Regulator Of Iron Homeostasis Research Articles

Alphafold2-Assisted Structure-Function Analysis of Human Erythroferrone Relevant for Its Iron-Regulatory Function

Erythroferrone (ERFE), an erythrokine that helps mobilize iron for developing erythrocytes, acts by decreasing the production of hepcidin, through sequestration of bone morphogenetic proteins (BMPs) that bind to BMP receptors to activate hepcidin transcription. We used Alphafold2 (AF2) to model the conformation of human erythroferrone monomers and various multimers, with or without interactions with BMPs, with emphasis on the principal ligand, the BMP2/6 heterodimer. We then tested the models by targeted mutations to disrupt key interactions suggested by the models, and assessed the effect of the mutations on bioactivity and BMP binding. ERFE is a member of the C1q/TNFa family of proteins known for containing N-terminal unstructured regions and C-terminal TNFa-like globular heads that promote the formation of a spectrum of biologically significant multimers. After cleavage by a prohormone convertase, the ERFE molecule consists of an unstructured N-terminal segment residues 43-185 and a globular TNF-like head 186-354. We found that a segment of the N-terminal region of the molecule-segment 43-148 of the 354 amino acid protein-was sufficient to bind BMPs and suppress hepcidin; the larger C-terminal region does neither. For bioassays, we prepared ERFE or its mutants in HEK293 mammalian cells and tested these without purification, so as not to disrupt their mixture of multimeric conformations. The Hep3B human liver cell line was used for bioactivity assays with the readout of hepcidin mRNA concentrations, measured by qRT-PCR. To avoid the confounding effects of ERFE multimerization driven by the TNFa-like globular regions on avidity, we purified untagged ERFE N-terminal (43-148) segments or their mutants from bacteria, and measured the binding between these and BMPs using surface plasmon resonance. These forms lacked TNFa-like heads and, unlike full length ERFE, did not form multimers in nondenaturing PAGE. We identified and explored several structural elements within the ERFE N-terminus. All models of BMP interaction with ERFE predicted the binding of a tryptophan-containing hydrophobic helix of ERFE to a deep groove formed at the interface of all BMP dimers (BMP2/6 in cyan/green, ERFE in magenta; interaction in callout box, left panel). The highly evolutionarily conserved hydrophobic region (residues 81-86) was required for bioactivity and for tight binding to BMPs. Deletion of the hydrophobic segment or W82A substitution of the strictly conserved tryptophan ablated bioactivity and greatly decreased the binding of ERFE to BMPs. In nearly all models, a positively-charged cationic segment and the collagen-like segment made no contact with BMPs. However, the cationic segment was required for activity (right panel) although its deletion had only minor effect on binding to BMP. The specific arrangement of arginines and lysines in this segment did not matter for bioactivity or binding. We showed that the segment was essential for heparin-binding and propose that it may serve to concentrate ERFE at the site of its activity. Deletion of the collagen segment or the more distal helical segment (green) impaired IC 50 much less (50-100fold) but also substantially disrupted monomeric binding. The collagen segment forms a triple helix in trimeric models of ERFE, but the helix is disrupted by BMP binding. Secondary binding contacts between ERFE and BMPs in AF2 varied depending on the oligomeric state of erythroferrone. In monomers and dimers, the helical segment made contact with the contralateral wing of the BMP dimer. Remarkably, both primary and secondary binding interactions of BMPs were structurally similar to the BMP interactions with their receptors or known antagonists. Nondenaturing PAGE suggested that the predominant form of ERFE was a hexamer. The AF2 structure of hexameric ERFE (magenta) with three molecules of BMP2/6 (cyan-green) showed that each BMP heterodimer bound two ERFE proximal helical segments including 81-86, within each of its two BMP2/6 interfacial grooves (left panel) but unlike in monomers and dimers, the secondary interaction did not involve the distal helical segment but the globular heads (red trimers on each side). Despite their reductionist nature, these studies provide useful structural predictions that should allow better pharmacologic targeting of this important erythroid regulator of iron homeostasis.

TroR is the primary regulator of the iron homeostasis transcription network in the halophilic archaeon Haloferax volcanii.

Maintaining theintracellular iron concentration within the homeostatic range is vital to meet cellular metabolic needs and reduce oxidative stress. Previous research revealed that the haloarchaeon Halobacterium salinarum encodes four diphtheria toxin repressor (DtxR) family transcription factors (TFs) that together regulate the iron response through an interconnected transcriptional regulatory network (TRN). However, the conservation of the TRN andthe metal specificity of DtxR TFsremained poorly understood. Here we identified and characterized the TRN of Haloferax volcanii for comparison. Genetic analysis demonstrated that Hfx. volcanii relies on three DtxR transcriptional regulators (Idr, SirR, and TroR), with TroR as the primary regulator of iron homeostasis. Bioinformatics and molecular approaches revealed that TroR binds a conserved cis-regulatory motif located ∼100 nt upstream of the start codon of iron-related target genes. Transcriptomics analysis demonstrated that, under conditions of iron sufficiency, TroR repressed iron uptake and induced iron storage mechanisms. TroR repressed the expression of one other DtxR TF, Idr. This reduced DtxR TRN complexity relative to that of Hbt. salinarum appeared correlated with natural variations in iron availability. Based on these data, we hypothesize that variable environmental conditions such as iron availability appear to select forincreasing TRN complexity.

Hepcidin's Antimalarial Arsenal: Safeguarding the Host

Malaria remains a significant global health threat, causing hundreds of thousands of deaths annually. Understanding the host's protective mechanisms against this parasitic infection is crucial for developing effective prevention and treatment strategies. Hepcidin, a central regulator of iron homeostasis, has emerged as a key player in the host's defense against malaria. This comprehensive review explores the multifaceted roles of hepcidin in safeguarding the host from malaria infection. We delve into the molecular mechanisms of hepcidin regulation and its impact on the intricate interplay between iron, the immune system, and Plasmodium species. Additionally, we discuss the potential therapeutic implications of targeting hepcidin to enhance antimalarial responses. By shedding light on hepcidin's antimalarial arsenal, this review aims to contribute to the development of innovative strategies for combating malaria. Keywords: Hepcidin, antimalaria, malaria and host

Restoring neuronal iron homeostasis revitalizes neurogenesis after spinal cord injury.

Spinal cord injury (SCI) can lead to iron overloading and subsequent neuronal ferroptosis, which hinders the recovery of locomotor function. However, it is still unclear whether the maintenance of neuronal iron homeostasis enables to revitalize intrinsic neurogenesis. Herein, we report the regulation of cellular iron homeostasis after SCI via the chelation of excess iron ions and modulation of the iron transportation pathway using polyphenol-based hydrogels for the revitalization of intrinsic neurogenesis. The reversed iron overloading can promote neural stem/progenitor cell differentiation into neurons and elicit the regenerative potential of newborn neurons, which is accompanied by improved axon reinnervation and remyelination. Notably, polyphenol-based hydrogels significantly increase the neurological motor scores from ~8 to 18 (out of 21) and restore the transmission of sensory and motor electrophysiological signals after SCI. Maintenance of iron homeostasis at the site of SCI using polyphenol-based hydrogels provides a promising paradigm to revitalize neurogenesis for the treatment of iron accumulation-related nervous system diseases.

The role of ferritin in liver disease assessment

Background. Ferritin is an important integral and diagnostic marker of liver diseases. In 1/3 of patients with nonalcoholic fatty liver disease (NAFLD), manifestations of hyperferritinemia are revealed. Increased ferritin level indicates the severity of the disease course and affects the prognosis.Objective: to determine the prevalence and character of hyperferritinemia manifestations in NAFLD patients and to evaluate the effectiveness of its correction with human placenta hydrolysate.Material and methods. We examined 158 patients aged from 20 to 63 years (92 men and 66 women). There were no significant differences in age between men and women. The control group consisted of 20 practically healthy individuals. Molecular mechanisms of peptide components of human placenta hydrolysate (Laennec®) impact on pathophysiological processes of serum ferritin disorders, iron metabolism indicators, and inflammation manifestations were analyzed.Results. Nineteen peptides potentially important for regulation of iron homeostasis were identified in Laennec® composition. These peptides contribute to the elimination of iron metabolism disorders by regulating the levels of hepcidin (the main hormone of iron homeostasis), reducing ferritin synthesis, as well as exhibiting anti-inflammatory, and immunomodulatory effects. The efficacy of the drug monotherapy in patients with hyperferritinemia was shown.Conclusion. Laennec® was found to be one of the medicines contributing to the reduction of hyperferritinemia manifestations, iron metabolism disorders, and systemic inflammatory process in NAFLD.

Fibroblast growth factor 21 inhibits ferroptosis following spinal cord injury by regulating heme oxygenase-1.

Abstract JOURNAL/nrgr/04.03/01300535-202407000-00037/figure1/v/2023-11-20T171125Z/r/image-tiff Interfering with the ferroptosis pathway is a new strategy for the treatment of spinal cord injury. Fibroblast growth factor 21 can inhibit ferroptosis and promote neurofunctional recovery, while heme oxygenase-1 is a regulator of iron and reactive oxygen species homeostasis. The relationship between heme oxygenase-1 and ferroptosis remains controversial. In this study, we used a spinal cord injury rat model to show that the levels of fibroblast growth factor 21 in spinal cord tissue decreased after spinal cord injury. In addition, there was a significant aggravation of ferroptosis and a rapid increase in heme oxygenase-1 expression after spinal cord injury. Further, heme oxygenase-1 aggravated ferroptosis after spinal cord injury, while fibroblast growth factor 21 inhibited ferroptosis by downregulating heme oxygenase-1. Thus, the activation of fibroblast growth factor 21 may provide a potential treatment for spinal cord injury. These findings could provide a new potential mechanistic explanation for fibroblast growth factor 21 in the treatment of spinal cord injury.

Iron homeostasis proteins Grx4 and Fra2 control activity of the Schizosaccharomyces pombe iron repressor Fep1 by facilitating [2Fe-2S] cluster removal

The Bol2 homolog Fra2 and monothiol glutaredoxin Grx4 together play essential roles in regulating iron homeostasis in Schizosaccharomyces pombe. In vivo studies indicate that Grx4 and Fra2 act as co-inhibitory partners that inactivate the transcriptional repressor Fep1 in response to iron deficiency. In Saccharomyces cerevisiae, Bol2 is known to form a [2Fe-2S]-bridged heterodimer with the monothiol glutaredoxins Grx3 and Grx4, with the cluster ligands provided by conserved residues in Grx3/4 and Bol2 as well as glutathione (GSH). In this study, we characterized this analogous [2Fe-2S]-bridged Grx4-Fra2 complex in S. pombe by identifying the specific residues in Fra2 that act as ligands for the Fe-S cluster and are required to regulate Fep1 activity. We present spectroscopic and biochemical evidence confirming the formation of a [2Fe-2S]-bridged Grx4-Fra2 heterodimer with His66 and Cys29 from Fra2 serving as Fe-S cluster ligands in S. pombe. In vivo transcription and growth assays confirm that both His66 and Cys29 are required to fully mediate the response of Fep1 to low iron conditions. Furthermore, we analyzed the interaction between Fep1 and Grx4-Fra2 using circular dichroism spectroscopy to monitor changes in Fe-S cluster coordination chemistry. These experiments demonstrate unidirectional [2Fe-2S] cluster transfer from Fep1 to Grx4-Fra2 in the presence of GSH, revealing the Fe-S cluster dependent mechanism of Fep1 inactivation mediated by Grx4 and Fra2 in response to iron deficiency.

Hepcidin from Onychostoma macrolepis: Response to Aeromonas hydrophila infection by down-regulating expression of inflammatory factor genes and regulating iron homeostasis potentially

Hepcidin, as an antimicrobial peptide, is associated with innate immunity and is considered a potential antibiotic substitute. In the present study, the hepcidin gene from the cavefish - Onychostoma macrolepis was identified and analyzed. The recombinant hepcidin protein (rOmhepc) was obtained by prokaryotic expression, evaluating the inhibitory effect of 5 pathogenic bacteria in vitro. Sixty O. macrolepis injected with 100 μL A. hydrophila (1.5 × 108 CFU/mL) were randomly divided into the therapeutic group and infection group, and therapeutic group was injected with 100 μL rOmhepc (100 μg/mL) at 6 and 18 h. The survival rates of O. macrolepis and bacterial load in liver were measured at 24 h. The liver tissues were collected at 0, 6, 12, and 24 h after A. hydrophila injection for investigating expression levels of immune-related, inflammatory factor genes and FPN1 gene. The results demonstrated that the hepcidin CDS contained 279 bp and encoded 93 aa. Hepcidin protein has a hydrophobic surface formed by multiple hydrophobic residues (CCGCCYC), and the theoretical pI was 7.53. Omhepc gene was expressed at varying levels in tested tissues, with the liver showing the highest expression, followed by the spleen. The expression of hepcidin gene following A. hydrophila infection was up-regulated and then down-regulated in liver, and the highest expression level was found at 12 h with a 10.93-fold. The rOmhepc remarkably inhibited the growth of A. hydrophila, Staphylococcus aureus, and Streptococcus agalactiae, with inhibition rates reaching 69.67 %, 42.97 %, and 65.74 % at 100 μg/mL. The mortality rates of O. macrolepis and bacterial load in liver were significantly decreased in the therapeutic group than that of infection group (p < 0.05). After the rOmhepc therapeutic, interleukin-1β (IL-1β) and interleukin-6 (IL-6) were significantly down-regulated with 14.4-fold and 106.07-fold at 24 h. Furthermore, the expression of immune-related genes (C3, TNF-α, IFN-γ) and Ferroportin gene (FPN1) significantly decreased (p < 0.05).The integrated analyses indicated that the rOmhepc could significantly inhibit the growth of A. hydrophila both in vitro and in vivo, attenuating the over-expression of inflammatory factor, FPN1 and immune-related genes.

Estrogens and Prolactin Do Not Regulate Maternal and Embryo Iron Homeostasis during Pregnancy

Iron is essential for maternal and fetal health but the molecular mechanisms ensuring increased iron availability during pregnancy are not well understood. Hepcidin is the key iron-regulatory hormone and functions by blocking iron absorption, recycling and mobilization from stores, effectively decreasing plasma iron levels. In healthy human and rodent pregnancies, maternal hepcidin decreases starting in the second trimester and is nearly undetectable by late pregnancy. This decrease in hepcidin allows for greater iron availability for transfer to the fetus. If hepcidin is not appropriately suppressed during pregnancy, it results in fetal anemia, intrauterine growth restriction, and even fetal demise in severe cases 1. Unrelated to pregnancy, hormones estrogen and prolactin have been previously implicated in hepcidin regulation 2,3, although with conflicting results on the direction of hepcidin change 4-6. The levels of both hormones increase dramatically over the course of pregnancy, thus we hypothesized that they may modulate iron homeostasis during pregnancy. Studying the role of estrogens and prolactin during pregnancy is challenging as both hormones are required for pregnancy. Additionally, mice with global knockout (KO) of either estrogen receptor alpha or prolactin receptor are infertile. Thus, to determine the contribution of estrogens and prolactin on maternal hepcidin suppression during pregnancy, we generated liver-specific knockouts of either estrogen receptor alpha ( Er- α f/f;Alb-Cre +) or prolactin receptor ( Prlr f/f;Alb-Cre +), here abbreviated as ER-α KO and PRLR KO. Female ER-α KO, PRLR KO and WT mice were set up for timed pregnancy, and liver hepcidin mRNA, as well as maternal and embryo iron and hematological parameters were measured at E18.5 . Hepcidin suppression in ER-α KO and PRLR KO dams was not different from that in WT dams, suggesting that estrogens and prolactin are not major mediators of maternal hepcidin suppression. Additionally, maternal and embryo hematological parameters and liver iron concentrations were similar between WT, ER-α KO, PRLR KO pregnancies, further supporting our observation that estrogens and prolactin do not regulate iron homeostasis in pregnancy. The mechanisms by which maternal hepcidin is suppressed during pregnancy remain to be determined. 1Sangkhae V et al, Blood, 2020 2Yang Q et al, Endocrinology, 2012 3Wang J et al, Nanomedicine: nanotechnology, biology and medicine, 2015 4Zhen et al, Hepatology, 2013 5Ikeda Y et al, PloS one, 2012 6Lehtihet M et al, PloS one, 2016

A Phase 1b Double-Blind, Placebo-Controlled Study of DISC-0974, an Anti-Hemojuvelin Antibody, in Patients with Non-Dialysis Dependent Chronic Kidney Disease and Anemia

Hepcidin, a central regulator of iron homeostasis, is pathologically elevated in patients with non-dialysis-dependent chronic kidney disease (NDD-CKD) and anemia. Elevations in hepcidin contribute to the onset, maintenance, and severity of anemia. DISC-0974 is an investigational, first-in-class, monoclonal antibody that blocks hemojuvelin (HJV), a co-receptor in the bone morphogenetic protein-signaling pathway regulating hepcidin expression. In a healthy volunteer study, DISC-0974 demonstrated dose-dependent reductions in serum hepcidin, increases in serum iron with doses up to 56 mg administered subcutaneously (SC), and increasing trends in reticulocyte count, reticulocyte hemoglobin, mean corpuscular hemoglobin, total hemoglobin (Hgb), and red blood cell count. In a rat model of anemia in CKD, DISC-0974 decreased hepcidin, increased serum iron, and normalized hemoglobin (Wu et al., ASH 2022). DISC-0974-103 is a Phase 1b, double-blind, placebo-controlled, single-ascending dose study (NCT05745883) to assess the safety, tolerability, pharmacokinetics (PK), and pharmacodynamics (PD) of DISC-0974 in patients with NDD-CKD and anemia. Eligible participants include patients over 18 years of age with NDD-CKD, stages 2-5, hemoglobin (Hgb) &amp;lt;10.5 g/dL in women and &amp;lt;11.0 g/dL in men, serum ferritin ≥100 μg/L, and transferrin saturation (TSAT) ≤30%. Major exclusionary criteria include: concomitant treatment with therapeutic oral iron, intravenous iron, erythropoietin-stimulating agents, or blood transfusions; splenectomy; and nutritional, genetic, infectious, or autoimmune causes of anemia. Approximately 32 participants will be enrolled. On Day 1, participants will receive a single dose of DISC-0974 SC with evaluations for safety, PK, and PD through 57 days of follow-up. Dose escalation is planned across 4 cohorts consisting of 8 participants each, randomized 3:1 active treatment to placebo, treated at single doses of 28, 40, 60, and 90 mg SC DISC-0974. Safety Review Committee assessments are planned to gate escalation decisions. Primary endpoints include adverse events (AEs), clinical laboratory assessments, vital signs, physical examinations, and electrocardiograms. Secondary endpoints include changes in serum hepcidin-25, iron, TSAT, ferritin, Hgb, and other hematology biomarkers. Enrollment is currently ongoing, and available study results will be presented at the meeting.

A Phase 1b Trial of DISC-0974, an Anti-Hemojuvelin Antibody, in Patients with Myelofibrosis and Anemia

Hepcidin, a central regulator of iron homeostasis, is pathologically elevated in patients with myelofibrosis (MF) and anemia. Chronic elevations in hepcidin contribute to the onset and severity of anemia. DISC-0974 is an investigational, first-in-class, monoclonal antibody that blocks hemojuvelin, a co-receptor in the bone morphogenetic protein-signaling pathway driving hepcidin expression. Preclinical studies have shown that DISC-0974 suppresses hepcidin and increases serum iron. A healthy volunteer study has demonstrated dose-dependent reductions in serum hepcidin, increases in serum iron with doses up to 56 mg administered subcutaneously, and increasing trends in reticulocyte count, reticulocyte hemoglobin, mean corpuscular hemoglobin, total hemoglobin (Hgb), and red blood cell count. In this Phase 1b/2a, open-label, multiple-ascending dose study (NCT05320198), we are assessing the safety, tolerability, pharmacokinetics (PK), and pharmacodynamics (PD) of DISC-0974 in patients with MF and anemia. Eligible participants include patients over 18 years of age with intermediate-2 or higher-risk MF and anemia. MF is required to be confirmed using the World Health Organization 2016 criteria. Anemia is defined per protocol as Hgb &amp;lt; 10 g/dL or transfusion dependence, as defined by the International Working Group for Myelofibrosis Research and Treatment (IWG-MRT). A stable dose of hydroxyurea and/or Janus kinase (JAK) inhibitor is allowed on trial. Major exclusion criteria include: liver iron concentration ≥ 7 mg/g dry weight; splenectomy; nutritional, genetic, infectious, or autoimmune causes of anemia. Dose escalation is based on a Bayesian optimal interval design with accelerated titration. Safety Review Committee assessments gate escalation decisions. Dosing occurs monthly for a total of 6 doses. Primary endpoints include safety and tolerability assessments of adverse events (AEs), clinical laboratory assessments, vital signs, physical examinations, and electrocardiograms. Secondary endpoints include anemia response as defined by the IWG-MRT, transfusion response as defined by a decrease of 50% in transfusion requirement over any 8-week period, standard PK parameters and PD metrics including serum hepcidin-25, iron, transferrin saturation, ferritin, and hematology biomarkers. All data are summarized using descriptive statistics. Initial data show a dose-related increase in pharmacokinetic parameters. DISC-0974 treatment led to dose-dependent reductions in serum hepcidin-25 and increases in serum iron levels. There have been no serious AEs or AEs leading to study withdrawal. Most AEs were mild, transient, and unrelated to DISC-0974. As of August 1, 2023, enrollment and dose escalation are ongoing. Safety, PK, and PD data from at least the first two cohorts will be presented.

ALK2 and JAK2 Inhibition for Improved Treatment of Anemia in Myelofibrosis Patients: Preclinical Profile of an ALK2 Inhibitor Zilurgisertib in Combination with Ruxolitinib

Introduction: Anemia is a common occurrence in patients with myelofibrosis (MF) and is associated with the need for red blood cell transfusion and poor clinical prognosis. JAK inhibitors such as ruxolitinib are used extensively to treat symptoms of MF and improve quality of life and overall survival. However, JAK inhibitors may also contribute to myelosuppression. Therapeutic interventions that allow for optimization of JAK inhibition with no concern of anemia would benefit patients with MF and potentially lower the rate of treatment discontinuation or suboptimal dosing. Recent studies have demonstrated that inhibition of ACVR1/ALK2, a bone morphogenetic protein receptor that is upstream of the transcriptional regulation of hepcidin, could reduce serum hepcidin levels in patients with MF and improve anemia (Verstovsek S et al. Lancet 2023; Oh S et al. Clin Lymphoma Myeloma Leuk 2022). Reducing levels of hepcidin, a key regulator of plasma iron levels, and restoring erythropoiesis would broadly benefit patients with MF. Based on these recent data, we developed zilurgisertib, a potent and selective ALK2 inhibitor that could be administered in combination with ruxolitinib at doses titrated to the needs of patients with MF. Methods and Results: In biochemical and cellular assays, zilurgisertib inhibited ALK2 kinase activity and SMAD1/5 phosphorylation with IC 50 values of 15 nM and 63 nM, respectively. In Huh-7 cells stimulated with BMP-6, zilurgisertib inhibited hepcidin production with an IC 50 of 20 nM, demonstrating the compound is a potent ALK2 inhibitor capable of regulating iron homeostasis via hepcidin. To assess possible off-target effects of zilurgisertib, kinome profiling at 10 μM ATP was performed at Reaction Biology (Malvern, PA) to determine the overall specificity across 356 kinases (see Figure). At 200 nM, zilurgisertib only inhibited ALK2, ALK1 (to 50%), and ALK6 (to 48%). In addition, zilurgisertib at 20 μM did not affect viability of HEK293 cells, a human cell line commonly used to assess general cell health and compound toxicity. Similarly, zilurgisertib did not affect viability of human fibroblasts or endothelial cells at concentrations up to 5 μM. To test whether the combination of ruxolitinib and zilurgisertib could suppress hepcidin and restore erythropoiesis in an in vivo mouse model of cancer-induced anemia, B16F10 cells were injected intraperitoneally, mimicking a metastatic tumor that leads to anemia 1 week after injection. In a dose-dependent manner, zilurgisertib improved hemoglobin by 2 to 3 g/dL and red blood cell counts, while reducing both liver pSMAD and circulating hepcidin levels by 50% or more compared with vehicle control. The combination of zilurgisertib with ruxolitinib had no effect on this activity, suggesting that inhibition of JAK2 does not inhibit erythropoiesis restored following ALK2 inhibition. Conclusion: Taken together, the potent and selective on-target activity of zilurgisertib suggests that ALK2 inhibition could reduce hepcidin and improve anemia, and that the combination of zilurgisertib with ruxolitinib is a rational and attractive approach to mitigate anemia in patients with MF. The combination of ruxolitinib and zilurgisertib is currently being evaluated in a phase 1 clinical trial in patients with anemia due to myeloproliferative disorders (NCT04455841).

Capsaicin alleviates doxorubicin-induced acute myocardial injury by regulating iron homeostasis and PI3K-Akt signaling pathway.

Capsaicin (CAP), a frequently occurring alkaloid component found in spicy peppers, has demonstrated therapeutic potential against tumors, metabolic disease, and cardiovascular disorders. Doxorubicin (DOX), a widely used anthracycline drug in chemotherapy, is notorious for its cardiotoxicity. This study aimed to investigate the potential of CAP in mitigating DOX toxicity in mouse hearts and H9C2 cells, as well as to explore the underlying mechanisms. In our study, we conducted experiments on both mice and H9C2 cells. The mice were divided into four groups and treated with different substances: normal saline, CAP, DOX and CAP+DOX. We evaluated the induction of ferroptosis by DOX and the remission of ferroptosis by CAP using various methods, including echocardiography, Hematoxylin and Eosin (H&E) staining, Masson's trichrome staining, and determination of ferroptosis metabolites, genes and proteins. Additionally, we employed RNA-seq to identify the inhibitory effect of CAP on DOX-induced myocardial apoptosis, which was further confirmed through western blotting. Similar approaches were applied to H9C2 cells, yielding reliable results. Our study demonstrated that treatment with CAP improved the survival rate of DOX-treated mice and reduced myocardial injury. Mechanistically, CAP downregulated transferrin (Trf) and upregulated solute carrier family 40 member 1 (SLC40A1), which helped maintain iron levels in the cells and prevent ferroptosis. Furthermore, CAP inhibited DOX-induced apoptosis by modulating the phosphoinositide 3-kinase (PI3K)- protein kinase B (Akt) signaling pathway. Specifically, CAP activated the PI3K-Akt pathway and regulated downstream BCL2 and BAX to mitigate DOX-induced apoptosis. Therefore, our results suggest that CAP effectively alleviates acute myocardial injury induced by DOX. Our findings demonstrate that CAP has the potential to alleviate DOX-induced ferroptosis by regulating iron homeostasis. Additionally, it can inhibit DOX-induced apoptosis by activating PI3K-Akt signaling pathway.

Evoking Ferroptosis by Synergistic Enhancement of a Cyclopentadienyl Iridium-Betulin Immune Agonist.

Ferroptosis is a form of programmed cell death driven by iron-dependent lipid peroxidation (LPO) with the potential for antitumor immunity activation. In this study, a nonferrous cyclopentadienyl metal-based ferroptosis inducer [Ir(Cp*)(Bet)Cl]Cl (Ir-Bet) was developed by a metal-ligand synergistic enhancement (MLSE) strategy involving the reaction of [Ir(Cp*)Cl]2 Cl2 with the natural product Betulin. The fusion of Betulin with iridium cyclopentadienyl (Ir-Cp*) species as Ir-Bet not only tremendously enhanced the antiproliferative activity toward cancer cells, but also activated ferritinophagy for iron homeostasis regulation by PI3K/Akt/mTOR cascade inhibition with a lower dosage of Betulin, and then evoked an immune response by nuclear factor kappa-B (NF-κB) activation of Ir-Cp* species. Further immunogenic cell death (ICD) occurred by remarkable ferroptosis through glutathione (GSH) depletion, glutathione peroxidase 4 (GPX4) deactivation and ferritinophagy. An in vivo vaccination experiment demonstrated desirable antitumor and immunogenic effects of Ir-Bet by increasing the ratio of cytotoxic T cells (CTLs)/regulatory T cells (Tregs).

Evoking Ferroptosis by Synergistic Enhancement of a Cyclopentadienyl Iridium‐Betulin Immune Agonist

AbstractFerroptosis is a form of programmed cell death driven by iron‐dependent lipid peroxidation (LPO) with the potential for antitumor immunity activation. In this study, a nonferrous cyclopentadienyl metal‐based ferroptosis inducer [Ir(Cp*)(Bet)Cl]Cl (Ir‐Bet) was developed by a metal‐ligand synergistic enhancement (MLSE) strategy involving the reaction of [Ir(Cp*)Cl]2Cl2 with the natural product Betulin. The fusion of Betulin with iridium cyclopentadienyl (Ir‐Cp*) species as Ir‐Bet not only tremendously enhanced the antiproliferative activity toward cancer cells, but also activated ferritinophagy for iron homeostasis regulation by PI3K/Akt/mTOR cascade inhibition with a lower dosage of Betulin, and then evoked an immune response by nuclear factor kappa‐B (NF‐κB) activation of Ir‐Cp* species. Further immunogenic cell death (ICD) occurred by remarkable ferroptosis through glutathione (GSH) depletion, glutathione peroxidase 4 (GPX4) deactivation and ferritinophagy. An in vivo vaccination experiment demonstrated desirable antitumor and immunogenic effects of Ir‐Bet by increasing the ratio of cytotoxic T cells (CTLs)/regulatory T cells (Tregs).

Lipocalin-2: A Nurturer of Tumor Progression and a Novel Candidate for Targeted Cancer Therapy

Within the tumor microenvironment (TME) exists a complex signaling network between cancer cells and stromal cells, which determines the fate of tumor progression. Hence, interfering with this signaling network forms the basis for cancer therapy. Yet, many types of cancer, in particular, solid tumors, are refractory to the currently used treatments, so there is an urgent need for novel molecular targets that could improve current anti-cancer therapeutic strategies. Lipocalin-2 (Lcn-2), a secreted siderophore-binding glycoprotein that regulates iron homeostasis, is highly upregulated in various cancer types. Due to its pleiotropic role in the crosstalk between cancer cells and stromal cells, favoring tumor progression, it could be considered as a novel biomarker for prognostic and therapeutic purposes. However, the exact signaling route by which Lcn-2 promotes tumorigenesis remains unknown, and Lcn-2-targeting moieties are largely uninvestigated. This review will (i) provide an overview on the role of Lcn-2 in orchestrating the TME at the level of iron homeostasis, macrophage polarization, extracellular matrix remodeling, and cell migration and survival, and (ii) discuss the potential of Lcn-2 as a promising novel drug target that should be pursued in future translational research.

Key players in the regulation of iron homeostasis at the host-pathogen interface.

Iron plays a crucial role in the biochemistry and development of nearly all living organisms. Iron starvation of pathogens during infection is a striking feature utilized by a host to quell infection. In mammals and some other animals, iron is essentially obtained from diet and recycled from erythrocytes. Free iron is cytotoxic and is readily available to invading pathogens. During infection, most pathogens utilize host iron for their survival. Therefore, to ensure limited free iron, the host's natural system denies this metal in a process termed nutritional immunity. In this fierce battle for iron, hosts win over some pathogens, but others have evolved mechanisms to overdrive the host barriers. Production of siderophores, heme iron thievery, and direct binding of transferrin and lactoferrin to bacterial receptors are some of the pathogens' successful strategies which are highlighted in this review. The intricate interplay between hosts and pathogens in iron alteration systems is crucial for understanding host defense mechanisms and pathogen virulence. This review aims to elucidate the current understanding of host and pathogen iron alteration systems and propose future research directions to enhance our knowledge in this field.

Hepcidin as a key regulator of iron homeostasis triggers inflammatory features in the normal endometrium

Menstrual blood, containing high iron levels, can undergo retrograde transport into the abdominal cavity. Excess iron causes oxidative stress and inflammation. Iron metabolism is regulated by hepcidin, and serum hepcidin levels are increased in patients with endometriosis; however, the functions of hepcidin in normal endometrium remain unclear. We therefore aimed to examine hepcidin concentrations in patients with endometriosis and to determine if iron accumulation and hepcidin increased the production of reactive oxygen species (ROS) and inflammation in normal endometrial cells. We determined hepcidin levels in peritoneal fluid and menstrual blood from patients with and without endometriosis (25/16 and 15/15 patients, respectively). We also examined the effects of hepcidin on ferroportin expression, iron accumulation, and ROS generation in normal endometrial stromal cells (NESCs) from 20 women who underwent surgery for uterine leiomyoma, using immunohistochemistry and immunofluorescence analyses and analyzed its effect on the expression of inflammatory cytokines by real-time polymerase chain reaction. There was no significant difference in iron concentrations in menstrual blood or peritoneal fluid between women with and without endometriosis; however, women with endometriosis had significantly higher hepcidin levels in menstrual blood. Hepcidin reduced the expression of ferroportin in NESCs and promoted the accumulation of ferrous iron. Hepcidin plus ferrous iron increased the production of ROS and inflammatory cytokines compared with ferrous iron alone. These results indicate that women with endometriosis have high hepcidin levels in menstrual blood, leading to increased iron production, oxidative stress, and inflammation, which may, in turn, promote the development of endometriosis.

NRF2 is essential for iron-overload stimulated osteoclast differentiation through regulation of redox and iron homeostasis.

Iron overload enhances osteoclastic bone resorption and induces osteoporosis. Excess iron is highly toxic. The modulation of redox and iron homeostasis is critical for osteoclast differentiation under iron-overload condition. Nuclear factor erythroid 2-related factor 2 (NRF2) is a transcription factor that regulates the cellular defense against oxidative stress and iron overload through the expression of genes involved in anti-oxidative processes and iron metabolism. Our studies demonstrated that NRF2 activation was suppressed during osteoclast differentiation. Under iron-overload condition, NRF2 and its mediated antioxidant and iron metabolism genes were activated by reactive oxygen species (ROS), which enhanced antioxidant capability. NRF2 mediated the upregulation of iron exporter ferroportin 1 (FPN1) and iron storage protein ferritin, contributing to decreased levels of intracellular iron. Nfe2l2 knockout induced oxidative stress and promoted osteoclast differentiation under normal condition, but induced ferroptosis under iron-overload condition. Nfe2l2 knockout alleviated iron overload induced bone loss by inhibiting osteoclast differentiation. Our results suggest that NRF2 activation is essential for osteoclast differentiation by enhancing antioxidant capability and reducing intracellular iron under iron-overload condition. Targeting NRF2 to induce ferroptosis could be a potential therapy for the treatment of iron-overload induced osteoporosis.

A systematic review of natural resistance-associated macrophage protein 1 (NRAMP1) gene and susceptibility to tuberculosis: What is the link for ocular tuberculosis?

Link of Video Abstract: https://youtu.be/hxFKCtWwc3c Introduction: The host genetic factors play a crucial role in determining susceptibility to tuberculosis (TB), and one such candidate gene is Natural Resistance-Associated Macrophage Protein 1 (NRAMP1). NRAMP1 is involved in the regulation of intracellular iron homeostasis, an essential nutrient for MTb survival within macrophages. Several polymorphisms of the NRAMP1 gene have been extensively studied for their association with TB susceptibility. However, the specific role of these NRAMP1 gene polymorphisms in the development of ocular tuberculosis, a rare but potentially severe form of TB, remains poorly understood. Methods: This study was a systematic review conducted according to the Preferred Reporting Items for Systematic Review and Meta-Analyses (PRISMA). A comprehensive search was performed in the PubMed, Cochrane, and Science Direct databases for original, full-text studies of NRAMP1 gene related to tuberculosis infection. Ethnic, polymorphisms of NRAMP1 evaluated, and its association to TB susceptibility were recorded and summarized qualitatively. Results: There were 41 studies included in our systematic review. No study of NRAMP1 gene polymorphism in regards to ocular tuberculosis was found. Polymorphisms of the NRAMP1 gene, such as 3'UTR TGTG ins/del, D543N, INT4, and (GT)n, have been investigated for their potential role in determining the susceptibility or immunity to Mycobacterium tuberculosis infection. However, the findings regarding the relationship between NRAMP1 gene polymorphism and tuberculosis are still inconsistent, with controversies and discrepancies among different studies. Conclusion: NRAMP1 gene polymorphisms showed important involvement to tuberculosis infection susceptibility, therefore its relation to ocular tuberculosis is suggested.