RICTOR/mTORC2 downregulation in BRAFV600E melanoma cells promotes resistance to BRAF/MEK inhibition.

BACKGROUND: The main drawback of BRAF/MEK inhibitors (BRAF/MEKi)-based targeted therapy in the management of BRAF-mutated cutaneous metastatic melanoma (MM) is the development of therapeutic resistance. We aimed to assess in this context the role of mTORC2, a signaling complex defined by the presence of the essential RICTOR subunit, regarded as an oncogenic driver in several tumor types, including MM. METHODS: After analyzing The Cancer Genome Atlas MM patients' database to explore both overall survival and molecular signatures as a function of intra-tumor RICTOR levels, we investigated the effects of RICTOR downregulation in BRAFV600E MM cell lines on their response to BRAF/MEKi. We performed proteomic screening to identify proteins modulated by changes in RICTOR expression, and Seahorse analysis to evaluate the effects of RICTOR depletion on mitochondrial respiration. The combination of BRAFi with drugs targeting proteins and processes emerged in the proteomic screening was carried out on RICTOR-deficient cells in vitro and in a xenograft setting in vivo. RESULTS: Low RICTOR levels in BRAF-mutated MM correlate with a worse clinical outcome. Gene Set Enrichment Analysis of low-RICTOR tumors display gene signatures suggestive of activation of the mitochondrial Electron Transport Chain (ETC) energy production. RICTOR-deficient BRAFV600E cells are intrinsically tolerant to BRAF/MEKi and anticipate the onset of resistance to BRAFi upon prolonged drug exposure. Moreover, in drug-naïve cells we observed a decline in RICTOR expression shortly after BRAFi exposure. In RICTOR-depleted cells, both mitochondrial respiration and expression of nicotinamide phosphoribosyltransferase (NAMPT) are enhanced, and their pharmacological inhibition restores sensitivity to BRAFi. CONCLUSIONS: Our work unveils an unforeseen tumor-suppressing role for mTORC2 in the early adaptation phase of BRAFV600E melanoma cells to targeted therapy and identifies the NAMPT-ETC axis as a potential therapeutic vulnerability of low RICTOR tumors. Importantly, our findings indicate that the evaluation of intra-tumor RICTOR levels has a prognostic value in metastatic melanoma and may help to guide therapeutic strategies in a personalized manner.

MicroRNA-22 Is a Key Regulator of Lipid and Metabolic Homeostasis.

Obesity is a growing public health problem associated with increased risk of type 2 diabetes, cardiovascular disease, nonalcoholic fatty liver disease (NAFLD) and cancer. Here, we identify microRNA-22 (miR-22) as an essential rheostat involved in the control of lipid and energy homeostasis as well as the onset and maintenance of obesity. We demonstrate through knockout and transgenic mouse models that miR-22 loss-of-function protects against obesity and hepatic steatosis, while its overexpression promotes both phenotypes even when mice are fed a regular chow diet. Mechanistically, we show that miR-22 controls multiple pathways related to lipid biogenesis and differentiation. Importantly, genetic ablation of miR-22 favors metabolic rewiring towards higher energy expenditure and browning of white adipose tissue, suggesting that modulation of miR-22 could represent a viable therapeutic strategy for treatment of obesity and other metabolic disorders.

Proteomics-Based Evidence for a Pro-Oncogenic Role of ESRP1 in Human Colorectal Cancer Cells.

The RNA-binding protein, Epithelial Splicing Regulatory Protein 1 (ESRP1) can promote or suppress tumorigenesis depending on the cell type and disease context. In colorectal cancer, we have previously shown that aberrantly high ESRP1 expression can drive tumor progression. In order to unveil the mechanisms by which ESRP1 can modulate cancer traits, we searched for proteins affected by modulation of Esrp1 in two human colorectal cancer cell lines, HCA24 and COLO320DM, by proteomics analysis. Proteins hosted by endogenous ESRP1 ribonucleoprotein complex in HCA24 cells were also analyzed following RNA-immunoprecipitation. Proteomics data were complemented with bioinformatics approach to exploit publicly available data on protein-protein interaction (PPI). Gene Ontology was analysed to identify a common molecular signature possibly explaining the pro-tumorigenic role of ESRP1. Interestingly, proteins identified herein support a role for ESRP1 in response to external stimulus, regulation of cell cycle and hypoxia. Our data provide further insights into factors affected by and entwined with ESRP1 in colorectal cancer.

Mice harbouring a SCA28 patient mutation in AFG3L2 develop late-onset ataxia associated with enhanced mitochondrial proteotoxicity.

Spinocerebellar ataxia 28 is an autosomal dominant neurodegenerative disorder caused by missense mutations affecting the proteolytic domain of AFG3L2, a major component of the mitochondrial m-AAA protease. However, little is known of the underlying pathogenetic mechanisms or how to treat patients with SCA28. Currently available Afg3l2 mutant mice harbour deletions that lead to severe, early-onset neurological phenotypes that do not faithfully reproduce the late-onset and slowly progressing SCA28 phenotype. Here we describe production and detailed analysis of a new knock-in murine model harbouring an Afg3l2 allele carrying the p.Met665Arg patient-derived mutation. Heterozygous mutant mice developed normally but adult mice showed signs of cerebellar ataxia detectable by beam test. Although cerebellar pathology was negative, electrophysiological analysis showed a trend towards increased spontaneous firing in Purkinje cells from heterozygous mutants with respect to wild-type controls. As homozygous mutants died perinatally with evidence of cardiac atrophy, for each genotype we generated mouse embryonic fibroblasts (MEFs) to investigate mitochondrial function. MEFs from mutant mice showed altered mitochondrial bioenergetics, with decreased basal oxygen consumption rate, ATP synthesis and mitochondrial membrane potential. Mitochondrial network formation and morphology was altered, with greatly reduced expression of fusogenic Opa1 isoforms. Mitochondrial alterations were also detected in cerebella of 18-month-old heterozygous mutants and may be a hallmark of disease. Pharmacological inhibition of de novo mitochondrial protein translation with chloramphenicol caused reversal of mitochondrial morphology in homozygous mutant MEFs, supporting the relevance of mitochondrial proteotoxicity for SCA28 pathogenesis and therapy development.

Integrating cardiac PIP3 and cAMP signaling through a PKA anchoring function of p110γ.

Adrenergic stimulation of the heart engages cAMP and phosphoinositide second messenger signaling cascades. Cardiac phosphoinositide 3-kinase p110γ participates in these processes by sustaining ß-adrenergic receptor internalization through its catalytic function and by controlling phosphodiesterase 3B (PDE3B) activity via an unknown kinase-independent mechanism. We have discovered that p110γ anchors protein kinase A (PKA) through a site in its N-terminal region. Anchored PKA activates PDE3B to enhance cAMP degradation and phosphorylates p110γ to inhibit PIP(3) production. This provides local feedback control of PIP(3) and cAMP signaling events. In congestive heart failure, p110γ is upregulated and escapes PKA-mediated inhibition, contributing to a reduction in ß-adrenergic receptor density. Pharmacological inhibition of p110γ normalizes ß-adrenergic receptor density and improves contractility in failing hearts.

Mutations in the Heme Exporter FLVCR1 Cause Sensory Neurodegeneration with Loss of Pain Perception.

Pain is necessary to alert us to actual or potential tissue damage. Specialized nerve cells in the body periphery, so called nociceptors, are fundamental to mediate pain perception and humans without pain perception are at permanent risk for injuries, burns and mutilations. Pain insensitivity can be caused by sensory neurodegeneration which is a hallmark of hereditary sensory and autonomic neuropathies (HSANs). Although mutations in several genes were previously associated with sensory neurodegeneration, the etiology of many cases remains unknown. Using next generation sequencing in patients with congenital loss of pain perception, we here identify bi-allelic mutations in the FLVCR1 (Feline Leukemia Virus subgroup C Receptor 1) gene, which encodes a broadly expressed heme exporter. Different FLVCR1 isoforms control the size of the cytosolic heme pool required to sustain metabolic activity of different cell types. Mutations in FLVCR1 have previously been linked to vision impairment and posterior column ataxia in humans, but not to HSAN. Using fibroblasts and lymphoblastoid cell lines from patients with sensory neurodegeneration, we here show that the FLVCR1-mutations reduce heme export activity, enhance oxidative stress and increase sensitivity to programmed cell death. Our data link heme metabolism to sensory neuron maintenance and suggest that intracellular heme overload causes early-onset degeneration of pain-sensing neurons in humans.

A composite enhancer regulates p63 gene expression in epidermal morphogenesis and in keratinocyte differentiation by multiple mechanisms.

p63 is a crucial regulator of epidermal development, but its transcriptional control has remained elusive. Here, we report the identification of a long-range enhancer (p63LRE) that is composed of two evolutionary conserved modules (C38 and C40), acting in concert to control tissue- and layer-specific expression of the p63 gene. Both modules are in an open and active chromatin state in human and mouse keratinocytes and in embryonic epidermis, and are strongly bound by p63. p63LRE activity is dependent on p63 expression in embryonic skin, and also in the commitment of human induced pluripotent stem cells toward an epithelial cell fate. A search for other transcription factors involved in p63LRE regulation revealed that the CAAT enhancer binding proteins Cebpa and Cebpb and the POU domain-containing protein Pou3f1 repress p63 expression during keratinocyte differentiation by binding the p63LRE enhancer. Collectively, our data indicate that p63LRE is composed of additive and partly redundant enhancer modules that act to direct robust p63 expression selectively in the basal layer of the epidermis.

Alteration of heme metabolism in a cellular model of Diamond-Blackfan anemia.

Diamond-Blackfan anemia (DBA) is a congenital pure red cell aplasia often associated with skeletal malformations. Mutations in ribosomal protein coding genes, mainly in RPS19, account for the majority of DBA cases. The molecular mechanisms underlying DBA pathogenesis are still not completely understood. Alternative spliced isoforms of FLVCR1 (feline leukemia virus subgroup C receptor 1) transcript coding for non-functional proteins have been reported in some DBA patients. Consistently, a phenotype very close to DBA has been described in animal models of FLVCR1 deficiency. FLVCR1 gene codes for two proteins: the plasma membrane heme exporter FLVCR1a and the mitochondrial heme exporter FLVCR1b. The coordinated expression of both FLVCR1 isoforms regulates an intracellular heme pool, necessary for proper expansion and differentiation of erythroid precursors. Here, we investigate the role of FLVCR1 isoforms in a cellular model of DBA. RPS19-downregulated TF1 cells show reduced FLVCR1a and FLVCR1b mRNA levels associated with heme overload. The downregulation of FLVCR1 isoforms affects cell cycle progression and apoptosis in differentiating K562 cells, a phenotype similar to DBA. Taken together, these data suggest that alteration of heme metabolism could play a role in the pathogenesis of DBA.

LSD1 Neurospecific Alternative Splicing Controls Neuronal Excitability in Mouse Models of Epilepsy.

Alternative splicing in the brain is dynamic and instrumental to adaptive changes in response to stimuli. Lysine-specific demethylase 1 (LSD1/KDM1A) is a ubiquitously expressed histone H3Lys4 demethylase that acts as a transcriptional co-repressor in complex with its molecular partners CoREST and HDAC1/2. In mammalian brain, alternative splicing of LSD1 mini-exon E8a gives rise to neuroLSD1, a neurospecific isoform that, upon phosphorylation, acts as a dominant-negative causing disassembly of the co-repressor complex and de-repression of target genes. Here we show that the LSD1/neuroLSD1 ratio changes in response to neuronal activation and such effect is mediated by neurospecific splicing factors NOVA1 and nSR100/SRRM4 together with a novel cis-silencer. Indeed, we found that, in response to epileptogenic stimuli, downregulation of NOVA1 reduces exon E8a splicing and expression of neuroLSD1. Using behavioral and EEG analyses we observed that neuroLSD1-specific null mice are hypoexcitable and display decreased seizure susceptibility. Conversely, in a mouse model of Rett syndrome characterized by hyperexcitability, we measured higher levels of NOVA1 protein and upregulation of neuroLSD1. In conclusion, we propose that, in the brain, correct ratio between LSD1 and neuroLSD1 contributes to excitability and, when altered, could represent a pathogenic event associated with neurological disorders involving altered E/I.

Hypoxia controls Flvcr1 gene expression in Caco2 cells through HIF2α and ETS1.

The tissue-specific gene expression changes mediated by the hypoxia inducible factors (HIFs) allow the adaptation of cells to low oxygen tension and control several processes including erythropoiesis, angiogenesis and vasculogenesis. The Feline Leukemia Virus, subgroup C, Receptor 1 (Flvcr1) gene encodes for two isoforms, Flvcr1a and 1b, involved in the export of heme out of the cell and of mitochondria respectively. Studies in mouse models demonstrated a crucial role of Flvcr1 isoforms in erythropoiesis and during embryo development. Here, we showed the modulation of Flvcr1 gene expression in different tissues and cell lines in response to hypoxia. Chromatin immunoprecipitation analysis demonstrated that HIF2α and HIF-dependent transcription factor ETS1 (v-ets avian erythroblastosis virus E26 oncogene homolog 1) bind at the region -318/+39 of the Flvcr1 promoter. Analysis of Caco2 cells in which HIF2α or ETS1 were silenced or overexpressed demonstrated that, both HIF2α and ETS1 are involved in the transcriptional regulation of Flvcr1a and that HIF2α is absolutely required for Flvcr1a induction upon hypoxia. The inclusion of the Flvcr1 gene in the group of HIF2α-responsive genes strengthens its role in hypoxia-stimulated processes like erythropoiesis, vasculogenesis and heme absorption.

Heme exporter FLVCR1a regulates heme synthesis and degradation and controls activity of cytochromes P450.

BACKGROUND & AIMS: The liver has one of the highest rates of heme synthesis of any organ. More than 50% of the heme synthesized in the liver is used for synthesis of P450 enzymes, which metabolize exogenous and endogenous compounds that include natural products, hormones, drugs, and carcinogens. Feline leukemia virus subgroup C cellular receptor 1a (FLVCR1a) is plasma membrane heme exporter that is ubiquitously expressed and controls intracellular heme content in hematopoietic lineages. We investigated the role of Flvcr1a in liver function in mice. METHODS: We created mice with conditional disruption of Mfsd7b, which encodes Flvcr1a, in hepatocytes (Flvcr1a(fl/fl);alb-cre mice). Mice were analyzed under basal conditions, after phenylhydrazine-induced hemolysis, and after induction of cytochromes P450 synthesis. Livers were collected and analyzed by histologic, quantitative real-time polymerase chain reaction, and immunoblot analyses. Hepatic P450 enzymatic activities were measured. RESULTS: Flvcr1a(fl/fl);alb-cre mice accumulated heme and iron in liver despite up-regulation of heme oxygenase 1, ferroportin, and ferritins. Hepatic heme export activity of Flvcr1a was closely associated with heme biosynthesis, which is required to sustain cytochrome induction. Upon cytochromes P450 stimulation, Flvcr1a(fl/fl);alb-cre mice had reduced cytochrome activity, associated with accumulation of heme in hepatocytes. The expansion of the cytosolic heme pool in these mice was likely responsible for the early inhibition of heme synthesis and increased degradation of heme, which reduced expression and activity of cytochromes P450. CONCLUSIONS: In livers of mice, Flvcr1a maintains a free heme pool that regulates heme synthesis and degradation as well as cytochromes P450 expression and activity. These findings have important implications for drug metabolism.

The heme exporter Flvcr1 regulates expansion and differentiation of committed erythroid progenitors by controlling intracellular heme accumulation.

Feline leukemia virus subgroup C receptor 1 (Flvcr1) encodes two heme exporters: FLVCR1a, which localizes to the plasma membrane, and FLVCR1b, which localizes to mitochondria. Here, we investigated the role of the two Flvcr1 isoforms during erythropoiesis. We showed that, in mice and zebrafish, Flvcr1a is required for the expansion of committed erythroid progenitors but cannot drive their terminal differentiation, while Flvcr1b contributes to the expansion phase and is required for differentiation. FLVCR1a-down-regulated K562 cells have defective proliferation, enhanced differentiation, and heme loading in the cytosol, while FLVCR1a/1b-deficient K562 cells show impairment in both proliferation and differentiation, and accumulate heme in mitochondria. These data support a model in which the coordinated expression of Flvcr1a and Flvcr1b contributes to control the size of the cytosolic heme pool required to sustain metabolic activity during the expansion of erythroid progenitors and to allow hemoglobinization during their terminal maturation. Consistently, reduction or increase of the cytosolic heme rescued the erythroid defects in zebrafish deficient in Flvcr1a or Flvcr1b, respectively. Thus, heme export represents a tightly regulated process that controls erythropoiesis.

Acute-phase protein hemopexin is a negative regulator of Th17 response and experimental autoimmune encephalomyelitis development.

Hemopexin (Hx) is an acute-phase protein synthesized by hepatocytes in response to the proinflammatory cytokines IL-6, IL-1ß, and TNF-α. Hx is the plasma protein with the highest binding affinity to heme and controls heme-iron availability in tissues and also in T lymphocytes, where it modulates their responsiveness to IFN-γ. Recent data have questioned regarding an anti-inflammatory role of Hx, a role that may be both heme-binding dependent and independent. The aim of this study was to investigate the role of Hx in the development of a T cell-mediated inflammatory autoimmune response. During experimental autoimmune encephalomyelitis (EAE), the mouse model of multiple sclerosis, Hx content in serum increased and remained high. When EAE was induced in Hx knockout (Hx(-/-)) mice, they developed a clinically earlier and exacerbated EAE compared with wild-type mice, associated to a higher amount of CD4(+)-infiltrating T cells. The severe EAE developed by Hx(-/-) mice could be ascribed to an enhanced expansion of Th17 cells accounting for both a higher disposition of naive T cells to differentiate toward the Th17 lineage and a higher production of Th17 differentiating cytokines IL-6 and IL-23 by APCs. When purified human Hx was injected in Hx(-/-) mice before EAE induction, Th17 expansion, as well as disease severity, were comparable with those of wild-type mice. Taken together, these data indicate that Hx has a negative regulatory role in Th17-mediated inflammation and prospect its pharmacological use to limit the expansion of this cell subset in inflammatory and autoimmune disease.

Renal cells from spermatogonial germline stem cells protect against kidney injury.

Spermatogonial stem cells reside in specific niches within seminiferous tubules and continuously generate differentiating daughter cells for production of spermatozoa. Although spermatogonial stem cells are unipotent, these cells are able to spontaneously convert to germline cell-derived pluripotent stem cells (GPSCs) in vitro. GPSCs have many properties of embryonic stem cells and are highly plastic, but their therapeutic potential in tissue regeneration has not been fully explored. Using a novel renal epithelial differentiation protocol, we obtained GPSC-derived tubular-like cells (GTCs) that were functional in vitro, as demonstrated through transepithelial electrical resistance analysis. In mice, GTCs injected after ischemic renal injury homed to the renal parenchyma, and GTC-treated mice showed reduced renal oxidative stress, tubular apoptosis, and cortical damage and upregulated tubular expression of the antioxidant enzyme hemeoxygenase-1. Six weeks after ischemic injury, kidneys of GTC-treated mice had less fibrosis and inflammatory infiltrate than kidneys of vehicle-treated mice. In conclusion, we show that GPSCs can be differentiated into functionally active renal tubular-like cells that therapeutically prevent chronic ischemic damage in vivo, introducing the potential utility of GPSCs in regenerative cell therapy.

Hemopexin therapy improves cardiovascular function by preventing heme-induced endothelial toxicity in mouse models of hemolytic diseases.

BACKGROUND: Hemolytic diseases are characterized by enhanced intravascular hemolysis resulting in heme-catalyzed reactive oxygen species generation, which leads to endothelial dysfunction and oxidative damage. Hemopexin (Hx) is a plasma heme scavenger able to prevent endothelial damage and tissue congestion in a model of heme overload. Here, we tested whether Hx could be used as a therapeutic tool to counteract heme toxic effects on the cardiovascular system in hemolytic diseases. METHODS AND RESULTS: By using a model of heme overload in Hx-null mice, we demonstrated that heme excess in plasma, if not bound to Hx, promoted the production of reactive oxygen species and the induction of adhesion molecules and caused the reduction of nitric oxide availability. Then, we used ß-thalassemia and sickle cell disease mice as models of hemolytic diseases to evaluate the efficacy of an Hx-based therapy in the treatment of vascular dysfunction related to heme overload. Our data demonstrated that Hx prevented heme-iron loading in the cardiovascular system, thus limiting the production of reactive oxygen species, the induction of adhesion molecules, and the oxidative inactivation of nitric oxide synthase/nitric oxide, and promoted heme recovery and detoxification by the liver mainly through the induction of heme oxygenase activity. Moreover, we showed that in sickle cell disease mice, endothelial activation and oxidation were associated with increased blood pressure and altered cardiac function, and the administration of exogenous Hx was found to almost completely normalize these parameters. CONCLUSIONS: Hemopexin treatment is a promising novel therapy to protect against heme-induced cardiovascular dysfunction in hemolytic disorders.

FLVCR1a Controls Cellular Cholesterol Levels through the Regulation of Heme Biosynthesis and Tricarboxylic Acid Cycle Flux in Endothelial Cells.

Feline leukemia virus C receptor 1a (FLVCR1a), initially identified as a retroviral receptor and localized on the plasma membrane, has emerged as a crucial regulator of heme homeostasis. Functioning as a positive regulator of δ-aminolevulinic acid synthase 1 (ALAS1), the rate-limiting enzyme in the heme biosynthetic pathway, FLVCR1a influences TCA cycle cataplerosis, thus impacting TCA flux and interconnected metabolic pathways. This study reveals an unexplored link between FLVCR1a, heme synthesis, and cholesterol production in endothelial cells. Using cellular models with manipulated FLVCR1a expression and inducible endothelial-specific Flvcr1a-null mice, we demonstrate that FLVCR1a-mediated control of heme synthesis regulates citrate availability for cholesterol synthesis, thereby influencing cellular cholesterol levels. Moreover, alterations in FLVCR1a expression affect membrane cholesterol content and fluidity, supporting a role for FLVCR1a in the intricate regulation of processes crucial for vascular development and endothelial function. Our results underscore FLVCR1a as a positive regulator of heme synthesis, emphasizing its integration with metabolic pathways involved in cellular energy metabolism. Furthermore, this study suggests that the dysregulation of heme metabolism may have implications for modulating lipid metabolism. We discuss these findings in the context of FLVCR1a's potential heme-independent function as a choline importer, introducing additional complexity to the interplay between heme and lipid metabolism.

Extracellular Vesicles as Delivery Vehicles for Non-Coding RNAs: Potential Biomarkers for Chronic Liver Diseases.

In recent years, EVs have emerged as promising vehicles for coding and non-coding RNAs (ncRNAs), which have demonstrated remarkable potential as biomarkers for various diseases, including chronic liver diseases (CLDs). EVs are small, membrane-bound particles released by cells, carrying an arsenal of ncRNAs, including microRNAs (miRNAs), long non-coding RNAs (lncRNAs), and other ncRNA species, such as piRNAs, circRNAs, and tsRNAs. These ncRNAs act as key regulators of gene expression, splicing, and translation, providing a comprehensive molecular snapshot of the cells of origin. The non-invasive nature of EV sampling, typically via blood or serum collection, makes them highly attractive candidates for clinical biomarker applications. Moreover, EV-encapsulated ncRNAs offer unique advantages over traditional cell-free ncRNAs due to their enhanced stability within the EVs, hence allowing for their detection in circulation for extended periods and enabling more sensitive and reliable biomarker measurements. Numerous studies have investigated the potential of EV-enclosed ncRNAs as biomarkers for CLD. MiRNAs, in particular, have gained significant attention due to their ability to rapidly respond to changes in cellular stress and inflammation, hallmarks of CLD pathogenesis. Elevated levels of specific miRNAs have been consistently associated with various CLD subtypes, including metabolic dysfunction-associated steatotic liver disease (MASLD), metabolic dysfunction-associated steatohepatitis (MASH), and chronic hepatitis B and C. LncRNAs have also emerged as promising biomarkers for CLD. These transcripts are involved in a wide range of cellular processes, including liver regeneration, fibrosis, and cancer progression. Studies have shown that lncRNA expression profiles can distinguish between different CLD subtypes, providing valuable insights into disease progression and therapeutic response. Promising EV-enclosed ncRNA biomarkers for CLD included miR-122 (elevated levels of miR-122 are associated with MASLD progression and liver fibrosis), miR-21 (increased expression of miR-21 is linked to liver inflammation and fibrosis in CLD patients), miR-192 (elevated levels of miR-192 are associated with more advanced stages of CLD, including cirrhosis and HCC), LncRNA HOTAIR (increased HOTAIR expression is associated with MASLD progression and MASH development), and LncRNA H19 (dysregulation of H19 expression is linked to liver fibrosis and HCC progression). In the present review, we focus on the EV-enclosed ncRNAs as promising tools for the diagnosis and monitoring of CLD of various etiologies.

Cell-specific regulation of Ferroportin transcription following experimentally-induced acute anemia in mice.

Ferroportin (FPN), the sole characterized iron exporter, is mainly controlled by the peptide hormone hepcidin in response to iron, erythroid factors, hypoxia, and inflammation. In addition, intracellular iron level controls FPN translation by modulating the binding of Iron Responsive Proteins at the 5'UTR of FPN mRNA. Recently, hypoxia inducible factor (HIF)2α has been shown to regulate FPN expression in intestinal cells. Here we show that, during experimentally-induced acute anemia in mice, FPN is regulated at transcriptional level in a cell-specific manner. FPN mRNA level increases in duodenum and spleen macrophages, whereas it does not change in liver and is strongly down-regulated in erythroid precursors. These results were confirmed in Caco2, Raw264.7 and K562 cells treated with a hypoxic stimulus. Moreover, we found a differential expression of HIF1α and HIF2α in cells and tissues that might account for the specificity of FPN regulation. Thus, hypoxia, by directly controlling hepcidin and its target FPN, orchestrates a complex regulatory network aimed at ensuring rapid iron recovery from the periphery and efficient iron utilization in the erythroid compartment.

The RacGAP ArhGAP15 is a master negative regulator of neutrophil functions.

In phagocytes, GTPases of the Rac family control crucial antimicrobial functions. The RacGAP ArhGAP15 negatively modulates Rac activity in leukocytes, but its in vivo role in innate immunity remains largely unknown. Here we show that neutrophils and macrophages derived from mice lacking ArhGAP15 presented higher Rac activity but distinct phenotypes. In macrophages, the loss of ArhGAP15 induced increased cellular elongation and membrane protrusions but did not modify chemotactic responses. Conversely, the lack of ArhGAP15 in neutrophils affected critical Rac-dependent antimicrobial functions, specifically causing enhanced chemotactic responses, straighter directional migration, amplified reactive oxygen species production, increased phagocytosis, and improved bacterial killing. In vivo, in a model of severe abdominal sepsis, these effects contributed to increase neutrophil recruitment to the site of infection, thereby limiting bacterial growth, controlling infection spread, reducing systemic inflammation, and ultimately improving survival in ArhGAP15-null mice. Altogether, these results demonstrate the relevance of ArhGAP15 in the selective regulation of multiple neutrophil functions, suggesting that ArhGAP15 targeting might be beneficial in specific pathologic settings like severe sepsis.

Comparison of 3 Tfr2-deficient murine models suggests distinct functions for Tfr2-alpha and Tfr2-beta isoforms in different tissues.

Transferrin receptor 2 (TFR2) is a transmembrane protein that is mutated in hemochromatosis type 3. The TFR2 gene is transcribed in 2 main isoforms: the full-length (alpha) and a shorter form (beta). alpha-Tfr2 is the sensor of diferric transferrin, implicated in the modulation of hepcidin, the main regulator of iron homeostasis. The function of the putative beta-Tfr2 protein is unknown. We have developed a new mouse model (KI) lacking beta-Tfr2 compared with Tfr2 knockout mice (KO). Adult Tfr2 KO mice show liver iron overload and inadequate hepcidin levels relative to body iron stores, even though they increase Bmp6 production. KI mice have normal transferrin saturation, liver iron concentration, hepcidin and Bmp6 levels but show a transient anemia at young age and severe spleen iron accumulation in adult animals. Fpn1 is strikingly decreased in the spleen of these animals. These findings and the expression of beta-Tfr2 in wild-type mice spleen suggest a role for beta-Tfr2 in Fpn1 transcriptional control. Selective inactivation of liver alpha-Tfr2 in KI mice (LCKO-KI) returned the phenotype to liver iron overload. Our results strengthen the function of hepatic alpha-Tfr2 in hepcidin activation, suggest a role for extrahepatic Tfr2 and indicate that beta-Tfr2 may specifically control spleen iron efflux.