Peter D. Buckett

The small molecule, genistein, increases hepcidin expression in human hepatocytes

Hepcidin, a peptide hormone that decreases intestinal iron absorption and macrophage iron release, is a potential drug target for patients with iron overload syndromes because its levels are inappropriately low in these individuals. Endogenous stimulants of Hepcidin transcription include bone morphogenic protein 6 (BMP6) and interleukin‐6 (IL‐6) by effects on mothers against decapentaplegic homolog (Smad)4 or signal transducer and activator of transcription (Stat)3, respectively. We conducted a small‐scale chemical screen in zebrafish embryos to identify small molecules that modulate hepcidin expression. We found that treatment with the isoflavone, genistein, from 28‐52 hours postfertilization in zebrafish embryos enhanced Hepcidin transcript levels, as assessed by whole‐mount in situ hybridization and quantitative real‐time reverse‐transcriptase polymerase chain reaction. Genisteins stimulatory effect was conserved in human hepatocytes: Genistein treatment of HepG2 cells increased both Hepcidin transcript levels and promoter activity. We found that genisteins effect on Hepcidin expression did not depend on estrogen receptor signaling or increased cellular iron uptake, but was impaired by mutation of either BMP response elements or the Stat3‐binding site in the Hepcidin promoter. RNA sequencing of transcripts from genistein‐treated hepatocytes indicated that genistein up‐regulated 68% of the transcripts that were up‐regulated by BMP6; however, genistein raised levels of several transcripts involved in Stat3 signaling that were not up‐regulated by BMP6. Chromatin immunoprecipitation and ELISA experiments revealed that genistein enhanced Stat3 binding to the Hepcidin promoter and increased phosphorylation of Stat3 in HepG2 cells. Conclusion: Genistein is the first small‐molecule experimental drug that stimulates Hepcidin expression in vivo and in vitro. These experiments demonstrate the feasibility of identifying and characterizing small molecules that increase Hepcidin expression. Genistein and other candidate molecules may subsequently be developed into new therapies for iron overload syndromes. (Hepatology 2013;58:1315–1325)

Iron-Responsive Olfactory Uptake of Manganese Improves Motor Function Deficits Associated with Iron Deficiency

Iron-responsive manganese uptake is increased in iron-deficient rats, suggesting that toxicity related to manganese exposure could be modified by iron status. To explore possible interactions, the distribution of intranasally-instilled manganese in control and iron-deficient rat brain was characterized by quantitative image analysis using T1-weighted magnetic resonance imaging (MRI). Manganese accumulation in the brain of iron-deficient rats was doubled after intranasal administration of MnCl2 for 1- or 3-week. Enhanced manganese level was observed in specific brain regions of iron-deficient rats, including the striatum, hippocampus, and prefrontal cortex. Iron-deficient rats spent reduced time on a standard accelerating rotarod bar before falling and with lower peak speed compared to controls; unexpectedly, these measures of motor function significantly improved in iron-deficient rats intranasally-instilled with MnCl2. Although tissue dopamine concentrations were similar in the striatum, dopamine transporter (DAT) and dopamine receptor D1 (D1R) levels were reduced and dopamine receptor D2 (D2R) levels were increased in manganese-instilled rats, suggesting that manganese-induced changes in post-synaptic dopaminergic signaling contribute to the compensatory effect. Enhanced olfactory manganese uptake during iron deficiency appears to be a programmed “rescue response” with beneficial influence on motor impairment due to low iron status.

Absorption of Manganese and Iron in a Mouse Model of Hemochromatosis

Hereditary hemochromatosis, an iron overload disease associated with excessive intestinal iron absorption, is commonly caused by loss of HFE gene function. Both iron and manganese absorption are regulated by iron status, but the relationships between the transport pathways of these metals and how they are affected by HFE-associated hemochromatosis remain poorly understood. Loss of HFE function is known to alter the intestinal expression of DMT1 (divalent metal transporter-1) and Fpn (ferroportin), transporters that have been implicated in absorption of both iron and manganese. Although the influence of HFE deficiency on dietary iron absorption has been characterized, potential effects on manganese metabolism have yet to be explored. To investigate the role of HFE in manganese absorption, we characterized the uptake and distribution of the metal in Hfe −/− knockout mice after intravenous, intragastric, and intranasal administration of 54Mn. These values were compared to intravenous and intragastric administration of 59Fe. Intestinal absorption of 59Fe was increased and clearance of injected 59Fe was also increased in Hfe−/− mice compared to controls. Hfe −/− mice displayed greater intestinal absorption of 54Mn compared to wild-type Hfe+/+ control mice. After intravenous injection, the distribution of 59Fe to heart and liver was greater in Hfe −/− mice but no remarkable differences were observed for 54Mn. Although olfactory absorption of 54Mn into blood was unchanged in Hfe −/− mice, higher levels of intranasally-instilled 54Mn were associated with Hfe−/− brain compared to controls. These results show that manganese transport and metabolism can be modified by HFE deficiency.

Small molecule inhibitors of divalent metal transporter-1

Divalent metal transporter-1 (DMT1) is a divalent cation transporter that plays a key role in iron metabolism by mediating ferrous iron uptake across the small intestine. We have previously identified several small molecule inhibitors of iron uptake (4). Using a cell line that stably overexpresses DMT1, we screened the ability of these inhibitors to specifically block this transporters activity. One compound, NSC306711, inhibited DMT1-mediated iron uptake in a reversible and competitive manner. This inhibitor is a polysulfonated dye containing two copper centers. Although one of these two sites could be chelated by Triethylenetetramine copper chelation did not perturb NSC306711 inhibition of DMT1 activity. Several other polysulfonated dyes with structural features similar to NSC306711 were identified as potential DMT1 transport inhibitors. This study characterizes important pharmacological tools that can be used to probe DMT1s mechanism of iron transport and its role in iron metabolism.

Chemical Genetic Screening Identifies Sulfonamides That Raise Organellar pH and Interfere with Membrane Traffic

Chemical genetics seeks to identify small molecules that afford functional dissection of cell biological pathways. Previous screens for small molecule inhibitors of exocytic membrane traffic yielded the identification and characterization of several compounds that block traffic from the Golgi to the cell surface as well as transport from the endoplasmic reticulum to the Golgi network [Feng et al. Proc Natl Acad Sci USA 2003;100:6469–6474; Yarrow et al. Comb Chem High Throughput Screen 2003;6:279–286; Feng et al. EMBO Reports 2004: in press]. Here, we screened these inhibitors for potential effects on endocytic membrane traffic. Two structurally related sulfonamides were found to be potent and reversible inhibitors of transferrin‐mediated iron uptake. These inhibitors do not block endoplasmic reticulum‐to‐Golgi transport, but do disrupt Golgi‐to‐cell surface traffic. The compounds are members of a novel class of sulfonamides that elevate endosomal and lysosomal pH, down‐regulate cell surface receptors, and impair recycling of internalized transferrin receptors to the plasma membrane. In vitro experiments revealed that the sulfonamides directly inhibit adenosine triphosphate (ATP) hydrolysis by the V‐ATPase and that they also possess a potent proton ionophore activity. While maintenance of organellar pH is known to be a critical factor in both endocytosis and exocytosis, the precise role of acidification, beyond the uncoupling of ligands from their receptors, remains largely unknown. Identification of this novel class of sulfonamide inhibitors provides new chemical tools to better understand the function of organelle pH in membrane traffic and the activity of V‐ATPases in particular.

Influence of DMT1 and iron status on inflammatory responses in the lung

Divalent metal transporter 1 (DMT1) is the major iron transporter responsible for duodenal dietary iron absorption and is required for erythropoiesis. Recent studies suggest that loss of DMT1 activity could be involved in metal-related lung injury, but little is known about the effects of iron status and DMT1 function on pulmonary inflammation. To better define the role of DMT1 and iron status in pulmonary inflammatory responses, we performed bronchoalveolar lavage (BAL) following intratracheal instillation of lipopolysaccharide (LPS) to the Belgrade rat, an animal model deficient in DMT1 function. In the basal state, the BAL fluid of Belgrade rats had more macrophages and higher lactate dehydrogenase, myeloperoxidase, albumin, and hemoglobin levels compared with heterozygote control rats. Following LPS instillation, the macrophage fraction relative to total BAL cell content and levels of albumin and IgM were increased in Belgrade rats compared with controls. In contrast, heterozygote Belgrade rats made anemic by diet-induced iron deficiency exhibited attenuated inflammatory responses to LPS. These combined results show that pulmonary inflammation can be modified by both DMT1 and iron status. Loss of DMT1 alters pulmonary responses necessary for lung homeostasis in the basal state and enhances LPS-induced inflammation and therefore would contribute to progression of lung injury.

Iron loading impairs lipoprotein lipase activity and promotes hypertriglyceridemia

Iron loading is associated with altered lipid metabolism, but underlying mechanisms remain unknown. We compared serum iron and triglycerides (TGs) in Belgrade rats, a genetic model of iron‐loading anemia. Homozygous b/b rats had greater serum iron (68 vs. 28 μM; P=0.0004) and TG levels (180 vs. 84 mg/dl; P=0.014) compared to +/b controls. To confirm the association between iron loading and high TGs, Fischer rats were fed chow containing 1% carbonyl iron. Compared to controls pair‐fed normal chow, carbonyl iron‐fed rats had elevated serum iron (42 vs. 21 (μM; P=0.007) and TGs (190 vs. 115 mg/dl; P=0.009). Despite normal hepatic production and secretion, TG clearance was lower in b/b than +/b rats due to reduced serum lipoprotein lipase (LPL) activity (3.1 vs. 5.0 mM/min; P=0.026). Likewise, LPL was lower in carbonyl iron‐fed rats compared to controls (2.4 vs. 3.7 mM/min; P=0.017). Direct addition of iron to serum ex vivo or recombinant LPL in vitro decreased enzymatic activity in a dose‐dependent manner. Lowering serum iron in Belgrade rats reduced TG levels (274 to 67 mg/dl, P=0.001). This study explains the relationship between iron status and lipid metabolism and provides mechanistic support for interventions that reduce serum iron levels in individuals at risk for hypertriglyceridemia.—Kim, J., Jia, X., Buckett, P. D., Liu, S., Lee, C.‐H., Wessling‐Resnick, M. Iron loading impairs lipoprotein lipase activity and promotes hypertriglyceridemia. FASEB J. 27, 1657–1663 (2013). www.fasebj.org

Ferristatin II Promotes Degradation of Transferrin Receptor-1 In Vitro and In Vivo

Previous studies have shown that the small molecule iron transport inhibitor ferristatin (NSC30611) acts by down-regulating transferrin receptor-1 (TfR1) via receptor degradation. In this investigation, we show that another small molecule, ferristatin II (NSC8679), acts in a similar manner to degrade the receptor through a nystatin-sensitive lipid raft pathway. Structural domains of the receptor necessary for interactions with the clathrin pathway do not appear to be necessary for ferristatin II induced degradation of TfR1. While TfR1 constitutively traffics through clathrin-mediated endocytosis, with or without ligand, the presence of Tf blocked ferristatin II induced degradation of TfR1. This effect of Tf was lost in a ligand binding receptor mutant G647A TfR1, suggesting that Tf binding to its receptor interferes with the drug’s activity. Rats treated with ferristatin II have lower TfR1 in liver. These effects are associated with reduced intestinal 59Fe uptake, lower serum iron and transferrin saturation, but no change in liver non-heme iron stores. The observed hypoferremia promoted by degradation of TfR1 by ferristatin II appears to be due to induced hepcidin gene expression.

Severe Postnatal Iron Deficiency Alters Emotional Behavior and Dopamine Levels in the Prefrontal Cortex of Young Male Rats

Iron deficiency in early human life is associated with abnormal neurological development. The objective of this study was to evaluate the effect of postnatal iron deficiency on emotional behavior and dopaminergic metabolism in the prefrontal cortex in a young male rodent model. Weanling, male, Sprague-Dawley rats were fed standard nonpurified diet (220 mg/kg iron) or an iron-deficient diet (2-6 mg/kg iron). After 1 mo, hematocrits were 0.42 ± 0.0043 and 0.16 ± 0.0068 (mean ± SEM; P < 0.05; n = 8), liver nonheme iron concentrations were 2.3 ± 0.24 and 0.21 ± 0.010 μmol/g liver (P < 0.05; n = 8), and serum iron concentrations were 47 ± 5.4 and 23 ± 7.1 μmol/L (P < 0.05; n = 8), respectively. An elevated plus maze was used to study emotional behavior. Iron-deficient rats displayed anxious behavior with fewer entries and less time spent in open arms compared to control rats (0.25 ± 0.25 vs. 1.8 ± 0.62 entries; 0.88 ± 0.88 vs. 13 ± 4.6 s; P < 0.05; n = 8). Iron-deficient rats also traveled with a lower velocity in the elevated plus maze (1.2 ± 0.15 vs. 1.7 ± 0.12 cm/s; P < 0.05; n = 8), behavior that reflected reduced motor function as measured on a standard accelerating rotarod device. Both the time on the rotarod bar before falling and the peak speed attained on rotarod by iron-deficient rats were lower than control rats (156 ± 12 vs. 194 ± 12 s; 23 ± 1.5 vs. 28 ± 1.6 rpm; P < 0.05; n = 7-8). Microdialysis experiments showed that these behavioral effects were associated with reduced concentrations of extracellular dopamine in the prefrontal cortex of the iron-deficient rats (79 ± 7.0 vs. 110 ± 14 ng/L; P < 0.05; n = 4). Altered dopaminergic signaling in the prefrontal cortex most likely contributes to the anxious behavior observed in young male rats with severe iron deficiency.

The small molecule ferristatin II induces hepatic hepcidin expression in vivo and in vitro

Previous studies have shown that administration of ferristatin II to rats is associated with decreased serum iron, reduced transferrin saturation, and increased hepatic hepcidin expression. BMP and IL-6 signaling act via Smad and Stat3 transcription factors, respectively, to increase expression of hepcidin, the master regulator of iron metabolism. In this study, we aimed to explore the underlying mechanism of ferristatin II action on hepcidin production. We found that ferristatin II greatly increased hepcidin expression both in vivo and in vitro. In the rat liver, ferristatin II treatment decreased expression of Smad downstream targets Smad7 and Id1 and increased expression of Stat3 downstream targets α-2-macroglobulin, α-1-acid glycoprotein, and C-reactive peptide. Ferristatin II also increased Stat3 phosphorylation in the rat liver without affecting serum or hepatic IL-6 levels. It is unclear whether the Stat3 activation observed in vivo is a cause or a consequence to hepcidin induction. Reporter gene expression studies demonstrated that ferristatin II synergized with BMP6 and IL-6 to enhance hepcidin expression in vitro. However, this synergy was not due to activation of either Smad or Stat3 signaling, raising the possibility that ferristatin II may activate a novel pathway for hepcidin regulation.