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Background: Microglial cell iron load and inflammatory activation are significant hallmarks of late-stage Alzheimer's disease (AD). In vitro, microglia preferentially upregulate the iron importer, divalent metal transporter 1 (DMT1, gene name Slc11a2) in response to inflammatory stimuli, and excess iron can augment cellular inflammation, suggesting a feed-forward loop between iron import mechanisms and inflammatory signaling. However, it is not understood whether microglial iron import mechanisms directly contribute to inflammatory signaling and chronic disease in vivo. These studies determined the effects of microglial-specific knockdown of Slc11a2 on AD-related cognitive decline and microglial transcriptional phenotype. Methods: In vitro experiments and RT-qPCR were used to assess a role for DMT1 in amyloid-β-associated inflammation. To determine the effects of microglial Slc11a2 knockdown on AD-related phenotypes in vivo, triple-transgenic Cx3cr1Cre−ERT2;Slc11a2flfl;APP/PS1+or – mice were generated and administered corn oil or tamoxifen to induce knockdown at 5–6 months of age. Both sexes underwent behavioral analyses to assess cognition and memory (12–15 months of age). Hippocampal CD11b+ microglia were magnetically isolated from female mice (15–17 months) and bulk RNA-sequencing analysis was conducted. Results: DMT1 inhibition in vitro robustly decreased Aβ-induced inflammatory gene expression and cellular iron levels in conditions of excess iron. In vivo, Slc11a2KDAPP/PS1 female, but not male, mice displayed a significant worsening of memory function in Morris water maze and a fear conditioning assay, along with significant hyperactivity compared to control WT and APP/PS1 mice. Hippocampal microglia from Slc11a2KDAPP/PS1 females displayed significant increases in Enpp2, Ttr, and the iron-export gene, Slc40a1, compared to control APP/PS1 cells. Slc11a2KD cells from APP/PS1 females also exhibited decreased expression of markers associated with subsets of disease-associated microglia (DAMs), such as Apoe, Ctsb, Ly9, Csf1, and Hif1α. Conclusions: This work suggests a sex-specific role for microglial iron import gene Slc11a2 in propagating behavioral and cognitive phenotypes in the APP/PS1 model of AD. These data also highlight an association between loss of a DAM-like phenotype in microglia and cognitive deficits in Slc11a2KDAPP/PS1 female mice. Overall, this work illuminates an iron-related pathway in microglia that may serve a protective role during disease and offers insight into mechanisms behind disease-related sex differences. [ABSTRACT FROM AUTHOR]

The hallmark pathological features of Alzheimer's disease (AD) consist of senile plaques, which are formed by extracellular β-amyloid (Aβ) deposition, and neurofibrillary tangles, which are formed by the hyperphosphorylation of intraneuronal tau proteins. With the increase in clinical studies, the in vivo imbalance of iron homeostasis and the dysfunction of synaptic plasticity have been confirmed to be involved in AD pathogenesis. All of these mechanisms are constituted by the abnormal accumulation of misfolded or conformationally altered protein aggregates, which in turn drive AD progression. Proteostatic imbalance has emerged as a key mechanism in the pathogenesis of AD. Ubiquitination modification is a major pathway for maintaining protein homeostasis, and protein degradation is primarily carried out by the ubiquitin-proteasome system (UPS). In this review, we provide an overview of the ubiquitination modification processes and related protein ubiquitination degradation pathways in AD, focusing on the microtubule-associated protein Tau, amyloid precursor protein (APP), divalent metal transporter protein 1 (DMT1), and α-amino-3-hyroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors. We also discuss recent advances in ubiquitination-based targeted therapy for AD, with the aim of contributing new ideas to the development of novel therapeutic interventions for AD. [ABSTRACT FROM AUTHOR]

Abstract Background Microglial cell iron load and inflammatory activation are significant hallmarks of late-stage Alzheimer’s disease (AD). In vitro, microglia preferentially upregulate the iron importer, divalent metal transporter 1 (DMT1, gene name Slc11a2) in response to inflammatory stimuli, and excess iron can augment cellular inflammation, suggesting a feed-forward loop between iron import mechanisms and inflammatory signaling. However, it is not understood whether microglial iron import mechanisms directly contribute to inflammatory signaling and chronic disease in vivo. These studies determined the effects of microglial-specific knockdown of Slc11a2 on AD-related cognitive decline and microglial transcriptional phenotype. Methods In vitro experiments and RT-qPCR were used to assess a role for DMT1 in amyloid-β-associated inflammation. To determine the effects of microglial Slc11a2 knockdown on AD-related phenotypes in vivo, triple-transgenic Cx3cr1 Cre−ERT2 ;Slc11a2 flfl ;APP/PS1 +or – mice were generated and administered corn oil or tamoxifen to induce knockdown at 5–6 months of age. Both sexes underwent behavioral analyses to assess cognition and memory (12–15 months of age). Hippocampal CD11b+ microglia were magnetically isolated from female mice (15–17 months) and bulk RNA-sequencing analysis was conducted. Results DMT1 inhibition in vitro robustly decreased Aβ-induced inflammatory gene expression and cellular iron levels in conditions of excess iron. In vivo, Slc11a2 KD APP/PS1 female, but not male, mice displayed a significant worsening of memory function in Morris water maze and a fear conditioning assay, along with significant hyperactivity compared to control WT and APP/PS1 mice. Hippocampal microglia from Slc11a2 KD APP/PS1 females displayed significant increases in Enpp2, Ttr, and the iron-export gene, Slc40a1, compared to control APP/PS1 cells. Slc11a2 KD cells from APP/PS1 females also exhibited decreased expression of markers associated with subsets of disease-associated microglia (DAMs), such as Apoe, Ctsb, Ly9, Csf1, and Hif1α. Conclusions This work suggests a sex-specific role for microglial iron import gene Slc11a2 in propagating behavioral and cognitive phenotypes in the APP/PS1 model of AD. These data also highlight an association between loss of a DAM-like phenotype in microglia and cognitive deficits in Slc11a2 KD APP/PS1 female mice. Overall, this work illuminates an iron-related pathway in microglia that may serve a protective role during disease and offers insight into mechanisms behind disease-related sex differences.

Natural resistance-associated macrophage protein 2 (NRAMP 2; also known as DMT1 and encoded by SLC11A2) is mainly known for its iron transport activity. Recently, the DMT1 isoform lacking the iron-response element (non-IRE) was associated with aberrant NOTCH pathway activity. In this report, we investigated the function of DMT1 nonIRE in normal and malignant hematopoiesis. Knockdown of Dmt1 nonIRE in mice showed that it has non-canonical functions in hematopoietic stem cell differentiation: its knockdown suppressed development along the myeloid and lymphoid lineages, while promoting the production of platelets. These phenotypic effects on the hematopoietic system induced by Dmt1 nonIRE knockdown were linked to suppression of Notch/Myc pathway activity. Conversely, our data indicate a non-canonical function for DMT1 nonIRE overexpression in boosting NOTCH pathway activity in T-cell leukemia homeobox protein 1 (TLX1)-defective leukemia. This work sets the stage for future investigation using a multiple-hit T-cell acute lymphoblastic leukemia (T-ALL) model to further investigate the function of DMT1 nonIRE in T-ALL disease development and progression. [ABSTRACT FROM AUTHOR]

The microbiota significantly impacts digestive epithelium functionality, especially in nutrient processing. Given the importance of iron for both the host and the microbiota, we hypothesized that host-microbiota interactions fluctuate with dietary iron levels. We compared germ-free (GF) and conventional mice (SPF) fed iron-containing (65 mg/Kg) or iron-depleted (

Objective: Neuronal precursor cells expressed developmentally down‐regulated 4 (Nedd4) are believed to play a critical role in promoting the degradation of substrate proteins and are involved in numerous biological processes. However, the role of Nedd4 in intracerebral hemorrhage (ICH) remains unknown. This study aims to investigate the regulatory role of Nedd4 in the ICH model. Methods: Male C57BL/6J mice were induced with ICH. Subsequently, the levels of glutathione peroxidase 4 (GPX4), malondialdehyde (MDA) concentration, iron content, mitochondrial morphology, as well as the expression of divalent metal transporter 1 (DMT1) and Nedd4 were assessed after ICH. Furthermore, the impact of Nedd4 overexpression was evaluated through analyses of hematoma area, ferroptosis, and neurobehavioral function. The mechanism underlying Nedd4‐mediated degradation of DMT1 was elecidated using immunoprecipitation (IP) after ICH. Results: Upon ICH, the level of DMT1 in the brain increased, but decreased when Nedd4 was overexpressed using Lentivirus, suggesting a negative correlation between Nedd4 and DMT1. Additionally, the degradation of DMT1 was inhibited after ICH. Furthermore, it was found that Nedd4 can interact with and ubiquitinate DMT1 at lysine residues 6, 69, and 277, facilitating the degradation of DMT1. Functional analysis indicated that overexpression of Nedd4 can alleviate ferroptosis and promote recovery following ICH. Conclusion: The results demonstrated that ferroptosis occurs via the Nedd4/DMT1 pathway during ICH, suggesting it potential as a valuable target to inhibit ferroptosis for the treatment of ICH. [ABSTRACT FROM AUTHOR]

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 HMGB1 could serve as a potential target for early-stage PD treatment. [ABSTRACT FROM AUTHOR]

Summary: Iron deficiency (ID) and iron‐deficiency anaemia (IDA) are global public health concerns, most commonly afflicting children, pregnant women and women of childbearing age. Pathological outcomes of ID include delayed cognitive development in children, adverse pregnancy outcomes and decreased work capacity in adults. IDA is usually treated by oral iron supplementation, typically using iron salts (e.g. FeSO4); however, dosing at several‐fold above the RDA may be required due to less efficient absorption. Excess enteral iron causes adverse gastrointestinal side effects, thus reducing compliance, and negatively impacts the gut microbiome. Recent research has sought to identify new iron formulations with better absorption so that lower effective dosing can be utilized. This article outlines emerging research on oral iron supplementation and focuses on molecular mechanisms by which different supplemental forms of iron are transported across the intestinal epithelium and whether these transport pathways are subject to regulation by the iron‐regulatory hormone hepcidin. [ABSTRACT FROM AUTHOR]

• Microglia upregulate expression of iron import gene, divalent metal transporter 1 (DMT1, gene name Slc11a2) in male, but not female, mice following systemic lipopolysaccharide (LPS) injection. • Microglial-specific Slc11a2 knockdown is associated with an acute improvement in LPS-induced sickness behavior only in male mice. • Microglial Slc11a2 knockdown did not affect longer-term behavior or cognitive capacity in males or females following recovery from LPS-induced sickness. • Slc11a2 knockdown in male microglia is associated with a significant blunting of acute LPS-provoked systemic cytokine responses. • Microglia from Slc11a2 knockdown male mice exhibit a more homeostatic, iron-releasing, and anti-oxidant transcriptional signature following LPS compared to LPS control male mice. Neuroinflammation and microglial iron load are significant hallmarks found in several neurodegenerative diseases. In in vitro systems, microglia preferentially upregulate the iron importer, divalent metal transporter 1 (DMT1, gene name Slc11a2) in response to inflammatory stimuli, and it has been shown that iron can augment cellular inflammation, suggesting a feed-forward loop between mechanisms involved in iron import and inflammatory signaling. However, it is not understood how microglial iron import mechanisms contribute to inflammation in vivo, or whether altering a microglial iron-related gene affects the inflammatory response. These studies aimed to determine the effect of knocking down microglial iron import gene Slc11a2 on the inflammatory response in vivo. We generated a novel model of tamoxifen-inducible, microglial-specific Slc11a2 knockdown using Cx3cr1Cre-ERT2 mice. Transgenic male and female mice were administered intraperitoneal saline or lipopolysaccharide (LPS) and assessed for sickness behavior post-injection. Plasma cytokines and microglial bulk RNA sequencing (RNASeq) analyses were performed at 4 h post-LPS, and microglia were collected for gene expression analysis after 24 h. A subset of mice was assessed in a behavioral test battery following LPS-induced sickness recovery. Control male, but not female, mice significantly upregulated microglial Slc11a2 at 4 and 24 h following LPS. In Slc11a2 knockdown mice, we observed an improvement in the acute behavioral sickness response post-LPS in male, but not female, animals. Microglia from male, but not female, knockdown animals exhibited a significant decrease in LPS-provoked pro-inflammatory cytokine expression after 24 h. RNASeq data from male knockdown microglia 4 h post-LPS revealed a robust downregulation in inflammatory genes including Il6, Tnfα, and Il1β , and an increase in anti-inflammatory and homeostatic markers (e.g., Tgfbr1, Cx3cr1, and Trem2). This corresponded with a profound decrease in plasma pro-inflammatory cytokines 4 h post-LPS. At 4 h, male knockdown microglia also upregulated expression of markers of iron export, iron recycling, and iron homeostasis and decreased iron storage and import genes, along with pro-oxidant markers such as Cybb, Nos2, and Hif1α. Overall, this work elucidates how manipulating a specific gene involved in iron import in microglia alters acute inflammatory signaling and overall cell activation state in male mice. These data highlight a sex-specific link between a microglial iron import gene and the pro-inflammatory response to LPS in vivo, providing further insight into the mechanisms driving neuroinflammatory disease. [ABSTRACT FROM AUTHOR]

Background: Obese patients have been found to be susceptible to iron deficiency, and malabsorption of dietary iron is the cause of obesity-related iron deficiency (ORID). Divalent metal transporter 1 (DMT1) and ferroportin (FPN), are two transmembrane transporter proteins expressed in the duodenum that are closely associated with iron absorption. However, there have been few studies on the association between these two proteins and the increased susceptibility to iron deficiency in obese patients. Chronic inflammation is also thought to be a cause of obesity-related iron deficiency, and both conditions can have an impact on spermatogenesis and impair male reproductive function. Based on previous studies, transgenerational epigenetic inheritance through gametes was observed in obesity. Results: Our results showed that obese mice had decreased blood iron levels (p < 0.01), lower protein and mRNA expression for duodenal DMT1 (p < 0.05), but no statistically significant variation in mRNA expression for duodenal FPN (p > 0.05); there was an increase in sperm miR-135b expression (p < 0.05). Bioinformatics revealed ninety overlapping genes and further analysis showed that they were primarily responsible for epithelial cilium movement, fatty acid beta-oxidation, protein dephosphorylation, fertilization, and glutamine transport, which are closely related to spermatogenesis, sperm development, and sperm viability in mice. Conclusions: In obese mice, we observed downregulation of DMT1 in the duodenum and upregulation of miR-135b in the spermatozoa. [ABSTRACT FROM AUTHOR]

Mitochondria are vital for energy production and redox homeostasis, yet knowledge of relevant mechanisms remains limited. Here, through a genome-wide CRISPR-Cas9 knockout screening, we have identified DMT1 as a major regulator of mitochondria membrane potential. Our findings demonstrate that DMT1 deficiency increases the activity of mitochondrial complex I and reduces that of complex III. Enhanced complex I activity leads to increased NAD+ production, which activates IDH2 by promoting its deacetylation via SIRT3. This results in higher levels of NADPH and GSH, which improve antioxidant capacity during Erastin-induced ferroptosis. Meanwhile, loss of complex III activity impairs mitochondrial biogenesis and promotes mitophagy, contributing to suppression of ferroptosis. Thus, DMT1 differentially regulates activities of mitochondrial complex I and III to cooperatly suppress Erastin-induced ferroptosis. Furthermore, NMN, an alternative method of increasing mitochondrial NAD+, exhibits similar protective effects against ferroptosis by boosting GSH in a manner similar to DMT1 deficiency, shedding a light on potential therapeutic strategy for ferroptosis-related pathologies. [Display omitted] • DMT1 is a key regulator of mitochondrial membrane potential. • DMT1 deficiency increases mitochondrial complex I activity and decreases complex III activity. • Hyperactive complex I raises mitochondrial NAD+, which elevates GSH via SIRT3/IDH2 axis and inhibits ferroptosis. • Administration of NMN elevates mitochondrial NAD+ and GSH, mimicking the ferroptosis-repression effect of DMT1 deletion. [ABSTRACT FROM AUTHOR]

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. 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. [ABSTRACT FROM AUTHOR]

Iron is an essential transition metal for its involvement in several crucial biological functions, the most notable being oxygen storage and transport. Due to its high reactivity and potential toxicity, intracellular and extracellular iron levels must be tightly regulated. This is achieved through transport systems that mediate cellular uptake and efflux both at the level of the plasma membrane and on the membranes of lysosomes, endosomes and mitochondria. Among these transport systems, the key players are ferroportin, the only known transporter mediating iron efflux from cells; DMT1, ZIP8 and ZIP14, which on the contrary, mediate iron influx into the cytoplasm, acting on the plasma membrane and on the membranes of lysosomes and endosomes; and mitoferrin, involved in iron transport into the mitochondria for heme synthesis and Fe-S cluster assembly. The focus of this review is to provide an updated view of the physiological role of these membrane proteins and of the pathologies that arise from defects of these transport systems. [ABSTRACT FROM AUTHOR]

Excessive iron accumulation in the heart causes iron overload cardiomyopathy (IOC), which initially presents as diastolic dysfunction and arrhythmia but progresses to systolic dysfunction and end-stage heart failure when left untreated. However, the mechanisms of iron-related cardiac injury and how iron accumulates in human cardiomyocytes are not well understood. Herein, using human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs), we model IOC and screen for drugs to rescue the iron overload phenotypes. Human iPSC-CMs under excess iron exposure recapitulate early-stage IOC, including oxidative stress, arrhythmia, and contractile dysfunction. We find that iron-induced changes in calcium kinetics play a critical role in dysregulation of CM functions. We identify that ebselen, a selective divalent metal transporter 1 (DMT1) inhibitor and antioxidant, could prevent the observed iron overload phenotypes, supporting the role of DMT1 in iron uptake into the human myocardium. These results suggest that ebselen may be a potential preventive and therapeutic agent for treating patients with secondary iron overload.

Background: According to several animal and human studies, Vitamin D appears to play a substantial role in the development of diabetic nephropathy, However, the possibility of vitamin D's Reno protective impact and influence on the reversal of already-existing renal damage remains speculative. Vitamin D deficiency and insufficiency are ubiquitous worldwide and have been linked to a variety of pathophysiological conditions, including diabetes, allergies, autoimmune illnesses, pregnancy difficulties, and, more recently, worse COVID-19 clinical outcomes. From a translational perspective, the goal of this review is to look into the potential function of vitamin D in the development of diabetic kidney diseases Aim of the study: to evaluate the role of vit. D on renal function in patients with DMT1. Patient and Method: The total number of study participants was 120, divided into three groups: Group A: Included 40 patients have DM with NP (stage1, stage 2, stage 3a), Group B included 40 patients who had DM without NP, and group C included 40 healthy participants (control). Samples were taken from the Diabetic control clinic of Endocrinology and Diabetes Center/Al-Kindy Hospital, Baghdad Teaching Hospital/ Medical City, and Al-Shaheed Al-Sadder General Hospital during the period from October 2021 to March 2022. Result: Statistically significant weak positive correlations were detected between vitamin D and GFR (r= 0.321, P= 0.001); while a significant moderate negative correlation was seen between vitamin D and HbA1c (r= -0.494, P= 0.001) and weak negative correlation was seen with B. Urea (r= -0.2, P= 0.028). No statistically significant correlations were detected between vitamin D and all of age, DM duration, and s. creatinine. Conclusion: Our data suggest a correlation between reduced levels of vitamin D3 and diabetes nephropathy and it may be a potential predictor for both the occurrence and severity of diabetic nephropathy

Hypertrophic scar is an unavoidable result of wound healing following burns and trauma, which remains a challenging problem for clinicians. Previously, we demonstrated that exosomal microRNAs (miRs) of human amniotic epithelial cells accelerated wound healing and inhibited scar formation. However, the underlying mechanism is still unclear. In this particular study, we found that miR-let-7d reduced collagen deposition, and this was accompanied by decreased level of iron content in myofibroblasts. Importantly, inhibition of miR-let-7d in myofibroblasts accelerated collagen deposition and promoted cell proliferation. In addition, bioinformatics prediction combined with classical dual-luciferase reporter gene assay demonstrated that the cellular iron importer divalent metal transporter 1 (DMT1) was a target gene of miR-let-7d, and the miR-let-7d mimics inhibited the expression of DMT1 in myofibroblasts. Moreover, silencing of DMT1 with small interfering RNA (siRNA) reduced the deposition of extracellular matrix. Consistent with the results in vitro, the miR-let-7d mimics effectively ameliorated hypertrophic scar fibrosis in a rabbit ear hypertrophic scar model. Taken together, our results indicated for the first time that miR-let-7d attenuated hypertrophic scar fibrosis through modulation of iron metabolism by reducing iron uptake through DMT1, which may provide a novel therapeutic strategy for hypertrophic scar. [ABSTRACT FROM AUTHOR]

Vascular calcification (VC) has been associated with a risk of cardiovascular diseases. Iron may play a critical role in progressive VC. Therefore, we investigated the effects of iron overload on the aorta of rats. A rat model of iron overload was established by intraperitoneal injection of Iron-Dextran. The levels of iron, calcium, and ALP activity were detected. Von Kossa staining and Perl's staining were conducted. The expression of iron metabolism-related and calcification related factors were examined in the aortic tissue of rats. The results showed serum and aortic tissue iron were increased induced by iron overload and excessive iron induced hepatic and renal damage. In iron overload rats, the expression of divalent metal transporter 1 (DMT1) and hepcidin were higher, but ferroportin1 (FPN1) was lower. Von Kossa staining demonstrated calcium deposition in the aorta of iron overload rats. The calcium content and ALP activity in serum and aortic tissue were increased and iron level in aortic tissue highly correlated with calcium content and ALP activity. The expressions of the osteogenic markers were increased while a decrease of Alpha-smooth muscle actin (α-SMA) in the aortic tissue of iron overload rats. IL-24 was increased during the calcification process induced by iron. Overall, we demonstrated excessive iron accumulation in the aortic tissue and induced organs damage. The iron metabolism-related factors were significantly changed during iron overload. Moreover, we found that iron overload leads to calcium deposition in aorta, playing a key role in the pathological process of VC by mediating osteoblast differentiation factors. [ABSTRACT FROM AUTHOR]

The SLC11/NRAMP proteins constitute a conserved family of metal ion transporters that are expressed in all kingdoms of life. In humans, the two paralogs DMT1 and NRMP1 play an important role in iron homeostasis and the defense against pathogens. SLC11 transporters have evolved an exquisite selectivity for transition metal ions, which facilitates their efficient transport from a large background of Ca2+ and Mg2+. This is accomplished by the evolution of a conserved binding site, which contains besides promiscuous hard ligands, a methionine acting as soft ligand that exclusively coordinates transition metals and thus contributes to the exclusion of alkaline earth metal ions. This site is altered in a branch of prokaryotic family members, which are capable of transporting Mg2+, where the removal of the coordinating methionine and the accompanying expansion of the binding pocket captures this small ion in a hydrated state. The disposition of titratable residues in H+-coupled transition metal ion transporters, that are absent in uncoupled Mg2+ transporters, sheds light on potential coupling mechanisms. In combination, the discussed work has revealed detailed insight into transition metal ion transport and provides a basis for the development of inhibitors of DMT1 as strategy against iron overload disorders.

The TransCure project entitled 'Iron Transporters DMT1 and FPN1' took an interdisciplinary approach combining structural biology, chemistry and physiology to gain new insights into iron transport. Proteins studied included Divalent Metal Transporter 1 (DMT1, SLC11A2), enabling the import of Fe2+ into the cytoplasm, and the iron efflux transporter Ferroportin (FPN1, SLC40A1). The physiology and pathophysiology, and the mechanisms underlying iron transport in the gut, across the placenta and in bone were investigated. Small molecule high-throughput screening was used to identify improved modulators of DMT1. The characterization of DMT1 inhibitors have provided first detailed insights into the pharmacology of a human iron transport protein. In placental physiology, the identification of the expressional and functional alterations and underlying mechanisms in trophoblast cells clarified the association between placental iron transport by DMT1/FPN1 and gestational diabetes mellitus. In bone, iron metabolism was found to differ between cells of the monocyte/ macrophage lineages, including osteoclasts. Osteoclast development and activity depended on exogenous iron, the expression of high levels of the transferrin receptor (TFR) and low levels of FPN1 suggesting the expression of an “iron storage” phenotype by these cells. The principles and main findings of the TransCure studies on transmembrane iron transport physiology are summarized in this review.

Mucosal block (MB) is induced by the oral administration of excess iron (10 mg) and suppresses intestinal iron absorption for 3–72 h. The inhibition of iron absorption is accompanied by the downregulation of molecules associated with intestinal iron absorption. Recently, we found that a smaller amount of iron (1 mg) also induced a transient suppression of iron uptake without affecting gene expression levels (short-acting mucosal block, SAMB), which is specific to iron-deficient rats. In this study, we investigated how the nonheme iron transporters divalent metal transporter 1 (DMT1) and ferroportin (FPN) are involved in the transient suppression of iron uptake in SAMB. To induce SAMB, a test solution containing 1 mg iron was infused into the duodenum loop in iron-sufficient and iron-deficient rats. Total duodenal DMT1 and DMT1-IRE expression were increased during iron deficiency. After 15 min of 1 mg iron loading, the fluorescence intensity of duodenal DMT1 in iron-deficient rats was decreased and was comparable to that in iron-sufficient rats. Internalized DMT1-IRE as puncta was observed at 15 and 60 min after 1 mg iron loading, and the number of punctas was significantly increased after 60 min compared with control. There was no effect of 1 mg iron loading on the intracellular distribution of duodenal FPN. Our results suggest that the decrease and internalization of DMT1-IRE protein may be related, at least in part, to iron uptake suppression in SAMB. [ABSTRACT FROM AUTHOR]

Objective To investigate the effects of bovine lactoferrin (bLF)in inhibiting iron metabolism and lipid peroxidation in rats with intracerebral hemorrhage, and to explore its mechanism of action. Methods Ninety adult healthy male SD rats were randomly divided into sham-operated group, model group and bLF intervention group, with 30 rats in each. The two-step autologous blood right caudate nucleus injection was used to establish the intracerebral hemorrhage models. After successful modeling, the rats in the bLF group were given bLF 85 mg/ (kg-d)by gavage, and the rats in the sham-operated group and the model group were given the same amount of normal saline by gavage for 7 d. The neurological deficits were evaluated by the modified Neurological Severity Score (mNSS)at 1, 3 and 7 d after modeling, respec⁃ tively. After executing rats to extract brain tissues, the histopathological changes were observed by HE staining; iron and reactive oxygen species (ROS)content were detected using a bioreagent kit. The expression levels of ferritin, transferrin receptor (TFR), divalent iron metal transporter (DMT1), iron regulatory protein 2 (IRP2)and the antioxidant glutathione peroxidase 4 (GPX4)in the IRE/IRP regulatory system were detected by Western blotting. The correlation between the iron metabolism-related proteins ferritin, TFR, DMT1, IRP2 and the antioxidant enzyme GPX4 in the bLF intervention group was analyzed by Pearson correlation analysis. Results Compared with the sham-operated group, brain histopathological damage was significantly more severe in the model group; mNSS increased (all P<0. 05)and decreased after reaching the highest score on day 3; iron and ROS content levels increased (all P<0. 05), but there was no statistically signifi⁃ cant difference between the third day and the seventh day; IRE/IRP regulatory system-related proteins (ferritin, TFR, DMT1, IRP2)expression increased (P<0. 05), and it increased to the highest level on day 3 and then decreased; the ex⁃ pression of antioxidant GPX4 decreased (P<0. 05), and it reached the lowest level on day 3 and then increased. Com⁃ pared with the model group, the bLF group showed and itreduced brain histopathological damage and decreased mNSS score (both P<0. 05), decreased expression of iron and ROS content, IRE/IRP regulatory system-related proteins (ferritin, TFR, DMT1, IRP2) (all P<0. 05), and increased expression of antioxidant GPX4 (P<0. 05). Correlation analysis showed that the antioxidant GPX4 was negatively correlated with the expression of ferritin, TFR, DMT1, and IRP2 in the bLF group (all P<0. 05). Conclusions Bovine lactoferrin may down-regulate the expression of ferritin, TFR, DMT1, and IRP2 in the IRE/IRP regulatory system and reduce iron and ROS content, and meanwhile, it up-regulates the expres⁃ sion of the antioxidant GPX4, thereby reducing lipid peroxidation and inhibiting ferroptosis after intracerebral hemorrhage and exerting neuroprotective effects. [ABSTRACT FROM AUTHOR]

The hallmark pathological features of Alzheimer's disease (AD) consist of senile plaques, which are formed by extracellular β-amyloid (Aβ) deposition, and neurofibrillary tangles, which are formed by the hyperphosphorylation of intra-neuronal tau proteins. With the increase in clinical studies, the in vivo imbalance of iron homeostasis and the dysfunction of synaptic plasticity have been confirmed to be involved in AD pathogenesis. All of these mechanisms are constituted by the abnormal accumulation of misfolded or conformationally altered protein aggregates, which in turn drive AD progression. Proteostatic imbalance has emerged as a key mechanism in the pathogenesis of AD. Ubiquitination modification is a major pathway for maintaining protein homeostasis, and protein degradation is primarily carried out by the ubiquitin-proteasome system (UPS). In this review, we provide an overview of the ubiquitination modification processes and related protein ubiquitination degradation pathways in AD, focusing on the microtubule-associated protein Tau, amyloid precursor protein (APP), divalent metal transporter protein 1 (DMT1), and α-amino-3-hyroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors. We also discuss recent advances in ubiquitination-based targeted therapy for AD, with the aim of contributing new ideas to the development of novel therapeutic interventions for AD., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Lin, Gao, Li and Chu.)

Iron is an essential transition metal for its involvement in several crucial biological functions, the most notable being oxygen storage and transport. Due to its high reactivity and potential toxicity, intracellular and extracellular iron levels must be tightly regulated. This is achieved through transport systems that mediate cellular uptake and efflux both at the level of the plasma membrane and on the membranes of lysosomes, endosomes and mitochondria. Among these transport systems, the key players are ferroportin, the only known transporter mediating iron efflux from cells; DMT1, ZIP8 and ZIP14, which on the contrary, mediate iron influx into the cytoplasm, acting on the plasma membrane and on the membranes of lysosomes and endosomes; and mitoferrin, involved in iron transport into the mitochondria for heme synthesis and Fe-S cluster assembly. The focus of this review is to provide an updated view of the physiological role of these membrane proteins and of the pathologies that arise from defects of these transport systems.

Encephalopathy is a frequent and lethal consequence of sepsis. Recently, a growing body of evidence has provided important insights into the role of iron dyshomeostasis in the context of inflammation. The molecular mechanisms underlying iron dyshomeostasis and its relationship with macrophage phenotypes are largely unknown. Here, we aimed to characterize the changes in iron-transporter and storage proteins and the microglia phenotype that occur during the course of sepsis, as well as their relationship with sepsis-induced encephalopathy. We used a cecal ligation and puncture (CLP) murine model that closely resembles sepsis-induced encephalopathy. Rats were subjected to CLP or sham laparotomy, then were neurologically assessed at 6 h, 24 h, and 3 days after sepsis induction. The serum and brain were collected for subsequent biochemical, histological, and immunohistochemical assessment. Here, an iron excess was observed at time points that followed the pro-inflammatory macrophage polarization in CLP-induced encephalopathy. Our results revealed that the upregulation of non-transferrin-bound iron uptake (NTBI) and ferritin reduction appeared to be partially responsible for the excess free iron detected within the brain tissues. We further demonstrated that the microglia were shifted toward the proinflammatory phenotype, leading to persistent neuro-inflammation and neuronal damage after CLP. Taken together, these findings led us to conclude that sepsis increased the susceptibility of the brain to the iron burden via the upregulation of NTBI and the reduction of ferritin, which was concomitantly and correlatively associated with dominance of proinflammatory microglia and could explain the neurological dysfunction observed during sepsis. [ABSTRACT FROM AUTHOR]

Macrophages are at the center of innate pathogen control and iron recycling. Divalent metal transporter 1 (DMT1) is essential for the uptake of non-transferrin-bound iron (NTBI) into macrophages and for the transfer of transferrin-bound iron from the endosome to the cytoplasm. As the control of cellular iron trafficking is central for the control of infection with siderophilic pathogens such as Salmonella Typhimurium, a Gram-negative bacterium residing within the phagosome of macrophages, we examined the potential role of DMT1 for infection control. Bone marrow derived macrophages lacking DMT1 (DMT1fl/flLysMCre(+)) present with reduced NTBI uptake and reduced levels of the iron storage protein ferritin, the iron exporter ferroportin and, surprisingly, of the iron uptake protein transferrin receptor. Further, DMT1-deficient macrophages have an impaired control of Salmonella Typhimurium infection, paralleled by reduced levels of the peptide lipocalin-2 (LCN2). LCN2 exerts anti-bacterial activity upon binding of microbial siderophores but also facilitates systemic and cellular hypoferremia. Remarkably, nifedipine, a pharmacological DMT1 activator, stimulates LCN2 expression in RAW264.7 macrophages, confirming its DMT1-dependent regulation. In addition, the absence of DMT1 increases the availability of iron for Salmonella upon infection and leads to increased bacterial proliferation and persistence within macrophages. Accordingly, mice harboring a macrophage-selective DMT1 disruption demonstrate reduced survival following Salmonella infection. This study highlights the importance of DMT1 in nutritional immunity and the significance of iron delivery for the control of infection with siderophilic bacteria. [ABSTRACT FROM AUTHOR]

Simple Summary: Diabetes mellitus is a disease that affects some metabolic processes, altering the metabolism of trace elements associated with the antioxidant defense system. The insulin-mimetic activity of vanadium has received attention in recent years. However, the use of vanadium as an antidiabetic agent is limited due to its pro-oxidant properties, which are probably caused by interactions with other trace elements. There is currently little information on the interactions between vanadium and zinc, copper and manganese in diabetic rats. For these reasons, the objective of this study was to examine whether diabetic rats treated with bis(maltolato)oxovanadium(IV) (BMOV(IV)) undergo alterations in zinc, copper and manganese homeostasis, and whether such changes are related to the gene expression of the divalent metal transporter 1 (DMT1). The results obtained from this study showed that the metabolism of Zn, Cu and, to a lesser extent, Mn is altered by diabetes and that BMOV(IV) administered to diabetic rats at a dose of 3 mg V/day reverted the alterations in zinc and copper homeostasis produced by diabetes. The effect of this supplementation was possibly mediated by a decrease in food intake and DMT1 gene expression. Our aim was to examine whether vanadium (IV) corrects alterations in zinc, copper and manganese homeostasis, observed in streptozotocin-induced hyperglycemic rats, and whether such changes are related to divalent metal transporter 1 (DMT1) mRNA expression, and antioxidant and proinflammatory parameters. Four groups of Wistar rats were examined: control; hyperglycemic (H); hyperglycemic treated with 1 mg V/day (HV); and hyperglycemic treated with 3 mg V/day (HVH). Vanadium was supplied in drinking water as bis(maltolato)oxovanadium(IV) for five weeks. Zinc, copper and manganese were measured in food, excreta, serum and tissues. DMT1 mRNA expression was quantified in the liver. Hyperglycemic rats showed increased Zn and Cu absorption and content in the liver, serum, kidneys and femurs; DMT1 expression also increased (p < 0.05 in all cases). HV rats showed no changes compared to H rats other than decreased DMT1 expression (p < 0.05). In the HVH group, decreased absorption and tissular content of studied elements (p < 0.05 in all cases) and DMT1 expression compared to H (p < 0.05) were observed. Liver zinc, copper and manganese content correlated positively with glutathione peroxidase activity and negatively with catalase activity (p < 0.05 in both cases). In conclusion, treatment with 3 mg V/d reverted the alterations in zinc and copper homeostasis caused by hyperglycemia, possibly facilitated by decreased DMT1 expression. [ABSTRACT FROM AUTHOR]

Encephalopathy is a frequent and lethal consequence of sepsis. Recently, a growing body of evidence has provided important insights into the role of iron dyshomeostasis in the context of inflammation. The molecular mechanisms underlying iron dyshomeostasis and its relationship with macrophage phenotypes are largely unknown. Here, we aimed to characterize the changes in iron-transporter and storage proteins and the microglia phenotype that occur during the course of sepsis, as well as their relationship with sepsis-induced encephalopathy. We used a cecal ligation and puncture (CLP) murine model that closely resembles sepsis-induced encephalopathy. Rats were subjected to CLP or sham laparotomy, then were neurologically assessed at 6 h, 24 h, and 3 days after sepsis induction. The serum and brain were collected for subsequent biochemical, histological, and immunohistochemical assessment. Here, an iron excess was observed at time points that followed the pro-inflammatory macrophage polarization in CLP-induced encephalopathy. Our results revealed that the upregulation of non-transferrin-bound iron uptake (NTBI) and ferritin reduction appeared to be partially responsible for the excess free iron detected within the brain tissues. We further demonstrated that the microglia were shifted toward the pro-inflammatory phenotype, leading to persistent neuro-inflammation and neuronal damage after CLP. Taken together, these findings led us to conclude that sepsis increased the susceptibility of the brain to the iron burden via the upregulation of NTBI and the reduction of ferritin, which was concomitantly and correlatively associated with dominance of pro-inflammatory microglia and could explain the neurological dysfunction observed during sepsis.

Atherosclerosis is the primary pathological basis of cardiovascular disease. Ferroptosis is a regulated form of cell death, a process of lipid peroxidation driven by iron, which can initiate and promote atherosclerosis. STAT6 is a signal transducer that shows a potential role in regulating ferroptosis, but, the exact role in ferroptosis during atherogenesis remains unclear. The Traditional Chinese Medicine Maijitong granule (MJT) is used for treating cardiovascular disease and shows a potential inhibitory effect on ferroptosis. However, the antiatherogenic effect and the underlying mechanism remain unclear. In this study, we determined the role of STAT6 in ferroptosis during atherogenesis, investigated the antiatherogenic effect of MJT, and determined whether its antiatherogenic effect was dependent on the inhibition of ferroptosis. 8-week-old male LDLR−/− mice were fed a high-fat diet (HFD) at 1st and 10th week, respectively, to assess the preventive and therapeutic effects of MJT on atherosclerosis and ferroptosis. Simultaneously, the anti-ferroptotic effects and mechanism of MJT were determined by evaluating the expression of genes responsible for lipid peroxidation and iron metabolism. Subsequently, we reanalyzed microarray data in the GSE28117 obtained from cells after STAT6 knockdown or overexpression and analyzed the correlation between STAT6 and ferroptosis. Finally, the STAT6−/− mice were fed HFD and injected with AAV-PCSK9 to validate the role of STAT6 in ferroptosis during atherogenesis and revealed the antiatherogenic and anti-ferroptotic effect of MJT. MJT attenuated atherosclerosis by reducing plaque lesion area and enhancing plaque stability in both preventive and therapeutic groups. MJT reduced inflammation via suppressing inflammatory cytokines and inhibited foam cell formation by lowering the LDL level and promoting ABCA1/G1-mediated lipid efflux. MJT ameliorated the ferroptosis by reducing lipid peroxidation and iron dysregulation during atherogenesis. Mechanistically, STAT6 negatively regulated ferroptosis by transcriptionally suppressing SOCS1/p53 and DMT1 pathways. MJT suppressed the DMT1 and SOCS1/p53 via stimulating STAT6 phosphorylation. In addition, STAT6 knockout exacerbated atherosclerosis and ferroptosis, which abolished the antiatherogenic and anti-ferroptotic effects of MJT. STAT6 acts as a negative regulator of ferroptosis and atherosclerosis via transcriptionally suppressing DMT1 and SOCS1 expression and MJT attenuates atherosclerosis and ferroptosis by activating the STAT6-mediated inhibition of DMT1 and SOCS1/p53 pathways, which indicated that STAT6 acts a novel promising therapeutic target to ameliorate atherosclerosis by inhibiting ferroptosis and MJT can serve as a new therapy for atherosclerosis treatment. STAT6 acts as a negative regulator of ferroptosis and atherosclerosis via transcriptionally suppressing DMT1 and SOCS1 expression and MJT attenuates atherosclerosis and ferroptosis by regulating the STAT6-mediated inhibition of DMT1 and SOCS1/p53 pathways. [Display omitted] [ABSTRACT FROM AUTHOR]

Divalent metal-iron transporter 1 (DMT1) is a mammalian iron transporter encoded by the SLC11A2 gene. DMT1 has a vital role in iron homeostasis by mediating iron uptake in the intestine and kidneys and by recovering iron from recycling endosomes after transferrin endocytosis. Mutations in SLC11A2 cause an ultra-rare hypochromic microcytic anemia with iron overload (AHMIO1), which has been described in eight patients so far. Here, we report two novel cases of this disease. The first proband is homozygous for a new SLC11A2 splicing variant (c.762 + 35A > G), becoming the first ever patient reported with a SLC11A2 splicing mutation in homozygosity. Splicing studies performed in this work confirm its pathogenicity. The second proband harbors the previously reported DMT1 G75R mutation in homozygosis. Functional studies with the G75R mutation in HuTu 80 cells demonstrate that this mutation results in improper DMT1 accumulation in lysosomes, which correlates with a significant decrease in DMT1 levels in patient-derived lymphoblast cell lines (LCLs). We also suggest that recombinant erythropoietin would be an adequate therapeutic approach for AHMIO1 patients as it improves their anemic state and may possibly contribute to mobilizing excessive hepatic iron. [ABSTRACT FROM AUTHOR]

Extracellular vesicles (EVs) are important mediators of intercellular communication. However, EV biogenesis remains poorly understood. We previously defined a role for Arrdc4 (Arrestin domain containing protein 4), an adaptor for Nedd4 family ubiquitin ligases, in the biogenesis of EVs. Here we report that ubiquitination of Arrdc4 is critical for its role in EV secretion. We identified five potential ubiquitinated lysine residues in Arrdc4 using mass spectrometry. By analysing Arrdc4 lysine mutants we discovered that lysine 270 (K270) is critical for Arrdc4 function in EV biogenesis. Arrdc4K270R mutation caused a decrease in the number of EVs released by cells compared to Arrdc4WT, and a reduction in trafficking of divalent metal transporter (DMT1) into EVs. Furthermore, we also observed a decrease in DMT1 activity and an increase in its intracellular degradation in the presence of Arrdc4K270R. K270 was found to be ubiquitinated with K‐29 polyubiquitin chains by the ubiquitin ligase Nedd4‐2. Thus, our results uncover a novel role of K‐29 polyubiquitin chains in Arrdc4‐mediated EV biogenesis and protein trafficking. [ABSTRACT FROM AUTHOR]

• DMT1 genotyping in lead-exposed may serve as a prognostic marker of lead toxicity. • Heterozygous CA carriers were predisposed to higher Pb-B levels. • Heterozygous CA presented a negative correlation of Pb-B levels and hemoglobin. Lead (Pb) is an environmental and public health toxicant. It affects various organ systems of the body, thereby disrupting their normal functions. To date, several genes that are known to influence the mechanism of action of lead and toxicity have been studied. Among them, the iron transporter gene, SLC11A2 (Solute Carrier 11 group A member 2) which codes for the transmembrane protein, DMT1 (Divalent Metal Transporter 1) has shown to transport other metals including zinc, copper, and lead. We investigated the influence of DMT1 polymorphism (rs224589) on blood lead (Pb-B) levels. In the present study, we enrolled 113 lead-exposed workers and performed a comprehensive biochemical analysis and genetic composition. The frequency of DMT1 variants observed in the total subjects (n = 113) was 42 % for homozygous CC wild type, 54 % for heterozygous CA, and 4 % for homozygous AA mutant. The heterozygous CA carriers presented higher Pb-B levels compared to wild type CC and mutant AA carriers. Further, a negative association was observed between Pb-B levels and hemoglobin in heterozygous CA carriers. Hence, C allele may be the risk allele that contributes to increased susceptibility to high Pb-B retention, and genotyping of DMT1 in lead exposed subjects might be used as a prognostic marker to impede organ damage due to lead toxicity. [ABSTRACT FROM AUTHOR]

Abstract Extracellular vesicles (EVs) are important mediators of intercellular communication. However, EV biogenesis remains poorly understood. We previously defined a role for Arrdc4 (Arrestin domain containing protein 4), an adaptor for Nedd4 family ubiquitin ligases, in the biogenesis of EVs. Here we report that ubiquitination of Arrdc4 is critical for its role in EV secretion. We identified five potential ubiquitinated lysine residues in Arrdc4 using mass spectrometry. By analysing Arrdc4 lysine mutants we discovered that lysine 270 (K270) is critical for Arrdc4 function in EV biogenesis. Arrdc4K270R mutation caused a decrease in the number of EVs released by cells compared to Arrdc4WT, and a reduction in trafficking of divalent metal transporter (DMT1) into EVs. Furthermore, we also observed a decrease in DMT1 activity and an increase in its intracellular degradation in the presence of Arrdc4K270R. K270 was found to be ubiquitinated with K‐29 polyubiquitin chains by the ubiquitin ligase Nedd4‐2. Thus, our results uncover a novel role of K‐29 polyubiquitin chains in Arrdc4‐mediated EV biogenesis and protein trafficking.

Background: Microglial cell iron load and inflammatory activation are significant hallmarks of late-stage Alzheimer's disease (AD). In vitro , microglia preferentially upregulate the iron importer, divalent metal transporter 1 (DMT1, gene name Slc11a2 ) in response to inflammatory stimuli, and excess iron can augment cellular inflammation, suggesting a feed-forward loop between iron import mechanisms and inflammatory signaling. However, it is not understood whether microglial iron import mechanisms directly contribute to inflammatory signaling and chronic disease in vivo . These studies determined the effects of microglial-specific knockdown of Slc11a2 on AD-related cognitive decline and microglial transcriptional phenotype., Methods: In vitro experiments and RT-qPCR were used to assess a role for DMT1 in amyloid-β-associated inflammation. To determine the effects of microglial Slc11a2 knockdown on AD-related phenotypes in vivo , triple-transgenic Cx3cr1 Cre - ERT2 ; Slc11a2 flfl ; APP/PS1 + or - mice were generated and administered corn oil or tamoxifen to induce knockdown at 5-6 months of age. Both sexes underwent behavioral analyses to assess cognition and memory (12-15 months of age). Hippocampal CD11b + microglia were magnetically isolated from female mice (15-17 months) and bulk RNA-sequencing analysis was conducted., Results: DMT1 inhibition in vitro robustly decreased Aβ-induced inflammatory gene expression and cellular iron levels in conditions of excess iron. In vivo , Slc11a2 KD APP/PS1 female, but not male, mice displayed a significant worsening of memory function in Morris water maze and a fear conditioning assay, along with significant hyperactivity compared to control WT and APP/PS1 mice. Hippocampal microglia from Slc11a2 KD APP/PS1 females displayed significant increases in Enpp2 , Ttr , and the iron-export gene, Slc40a1 , compared to control APP/PS1 cells. Slc11a2 KD cells from APP/PS1 females also exhibited decreased expression of markers associated with disease-associated microglia (DAMs), such as Apoe , Ctsb, Csf1 , and Hif1α ., Conclusions: This work suggests a sex-specific role for microglial iron import gene Slc11a2 in propagating behavioral and cognitive phenotypes in the APP/PS1 model of AD. These data also highlight an association between loss of a DAM-like phenotype in microglia and cognitive deficits in Slc11a2 KD APP/PS1 female mice. Overall, this work illuminates an iron-related pathway in microglia that may serve a protective role during disease and offers insight into mechanisms behind disease-related sex differences., Competing Interests: Competing Interests The authors declare that they have no competing interests.

The aim of this study was to determine the iron (Fe) concentration profile within the lumen of the S2 renal proximal convoluted tubule (PCT) and to resolve whether this nephron segment transported Fe. To do this, we performed in vivo renal micropuncture on Wistar rats, collected PCT tubular fluid from superficial nephrons, and measured Fe concentration. The Fe concentration profile along the S2 PCT suggested significant Fe reabsorption. Proximal tubules were also microperfused in vivo with physiological solutions containing Fe and Zn, Cu, Mn, or Cd. PCTs perfused with 12μmol.l−1 55FeCl3 reabsorbed 105.2±12.7 fmol.mm−1.min−1 Fe, 435±52pmol.mm-1.min−1 Na, and 2.7±0.2nl.mm−1.min−1 water (mean ± SEM; n =19). Addition of ascorbate (1mmol.l−1) to the perfusate did not significantly alter Fe, Na, or water reabsorption. Supplementing the control perfusate with 60μmol.l−1 FeSO4 significantly decreased 55Fe uptake. Recalculating for the altered molar activity following addition of unlabeled Fe revealed a three-fold increase in Fe flux. Addition to the perfusate 12μmol.l−1 CuSO4, MnSO4, CdSO4, or ZnSO4 did not affect Fe, Na, or water flux. In conclusion, (1) in vivo , S2 PCTs of rat reabsorb Fe and (2) Fe is reabsorbed along the PCT via a pathway that is insensitive to Cu, Mn, Cd, or Zn. Together, these data demonstrate for the first time the hitherto speculated process of renal Fe filtration and subsequent tubular Fe reabsorption in a living mammal. [ABSTRACT FROM AUTHOR]

Purpose: Temozolomide is used in first-line treatment for glioblastoma. However, chemoresistance to temozolomide is common in glioma patients. In addition, mechanisms for the anti-tumor effects of temozolomide are largely unknown. Ferroptosis is a form of programmed cell death triggered by disturbed redox homeostasis, overloaded iron, and increased lipid peroxidation. The present study was performed to elucidate the involvement of ferroptosis in the anti-tumor mechanisms of temozolomide. Materials and Methods: We utilized the CCK8 assay to evaluate cytotoxicity. Levels of lactate dehydrogenase (LDH), malondialdehyde (MDA), iron, and glutathione (GSH) were measured. Flow cytometry and fluorescence microscope were used to detect the production of reactive oxygen species (ROS). Western blotting, RT-PCR and siRNA transfection were used to investigate molecular mechanisms. Results: Temozolomide increased the levels of LDH, MDA, and iron and reduced GSH levels in TG905 cells. Furthermore, we found that ROS levels and DMT1 expression were elevated in TG905 cells treated with temozolomide and were accompanied by a decrease in the expression of glutathione peroxidase 4, indicating an iron-dependent cell death, ferroptosis. Our results also showed that temozolomide-induced ferroptosis is associated with regulation of the Nrf2/HO-1 pathway. Conversely, DMT1 knockdown by siRNA evidently blocked temozolomide-induced ferroptosis in TG905 cells. Conclusion: Taken together, our findings indicate that temozolomide may suppress cell growth partly by inducing ferroptosis by targeting DMT1 expression in glioblastoma cells. [ABSTRACT FROM AUTHOR]

The aim of this study was to determine the iron (Fe) concentration profile within the lumen of the S2 renal proximal convoluted tubule (PCT) and to resolve whether this nephron segment transported Fe. To do this, we performed in vivo renal micropuncture on Wistar rats, collected PCT tubular fluid from superficial nephrons, and measured Fe concentration. The Fe concentration profile along the S2 PCT suggested significant Fe reabsorption. Proximal tubules were also microperfused in vivo with physiological solutions containing Fe and Zn, Cu, Mn, or Cd. PCTs perfused with 12μmol.l−1 55FeCl3 reabsorbed 105.2±12.7 fmol.mm−1.min−1 Fe, 435±52pmol.mm-1.min−1 Na, and 2.7±0.2nl.mm−1.min−1 water (mean ± SEM; n=19). Addition of ascorbate (1mmol.l−1) to the perfusate did not significantly alter Fe, Na, or water reabsorption. Supplementing the control perfusate with 60μmol.l−1 FeSO4 significantly decreased 55Fe uptake. Recalculating for the altered molar activity following addition of unlabeled Fe revealed a three-fold increase in Fe flux. Addition to the perfusate 12μmol.l−1 CuSO4, MnSO4, CdSO4, or ZnSO4 did not affect Fe, Na, or water flux. In conclusion, (1) in vivo, S2 PCTs of rat reabsorb Fe and (2) Fe is reabsorbed along the PCT via a pathway that is insensitive to Cu, Mn, Cd, or Zn. Together, these data demonstrate for the first time the hitherto speculated process of renal Fe filtration and subsequent tubular Fe reabsorption in a living mammal.

The liver hormone hepcidin regulates systemic iron homeostasis. Hepcidin is also expressed by the kidney, but exclusively in distal nephron segments. Several studies suggest hepcidin protects against kidney damage involving Fe2+ overload. The nephrotoxic non-essential metal ion Cd2+ can displace Fe2+ from cellular biomolecules, causing oxidative stress and cell death. The role of hepcidin in Fe2+ and Cd2+ toxicity was assessed in mouse renal cortical [mCCD(cl.1)] and inner medullary [mIMCD3] collecting duct cell lines. Cells were exposed to equipotent Cd2+ (0.5–5 μmol/l) and/or Fe2+ (50–100 μmol/l) for 4–24 h. Hepcidin (Hamp1) was transiently silenced by RNAi or overexpressed by plasmid transfection. Hepcidin or catalase expression were evaluated by RT-PCR, qPCR, immunoblotting or immunofluorescence microscopy, and cell fate by MTT, apoptosis and necrosis assays. Reactive oxygen species (ROS) were detected using CellROX™ Green and catalase activity by fluorometry. Hepcidin upregulation protected against Fe2+-induced mIMCD3 cell death by increasing catalase activity and reducing ROS, but exacerbated Cd2+-induced catalase dysfunction, increasing ROS and cell death. Opposite effects were observed with Hamp1 siRNA. Similar to Hamp1 silencing, increased intracellular Fe2+ prevented Cd2+ damage, ROS formation and catalase disruption whereas chelation of intracellular Fe2+ with desferrioxamine augmented Cd2+ damage, corresponding to hepcidin upregulation. Comparable effects were observed in mCCD(cl.1) cells, indicating equivalent functions of renal hepcidin in different collecting duct segments. In conclusion, hepcidin likely binds Fe2+, but not Cd2+. Because Fe2+ and Cd2+ compete for functional binding sites in proteins, hepcidin affects their free metal ion pools and differentially impacts downstream processes and cell fate. [ABSTRACT FROM AUTHOR]

Metal implants are used worldwide, with millions of nails, plates, and fixtures grafted during orthopedic surgeries. Iron is the most common element of these metal implants. As time passes, implants can be corroded and iron can be released. Ionized iron permeates the surrounding tissues and enters circulation; importantly, iron ions pass through the blood–brain barrier. Can iron from implants represent a risk factor for neurological diseases? This remains an unanswered question. In this study, we discovered that patients with metal implants delivered through orthopedic surgeries have higher incidence of Parkinson's disease or ischemic stroke compared to patients who underwent similar surgeries but did not have implants. Concentration of serum iron and ferritin was increased in subjects with metal implants. In experiments in vivo, we found that injection of iron dextran selectively decreased the presence of divalent metal transporter 1 (DMT1) in neurons through increasing the expression of Ndfip1, which degrades DMT1 and does not exist in glial cells. At the same time, excess of iron increased expression of DMT1 in astrocytes and microglial cells and triggered reactive astrogliosis and microgliosis. Facing the attack of excess iron, glial cells act as neuroprotectors to accumulate more extracellular iron by upregulating DMT1, whereas neurons limit iron uptake through increasing DMT1 degradation. Cerebral accumulation of iron in animals is associated with impaired cognition, locomotion, and mood. Excess iron from surgical implants thus can affect neural cells and may be regarded as a risk factor for neurodegeneration. Graphical Abstract [ABSTRACT FROM AUTHOR]

Excess iron contributes to Hemophilic Arthropathy (HA) development. Divalent metal transporter 1 (DMT1) delivers iron into the cytoplasm, thus regulating iron homeostasis. We aimed to investigate whether DMT1-mediated iron homeostasis is involved in bleeding-induced cartilage degeneration and the molecular mechanisms underlying iron overload-induced chondrocyte damage. This study established an in vivo HA model by puncturing knee joints of coagulation factor VIII gene knockout mice with a needle, and mimicked iron overload conditions in vitro by treatment of Ferric ammonium citrate (FAC). We demonstrated that blood exposure caused iron overload and cartilage degeneration, as well as elevated expression of DMT1. Furthermore, DMT1 silencing alleviated blood-induced iron overload and cartilage degeneration. In hemophilic mice, articular cartilage degeneration was also suppressed by intro-articularly injection of DMT1 adeno-associated virus 9 (AAV9). Mechanistically, RNA-sequencing analysis indicated the association between iron overload and cGAS-STING pathway. Further, iron overload triggered mtDNA-cGAS-STING pathway activation, which could be effectively mitigated by DMT1 silencing. Additionally, we discovered that RU.521, a potent Cyclic GMP-AMP Synthase (cGAS) inhibitor, successfully suppressed the downward cascades of cGAS-STING, thereby protecting against chondrocyte damage. Taken together, DMT1-mediated iron overload promotes chondrocyte damage and murine HA development, and targeted DMT1 may provide therapeutic and preventive approaches in HA. Schematic representation for the role of DMT1-mediated iron overload and mtDNA-cGAS-STING axis in cartilage degeneration during HA pathogenesis. In hemophilic arthropathy (HA), after joint bleeding, iron is released from ruptured red blood cells. Mediated by upregulated DMT1 expression, iron enters chondrocytes, leading to cellular iron overload. Excessive iron causes mitochondrial dysfunction including mitochondrial DNA leakage. After mitochondrial DNA was released into the cytoplasm, cGAS binds to it and activates STING as well as downstream signaling pathways including the NF-κB pathway, leading to the production of pro-inflammatory cytokines. [Display omitted] • Blood exposure directly caused iron overload and cartilage degeneration. • Blood exposure increased the expression of DMT1 in chondrocytes. • Inhibiting iron overload by silencing DMT1 alleviated cartilage degeneration caused by blood exposure. • DMT1-mediated iron overload contributed to mitochondrial dysfunction and activation of mtDNA-cGAS-STING pathway. • Inhibition of mtDNA-cGAS-STING axis with RU.521 protected against chondrocyte damage induced by iron overload. [ABSTRACT FROM AUTHOR]

Macrophages are at the center of innate pathogen control and iron recycling. Divalent metal transporter 1 (DMT1) is essential for the uptake of non-transferrin-bound iron (NTBI) into macrophages and for the transfer of transferrin-bound iron from the endosome to the cytoplasm. As the control of cellular iron trafficking is central for the control of infection with siderophilic pathogens such as Salmonella Typhimurium, a Gram-negative bacterium residing within the phagosome of macrophages, we examined the potential role of DMT1 for infection control. Bone marrow derived macrophages lacking DMT1 (DMT1fl/flLysMCre(+)) present with reduced NTBI uptake and reduced levels of the iron storage protein ferritin, the iron exporter ferroportin and, surprisingly, of the iron uptake protein transferrin receptor. Further, DMT1-deficient macrophages have an impaired control of Salmonella Typhimurium infection, paralleled by reduced levels of the peptide lipocalin-2 (LCN2). LCN2 exerts anti-bacterial activity upon binding of microbial siderophores but also facilitates systemic and cellular hypoferremia. Remarkably, nifedipine, a pharmacological DMT1 activator, stimulates LCN2 expression in RAW264.7 macrophages, confirming its DMT1-dependent regulation. In addition, the absence of DMT1 increases the availability of iron for Salmonella upon infection and leads to increased bacterial proliferation and persistence within macrophages. Accordingly, mice harboring a macrophage-selective DMT1 disruption demonstrate reduced survival following Salmonella infection. This study highlights the importance of DMT1 in nutritional immunity and the significance of iron delivery for the control of infection with siderophilic bacteria.

Our aim was to examine whether vanadium (IV) corrects alterations in zinc, copper and manganese homeostasis, observed in streptozotocin-induced hyperglycemic rats, and whether such changes are related to divalent metal transporter 1 (DMT1) mRNA expression, and antioxidant and proinflammatory parameters. Four groups of Wistar rats were examined: control; hyperglycemic (H); hyperglycemic treated with 1 mg V/day (HV); and hyperglycemic treated with 3 mg V/day (HVH). Vanadium was supplied in drinking water as bis(maltolato)oxovanadium(IV) for five weeks. Zinc, copper and manganese were measured in food, excreta, serum and tissues. DMT1 mRNA expression was quantified in the liver. Hyperglycemic rats showed increased Zn and Cu absorption and content in the liver, serum, kidneys and femurs; DMT1 expression also increased (p < 0.05 in all cases). HV rats showed no changes compared to H rats other than decreased DMT1 expression (p < 0.05). In the HVH group, decreased absorption and tissular content of studied elements (p < 0.05 in all cases) and DMT1 expression compared to H (p < 0.05) were observed. Liver zinc, copper and manganese content correlated positively with glutathione peroxidase activity and negatively with catalase activity (p < 0.05 in both cases). In conclusion, treatment with 3 mg V/d reverted the alterations in zinc and copper homeostasis caused by hyperglycemia, possibly facilitated by decreased DMT1 expression.

Divalent metal-iron transporter 1 (DMT1) is a mammalian iron transporter encoded by the SLC11A2 gene. DMT1 has a vital role in iron homeostasis by mediating iron uptake in the intestine and kidneys and by recovering iron from recycling endosomes after transferrin endocytosis. Mutations in SLC11A2 cause an ultra-rare hypochromic microcytic anemia with iron overload (AHMIO1), which has been described in eight patients so far. Here, we report two novel cases of this disease. The first proband is homozygous for a new SLC11A2 splicing variant (c.762 + 35A > G), becoming the first ever patient reported with a SLC11A2 splicing mutation in homozygosity. Splicing studies performed in this work confirm its pathogenicity. The second proband harbors the previously reported DMT1 G75R mutation in homozygosis. Functional studies with the G75R mutation in HuTu 80 cells demonstrate that this mutation results in improper DMT1 accumulation in lysosomes, which correlates with a significant decrease in DMT1 levels in patient-derived lymphoblast cell lines (LCLs). We also suggest that recombinant erythropoietin would be an adequate therapeutic approach for AHMIO1 patients as it improves their anemic state and may possibly contribute to mobilizing excessive hepatic iron.

The SARS-CoV-2 virus that is the cause of coronavirus disease 2019 (COVID-19) affects not only peripheral organs such as the lungs and blood vessels, but also the central nervous system (CNS)—as seen by effects on smell, taste, seizures, stroke, neuropathological findings and possibly, loss of control of respiration resulting in silent hypoxemia. COVID-19 induces an inflammatory response and, in severe cases, a cytokine storm that can damage the CNS. Antimalarials have unique properties that distinguish them from other anti-inflammatory drugs. (A) They are very lipophilic, which enhances their ability to cross the blood-brain barrier (BBB). Hence, they have the potential to act not only in the periphery but also in the CNS, and could be a useful addition to our limited armamentarium against the SARS-CoV-2 virus. (B) They are non-selective inhibitors of phospholipase A2 isoforms, including cytosolic phospholipase A2 (cPLA2). The latter is not only activated by cytokines but itself generates arachidonic acid, which is metabolized by cyclooxygenase (COX) to pro-inflammatory eicosanoids. Free radicals are produced in this process, which can lead to oxidative damage to the CNS. There are at least 4 ways that antimalarials could be useful in combating COVID-19. (1) They inhibit PLA2. (2) They are basic molecules capable of affecting the pH of lysosomes and inhibiting the activity of lysosomal enzymes. (3) They may affect the expression and Fe2+/H+ symporter activity of iron transporters such as divalent metal transporter 1 (DMT1), hence reducing iron accumulation in tissues and iron-catalysed free radical formation. (4) They could affect viral replication. The latter may be related to their effect on inhibition of PLA2 isoforms. Inhibition of cPLA2 impairs an early step of coronavirus replication in cell culture. In addition, a secretory PLA2 (sPLA2) isoform, PLA2G2D, has been shown to be essential for the lethality of SARS-CoV in mice. It is important to take note of what ongoing clinical trials on chloroquine and hydroxychloroquine can eventually tell us about the use of antimalarials and other anti-inflammatory agents, not only for the treatment of COVID-19, but also for neurovascular disorders such as stroke and vascular dementia. [ABSTRACT FROM AUTHOR]

Iron deficiency (ID) has been shown to affect central nervous system (CNS) development and induce hypomyelination. Previous work from our laboratory in a gestational ID model showed that both oligodendrocyte (OLG) and astrocyte (AST) maturation was impaired. To explore the contribution of AST iron to the myelination process, we generated an in vitro ID model by silencing divalent metal transporter 1 (DMT1) in AST (siDMT1 AST) or treating AST with Fe 3+ chelator deferoxamine (DFX; DFX AST). siDMT1 AST showed no changes in proliferation but remained immature. Co-cultures of oligodendrocyte precursors cells (OPC) with siDMT1 AST and OPC cultures incubated with siDMT1 AST-conditioned media (ACM) rendered a reduction in OPC maturation. These findings correlated with a decrease in the expression of AST-secreted factors IGF-1, NRG-1, and LIF, known to promote OPC differentiation. siDMT1 AST also displayed increased mitochondrial number and reduced mitochondrial size as compared to control cells. DFX AST also remained immature and DFX AST-conditioned media also hampered OPC maturation in culture, in keeping with a decrease in the expression of AST-secreted growth factors IGF-1, NRG-1, LIF, and CNTF. DFX AST mitochondrial morphology and number showed results similar to those observed in siDMT1 AST. In sum, our results show that ID, induced through two different methods, impacts AST maturation and mitochondrial functioning, which in turn hampers OPC differentiation., (© 2024 Wiley Periodicals LLC.)

Summary: Excessive iron accumulation in the heart causes iron overload cardiomyopathy (IOC), which initially presents as diastolic dysfunction and arrhythmia but progresses to systolic dysfunction and end-stage heart failure when left untreated. However, the mechanisms of iron-related cardiac injury and how iron accumulates in human cardiomyocytes are not well understood. Herein, using human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs), we model IOC and screen for drugs to rescue the iron overload phenotypes. Human iPSC-CMs under excess iron exposure recapitulate early-stage IOC, including oxidative stress, arrhythmia, and contractile dysfunction. We find that iron-induced changes in calcium kinetics play a critical role in dysregulation of CM functions. We identify that ebselen, a selective divalent metal transporter 1 (DMT1) inhibitor and antioxidant, could prevent the observed iron overload phenotypes, supporting the role of DMT1 in iron uptake into the human myocardium. These results suggest that ebselen may be a potential preventive and therapeutic agent for treating patients with secondary iron overload.

Clinical studies suggest that pregnant women with elevated iron levels are more vulnerable to develop gestational diabetes mellitus (GDM), but the causes and underlying mechanisms are unknown. We hypothesized that hyperglycemia induces cellular stress responses leading to dysregulated placental iron homeostasis. Hence, we compared the expression of genes/proteins involved in iron homeostasis in placentae from GDM and healthy pregnancies (n = 11 each). RT‐qPCR and LC‐MS/MS analyses revealed differential regulation of iron transporters/receptors (DMT1/FPN1/ZIP8/TfR1), iron sensors (IRP1), iron regulators (HEPC), and iron oxidoreductases (HEPH/Zp). To identify the underlying mechanisms, we adapted BeWo trophoblast cells to normoglycemic (N), hyperglycemic (H), and hyperglycemic‐hyperlipidemic (HL) conditions and assessed Fe3+‐uptake, expression patterns, and cellular pathways involving oxidative stress (OS), ER‐stress, and autophagy. H and HL induced alterations in cellular morphology, differential iron transporter expression, and reduced Fe3+‐uptake confirming the impact of hyperglycemia on iron transport observed in GDM patients. Pathway analysis and rescue experiments indicated that dysregulated OS and disturbed autophagy processes contribute to the reduced placental iron transport under hyperglycemic conditions. These adaptations could represent a protective mechanism preventing the oxidative damage for both fetus and placenta caused by highly oxidative iron. In pregnancies with risk for GDM, antioxidant treatment, and controlled iron supplementation could help to balance placental OS levels protecting mother and fetus from impaired iron homeostasis. [ABSTRACT FROM AUTHOR]

Iron is an essential cofactor for many cellular enzymes involved in myelin synthesis, and iron homeostasis unbalance is a central component of peripheral neuropathies. However, iron absorption and management in the PNS are poorly understood. To study iron metabolism in Schwann cells (SCs), we have created 3 inducible conditional KO mice in which three essential proteins implicated in iron uptake and storage, the divalent metal transporter 1 (DMT1), the ferritin heavy chain (Fth), and the transferrin receptor 1 (Tfrl), were postnatally ablated specifically in SCs. Deleting DMT1, Fth, or Tfrl in vitro significantly reduce SC proliferation, maturation, and the myelination of DRG axons. This was accompanied by an important reduction in iron incorporation and storage. When these proteins were KO in vivo during the first postnatal week, the sciatic nerve of all 3 conditional KO animals displayed a significant reduction in the synthesis of myelin proteins and in the percentage of myelinated axons. Knocking out Fth produced the most severe phenotype, followed by DMT1 and, last, Tfrl. Importantly, DMT1 as well as Fth KO mice showed substantial motor coordination deficits. In contrast, deleting these proteins in mature myelinating SCs results in milder phenotypes characterized by small reductions in the percentage of myelinated axons and minor changes in they-ratio of myelinated axons. These results indicate that DMT1, Fth, and Tfrl are critical proteins for early postnatal iron uptake and storage in SCs and, as a consequence, for the normal myelination of the PNS. [ABSTRACT FROM AUTHOR]

Osteoporosis (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. RAW264.7 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. We 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. Therefore, the study revealed that SPI1 could inhibit Hepcidin expression contribute to iron accumulation and osteoclast formation via DMT1 signaling activation in mouse with OVX. • SPI1 transcriptionally repressed the expression of Hepcidin. • SPI1 increased DMT1 and facilitated the iron accumulation and osteoclast formation. • Overexpression of DMT1 facilitated osteoclast differentiation and iron accumulation in OVX mice. [ABSTRACT FROM AUTHOR]

A high-mineral polysaccharide (LP) was extracted from Lycium barbarum L. leaves, and the absorption of endogenous calcium, iron, and zinc was evaluated in Caco-2 cells and rat models. The absorption of these minerals ranged from 2.7 to 5.9% in Caco-2 cells, compared with 60.5–81.9% in rat models. LP had a compact globular conformation at pH ∼ 7.2 when co-cultured with Caco-2 cells, and degraded to a loosely linear structure at pH ∼ 6.7 in rat cecum. The degraded polysaccharides didn’t change the mucosal morphometry but caused higher expression of calcium transporter 1 (CAT1) and zinc transporter 1 (ZIP1) proteins in rat cecum. Furthermore, fermentation of polysaccharides in cecum also caused elevated levels of serum cytokines, including TNF-α and IL-6. GL polysaccharides could increase the bioavailability of endogenous calcium via changes in the molecular conformation and through upregulation of the expression of mineral transporter proteins.