The weighted quintile sum regression result showed that higher blood concentrations of a metal mixture were associated with poorer iron status represented by sTfR, ferritin, and hepcidin, mainly driven by Co and Mn. ConclusionsOur study findings suggest that poorer iron status is associated with overall heavy metal burden, predominantly Co and Mn, among Ugandan children. Further prospective studies should confirm our primary findings and investigate the combined effects of coexposures to neurotoxicants on the neurodevelopment of young children.

Alpha-synuclein Affects Certain Iron Transporters of BV2 Microglia Cell through its ferric reductase activity.

Alpha-synuclein (α-syn) is a major component of lewy bodies, which is biomarker of Parkinson's disease (PD). It accumulates in substantia nigra pars compacts (SNpc) to form insoluble aggregates and cause neurotoxicity, which is often accompanied by iron deposition. In this study, we compared the iron reductase activity between monomeric α-syn (M-α-syn) and oligomeric α-syn (O-α-syn), investigated the effect of α-syn on iron metabolism of BV2 microglia cells as well. We found that α-syn had ferric reductase activity, and O-α-syn had stronger enzyme activity than M-α-syn. M-α-syn upregulated iron uptake protein, divalent metal transporter1 (DMT1) expression and iron influx, but did not regulate iron release protein, ferroportin1 (FPN1) expression and iron efflux. O-α-syn elevated the expression of both DMT1 and FPN1, thus increased the iron influx and efflux in BV2 microglial cells, but the expressions of iron regulatory protein1 and hypoxia inducible factor2α have no significant change. Moreover, both M-α-syn and O-α-syn could increase the mRNA expressions of TNF-α and IL-1β in BV2 microglia cells. Taken together, our data suggest that both types of α-syn can activate microglia, which leads to increased expressions of pro-inflammatory factors. α-syn can affect DMT1 and FPN1 expressions in BV2 microglia cells, which might be through its ferric reductase activity.

Group A Streptococcus cation diffusion facilitator proteins contribute to immune evasion by regulating intracellular metal concentrations

Cation diffusion facilitators (CDFs) are a large family of divalent metal transporters with broad specificities that contribute to intracellular metal homeostasis and toxicity in bacterial pathogens. Streptococcus pyogenes (Group A Streptococcus [GAS]) expresses two homologous CDF efflux transporters, MntE and CzcD, which selectively transport Mn and Zn, respectively. We discovered that the MntE- and CzcD-deficient strains exhibited a marked decrease in the viability of macrophage-differentiated THP-1 cells and neutrophils. In addition, the viability of mice infected with both deficient strains markedly increased. Consistent with a previous study, our results suggest that MntE regulates the PerR-dependent oxidative stress response by maintaining intracellular Mn levels and contributing to the growth of GAS. The maturation and proteolytic activity of streptococcal cysteine protease (SpeB), an important virulence factor in GAS, has been reported to be abrogated by zinc and copper. Zn inhibited the maturation and proteolytic activity of SpeB in the culture supernatant of the CzcD-deficient strain. Furthermore, Mn inhibited SpeB maturation and proteolytic activity in a MntE-deficient strain. Since the host pathogenicity of the SpeB-deficient strain was significantly reduced, maintenance of intracellular manganese and zinc levels in the GAS via MntE and CzcD may not only confer metal resistance to the bacterium, but may also play an essential role in its virulence. These findings provide new insights into the molecular mechanisms of pathogenicity, which allow pathogens to survive under stressful conditions associated with elevated metal ion concentrations during host infection.

Sesamol Mitigates Chronic Iron Overload-Induced Cognitive Impairment and Systemic Inflammation via IL-6 and DMT1 Regulation.

Excessive iron contributes to oxidative damage and cognitive decline in Alzheimer's disease. Sesamol, a compound in sesame oil that exhibits both anti-inflammatory and neuroprotective properties, is examined in this study for its ability to alleviate cognitive impairments in iron overload mice model. An iron overload model is established by intraperitoneally injecting dextran iron (250mgkg-1 body weight) twice a week for 6 weeks, while sesamol (100mgkg-1 body weight) is administered daily for the same length of time. The results demonstrate that sesamol protects spatial working memory and learning ability in iron overload mice, and inhibits neuronal loss and brain atrophy induced by iron overload. Moreover, sesamol significantly decreases interleukin-6 and malondialdehyde, and increases glutathione peroxidase 4 in the brains of iron overload mice. Additionally, sesamol maintains iron homeostasis in the brain by regulating the expressions of transferrin receptors, divalent metal transporter 1, and hepcidin, and reducing iron accumulation. Furthermore, sesamol suppresses disturbed systemic iron homeostasis and inflammation, particularly liver interleukin-6 expression. These findings suggest that sesamol may be effective in mitigating neuroinflammatory responses and cognitive impairments induced by iron overload, potentially through its involvement in mediating the liver-brain axis.

Role of mass effect on neuronal iron deposition after intracerebral hemorrhage

Mass effect after intracerebral hemorrhage (ICH) not only mechanically induces the brain damage, but also influences the progress of secondary brain damage. However, the influence of mass effect on the iron overload after ICH is still unclear. Here, a fixed volume of ferrous chloride solution and different volumes of poly(N-isopropylacrylamide) (PNIPAM) hydrogel were co-injected into the right basal ganglia of rats to establish the ICH model with certain degree of iron deposition but different degrees of mass effect. We found that mass effect significantly increased the iron deposition on neuronal cells at 6 h after ICH in a volume-dependent manner. Furthermore, the upregulation of Piezo-2, divalent metal transporter 1 (DMT1), transferrin receptor (TfR), and ferroptosis expressions were noted as the increase of mass effect. In addition, the pERK1/2 inhibitor PD98059 treated ICH rats reversed the upregulation of iron uptake protein and ferroptosis. Our findings revealed the relationship between mass effect and the iron uptake and ferroptosis, which are benefit to understand the brain damage process after ICH.

Ebselen: A promising therapy protecting cardiomyocytes from excess iron in iron-overloaded thalassemia patients.

Iron-overload-associated cardiomyopathy has been one of the primary causes of mortality in thalassemia patients with iron burden. There is growing evidence citing the beneficial effects of ebselen as an antioxidant selectively blocking the divalent metal transporter 1 (DMT-1) to deter iron ingress into cardiomyocytes, raising internets in viewing this component in this population in order to treat and even prevent cardiomyopathy occurring from iron surplus. In this article, we reviewed the potential advantageous effects of ebselen in thalassemia patients who suffer from iron excess, susceptible to cardiomyopathy induced by iron overload. A systematic search in several databases, including PubMed, Scopus, and Web of Science, was conducted to explore the role of ebselen in controlling iron-overload-related cardiomyopathy in thalassemia patients by the keywords of Ebselen AND iron. The inclusion criteria were English-written preclinical and clinical studies investigating the efficacy and side effects of ebselen in an iron-overload context. After searching the databases, 44 articles were found. Next, of 19 published articles, 3 were included in this article. After reviewing the references of the included studies, no articles were added. In conclusion ebselen can be a promising adjuvant therapy in patients with thalassemia alongside the standard treatment with iron chelators, particularly in severe cases with cardiomyopathy, due to falling iron inflow by inhibiting DMT-1 and increasing ferroportin-1 expression and antioxidant properties. However, clinical studies need to be carried out to reach a definite conclusion.