Marco Rausa
Vita-Salute San Raffaele University
Network
Latest external collaboration on country level. Dive into details by clicking on the dots.
Publication
Featured researches published by Marco Rausa.
Blood | 2015
Antonella Nai; Maria Rosa Lidonnici; Marco Rausa; Giacomo Mandelli; Alessia Pagani; Laura Silvestri; Giuliana Ferrari; Clara Camaschella
Transferrin receptor 2 (TFR2) contributes to hepcidin regulation in the liver and associates with erythropoietin receptor in erythroid cells. Nevertheless, TFR2 mutations cause iron overload (hemochromatosis type 3) without overt erythroid abnormalities. To clarify TFR2 erythroid function, we generated a mouse lacking Tfr2 exclusively in the bone marrow (Tfr2(BMKO)). Tfr2(BMKO) mice have normal iron parameters, reduced hepcidin levels, higher hemoglobin and red blood cell counts, and lower mean corpuscular volume than normal control mice, a phenotype that becomes more evident in iron deficiency. In Tfr2(BMKO) mice, the proportion of nucleated erythroid cells in the bone marrow is higher and the apoptosis lower than in controls, irrespective of comparable erythropoietin levels. Induction of moderate iron deficiency increases erythroblasts number, reduces apoptosis, and enhances erythropoietin (Epo) levels in controls, but not in Tfr2(BMKO) mice. Epo-target genes such as Bcl-xL and Epor are highly expressed in the spleen and in isolated erythroblasts from Tfr2(BMKO) mice. Low hepcidin expression in Tfr2(BMKO) is accounted for by erythroid expansion and production of the erythroid regulator erythroferrone. We suggest that Tfr2 is a component of a novel iron-sensing mechanism that adjusts erythrocyte production according to iron availability, likely by modulating the erythroblast Epo sensitivity.
Human Mutation | 2014
Luigia De Falco; Laura Silvestri; Caroline Kannengiesser; Erica Morán; Claire Oudin; Marco Rausa; Mariasole Bruno; Jessica Aranda; Bienvenida Argiles; Idil Yenicesu; Maria Falcon-Rodriguez; Ebru Yilmaz-Keskin; Ulker Kocak; Carole Beaumont; Clara Camaschella; Achille Iolascon; Bernard Grandchamp; Mayka Sanchez
Iron‐refractory iron‐deficiency anemia (IRIDA) is a rare autosomal‐recessive disorder characterized by hypochromic microcytic anemia, low transferrin saturation, and inappropriate high levels of the iron hormone hepcidin. The disease is caused by variants in the transmembrane protease serine 6 (TMPRSS6) gene that encodes the type II serine protease matriptase‐2, a negative regulator of hepcidin transcription. Sequencing analysis of the TMPRSS6 gene in 21 new IRIDA patients from 16 families with different ethnic origin reveal 17 novel mutations, including the most frequent mutation in Southern Italy (p.W590R). Eight missense mutations were analyzed in vitro. All but the p.T287N variant impair matriptase‐2 autoproteotylic activation, decrease the ability to cleave membrane HJV and inhibit the HJV‐dependent hepcidin activation. Genotype–phenotype studies in IRIDA patients have been so far limited due to the relatively low number of described patients. Our genotype–phenotype correlation analysis demonstrates that patients carrying two nonsense mutations present a more severe anemia and microcytosis and higher hepcidin levels than the other patients. We confirm that TMPRSS6 mutations are spread along the gene and that mechanistically they fully or partially abrogate hepcidin inhibition. Genotyping IRIDA patients help in predicting IRIDA severity and may be useful for predicting response to iron treatment.
Haematologica | 2015
Alessia Pagani; Maud Vieillevoye; Antonella Nai; Marco Rausa; Meriem Ladli; Catherine Lacombe; Patrick Mayeux; Frédérique Verdier; Clara Camaschella; Laura Silvestri
Transferrin receptor-2 is a transmembrane protein whose expression is restricted to hepatocytes and erythroid cells. Transferrin receptor-2 has a regulatory function in iron homeostasis, since its inactivation causes systemic iron overload. Hepatic transferrin receptor-2 participates in iron sensing and is involved in hepcidin activation, although the mechanism remains unclear. Erythroid transferrin receptor-2 associates with and stabilizes erythropoietin receptors on the erythroblast surface and is essential to control erythrocyte production in iron deficiency. We identified a soluble form of transferrin receptor-2 in the media of transfected cells and showed that cultured human erythroid cells release an endogenous soluble form. Soluble transferrin receptor-2 originates from a cleavage of the cell surface protein, which is inhibited by diferric transferrin in a dose-dependent manner. Accordingly, the shedding of the transferrin receptor-2 variant G679A, mutated in the Arginine-Glycine-Aspartic acid motif and unable to bind diferric transferrin, is not modulated by the ligand. This observation links the process of transferrin receptor-2 removal from the plasma membrane to iron homeostasis. Soluble transferrin receptor-2 does not affect the binding of erythropoietin to erythropoietin receptor or the consequent signaling and partially inhibits hepcidin promoter activation only in vitro. Whether it is a component of the signals released by erythropoiesis in iron deficiency remains to be investigated. Our results indicate that membrane transferrin receptor-2, a sensor of circulating iron, is released from the cell membrane in iron deficiency.
PLOS ONE | 2015
Marco Rausa; Alessia Pagani; Antonella Nai; Alessandro Campanella; Maria Enrica Gilberti; Pietro Apostoli; Clara Camaschella; Laura Silvestri
Bmp6 is the main activator of hepcidin, the liver hormone that negatively regulates plasma iron influx by degrading the sole iron exporter ferroportin in enterocytes and macrophages. Bmp6 expression is modulated by iron but the molecular mechanisms are unknown. Although hepcidin is expressed almost exclusively by hepatocytes (HCs), Bmp6 is produced also by non-parenchymal cells (NPCs), mainly sinusoidal endothelial cells (LSECs). To investigate the regulation of Bmp6 in HCs and NPCs, liver cells were isolated from adult wild type mice whose diet was modified in iron content in acute or chronic manner and in disease models of iron deficiency (Tmprss6 KO mouse) and overload (Hjv KO mouse). With manipulation of dietary iron in wild-type mice, Bmp6 and Tfr1 expression in both HCs and NPCs was inversely related, as expected. When hepcidin expression is abnormal in murine models of iron overload (Hjv KO mice) and deficiency (Tmprss6 KO mice), Bmp6 expression in NPCs was not related to Tfr1. Despite the low Bmp6 in NPCs from Tmprss6 KO mice, Tfr1 mRNA was also low. Conversely, despite body iron overload and high expression of Bmp6 in NPCs from Hjv KO mice, Tfr1 mRNA and protein were increased. However, in the same cells ferritin L was only slightly increased, but the iron content was not, suggesting that Bmp6 in these cells reflects the high intracellular iron import and export. We propose that NPCs, sensing the iron flux, not only increase hepcidin through Bmp6 with a paracrine mechanism to control systemic iron homeostasis but, controlling hepcidin, they regulate their own ferroportin, inducing iron retention or release and further modulating Bmp6 production in an autocrine manner. This mechanism, that contributes to protect HC from iron loading or deficiency, is lost in disease models of hepcidin production.
Haematologica | 2014
Antonella Nai; Rosa Maria Pellegrino; Marco Rausa; Alessia Pagani; Martina Boero; Laura Silvestri; Giuseppe Saglio; Antonella Roetto; Clara Camaschella
Transferrin receptor 2 (TFR2) is a transmembrane glycoprotein expressed in the liver and in the erythroid compartment, mutated in a form of hereditary hemochromatosis. Hepatic TFR2, together with HFE, activates the transcription of the iron-regulator hepcidin, while erythroid TFR2 is a member of the erythropoietin receptor complex. The TMPRSS6 gene, encoding the liver-expressed serine protease matriptase-2, is the main inhibitor of hepcidin and inactivation of TMPRSS6 leads to iron deficiency with high hepcidin levels. Here we evaluate the phenotype resulting from the genetic loss of Tmprss6 in Tfr2 total (Tfr2−/−) and liver-specific (Tfr2LCKO) knockout mice. Tmprss6−/−Tfr2−/− and Tmprss6−/−Tfr2LCKO mice have increased hepcidin levels and show iron-deficiency anemia like Tmprss6−/−mice. However, while Tmprss6−/−Tfr2LCKO are phenotypically identical to Tmprss6−/− mice, Tmprss6−/−Tfr2−/− mice have increased red blood cell count and more severe microcytosis than Tmprss6−/− mice. In addition hepcidin expression in Tmprss6−/−Tfr2−/− mice is higher than in the wild-type animals, but lower than in Tmprss6−/− mice, suggesting partial inhibition of the hepcidin activating pathway. Our results prove that hepatic TFR2 acts upstream of TMPRSS6. In addition Tfr2 deletion causes a relative erythrocytosis in iron-deficient mice, which likely attenuates the effect of over-expression of hepcidin in Tmprss6−/− mice. Since liver-specific deletion of Tfr2 in Tmprss6−/− mice does not modify the erythrocyte count, we speculate that loss of Tfr2 in the erythroid compartment accounts for the hematologic phenotype of Tmprss6−/−Tfr2−/− mice. We propose that TFR2 is a limiting factor for erythropoiesis, particularly in conditions of iron restriction.
PLOS ONE | 2013
Michela Riba; Marco Rausa; Melissa Sorosina; Davide Cittaro; Jose Manuel Garcia Manteiga; Antonella Nai; Alessia Pagani; Filippo Martinelli-Boneschi; Elia Stupka; Clara Camaschella; Laura Silvestri
Control of systemic iron homeostasis is interconnected with the inflammatory response through the key iron regulator, the antimicrobial peptide hepcidin. We have previously shown that mice with iron deficiency anemia (IDA)-low hepcidin show a pro-inflammatory response that is blunted in iron deficient-high hepcidin Tmprss6 KO mice. The transcriptional response associated with chronic hepcidin overexpression due to genetic inactivation of Tmprss6 is unknown. By using whole genome transcription profiling of the liver and analysis of spleen immune-related genes we identified several functional pathways differentially expressed in Tmprss6 KO mice, compared to IDA animals and thus irrespective of the iron status. In the effort of defining genes potentially targets of Tmprss6 we analyzed liver gene expression changes according to the genotype and independently of treatment. Tmprss6 inactivation causes down-regulation of liver pathways connected to immune and inflammatory response as well as spleen genes related to macrophage activation and inflammatory cytokines production. The anti-inflammatory status of Tmprss6 KO animals was confirmed by the down-regulation of pathways related to immunity, stress response and intracellular signaling in both liver and spleen after LPS treatment. Opposite to Tmprss6 KO mice, Hfe−/− mice are characterized by iron overload with inappropriately low hepcidin levels. Liver expression profiling of Hfe−/− deficient versus iron loaded mice show the opposite expression of some of the genes modulated by the loss of Tmprss6. Altogether our results confirm the anti-inflammatory status of Tmprss6 KO mice and identify new potential target pathways/genes of Tmprss6.
Journal of Cellular and Molecular Medicine | 2015
Marco Rausa; Michela Ghitti; Alessia Pagani; Antonella Nai; Alessandro Campanella; Giovanna Musco; Clara Camaschella; Laura Silvestri
Hemojuvelin (HJV), the coreceptor of the BMP‐SMAD pathway that up‐regulates hepcidin transcription, is a repulsive guidance molecule (RGMc) which undergoes a complex intracellular processing. Following autoproteolysis, it is exported to the cell surface both as a full‐length and a heterodimeric protein. In vitro membrane HJV (m‐HJV) is cleaved by the transmembrane serine protease TMPRSS6 to attenuate signalling and to inhibit hepcidin expression. In this study, we investigated the number and position of HJV cleavage sites by mutagenizing arginine residues (R), potential TMPRSS6 targets, to alanine (A). We analysed translation and membrane expression of HJV R mutants and the pattern of fragments they release in the culture media in the presence of TMPRSS6. Abnormal fragments were observed for mutants at arginine 121, 176, 218, 288 and 326. Considering that all variants, except HJVR121A, lack autoproteolytic activity and some (HJVR176A and HJVR288A) are expressed at reduced levels on cell surface, we identified the fragments originating from either full‐length or heterodimeric proteins and defined the residues 121 and 326 as the TMPRSS6 cleavage sites in both isoforms. Using the N‐terminal FLAG‐tagged HJV, we showed that residue 121 is critical also in the rearrangement of the N‐terminal heterodimeric HJV. Exploiting the recently reported RGMb crystallographic structure, we generated a model of HJV that was used as input structure for all‐atoms molecular dynamics simulation in explicit solvent. As assessed by in silico studies, we concluded that some arginines in the von Willebrand domain appear TMPRSS6 insensitive, likely because of partial protein structure destabilization.
Human Mutation | 2013
Laura Silvestri; Marco Rausa; Alessia Pagani; Antonella Nai; Clara Camaschella
We have read with interest Guillem et al. (2012), which reports and characterizes new TMPRSS6 mutations in patients with iron refractory iron deficiency anemia (IRIDA) [Finberg et al., 2008]. The authors propose a method to assess the causality of novel mutations. To this end, the authors compare the results obtained by using a hepcidin–luciferase-based assay and the determination of the serine protease activity in the culture media of cells transfected with IRIDA-associated mutations in comparison with wild type (wt) and artificial, inactive TMPRSS6 variants. They conclude that the serine protease-based test, which uses a chromogenic substrate, is a simple and preferential method to assess the pathogenic role of novel mutations. The authors showed that in the luciferase assay some IRIDAassociated mutants (E114K; L235P; Y418C and P765A) retain the ability to suppress hepcidin transcription. In addition, the artificial variants R576A and S762A, expected to be inactive as they affect the autocatalytic cleavage site and the protease catalytic triad, respectively, also inhibit hepcidin (see Fig. 3 of Guillem et al., 2012]. For this reason, the authors concluded that the hepcidin–luciferasebased assay is inappropriate to evaluate the functionality of TMPRSS6 mutants. We were surprised by this conclusion, as we have extensively used a similar assay to characterize TMPRSS6 mutations identified in IRIDA patients [De Falco et al., 2010; Silvestri et al., 2008; 2009]. In our hands, all the mutations tested showed reduced inhibitory activities, except R271Q, that in vivo is associated with the I212T causal mutation [De Falco et al., 2010]. Other defective TMPRSS6 variants were characterized by this assay [Altamura et al., 2010; Ramsay et al., 2009]. Furthermore, using this approach, we were able to detect the small difference in the inhibitory activity of the common TMPRSS6 polymorphic variant (rs855791 or V736A) and to show that the difference was well correlated to serum hepcidin level variations in normal subjects [Nai et al., 2011]. We had the opportunity to analyze the new TMPRSS6 variants described by Guillem et al. (2012), which were kindly released to us by the authors. Using the Hep3B cells (Fig. 1A) and the controlled conditions of the transfection assay as previously described [Pagani et al., 2008; Silvestri et al., 2008], all the variants studied had significantly impaired ability to repress the HJV-dependent hepcidin activation (Fig. 1A). All mutants were less inactive compared with the truncated MASK variant [Du et al., 2008]. Their inhibition ability was significantly lower than that of wt and similar to the inhibition shown by the well-characterized serine protease R774C mutant [Silvestri et al., 2008; 2009]. The only exception was L235P, whose inhibitory activity was still decreased compared with the wt isoform, although the difference was not statistically significant. Interestingly, heterozygous L235P was found in a patient in combination with Y418C causal variant [Guillem et al., 2012]. Consistent with previous results [Guillem et al., 2012], R576A and S762A inhibited hepcidin activation even in our assay (Fig. 1A). Using the HuH7 cells, that are hemochromatosis cell lines [Vecchi et al., 2010], a trend toward an impaired inhibitory activity of MT2 mutants in the hepcidin promoter luciferase-based assay was observed (Fig. 1B), although no statistical significance was reached for almost all the MT2 mutants analyzed, with the exception of Y418C and the MASK variant. As TMPRSS6 cleaves membrane-HJV [Silvestri et al., 2008], we then moved to study the HJV proteolytic cleavage. R576A and S762A were both unable to cleave HJV, as they did not release soluble fragments in the medium (Fig. 1C, upper panel). In accordance, they did not release the serine protease domain, confirming that they are proteolytically inactive. These results were confirmed using the HJV variant (data not shown), which does not release soluble HJV but localizes to the cell surface as the wt isoform [Silvestri et al., 2008]. To investigate why proteolytically inactive variants suppress hepcidin transcription in the promoter assay, we analyzed the plasma membrane localization of TMPRSS6 artificial variants and HJV by using both cell surface biotinylation and binding assay [Silvestri et al., 2007]. Both TMPRSS6 variants are expressed on the cell surface as the wt protein (Fig. 1C, lower panel). When coexpressed with the proteolytically inactive MASK, HJV is found in large proportion on cell membrane (Fig. 1C, panel “m-HJV” and Fig. 1D), whereas the amount of membrane HJV in coexpression with R576A and S762A TMPRSS6 variants was highly reduced at the level reached by the wt serine protease (Fig. 1C, panel “m-HJV” and Fig. 1D). The same results were confirmed in HuH7 cells (data not shown). Because this reduction cannot be ascribed to HJV cleavage, we suggest that the two artificial mutants interfere with the HJV plasma membrane localization, although the mechanism remains to be clarified. These results may explain the lower degree of hepcidin activation of R576A and S762A compared with the truncated variant MASK observed in Figure 1A and B. Overall, we suggest a note of caution in the interpretation of the data obtained by the luciferase promoter assay when using insufficiently characterized TMPRSS6 variants. Under controlled experimental conditions (i.e., low concentrations of HJV and TMPRSS6 expressing vectors) and the appropriate (Hep3B) cell line the hepcidin–luciferase-based method seems to be more sensitive compared with the proposed biochemical assay. The proposed serine protease chromogenic assay may have drawbacks. Small but significant amounts of serine protease domain are released from E114K and L235P variants (Figure 1E); however, no activity was detected by the biochemical assay and the cut off of sensitivity of this method remains to be defined. Our conclusion is that neither the chromogenic method nor the hepcidin–luciferase-based assay is suitable for routine studies. An ideal simple method to assess the function of TMPRSS6 variants has not likely been identified. Considering that IRIDA patients have normal/high serum hepcidin levels despite the condition of iron deficiency that is usually associated with low/undetectable hepcidin [Traglia et al., 2011], if assays to measure serum hepcidin
American Journal of Hematology | 2015
Giulia Ravasi; Marco Rausa; Sara Pelucchi; Cristina Arosio; Federico Greni; Raffaella Mariani; Irene Pelloni; Laura Silvestri; Pedro Pineda; Clara Camaschella; Alberto Piperno
MILENA CAU FABRICE DANJOU ROBERTA CHESSA MARIANNA SERRENTI MARIA ADDIS SUSANNA BARELLA RAFFAELLA ORIGA* Dipartimento Di Sanit a Pubblica, Medicina Clinica E Molecolare, Universit a Di Cagliari, Via Jenner s/n, Cagliari, Italy Ospedale Microcitemico, Via Jenner s/n Cagliari, Italy Additional Supporting Information may be found in the online version of this article. Conflict of Interest: The authors declare no competing financial interests. M.C. and F.D. contributed equally to this study This work is dedicated to the memory and in honor of Renzo Galanello, who had conceived the study and continues to inspire us every day. *Correspondence to: Raffaella Origa, MD, PhD, Struttura Complessa Talassemie ed Altre Malattie Ematologiche, Via Jenner sn 09121 Dipartimento di Sanit a Pubblica, Medicina Clinica e Molecolare, Universit a di Cagliari, Italy. E-mail: [email protected] Received for publication: 8 July 2015; Revised: 9 September 2015; Accepted: 15 September 2015 Published online: 18 September 2015 in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/ajh.24197
American Journal of Hematology | 2015
Giulia Ravasi; Marco Rausa; Sara Pelucchi; Cristina Arosio; Federico Greni; Raffaella Mariani; Irene Pelloni; Laura Silvestri; Pedro Pineda; Clara Camaschella; Alberto Piperno
MILENA CAU FABRICE DANJOU ROBERTA CHESSA MARIANNA SERRENTI MARIA ADDIS SUSANNA BARELLA RAFFAELLA ORIGA* Dipartimento Di Sanit a Pubblica, Medicina Clinica E Molecolare, Universit a Di Cagliari, Via Jenner s/n, Cagliari, Italy Ospedale Microcitemico, Via Jenner s/n Cagliari, Italy Additional Supporting Information may be found in the online version of this article. Conflict of Interest: The authors declare no competing financial interests. M.C. and F.D. contributed equally to this study This work is dedicated to the memory and in honor of Renzo Galanello, who had conceived the study and continues to inspire us every day. *Correspondence to: Raffaella Origa, MD, PhD, Struttura Complessa Talassemie ed Altre Malattie Ematologiche, Via Jenner sn 09121 Dipartimento di Sanit a Pubblica, Medicina Clinica e Molecolare, Universit a di Cagliari, Italy. E-mail: [email protected] Received for publication: 8 July 2015; Revised: 9 September 2015; Accepted: 15 September 2015 Published online: 18 September 2015 in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/ajh.24197