Hubert de Verneuil

Publications of Hubert de Verneuil

• Loss of epidermal hypoxia-inducible factor-1α accelerates epidermal aging and affects re-epithelialization in human and mouse.

  Authors: Hamid Reza Rezvani, Nsrein Ali, Martin Serrano-Sanchez, Pierre Dubus, Christine Varon, Cécile Ged, Catherine Pain, Muriel Cario-André, Julien Seneschal, Alain Taïeb, Hubert de Verneuil, Frédéric Mazurier

  Journal of cell science. 12/2011; 124(Pt 24):4172-83.

  In mouse and human skin, HIF-1α is constitutively expressed in the epidermis, mainly in the basal layer. HIF-1α has been shown to have crucial systemic functions: regulation of kidney erythropoietinIn mouse and human skin, HIF-1α is constitutively expressed in the epidermis, mainly in the basal layer. HIF-1α has been shown to have crucial systemic functions: regulation of kidney erythropoietin production in mice with constitutive HIF-1α epidermal deletion, and hypervascularity following epidermal HIF-1α overexpression. However, its local role in keratinocyte physiology has not been clearly defined. To address the function of HIF-1α in the epidermis, we used the mouse model of HIF-1α knockout targeted to keratinocytes (K14-Cre/Hif1a(flox/flox)). These mice had a delayed skin phenotype characterized by skin atrophy and pruritic inflammation, partly mediated by basement membrane disturbances involving laminin-332 (Ln-332) and integrins. We also investigated the relevance of results of studies in mice to human skin using reconstructed epidermis and showed that HIF-1α knockdown in human keratinocytes impairs the formation of a viable reconstructed epidermis. A diminution of keratinocyte growth potential, following HIF-1α silencing, was associated with a decreased expression of Ln-322 and α6 integrin and β1 integrin. Overall, these results indicate a role of HIF-1α in skin homeostasis especially during epidermal aging.

• ALAS2 acts as a modifier gene in patients with congenital erythropoietic porphyria.

  Authors: Jordi To-Figueras, Sarah Ducamp, Jerome Clayton, Celia Badenas, Constance Delaby, Cecile Ged, Said Lyoumi, Laurent Gouya, Hubert de Verneuil, Carole Beaumont, Gloria C Ferreira, Jean-Charles Deybach, Carmen Herrero, Herve Puy

  Blood. 06/2011; 118(6):1443-51.

  Mutations in the uroporphyrinogen III synthase (UROS) gene cause congenital erythropoietic porphyria (CEP), an autosomal-recessive inborn error of erythroid heme biosynthesis. Clinical features ofMutations in the uroporphyrinogen III synthase (UROS) gene cause congenital erythropoietic porphyria (CEP), an autosomal-recessive inborn error of erythroid heme biosynthesis. Clinical features of CEP include dermatologic and hematologic abnormalities of variable severity. The discovery of a new type of erythroid porphyria, X-linked dominant protoporphyria (XLDPP), which results from increased activity of 5-aminolevulinate synthase 2 (ALAS2), the rate-controlling enzyme of erythroid heme synthesis, led us to hypothesize that the CEP phenotype may be modulated by sequence variations in the ALAS2 gene. We genotyped ALAS2 in 4 unrelated CEP patients exhibiting the same C73R/P248Q UROS genotype. The most severe of the CEP patients, a young girl, proved to be heterozygous for a novel ALAS2 mutation: c.1757 A > T in exon 11. This mutation is predicted to affect the highly conserved and penultimate C-terminal amino acid of ALAS2 (Y586). The rate of 5-aminolevulinate release from Y586F was significantly increased over that of wild-type ALAS2. The contribution of the ALAS2 gain-of-function mutation to the CEP phenotype underscores the importance of modifier genes underlying CEP. We propose that ALAS2 gene mutations should be considered not only as causative of X-linked sideroblastic anemia (XLSA) and XLDPP but may also modulate gene function in other erythropoietic disorders.

• HIF-1α in epidermis: oxygen sensing, cutaneous angiogenesis, cancer, and non-cancer disorders.

  Authors: Hamid R Rezvani, Nsrein Ali, Lars J Nissen, Ghida Harfouche, Hubert de Verneuil, Alain Taïeb, Frédéric Mazurier

  The Journal of investigative dermatology. 06/2011; 131(9):1793-805.

  Besides lung, postnatal human epidermis is the only epithelium in direct contact with atmospheric oxygen. Skin epidermal oxygenation occurs mostly through atmospheric oxygen rather than tissueBesides lung, postnatal human epidermis is the only epithelium in direct contact with atmospheric oxygen. Skin epidermal oxygenation occurs mostly through atmospheric oxygen rather than tissue vasculature, resulting in a mildly hypoxic microenvironment that favors increased expression of hypoxia-inducible factor-1α (HIF-1α). Considering the wide spectrum of biological processes, such as angiogenesis, inflammation, bioenergetics, proliferation, motility, and apoptosis, that are regulated by this transcription factor, its high expression level in the epidermis might be important to HIF-1α in skin physiology and pathophysiology. Here, we review the role of HIF-1α in cutaneous angiogenesis, skin tumorigenesis, and several skin disorders.

• XPC silencing in normal human keratinocytes triggers metabolic alterations that drive the formation of squamous cell carcinomas.

  Authors: Hamid Reza Rezvani, Arianna L Kim, Rodrigue Rossignol, Nsrein Ali, Meaghan Daly, Walid Mahfouf, Nadège Bellance, Alain Taïeb, Hubert de Verneuil, Frédéric Mazurier, David R Bickers

  The Journal of clinical investigation. 01/2011; 121(1):195-211.

  DNA damage is a well-known initiator of tumorigenesis. Studies have shown that most cancer cells rely on aerobic glycolysis for their bioenergetics. We sought to identify a molecular link betweenDNA damage is a well-known initiator of tumorigenesis. Studies have shown that most cancer cells rely on aerobic glycolysis for their bioenergetics. We sought to identify a molecular link between genomic mutations and metabolic alterations in neoplastic transformation. We took advantage of the intrinsic genomic instability arising in xeroderma pigmentosum C (XPC). The XPC protein plays a key role in recognizing DNA damage in nucleotide excision repair, and patients with XPC deficiency have increased incidence of skin cancer and other malignancies. In cultured human keratinocytes, we showed that lentivirus-mediated knockdown of XPC reduced mitochondrial oxidative phosphorylation and increased glycolysis, recapitulating cancer cell metabolism. Accumulation of unrepaired DNA following XPC silencing increased DNA-dependent protein kinase activity, which subsequently activated AKT1 and NADPH oxidase-1 (NOX1), resulting in ROS production and accumulation of specific deletions in mitochondrial DNA (mtDNA) over time. Subcutaneous injection of XPC-deficient keratinocytes into immunodeficient mice led to squamous cell carcinoma formation, demonstrating the tumorigenic potential of transduced cells. Conversely, simultaneous knockdown of either NOX1 or AKT1 blocked the neoplastic transformation induced by XPC silencing. Our results demonstrate that genomic instability resulting from XPC silencing results in activation of AKT1 and subsequently NOX1 to induce ROS generation, mtDNA deletions, and neoplastic transformation in human keratinocytes.

• XPC silencing in normal human keratinocytes triggers metabolic alterations through NOX-1 activation-mediated reactive oxygen species.

  Authors: Hamid Reza Rezvani, Rodrigue Rossignol, Nsrein Ali, Giovanni Benard, Xiuwei Tang, Hee Seung Yang, Thomas Jouary, Hubert de Verneuil, Alain Taïeb, Arianna L Kim, Frédéric Mazurier

  Biochimica et biophysica acta. 12/2010; 1807(6):609-19.

  Cancer cells utilize complex mechanisms to remodel their bioenergetic properties. We exploited the intrinsic genomic stability of xeroderma pigmentosum C (XPC) to understand the inter-relationshipsCancer cells utilize complex mechanisms to remodel their bioenergetic properties. We exploited the intrinsic genomic stability of xeroderma pigmentosum C (XPC) to understand the inter-relationships between genomic instability, reactive oxygen species (ROS) generation, and metabolic alterations during neoplastic transformation. We showed that knockdown of XPC (XPC(KD)) in normal human keratinocytes results in metabolism remodeling through NADPH oxidase-1 (NOX-1) activation, which in turn leads to increased ROS levels. While enforcing antioxidant defenses by overexpressing catalase, CuZnSOD, or MnSOD could not block the metabolism remodeling, impaired NOX-1 activation abrogates both alteration in ROS levels and modifications of energy metabolism. As NOX-1 activation is observed in human squamous cell carcinomas (SCCs), the blockade of NOX-1 could be a target for the prevention and the treatment of skin cancers.

• Neonatal bone marrow transplantation prevents liver disease in a murine model of erythropoietic protoporphyria.

  Authors: Yann Duchartre, Nicolas Petit, Corrine Moya, Magalie Lalanne, Pierre Dubus, Hubert de Verneuil, François Moreau-Gaudry, Emmanuel Richard

  Journal of hepatology. 10/2010; 55(1):162-70.

  Erythropoietic protoporphyria (EPP) is an inherited disorder of heme biosynthesis caused by partial ferrochelatase deficiency, resulting in protoporphyrin IX (PPIX) accumulation in erythrocytes,Erythropoietic protoporphyria (EPP) is an inherited disorder of heme biosynthesis caused by partial ferrochelatase deficiency, resulting in protoporphyrin IX (PPIX) accumulation in erythrocytes, responsible for skin photosensitivity. In some EPP patients, the development of cholestatic liver injury due to PPIX accumulation can lead to hepatic failure. In adult EPP mice, bone marrow transplantation (BMT) leads to skin photosensitivity correction but fails to reverse liver damages, probably because of the irreversible nature of liver fibrosis. Our aim was to determine the time course of liver disease progression in EPP mice and to evaluate the protective effect of BMT into neonates. We studied the development of liver disease from birth in EPP mice, in relation with erythroid and hepatic PPIX accumulation. To prevent the development of liver disease, BMT was performed into newborn mice using a novel busulfan-mediated preconditioning assay. We showed that hepatic PPIX accumulates during the first 2 weeks and correlates with the onset of a progressive liver fibrosis in 12-day-old EPP mice. Transplantation of normal congenic hematopoietic stem cells into EPP neonates led to long-term donor hematopoiesis recovery. A full correction of erythroid PPIX accumulation and skin photosensitivity was obtained. Furthermore, five months after neonatal BMT, liver damage was almost completely prevented. We demonstrated for the first time that BMT could be successfully used to prevent liver disease in EPP mice and suggested that BMT would be an attractive therapeutic option to prevent severe liver dysfunction in EPP patients.

• Modeling of congenital erythropoietic porphyria by RNA interference: a new tool for preclinical gene therapy evaluation.

  Authors: Elodie Robert-Richard, Magalie Lalanne, Isabelle Lamrissi-Garcia, Véronique Guyonnet-Duperat, Emmanuel Richard, Vincent Pitard, Fréderic Mazurier, François Moreau-Gaudry, Cécile Ged, Hubert de Verneuil

  The journal of gene medicine. 08/2010; 12(8):637-46.

  Congenital erythropoietic porphyria (CEP) is a severe autosomal recessive disorder characterized by a deficiency in uroporphyrinogen III synthase (UROS), the fourth enzyme of the heme biosyntheticCongenital erythropoietic porphyria (CEP) is a severe autosomal recessive disorder characterized by a deficiency in uroporphyrinogen III synthase (UROS), the fourth enzyme of the heme biosynthetic pathway. We recently demonstrated the definitive cure of a murine model of CEP by lentiviral vector-mediated hematopoietic stem cell (HSC) gene therapy. In the perspective of a gene therapy clinical trial, human cellular models are required to evaluate the therapeutic potential of lentiviral vectors in UROS-deficient cells. However, the rare incidence of the disease makes difficult the availability of HSCs derived from patients. RNA interference (RNAi) has been used to develop a new human model of the disease from normal cord blood HSCs. Lentivectors were developed for this purpose. We were able to down-regulate the level of human UROS in human cell lines and primary hematopoietic cells. A 97% reduction of UROS activity led to spontaneous uroporphyrin accumulation in human erythroid bone marrow cells of transplanted immune-deficient mice, recapitulating the phenotype of cells derived from patients. A strong RNAi-induced UROS inhibition allowed us to test the efficiency of different lentiviral vectors with the aim of selecting a safer vector. Restoration of UROS activity in these small hairpin RNA-transduced CD34(+) cord blood cells by therapeutic lentivectors led to a partial correction of the phenotype in vivo. The RNAi strategy is an interesting new tool for preclinical gene therapy evaluation.

• A prevalent mutation with founder effect in xeroderma pigmentosum group C from north Africa.

  Authors: Nadem Soufir, Cecile Ged, Agnes Bourillon, Frederic Austerlitz, Cécile Chemin, Anne Stary, Jacques Armier, Daniele Pham, Khadija Khadir, Joelle Roume, Smail Hadj-Rabia, Bakar Bouadjar, Alain Taieb, Hubert de Verneuil, Hakima Benchiki, Bernard Grandchamp, Alain Sarasin

  The Journal of investigative dermatology. 06/2010; 130(6):1537-42.

  Xeroderma pigmentosum (XP) is a rare autosomal recessive disorder that is associated with an inherited defect of the nucleotide excision repair pathway (NER). In this study, we investigated theXeroderma pigmentosum (XP) is a rare autosomal recessive disorder that is associated with an inherited defect of the nucleotide excision repair pathway (NER). In this study, we investigated the involvement of XP genes in 86 XP patients belonging to 66 unrelated families, most of them consanguineous and originating from Maghreb. Sequencing analysis was performed either directly (44 probands) or after having previously characterized the involved XP gene by complementation assay (22 families). XPC and XPA mutations were respectively present in 56/66 and 8/66 probands. Strikingly, we identified the same homozygous frameshift mutation c.1643_1644delTG (p.Val548AlafsX25) in 87% of XP-C patients. Haplotype analysis showed a common founder effect for this mutation in the Mediterranean region, with an estimated age of 50 generations or 1,250 years. Among 7/8 XP-A patients, we found the previously reported nonsense homozygous XPA mutation (p.Arg228X). Six mutations--to our knowledge previously unreported--(five in XPC, one in XPA) were also identified. In conclusion, XPC appears to be the major disease-causing gene concerning xeroderma pigmentosum in North Africa. As the (p.Val548AlafsX25) XPC mutation is responsible for a huge proportion of XP cases, our data imply an obvious simplification of XP molecular diagnosis, at least in North Africa.

• Hypoxia-inducible factor-1{alpha} regulates the expression of nucleotide excision repair proteins in keratinocytes.

  Authors: Hamid Reza Rezvani, Walid Mahfouf, Nsrein Ali, Cecile Chemin, Cecile Ged, Arianna L Kim, Hubert de Verneuil, Alain Taïeb, David R Bickers, Frédéric Mazurier

  Nucleic acids research. 11/2009;

  The regulation of DNA repair enzymes is crucial for cancer prevention, initiation, and therapy. We have studied the effect of ultraviolet B (UVB) radiation on the expression of the two nucleotideThe regulation of DNA repair enzymes is crucial for cancer prevention, initiation, and therapy. We have studied the effect of ultraviolet B (UVB) radiation on the expression of the two nucleotide excision repair factors (XPC and XPD) in human keratinocytes. We show that hypoxia-inducible factor-1alpha (HIF-1alpha) is involved in the regulation of XPC and XPD. Early UVB-induced downregulation of HIF-1alpha increased XPC mRNA expression due to competition between HIF-1alpha and Sp1 for their overlapping binding sites. Late UVB-induced enhanced phosphorylation of HIF-1alpha protein upregulated XPC mRNA expression by direct binding to a separate hypoxia response element (HRE) in the XPC promoter region. HIF-1alpha also regulated XPD expression by binding to a region of seven overlapping HREs in its promoter. Quantitative chromatin immunoprecipitation assays further revealed putative HREs in the genes encoding other DNA repair proteins (XPB, XPG, CSA and CSB), suggesting that HIF-1alpha is a key regulator of the DNA repair machinery. Analysis of the repair kinetics of 6-4 photoproducts and cyclobutane pyrimidine dimers also revealed that HIF-1alpha downregulation led to an increased rate of immediate removal of both photolesions but attenuated their late removal following UVB irradiation, indicating the functional effects of HIF-1alpha in the repair of UVB-induced DNA damage.

• The hematopoietic stem cell compartment of JAK2V617F positive myeloproliferative disorders is a reflection of disease heterogeneity.

  Authors: Chloe James, Frederic Mazurier, Sabrina Dupont, Ronan Chaligne, Isabelle Lamrissi-Garcia, Micheline Tulliez, Eric Lippert, Francois-Xavier Mahon, Jean-Max Pasquet, Gabriel Etienne, Francois Delhommeau, Stephane Giraudier, William Vainchenker, Hubert de Verneuil

  Blood. 08/2008;

  The JAK2V617F somatic point mutation has been described in patients with myeloproliferative disorders (MPD). Despite this progress, it remains unknown how a single JAK2 mutation causes threeThe JAK2V617F somatic point mutation has been described in patients with myeloproliferative disorders (MPD). Despite this progress, it remains unknown how a single JAK2 mutation causes three different MPD phenotypes, polycythemia vera (PV), essential thrombocythemia and primitive myelofibrosis (PMF). Using an in vivo xenotransplantation assay in nonobese diabetic-severe combined immunodeficient (NOD/SCID) mice, we tested whether disease heterogeneity was associated with quantitative or qualitative differences in the hematopoietic stem cell (HSC) compartment. We show that the HSC compartment of PV and PMF patients contains JAK2V617F-positive long-term, multipotent and self-renewing cells. However, the proportion of JAK2V617F and JAK2 wild type SCID repopulating cells (SRC) was dramatically different in these diseases, without major modifications of the self-renewal and proliferation capacities for JAK2V617F SRC. These experiments provide new insights into the pathogenesis of JAK2V617F MPD and demonstrate that a JAK2 inhibitor needs to target the HSC compartment for optimal disease control in classical MPD.

• Erythropoietic porphyrias: animal models and update in gene-based therapies.

  Authors: Emmanuel Richard, Elodie Robert-Richard, Cécile Ged, François Moreau-Gaudry, Hubert de Verneuil

  Current gene therapy. 07/2008; 8(3):176-86.

  The inherited porphyrias are inborn errors of haem biosynthesis, each resulting from the deficient activity of a specific enzyme of the haem biosynthetic pathway. Porphyrias are divided intoThe inherited porphyrias are inborn errors of haem biosynthesis, each resulting from the deficient activity of a specific enzyme of the haem biosynthetic pathway. Porphyrias are divided into erythropoietic and hepatic according to the predominant porphyrin-accumulating tissue. Three different erythropoietic porphyrias (EP) have been described: erythropoietic protoporphyria (EPP, MIM 177000) the most frequent, congenital erythropoietic porphyria (CEP, MIM 263700), and the very rare hepatoerythropoietic porphyria (HEP, MIM 176100). Bone marrow transplantation is considered as the only curative treatment for severe cases of erythropoietic porphyria (especially CEP), if donors are available. Some EPP patients who undergo liver failure may require hepatic transplantation. Murine models of EPP and CEP have been developed and mimic most of the human disease features. These models allow a better understanding of the pathophysiological mechanisms involved in EP as well as the development of new therapeutic strategies. The restoration of deficient enzymatic activity in the bone marrow compartment following gene therapy has been extensively studied. Murine oncoretroviral, and recently, lentiviral vectors have been successfully used to transduce hematopoietic stem cells, allowing full metabolic and phenotypic correction of both EPP and CEP mice. In CEP, a selective survival advantage of corrected cells was demonstrated in mice, reinforcing the arguments for a gene therapy approach in the human disease. These successful results form the basis for gene therapy clinical trials in severe forms of erythropoietic porphyrias.

• Effective gene therapy of mice with congenital erythropoietic porphyria is facilitated by a survival advantage of corrected erythroid cells.

  Authors: Elodie Robert-Richard, François Moreau-Gaudry, Magalie Lalanne, Isabelle Lamrissi-Garcia, Muriel Cario-André, Véronique Guyonnet-Dupérat, Laurence Taine, Cécile Ged, Hubert de Verneuil

  American journal of human genetics. 02/2008; 82(1):113-24.

  Achieving long-term expression of a therapeutic gene in a given hematopoietic lineage remains an important goal of gene therapy. Congenital erythropoietic porphyria (CEP) is a severeAchieving long-term expression of a therapeutic gene in a given hematopoietic lineage remains an important goal of gene therapy. Congenital erythropoietic porphyria (CEP) is a severe autosomal-recessive disorder characterized by a deficiency in uroporphyrinogen III synthase (UROS), the fourth enzyme of the heme biosynthetic pathway. We used a recently obtained murine model to check the feasibility of gene therapy in this disease. Lentivirus-mediated transfer of the human UROS cDNA into hematopoietic stem cells (HSCs) from Uros(mut248) mice resulted in a complete and long-term enzymatic, metabolic, and phenotypic correction of the disease, favored by a survival advantage of corrected red blood cells. These results demonstrate that the cure of this mouse model of CEP at a moderate transduction level supports the proof of concept of a gene therapy in this disease by transplantation of genetically modified hematopoietic stem cells.

• Hypoxia-inducible factor-1alpha, a key factor in the keratinocyte response to UVB exposure.

  Authors: Hamid Reza Rezvani, Sophie Dedieu, Sophie North, Francis Belloc, Rodrigue Rossignol, Thierry Letellier, Hubert de Verneuil, Alain Taïeb, Frédéric Mazurier

  The Journal of biological chemistry. 07/2007; 282(22):16413-22.

  Hypoxia-inducible factor-1 (HIF-1) is a major transcription factor sensitive to oxygen levels, which responds to stress factors under both hypoxic and nonhypoxic conditions. UV irradiation being aHypoxia-inducible factor-1 (HIF-1) is a major transcription factor sensitive to oxygen levels, which responds to stress factors under both hypoxic and nonhypoxic conditions. UV irradiation being a common stressor of skin, we looked at the effect of UVB on HIF-1alpha expression in keratinocytes. We found that UVB induces a biphasic HIF-1alpha variation through reactive oxygen species (ROS) generation. Whereas rapid production of cytoplasmic ROS down-regulates HIF-1alpha expression, delayed mitochondrial ROS generation results in its up-regulation. Indeed, activation of p38 MAPK and JNK1 mediated by mitochondrial ROS were required for HIF-1alpha phosphorylation and accumulation after UVB irradiation. Our experiments also revealed a key role of HIF-1alpha in mediating UVB-induced apoptosis. We conclude that the broad impact of the HIF-1 transcription factor on gene expression could make it a key regulator of UV-responsive genes and photocarcinogenesis.

• Proteasome inhibition specifically sensitizes leukemic cells to anthracyclin-induced apoptosis through the accumulation of Bim and Bax pro-apoptotic proteins.

  Authors: Arnaud Pigneux, François-Xavier Mahon, François Moreau-Gaudry, Maialene Uhalde, Hubert de Verneuil, Francis Lacombe, Josy Reiffers, Noel Milpied, Vincent Praloran, Francis Belloc

  Cancer biology & therapy. 05/2007; 6(4):603-11.

  Proteasome inhibitors are a novel class of compounds that might increase sensitivity to chemotherapy for acute myeloid leukemia (AML). We quantified apoptosis in THP-1 cells incubated with idarubicinProteasome inhibitors are a novel class of compounds that might increase sensitivity to chemotherapy for acute myeloid leukemia (AML). We quantified apoptosis in THP-1 cells incubated with idarubicin (IDA) alone or together with a low concentration of MG132 or bortezomib. The combination of both drugs yielded a percentage of apoptotic cells that was significantly higher than the additive effect of both drugs administered separately (p < 0.01). Isobologram analysis showed that both MG132 and bortezomib interacted synergistically with IDA to induce apoptosis of THP1 cells. Western blot analysis of Bax and Bim show an acumulation of these pro-apoptotic proteins in THP1 treated cells. This increase in Bim preceded the induction of apoptosis and participated in idarubicin-induced apoptosis. Proteasome inhibition also potentiated IDA-induced apoptosis in primary blast cells from 22 AML patients while no such effect was found on normal lymphocytes, PHA-stimulated lymphocytes, normal cord blood CD34+ cells or bone marrow normal myeloid cells. These data show that MG132 and bortezomib specifically sensitize leukemic cells to IDA through an increase in BIM and Bax pro-apoptotic Bcl-2 family proteins.

• Increased plasma transferrin, altered body iron distribution, and microcytic hypochromic anemia in ferrochelatase-deficient mice.

  Authors: Saïd Lyoumi, Marie Abitbol, Valérie Andrieu, Dominique Henin, Elodie Robert, Caroline Schmitt, Laurent Gouya, Hubert de Verneuil, Jean-Charles Deybach, Xavier Montagutelli, Carole Beaumont, Hervé Puy

  Blood. 02/2007; 109(2):811-8.

  Patients with deficiency in ferrochelatase (FECH), the last enzyme of the heme biosynthetic pathway, experience a painful type of skin photosensitivity called erythropoietic protoporphyria (EPP),Patients with deficiency in ferrochelatase (FECH), the last enzyme of the heme biosynthetic pathway, experience a painful type of skin photosensitivity called erythropoietic protoporphyria (EPP), which is caused by the excessive production of protoporphyrin IX (PPIX) by erythrocytes. Controversial results have been reported regarding hematologic status and iron status of patients with EPP. We thoroughly explored these parameters in Fechm1Pas mutant mice of 3 different genetic backgrounds. FECH deficiency induced microcytic hypochromic anemia without ringed sideroblasts, little or no hemolysis, and no erythroid hyperplasia. Serum iron, ferritin, hepcidin mRNA, and Dcytb levels were normal. The homozygous Fechm1Pas mutant involved no tissue iron deficiency but showed a clear-cut redistribution of iron stores from peripheral tissues to the spleen, with a concomitant 2- to 3-fold increase in transferrin expression at the mRNA and the protein levels. Erythrocyte PPIX levels strongly correlated with serum transferrin levels. At all stages of differentiation in our study, transferrin receptor expression in bone marrow erythroid cells in Fech(m1Pas) was normal in mutant mice but not in patients with iron-deficiency anemia. Based on these observations, we suggest that oral iron therapy is not the therapy of choice for patients with EPP and that the PPIX-liver transferrin pathway plays a role in the orchestration of iron distribution between peripheral iron stores, the spleen, and the bone marrow.

• Murine retroviral but not human cellular promoters induce in vivo erythroid-specific deregulation that can be partially prevented by insulators.

  Authors: Elodie Robert-Richard, Emmanuel Richard, Punam Malik, Cécile Ged, Hubert de Verneuil, François Moreau-Gaudry

  Molecular therapy : the journal of the American Society of Gene Therapy. 02/2007; 15(1):173-82.

  We are developing lentiviral vectors for gene therapy of red blood cell disorders that co-express a transgene in an erythroid-specific manner and the O(6)-methylguanine-DNA-methyltransferase (MGMT)We are developing lentiviral vectors for gene therapy of red blood cell disorders that co-express a transgene in an erythroid-specific manner and the O(6)-methylguanine-DNA-methyltransferase (MGMT) selective gene in a constitutive way. We report that transduction of murine hematopoietic stem cells (HSCs) with a human phosphoglycerate kinase promoter-based vector at low multiplicity of infection (MOI) does not result in a selective in vivo expansion in the presence of alkylating agents. In contrast, by replacing this cellular promoter with the powerful retroviral-derived myeloproliferative sarcoma virus enhancer, negative control region-deleted, dl587rev primer-binding site substituted promoter, the vector allowed efficient chemoprotection of transduced HSCs at low MOI. However, this promoter interacted with the erythroid HS40/ankyrin enhancer/promoter driving green fluorescent protein, leading to an unexpected loss of erythroid specificity. A partial restoration of tissue-specific expression was obtained by interposition of insulator sequences between the expression units. Alternatively, we found that the strong human cellular elongation factor1-alpha promoter allows similar chemoprotection but without any deregulation of the erythroid-specific promoter in the absence of insulators. These data demonstrate that the level of in vivo deregulation induced by a promoter is not correlated with its transcriptional activity.

• Analysis of SPINK 5, KLK 7 and FLG genotypes in a French atopic dermatitis cohort.

  Authors: Thomas Hubiche, Cécile Ged, Antoine Benard, Christine Léauté-Labrèze, Ken McElreavey, Hubert de Verneuil, Alain Taïeb, Franck Boralevi

  Acta dermato-venereologica. 02/2007; 87(6):499-505.

  The role of a genetically impaired epidermal barrier as a major predisposing factor in the pathogenesis of atopic disorders is currently under closer investigation. Variants on three candidate genesThe role of a genetically impaired epidermal barrier as a major predisposing factor in the pathogenesis of atopic disorders is currently under closer investigation. Variants on three candidate genes (SPINK5, KLK7 and FLG) have been associated with atopic dermatitis. A functional relevance has already been established for filaggrin variants, but not for SPINK5 and KLK7 polymorphisms. The objectives of this study were to confirm the association between SPINK5, KLK7, FLG variants and atopic dermatitis and to assess how variants influence selected phenotypic traits. This cross-sectional study was carried out over 20 months in 99 children and adults with atopic dermatitis (median age 7 years). The following items were analysed: SCORAD, TEWL, ichthyosis vulgaris, presence of asthma, total IgE serum levels. The SPINK5 E420K SNP, the KLK7 4bp insertion polymorphism and the filaggrin mutants (R510X and 2282del4) were analysed as described previously. The control group for genetic analysis was recruited in an ethnically matched, phenotypically anonymous cohort (n=102). The allelic frequencies were 0.525 for SPINK5, 0.26 for KLK7 polymorphisms, 0.101 and 0.075 for 2282del4 and R501X FLG mutants, respectively. The association of atopic dermatitis with filaggrin variants was confirmed, but not that of SPINK5 or KLK7 polymorphisms. SCORAD and TEWL measurements were not influenced by any of the variants. The SPINK5 polymorphism was associated with high IgE serum levels (p=0.011). Abnormal barrier genes do not influence the severity of atopic dermatitis. The SPINK5 gene polymorphism may modulate systemic immune effects favouring the IgE response to atopens. TEWL does not allow the characterization of subsets of patients with or without abnormal barrier genes.

• Human cell engraftment after busulfan or irradiation conditioning of NOD/SCID mice.

  Authors: Elodie Robert-Richard, Cécile Ged, Jacqueline Ortet, Xavier Santarelli, Isabelle Lamrissi-Garcia, Hubert de Verneuil, Frédéric Mazurier

  Haematologica. 11/2006; 91(10):1384.

  Human hematopoietic stem cell (HSC) xenotransplantation in NOD/SCID mice requires recipient conditioning, classically achieved by sublethal irradiation. Pretreatment with immunosuppressive andHuman hematopoietic stem cell (HSC) xenotransplantation in NOD/SCID mice requires recipient conditioning, classically achieved by sublethal irradiation. Pretreatment with immunosuppressive and alkylating agents has been reported, but has not been rigorously tested against standard irradiation protocols. Here, we report that treatment of mice with a single dose (35 mg/kg) of Busilvex, an injectable form of busulfan, enables equivalent engraftment compared to 3.5 Gy irradiation. Mice treated with two doses of 25 mg/kg to reduce busulfan toxicity showed increased chimerism. Busulfan conditioning and irradiation resulted in comparable sensitivity of HSC detection as evaluated by limiting dilution analysis.

• Protective effects of catalase overexpression on UVB-induced apoptosis in normal human keratinocytes.

  Authors: Hamid Reza Rezvani, Frédéric Mazurier, Muriel Cario-André, Catherine Pain, Cécile Ged, Alain Taïeb, Hubert de Verneuil

  The Journal of biological chemistry. 07/2006; 281(26):17999-8007.

  UV-induced apoptosis in keratinocytes is a highly complex process in which various molecular pathways are involved. These include the extrinsic pathway via triggering of death receptors and theUV-induced apoptosis in keratinocytes is a highly complex process in which various molecular pathways are involved. These include the extrinsic pathway via triggering of death receptors and the intrinsic pathway via DNA damage and reactive oxygen species (ROS) formation. In this study we investigated the effect of catalase and CuZn-superoxide dismutase (SOD) overexpression on apoptosis induced by UVB exposure at room temperature or 4 degrees C on normal human keratinocytes. Irradiation at low temperature reduced UV-induced apoptosis by 40% in normal keratinocytes independently of any change in p53 and with a decrease in caspase-8 activation. Catalase overexpression decreased apoptosis by 40% with a reduction of caspase-9 activation accompanied by a decrease in p53. Keeping cells at low temperature and catalase overexpression had additive effects. CuZn-SOD overexpression had no significant effect on UVB-induced apoptosis. UVB induced an increase in ROS levels at two distinct stages: immediately following irradiation and around 3 h after irradiation. Catalase overexpression inhibited only the late increase in ROS levels. We conclude that catalase overexpression has a protective role against UVB irradiation by preventing DNA damage mediated by the late ROS increase.

• Polyunsaturated fatty acids partially reproduce the role of melanocytes in the epidermal melanin unit.

  Authors: Muriel Cario-André, Stefania Briganti, Mauro Picardo, Osamu Nikaido, Hubert de Verneuil, Alain Taïeb

  Experimental dermatology. 04/2005; 14(3):194-201.

  The incidence rate of melanoma is higher in fair-skinned than in dark-skinned individuals. In negroid skin there is more eumelanin which is present in all skin layers and fewer polyunsaturated fattyThe incidence rate of melanoma is higher in fair-skinned than in dark-skinned individuals. In negroid skin there is more eumelanin which is present in all skin layers and fewer polyunsaturated fatty acids (PUFA) than in caucasoid skin. The western diet, which is rich in omega-6 polyunsaturated fatty acids, is associated with more proneness to cancer including cutaneous melanoma. To study the respective influence of omega-6 PUFA and low phototype melanocytes on redox status -basal and following UV irradiation-, we used epidermal reconstructs. The addition of polyunsaturated fatty acids as well as the presence of low phototype melanocytes affected basal status similarly except for catalase activity, which decreased significantly in polyunsaturated fatty acid-supplemented reconstructs. Following UV, polyunsaturated fatty acids and low phototype melanocytes increased lipid and protein oxidative damage without affecting direct DNA damage. However, polyunsaturated fatty acids increased epidermal apoptosis whereas low phototype melanocytes decreased it. Since our data suggest that an omega-6 PUFA rich-diet may increase oxidative damage in melanocytes without inducing apoptosis, the long-term net outcome could be cumulated mutations and an increased risk of skin cancer, especially melanoma.