Intracellular Iron Status Research Articles

Visceral adiposity is associated with iron deposition and myelin loss in the brains of aged mice

Iron deposition and myelin loss are observed in the brain with aging, and iron accumulation is suggested to be involved in myelin damage. However, the exact mechanism of iron deposition with aging remains unclear. This study was aimed to determine whether expanded visceral adipose tissue contributes to iron deposition and myelin loss by inducing hepcidin in the brains of aged male mice. Compared with young adult mice, levels of hepcidin in the brain, epididymal adipose tissue, and circulation were increased in aged mice, which had expanded visceral adipose tissue with inflammation. An increase in expressions of ferritin, an indicator of intracellular iron status, was accompanied by decreased levels of proteins related to myelin sheath in the brains of aged mice. These age-related changes in the brain were improved by visceral fat removal. In addition, IL-6 level, activation of microglia/macrophages, and nuclear translocation of phosphorylated Smad1/5 (pSmad1/5) inducing hepcidin expression were reduced in the brains of aged mice after visceral fat removal, accompanied by decreases of pSmad1/5- and ferritin-positive microglia/macrophages and mature oligodendrocytes. These findings indicate that visceral adiposity contributes to hepcidin-mediated iron deposition and myelin loss with inflammation in the aged brain. Our results support the importance of preventing visceral adiposity for maintaining brain health in older individuals.

Ferrodifferentiation regulates neurodevelopment via ROS generation.

Iron is important for life, and iron deficiency impairs development, but whether the iron level regulates neural differentiation remains elusive. In this study, with iron-regulatory proteins (IRPs) knockout embryonic stem cells (ESCs) that showed severe iron deficiency, we found that the Pax6- and Sox2-positive neuronal precursor cells and Tuj1 fibers in IRP1-/-IRP2-/- ESCs were significantly decreased after inducing neural differentiation. Consistently, in vivo study showed that the knockdown of IRP1 in IRP2-/- fetal mice remarkably affected the differentiation of neuronal precursors and the migration of neurons. These findings suggest that low intracellular iron status significantly inhibits neurodifferentiation. When supplementing IRP1-/-IRP2-/- ESCs with iron, these ESCs could differentiate normally. Further investigations revealed that the underlying mechanism was associated with an increase in reactive oxygen species (ROS) production caused by the substantially low level of iron and the down-regulation of iron-sulfur cluster protein ISCU, which, in turn, affected the proliferation and differentiation of stem cells. Thus, the appropriate amount of iron is crucial for maintaining normal neural differentiation that is termed ferrodifferentiation.

Iron therapy mitigates chronic kidney disease progression by regulating intracellular iron status of kidney macrophages.

Systemic iron metabolism is disrupted in chronic kidney disease (CKD). However, little is known about local kidney iron homeostasis and its role in kidney fibrosis. Kidney-specific effects of iron therapy in CKD also remain elusive. Here, we elucidate the role of macrophage iron status in kidney fibrosis and demonstrate that it is a potential therapeutic target. In CKD, kidney macrophages exhibited depletion of labile iron pool (LIP) and induction of transferrin receptor 1, indicating intracellular iron deficiency. Low LIP in kidney macrophages was associated with their defective antioxidant response and proinflammatory polarization. Repletion of LIP in kidney macrophages through knockout of ferritin heavy chain (Fth1) reduced oxidative stress and mitigated fibrosis. Similar to Fth1 knockout, iron dextran therapy, through replenishing macrophage LIP, reduced oxidative stress, decreased the production of proinflammatory cytokines, and alleviated kidney fibrosis. Interestingly, iron markedly decreased TGF-β expression and suppressed TGF-β-driven fibrotic response of macrophages. Iron dextran therapy and FtH suppression had an additive protective effect against fibrosis. Adoptive transfer of iron-loaded macrophages alleviated kidney fibrosis, validating the protective effect of iron-replete macrophages in CKD. Thus, targeting intracellular iron deficiency of kidney macrophages in CKD can serve as a therapeutic opportunity to mitigate disease progression.

Iron Therapy Mitigates Kidney Fibrosis by Regulating Intracellular Iron Status of Kidney Macrophages

Iron Therapy Mitigates Kidney Fibrosis by Regulating Intracellular Iron Status of Kidney Macrophages

12 T high static magnetic field suppresses osteosarcoma cells proliferation by regulating intracellular ROS and iron status

Many studies indicated that static magnetic fields (SMFs) have anti-cancer effects. However, effect of SMFs on cancer cells with strength exceeding 12 T are rarely reported. The intracellular iron could participate in the reactive oxygen species (ROS) production and affect cell proliferation. This study aimed to investigate the effect of 12 T high static magnetic field (HiSMF) on osteosarcoma cells and the relationship with intracellular iron. The 12 T HiSMF was generated by a superconducting magnet. The proliferation was evaluated by CCK-8 assays and cell counting. The apoptosis, cell cycle distribution, and ROS were evaluated by flow cytometry. Intracellular iron status was evaluated by atomic absorption spectroscopy and Calcein-AM/2,2′-bipyridyl. The expression of cell cycle and iron metabolism-related genes were analyzed by Western Blot. The result showed that 12 T HiSMF exposure suppressed the proliferation of osteosarcoma cell lines MNNG/HOS, U-2 OS, and MG63 via cell cycle arrest in S and G2/M. Meanwhile, 12 T HiSMF increasing intracellular ROS, and its antitumor effect was reduced by antioxidant. Furthermore, the intracellular total and free iron levels, the expression of FTH1 and DMT1 were increased by 12 HiSMF. The iron chelator (DFO) could reduce the cytotoxicity of 12 T HiSMF on osteosarcoma cells. Moreover, 12 T HiSMF could enhance the cytotoxicity of cisplatin and sorafenib in osteosarcoma cells. In Conclusion, 12 T HiSMF could suppress osteosarcoma cells proliferation via intracellular iron and ROS related cell cycle arrest, and have application potential in osteosarcoma therapy combined with sorafenib and cisplatin.

Hepcidin-to-Ferritin Ratio Is Decreased in Astrocytes With Extracellular Alpha-Synuclein and Iron Exposure.

Astrocytes are the most abundant glial cells in the central nervous system (CNS). As indispensable elements of the neurovascular unit, they are involved in the inflammatory response and disease-associated processes. Alpha-synuclein (α-syn) is released into the extracellular space by neurons and can be internalized by adjacent astrocytes, which activates glial cells to induce neuroinflammation. We were interested in whether astrocyte-mediated neuroinflammation is modulated by intracellular iron status and extracellular α-syn. Our results showed that recombinant α-syn (1 μg/ml and 5 μg/ml) treatment for 24 h did not affect the expression of the iron transporters divalent metal transporter 1 (DMT1) and ferroportin 1 (FPN1), nor those of iron regulatory protein (IRP) 1 or IRP2. Several proinflammatory cytokines, including tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, and IL-6 exhibited up-regulated mRNA levels in 5 μg/ml α-syn-treated astrocytes. TNF-α release was increased, indicating that inflammatory responses were triggered in these cells. Pretreatment with the iron-overload reagent ferric ammonium citrate (FAC, 100 μmol/L) for 24 h had no effects on mRNA levels and release of proinflammatory cytokines. Inflammatory responses triggered by α-syn were not affected by iron overload. The iron chelator desferrioxamine (DFO, 100 μmol/L) exerted suppressive effects on TNF-α mRNA levels, although no change was observed for TNF-α release. Hepcidin mRNA levels were down-regulated significantly in astrocytes co-treated with FAC and α-syn, although independent treatment with either FAC or α-syn did not alter hepcidin levels. In contrast, hepcidin mRNA levels were up-regulated in DFO and α-syn co-treated cells. As expected, ferritin protein levels were up-regulated or down-regulated with FAC or DFO treatment, respectively. Following the up-regulation of ferritin mediated by α-syn, hepcidin-to-ferritin levels were indicative of modulatory effects in α-syn-treated astrocytes with altered iron status. Therefore, we propose that the hepcidin-to-ferritin ratio is indicative of a detrimental response in primary cultured astrocytes experiencing iron and extracellular α-syn.

P935 Association of intracellular iron status with left ventricular systolic function response to exercise in heart failure with preserved ejection fraction

Abstract The intracellular iron depletion has been recognized to contribute to the dysregulation of cell energetics. The soluble transferrin receptor (sTfR) is regarded as a marker of cellular iron balance, and its elevated level reflects an insufficient iron delivery to target tissues. Despite the strong pathophysiological link, there is a scarcity of data on the impact of intracellular iron status on myocardial performance. Aim To investigate the association between the intracellular iron status, as assessed by sTfR, and left ventricular (LV) function in a well-characterized population with heart failure and preserved ejection fraction (HFpEF). Methods A complete echocardiogram including evaluation of LV global longitudinal deformation by speckle tracking (GLS) was performed at rest and immediately post-exercise in 83 pts (age 66 ± 8 yrs) with symptomatic HFpEF. Results Pts with the highest sTfR concentrations (from the 3rd sTfR tertile) demonstrated significantly lower exertional GLS than their peers from the other 2 tertiles and lower resting GLS vs. the 2nd tertile (Table). Exercise GLS was inversely correlated with sTfR (r=-0.27, p = 0.01), and this association remained significant after adjustment for age, sex, BMI, LV mass, exercise blood pressure, hemoglobin and serum galectin-3 – a marker of fibrosis (beta=-0.24, p = 0.04). Conclusions In HFpEF, higher sTfR reflecting a decreased global intracellular iron content is independently associated with reduced LV longitudinal contractility response to exertion. This might represent another mechanism of exercise intolerance and should be considered in management strategies in this condition. Abstract P935 Figure.

Estrogen-dependent disruption of intracellular iron metabolism augments the cytotoxic effects of doxorubicin in select breast and ovarian cancer cells.

Introduction: Increased iron content in cancer cells is associated with resistance to chemotherapy. Recent studies have demonstrated that estrogen (E2) suppresses hepcidin synthesis and enhances intracellular iron efflux. Herein, we investigated whether E2-driven intracellular iron efflux renders cancer cells more susceptible to doxorubicin (Dox)-induced cytotoxicity.Methods: Breast, ovarian, and liver cancer cell lines treated with E2, Dox, or a combination of both were assessed for intracellular iron status, mitochondrial function, cell cycle, and apoptosis.Results: E2+Dox treatment in MCF7, SKOV3 and MDA-MB231 cells resulted in enhanced apoptosis compared with Dox-treated cells. Expression of γH2AX was significantly higher and that of survivin significantly lower in E2+Dox-treated cells than Dox-treated cells. At 48 hours, E2+Dox had induced a significant increase in the percentage of sub-G1 apoptotic cells, increased CHK1 expression, and decreased cyclin D1, CDK4, and CDK6 expression. Ferroportin and ferritin expression was significantly higher and that of TfR1 significantly lower in E2+Dox-treated cells than Dox-treated cells. Intracellular iron content was significantly reduced in E2+Dox-treated cells at 48 hours posttreatment. Lastly, E2+Dox-treated cells showed higher levels of mitochondrial membrane hyperpolarization than Dox-treated cells.Conclusion: These findings suggest that E2 disrupts intracellular iron metabolism in such a way that increases cell susceptibility to Dox-induced cytotoxicity.

Elevated lactate in acute heart failure patients with intracellular iron deficiency as identifier of poor outcome.

We believe that there is a physiological link between intracellular iron status (assessed by soluble transferrin receptor [sTfR]) and efficiency of energy production/consumption (assessed by lactate, a product of anaerobic cell metabolism), which may further impact the outcome of patients with acute heart failure (AHF). To examine if elevated levels of lactate (> 2 mmol/L) accompanied by unmet cellular iron requirements (defined as sTfR > 1.59 mg/L) identify AHF patients with an unfavourable outcome. The study is a single-centre, retrospective analysis of AHF patients in whom lactate and iron status were assessed on admission. The endpoint of the study was one-year mortality. The study population consisted of 89 patients at a mean age of 65 ± 13 years. Mean systolic blood pressure and creatinine level were 135 ± 36 mmHg and 1.3 ± 0.6 mg/dL, respectively, and median [25th-75th quartiles] lactate level on admission was 2.0 [1.6-2.6] mmol/L. In 17 (19%) patients, both lactate and sTfR were below the cut-off values (group 1). In 38 (43%) individuals one of the markers was elevated (group 2) and in the remaining 34 (38%) patients both markers were above the predefined cut-off values (group 3). There was no difference in clinical and laboratory characteristics between the groups. During one-year follow-up 23 (26%) patients died. Mortality risk in group 3 was higher compared to the rest of the population (hazard ratio 5.6, 95% confidence interval 2.2-14, p = 0.0003), even after adjustments for well-defined prognostic factors. Patients with unmet iron cell requirements and hyperlactataemia on admission have significantly higher mortality risk compared to individuals without those pathologies.

High-Dose Deferoxamine Treatment Disrupts Intracellular Iron Homeostasis, Reduces Growth, and Induces Apoptosis in Metastatic and Nonmetastatic Breast Cancer Cell Lines.

Mounting evidence suggest that iron overload enhances cancer growth and metastasis; hence, iron chelation is being increasingly used as part of the treatment regimen in patients with cancer. Now whether iron chelation depletes intracellular iron and/or disrupts intracellular iron homeostasis is yet to be fully addressed. MCF-7 and MDA-MB-231 breast cancer cells treated with increasing concentrations of the iron chelator deferoxamine were assessed for intracellular iron status, the expression of key proteins involved in iron metabolism, cell viability, growth potential, and apoptosis at different time points following treatment. Treatment with deferoxamine at 1, 5, or 10 μM for 24 or 48 hours, while not leading to significant changes in intracellular labile iron content, upregulated the expression of hepcidin, ferroportin, and transferrin receptors 1 and 2. In contrast, deferoxamine at 30, 100, or 300 μM for 24 hours induced a significant decrease in intracellular labile iron, which was associated with increased expression of hepcidin, ferritin, and transferrin receptors 1 and 2. At 48 hours, there was an increase in intracellular labile iron, which was associated with a significant reduction in hepcidin and ferritin expression and a significant increase in ferroportin expression. Although low-dose deferoxamine treatment resulted in a low to moderate decrease in MCF-7 cell growth, high-dose treatment resulted in a significant and precipitous decrease in cell viability and growth, which was associated with increased expression of phosphorylated Histone 2A family member X and near absence of survivin. High-dose deferoxamine treatment also resulted in a very pronounced reduction in wound healing and growth in MDA-MB-231 cells. These findings suggest that high-dose deferoxamine treatment disrupts intracellular iron homeostasis, reduces cell viability and growth, and enhances apoptosis in breast cancer cells. This is further evidence to the potential utility of iron chelation as an adjunctive therapy in iron-overloaded cancers.

Estrogen-induced disruption of intracellular iron metabolism leads to oxidative stress, membrane damage, and cell cycle arrest in MCF-7 cells.

It is well established that several forms of cancer associate with significant iron overload. Recent studies have suggested that estrogen (E2) disrupts intracellular iron homeostasis by reducing hepcidin synthesis and maintaining ferroportin integrity. Here, the ability of E2 to alter intracellular iron status and cell growth potential was investigated in MCF-7 cells treated with increasing concentrations of E2. Treated cells were assessed for intracellular iron status, the expression of key proteins involved in iron metabolism, oxidative stress, cell survival, growth, and apoptosis. E2 treatment resulted in a significant reduction in hepcidin expression and a significant increase in hypoxia-inducible factor 1 alpha, ferroportin, transferrin receptor, and ferritin expression; a transient decrease in labile iron pool; and a significant increase in total intracellular iron content mainly at 20 nM/48 h E2 dose. Treated cells also showed increased total glutathione and oxidized glutathione levels, increased superoxide dismutase activity, and increased hemoxygenase 1 expression. Treatment with E2 at 20 nM for 48 h resulted in a significant reduction in cell growth (0.35/1 migration rate) and decreased cell survival (<80%) as compared with controls. Survivin expression significantly increased at 24 h post treatment with 5, 10, or 20 nM; however, that of γ-H2AX increased only after survivin levels dropped and only at the 20 nM E2 dose. Minimal upregulation and splitting of caspase 9 was only evident in cells treated with 20 nM E2; no changes in caspase 3 expression were evident. Although Annexin V staining studies showed that E2 treatment did not induce apoptosis, scanning electron microscopy studies showed marked membrane blebbing at 20 nM/48 h of E2. These findings suggest that estrogen treatment disrupts intracellular iron metabolism and precipitates adverse effects concerning cell viability, membrane integrity, and growth potential.

Characterization of Staphylococcus aureus Responses to Spermine Stress

Spermine (Spm), a potent bactericidal polyamine, exerts a strong synergistic effect with β-lactams against methicillin-resistant Staphylococcus aureus (MRSA). To explore the Spm-based antibacterial targets in S. aureus, time course-dependent transcriptome analysis was conducted on Mu50 (MRSA) in the absence and presence of Spm. Genes in the sigB regulon and most ATP-producing pathways were found down-regulated when exposure to high dose Spm. In contrast, a number of genes for iron acquisition and regulation showed significant induction, indicating a specific connection between Spm and iron-depletion. The tetM gene for tetracycline (Tc) resistance exhibited most significant fold change among the listed genes. It was specifically upregulated by Tc and Spm but not by other ribosome-targeted drugs or other polyamines; however, such induction of tetM cannot confer resistance to Spm. A set of genes for osmotic balance, including kdpABCDE for potassium ion uptake and regulation, was also induced by Spm stress. Addition of KCl or NaCl, but not high concentration sucrose, was found to increase Spm MIC over 30-fold. In summary, transcriptome analysis demonstrated a specific pattern of response upon Spm exposure, suggesting Spm may alter the intracellular iron status and suppress the SigB regulon to exert its toxicity.

Silencing airway epithelial cell-derived hepcidin exacerbates acute lung injury by regulating the intracellular iron status in alveolar macrophages (INM7P.434)

Abstract Objective: Hepcidin is a β-defensin-like antimicrobial peptide and acts as a principal iron regulatory hormone. Hepcidin is mostly produced by hepatocytes, but is also expressed by other cells, such as airway epithelial cells. However, nothing is known about its function in lung infectious and inflammatory disease such as acute lung injury. Methods: Acute lung injury was induced by polymicrobial sepsis via cecal ligation and puncture surgery. Adenovirus-mediated short hairpin RNA specific for the mouse hepcidin gene hepc1 and control adenovirus were intratracheally injected into mice twice. The adenovirus-mediated knockdown of hepcidin in airway epithelial cells was evaluated in vivo. Lung injury and the 7-day survival rate were assessed. The local and systemic iron content was assayed. Results: The hepcidin level in airway epithelial cells was upregulated during polymicrobial sepsis. The knockdown of airway epithelial cell-derived hepcidin aggravated the polymicrobial sepsis-induced lung injury and pulmonary bacterial infection and increased the mortality. Moreover, the knockdown of hepcidin in airway epithelial cells led to reduced ferroportin degradation and a low intracellular iron content in alveolar macrophages, but did not affect the STAT3/SOCS3 axis. Conclusions: Airway epithelial cell-derived hepcidin plays an important role in protection against acute lung injury via regulating the intracellular iron status in alveolar macrophages.

Splicing Factor 3b Subunit 1 (SF3B1) mediates Mitochondrial Iron Overload In Myelodysplastic Syndromes With Ring Sideroblasts By Alternative Splicing Of Mitoferrin-1 (SLC25A37)

Splicing factor 3b subunit 1, SF3B1 contributes to the formation of ring sideroblasts (RS) in Myelodysplastic syndromes (MDS). Abnormalities in iron trafficking have been implicated in the pathogenesis of refractory anemia with RS (RARS) and RARS with thrombocytosis (RARS-T). In RARS/-T, light microscopy and traditional iron profiles do not accurately reflect intracellular iron status. SF3B1 mutant (MUT, n=25) and wild type (WT, n=8) RARS patients (pts) have no difference in iron profiles (ferritin (ng/mL): 1244 ng/mL ± 926 vs 1215 ± 1065; total iron binding capacity (ug/dL): 252 ± 80 vs 234 ± 50). We previously reported distinct differences in iron distribution between SF3B1 MUT and WT pts based on transmission electron microscopy (EM). Coarse iron deposits are present in the mitochondria of SF3B1 MUT while smaller iron deposits are found in WT. We hypothesized that SF3B1 mutations affect distinct downstream targets leading to the iron phenotype differences in MUT vs WT. To study these differences, we performed a series of EM, flow cytometric (FC) and RNA-sequencing experiments. Quantification of iron by scoring deposits of iron/grid showed higher amount of mitochondrial iron in MUT vs WT by EM. We used a FC based-approach to quantify the cytoplasmic and mitochondrial iron utilizing the properties of two cell-permeant compounds (calcein-AM and rhodamine-B) being retained in the cytoplasm and the mitochondria, respectively. In keeping with our EM studies, we found that SF3B1 MUT have a higher % of rhodamine-B+ cells compared to WT RARS pts and healthy subjects (77 vs 19 vs 0.87%; n=6) indicating that MUT accumulates more mitochondrial iron compared to WT. Cytoplasmic iron was not different between MUT and WT (57 vs 43%) but higher than healthy subjects (1.5%; p=0.02), suggesting that MUT store more mitochondrial iron compared to WT pts. Sf3b1+/- mice also showed higher iron stores in the mitochondria rather than in the cytosplasm (85 vs 16%; n=4). To understand the factors leading to increased mitochondrial iron, we analyzed the transcriptome of BM cells derived from a homogeneous group of SF3B1 MUT (n=3), WT RARS (n=3), and healthy subjects (n=3). Total RNA (1.5-3ug) was subjected to RNA-sequencing using Illumina HiSeq2000. Twenty-million sequencing reads were interrogated. A comprehensive bioinformatic analysis was conducted on three levels (exon usage, gene expression, pathway analysis). Global gene expression analysis detected significant gene expression changes in 59 genes (FDR < 0.2) between MUT and healthy subjects. Mitochondrial genes linked to iron pathophysiology were well represented in the analysis. A total of 354 genes with exon usage/ gene expression difference in at least 1 exon were investigated. One of the top candidate genes showing alternative splicing is SLC25A37 (chr:8p21.2), a mitochondrial iron importer that mediates Fe2+ incorporation into the mitochondria. SLC25A37 was associated with a 2-4 fold higher expression in MUT vs WT and healthy individuals (mean base=4145 vs 2451 vs 1058). We also investigated other genes important in iron metabolism including SLC25A38, PUS1, and GLRX5 but no differences in both exon usage and gene expression were noted supporting the different nature of acquired and congenital sideroblastic anemia (SA). Some mitochondrial genes important in iron trafficking showed no changes in exon usage but exhibited differences in gene expression suggesting that they are pathways independent of SF3B1 mutations but still contributing to iron metabolism. ALAS2 was down-regulated in MUT vs WT RARS (fold change (FC): 0.39) while both MUT and WT pts exhibited an increased level compared to healthy subjects (FC: 5.0 and 2.0). ABCB7 was down-regulated compared to healthy subjects in both groups (FC:0.45 and 0.42). Using bisulfite sequencing we found that MUT have significantly higher hypermethylation of ALAS2 compared to WT RARS pts (FDR<.01; p<.00036) suggesting that epigenetic modification may explain the reduced ALAS2 expression instead of a splicing defect. SLC25A37 has been linked to an erythropoietic protoporphyria variant but not to congenital or acquired SA. In summary, SF3B1 MUT have increased mitochondrial iron as shown by EM and FC vs WT RARS pts. SF3B1 mutations mediate increased mitochondrial iron and RS formation by the alternative splicing of an iron transporter gene, SLC25A37, a novel pathway of iron-overload in the pathogenesis of MDS with RS. Disclosures:No relevant conflicts of interest to declare.

Iron plays a key role in the cytodifferentiation of human periodontal ligament cells

The periodontal ligament (PDL) is vital to maintaining the homeostasis of the tooth and periodontal tissue. The influence of iron levels on the cytodifferentiation of PDL cells has not been studied, despite evidence that iron overload or deficiency can have adverse effects on alveolar bone density. The purpose of this study was to examine the effects of altered iron levels on cytodifferentiation in human PDL cells. Human PDL cells were incubated with culture media supplemented with 10-50 μm ammonium ferric citrate or 5 μm deferoxamine (an iron chelator) during differentiation. Intracellular iron status was assessed by measuring changes in the expression of ferritin RNA and protein. PDL cell differentiation and function were evaluated by measuring osteoblast differentiation gene markers and the capacity of cultures to form mineralized nodules. Iron accumulation resulted in upregulation of light and heavy chain ferritin proteins. Concurrently, osteoblast differentiation gene markers and mineralized nodule formation were suppressed. Iron deficiency resulted in downregulation of light and heavy chain ferritin proteins, suppression of alkaline phosphatase activity and formation of mineralized nodules during PDL cell differentiation. We conclude that iron is critical for normal cell differentiation of human PDL cells.

Iron chelator deferoxamine alters iron-regulatory genes and proteins and suppresses osteoblast phenotype in fetal rat calvaria cells

There are few studies describing the extent to which low iron status affects osteoblastogenesis, despite evidence that iron deficiency produces adverse effects on bone density. The purpose of this study was to evaluate alterations in intracellular iron status by measuring iron-regulated gene and protein expression and to describe development of osteoblast phenotype in primary cells treated with iron chelator deferoxamine (DFOM) during differentiation. Using the well-described fetal rat calvaria model, cells were incubated with 0–8µM DFOM throughout differentiation (confluence to day (D) 21), or only during early differentiation (confluence to D13–15) or late differentiation (D13–15 to D21). Changes in intracellular iron status were determined by measuring alterations in gene and protein expression of transferrin receptor and ferritin light chain and heavy chain. Development of osteoblast phenotype was monitored by measuring expression of genes that are known to be up-regulated during differentiation, analyzing the percentage of mineralized surface area, and counting the number of multi-layered bone nodules at the end of culture. Results indicate that treatment throughout differentiation with 8µM DFOM alters iron-regulated genes and proteins by mid-differentiation (D13–15) in a pattern consistent with iron deficiency with concomitant down-regulation of osteoblast phenotype genes, especially osteocalcin. Additionally, alkaline phosphatase staining was lower and there was about 70% less mineralized surface area (p<0.05) by D21 in wells treated throughout differentiation with 8µM DFOM compared to control. Down-regulation of osteocalcin and alkaline phosphatase mRNA (p<0.05) and suppressed mineralization (p<0.05) was also evident at D21 in cells treated only during early differentiation. In contrast, treatment during late differentiation did not alter osteoblastic outcomes by D21. In conclusion, it appears that iron is required for normal osteoblast phenotype development, and that early rather than late differentiation events may be more sensitive to iron availability.

Iron and Parenteral Nutrition

There is ample evidence that iron is an essential trace element, but assessment of iron status and decisions on the amounts needed and the means of delivery in patients on parenteral nutrition (PN) have been unclear. Although iron requirements may diminish during acute illness, the frequent concurrence of blood loss and iron deficiency argue strongly for maintenance of levels of delivery in line with basal requirements. Maintenance of iron delivery has not been thought likely to present risk, but new data are questioning this. The needs of menstruating, pregnant, and lactating women are greater than those of adult men. The evidence favors an intravenous dose of around 1 mg of elemental iron per day in adult men and postmenopausal women. Doses of 1.5 mg/d [DOSAGE ERROR CORRECTED] in menstruating women and 2.0 mg/d [DOSAGE ERROR CORRECTED] for those in the later stages of pregnancy or lactating can be supported. A calibrated response is required in the growing child. Continued monitoring of iron status is recommended. Iron is an essential component for most PN regimens. The quantity of iron to be included should take account of predicted requirements.

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

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

Influence of HFE variants and cellular iron on monocyte chemoattractant protein-1

BackgroundPolymorphisms in the MHC class 1-like gene known as HFE have been proposed as genetic modifiers of neurodegenerative diseases that include neuroinflammation as part of the disease process. Variants of HFE are relatively common in the general population and are most commonly associated with iron overload, but can promote subclinical cellular iron loading even in the absence of clinically identified disease. The effects of the variants as well as the resulting cellular iron dyshomeostasis potentially impact a number of disease-associated pathways. We tested the hypothesis that the two most common HFE variants, H63D and C282Y, would affect cellular secretion of cytokines and trophic factors.MethodsWe screened a panel of cytokines and trophic factors using a multiplexed immunoassay in human neuroblastoma SH-SY5Y cells expressing different variants of HFE. The influence of cellular iron secretion on the potent chemokine monocyte chemoattractant protein-1 (MCP-1) was assessed using ferric ammonium citrate and the iron chelator, desferroxamine. Additionally, an antioxidant, Trolox, and an anti-inflammatory, minocycline, were tested for their effects on MCP-1 secretion in the presence of HFE variants.ResultsExpression of the HFE variants altered the labile iron pool in SH-SY5Y cells. Of the panel of cytokines and trophic factors analyzed, only the release of MCP-1 was affected by the HFE variants. We further examined the relationship between iron and MCP-1 and found MCP-1 secretion tightly associated with intracellular iron status. A potential direct effect of HFE is considered because, despite having similar levels of intracellular iron, the association between HFE genotype and MCP-1 expression was different for the H63D and C282Y HFE variants. Moreover, HFE genotype was a factor in the effect of minocycline, a multifaceted antibiotic used in treating a number of neurologic conditions associated with inflammation, on MCP-1 secretion.ConclusionOur results demonstrate that HFE polymorphisms influence the synthesis and release of MCP-1. The mechanism of action involves cellular iron status but it appears there could be additional influences such as ER stress. Finally, these data demonstrate a pharmacogenetic effect of HFE polymorphisms on the ability of minocycline to inhibit MCP-1 secretion.

Plantaris muscle of aged rats demonstrates iron accumulation and altered expression of iron regulation proteins

Increased free radical production and oxidative damage in ageing muscle may be a contributing factor to the development of sarcopenia. It has been suggested that the accumulation of iron may be an underlying factor in the development of oxidative stress in ageing tissues, including skeletal muscle. At present, however, the mechanisms responsible for ageing-associated muscle iron accumulation are unknown. These experiments tested the hypothesis that ageing-associated elevations in skeletal muscle iron are accompanied by altered expression of key regulators of intracellular iron status. We determined non-haem iron, oxidative injury, and expression levels of iron regulation proteins in plantaris muscles harvested from 6- and 24- to 26-month-old Fisher 344 rats (n = 10 per group). Ageing resulted in a 62% elevation in skeletal muscle non-haem iron (P < 0.05) and higher protein oxidative damage (P < 0.05). Notably, ageing was associated with elevated expression of ferritin (heavy chain, +56.2-fold; light chain, +7.3-fold), an important iron storage protein. Conversely, the iron transport protein transferrin receptor-1 demonstrated dramatic downregulation (-10.8-fold; P < 0.05) in old muscle, whereas the level of divalent metal transporter-1 protein expression was unaltered. No change in protein level of iron regulatory protein-1 was observed. In summary, these results demonstrate the occurrence of altered iron regulation concomitant with iron accumulation and oxidative stress in aged skeletal muscle. Importantly, the maintenance of divalent metal transporter-1 protein expression into old age could play a role in the accumulation of skeletal muscle iron.