Zoubida Karim

Intestinal DMT1 Cotransporter Is Down-regulated by Hepcidin via Proteasome Internalization and Degradation

BACKGROUNDS & AIMSnThe mechanism by which hepcidin regulates iron export from macrophages has been well established and is believed to involve degradation of ferroportin. However, in the small intestine, hepcidins mechanisms of action are not known. We studied human polarized intestinal (Caco-2/TC7) cells and mouse duodenal segments, ex vivo, to investigate the molecular mechanisms by which hepcidin down-regulates intestinal transepithelial iron transport.nnnMETHODSnIron transport was analyzed using ⁵⁵FeNTA. Expression of Divalent Metal Transporter 1 (DMT1) and ferroportin was evaluated by reverse-transcription quantitative polymerase chain reaction and immunoblotting. Videomicroscopy analysis was performed on live cells that expressed either DMT1 or ferroportin fused to green fluorescent protein.nnnRESULTSnIn Caco-2/TC7 cells, physiologic doses of hepcidin (50-1000 nmol/L) inhibited transport of ⁵⁵Fe in a dose-dependent manner; a half-maximum effect was observed at 75-100 nmol/L. However, 200 nmol/L hepcidin induced a significant decrease in DMT1 protein expression but no change in ferroportin protein levels, unlike macrophages. This result was confirmed ex vivo in isolated duodenal segments: 200 nmol/L hepcidin induced a significant reduction in iron transport and DMT1 protein levels but no change in ferroportin levels. In Caco-2/TC7 cells, the effect of hepcidin on the DMT1 protein level was completely abolished in the presence of a proteasome inhibitor (MG-132); DMT1 ubiquitination was induced by the addition of hepcidin.nnnCONCLUSIONSnAn acute increase in hepcidin concentration reduces intestinal iron absorption through ubiquitin-dependent proteasome degradation of DMT1.

Hepcidin regulates intrarenal iron handling at the distal nephron

Hepcidin, the key regulatory hormone of iron homeostasis, and iron carriers such as transferrin receptor1 (TFR1), divalent metal transporter1 (DMT1), and ferroportin (FPN) are expressed in kidney. Whether hepcidin plays an intrinsic role in the regulation of renal iron transport is unknown. Here, we analyzed the renal handling of iron in hemochromatosis Hepc(-/-) and Hjv(-/-) mouse models, as well as in phenylhydrazine (PHZ)-treated mice. We found a marked medullary iron deposition in the kidneys of Hepc(-/-) mice, and iron leak in the urine. The kidneys of Hepc(-/-) mice exhibited a concomitant decrease in TFR1 and increase in ferritin and FPN expression. Increased FPN abundance was restricted to the thick ascending limb (TAL). DMT1 protein remained unaffected despite a significant decrease of its mRNA level, suggesting that DMT1 protein is stabilized in the absence of hepcidin. Treatment of kidney sections from Hepc(-/-) mice with hepcidin decreased DMT1 protein, an effect confirmed in renal cell lines where hepcidin markedly decreased (55)Fe transport. In the kidneys of Hjv(-/-) mice exhibiting low hepcidin expression, the iron overload was similar to that in the kidneys of Hepc(-/-) mice. However, in PHZ mice, iron accumulation resulting from hemoglobin leak was detected in the proximal tubule. Thus, kidneys exhibit a tissue-specific handling of iron that depends on the extra iron source. Hepcidin may control the expression of iron transporters to prevent renal iron overload.

Iron Regulatory Protein 1 Sustains Mitochondrial Iron Loading and Function in Frataxin Deficiency

Mitochondrial iron accumulation is a hallmark of diseases associated with impaired iron-sulfur cluster (Fe-S) biogenesis, such as Friedreich ataxia linked to frataxin (FXN) deficiency. The pathophysiological relevance of the mitochondrial iron loading and the underlying mechanisms are unknown. Using a mouse model of hepatic FXN deficiency in combination with mice deficient for iron regulatory protein 1 (IRP1), a key regulator of cellular iron metabolism, we show that IRP1 activation in conditions of Fe-S deficiency increases the available cytosolic labile iron pool. Surprisingly, our data indicate that IRP1 activation sustains mitochondrial iron supply and function rather than driving detrimental iron overload. Mitochondrial iron accumulation is shown to depend on mitochondrial dysfunction and heme-dependent upregulation of the mitochondrial iron importer mitoferrin-2. Our results uncover an unexpected protective role of IRP1 in pathological conditions associated with altered Fe-S metabolism.

Acute intermittent porphyria causes hepatic mitochondrial energetic failure in a mouse model.

Acute intermittent porphyria (AIP), an inherited hepatic disorder, is due to a defect of hydroxymethylbilane synthase (HMBS), an enzyme involved in heme biosynthesis. AIP is characterized by recurrent, life-threatening attacks at least partly due to the increased hepatic production of 5-aminolaevulinic acid (ALA). Both the mitochondrial enzyme, ALA synthase (ALAS) 1, involved in the first step of heme biosynthesis, which is closely linked to mitochondrial bioenergetic pathways, and the promise of an ALAS1 siRNA hepatic therapy in humans, led us to investigate hepatic energetic metabolism in Hmbs KO mice treated with phenobarbital. The mitochondrial respiratory chain (RC) and the tricarboxylic acid (TCA) cycle were explored in the Hmbs(-/-) mouse model. RC and TCA cycle were significantly affected in comparison to controls in mice treated with phenobarbital with decreased activities of RC complexes I (-52%, (**)p<0.01), II (-50%, (**)p<0.01) and III (-55%, (*)p<0.05), and decreased activity of α-ketoglutarate dehydrogenase (-64%, (*)p<0.05), citrate synthase (-48%, (**)p<0.01) and succinate dehydrogenase (-53%, (*)p<0.05). Complex II-driven succinate respiration was also significantly affected. Most of these metabolic alterations were at least partially restored after the phenobarbital arrest and heme arginate administration. These results suggest a cataplerosis of the TCA cycle induced by phenobarbital, caused by the massive withdrawal of succinyl-CoA by ALAS induction, such that the TCA cycle is unable to supply the reduced cofactors to the RC. This profound and reversible impact of AIP on mitochondrial energetic metabolism offers new insights into the beneficial effect of heme, glucose and ALAS1 siRNA treatments by limiting the cataplerosis of TCA cycle.

Protoporphyrin Retention in Hepatocytes and Kupffer Cells Prevents Sclerosing Cholangitis in Erythropoietic Protoporphyria Mouse Model

BACKGROUND & AIMSnChronic, progressive hepatobiliary disease is the most severe complication of erythropoietic protoporphyria (EPP) and can require liver transplantation, although the mechanisms that lead to liver failure are unknown. We characterized protoporphyrin-IX (PPIX)-linked hepatobiliary disease in BALB/c and C57BL/6 (Fechm1Pas) mice with mutations in ferrochelatase as models for EPP.nnnMETHODSnFechm1Pas and wild-type (control) mice were studied at 12-14 weeks of age. PPIX was quantified; its distribution in the liver, serum levels of lipoprotein-X, liver histology, contents of bile salt and cholesterol phospholipids, and expression of genes were compared in mice of the BALB/c and C57BL/6 backgrounds. The in vitro binding affinity of PPIX for bile components was determined.nnnRESULTSnCompared with mice of the C57BL/6 background, BALB/c Fechm1Pas mice had a more severe pattern of cholestasis, fibrosis with portoportal bridging, bile acid regurgitation, sclerosing cholangitis, and hepatolithiasis. In C57BL/6 Fechm1Pas mice, PPIX was sequestrated mainly in the cytosol of hepatocytes and Kupffer cells, whereas, in BALB/c Fechm1Pas mice, PPIX was localized within enlarged bile canaliculi. Livers of C57BL/6 Fechm1Pas mice were protected through a combination of lower efflux of PPIX and reduced synthesis and export of bile acid.nnnCONCLUSIONSnPPIX binds to bile components and disrupts the physiologic equilibrium of phospholipids, bile acids, and cholesterol in bile. This process might be involved in pathogenesis of sclerosing cholangitis from EPP; a better understanding might improve diagnosis and development of reagents to treat or prevent liver failure in patients with EPP.

Iron is a substrate of the Plasmodium falciparum chloroquine resistance transporter PfCRT in Xenopus oocytes

The chloroquine resistance transporter of the human malaria parasite Plasmodium falciparum, PfCRT, is an important determinant of resistance to several quinoline and quinoline-like antimalarial drugs. PfCRT also plays an essential role in the physiology of the parasite during development inside erythrocytes. However, the function of this transporter besides its role in drug resistance is still unclear. Using electrophysiological and flux experiments conducted on PfCRT-expressing Xenopus laevis oocytes, we show here that both wild-type PfCRT and a PfCRT variant associated with chloroquine resistance transport both ferrous and ferric iron, albeit with different kinetics. In particular, we found that the ability to transport ferrous iron is reduced by the specific polymorphisms acquired by the PfCRT variant as a result of chloroquine selection. We further show that iron and chloroquine transport via PfCRT is electrogenic. If these findings in the Xenopus model extend to P. falciparum in vivo, our data suggest that PfCRT might play a role in iron homeostasis, which is essential for the parasites development in erythrocytes.

Mutation in human CLPX elevates levels of δ-aminolevulinate synthase and protoporphyrin IX to promote erythropoietic protoporphyria

Significance Although heme synthesis is ubiquitous, specific regulatory mechanisms couple heme production to cellular demand and environmental conditions. The importance of these regulatory mechanisms is highlighted by clinical variability in porphyrias caused by loss-of-function mutations in heme synthesis enzymes. Heme synthesis is also controlled by the mitochondrial AAA+ unfoldase ClpX, which participates in both heme-dependent degradation of δ-aminolevulinate synthase (ALAS) and ALAS activation. This study reports a human familial mutation in CLPX that contributes to erythropoietic protoporphyria (EPP) by partially inactivating CLPX. Reduced CLPX activity increases ALAS post-translational stability, causing pathological accumulation of protoporphyrin IX (PPIX) in human patients. Our results thus identify an additional gene that promotes PPIX overproduction and EPP and highlight the complex gene network contributing to disorders of heme metabolism. Loss-of-function mutations in genes for heme biosynthetic enzymes can give rise to congenital porphyrias, eight forms of which have been described. The genetic penetrance of the porphyrias is clinically variable, underscoring the role of additional causative, contributing, and modifier genes. We previously discovered that the mitochondrial AAA+ unfoldase ClpX promotes heme biosynthesis by activation of δ-aminolevulinate synthase (ALAS), which catalyzes the first step of heme synthesis. CLPX has also been reported to mediate heme-induced turnover of ALAS. Here we report a dominant mutation in the ATPase active site of human CLPX, p.Gly298Asp, that results in pathological accumulation of the heme biosynthesis intermediate protoporphyrin IX (PPIX). Amassing of PPIX in erythroid cells promotes erythropoietic protoporphyria (EPP) in the affected family. The mutation in CLPX inactivates its ATPase activity, resulting in coassembly of mutant and WT protomers to form an enzyme with reduced activity. The presence of low-activity CLPX increases the posttranslational stability of ALAS, causing increased ALAS protein and ALA levels, leading to abnormal accumulation of PPIX. Our results thus identify an additional molecular mechanism underlying the development of EPP and further our understanding of the multiple mechanisms by which CLPX controls heme metabolism.

Predominant role of microglia in brain iron retention in Sanfilippo syndrome, a pediatric neurodegenerative disease

Neuroinflammation and iron accumulation are hallmarks of a variety of adult neurodegenerative diseases. In Sanfilippo syndrome (mucopolysaccharidosis type III, MPSIII, a pediatric neurodegenerative disease that shares some features with adult neurodegenerative diseases), the progressive accumulation of heparan sulfate oligosaccharides (HSOs) induces microglia and astrocytes to produce pro‐inflammatory cytokines leading to severe neuroinflammation. The objectives of the present study were (1) to measure the local iron concentration and to assess iron metabolism in the brain of a MPSIIIB murine model and (2) to identify the brain cells involved in this accumulation. We found that iron accumulation in MPSIIIB mice primarily affected the cerebral cortex where hepcidin levels were higher than in wild‐type mice, and increased with aging. This increase was correlated with low expression of ferroportin 1 (FPN1), and thus brain iron retention. Moreover, we showed in vitro that HSOs are directly responsible for the production of hepcidin and the relative decrease in FPN1 expression when added to cultures of microglia and, to a lesser extent, to cultures of astrocytes. In contrast, no significant differences were observed in neurons. Hepcidin induction results from activation of the TLR4 pathway and STAT3 signaling, and leads to iron retention within microglia. Our results show that microglia have a key role in cerebral hepcidin overexpression and thus in the brain iron accumulation observed in the MPSIIIB model.

PXR-ALAS1: a key regulatory pathway in liver toxicity induced by isoniazid-rifampicin antituberculosis treatment.

u x a s U d t h Tuberculosis remains a global public health problem whose effects have major impact in developing countries. The World Health Report (WHO 2012) estimates that more than one third of the world’s population has been exposed to M. tuberculosis, of which approximately 10% will develop active tuberculosis during their lifetime [1]. In 2011 alone, there were 8.7 million new tuberculosis cases reported, of which 1.4 million patients succumbed to the disease [1]. Rifampicin (RIF) and isoniazid (INH) are first-line antituberculosis drugs. However, the combination of these two highly effective drugs is frequently complicated by serious side effects. Progressive hepatobiliary disease is the most severe complication of the INH-RIF treatment [2]. The mechanisms leading to liver failure in humans were poorly understood. The study by Li et al. in nature medicine brings an outstanding and substantial contribution towards the characterization of the mechanisms involved in this hepatotoxicity. The authors underlined the species differences between animal models and humans. Using a pregnant X receptor (PXR)-humanized mouse model (hPXR), they showed that the INH-RIF co-treatment impaired heme-biosynthetic pathway in the liver. The co-therapy caused accumulation of the endogenous hepatotoxin protoporphyrin IX (PPIX) through PXR-mediated transcriptional activations of both cytochromes P450 (CYP450) and aminolevulinic synthase-1 (ALAS1) genes [3]. All these activations directly affected the heme biosynthetic pathway. Although heme is synthesised in every human cell for respiratory and oxido-reduction reactions, it is mostly produced in the erythropoietic cells for hemoglobin synthesis and in the

High urinary ferritin reflects myoglobin iron evacuation in DMD patients

Duchenne muscular dystrophy (DMD) is an X-linked disease caused by mutations in the dystrophin gene leading to the absence of the normal dystrophin protein. The efforts of many laboratories brought new treatments of DMD to the reality, but ongoing and forthcoming clinical trials suffer from absence of valuable biomarkers permitting to follow the outcome of the treatment day by day and to adjust the treatment if needed. In the present study the levels of 128 urinary proteins including growth factors, cytokines and chemokines were compared in urine of DMD patients and age related control subjects by antibody array approach. Surprisingly, statistically significant difference was observed only for urinary ferritin whose level was 50 times higher in young DMD patients. To explain the observed high urinary ferritin content we analysed the levels of iron, iron containing proteins and proteins involved in regulation of iron metabolism in serum and urine of DMD patients and their age-matched healthy controls. Obtained data strongly suggest that elevated level of urinary ferritin is functionally linked to the renal management of myoglobin iron derived from leaky muscles of DMD patients. This first observation of the high level of ferritin in urine of DMD patients permits to consider this protein as a new urinary biomarker in muscular dystrophies and sheds light on the mechanisms of iron metabolism and kidney functioning in DMD.