UCAGenomiX related publications

Du to our strong expertise in "omics" experiments and in microRNAs topics we decided to separate into 3 categories the related publications into which the Functional genomics Platform of Nice-Sophia-Antipolis is involved :
  1. Expression studies (DNA microarrays and high-throughput sequencing experiments)
  2. MicroRNA studies
  3. Miscellaneous

Barbry Pascal

 04 93 95 77 00
 660 route des lucioles 06560 Valbonne - Sophia-Antipolis

82 publications found

1. CDC20B is required for deuterosome-mediated centriole production in multiciliated cells
Nat Commun. 2018 Nov 7;9(1):4668. doi: 10.1038/s41467-018-06768-z.
Revinski DR, Zaragosi LE, Boutin C, Ruiz-Garcia S, Deprez M, Thomé V, Rosnet O, Gay AS, Mercey O, Paquet A, Pons N, Ponzio G, Marcet B, Kodjabachian L, Barbry P
Aix Marseille Univ, CNRS, IBDM, Marseille, France. Université Côte d'Azur, CNRS, IPMC, Sophia-Antipolis, France. Université Côte d'Azur, CNRS, IPMC, Sophia-Antipolis, France. marcet@ipmc.cnrs.fr. Aix Marseille Univ, CNRS, IBDM, Marseille, France. laurent.kodjabachian@univ-amu.fr. Université Côte d'Azur, CNRS, IPMC, Sophia-Antipolis, France. barbry@ipmc.cnrs.fr.

Multiciliated cells (MCCs) harbor dozens to hundreds of motile cilia, which generate hydrodynamic forces important in animal physiology. In vertebrates, MCC differentiation involves massive centriole production by poorly characterized structures called deuterosomes. Here, single-cell RNA sequencing reveals that human deuterosome stage MCCs are characterized by the expression of many cell cycle-related genes. We further investigated the uncharacterized vertebrate-specific cell division cycle 20B (CDC20B) gene, which hosts microRNA-449abc. We show that CDC20B protein associates to deuterosomes and is required for centriole release and subsequent cilia production in mouse and Xenopus MCCs. CDC20B interacts with PLK1, a kinase known to coordinate centriole disengagement with the protease Separase in mitotic cells. Strikingly, over-expression of Separase rescues centriole disengagement and cilia production in CDC20B-deficient MCCs. This work reveals the shaping of deuterosome-mediated centriole production in vertebrate MCCs, by adaptation of canonical and recently evolved cell cycle-related molecules.
Pubmed link : 30405130

2. The "one airway, one disease" concept in light of Th2 inflammation.
Eur Respir J. 2018 Sep 6. pii: 1800437. doi: 10.1183/13993003.00437-2018.
Giovannini-Chami L, Paquet A, Sanfiorenzo C, Pons N, Cazareth J, Magnone V, Lebrigand K, Chevalier B, Vallauri A, Julia V, Marquette CH, Marcet B, Leroy S, Barbry P
Université Côte d'Azur, Hôpitaux pédiatriques de Nice CHU-Lenval, Pediatric Pulmonology and Allergology Department, Nice, France. Université Côte d'Azur, CNRS, Institut de Pharmacologie Moléculaire et Cellulaire, Sophia Antipolis, France. Université Côte d'Azur, CHU de Nice, FHU Oncoage, Pulmonology Department, Nice, France.

In line with the pathophysiological continuum described between nose and bronchus in allergic respiratory diseases, we assessed whether nasal epithelium could mirror the Th2 status of bronchial epithelium.Nasal and bronchial cells were collected by brushings from patients with allergic rhinitis and asthma (AR, n=12), isolated allergic rhinitis (R, n=14) and healthy controls (C, n=13). Cellular composition was assessed by flow cytometry. Gene expression was analysed by RNA sequencing. Th2, Th17 and interferon signatures were derived from the literature.Infiltration by polymorphonuclear neutrophils in nose excluded 30% of the initial cohort. All bronchial samples from AR group were Th2-high. Nasal samples gene expression profile from the AR group correctly predicted the paired bronchial sample Th2 status in 71% of cases. Nevertheless, nasal cells did not appear as a reliable surrogate of the Th2 response, in particular due to a more robust influence of the interferon response in 14/26 nasal samples. Th2 scores correlated with mast cells counts (p<0.001) and numbers of sensitizations (p=0.006 and 0.002), while Th17 scores correlated with PMN counts (p<0.014).The large variability in nasal cell composition and type of inflammation restricts its use as a surrogate for assessing bronchial Th2 inflammation in AR patients.
Pubmed link : 30190271

3. Genome evolution across 1,011 Saccharomyces cerevisiae isolates
Nature. 2018 Apr;556(7701):339-344. doi: 10.1038/s41586-018-0030-5. Epub 2018 Apr 11.
Peter J, De Chiara M, Friedrich A, Yue JX, Pflieger D, Bergström A, Sigwalt A, Barre B, Freel K, Llored A, Cruaud C, Labadie K, Aury JM, Istace B, Lebrigand K, Barbry P, Engelen S, Lemainque A, Wincker P, Liti G, Schacherer J
Université de Strasbourg, CNRS, GMGM UMR 7156, Strasbourg, France. Université Côte d'Azur, CNRS, INSERM, IRCAN, Nice, France. Commissariat à l'Energie Atomique (CEA), Genoscope, Institut de Biologie François-Jacob, Evry, France. Université Côte d'Azur, CNRS, IPMC, Sophia Antipolis, Valbonne, France. CNRS UMR 8030, Université d'Evry Val d'Essonne, Evry, France. Université Côte d'Azur, CNRS, INSERM, IRCAN, Nice, France. gianni.liti@unice.fr. Université de Strasbourg, CNRS, GMGM UMR 7156, Strasbourg, France. schacherer@unistra.fr.

Large-scale population genomic surveys are essential to explore the phenotypic diversity of natural populations. Here we report the whole-genome sequencing and phenotyping of 1,011 Saccharomyces cerevisiae isolates, which together provide an accurate evolutionary picture of the genomic variants that shape the species-wide phenotypic landscape of this yeast. Genomic analyses support a single 'out-of-China' origin for this species, followed by several independent domestication events. Although domesticated isolates exhibit high variation in ploidy, aneuploidy and genome content, genome evolution in wild isolates is mainly driven by the accumulation of single nucleotide polymorphisms. A common feature is the extensive loss of heterozygosity, which represents an essential source of inter-individual variation in this mainly asexual species. Most of the single nucleotide polymorphisms, including experimentally identified functional polymorphisms, are present at very low frequencies. The largest numbers of variants identified by genome-wide association are copy-number changes, which have a greater phenotypic effect than do single nucleotide polymorphisms. This resource will guide future population genomics and genotype-phenotype studies in this classic model system.
Pubmed link : 29643504

4. A new long noncoding RNA (LncRNA) is induced in cutaneous squamous cell carcinoma and downregulates several anticancer and cell-differentiation genes in mouse.
J Biol Chem. 2017 Jun 8. pii: jbc.M117.776260. doi: 10.1074/jbc.M117.776260. [Epub ahead of print]
Ponzio G, Rezzonico R, Bourget I, Allan R, Nottet N, Popa A, Magnone V, Rios G, Mari B, Barbry P
Université Côte d’Azur, CNRS, IPMC, France. Université Côte d'Azur, CNRS, INSERM, IRCAN, France.

Keratinocyte-derived cutaneous squamous cell carcinoma (cSCC) is the most common metastatic skin cancer. Although some of the early events involved in this pathology have been identified, the subsequent steps leading to tumor development are poorly defined. We demonstrate here that the development of mouse tumors induced by the concomitant application of a carcinogen and a tumor promoter (7,12 dimethylbenz[a]anthracene [DMBA] and 12-O-tetradecanoylphorbol-13-acetate [TPA], respectively) is associated with the upregulation of a previously uncharacterized long noncoding RNA (lncRNA), termed AK144841. We found that AK144841 expression was absent from normal skin and was specifically stimulated in tumors and highly tumorigenic cells. We also found that AK144841 exists in two variants, one consisting of a large 2-kb transcript composed of four exons and one of a 1.8-kb transcript lacking the second exon. Gain- and loss-of-function studies indicated that AK144841 mainly inhibited gene expression, specifically downregulating the expression of genes of the late-cornified-envelope-1 (Lce1) family involved in epidermal terminal differentiation and of anticancer genes such as Cgref1, Brsk1, Basp1, Dusp5, Btg2, Anpep, Dhrs9, Stfa2, Tpm1, SerpinB2, Cpa4, Crct1, Cryab, Il24, Csf2, and Rgs16. Interestingly, the lack of the second exon significantly decreased AK144841's inhibitory effect on gene expression. We also noted that high AK144841 expression correlated with a low expression of the aforementioned genes and with the tumorigenic potential of cell lines. These findings suggest that AK144841 could contribute to the dedifferentiation program of tumor-forming keratinocytes and to molecular cascades leading to tumor development.
Pubmed link : 28596382

5. Characterizing isomiR variants within the microRNA-34/449 family
FEBS Lett. 2017 Mar;591(5):693-705. doi: 10.1002/1873-3468.12595. Epub 2017 Feb 28
Mercey O, Popa A, Cavard A, Paquet A, Chevalier B, Pons N, Magnone V, Zangari J, Brest P, Zaragosi LE, Ponzio G, Lebrigand K, Barbry P, Marcet B
CNRS, IPMC, Université Côte d'Azur, Sophia-Antipolis, Valbonne, France. CNRS, INSERM, IRCAN, FHU-OncoAge, Université Côte d'Azur, Sophia-Antipolis, Valbonne, France.

miR-34/449 microRNAs are conserved regulators of multiciliated cell differentiation. Here, we evidence and characterize expression of two isomiR variant sequences from the miR-34/449 family in human airway epithelial cells. These isomiRs differ from their canonical counterparts miR-34b and miR-449c by one supplemental uridine at their 5'-end, leading to a one-base shift in their seed region. Overexpression of canonical miR-34/449 or 5'-isomiR-34/449 induces distinct gene expression profiles and biological effects. However, some target transcripts and functional activities are shared by both canonical microRNAs and isomiRs. Indeed, both repress important targets that result in cell cycle blockage and Notch pathway inhibition. Our findings suggest that 5'-isomiR-34/449 may represent additional mechanisms by which miR-34/449 family finely controls several pathways to drive multiciliogenesis.
Pubmed link : 28192603

6. A cost effective 5' selective single cell transcriptome profiling approach with improved UMI design
Nucleic Acids Res. 2016 Dec 9. pii: gkw1242.
Arguel MJ, Lebrigand K, Paquet A, Ruiz Garcia S, Zaragosi LE, Barbry P, Waldmann R
Université Côte d'Azur, CNRS, Institut de Pharmacologie Moléculaire et Cellulaire, F06560 Sophia Antipolis, France. Université Côte d'Azur, CNRS, Institut de Pharmacologie Moléculaire et Cellulaire, F06560 Sophia Antipolis, France

Single cell RNA sequencing approaches are instrumental in studies of cell-to-cell variability. 5' selective transcriptome profiling approaches allow simultaneous definition of the transcription start size and have advantages over 3' selective approaches which just provide internal sequences close to the 3' end. The only currently existing 5' selective approach requires costly and labor intensive fragmentation and cell barcoding after cDNA amplification. We developed an optimized 5' selective workflow where all the cell indexing is done prior to fragmentation. With our protocol, cell indexing can be performed in the Fluidigm C1 microfluidic device, resulting in a significant reduction of cost and labor. We also designed optimized unique molecular identifiers that show less sequence bias and vulnerability towards sequencing errors resulting in an improved accuracy of molecule counting. We provide comprehensive experimental workflows for Illumina and Ion Proton sequencers that allow single cell sequencing in a cost range comparable to qPCR assays.
Pubmed link : 27940562

7. Copy-number analysis identified new prognostic marker in acute myeloid leukemia.
Leukemia. 2016 Nov 4. doi: 10.1038/leu.2016.265.
Nibourel O, Guihard S, Roumier C, Pottier N, Terre C, Paquet A, Peyrouze P, Geffroy S, Quentin S, Alberdi A, Abdelali RB, Renneville A, Demay C, Celli-Lebras K, Barbry P, Quesnel B, Castaigne S, Dombret H, Soulier J, Preudhomme C, Cheok MH
CHU Lille University Hospital, Department of Hematology, Lille, France. INSERM UMR-S1172, Institute for Cancer Research of Lille, Factors of Leukemia Cell Persistance, Lille Cedex, France. CHU Lille University Hospital, Department of Biochemistry and Molecular Biology, Lille, France. Hospital of Versailles, Department of Hematology, Chesnay, France. University Côte d'Azur, CNRS Institute of Molecular and Cellular Pharmacology, Sophia-Antipolis, Nice, France. University Paris Diderot, INSERM U944 Saint-Louis Hospital, Department of Hematology, Paris, France. 7University Paris 7, Department of Hematology, Paris, France.

Recent advances in genomic technologies have revolutionized acute myeloid leukemia (AML) understanding by identifying potential novel actionable genomic alterations. Consequently, current risk stratification at diagnosis not only relies on cytogenetics, but also on the inclusion of several of these abnormalities. Despite this progress, AML remains a heterogeneous and complex malignancy with variable response to current therapy. Although copy-number alterations (CNAs) are accepted prognostic markers in cancers, large-scale genomic studies aiming at identifying specific prognostic CNA-based markers in AML are still lacking. Using 367 AML, we identified four recurrent CNA on chromosomes 11 and 21 that predicted outcome even after adjusting for standard prognostic risk factors and potentially delineated two new subclasses of AML with poor prognosis. ERG amplification, the most frequent CNA, was related to cytarabine resistance, a cornerstone drug of AML therapy. These findings were further validated in The Cancer Genome Atlas data. Our results demonstrate that specific CNA are of independent prognostic relevance, and provide new molecular information into the genomic basis of AML and cytarabine response. Finally, these CNA identified two potential novel risk groups of AML, which when confirmed prospectively, may improve the clinical risk stratification and potentially the AML outcome.Leukemia advance online publication, 4 November 2016; doi:10.1038/leu.2016.265.
Pubmed link : 27686867

8. miR-200 family controls late steps of postnatal forebrain neurogenesis via Zeb2 inhibition.
Sci Rep. 2016 Oct 21;6:35729. doi: 10.1038/srep35729.
Beclin C, Follert P, Stappers E, Barral S, Nathalie C, de Chevigny A, Magnone V, Lebrigand K, Bissels U, Huylebroeck D, Bosio A, Barbry P, Seuntjens E, Cremer H
IBDM, Aix-Marseille Université, CNRS, UMR7288, 13288 Marseille, France. Laboratory of Molecular Biology, Dept Development and Regeneration, KULeuven, 3000 Leuven, Belgium. Miltenyi Biotec GmbH, Bergisch Gladbach, Germany. CNRS and University Nice Sophia Antipolis, IPMC, Sophia Antipolis, France. Dept Cell Biology, Erasmus MC, 3015 CN Rotterdam, The Netherlands. GIGA-Neurosciences, Université de Liège, 4000 Liège, Belgium.

During neurogenesis, generation, migration and integration of the correct numbers of each neuron sub-type depends on complex molecular interactions in space and time. MicroRNAs represent a key control level allowing the flexibility and stability needed for this process. Insight into the role of this regulatory pathway in the brain is still limited. We performed a sequential experimental approach using postnatal olfactory bulb neurogenesis in mice, starting from global expression analyses to the investigation of functional interactions between defined microRNAs and their targets. Deep sequencing of small RNAs extracted from defined compartments of the postnatal neurogenic system demonstrated that the miR-200 family is specifically induced during late neuronal differentiation stages. Using in vivo strategies we interfered with the entire miR-200 family in loss- and gain-of-function settings, showing a role of miR-200 in neuronal maturation. This function is mediated by targeting the transcription factor Zeb2. Interestingly, so far functional interaction between miR-200 and Zeb2 has been exclusively reported in cancer or cultured stem cells. Our data demonstrate that this regulatory interaction is also active during normal neurogenesis.
Pubmed link : 27767083

9. RiboProfiling: a Bioconductor package for standard Ribo-seq pipeline processing.
F1000Res. 2016 Jun 9;5:1309. doi: 10.12688/f1000research.8964.1. eCollection 2016.
Popa A, Lebrigand K, Paquet A, Nottet N, Robbe-Sermesant K, Waldmann R, Barbry P
Institut de Pharmacologie Moleculaire et Cellulaire, University Nice Sophia Antipolis and CNRS, Sophia- Antipolis, 06560, France.

The ribosome profiling technique (Ribo-seq) allows the selective sequencing of translated RNA regions. Recently, the analysis of genomic sequences associated to Ribo-seq reads has been widely employed to assess their coding potential. These analyses led to the identification of differentially translated transcripts under different experimental conditions, and/or ribosome pausing on codon motifs. In the context of the ever-growing need for tools analyzing Ribo-seq reads, we have developed 'RiboProfiling', a new Bioconductor open-source package. 'RiboProfiling' provides a full pipeline to cover all key steps for the analysis of ribosome footprints. This pipeline has been implemented in a single R workflow. The package takes an alignment (BAM) file as input and performs ribosome footprint quantification at a transcript level. It also identifies footprint accumulation on particular amino acids or multi amino-acids motifs. Report summary graphs and data quantification are generated automatically. The package facilitates quality assessment and quantification of Ribo-seq experiments. Its implementation in Bioconductor enables the modeling and statistical analysis of its output through the vast choice of packages available in R. This article illustrates how to identify codon-motifs accumulating ribosome footprints, based on data from Escherichia coli.
Pubmed link : 27347386

10. MicroRNA-375/SEC23A as biomarkers of the in vitro efficacy of vandetanib.
Oncotarget. 2016 May 24;7(21):30461-78. doi: 10.18632/oncotarget.8458.
Lassalle S, Zangari J, Popa A, Ilie M, Hofman V, Long E, Patey M, Tissier F, Belléannée G, Trouette H, Catargi B, Peyrottes I, Sadoul JL, Bordone O, Bonnetaud C, Butori C, Bozec A, Guevara N, Santini J, Hénaoui IS, Lemaire G, Blanck O, Vielh P, Barbry P, Mari B, Brest P, Hofman P
1Centre Hospitalier Universitaire de Nice, Laboratory of Clinical and Experimental Pathology, Nice, France. 2Institute of Research on Cancer and Ageing of Nice (IRCAN), INSERM U1081/CNRS UMR7284, Nice, France. 3University of Nice Sophia-Antipolis, Nice, France. 4Centre Hospitalier Universitaire de Nice, Hospital Integrated Biobank (BB 0033-00025), Nice, France. 5Fédération Hospitalo-Universitaire "OncoAge", University of Nice Sophia Antipolis, Nice, France. 6Institut de Pharmacologie Moléculaire et Cellulaire IPMC, CNRS UMR7275, Sophia-Antipolis, France. 7Hôpital Universitaire de Reims - Hôpital Robert Debré, Department of Pathology, Institut Jean Godinot, Reims, France. 8Assistance Publique - Hôpitaux de Paris (AP-HP), Groupe Hospitalier Pitié-Salpêtrière, Laboratory of Pathology, Paris, France. 9Centre Hospitalier Universitaire de Bordeaux, Hôpital Universitaire de Pessac-Haut Lévêque, Laboratory of Pathology, Pessac, France. 10Centre Hospitalier Universitaire de Bordeaux, Department of Endocrinology, Pessac, France. 11Centre Antoine Lacassagne, Laboratory of Pathology, Nice, France. 12Centre Hospitalier Universitaire de Nice, Hôpital de l'Archet, Department of Endocrinology, Nice, France. 13Centre Antoine Lacassagne, Head and Neck Institute, Surgery and Otorhinolaryngology Department, Nice, France. 14Bayer CropScience SA, Research Center, Sophia Antipolis, Valbonne, France. 15Institut Gustave Roussy, Translational Research Laboratory, Department of Pathology, Villejuif, France.

In this study, we performed microRNA (miRNA) expression profiling on a large series of sporadic and hereditary forms of medullary thyroid carcinomas (MTC). More than 60 miRNAs were significantly deregulated in tumor vs adjacent non-tumor tissues, partially overlapping with results of previous studies. We focused our attention on the strongest up-regulated miRNA in MTC samples, miR-375, the deregulation of which has been previously observed in a variety of human malignancies including MTC. We identified miR-375 targets by combining gene expression signatures from human MTC (TT) and normal follicular (Nthy-ori 3-1) cell lines transfected with an antagomiR-375 inhibitor or a miR-375 mimic, respectively, and from an in silico analysis of thyroid cell lines of Cancer Cell Line Encyclopedia datasets. This approach identified SEC23A as a bona fide miR-375 target, which we validated by immunoblotting and immunohistochemistry of non-tumor and pathological thyroid tissue. Furthermore, we observed that miR-375 overexpression was associated with decreased cell proliferation and synergistically increased sensitivity to vandetanib, the clinically relevant treatment of metastatic MTC. We found that miR-375 increased PARP cleavage and decreased AKT phosphorylation, affecting both cell proliferation and viability. We confirmed these results through SEC23A direct silencing in combination with vandetanib, highlighting the importance of SEC23A in the miR-375-associated increased sensitivity to vandetanib.Since the combination of increased expression of miR-375 and decreased expression of SEC23A point to sensitivity to vandetanib, we question if the expression levels of miR-375 and SEC23A should be evaluated as an indicator of eligibility for treatment of MTC patients with vandetanib.
Pubmed link : 27036030

11. Comparative Genomic Analysis of Drechmeria coniospora Reveals Core and Specific Genetic Requirements for Fungal Endoparasitism of Nematodes.
PLoS Genet. 2016 May 6;12(5):e1006017. doi: 10.1371/journal.pgen.1006017. eCollection 2016.
Lebrigand K, He le D, Thakur N, Arguel MJ, Polanowska J, Henrissat B, Record E, Magdelenat G, Barbe V, Raffaele S, Barbry P, Ewbank JJ
CNRS and University Nice Sophia Antipolis, Institute of Molecular and Cellular Pharmacology, Sophia Antipolis, France. Centre d'Immunologie de Marseille-Luminy, Aix Marseille Université UM2, Inserm, U1104, CNRS UMR7280, Marseille, France. CNRS UMR 7257, Aix-Marseille University, Marseille, France. INRA, USC 1408 AFMB, Marseille, France. Department of Biological Sciences, King Abdulaziz University, Jeddah, Saudi Arabia. INRA, UMR1163 Biodiversité et Biotechnologie Fongiques, Aix-Marseille Université, Polytech Marseille, CP 925, Marseille, France. Aix-Marseille Université, UMR1163 Biodiversité et Biotechnologie Fongiques, Faculté des Sciences de Luminy-Polytech, CP 925, Marseille, France. Commissariat à l'Energie Atomique, Institut de Génomique, Génoscope, Laboratoire de Biologie Moleculaire pour l'Etude des Génomes (LBioMEG), Evry, France. INRA, Laboratoire des Interactions Plantes-Microorganismes (LIPM), UMR441, Castanet Tolosan, France. CNRS, Laboratoire des Interactions Plantes-Microorganismes (LIPM), UMR2594, Castanet Tolosan, France.

Drechmeria coniospora is an obligate fungal pathogen that infects nematodes via the adhesion of specialized spores to the host cuticle. D. coniospora is frequently found associated with Caenorhabditis elegans in environmental samples. It is used in the study of the nematode's response to fungal infection. Full understanding of this bi-partite interaction requires knowledge of the pathogen's genome, analysis of its gene expression program and a capacity for genetic engineering. The acquisition of all three is reported here. A phylogenetic analysis placed D. coniospora close to the truffle parasite Tolypocladium ophioglossoides, and Hirsutella minnesotensis, another nematophagous fungus. Ascomycete nematopathogenicity is polyphyletic; D. coniospora represents a branch that has not been molecularly characterized. A detailed in silico functional analysis, comparing D. coniospora to 11 fungal species, revealed genes and gene families potentially involved in virulence and showed it to be a highly specialized pathogen. A targeted comparison with nematophagous fungi highlighted D. coniospora-specific genes and a core set of genes associated with nematode parasitism. A comparative gene expression analysis of samples from fungal spores and mycelia, and infected C. elegans, gave a molecular view of the different stages of the D. coniospora lifecycle. Transformation of D. coniospora allowed targeted gene knock-out and the production of fungus that expresses fluorescent reporter genes. It also permitted the initial characterisation of a potential fungal counter-defensive strategy, involving interference with a host antimicrobial mechanism. This high-quality annotated genome for D. coniospora gives insights into the evolution and virulence of nematode-destroying fungi. Coupled with genetic transformation, it opens the way for molecular dissection of D. coniospora physiology, and will allow both sides of the interaction between D. coniospora and C. elegans, as well as the evolutionary arms race that exists between pathogen and host, to be studied.
Pubmed link : 27153332

12. MicroRNAs as key regulators of GTPase-mediated apical actin reorganization in multiciliated epithelia
Small GTPases. 2016 Apr 2;7(2):54-8. doi: 10.1080/21541248.2016.1151099.
Mercey O, Kodjabachian L, Barbry P, Marcet B
CNRS-IPMC, UMR-7275 , Sophia-Antipolis , France. University of Nice-Sophia-Antipolis (UNS) , Sophia-Antipolis , France. Aix-Marseille Université, CNRS, IBDM UMR7288 , Marseille , France.

Multiciliated cells (MCCs), which are present in specialized vertebrate tissues such as mucociliary epithelia, project hundreds of motile cilia from their apical membrane. Coordinated ciliary beating in MCCs contributes to fluid propulsion in several biological processes. In a previous work, we demonstrated that microRNAs of the miR-34/449 family act as new conserved regulators of MCC differentiation by specifically repressing cell cycle genes and the Notch pathway. Recently, we have shown that miR-34/449 also modulate small GTPase pathways to promote, in a later stage of differentiation, the assembly of the apical actin network, a prerequisite for proper anchoring of centrioles-derived neo-synthesized basal bodies. We characterized several miR-34/449 targets related to small GTPase pathways including R-Ras, which represents a key and conserved regulator during MCC differentiation. Direct RRAS repression by miR-34/449 is necessary for apical actin meshwork assembly, notably by allowing the apical relocalization of the actin binding protein Filamin-A near basal bodies. Our studies establish miR-34/449 as central players that orchestrate several steps of MCC differentiation program by regulating distinct signaling pathways.
Pubmed link : 27144998

13. SENS-IS, a 3D reconstituted epidermis based model for quantifying chemical sensitization potency: Reproducibility and predictivity results from an inter-laboratory study.
Toxicol In Vitro. 2016 Jan 18;32:248-260. doi: 10.1016/j.tiv.2016.01.007.
Cottrez F, Boitel E, Ourlin JC, Peiffer JL, Fabre I, Henaoui IS, Mari B, Vallauri A, Paquet A, Barbry P, Auriault C, Aeby P, Groux H
1ImmunoSearch, Grasse, France. 2Agence nationale de sécurité du médicament, Vendargues, France. 3CNRS, Institute of Molecular and Cellular Pharmacology, Sophia Antipolis, France; University of Nice Sophia Antipolis, Nice, France. 4Independant Consultant, Marly, Switzerland. 5ImmunoSearch, Grasse, France. Electronic address: hgroux@immunosearch.fr.

The SENS-IS test protocol for the in vitro detection of sensitizers is based on a reconstructed human skin model (Episkin) as the test system and on the analysis of the expression of a large panel of genes. Its excellent performance was initially demonstrated with a limited set of test chemicals. Further studies (described here) were organized to confirm these preliminary results and to obtain a detailed statistical analysis of the predictive capacity of the assay. A ring-study was thus organized and performed within three laboratories, using a test set of 19 blind coded chemicals. Data analysis indicated that the assay is robust, easily transferable and offers high predictivity and excellent within- and between-laboratories reproducibility. To further evaluate the predictivity of the test protocol according to Cooper statistics a comprehensive test set of 150 chemicals was then analyzed. Again, data analysis confirmed the excellent capacity of the SENS-IS assay for predicting both hazard and potency characteristics, confirming that this assay should be considered as a serious alternative to the available in vivo sensitization tests.
Pubmed link : 26795242

14. Pateamine A-sensitive ribosome profiling reveals the scope of translation in mouse embryonic stem cells.
BMC Genomics. 2016 Jan 14;17(1):52. doi: 10.1186/s12864-016-2384-0.
Popa A, Lebrigand K, Barbry P, Waldmann R
1Institut de Pharmacologie Moléculaire et Cellulaire (IPMC), University Nice Sophia Antipolis, CNRS, F06560, Sophia-Antipolis, France. 2Institut de Pharmacologie Moléculaire et Cellulaire (IPMC), University Nice Sophia Antipolis, CNRS, F06560, Sophia-Antipolis, France. barbry@ipmc.cnrs.fr.

BACKGROUND: Open reading frames are common in long noncoding RNAs (lncRNAs) and 5'UTRs of protein coding transcripts (uORFs). The question of whether those ORFs are translated was recently addressed by several groups using ribosome profiling. Most of those studies concluded that certain lncRNAs and uORFs are translated, essentially based on computational analysis of ribosome footprints. However, major discrepancies remain on the scope of translation and the translational status of individual ORFs. In consequence, further criteria are required to reliably identify translated ORFs from ribosome profiling data. RESULTS: We examined the effect of the translation inhibitors pateamine A, harringtonine and puromycin on murine ES cell ribosome footprints. We found that pateamine A, a drug that targets eIF4A, allows a far more accurate identification of translated sequences than previously used drugs and computational scoring schemes. Our data show that at least one third but less than two thirds of ES cell lncRNAs are translated. We also identified translated uORFs in hundreds of annotated coding transcripts including key pluripotency transcripts, such as dicer, lin28, trim71, and ctcf. CONCLUSION: Pateamine A inhibition data clearly increase the precision of the detection of translated ORFs in ribosome profiling experiments. Our data show that translation of lncRNAs and uORFs in murine ES cells is rather common although less pervasive than previously suggested. The observation of translated uORFs in several key pluripotency transcripts suggests that translational regulation by uORFs might be part of the network that defines mammalian stem cell identity.
Pubmed link : 26764022

15. RNY-derived small RNAs as a signature of coronary artery disease.
BMC Med. 2015 Oct 8;13:259. doi: 10.1186/s12916-015-0489-y.
Repetto E, Lichtenstein L, Hizir Z, Tekaya N, Benahmed M, Ruidavets JB, Zaragosi LE, Perret B, Bouchareychas L, Genoux A, Lotte R, Ruimy R, Ferrières J, Barbry P, Martinez LO, Trabucchi M
1INSERM U1065 and University of Nice Sophia Antipolis, Centre Méditerranéen de Médecine Moléculaire (C3M), Team 10 "Control of Gene Expression", F-06204, Nice, France. erepetto@unice.fr. 2INSERM UMR 1048, Toulouse, 31000, France. 3Université de Toulouse III, UMR 1048, Toulouse, 31300, France. 4INSERM U1065 and University of Nice Sophia Antipolis, Centre Méditerranéen de Médecine Moléculaire (C3M), Team 10 "Control of Gene Expression", F-06204, Nice, France. 5INSERM U1065, Team 5, F-06204, Nice, France. 6INSERM U1027, Faculté de Médecine, Toulouse, 31073, France. 7CNRS and University of Nice Sophia Antipolis, IPMC, Sophia Antipolis, Nice, France. 8CHU de Toulouse, Hôpital Purpan, Toulouse, France. 9Sorbonne Universités, UPMC Université Paris 06, UMR_S 1166, ICAN, Integrative Biology of Atherosclerosis Team, Paris, F-75005, France. 10Hôpital Archet, Nice, France. 11INSERM U1065 and University of Nice Sophia Antipolis, Centre Méditerranéen de Médecine Moléculaire (C3M), Team 10 "Control of Gene Expression", F-06204, Nice, France. michele.trabucchi@unice.fr.

Data from next generation sequencing technologies uncovered the existence of many classes of small RNAs. Recent studies reported that small RNAs are released by cells and can be detected in the blood. In this report, we aimed to discover the occurrence of novel circulating small RNAs in coronary artery disease (CAD). METHODS: We used high-throughput sequencing of small RNAs from human and mouse apoptotic primary macrophages, and analyzed the data by empirical Bayes moderated t-statistics to assess differential expression and the Benjamini and Hochberg method to control the false discovery rate. Results were then confirmed by Northern blot and RT-qPCR in foam cells and in two animal models for atherosclerosis, namely ApoE(-/-) and Ldlr(-/-) mouse lines. Quantitative RT-PCR to detect identified small RNAs, the RNY-derived small RNAs, was performed using sera of 263 patients with CAD compared to 514 matched healthy controls; the Student t-test was applied to statistically assess differences. Associations of small RNAs with clinical characteristics and biological markers were tested using Spearman's rank correlations, while multivariate logistic regressions were performed to test the statistical association of small RNA levels with CAD. RESULTS: Here, we report that, in macrophages stimulated with pro-apoptotic or pro-atherogenic stimuli, the Ro-associated non-coding RNAs, called RNYs or Y-RNAs, are processed into small RNAs (~24-34 nt) referred to as small-RNYs (s-RNYs), including s-RNY1-5p processed from RNY1. A significant upregulation of s-RNY expression was found in aortic arches and blood plasma from ApoE(-/-) and Ldlr(-/-) mice and in serum from CAD patients (P <0.001). Biostatistical analysis revealed a positive association of s-RNY1-5p with hs-CRP and ApoB levels; however, no statistical interaction was found between either of these two markers and s-RNY1-5p in relation to the CAD status. Levels of s-RNY1-5p were also independent from statin and fibrate therapies. CONCLUSION: Our results position the s-RNY1-5p as a relevant novel independent diagnostic biomarker for atherosclerosis-related diseases. Measurement of circulating s-RNY expression would be a valuable companion diagnostic to monitor foam cell apoptosis during atherosclerosis pathogenesis and to evaluate patient's responsiveness to future therapeutic strategies aiming to attenuate apoptosis in foam cells in advanced atherosclerotic lesions.
Pubmed link : 26449324

16. miR-34/449 control apical actin network formation during multiciliogenesis through small GTPase pathways.
Nat Commun. 2015 Sep 18;6:8386. doi: 10.1038/ncomms9386.
Chevalier B, Adamiok A, Mercey O, Revinski DR, Zaragosi LE, Pasini A, Kodjabachian L, Barbry P, Marcet B
1CNRS, Institut de Pharmacologie Moléculaire et Cellulaire (IPMC), UMR-7275, 660 route des Lucioles, 06560 Sophia-Antipolis, France. 2University of Nice-Sophia-Antipolis (UNS), Institut de Pharmacologie Moléculaire et Cellulaire, 660 route des Lucioles, Valbonne, 06560 Sophia-Antipolis, France. 3Aix-Marseille Université, CNRS, UMR7288, Institut de Biologie du Développement de Marseille (IBDM), 13288 Marseille, France.

Vertebrate multiciliated cells (MCCs) contribute to fluid propulsion in several biological processes. We previously showed that microRNAs of the miR-34/449 family trigger MCC differentiation by repressing cell cycle genes and the Notch pathway. Here, using human and Xenopus MCCs, we show that beyond this initial step, miR-34/449 later promote the assembly of an apical actin network, required for proper basal bodies anchoring. Identification of miR-34/449 targets related to small GTPase pathways led us to characterize R-Ras as a key regulator of this process. Protection of RRAS messenger RNA against miR-34/449 binding impairs actin cap formation and multiciliogenesis, despite a still active RhoA. We propose that miR-34/449 also promote relocalization of the actin binding protein Filamin-A, a known RRAS interactor, near basal bodies in MCCs. Our study illustrates the intricate role played by miR-34/449 in coordinating several steps of a complex differentiation programme by regulating distinct signalling pathways.
Pubmed link : 26381333

17. BMP signalling controls the construction of vertebrate mucociliary epithelia.
Development. 2015 Jul 1;142(13):2352-63. doi: 10.1242/dev.118679. Epub 2015 Jun 19.
Cibois M, Luxardi G, Chevalier B, Thomé V, Mercey O, Zaragosi LE, Barbry P, Pasini A, Marcet B, Kodjabachian L
1Aix-Marseille Université, CNRS, IBDM, Marseille 13288, France. 2CNRS, IPMC, Sophia-Antipolis 06560, France. 3CNRS, IPMC, Sophia-Antipolis 06560, France University of Nice Sophia Antipolis (UNS), IPMC, Sophia-Antipolis 06560, France. 4Aix-Marseille Université, CNRS, IBDM, Marseille 13288, France laurent.kodjabachian@univ-amu.fr.

Despite the importance of mucociliary epithelia in animal physiology, the mechanisms controlling their establishment are poorly understood. Using the developing Xenopus epidermis and regenerating human upper airways, we reveal the importance of BMP signalling for the construction of vertebrate mucociliary epithelia. In Xenopus, attenuation of BMP activity is necessary for the specification of multiciliated cells (MCCs), ionocytes and small secretory cells (SSCs). Conversely, BMP activity is required for the proper differentiation of goblet cells. Our data suggest that the BMP and Notch pathways interact to control fate choices in the developing epidermis. Unexpectedly, BMP activity is also necessary for the insertion of MCCs, ionocytes and SSCs into the surface epithelium. In human, BMP inhibition also strongly stimulates the formation of MCCs in normal and pathological (cystic fibrosis) airway samples, whereas BMP overactivation has the opposite effect. This work identifies the BMP pathway as a key regulator of vertebrate mucociliary epithelium differentiation and morphogenesis.
Pubmed link : 26092849

18. An ABC of ciliogenesis.
Nat Cell Biol. 2014 Sep;16(9):826-7. doi: 10.1038/ncb3034.
Barbry P, Zaragosi LE
CNRS and University Nice Sophia Antipolis, Institute of Molecular and Cellular Pharmacology, 660 Route des Lucioles, F6560 Sophia Antipolis, France.

ABCC4 is a member of the ATP-binding cassette transporter family known to transport prostaglandin E2 and other molecules across cellular membranes. A mutation in ABCC4 is now shown to cause defects in ciliogenesis, revealing a role for prostaglandin signalling in regulating cilia dynamics.
Pubmed link : 25174819

19. MicroRNA target identification: lessons from hypoxamiRs.
Antioxid Redox Signal. 2014 Sep 10;21(8):1249-68. doi: 10.1089/ars.2013.5648. Epub 2014 Feb 3.
Bertero T, Robbe-Sermesant K, Le Brigand K, Ponzio G, Pottier N, Rezzonico R, Mazure NM, Barbry P, Mari B
Institut de Pharmacologie Moléculaire et Cellulaire (IPMC) , Centre National de la Recherche Scientifique, CNRS UMR 7275, Sophia Antipolis, France .

SIGNIFICANCE: MicroRNAs (miRNAs) are small noncoding RNAs that have emerged as key regulators of many physiological and pathological processes, including those relevant to hypoxia such as cancer, neurological dysfunctions, myocardial infarction, and lung diseases. RECENT ADVANCES: During the last 5 years, miRNAs have been shown to play a role in the regulation of the cellular response to hypoxia. The identification of several bona fide targets of these hypoxamiRs has underlined their pleiotropic functions and the complexity of the molecular rules directing miRNA::target transcript pairing. CRITICAL ISSUES: This review outlines the main in silico and experimental approaches used to identify the targetome of hypoxamiRs and presents new recent relevant methodologies for future studies. FUTURE DIRECTIONS: Since hypoxia plays key roles in many pathophysiological conditions, the precise characterization of regulatory hypoxamiRs networks will be instrumental both at a fundamental level and for their future potential therapeutic applications.
Pubmed link : 24111877

20. miR-193b/365a cluster controls progression of epidermal squamous cell carcinoma.
Carcinogenesis. 2014 May;35(5):1110-20. doi: 10.1093/carcin/bgt490. Epub 2013 Dec 28.
Gastaldi C, Bertero T, Xu N, Bourget-Ponzio I, Lebrigand K, Fourre S, Popa A, Cardot-Leccia N, Meneguzzi G, Sonkoly E, Pivarcsi A, Mari B, Barbry P, Ponzio G, Rezzonico R
UMR 7275, Centre National de la Recherche Scientifique, Institut de Pharmacologie Moléculaire et Cellulaire, 660 route des Lucioles, F-06560 Valbonne, France.

Incidence of cutaneous squamous cell carcinomas (cSCCs) constantly increases in the Caucasian population. Developing preferentially on precancerous lesions such as actinic keratoses due to chronic sunlight exposure, cSCCs result from the malignant transformation of keratinocytes. Although a resection of the primary tumor is usually curative, a subset of aggressive cSCCs shows a high risk of recurrence and metastases. The characterization of the molecular dysfunctions involved in cSCC development should help to identify new relevant targets against these aggressive cSCCs. In that context, we have used small RNA sequencing to identify 100 microRNAs (miRNAs) whose expression was altered during chemically induced mouse skin tumorigenesis. The decreased expression of the miR-193b/365a cluster during tumor progression suggests a tumor suppressor role. Ectopic expression of these miRNAs in tumor cells indeed inhibited their proliferation, clonogenic potential and migration, which were stimulated in normal keratinocytes when these miRNAs were blocked with antisense oligonucleotides. A combination of in silico predictions and transcriptome analyses identified several target genes of interest. We validated KRAS and MAX as direct targets of miR-193b and miR-365a. Repression of these targets using siRNAs mimicked the effects of miR-193b and miR-365a, suggesting that these genes might mediate, at least in part, the tumor-suppressive action of these miRNAs.
Pubmed link : 24374827

21. FibromiRs: translating molecular discoveries into new anti-fibrotic drugs.
Trends Pharmacol Sci. 2014 Mar;35(3):119-26. doi: 10.1016/j.tips.2014.01.003. Epub 2014 Feb 19.
Pottier N, Cauffiez C, Perrais M, Barbry P, Mari B
1EA4483, Department of Biochemistry and Molecular Biology, Lille 2 University School of Medicine, Lille, France. Electronic address: nico_pottier@yahoo.fr. 2EA4483, Department of Biochemistry and Molecular Biology, Lille 2 University School of Medicine, Lille, France. 3INSERM U837, Jean-Pierre Aubert Research Center, Lille, France. 4CNRS, Institute of Molecular and Cellular Pharmacology, Sophia Antipolis, France; University of Nice Sophia Antipolis, Nice, France.

Fibrosis, or tissue scarring, is defined as excessive and persistent accumulation of extracellular matrix components in response to chronic tissue injury. Fibrosis is a pathological feature characterizing nearly all forms of chronic organ failure. Fibroproliferative disorders of liver, kidney, heart, and lung are frequently associated with considerable morbidity and mortality worldwide. Limited therapeutic options are available; none is yet effective in stopping the ultimate progression of the disease. This has prompted investigations for new molecular targets. Recent studies have shown aberrant expression of miRNAs (fibromiRs) during the development of fibrosis. The challenge now is to understand how these aberrantly expressed miRNAs collaborate to drive fibrogenesis. Progress in understanding how fibromiRs contribute to tissue fibrosis is necessary to translate molecular discoveries into new therapeutics for fibroproliferative diseases.
Pubmed link : 24560301

22. miR-199a-5p in idiopathic pulmonary fibrosis
Med Sci (Paris). 2013 May;29(5):461-3. doi: 10.1051/medsci/2013295006. Epub 2013 May 28.
Henaoui IS, Cauffiez C, Aubert S, Buscot M, Dewaeles E, Copin MC, Marquette CH, Barbry P, Perrais M, Pottier N, Mari B
UMR-7275 CNRS, université de Nice Sophia-Antipolis, institut de pharmacologie moléculaire et cellulaire, 660, route des Lucioles Sophia-Antipolis, 06560 Valbonne, Nice, France.

Pubmed link : 23732092

23. A novel role for the RNA-binding protein FXR1P in myoblasts cell-cycle progression by modulating p21/Cdkn1a/Cip1/Waf1 mRNA stability.
PLoS Genet. 2013 Mar;9(3):e1003367. doi: 10.1371/journal.pgen.1003367. Epub 2013 Mar 21.
Davidovic L, Durand N, Khalfallah O, Tabet R, Barbry P, Mari B, Sacconi S, Moine H, Bardoni B
Institut de Pharmacologie Moléculaire et Cellulaire, CNRS UMR 7275, Valbonne, France.

The Fragile X-Related 1 gene (FXR1) is a paralog of the Fragile X Mental Retardation 1 gene (FMR1), whose absence causes the Fragile X syndrome, the most common form of inherited intellectual disability. FXR1P plays an important role in normal muscle development, and its absence causes muscular abnormalities in mice, frog, and zebrafish. Seven alternatively spliced FXR1 transcripts have been identified and two of them are skeletal muscle-specific. A reduction of these isoforms is found in myoblasts from Facio-Scapulo Humeral Dystrophy (FSHD) patients. FXR1P is an RNA-binding protein involved in translational control; however, so far, no mRNA target of FXR1P has been linked to the drastic muscular phenotypes caused by its absence. In this study, gene expression profiling of C2C12 myoblasts reveals that transcripts involved in cell cycle and muscular development pathways are modulated by Fxr1-depletion. We observed an increase of p21--a regulator of cell-cycle progression--in Fxr1-knocked-down mouse C2C12 and FSHD human myoblasts. Rescue of this molecular phenotype is possible by re-expressing human FXR1P in Fxr1-depleted C2C12 cells. FXR1P muscle-specific isoforms bind p21 mRNA via direct interaction with a conserved G-quadruplex located in its 3' untranslated region. The FXR1P/G-quadruplex complex reduces the half-life of p21 mRNA. In the absence of FXR1P, the upregulation of p21 mRNA determines the elevated level of its protein product that affects cell-cycle progression inducing a premature cell-cycle exit and generating a pool of cells blocked at G0. Our study describes a novel role of FXR1P that has crucial implications for the understanding of its role during myogenesis and muscle development, since we show here that in its absence a reduced number of myoblasts will be available for muscle formation/regeneration, shedding new light into the pathophysiology of FSHD.
Pubmed link : 23555284

24. MiR-210 promotes a hypoxic phenotype and increases radioresistance in human lung cancer cell lines.
Cell Death Dis. 2013 Mar 14;4:e544. doi: 10.1038/cddis.2013.71.
Grosso S, Doyen J, Parks SK, Bertero T, Paye A, Cardinaud B, Gounon P, Lacas-Gervais S, Noel A, Pouysségur J, Barbry P, Mazure NM, Mari B
Institut de Pharmacologie Moléculaire et Cellulaire (IPMC), Centre National de la Recherche Scientifique, CNRS UMR 7275, Sophia Antipolis, France.

The resistance of hypoxic cells to radiotherapy and chemotherapy is a major problem in the treatment of cancer. Recently, an additional mode of hypoxia-inducible factor (HIF)-dependent transcriptional regulation, involving modulation of a specific set of micro RNAs (miRNAs), including miR-210, has emerged. We have recently shown that HIF-1 induction of miR-210 also stabilizes HIF-1 through a positive regulatory loop. Therefore, we hypothesized that by stabilizing HIF-1 in normoxia, miR-210 may protect cancer cells from radiation. We developed a non-small cell lung carcinoma (NSCLC)-derived cell line (A549) stably expressing miR-210 (pmiR-210) or a control miRNA (pmiR-Ctl). The miR-210-expressing cells showed a significant stabilization of HIF-1 associated with mitochondrial defects and a glycolytic phenotype. Cells were subjected to radiation levels ranging from 0 to 10 Gy in normoxia and hypoxia. Cells expressing miR-210 in normoxia had the same level of radioresistance as control cells in hypoxia. Under hypoxia, pmiR-210 cells showed a low mortality rate owing to a decrease in apoptosis, with an ability to grow even at 10 Gy. This miR-210 phenotype was reproduced in another NSCLC cell line (H1975) and in HeLa cells. We have established that radioresistance was independent of p53 and cell cycle status. In addition, we have shown that genomic double-strand breaks (DSBs) foci disappear faster in pmiR-210 than in pmiR-Ctl cells, suggesting that miR-210 expression promotes a more efficient DSB repair. Finally, HIF-1 invalidation in pmiR-210 cells removed the radioresistant phenotype, showing that this mechanism is dependent on HIF-1. In conclusion, miR-210 appears to be a component of the radioresistance of hypoxic cancer cells. Given the high stability of most miRNAs, this advantage could be used by tumor cells in conditions where reoxygenation has occurred and suggests that strategies targeting miR-210 could enhance tumor radiosensitization.
Pubmed link : 23492775

25. CDC25A targeting by miR-483-3p decreases CCND-CDK4/6 assembly and contributes to cell cycle arrest.
Cell Death Differ. 2013 Jun;20(6):800-11. doi: 10.1038/cdd.2013.5. Epub 2013 Feb 22.
Bertero T, Gastaldi C, Bourget-Ponzio I, Mari B, Meneguzzi G, Barbry P, Ponzio G, Rezzonico R
1] CNRS UMR 7275, IPMC, Physiological Genomics of the Eukaryotes, Valbonne, France [2] Université de Nice Sophia-Antipolis, Nice, France.

Abstract Disruption of contact inhibition and serum afflux that occur after a tissue injury activate cell cycle, which then stops when confluence is reached again. Although the events involved in cell cycle entry have been widely documented, those managing cell cycle exit have remained so far ill defined. We have identified that the final stage of wound closure is preceded in keratinocytes by a strong accumulation of miR-483-3p, which acts as a mandatory signal triggering cell cycle arrest when confluence is reached. Blocking miR-483-3p accumulation strongly delays cell cycle exit, maintains cells into a proliferative state and retards their differentiation program. Using two models of cell cycle synchronization (i.e. mechanical injury and serum addition), we show that an ectopic upregulation of miR-483-3p blocks cell cycle progression in early G1 phase. This arrest results from a direct targeting of the CDC25A phosphatase by miR-483-3p, which can be impeded using an anti-miRNA against miR-483-3p or a protector that blocks the complex formation between miR-483-3p and the 3'-untranslated region (UTR) of CDC25A transcript. We show that the miRNA-induced silencing of CDC25A increases the tyrosine phosphorylation status of CDK4/6 cyclin-dependent kinases which, in turn, abolishes CDK4/6 capacity to associate with D-type cyclins. This prevents CDK4/6 kinases' activation, impairs downstream events such as cyclin E stimulation and sequesters cells in early G1. We propose this new regulatory process of cyclin-CDK association as a general mechanism coupling miRNA-mediated CDC25A invalidation to CDK post-transcriptional modifications and cell cycle control.
Pubmed link : 23429262

26. miR-199a-5p Is upregulated during fibrogenic response to tissue injury and mediates TGFbeta-induced lung fibroblast activation by targeting caveolin-1.
PLoS Genet. 2013 Feb;9(2):e1003291. doi: 10.1371/journal.pgen.1003291. Epub 2013 Feb 14.
Lino Cardenas CL, Henaoui IS, Courcot E, Roderburg C, Cauffiez C, Aubert S, Copin MC, Wallaert B, Glowacki F, Dewaeles E, Milosevic J, Maurizio J, Tedrow J, Marcet B, Lo-Guidice JM, Kaminski N, Barbry P, Luedde T, Perrais M, Mari B, Pottier N
EA4483, Faculté de Médecine de Lille, Pole Recherche, Lille, France.

As miRNAs are associated with normal cellular processes, deregulation of miRNAs is thought to play a causative role in many complex diseases. Nevertheless, the precise contribution of miRNAs in fibrotic lung diseases, especially the idiopathic form (IPF), remains poorly understood. Given the poor response rate of IPF patients to current therapy, new insights into the pathogenic mechanisms controlling lung fibroblasts activation, the key cell type driving the fibrogenic process, are essential to develop new therapeutic strategies for this devastating disease. To identify miRNAs with potential roles in lung fibrogenesis, we performed a genome-wide assessment of miRNA expression in lungs from two different mouse strains known for their distinct susceptibility to develop lung fibrosis after bleomycin exposure. This led to the identification of miR-199a-5p as the best miRNA candidate associated with bleomycin response. Importantly, miR-199a-5p pulmonary expression was also significantly increased in IPF patients (94 IPF versus 83 controls). In particular, levels of miR-199a-5p were selectively increased in myofibroblasts from injured mouse lungs and fibroblastic foci, a histologic feature associated with IPF. Therefore, miR-199a-5p profibrotic effects were further investigated in cultured lung fibroblasts: miR-199a-5p expression was induced upon TGFβ exposure, and ectopic expression of miR-199a-5p was sufficient to promote the pathogenic activation of pulmonary fibroblasts including proliferation, migration, invasion, and differentiation into myofibroblasts. In addition, we demonstrated that miR-199a-5p is a key effector of TGFβ signaling in lung fibroblasts by regulating CAV1, a critical mediator of pulmonary fibrosis. Remarkably, aberrant expression of miR-199a-5p was also found in unilateral ureteral obstruction mouse model of kidney fibrosis, as well as in both bile duct ligation and CCl4-induced mouse models of liver fibrosis, suggesting that dysregulation of miR-199a-5p represents a general mechanism contributing to the fibrotic process. MiR-199a-5p thus behaves as a major regulator of tissue fibrosis with therapeutic potency to treat fibroproliferative diseases.
Pubmed link : 23459460

27. The 3' UTR of FMR1 mRNA is a target of miR-101, miR-129-5p and miR-221: implications for the molecular pathology of FXTAS at the synapse.
Hum Mol Genet. 2013 May 15;22(10):1971-82. doi: 10.1093/hmg/ddt044. Epub 2013 Feb 5.
Zongaro S, Hukema R, D'Antoni S, Davidovic L, Barbry P, Catania MV, Willemsen R, Mari B, Bardoni B
CNRS UMR 7275, Institute of Molecular and Cellular Pharmacology, Valbonne Sophia-Antipolis, France.

While FMR1 is silenced in Fragile X syndrome (FXS) patients carrying the full mutation, its expression is elevated (2-8 fold) in premutated individuals. These people may develop the Fragile X-associated Tremor/Ataxia syndrome (FXTAS), a late onset neurodegenerative disorder characterized by ataxia and parkinsonism. In addition, people carrying the premutation can be affected by a set of neurological and behavioral disorders during young age. Problems of memory have been detected in these patients as well as in the mouse models for FXTAS. To date little is known concerning the metabolism of FMR1 mRNA, notwithstanding the importance of the finely tuned regulation of the expression of this gene. In the present study, we identified three microRNAs that specifically target the 3' UTR of FMR1 and can modulate its expression throughout the brain particularly at the synapse where their expression is very high. The expression level of miR-221 is reduced in synaptosomal preparations of young FXTAS mice suggesting a general deregulation of transcripts located at the synapse of these mice. By transcriptome analysis, we show here a robust deregulation of the expression levels of genes involved in learning, memory and autistic behavior, Parkinson disease and neurodegeneration. These findings suggest the presence of a synaptopathy in these animals. Interestingly, many of those deregulated mRNAs are target of the same microRNAs that modulate the expression of FMR1 at the synapse.
Pubmed link : 23390134

28. "Seed-Milarity" confers to hsa-miR-210 and hsa-miR-147b similar functional activity.
PLoS One. 2012;7(9):e44919. doi: 10.1371/journal.pone.0044919. Epub 2012 Sep 13.
Bertero T, Grosso S, Robbe-Sermesant K, Lebrigand K, Henaoui IS, Puissegur MP, Fourre S, Zaragosi LE, Mazure NM, Ponzio G, Cardinaud B, Barbry P, Rezzonico R, Mari B
Institut de Pharmacologie Moléculaire et Cellulaire-IPMC, Centre National de la Recherche Scientifique, CNRS UMR 7275, Sophia Antipolis, France.

Specificity of interaction between a microRNA (miRNA) and its targets crucially depends on the seed region located in its 5'-end. It is often implicitly considered that two miRNAs sharing the same biological activity should display similarity beyond the strict six nucleotide region that forms the seed, in order to form specific complexes with the same mRNA targets. We have found that expression of hsa-miR-147b and hsa-miR-210, though triggered by different stimuli (i.e. lipopolysaccharides and hypoxia, respectively), induce very similar cellular effects in term of proliferation, migration and apoptosis. Hsa-miR-147b only shares a "minimal" 6-nucleotides seed sequence with hsa-miR-210, but is identical with hsa-miR-147a over 20 nucleotides, except for one base located in the seed region. Phenotypic changes induced after heterologous expression of miR-147a strikingly differ from those induced by miR-147b or miR-210. In particular, miR-147a behaves as a potent inhibitor of cell proliferation and migration. These data fit well with the gene expression profiles observed for miR-147b and miR-210, which are very similar, and the gene expression profile of miR-147a, which is distinct from the two others. Bioinformatics analysis of all human miRNA sequences indicates multiple cases of miRNAs from distinct families exhibiting the same kind of similarity that would need to be further characterized in terms of putative functional redundancy. Besides, it implies that functional impact of some miRNAs can be masked by robust expression of miRNAs belonging to distinct families.
Pubmed link : 23028679

29. miR-7a regulation of Pax6 controls spatial origin of forebrain dopaminergic neurons.
Nat Neurosci. 2012 Jun 24;15(8):1120-6. doi: 10.1038/nn.3142.
de Chevigny A, Coré N, Follert P, Gaudin M, Barbry P, Béclin C, Cremer H
Institut de Biologie de Développement de Marseille-Luminy, Aix-Marseille University, Marseille, France.

In the postnatal and adult mouse forebrain, a mosaic of spatially separated neural stem cells along the lateral wall of the ventricles generates defined types of olfactory bulb neurons. To understand the mechanisms underlying the regionalization of the stem cell pool, we focused on the transcription factor Pax6, a determinant of the dopaminergic phenotype in this system. We found that, although Pax6 mRNA was transcribed widely along the ventricular walls, Pax6 protein was restricted to the dorsal aspect. This dorsal restriction was a result of inhibition of protein expression by miR-7a, a microRNA (miRNA) that was expressed in a gradient opposing Pax6. In vivo inhibition of miR-7a in Pax6-negative regions of the lateral wall induced Pax6 protein expression and increased dopaminergic neurons in the olfactory bulb. These findings establish miRNA-mediated fine-tuning of protein expression as a mechanism for controlling neuronal stem cell diversity and, consequently, neuronal phenotype.
Pubmed link : 22729175

30. Distinct epithelial gene expression phenotypes in childhood respiratory allergy.
Eur Respir J. 2012 May;39(5):1197-205. Epub 2011 Oct 17.
Giovannini-Chami L, Marcet B, Moreilhon C, Chevalier B, Illie MI, Lebrigand K, Robbe-Sermesant K, Bourrier T, Michiels JF, Mari B, Crénesse D, Hofman P, de Blic J, Castillo L, Albertini M, Barbry P
CNRS and University of Nice Sophia Antipolis, Institut de Pharmacologie Moléculaire et Cellulaire, UMR 7275, Sophia Antipolis, 06560 Sophia Antipolis, France.

Epithelial cell contribution to the natural history of childhood allergic respiratory disease remains poorly understood. Our aims were to identify epithelial pathways that are dysregulated in different phenotypes of respiratory allergy. We established gene expression signatures of nasal brushings from children with dust mite-allergic rhinitis, associated or not associated with controlled or uncontrolled asthma. Supervised learning and unsupervised clustering were used to predict the different subgroups of patients and define altered signalling pathways. These profiles were compared with those of primary cultures of human nasal epithelial cells stimulated with either interleukin (IL)-4, IL-13, interferon (IFN)-α, IFN-β or IFN-γ, or during in vitro differentiation. A supervised method discriminated children with allergic rhinitis from healthy controls (prediction accuracy 91%), based on 61 transcripts, including 21 T-helper cell (Th) type 2-responsive genes. This method was then applied to predict children with controlled or uncontrolled asthma (prediction accuracy 75%), based on 41 transcripts: nine of them, which were down-regulated in uncontrolled asthma, are directly linked to IFN. This group also included GSDML, which is genetically associated with asthma. Our data revealed a Th2-driven epithelial phenotype common to all children with dust mite allergic rhinitis. It highlights the influence of epithelially expressed molecules on the control of asthma, in association with atopy and impaired viral response.
Pubmed link : 22005912

31. MiR-129-5p is required for histone deacetylase inhibitor-induced cell death in thyroid cancer cells.
Endocr Relat Cancer. 2011 Nov 14;18(6):711-9. doi: 10.1530/ERC-10-0257. Print 2011 Dec.
Brest P, Lassalle S, Hofman V, Bordone O, Gavric Tanga V, Bonnetaud C, Moreilhon C, Rios G, Santini J, Barbry P, Svanborg C, Mograbi B, Mari B, Hofman P
Nice, France.

The molecular mechanism responsible for the antitumor activity of histone deacetylase inhibitors (HDACi) remains elusive. As HDACi have been described to alter miRNA expression, the aim of this study was to characterize HDACi-induced miRNAs and to determine their functional importance in the induction of cell death alone or in combination with other cancer drugs. Two HDACi, trichostatin A and vorinostat, induced miR-129-5p overexpression, histone acetylation and cell death in BCPAP, TPC-1, 8505C, and CAL62 cell lines and in primary cultures of papillary thyroid cancer (PTC) cells. In addition, miR-129-5p alone was sufficient to induce cell death and knockdown experiments showed that expression of this miRNA was required for HDACi-induced cell death. Moreover, miR-129-5p accentuated the anti-proliferative effects of other cancer drugs such as etoposide or human α-lactalbumin made lethal for tumor cells (HAMLET). Taken together, our data show that miR-129-5p is involved in the antitumor activity of HDACi and highlight a miRNA-driven cell death mechanism.
Pubmed link : 21946411

32. Genomotyping of Coxiella burnetii using microarrays reveals a conserved genomotype for hard tick isolates.
PLoS One. 2011;6(10):e25781. Epub 2011 Oct 25.
Leroy Q, Armougom F, Barbry P, Raoult D
Unité de Recherche en Maladies Infectieuses et Tropicales Emergentes, CNRS-IRD, UMR 6236, Faculté de Médecine, Université de la Méditerranée, Marseille, France.

C. burnetii is a Gram-negative intracellular Y-proteobacteria that causes the zoonotic disease Q fever. Q fever can manifest as an acute or chronic illness. Different typing methods have been previously developed to classify C. burnetii isolates to explore its pathogenicity. Here, we report a comprehensive genomotyping method based on the presence or absence of genes using microarrays. The genomotyping method was then tested in 52 isolates obtained from different geographic areas, different hosts and patients with different clinical manifestations. The analysis revealed the presence of 10 genomotypes organized into 3 groups, with a topology congruent with that obtained through multi-spacer typing. We also found that only 4 genomotypes were specifically associated with acute Q fever, whereas all of the genomotypes could be associated to chronic human infection. Serendipitously, the genomotyping results revealed that all hard tick isolates, including the Nine Mile strain, belong to the same genomotype.
Pubmed link : 22046248

33. Can the microRNA signature distinguish between thyroid tumors of uncertain malignant potential and other well-differentiated tumors of the thyroid gland?
Endocr Relat Cancer. 2011 Sep 13;18(5):579-94. Print 2011 Oct.
Lassalle S, Hofman V, Ilie M, Bonnetaud C, Puisségur MP, Brest P, Loubatier C, Guevara N, Bordone O, Cardinaud B, Lebrigand K, Rios G, Santini J, Franc B, Mari B, Al Ghuzlan A, Vielh P, Barbry P, Hofman P
INSERM ERI-21/EA4319, University of Nice Sophia Antipolis, 06107 Nice, France.

The term 'thyroid tumors of uncertain malignant potential' (TT-UMP) was coined by surgical pathologists to define well-differentiated tumors (WDT) showing inconclusive morphological evidence of malignancy or benignity. We have analyzed the expression of microRNA (miRNA) in a training set of 42 WDT of different histological subtypes: seven follicular tumors of UMP (FT-UMP), six WDT-UMP, seven follicular thyroid adenomas (FTA), 11 conventional papillary thyroid carcinomas (C-PTC), five follicular variants of PTC (FV-PTC), and six follicular thyroid carcinomas (FTC), which led to the identification of about 40 deregulated miRNAs. A subset of these altered miRNAs was independently validated by qRT-PCR, which included 18 supplementary TT-UMP (eight WDT-UMP and ten FT-UMP). Supervised clustering techniques were used to predict the first 42 samples. Based on the four possible outcomes (FTA, C-PTC, FV-PTC, and FTC), about 80% of FTA and C-PTC and 50% of FV-PTC and FTC samples were correctly assigned. Analysis of the independent set of 18 WDT-UMP by quantitative RT-PCR for the selection of the six most discriminating miRNAs was unable to separate FT-UMP from WDT-UMP, suggesting that the miRNA signature is insufficient in characterizing these two clinical entities. We conclude that considering FT-UMP and WDT-UMP as distinct and specific clinical entities may improve the diagnosis of WDT of the thyroid gland. In this context, a small set of miRNAs (i.e. miR-7, miR-146a, miR-146b, miR-200b, miR-221, and miR-222) appears to be useful, though not sufficient per se, in distinguishing TT-UMP from other WDT of the thyroid gland.
Pubmed link : 21778212

34. miR-483-3p controls proliferation in wounded epithelial cells.
FASEB J. 2011 Sep;25(9):3092-105. Epub 2011 Jun 15.
Bertero T, Gastaldi C, Bourget-Ponzio I, Imbert V, Loubat A, Selva E, Busca R, Mari B, Hofman P, Barbry P, Meneguzzi G, Ponzio G, Rezzonico R
INSERM U634, IFR50, Faculté de Médecine, France.

The mechanisms that regulate keratinocyte migration and proliferation in wound healing remain largely unraveled, notably regarding possible involvements of microRNAs (miRNAs). Here we disclose up-regulation of miR-483-3p in 2 distinct models of wound healing: scratch-injured cultures of human keratinocytes and wounded skin in mice. miR-483-3p accumulation peaks at the final stage of the wound closure process, consistent with a role in the arrest of "healing" progression. Using an in vitro wound-healing model, videomicroscopy, and 5-bromo-2'-uridine incorporation, we observed that overexpression of miR-483-3p inhibits keratinocyte migration and proliferation, whereas delivery of anti-miR-483-3p oligonucleotides sustains keratinocyte proliferation beyond the closure of the wound, compared with irrelevant anti-miR treatment. Expression profiling of keratinocytes transfected with miR-483-3p identified 39 transcripts that were both predicted targets of miR-483-3p and down-regulated after miR-483-3p overexpression. Luciferase reporter assays, Western blot analyses, and silencing by specific siRNAs finally established that kinase MK2, cell proliferation marker MKI67, and transcription factor YAP1 are direct targets of miR-483-3p that control keratinocyte proliferation. miR-483-3p-mediated down-regulation of MK2, MKI67, and YAP1 thus represents a novel mechanism controlling keratinocyte growth arrest at the final steps of reepithelialization.
Pubmed link : 21676945

35. MicroRNA-based silencing of Delta/Notch signaling promotes multiple cilia formation.
Cell Cycle. 2011 Sep 1;10(17):2858-64. Epub 2011 Sep 1.
Marcet B, Chevalier B, Coraux C, Kodjabachian L, Barbry P
Centre National de la Recherche Scientifique (CNRS), Institut de Pharmacologie Moléculaire et Cellulaire, UMR-6097, Sophia-Antipolis, France. marcet@ipmc.cnrs.fr

Multiciliated cells lining the surface of some vertebrate epithelia are essential for various physiological processes, such as airway cleansing. Their apical surface is constituted by hundreds of motile cilia, which beat in a coordinated manner to generate directional fluid flow. We recently reported the identification of microRNAs of the miR-449 family as evolutionary conserved key regulators of vertebrate multiciliogenesis. This novel function of miR-449 was established using in vivo and in vitro antisense approaches in two distinct experimental models. miR-449 strongly accumulated in multiciliated cells in human airway epithelium and Xenopus laevis embryonic epidermis, where it triggered centriole multiplication and multiciliogenesis by directly repressing the Delta/Notch pathway. Our data complement previous reports that showed the blocking action of miR-449 on the cell cycle, and unraveled a novel conserved mechanism whereby Notch signaling must undergo microRNA-mediated inhibition to permit differentiation of ciliated cell progenitors. We review here several important questions regarding the links between microRNAs and the Notch pathway in the control of cell fate.
Pubmed link : 21857154

36. CYR61 downregulation reduces osteosarcoma cell invasion, migration, and metastasis.
J Bone Miner Res. 2011 Jul;26(7):1533-42. doi: 10.1002/jbmr.343.
Fromigue O, Hamidouche Z, Vaudin P, Lecanda F, Patino A, Barbry P, Mari B, Marie PJ
Laboratory of Osteoblast Biology and Pathology, INSERM, Paris, France. olivia.fromigue@inserm.fr

Osteosarcoma is the most common primary tumor of bone. The rapid development of metastatic lesions and resistance to chemotherapy remain major mechanisms responsible for the failure of treatments and the poor survival rate for patients. We showed previously that the HMGCoA (3-hydroxy-3-methylglutaryl-coenzyme A) reductase inhibitor statin exhibits antitumoral effects on osteosarcoma cells. Here, using microarray analysis, we identify Cyr61 as a new target of statins. Transcriptome and molecular analyses revealed that statins downregulate Cyr61 expression in human and murine osteosarcoma cells. Cyr61 silencing in osteosarcoma cell lines enhanced cell death and reduced cell migration and cell invasion compared with parental cells, whereas Cyr61 overexpression had opposite effects. Cyr61 expression was evaluated in 231 tissue cores from osteosarcoma patients. Tissue microarray analysis revealed that Cyr61 protein expression was higher in human osteosarcoma than in normal bone tissue and was further increased in metastatic tissues. Finally, tumor behavior and metastasis occurrence were analyzed by intramuscular injection of modified osteosarcoma cells into BALB/c mice. Cyr61 overexpression enhanced lung metastasis development, whereas cyr61 silencing strongly reduced lung metastases in mice. The results reveal that cyr61 expression increases with tumor grade in human osteosarcoma and demonstrate that cyr61 silencing inhibits in vitro osteosarcoma cell invasion and migration as well as in vivo lung metastases in mice. These data provide a novel molecular target for therapeutic intervention in metastatic osteosarcoma.
Pubmed link : 21312266

37. MicroRNA control biosynthesis of motile cilia in vertebrates.
Med Sci (Paris). 2011 Jun-Jul;27(6-7):671-3. Epub 2011 Jul 1.
Chevalier B, Kodjabachian L, Coraux C, Barbry P, Marcet B

Article in French
Pubmed link : 21718654

38. Small RNA sequencing reveals miR-642a-3p as a novel adipocyte-specific microRNA and miR-30 as a key regulator of human adipogenesis.
Genome Biol. 2011 Jul 18;12(7):R64.
Zaragosi LE, Wdziekonski B, Brigand KL, Villageois P, Mari B, Waldmann R, Dani C, Barbry P
Centre National de la Recherche Scientifique, Institut de Pharmacologie Moléculaire et Cellulaire, UMR-6097, 660 Route des Lucioles, Valbonne Sophia-Antipolis 06560, France.

BACKGROUND: In severe obesity, as well as in normal development, the growth of adipose tissue is the result of an increase in adipocyte size and numbers, which is underlain by the stimulation of adipogenic differentiation of precursor cells. A better knowledge of the pathways that regulate adipogenesis is therefore essential for an improved understanding of adipose tissue expansion. As microRNAs (miRNAs) have a critical role in many differentiation processes, our study aimed to identify the role of miRNA-mediated gene silencing in the regulation of adipogenic differentiation. RESULTS: We used deep sequencing to identify small RNAs that are differentially expressed during adipogenesis of adipose tissue-derived stem cells. This approach revealed the un-annotated miR-642a-3p as a highly adipocyte-specific miRNA. We then focused our study on the miR-30 family, which was also up-regulated during adipogenic differentiation and for which the role in adipogenesis had not yet been elucidated. Inhibition of the miR-30 family blocked adipogenesis, whilst over-expression of miR-30a and miR-30d stimulated this process. We additionally showed that both miR-30a and miR-30d target the transcription factor RUNX2, and stimulate adipogenesis via the modulation of this major regulator of osteogenesis. CONCLUSIONS: Overall, our data suggest that the miR-30 family plays a central role in adipocyte development. Moreover, as adipose tissue-derived stem cells can differentiate into either adipocytes or osteoblasts, the down-regulation of the osteogenesis regulator RUNX2 represents a plausible mechanism by which miR-30 miRNAs may contribute to adipogenic differentiation of adipose tissue-derived stem cells.
Pubmed link : 21767385

39. Control of vertebrate multiciliogenesis by miR-449 through direct repression of the Delta/Notch pathway.
Nat Cell Biol. 2011 Jun;13(6):693-9. Epub 2011 May 22.
Marcet B, Chevalier B, Luxardi G, Coraux C, Zaragosi LE, Cibois M, Robbe-Sermesant K, Jolly T, Cardinaud B, Moreilhon C, Giovannini-Chami L, Nawrocki-Raby B, Birembaut P, Waldmann R, Kodjabachian L, Barbry P
CNRS, Institut de Pharmacologie Moléculaire et Cellulaire, F06560 Sophia Antipolis, France.

Multiciliated cells lining the surface of some vertebrate epithelia are essential for various physiological processes, such as airway cleansing. However, the mechanisms governing motile cilia biosynthesis remain poorly elucidated. We identify miR-449 microRNAs as evolutionarily conserved key regulators of vertebrate multiciliogenesis. In human airway epithelium and Xenopus laevis embryonic epidermis, miR-449 microRNAs strongly accumulated in multiciliated cells. In both models, we show that miR-449 microRNAs promote centriole multiplication and multiciliogenesis by directly repressing the Delta/Notch pathway. We established Notch1 and its ligand Delta-like 1(DLL1) as miR-449 bona fide targets. Human DLL1 and NOTCH1 protein levels were lower in multiciliated cells than in surrounding cells, decreased after miR-449 overexpression and increased after miR-449 inhibition. In frog, miR-449 silencing led to increased Dll1 expression. Consistently, overexpression of Dll1 mRNA lacking miR-449 target sites repressed multiciliogenesis, whereas both Dll1 and Notch1 knockdown rescued multiciliogenesis in miR-449-deficient cells. Antisense-mediated protection of miR-449-binding sites of endogenous human Notch1 or frog Dll1 strongly repressed multiciliogenesis. Our results unravel a conserved mechanism whereby Notch signalling must undergo miR-449-mediated inhibition to permit differentiation of ciliated cell progenitors.
Pubmed link : 21602795

40. Impact of microRNA in normal and pathological respiratory epithelia.
Methods Mol Biol. 2011;741:171-91.
Giovannini-Chami L, Grandvaux N, Zaragosi LE, Robbe-Sermesant K, Marcet B, Cardinaud B, Coraux C, Berthiaume Y, Waldmann R, Mari B, Barbry P
CNRS, Université de Nice Sophia Antipolis, IPMC, UMR6097, Sophia Antipolis, France. chami@ipmc.cnrs.fr

Extensive sequencing efforts, combined with ad hoc bioinformatics developments, have now led to the identification of 1222 distinct miRNAs in human (derived from 1368 distinct genomic loci) and of many miRNAs in other multicellular organisms. The present chapter is aimed at describing a general experimental strategy to identify specific miRNA expression profiles and to highlight the functional networks operating between them and their mRNA targets, including several miRNAs deregulated in cystic fibrosis and during differentiation of airway epithelial cells.
Pubmed link : 21594785

41. The human TTAGGG repeat factors 1 and 2 bind to a subset of interstitial telomeric sequences and satellite repeats.
Cell Res. 2011 Jul;21(7):1028-38. doi: 10.1038/cr.2011.40. Epub 2011 Mar 22.
Simonet T, Zaragosi LE, Philippe C, Lebrigand K, Schouteden C, Augereau A, Bauwens S, Ye J, Santagostino M, Giulotto E, Magdinier F, Horard B, Barbry P, Waldmann R, Gilson E
Laboratoire de Biologie Moléculaire de la Cellule-UMR 5239 CNRS/ENS Lyon/ Université Lyon, Ecole Normale Supérieure de Lyon, 46 allée d'Italie, Lyon 69364, France.

The study of the proteins that bind to telomeric DNA in mammals has provided a deep understanding of the mechanisms involved in chromosome-end protection. However, very little is known on the binding of these proteins to nontelomeric DNA sequences. The TTAGGG DNA repeat proteins 1 and 2 (TRF1 and TRF2) bind to mammalian telomeres as part of the shelterin complex and are essential for maintaining chromosome end stability. In this study, we combined chromatin immunoprecipitation with high-throughput sequencing to map at high sensitivity and resolution the human chromosomal sites to which TRF1 and TRF2 bind. While most of the identified sequences correspond to telomeric regions, we showed that these two proteins also bind to extratelomeric sites. The vast majority of these extratelomeric sites contains interstitial telomeric sequences (or ITSs). However, we also identified non-ITS sites, which correspond to centromeric and pericentromeric satellite DNA. Interestingly, the TRF-binding sites are often located in the proximity of genes or within introns. We propose that TRF1 and TRF2 couple the functional state of telomeres to the long-range organization of chromosomes and gene regulation networks by binding to extratelomeric sequences.
Pubmed link : 21423270

42. A synonymous variant in IRGM alters a binding site for miR-196 and causes deregulation of IRGM-dependent xenophagy in Crohn's disease.
Nat Genet. 2011 Mar;43(3):242-5. Epub 2011 Jan 30.
Brest P, Lapaquette P, Souidi M, Lebrigand K, Cesaro A, Vouret-Craviari V, Mari B, Barbry P, Mosnier JF, Hébuterne X, Harel-Bellan A, Mograbi B, Darfeuille-Michaud A, Hofman P
INSERM ERI-21, EA4319, Faculty of Medicine, Nice, France.

Susceptibility to Crohn's disease, a complex inflammatory disease, is influenced by common variants at many loci. The common exonic synonymous SNP (c.313C>T) in IRGM, found in strong linkage disequilibrium with a deletion polymorphism, has been classified as non-causative because of the absence of an alteration in the IRGM protein sequence or splice sites. Here we show that a family of microRNAs (miRNAs), miR-196, is overexpressed in the inflammatory intestinal epithelia of individuals with Crohn's disease and downregulates the IRGM protective variant (c.313C) but not the risk-associated allele (c.313T). Subsequent loss of tight regulation of IRGM expression compromises control of intracellular replication of Crohn's disease-associated adherent invasive Escherichia coli by autophagy. These results suggest that the association of IRGM with Crohn's disease arises from a miRNA-based alteration in IRGM regulation that affects the efficacy of autophagy, thereby implicating a synonymous polymorphism as a likely causal variant.
Pubmed link : 21278745

43. Coxiella burnetii transcriptional analysis reveals serendipity clusters of regulation in intracellular bacteria.
PLoS One. 2010 Dec 21;5(12):e15321.
Leroy Q, Lebrigand K, Armougom F, Barbry P, Thiéry R, Raoult D
Unité de Recherche en Maladies Infectieuses et Tropicales Emergentes, Faculté de Médecine, CNRS-IRD, UMR 6236, Université de la Méditerranée, Marseille, France.

Coxiella burnetii, the causative agent of the zoonotic disease Q fever, is mainly transmitted to humans through an aerosol route. A spore-like form allows C. burnetii to resist different environmental conditions. Because of this, analysis of the survival strategies used by this bacterium to adapt to new environmental conditions is critical for our understanding of C. burnetii pathogenicity. Here, we report the early transcriptional response of C. burnetii under temperature stresses. Our data show that C. burnetii exhibited minor changes in gene regulation under short exposure to heat or cold shock. While small differences were observed, C. burnetii seemed to respond similarly to cold and heat shock. The expression profiles obtained using microarrays produced in-house were confirmed by quantitative RT-PCR. Under temperature stresses, 190 genes were differentially expressed in at least one condition, with a fold change of up to 4. Globally, the differentially expressed genes in C. burnetii were associated with bacterial division, (p)ppGpp synthesis, wall and membrane biogenesis and, especially, lipopolysaccharide and peptidoglycan synthesis. These findings could be associated with growth arrest and witnessed transformation of the bacteria to a spore-like form. Unexpectedly, clusters of neighboring genes were differentially expressed. These clusters do not belong to operons or genetic networks; they have no evident associated functions and are not under the control of the same promoters. We also found undescribed but comparable clusters of regulation in previously reported transcriptomic analyses of intracellular bacteria, including Rickettsia sp. and Listeria monocytogenes. The transcriptomic patterns of C. burnetii observed under temperature stresses permits the recognition of unpredicted clusters of regulation for which the trigger mechanism remains unidentified but which may be the result of a new mechanism of epigenetic regulation.
Pubmed link : 21203564

44. MiRonTop: mining microRNAs targets across large scale gene expression studies
Bioinformatics. 2010 Oct 19
Le Brigand K, Robbe-Sermesant K, Mari B, Barbry P
CNRS, Institut de Pharmacologie Moleculaire et Cellulaire, UMR6097, 06560 Sophia-Antipolis, France.

SUMMARY: Current challenges in microRNA (miRNA) research are to improve the identification of in vivo mRNA targets and clarify the complex interplay existing between a specific miRNA and multiple biological networks. MiRonTop is an online java web tool that integrates DNA microarrays or high-throughput sequencing data to identify the potential implication of miRNAs on a specific biological system. It allows a rapid characterization of the most pertinent mRNA targets according to several existing miRNA target prediction approaches. It also provides useful representations of the enrichment scores according to the position of the target site along the 3'- UTR, where the contribution of the sites located in the vicinity of the stop codon and of the polyA tail can be clearly highlighted. It provides different graphs of miRNA enrichment associated with up- or down-regulated transcripts and different summary tables about selections of mRNA targets and their functional annotations by Gene Ontology. AVAILABILITY: http://www.microarray.fr:8080/miRonTop/index
Pubmed link : 20959382

45. In utero exposure to maternal diabetes impairs vascular expression of prostacyclin receptor in rat offspring.
Diabetes. 2010 Oct;59(10):2597-602.
Duong Van Huyen JP, Vessieres E, Perret C, Troise A, Prince S, Guihot AL, Barbry P, Henrion D, Bruneval P, Laurent S, Lelievre-Pegorier M, Fassot C
INSERM U872, Centre de Recherche des Cordeliers, Universite's Pierre et Marie Curie et Paris-Descartes, Paris, France.

OBJECTIVE: To evaluate modifications of arterial structure, gene expression, and function in our model of rats exposed to maternal diabetes. RESEARCH DESIGN AND METHODS: Morphometric analyses of elastic vessels structure and determination of thoracic aortic gene expression profile with oligonucleotide chips (Agilent, G4130, 22k) were performed before the onset of established hypertension (3 months). RESULTS: Arterial parameters of in situ fixed thoracic aorta were not significantly different between control mother offspring and diabetic mother offspring (DMO). The aortic gene expression profile of DMO is characterized by modifications of several members of the arachidonic acid metabolism including a twofold underexpression of prostacyclin receptor, which could contribute to decreased vasodilatation. This was confirmed by ex vivo experiments on isolated aortic rings. Pharmacological studies on conscious rats showed that systolic blood pressure decline in response to a PGI(2) analog was impaired in DMO rats. CONCLUSIONS: These results suggest an abnormal vascular fetal programming of prostacyclin receptor in rats exposed in utero to maternal hyperglycemia that is associated with impaired vasodilatation and may be involved in the pathophysiology of hypertension in this model.
Pubmed link : 20622163

46. miR-210 is overexpressed in late stages of lung cancer and mediates mitochondrial alterations associated with modulation of HIF-1 activity.
Cell Death Differ. 2010 Oct 1.
Puissegur MP, Mazure NM, Bertero T, Pradelli L, Grosso S, Robbe-Sermesant K, Maurin T, Lebrigand K, Cardinaud B, Hofman V, Fourre S, Magnone V, Ricci JE, Pouyssegur J, Gounon P, Hofman P, Barbry P, Mari B
[1] Institut de Pharmacologie Moleculaire et Cellulaire, CNRS UMR6097, Sophia Antipolis, France [2] University of Nice Sophia-Antipolis, Nice, France.

Following the identification of a set of hypoxia-regulated microRNAs (miRNAs), recent studies have highlighted the importance of miR-210 and of its transcriptional regulation by the transcription factor hypoxia-inducible factor-1 (HIF-1). We report here that miR-210 is overexpressed at late stages of non-small cell lung cancer. Expression of miR-210 in lung adenocarcinoma A549 cells caused an alteration of cell viability associated with induction of caspase-3/7 activity. miR-210 induced a loss of mitochondrial membrane potential and the apparition of an aberrant mitochondrial phenotype. The expression profiling of cells overexpressing miR-210 revealed a specific signature characterized by enrichment for transcripts related to 'cell death' and 'mitochondrial dysfunction', including several subunits of the electron transport chain (ETC) complexes I and II. The transcript coding for one of these ETC components, SDHD, subunit D of succinate dehydrogenase complex (SDH), was validated as a bona fide miR-210 target. Moreover, SDHD knockdown mimicked miR-210-mediated mitochondrial alterations. Finally, miR-210-dependent targeting of SDHD was able to activate HIF-1, in line with previous studies linking loss-of-function SDH mutations to HIF-1 activation. miR-210 can thus regulate mitochondrial function by targeting key ETC component genes with important consequences on cell metabolism, survival and modulation of HIF-1 activity. These observations help explain contradictory data regarding miR-210 expression and its putative function in solid tumors.
Pubmed link : 20885442

47. Functional characterization of ATAD2 as a new cancer/testis factor and a predictor of poor prognosis in breast and lung cancers.
Oncogene. 2010 Sep 16;29(37):5171-81.
Caron C, Lestrat C, Marsal S, Escoffier E, Curtet S, Virolle V, Barbry P, Debernardi A, Brambilla C, Brambilla E, Rousseaux S, Khochbin S
INSERM, U823, France

Cancer cells frequently express genes normally active in male germ cells. ATAD2 is one of them encoding a conserved factor harbouring an AAA type ATPase domain and a bromodomain. We show here that ATAD2 is highly expressed in testis as well as in many cancers of different origins and that its high expression is a strong predictor of rapid mortality in lung and breast cancers. These observations suggest that ATAD2 acts on upstream and basic cellular processes to enhance oncogenesis in a variety of unrelated cell types. Accordingly, our functional studies show that ATAD2 controls chromatin dynamics, genome transcriptional activities and apoptotic cell response. We could also highlight some of the important intrinsic properties of its two regulatory domains, including a functional cross-talk between the AAA ATPase domain and the bromodomain. Altogether, these data indicate that ATAD2 overexpression in somatic cells, by acting on basic properties of chromatin, may contribute to malignant transformation.
Pubmed link : 20581866

48. A Western-like fat diet is sufficient to induce a gradual enhancement in fat mass over generations.
J Lipid Res. 2010 Aug;51(8):2352-61
Massiera F, Barbry P, Guesnet P, Joly A, Luquet S, Moreilhon-Brest C, Mohsen-Kanson T, Amri EZ, Ailhaud G
Universite de Nice Sophia-Antipolis, CNRS, IBDC, UMR 6543, 06107 Nice, France.

The prevalence of obesity has steadily increased over the last few decades. During this time, populations of industrialized countries have been exposed to diets rich in fat with a high content of linoleic acid and a low content of alpha-linolenic acid compared with recommended intake. To assess the contribution of dietary fatty acids, male and female mice fed a high-fat diet (35% energy as fat, linoleic acid:alpha-linolenic acid ratio of 28) were mated randomly and maintained after breeding on the same diet for successive generations. Offspring showed, over four generations, a gradual enhancement in fat mass due to combined hyperplasia and hypertrophy with no change in food intake. Transgenerational alterations in adipokine levels were accompanied by hyperinsulinemia. Gene expression analyses of the stromal vascular fraction of adipose tissue, over generations, revealed discrete and steady changes in certain important players, such as CSF3 and Nocturnin. Thus, under conditions of genome stability and with no change in the regimen over four generations, we show that a Western-like fat diet induces a gradual fat mass enhancement, in accordance with the increasing prevalence of obesity observed in humans.
Pubmed link : 20410018

49. A purified population of multipotent cardiovascular progenitors derived from primate pluripotent stem cells engrafts in postmyocardial infarcted nonhuman primates.
J Clin Invest. 2010 Apr 1;120(4):1125-39
Blin G, Nury D, Stefanovic S, Neri T, Guillevic O, Brinon B, Bellamy V, Rucker-Martin C, Barbry P, Bel A, Bruneval P, Cowan C, Pouly J, Mitalipov S, Gouadon E, Binder P, Hagege A, Desnos M, Renaud JF, Menasche P, Puceat M
INSERM U633, Avenir Program, Embryonic Stem Cells and Cardiogenesis, Evry, France.

Cell therapy holds promise for tissue regeneration, including in individuals with advanced heart failure. However, treatment of heart disease with bone marrow cells and skeletal muscle progenitors has had only marginal positive benefits in clinical trials, perhaps because adult stem cells have limited plasticity. The identification, among human pluripotent stem cells, of early cardiovascular cell progenitors required for the development of the first cardiac lineage would shed light on human cardiogenesis and might pave the way for cell therapy for cardiac degenerative diseases. Here, we report the isolation of an early population of cardiovascular progenitors, characterized by expression of OCT4, stage-specific embryonic antigen 1 (SSEA-1), and mesoderm posterior 1 (MESP1), derived from human pluripotent stem cells treated with the cardiogenic morphogen BMP2. This progenitor population was multipotential and able to generate cardiomyocytes as well as smooth muscle and endothelial cells. When transplanted into the infarcted myocardium of immunosuppressed nonhuman primates, an SSEA-1+ progenitor population derived from Rhesus embryonic stem cells differentiated into ventricular myocytes and reconstituted 20% of the scar tissue. Notably, primates transplanted with an unpurified population of cardiac-committed cells, which included SSEA-1- cells, developed teratomas in the scar tissue, whereas those transplanted with purified SSEA-1+ cells did not. We therefore believe that the SSEA-1+ progenitors that we have described here have the potential to be used in cardiac regenerative medicine.
Pubmed link : 20335662

50. Innovative approach for transcriptomic analysis of obligate intracellular pathogen: selective capture of transcribed sequences of Ehrlichia ruminantium.
BMC Mol Biol. 2009 Dec 24;10:111.
Emboule L, Daigle F, Meyer DF, Mari B, Pinarello V, Sheikboudou C, Magnone V, Frutos R, Viari A, Barbry P, Martinez D, Lefrançois T, Vachiery N
UMR 15 CIRAD-INRA, Controle des maladies animales exotiques et emergentes, Site de Duclos, Prise d'Eau 97170, Petit Bourg, Guadeloupe. loic.emboule@cirad.fr

BACKGROUND: Whole genome transcriptomic analysis is a powerful approach to elucidate the molecular mechanisms controlling the pathogenesis of obligate intracellular bacteria. However, the major hurdle resides in the low quantity of prokaryotic mRNAs extracted from host cells. Our model Ehrlichia ruminantium (ER), the causative agent of heartwater, is transmitted by tick Amblyomma variegatum. This bacterium affects wild and domestic ruminants and is present in Sub-Saharan Africa and the Caribbean islands. Because of its strictly intracellular location, which constitutes a limitation for its extensive study, the molecular mechanisms involved in its pathogenicity are still poorly understood. RESULTS: We successfully adapted the SCOTS method (Selective Capture of Transcribed Sequences) on the model Rickettsiales ER to capture mRNAs. Southern Blots and RT-PCR revealed an enrichment of ER's cDNAs and a diminution of ribosomal contaminants after three rounds of capture. qRT-PCR and whole-genome ER microarrays hybridizations demonstrated that SCOTS method introduced only a limited bias on gene expression. Indeed, we confirmed the differential gene expression between poorly and highly expressed genes before and after SCOTS captures. The comparative gene expression obtained from ER microarrays data, on samples before and after SCOTS at 96 hpi was significantly correlated (R2 = 0.7). Moreover, SCOTS method is crucial for microarrays analysis of ER, especially for early time points post-infection. There was low detection of transcripts for untreated samples whereas 24% and 70.7% were revealed for SCOTS samples at 24 and 96 hpi respectively. CONCLUSIONS: We conclude that this SCOTS method has a key importance for the transcriptomic analysis of ER and can be potentially used for other Rickettsiales. This study constitutes the first step for further gene expression analyses that will lead to a better understanding of both ER pathogenicity and the adaptation of obligate intracellular bacteria to their environment.
Pubmed link : 20034374

51. miR-34b/miR-34c: a regulator of TCL1 expression in 11q- chronic lymphocytic leukaemia?
Leukemia. 2009 Nov;23(11):2174-7.
Cardinaud B, Moreilhon C, Marcet B, Robbe-Sermesant K, Lebrigand K, Mari B, Eclache V, Cymbalista F, Raynaud S, Barbry P
[1] CNRS, IPMC, UMR6097, Sophia Antipolis, France [2] Universite de Nice Sophia-Antipolis, IPMC, UMR6097, Sophia Antipolis, France [3] Universite Victor Segalen Bordeaux 2, EA 4135, Bordeaux, France E-mail: barbry@ipmc.cnrs.fr.

Pubmed link : 19536169

52. Identification of keratinocyte growth factor as a target of microRNA-155 in lung fibroblasts: implication in epithelial-mesenchymal interactions.
PLoS One. 2009 Aug 24;4(8):e6718.
Pottier N, Maurin T, Chevalier B, Puissegur MP, Lebrigand K, Robbe-Sermesant K, Bertero T, Lino Cardenas CL, Courcot E, Rios G, Fourre S, Lo-Guidice JM, Marcet B, Cardinaud B, Barbry P, Mari B
CNRS, Institut de Pharmacologie Moleculaire et Cellulaire, UMR6097, Sophia Antipolis, France.

BACKGROUND: Epithelial-mesenchymal interactions are critical in regulating many aspects of vertebrate embryo development, and for the maintenance of homeostatic equilibrium in adult tissues. The interactions between epithelium and mesenchyme are believed to be mediated by paracrine signals such as cytokines and extracellular matrix components secreted from fibroblasts that affect adjacent epithelia. In this study, we sought to identify the repertoire of microRNAs (miRNAs) in normal lung human fibroblasts and their potential regulation by the cytokines TNF-alpha, IL-1beta and TGF-beta. METHODOLOGY/PRINCIPAL FINDINGS: MiR-155 was significantly induced by inflammatory cytokines TNF-alpha and IL-1beta while it was down-regulated by TGF-beta. Ectopic expression of miR-155 in human fibroblasts induced modulation of a large set of genes related to "cell to cell signalling", "cell morphology" and "cellular movement". This was consistent with an induction of caspase-3 activity and with an increase in cell migration in fibroblasts tranfected with miR-155. Using different miRNA bioinformatic target prediction tools, we found a specific enrichment for miR-155 predicted targets among the population of down-regulated transcripts. Among fibroblast-selective targets, one interesting hit was keratinocyte growth factor (KGF, FGF-7), a member of the fibroblast growth factor (FGF) family, which owns two potential binding sites for miR-155 in its 3'-UTR. Luciferase assays experimentally validated that miR-155 can efficiently target KGF 3'-UTR. Site-directed mutagenesis revealed that only one out of the 2 potential sites was truly functional. Functional in vitro assays experimentally validated that miR-155 can efficiently target KGF 3'-UTR. Furthermore, in vivo experiments using a mouse model of lung fibrosis showed that miR-155 expression level was correlated with the degree of lung fibrosis. CONCLUSIONS/SIGNIFICANCE: Our results strongly suggest a physiological function of miR-155 in lung fibroblasts. Altogether, this study implicates this miRNA in the regulation by mesenchymal cells of surrounding lung epithelium, making it a potential key player during tissue injury.
Pubmed link : 19701459

53. Gene expression profiling of imatinib and PD166326-resistant CML cell lines identifies Fyn as a gene associated with resistance to BCR-ABL inhibitors.
Mol Cancer Ther. 2009 Jul;8(7):1924-33. Epub 2009 Jun 30.
Grosso S, Puissant A, Dufies M, Colosetti P, Jacquel A, Lebrigand K, Barbry P, Deckert M, Cassuto JP, Mari B, Auberger P
INSERM U895, Cell Death, Differentiation and Cancer Team, Faculte de Medecine de Nice, Nice Cedex 2, France.

Imatinib is used to treat chronic myelogenous leukemia (CML), but resistance develops in all phases of this disease. The purpose of the present study was to identify the mode of resistance of newly derived imatinib-resistant (IM-R) and PD166326-resistant (PD-R) CML cells. IM-R and PD-R clones exhibited an increase in viability and a decrease in caspase activation in response to various doses of imatinib and PD166326, respectively, as compared with parental K562 cells. Resistance involved neither mutations in BCR-ABL nor increased BCR-ABL, MDR1 or Lyn expression, all known modes of resistance. To gain insight into the resistance mechanisms, we used pangenomic microarrays and identified 281 genes modulated in parental versus IM-R and PD-R cells. The gene signature was similar for IM-R and PD-R cells, accordingly with the cross-sensitivity observed for both inhibitors. These genes were functionally associated with pathways linked to development, cell adhesion, cell growth, and the JAK-STAT cascade. Especially relevant were the increased expression of the tyrosine kinases AXL and Fyn as well as CD44 and HMGA2. Small interfering RNA experiments and pharmacologic approaches identified FYN as a candidate for resistance to imatinib. Our findings provide a comprehensive picture of the transcriptional events associated with imatinib and PD166326 resistance and identify Fyn as a new potential target for therapeutic intervention in CML.
Pubmed link : 19567819

54. Global analysis of DNA methylation and transcription of human repetitive sequences.
Epigenetics. 2009 Jul 17;4(5).
Horard B, Eymery A, Fourel G, Vassetzky N, Puechberty J, Roizes G, Lebrigand K, Barbry P, Laugraud A, Gautier C, Simon EB, Devaux F, Magdinier F, Vourc'h C, Gilson E
Laboratoire de Biologie Moleculaire de la Cellule UMR CNRS 5239, ENS Lyon, Universite, LYON 1, HCL, Lyon, France.

Half of the human genome consists of repetitive DNA sequences. Recent studies in various organisms highlight the role of chromatin regulation of repetitive DNA in gene regulation as well as in maintainance of chromosomes and genome integrity. Hence, repetitive DNA sequences might be potential "sensors" for chromatin changes associated with pathogenesis. Therefore, we developed a new genomic tool called RepArray. RepArray is a repeat-specific microarray composed of a representative set of human repeated sequences including transposon-derived repeats, simple sequences repeats, tandemly repeated sequences such as centromeres and telomeres. We showed that combined to anti-methylcytosine immunoprecipitation assay, the RepArray can be used to generate repeat-specific methylation maps. Using cell lines impaired chemically or genetically for DNA methyltransferases activities, we were able to distinguish different epigenomes demonstrating that repeats can be used as markers of genome-wide methylation changes. Besides, using a well-documented system model, the thermal stress, we demonstrated that RepArray is also a fast and reliable tool to obtain an overview of overall transcriptional activity on whole repetitive compartment in a given cell type. Thus, the RepArray represents the first valuable tool for systematic and genome-wide analyses of the methylation and transcriptional status of the repetitive counterpart of the human genome.
Pubmed link : 19633427

55. Dax-1 knockdown in mouse embryonic stem cells induces loss of pluripotency and multilineage differentiation.
Stem Cells. 2009 Jul;27(7):1529-37.
Khalfallah O, Rouleau M, Barbry P, Bardoni B, Lalli E
Institut de Pharmacologie Moleculaire et Cellulaire CNRS UMR 6097, Valbonne, France.

Dax-1 (Nr0b1) is an orphan member of the nuclear hormone receptor superfamily that has a key role in adrenogonadal development and function. Recent studies have also implicated Dax-1 in the transcriptional network controlling embryonic stem (ES) cell pluripotency. Here, we show that Dax-1 expression is affected by differentiating treatments and pharmacological activation of beta-catenin-dependent transcription in mouse ES cells. Furthermore, Dax-1 knockdown induced upregulation of multilineage differentiation markers, and produced enhanced differentiation and defects in ES viability and proliferation. Through RNA interference and transcriptome analysis, we have identified genes regulated by Dax-1 in mouse ES cells at 24 and 48 hours after knockdown. Strikingly, the great majority of these genes are upregulated, showing that the prevalent function of Dax-1 is to act as a transcriptional repressor in mouse ES cells, as confirmed by experiments using the Gal4 system. Genes involved in tissue differentiation and control of proliferation are significantly enriched among Dax-1-regulated transcripts. These data show that Dax-1 is an essential element in the molecular circuit involved in the maintenance of ES cell pluripotency and have implications for the understanding of stem cell function in both physiological (adrenal gland) and clinical (Ewing tumors) settings where Dax-1 plays a pivotal role in development and pathogenesis, respectively.
Pubmed link : 19530134

56. MicroRNAs and lung cancer: new oncogenes and tumor suppressors, new prognostic factors and potential therapeutic targets.
Curr Med Chem. 2009;16(9):1047-61.
Ortholan C, Puissegur MP, Ilie M, Barbry P, Mari B, Hofman P
Laboratory of Clinical and Experimental Pathology, Pasteur Hospital, Nice cedex 02, France.

MicroRNAs (miRNAs) are small non-protein-coding RNA that negatively control mRNA expression at a post-transcriptional level. They regulate various cellular functions and bioinformatic data suggest that they collectively control about 30% of human mRNAs. MiRNAs have been recently implicated in several carcinogenic processes, where they can act either as oncogenes or as tumor suppressors. This is the case in lung cancer, i.e. the leading cause of cancer deaths in Western countries, in which about 40-45 miRNAs have been found to be aberrantly expressed, thereby constituting a specific miRNA signature. Some of these miRNAs can play an important role in lung carcinogenesis. Indeed, some transcripts of the let-7 family that are significantly down-regulated in lung tumors have been identified as tumor suppressors through their ability to control several oncogenic pathways, including the RAS pathway. Identification of a growing number of other potential oncogenic or tumor suppressor miRNAs in lung cancers is in constant progress. Recent evidence supports the use of specific miRNA signatures to predict clinical outcome. This review aims to report the current knowledge about the role of miRNAs in lung cancer carcinogenesis, their potential for improving diagnosis and prognosis and their impact on future therapeutic strategies.
Pubmed link : 19275611

57. Transcriptional repression of microRNA genes by PML-RARA increases expression of key cancer proteins in acute promyelocytic leukemia.
Blood. 2009 Jan 8;113(2):412-21. Epub 2008 Oct 21.
Saumet A, Vetter G, Bouttier M, Portales-Casamar E, Wasserman WW, Maurin T, Mari B, Barbry P, Vallar L, Friederich E, Arar K, Cassinat B, Chomienne C, Lecellier CH
Institut de Genetique Humaine, CNRS UPR1142, Montpellier, France.

Micro(mi)RNAs are small noncoding RNAs that orchestrate many key aspects of cell physiology and their deregulation is often linked to distinct diseases including cancer. Here, we studied the contribution of miRNAs in a well-characterized human myeloid leukemia, acute promyelocytic leukemia (APL), targeted by retinoic acid and trioxide arsenic therapy. We identified several miRNAs transcriptionally repressed by the APL-associated PML-RAR oncogene which are released after treatment with all-trans retinoic acid. These coregulated miRNAs were found to control, in a coordinated manner, crucial pathways linked to leukemogenesis, such as HOX proteins and cell adhesion molecules whose expressions are thereby repressed by the chemotherapy. Thus, APL appears linked to transcriptional perturbation of miRNA genes, and clinical protocols able to successfully eradicate cancer cells may do so by restoring miRNA expression. The identification of abnormal miRNA biogenesis in cancer may therefore provide novel biomarkers and therapeutic targets in myeloid leukemias.
Pubmed link : 18941112

58. Genetic differences among Staphylococcus aureus isolates from dairy ruminant species: a single-dye DNA microarray approach
Vet Microbiol 133, 105-114
Vautor E, Magnone V, Rios G, Le Brigand K, Bergonier D, Lina G, Meugnier H, Barbry P, Thiery R, Pepin M
Agence Française de Securite Sanitaire des Aliments (AFSSA), Unite Pathologie des Petits Ruminants, Sophia-Antipolis, France. e.vautor@sophia.afssa.fr

Staphylococcus aureus is recognized worldwide as a major pathogen causing clinical or subclinical intramammary infections in lactating sheep, goats and cows. The present study was carried out to compare 65 S. aureus isolates mainly obtained from nasal carriage and subclinical mastitis in dairy sheep and 43 isolates obtained from subclinical mastitis from 22 goats and 21 cows. A DNA microarray, containing probes against 190 true or putative virulence factors, was used to detect the presence of the virulence genes. Their presence/absence was independently assessed by PCR for the genes of interest. Sheep isolates obtained from the nostrils or the udders did not show any significant tissue specific virulence factor. The dominant pulse-field electrophoresis profile (OV/OV'), associated with spa clonal complex spa-CC 1773, matched mainly with the agr group III and was only found in ovine and caprine isolates. This clone was more specifically characterized by the prevalence of the following virulence genes: lpl4, ssl6, bsaA1, bsaB, bsaP, SAV0812. Moreover, seven virulence-associated genes (lpl1, sel, sec, tst, lukF-PV-like component, lukM, SAV0876) were associated with isolates from small ruminants, while the egc cluster, fhuD1, abiF and SAV2496 with bovine isolates. This genomic study suggests the existence of lineage- and host-specific genes leading to the development of host-specific pathogenic traits of S. aureus isolates.
Pubmed link : 18640795

59. Gene expression profiling of human liver transplants identifies an early transcriptional signature associated with initial poor graft function.
Am J Transplant. 2008 Jun;8(6):1221-36.
Defamie V, Cursio R, Le Brigand K, Moreilhon C, Saint-Paul MC, Laurens M, Crenesse D, Cardinaud B, Auberger P, Gugenheim J, Barbry P, Mari B
CNRS, Institut de Pharmacologie Moleculaire et Cellulaire, UMR6097, 660, Route des Lucioles F-06560 Sophia Antipolis, France.

Liver ischemia-reperfusion injury occurring in orthotopic liver transplantation (OLT) may be responsible for early graft failure. Molecular mechanisms underlying initial poor graft function (IPGF) have been poorly documented in human. The purpose of this study was to identify the major transcriptional alterations occurring in human livers during OLT. Twenty-one RNA extracts derived from liver transplant biopsies taken after graft reperfusion were compared with 7 RNA derived from normal control livers. Three hundred seventy-one genes were significantly modulated and classified in molecular pathways relevant to liver metabolism, inflammatory response, cell proliferation and liver protection. Grafts were then subdivided into two groups based on their peak levels of serum aspartate amino transferase within 72 h after OLT (group 1, non-IPGF: 14 patients; group 2, IPGF: 7 patients). The two corresponding data sets were compared using a supervised prediction method. A new set of genes able to correctly classify 71% of the patients was defined. These genes were functionally associated with oxidative stress, inflammation and inhibition of cell proliferation. This study provides a comprehensive picture of the transcriptional events associated with human OLT and IPGF. We anticipate that such alterations provide a framework for the elucidation of the molecular mechanisms leading to IPGF.
Pubmed link : 18522548

60. Accelerated arterial stiffening and gene expression profile of the aorta in patients with coronary artery disease.
J Hypertens. 2008 Apr;26(4):747-57
Fassot C, Briet M, Rostagno P, Barbry P, Perret C, Laude D, Boutouyrie P, Bozec E, Bruneval P, Latremouille C, Laurent S
Faculty of Medicine, University Paris-Descartes, Department of Pharmacology, Georges Pompidou European Hospital, Paris, France.

Hypertension and chronic renal failure (CRF) are considered models of accelerated arterial stiffening. Arterial stiffness increases further when CRF is associated with hypertension. We hypothesized that, in patients with mild CRF, aortic gene expression profile would include genes involved in arterial calcifications and enlargement. METHOD: We analysed human aorta with the 'GeneChip Microarray' technology, in patients with or without CRF, scheduled for a coronary artery bypass graft. RESULTS: Nine of 25 patients had high-quality RNA and were included in the study. Among the 101 transcripts differentially expressed between CRF patients and controls, 97 transcripts were overexpressed in CRF patients. Two genes had the highest overexpression in CRF patients: lumican (LUM), involved in the regulation of collagen fibrillogenesis; and ornithine decarboxylase (ODC1), involved in polyamine biosynthesis, smooth muscle cell growth and proliferation. Immunohistochemical staining revealed an increased amount of LUM and ODC1 in the vascular smooth muscle cells (VSMCs) of CRF compared to non-CRF aortic sections. Eight genes were implicated in the regulation of the cytoskeleton (including capping protein muscle Z-line 1 alpha and moesin) and cell migration, and five genes were implicated in extracellular matrix function and apoptosis. A trend towards an upregulation of candidate genes involved in arterial calcifications was observed in CRF patients, but did not reach statistical significance. Carotid-femoral pulse wave velocity was not correlated with gene expression level. CONCLUSION: In conclusion, these results show that patients at an early stage of CRF have a specific gene expression profile of aortic tissue and suggest that genes implicated in collagen fibrillogenesis, and VSMCs migration and proliferation, particularly LUM and ODC1, may play a role.
Pubmed link : 18327085

61. Matrix metalloproteinase inhibition protects rat livers from prolonged cold ischemia-warm reperfusion injury.
Hepatology. 2008 Jan;47(1):177-85.
Defamie V, Laurens M, Patrono D, Devel L, Brault A, Saint-Paul MC, Yiotakis A, Barbry P, Gugenheim J, Crenesse D, Dive V, Huet, PM, Mari B
Institut de Pharmacologie Moleculaire et Cellulaire, CNRS, UMR6097, Universite de Nice-Sophia Antipolis, France.

Matrix metalloproteinases (MMPs) have been implicated in the hepatic injury induced after cold ischemia-warm reperfusion (CI-WR), by altering the extracellular matrix (ECM), but their precise role remains unknown. The hepatic MMP expression was evaluated after two conditions of CI (4oC for 24 and 42 hours: viable and nonviable livers) followed by different periods of WR, using isolated perfused rat livers. CI-WR induced moderate changes in hepatic MMP transcript levels not influenced by CI duration, while gelatinase activities accumulated in liver effluents. Therefore, the protective effect of a new phosphinic MMP inhibitor, RXP409, was tested after prolonged CI. RXP409 (10µM) was added to the UW solution and livers were preserved for 42 hours (4oC), then reperfused for 1 hour in Krebs solution (37oC), containing 20% erythrocytes. Liver viability parameters were recorded and the extent of cell necrosis was evaluated on liver biopsies, using trypan blue nuclear uptake. Treatment with RXP409 significantly improved liver function (transaminase release and bile secretion) and liver injury. In particular, the MMP inhibitor significantly modified the extent of cell death from large clusters of necrotic hepatocytes as found in control livers (2 to 60% of liver biopsies, mean: 26 ± 9%) to isolated necrotic hepatocytes as found in treated livers (0.2 to 12%, mean 3 ± 2%) (p<0.05). In conclusion, these data demonstrate that MMPs, by altering the ECM, play a major role in liver CI-WR injury leading to extensive hepatocyte necrosis and that their inhibition might prove to be a new strategy in improving preservation solutions.
Pubmed link : 18008367

62. A comparative analysis of perturbations caused by a gene knockout, a dominant negative allele, and a set of peptide aptamers.
Mol Cell Proteomics. 2007 Sep 4;
Abed N, Bickle M, Mari B, Schapira M, Sanjuan-Espana R, Robbe-Sermesant K, Moncorge O, Mouradian-Garcia S, Barbry P, Rudkin BB, Fauvarque MO, Michaud-Soret I, Colas P
CNRS UPS 2682, Station Biologique, Roscoff cedex 29682.

The study of protein function mostly relies on perturbing regulatory networks by acting upon protein expression levels or using transdominant negative agents. Here, we used the E.coli global transcription regulator Fur (ferric uptake regulator) as a case study to compare the perturbations exerted by a gene knockout, the expression of a dominant negative allele of a gene and the expression of peptide aptamers that bind a gene product. These three perturbations caused phenotypes that differed quantitatively and qualitatively from one another. The Fur peptide aptamers inhibited the activity of their target to various extents and reduced the virulence of a pathogenic E.coli strain in Drosophila. A genome-wide transcriptome analysis revealed that the "penetrance" of a peptide aptamer was comparable to that of a dominant-negative allele but lower than the "penetrance" of the gene knockout. Our work shows that comparative analysis of phenotypic and transcriptome responses to different types of perturbation can help decipher complex regulatory networks that control various biological processes.
Pubmed link : 17785351

63. Gene expression profiling in human gastric mucosa infected with Helicobacter pylori.
Mod Pathol. 2007 Sep;20(9):974-89. Epub 2007 Jul 20.
Hofman VJ, Moreilhon C, Brest PD, Lassalle S, Le Brigand K, Sicard D, Raymond J, Lamarque D, Hebuterne XA, Mari B, Barbry PJ, Hofman PM
1Faculty of Medicine, INSERM ERI-21, Nice Cedex, France [2] 2Laboratory of Clinical and Experimental Pathology, Pasteur Hospital, Nice, France.

Pathogenic mechanisms associated with Helicobacter pylori infection enhance susceptibility of the gastric epithelium to carcinogenic conversion. We have characterized the gene expression profiles of gastric biopsies from 69 French Caucasian patients, of which 43 (62%) were infected with H. pylori. The bacterium was detected in 27 of the 42 antral biopsies examined and in 16 of the 27 fundic biopsies. Infected biopsies were selected for the presence of chronic active gastritis, in absence of metaplasia and dysplasia of the gastric mucosa. Infected antral and fundic biopsies exhibited distinct transcriptional responses. Altered responses were linked with: (1) the extent of polymorphonuclear leukocyte infiltration, (2) bacterial density, and (3) the presence of the virulence factors vacA, babA2, and cagA. Robust modulation of transcripts associated with Toll-like receptors, signal transduction, the immune response, apoptosis, and the cell cycle was consistent with expected responses to Gram-negative bacterial infection. Altered expression of interferon-regulated genes (IFITM1, IRF4, STAT6), indicative of major histocompatibility complex (MHC) II-mediated and Th1-specific responses, as well as altered expression of GATA6, have previously been described in precancerous states. Upregulation of genes abundantly expressed in cancer tissues (UBD, CXCL13, LY96, MAPK8, MMP7, RANKL, CCL18) or in stem cells (IFITM1 and WFDC2) may reveal a molecular switch towards a premalignant state in infected tissues. Tissue microarray analysis of a large number of biopsies, which were either positive or negative for the cag-A virulence factor, when compared to each other and to noninfected controls, confirmed observed gene alterations at the protein level, for eight key transcripts. This study provides 'proof-of-principle' data for identifying molecular mechanisms driving H. pylori-associated carcinogenesis before morphological evidence of changes along the neoplastic progression pathway.Modern Pathology (2007) 20, 974-989; doi:10.1038/modpathol.3800930; published online 20 July 2007.
Pubmed link : 17643099

64. Relationships Between Early Inflammatory Response to Bleomycin and Sensitivity to Lung Fibrosis.
Am J Respir Crit Care Med. 2007 Aug 2;
Pottier N, Chupin C, Defamie V, Cardinaud B, Sutherland R, Rios G, Gauthier F, Wolters PJ, Berthiaume Y, Barbry P, Mari B
Institut de Pharmacologie Moleculaire et Cellulaire, UMR6097, CNRS and University of Nice Sophia Antipolis, Sophia Antipolis, France.

RATIONALE. Different sensitivities to pro-fibrotic compounds such as bleomycin are observed among mouse strains. OBJECTIVES. To identify genetic factors contributing to the outcome of lung injury. METHODS. Physiological comparison of C57BL/6 sensitive and Balb/C resistant mice challenged with intra tracheal bleomycin instillation revealed several early differences: global gene expression profiles were thus established from lungs derived from the two strains, in the absence of any bleomycin administration. MEASUREMENTS AND MAIN RESULTS. Expression of 25 genes differed between the two strains. Among them, two molecules, not previously associated with pulmonary fibrosis, were identified. The first one corresponds to dipeptidyl peptidase I (DPPI), a cysteine dipeptidyl peptidase (also known as cathepsin C) essential for the activation of serine proteinases produced by immune/inflammatory cells. The second corresponds to TIMP-3, an inhibitor of matrix metalloproteases and of ADAMs such as the TNFconverting enzyme. In functional studies performed in the bleomycin induced lung fibrosis model, the level of expression of these two genes was closely correlated with specific early events associated with lung fibrosis, namely activation of PMN-derived serine proteases and TNFalpha-dependent inflammatory syndrome. Surprisingly, genetic deletion of DPPI in the context of a C57BL/6 genetic background did not protect against bleomycin-mediated fibrosis, suggesting additional function(s) for this key enzyme. CONCLUSIONS. This study highlights the importance of the early inflammatory events that follow bleomycin instillation in the development of lung fibrosis, and describes for the first time the roles that DPPI and TIMP-3 may play in this process.
Pubmed link : 17673693

65. GAPDH and autophagy preserve survival after apoptotic cytochrome c release in the absence of caspase activation.
Cell. 2007 Jun 1;129(5):861-3.
Colell A, Ricci JE, Tait S, Milasta S, Maurer U, Bouchier-Hayes L, Fitzgerald P, Guio-Carrion A, Waterhouse NJ, Li CW, Mari B, Barbry P, Newmeyer DD, Beere HM, Green DR
Department of Cell Death and Proliferation, Institut d'Investigacions Biomediques de Barcelona (IIBB-CSIC), IDIBAPS, 08036 Barcelona, Spain.

In cells undergoing apoptosis, mitochondrial outer-membrane permeabilization (MOMP) is followed by caspase activation promoted by released cytochrome c. Although caspases mediate the apoptotic phenotype, caspase inhibition is generally not sufficient for survival following MOMP; instead cells undergo a "caspase-independent cell death" (CICD). Thus, MOMP may represent a point of commitment to cell death. Here, we identify glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a critical regulator of CICD. GAPDH-expressing cells preserved their clonogenic potential following MOMP, provided that caspase activation was blocked. GAPDH-mediated protection of cells from CICD involved an elevation in glycolysis and a nuclear function that correlated with and was replaced by an increase in Atg12 expression. Consistent with this, protection from CICD reflected an increase in and a dependence upon autophagy, associated with a transient decrease in mitochondrial mass. Therefore, GAPDH mediates an elevation in glycolysis and enhanced autophagy that cooperate to protect cells from CICD.
Pubmed link : 17540177

66. Transcriptional signature of epidermal keratinocytes subjected to in vitro scratch wounding, reveals selective roles for ERK1/2, P38 and PI3K signalling pathways.
J Biol Chem. 2007 May 18;282(20):15090-102.
Fitsialos G, Chassot AA, Turchi L, Dayem MA, Lebrigand K, Moreilhon C, Meneguzzi G, Busca R, Mari B, Barbry P, Ponzio G
Faculte de Medecine, INSERM U634, Nice 06107.

Covering denuded dermal surfaces after injury requires migration, proliferation and differentiation of skin keratinocytes. To clarify the major traits controlling these intermingled biological events, we surveyed the genomic modifications occurring during the course of a scratch wound closure of cultured human keratinocytes. Using a DNA microarray approach, we report the identification of 161 new markers of epidermal repair. Expression data, combined with functional analysis performed with specific inhibitors of ERK, p38[MAPK] and PI3 kinases, demonstrate that kinase pathways exert very selective functions by precisely controlling the expression of specific genes. Inhibition of the ERK pathway totally blocks the wound closure and inactivates many early transcription factors and EGF-type growth factors. P38[MAPK] inhibition only delays "healing", probably in line with the control of genes involved in the propagation of injury-initiated signalling. In contrast, PI3 kinase inhibition accelerates the scratch closure and potentiates the scratch-dependent stimulation of three genes related to epithelial cell transformation, namely HAS3, HBEGF and Ets1. Our results define in vitro human keratinocyte wound closure as a repair process resulting from a fine balance between positive signals controlled by ERK and p38[MAPK], and negative ones triggered by PI3 kinase. The perturbation of any of these pathways might lead to dysfunction in the healing process, similar to those observed in pathological wounding phenotypes, such as hypertrophic scars or keloids.
Pubmed link : 17363378

67. Mediante: a Web-based Microarray Data manager
Bioinformatics, doi:10.1093/bioinformatics/btm106
Le Brigand K, Barbry P
CNRS, Institut de Pharmacologie Moleculaire et Cellulaire, UMR6097, 660, route des Lucioles, F-06560 Sophia Antipolis, France.

Summary : Mediante is a MIAME-compliant microarray data manager that links together annotations and experimental data. Developed as a J2EE three-tier application, Mediante integrates a management system for production of long oligonucleotide microarrays, an experimental data repository suitable for home made or commercial microarrays, and a user interface dedicated to the management of microarrays projects. Several tools allow quality control of hybridizations and submission of validated data to public repositories.
Availability : http://www.microarray.fr.
Supplementary data : http://www.microarray.fr/SP/lebrigand2007/
Pubmed link : 16855282

68. Suppression of microRNA-silencing pathway by HIV-1 during virus replication.
Science. 2007 Mar 16;315(5818):1579-82.
Triboulet R, Mari B, Lin YL, Chable-Bessia C, Bennasser Y, Lebrigand K, Cardinaud B, Maurin T, Barbry P, Baillat V, Reynes J, Corbeau P, Jeang KT, Benkirane M
Laboratoire de Virologie Moleculaire, Institut de Genetique Humaine, Montpellier, France.

MicroRNAs (miRNAs) are single-stranded noncoding RNAs of 19 to 25 nucleotides that function as gene regulators and as a host cell defense against both RNA and DNA viruses. We provide evidence for a physiological role of the miRNA-silencing machinery in controlling HIV-1 replication. Type III RNAses Dicer and Drosha, responsible for miRNA processing, inhibited virus replication both in peripheral blood mononuclear cells from HIV-1-infected donors and in latently infected cells. In turn, HIV-1 actively suppressed the expression of the polycistronic miRNA cluster miR-17/92. This suppression was found to be required for efficient viral replication and was dependent on the histone acetyltransferase Tat cofactor PCAF. Our results highlight the involvement of the miRNA-silencing pathway in HIV-1 replication and latency.
Pubmed link : 17322031

69. Molecular basis of Tropheryma whipplei doxycycline susceptibility examined by transcriptional profiling.
J Antimicrob Chemother. 2007 Mar;59(3):370-7. Epub 2007 Feb 8.
Van La M, Barbry P, Raoult D, Renesto P
Unite des Rickettsies, CNRS-UMR6020, IFR48, Faculte de Medecine, 27 Bd Jean Moulin, Marseille, F13385, France.

Objectives and methods Tropheryma whipplei is a poorly studied bacterium responsible for Whipple's disease. In this study, its susceptibility to doxycycline was investigated at a transcriptional level using a whole-genome DNA microarray. Results Exposure of T. whipplei to the MIC of doxycycline (0.5 mg/L) induced antibiotic-specific primary expression profiles, while indirect effects were detected at 10 x MIC. In contrast to what was observed for several microorganisms exposed to antibiotics, the heat-shock proteins were not affected. Consistent with the mode of action of this translation inhibitor, genes encoding for ribosomal proteins and translation factors were differentially transcribed. This analysis also evidenced the regulation of genes that should account for cell growth arrest. Long-term survival of non-replicating bacteria is likely to be ensured by an increased level of ppGpp, the nucleotide effector of the stringent response. The gene expression profile observed with 10 x MIC was mainly characterized by the up-regulation of ABC transporters that possibly form efflux and detoxification systems, through which T. whipplei may limit the effects of this bacteriostatic compound. Obtained microarray data showed good agreement with real-time quantitative PCR (R(2) = 0.969). Conclusions This work represents the first comprehensive genomic approach providing insights into the expression signature triggered by the exposure of T. whipplei to antibiotics.
Pubmed link : 17289769

70. Mycobacterial lipomannan induces granuloma macrophage fusion via a TLR2-dependent, ADAM9- and beta1 integrin-mediated pathway.
J Immunol. 2007 Mar 1;178(5):3161-9.
Puissegur MP, Lay G, Gilleron M, Botella L, Nigou J, Marrakchi H, Mari B, Duteyrat JL, Guerardel Y, Kremer L, Barbry P, Puzo G, Altare F
Laboratory of Molecular Physiology of Mycobacterial Granulomas, Department of Molecular Mechanisms of Mycobacterial Infections, Institut de Pharmacologie et Biologie Structurale, CNRS/UMR 5089, 205 Route de Narbonne, Toulouse, France.

Tuberculous granulomas are the sites of interaction between the host response and the tubercle bacilli within infected individuals. They mainly consist of organized aggregations of lymphocytes and macrophages (Mf). A predominant role of mycobacterial envelope glycolipids in granulomas formation has been recently emphasized, yet the signaling events interfering with granuloma cell differentiation remain elusive. To decipher this molecular machinery, we have recently developed an in vitro human model of mycobacterial granulomas. In this study, we provide evidence that the mycobacterial proinflammatory phosphatidyl-myo-inositol mannosides and lipomannans (LM), as well as the anti-inflammatory lipoarabinomannan induce granuloma formation, yet only the proinflammatory glycolipids induce the fusion of granuloma Mf into multinucleated giant cells (MGC). We also demonstrate that LM induces large MGC resembling those found in vivo within the granulomas of tuberculosis patients, and that this process is mediated by TLR2 and is dependent on the beta(1) integrin/ADAM9 cell fusion machinery. Our results demonstrate for the first time that the Mf differentiation stage specifically occurring within granulomatous structures (i.e., MGC formation) is triggered by mycobacterial envelope glycolipids, which are capable of inducing the cell fusion machinery. This provides the first characterization of the ontogeny of human granuloma MGC, thus resulting in a direct modulation by a particular mycobacterial envelope glycolipid of the differentiation process of granuloma Mf.
Pubmed link : 17312164

71. Omega 3 polyunsaturated fatty acids improve host response in chronic Pseudomonas aeruginosa lung infection in mice.
Am J Physiol Lung Cell Mol Physiol. 2007 Feb 23
Pierre M, Husson MO, Leberre R, Desseyn JL, Galabert C, Beghin L, Beermann C, Dagenais A, Berthiaume Y, Cardinaud B, Barbry P, Gottrand F, Guery BP
EA 2689, IFR 14, Universite de Lille 2, Lille, France; EA 3925, IFR 14, Clinique de Pediatrie, Universite de Lille 2, Lille, France.

Pseudomonas aeruginosa is a Gram negative bacilli frequently encountered in human pathology. This pathogen is involved in a large number of nosocomial infections and chronic diseases. Herein we investigated the effects of polyunsaturated fatty acids (PUFA) in chronic Pseudomonas aeruginosa lung infection. C57BL/6 mice were fed for 5 weeks with specifically designed diets with high contents in either omega3, or omega6 PUFA and compared to a control diet. P. aeruginosa included in agarose beads was then instilled intratracheally and the animals studied for 7 days. On the 4(th) day, the mice fed with the omega3 diet had a higher lean body mass gain and a lower omega6/omega3 ratio of fatty acids extracted from the lung tissue compared to the other groups (p<0.05). The omega3 group had the lowest mortality. Distal alveolar fluid clearance (DAFC) as well as the inflammatory response and the cellular recruitment were higher in the omega3 group on the 4(th) day. The effect on DAFC was independent of alpha, betaENaC, and alphaNa, K-ATPase mRNA expressions, which were not altered by the different diets. In conclusion, a diet enriched in omega3 PUFA can change lung membrane composition and improve survival in chronic pneumonia. This effect on survival is probably multifactorial involving the increased DAFC capacity as well as the optimization of the initial inflammatory response. This work suggests that a better control of the omega6/omega3 PUFA balance may represent an interesting target in the prevention and/or control of P aeruginosa infection in patients. Key words: polyunsaturated fatty acids, pneumonia, pseudomonas, distal alveolar fluid clearance.
Pubmed link : 17322280

72. Easy and fast detection and genotyping of high-risk human papillomavirus by dedicated DNA microarrays.
J Virol Methods. 2006 Nov;137(2):236-44. Epub 2006 Aug 1.
Albrecht V, Chevallier A, Magnone V, Barbry P, Vandenbos F, Bongain A, Lefebvre JC, Giordanengo V
Laboratoire de Virologie, Hopital de l'Archet, BP 3079, 151 route de Saint-Antoine de Ginestiere, 06200 Nice Cedex 3, France.

Persistent cervical high-risk human papillomavirus (HPV) infection is correlated with an increased risk of developing a high-grade cervical intraepithelial lesion. A two-step method was developed for detection and genotyping of high-risk HPV. DNA was firstly amplified by asymmetrical PCR in the presence of Cy3-labelled primers and dUTP. Labelled DNA was then genotyped using DNA microarray hybridization. The current study evaluated the technical efficacy of laboratory-designed HPV DNA microarrays for high-risk HPV genotyping on 57 malignant and non-malignant cervical smears. The approach was evaluated for a broad range of cytological samples: high-grade squamous intraepithelial lesions (HSIL), low-grade squamous intraepithelial lesions (LSIL) and atypical squamous cells of high-grade (ASC-H). High-risk HPV was also detected in six atypical squamous cells of undetermined significance (ASC-US) samples; among them only one cervical specimen was found uninfected, associated with no histological lesion. The HPV oligonucleotide DNA microarray genotyping detected 36 infections with a single high-risk HPV type and 5 multiple infections with several high-risk types. Taken together, these results demonstrate the sensitivity and specificity of the HPV DNA microarray approach. This approach could improve clinical management of patients with cervical cytological abnormalities.
Pubmed link : 16879879

73. Global transcriptome analysis of Tropheryma whipplei in response to temperature stresses.
J Bacteriol. 2006 Jul;188(14):5228-39.
Crapoulet N, Barbry P, Raoult D, Renesto P
Unite des Rickettsies, CNRS UMR6020, IFR48, Faculte de Medecine, 27, Boulevard Jean Moulin, 13385 Marseille, France.

Tropheryma whipplei, the agent responsible for Whipple disease, is a poorly known pathogen suspected to have an environmental origin. The availability of the sequence of the 0.92-Mb genome of this organism made a global gene expression analysis in response to thermal stresses feasible, which resulted in unique transcription profiles. A few genes were differentially transcribed after 15 min of exposure at 43 degrees C. The effects observed included up-regulation of the dnaK regulon, which is composed of six genes and is likely to be under control of two HspR-associated inverted repeats (HAIR motifs) found in the 5' region. Putative virulence factors, like the RibC and IspDF proteins, were also overexpressed. While it was not affected much by heat shock, the T. whipplei transcriptome was strongly modified following cold shock at 4 degrees C. For the 149 genes that were differentially transcribed, eight regulons were identified, and one of them was composed of five genes exhibiting similarity with genes encoding ABC transporters. Up-regulation of these genes suggested that there was an increase in nutrient uptake when the bacterium was exposed to cold stress. As observed for other bacterial species, the major classes of differentially transcribed genes encode membrane proteins and enzymes involved in fatty acid biosynthesis, indicating that membrane modifications are critical. Paradoxically, the heat shock proteins GroEL2 and ClpP1 were up-regulated. Altogether, the data show that despite the lack of classical regulation pathways, T. whipplei exhibits an adaptive response to thermal stresses which is consistent with its specific environmental origin and could allow survival under cold conditions.
Pubmed link : 16816195

74. An open-access long oligonucleotide microarray resource for analysis of the human and mouse transcriptomes.
Nucleic Acids Res. 2006 Jul 19;34(12):e87
Le Brigand K, Russell R, Moreilhon C, Rouillard JM, Jost B, Amiot F, Magnone V, Bole-Feysot C, Rostagno P, Virolle V, Defamie V, Dessen P, Williams G, Lyons P, Rios G, Mari B, Gulari E, Kastner P, Gidrol X, Freeman TC, Barbry P
CNRS, Institut de Pharmacologie Moleculaire et Cellulaire, UMR6097, 660, route des Lucioles, F-06560 Sophia Antipolis, France.

Two collections of oligonucleotides have been designed for preparing pangenomic human and mouse microarrays. A total of 148,993 and 121,703 oligonucleotides were designed against human and mouse transcripts. Quality scores were created in order to select 25,342 human and 24,109 mouse oligonucleotides. They correspond to: (i) a BLAST-specificity score; (ii) the number of expressed sequence tags matching each probe; (iii) the distance to the 3' end of the target mRNA. Scores were also used to compare in silico the two microarrays with commercial microarrays. The sets described here, called RNG/MRC collections, appear at least as specific and sensitive as those from the commercial platforms. The RNG/MRC collections have now been used by an Anglo-French consortium to distribute more than 3500 microarrays to the academic community. Ad hoc identification of tissue-specific transcripts and a approximately 80% correlation with hybridizations performed on Affymetrix GeneChiptrade mark suggest that the RNG/MRC microarrays perform well. This work provides a comprehensive open resource for investigators working on human and mouse transcriptomes, as well as a generic method to generate new microarray collections in other organisms. All information related to these probes, as well as additional information about commercial microarrays have been stored in a freely-accessible database called MEDIANTE.
Pubmed link : 17384016

75. Delta-subunit confers novel biophysical features to alpha beta gamma-human epithelial sodium channel (ENaC) via a physical interaction.
J Biol Chem. 2006 Mar 24;281(12):8233-41. Epub 2006 Jan 19.
Ji HL, Su XF, Kedar S, Li J, Barbry P, Smith PR, Matalon S, Benos DJ
Department of Anesthesiology, University of Alabama at Birmingham, Alabama 35205, USA.

Native amiloride-sensitive Na+ channels exhibit a variety of biophysical properties, including variable sensitivities to amiloride, different ion selectivities, and diverse unitary conductances. The molecular basis of these differences has not been elucidated. We tested the hypothesis that co-expression of delta-epithelial sodium channel (ENaC) underlies, at least in part, the multiplicity of amiloride-sensitive Na+ conductances in epithelial cells. For example, the delta-subunit may form multimeric channels with alpha beta gamma-ENaC. Reverse transcription-PCR revealed that delta-ENaC is co-expressed with alpha beta gamma-subunits in cultured human lung (H441 and A549), pancreatic (CFPAC), and colonic epithelial cells (Caco-2). Indirect immunofluorescence microscopy revealed that delta-ENaC is co-expressed with alpha-, beta-, and gamma-ENaC in H441 cells at the protein level. Measurement of current-voltage that cation selectivity ratios for the revealed relationships Na+/Li+/K+/Cs+/Ca2+/Mg2+, the apparent dissociation constant (Ki) for amiloride, and unitary conductances for delta alpha beta gamma-ENaC differed from those of both alpha beta gamma- and delta beta gamma-ENaC (n = 6). The contribution of the delta subunit to P(Li)/P(Na) ratio and unitary Na+ conductance under bi-ionic conditions depended on the injected cRNA concentration. In addition, the EC50 for proton activation, mean open and closed times, and the self-inhibition time of delta alpha beta gamma-ENaC differed from those of alpha beta gamma- and delta beta gamma-ENaC. Co-immunoprecipitation of delta-ENaC with alpha- and gamma-subunits in H441 and transfected COS-7 cells suggests an interaction among these proteins. We, therefore, concluded that the interactions of delta-ENaC with other subunits could account for heterogeneity of native epithelial channels.
Pubmed link : 16423824

76. Hypoxia-inducible factor 1{alpha} is a new target of microphthalmia-associated transcription factor (MITF) in melanoma cells.
J Cell Biol. 2005 Jul 4;170(1):49-59. Epub 2005 Jun 27.
Busca R, Berra E, Gaggioli C, Khaled M, Bille K, Marchetti B, Thyss R, Fitsialos G, Larribere L, Bertolotto C, Virolle T, Barbry P, Pouyssegur J, Ponzio G, Ballotti R
INSERM U597, Biologie et physiopathologie des cellules melanocytaires, Faculty of Medicine, 06107 Nice cedex 2, France.

In melanocytes and melanoma cells alpha-melanocyte stimulating hormone (alpha-MSH), via the cAMP pathway, elicits a large array of biological responses that control melanocyte differentiation and influence melanoma development or susceptibility. In this work, we show that cAMP transcriptionally activates Hif1a gene in a melanocyte cell-specific manner and increases the expression of a functional hypoxia-inducible factor 1alpha (HIF1alpha) protein resulting in a stimulation of Vegf expression. Interestingly, we report that the melanocyte-specific transcription factor, microphthalmia-associated transcription factor (MITF), binds to the Hif1a promoter and strongly stimulates its transcriptional activity. Further, MITF "silencing" abrogates the cAMP effect on Hif1a expression, and overexpression of MITF in human melanoma cells is sufficient to stimulate HIF1A mRNA. Our data demonstrate that Hif1a is a new MITF target gene and that MITF mediates the cAMP stimulation of Hif1a in melanocytes and melanoma cells. Importantly, we provide results demonstrating that HIF1 plays a pro-survival role in this cell system. We therefore conclude that the alpha-MSH/cAMP pathway, using MITF as a signal transducer and HIF1alpha as a target, might contribute to melanoma progression.
Pubmed link : 15983061

77. CFTR fails to inhibit the epithelial sodium channel ENaC expressed in Xenopus laevis oocytes.
J Physiol. 2005 May 1;564(Pt 3):671-82. Epub 2005 Mar 3.
Nagel G, Barbry P, Chabot H, Brochiero E, Hartung K, Grygorczyk R
Max-Planck-Institute of Biophysics, Frankfurt am Main, Germany.

The cystic fibrosis transmembrane conductance regulator (CFTR) plays a crucial role in regulating fluid secretion by the airways, intestines, sweat glands and other epithelial tissues. It is well established that the CFTR is a cAMP-activated, nucleotide-dependent anion channel, but additional functions are often attributed to it, including regulation of the epithelial sodium channel (ENaC). The absence of CFTR-dependent ENaC inhibition and the resulting sodium hyperabsorption were postulated to be a major electrolyte transport abnormality in cystic fibrosis (CF)-affected epithelia. Several ex vivo studies, including those that used the Xenopus oocyte expression system, have reported ENaC inhibition by activated CFTR, but contradictory results have also been obtained. Because CFTR-ENaC interactions have important implications in the pathogenesis of CF, the present investigation was undertaken by our three independent laboratories to resolve whether CFTR regulates ENaC in oocytes and to clarify potential sources of previously reported dissimilar observations. Using different experimental protocols and a wide range of channel expression levels, we found no evidence that activated CFTR regulates ENaC when oocyte membrane potential was carefully clamped. We determined that an apparent CFTR-dependent ENaC inhibition could be observed when resistance in series with the oocyte membrane was not low enough or the feedback voltage gain was not high enough. We suggest that the inhibitory effect of CFTR on ENaC reported in some earlier oocyte studies could be attributed to problems arising from high levels of channel expression and suboptimal recording conditions, that is, large series resistance and/or insufficient feedback voltage gain.
Pubmed link : 15746174

78. Live Staphylococcus aureus and bacterial soluble factors induce different transcriptional responses in human airway cells.
Physiol Genomics. 2005 Feb 10;20(3):244-55. Epub 2004 Dec 14.Click here to read
Moreilhon C, Gras D, Hologne C, Bajolet O, Cottrez F, Magnone V, Merten M, Groux H, Puchelle E, Barbry P
Institut de Pharmacologie Moleculaire et Cellulaire UMR 6097 Centre National de la Recherche Scientifique, Universite de Nice-Sophia Antipolis, Valbonne, France.

To characterize the response of respiratory epithelium to infection by Staphylococcus aureus (S. aureus), human airway cells were incubated for 1 to 24 h with a supernatant of a S. aureus culture (bacterial supernatant), then profiled with a pangenomic DNA microarray. Because an upregulation of many genes was noticed around 3 h, three independent approaches were then used to characterize the host response to a 3-h contact either with bacterial supernatant or with live bacteria: 1) a DNA microarray containing 4,200 sequence-verified probes, 2) a semiquantitative RT-PCR with a set of 537 pairs of validated primers, or 3) ELISA assay of IL-8, IL-6, TNFalpha, and PGE(2). Among others, Fos, Jun, and EGR-1 were upregulated by the bacterial supernatant and by live bacteria. Increased expression of bhlhb2 and Mig-6, promoter regions which harbor HIF responding elements, was explained by an increased expression of the HIF-1alpha protein. Activation of the inducible form of cyclooxygenase, COX-2, and of the interleukins IL-1, IL-6, and IL-8, as well as of the NF-kappaB pathway, was observed preferentially in cells in contact with bacterial supernatant. Early infection was characterized by an upregulation of anti-apoptotic genes and a downregulation of pro-apoptotic genes. This correlated with a necrotic, rather than apoptotic cell death. Overall, this first global description of an airway epithelial infection by S. aureus demonstrates a larger global response to bacterial supernatant (in term of altered genes and variation factors) than to exponentially growing live bacteria.
Pubmed link : 15598879

79. Coordinated regulation of genes for secretion in tobacco at late developmental stages: association with resistance against oomycetes.
Plant Physiol. 2004 Feb;134(2):858-70. Epub 2004 Feb 5.
Hugot K, Riviere MP, Moreilhon C, Dayem MA, Cozzitorto J, Arbiol G, Barbry P, Weiss C, Galiana E
Institut National de la Recherche Agronomique, Unite Interactions Plantes-Microorganismes, Villa Thuret, Boite postale 2078, F-06606 Antibes cedex, France.

Besides the systemic acquired resistance (SAR) induced in response to microbial stimulation, host plants may also acquire resistance to pathogens in response to endogenous stimuli associated with their own development. In tobacco (Nicotiana tabacum), the vegetative-to-flowering transition comes along with a susceptibility-to-resistance transition to the causal agent of black shank disease, the oomycete Phytophthora parasitica. This resistance affects infection effectiveness and hyphal expansion and is associated with extracellular accumulation of a cytotoxic activity that provokes in vitro cell death of P. parasitica zoospores. As a strategy to determine the extracellular events important for restriction of pathogen growth, we screened the tobacco genome for genes encoding secreted or membrane-bound proteins expressed in leaves of flowering plants. Using a signal sequence trap approach in yeast (Saccharomyces cerevisiae), 298 clones were selected that appear to encode for apoplastic, cell wall, or membrane-bound proteins involved in stress response, in plant defense, or in cell wall modifications. Microarray and northern-blot analyses revealed that, at late developmental stages, leaves were characterized by the coordinate up-regulation of genes involved in SAR and in peroxidative cross-linking of structural proteins to cell wall. This suggests the potential involvement of these genes in extracellular events that govern the expression of developmental resistance. The analysis of the influence of salicylic acid on mRNA accumulation also indicates a more complex network for regulation of gene expression at a later stage of tobacco development than during SAR. Further characterization of these genes will permit the formulation of hypotheses to explain resistance and to establish the connection with development.
Pubmed link : 14764907

80. Gene expression profiling of normal human pulmonary fibroblasts following coculture with non-small-cell lung cancer cells reveals alterations related to matrix degradation, angiogenesis, cell growth and survival.
Oncogene. 2003 Nov 20;22(52):8487-97.
Fromigue O, Louis K, Dayem M, Milanini J, Pages G, Tartare-Deckert S, Ponzio G, Hofman P, Barbry P, Auberger P, Mari B
INSERM U526, IFR50, Faculte de Medecine Pasteur, Nice, France.

Increasing evidence supports a major role for the microenvironment in carcinoma formation and progression. The influence of the stroma is partly mediated by signalling between epithelial tumor cells and neighboring fibroblasts. However, the molecular mechanisms underlying these interactions are largely unknown. To mimic the initial steps of invasive carcinoma in which tumor cells come in contact with normal stromal cells, we used a coculture model of non-small-cell lung cancer tumor cells and normal pulmonary fibroblasts. Using DNA filter arrays, we first analysed the overall modification of gene expression profile after a 24 h period of coculture. Next, we focused our interest on the transcriptome of the purified fibroblastic fraction of coculture using both DNA filter arrays and a laboratory-made DNA microarray. These experiments allowed the identification of a set of modulated genes coding for growth and survival factors, angiogenic factors, proteases and protease inhibitors, transmembrane receptors, kinases and transcription regulators that can potentially affect the regulation of matrix degradation, angiogenesis, invasion, cell growth and survival. This study represents to our knowledge the first attempt to dissect early global gene transcription occurring in a tumor-stroma coculture model and should help to understand better some of the molecular mechanisms involved in heterotypic signalling between epithelial tumor cells and fibroblasts.
Pubmed link : 14627989

81. Alveolar liquid clearance and sodium channel expression are decreased in transplanted canine lungs.
Am J Respir Crit Care Med. 2003 May 15;167(10):1440-50. Epub 2003 Jan 24.
Sugita M, Ferraro P, Dagenais A, Clermont ME, Barbry P, Michel RP, Berthiaume Y
Centre de Recherche, Hotel-Dieu, Montreal, Quebec H2W 1T7, Canada.

To determine the impact of transplantation-associated injury on the clearance mechanisms of pulmonary edema, we created a canine single lung transplant model. After 3 hours of preservation and 4 hours of reperfusion, right native lungs and left transplanted lungs were used to measure alveolar liquid clearance (ALC) in ex vivo liquid-filled lung preparations. We also examined the role of the pulmonary circulation in edema clearance in in vivo liquid-filled lungs between 4 and 8 hours of reperfusion. To study molecular modifications in ALC, we also measured expression levels of the epithelial sodium channel (ENaC) and sodium-potassium-adenosine triphosphatase (ATPase). We found that ALC was significantly lower in transplanted than in right native lungs ex vivo (p < 0.05) and that transplanted lungs did not respond to the beta-adrenergic agonist terbutaline. Our in vivo study confirmed the ex vivo results. Molecular analyses revealed that ENaC messenger RNA but not sodium-potassium-ATPase was significantly decreased in transplanted lungs (p < 0.01). Furthermore, there was a significant decrease in ENaC protein expression. Therefore, we conclude that the current investigation indicates that the lung injury caused by lung preservation and transplantation significantly reduces the edema clearance ability of transplanted lungs.
Pubmed link : 12738601

82. Early gene expression in wounded human keratinocytes revealed by DNA microarray analysis.
Comp Funct Genomics. 2003;4(1):47-55.
Dayem MA, Moreilhon C, Turchi L, Magnone V, Christen R, Ponzio G, Barbry P
Laboratoire de Physiologie Genomique des Eucaryotes CNRS/UNSA UMR 6097 IPMC F-06560 Sophia Antipolis France.

WOUND HEALING INVOLVES SEVERAL STEPS: spreading of the cells, migration and proliferation. We have profiled gene expression during the early events of wound healing in normal human keratinocytes with a home-made DNA microarray containing about 1000 relevant human probes. An original wounding machine was used, that allows the wounding of up to 40% of the surface of a confluent monolayer of cultured cells grown on a Petri dish (compared with 5% with a classical 'scratch' method). The two aims of the present study were: (a) to validate a limited number of genes by comparing the expression levels obtained with this technique with those found in the literature; (b) to combine the use of the wounding machine with DNA microarray analysis for large-scale detection of the molecular events triggered during the early stages of the wound-healing process. The time-courses of RNA expression observed at 0.5, 1.5, 3, 6 and 15 h after wounding for genes such as c-Fos, c-Jun, Egr1, the plasminogen activator PLAU (uPA) and the signal transducer and transcription activator STAT3, were consistent with previously published data. This suggests that our methodologies are able to perform quantitative measurement of gene expression. Transcripts encoding two zinc finger proteins, ZFP36 and ZNF161, and the tumour necrosis factor alpha-induced protein TNFAIP3, were also overexpressed after wounding. The role of the p38 mitogen-activated protein kinase (p38MAPK) in wound healing was shown after the inhibition of p38 by SB203580, but our results also suggest the existence of surrogate activating pathways.
Pubmed link : 18629100