Brice Marcet

Identification of Keratinocyte Growth Factor as a Target of microRNA-155 in Lung Fibroblasts: Implication in Epithelial-Mesenchymal Interactions

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-α, IL-1β and TGF-β. Methodology/Principal Findings MiR-155 was significantly induced by inflammatory cytokines TNF-α and IL-1β while it was down-regulated by TGF-β. 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.

miR-199a-5p Is upregulated during fibrogenic response to tissue injury and mediates TGFbeta-induced lung fibroblast activation by targeting caveolin-1.

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.

Distinct epithelial gene expression phenotypes in childhood respiratory allergy.

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)-&agr;, IFN-&bgr; or IFN-&ggr;, 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.

MicroRNA-based silencing of Delta/Notch signaling promotes multiple cilia formation

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.

miR-34b/miR-34c: a regulator of TCL1 expression in 11q− chronic lymphocytic leukaemia?

TCL1 (T-cell leukaemia/lymphoma 1) is a 114-amino-acid protein normally expressed in CD3 CD4 CD8 thymic precursors, some dendritic cells and developing B cells. In B cells, TCL1 exerts indirect transcriptional effects through (i) an association with c-Jun, JunB and c-Fos, which results in the inhibition of AP-1-dependent transcription and (ii) by interacting with the nuclear factor-kB coactivator p300/CREB-binding protein. This leads to an activation of the nuclear factorkB-dependent transcriptional programme, and to the inhibition of cell death. TCL1 is overexpressed in most cases of chronic lymphocytic leukaemias (CLLs). Several studies have delineated the links existing between overexpression of TCL1 in CLL and some specific chromosomal abnormalities. Expression of TCL1 in CLL samples varied from complete absence to strong expression, and was dependent of the immunophenotypic and molecular CLL subgroups. Cases that strongly expressed TCL1 were more frequently found in cohorts having either (i) higher expression levels of ZAP70, (ii) unmutated IgVH genes and/or (iii) loss or monosomy of the 11q22/ATM locus. A correlation between the high expression of TCL1 and 11q deletion was also described by Pekarsky et al., who showed that a high expression of TCL1 was frequently observed, whereas a low expression was very rare in aggressive CLLs with 11q deletions (11q ) (24 out of 32 and 1 case out of 32, respectively). Taken together, these results suggest that the overexpression of TCL1 in CLL can be linked with a deletion of 11q. The mechanisms underlying TCL1 overexpression in B-cell pathologies, especially in 11q CLL, are poorly understood. MicroRNAs (miRNAs) are key regulators of protein expression in many situations. These highly conserved 21–23-mer RNAs regulate the expression of genes by binding to the 30-untranslated regions (30-UTR) of specific mRNAs and block translation of the target proteins and/or shorten half-lives of the transcripts. Two miRNAs (that is, miR-29 and miR-181) are downregulated in aggressive 11q CLL and can regulate TCL1 protein expression. By itself, this finding is not sufficient to explain why 11q CLLs overexpress TCL1, as miR-29 and miR-181 genes are located on other chromosomal regions. We hypothesized that negative regulator(s) of TCL1 expression, located in the 11q chromosomal region, might be deleted in aggressive 11q CLL, and that these negative regulators could correspond to miRNAs. Single-nucleotide polymorphism-chip analysis allows singlenucleotide polymorphism genotyping as well as detection of copy number changes and acquired uniparental disomies. We analysed 13 CLL samples carrying a deletion at 11q22 previously detected by fluorescence in situ hybridization with an ATM probe. Single-nucleotide polymorphism-chip analysis using Affymetrix GeneChip Human Mapping 250K Nsp singlenucleotide polymorphism arrays detected 11q hemizygous deletions in all of these ATM /þ samples (copy number1⁄4 1). Figure 1 delineates precisely the extent of the deletion in the 13 samples. We found that the genomic region encoding the miR-34b and miR-34c miRNAs was deleted in 12 of the 13 cases. This miR-34b/c genomic region was also deleted in all of the six previous 11q CLLs that we had previously described. Interestingly, one sample described by Lehmann et al. had two small deletions in 11q22 and 11q23.1. The first deletion (11q22) included the ATM gene. The second deletion (11q23.1) contained six genes including BTG4, which is indeed antisense to the gene coding for the miRNAs miR-34b and miR-34c. None of the other miRNAs located in the same region (that is, miR1261, miR-1304, miR-548l, miR-100 and miR-125b-1) was deleted more than five times. The miRNAs of the miR-34 family (namely miR-34a, miR-34b and miR-34c) are important components of the p53 network. Their expression is induced by p53 and they mediate some of the pro-apoptotic effects of this key cellular factor. It is worth noting that an ambiguity in nomenclature exists for the miR-34s in miRBase, the reference database of all known miRNAs (http://microrna.sanger.ac.uk/). MiRBase (version 13) describes for the human miR-34a and miR-34c two homologous sequences, derived from the 50-arm (called the ‘5p’ arm) of their respective pre-miRNAs. On the other hand, the sequence for miR-34b stored in miRBase corresponds to the 30 (‘3p’) arm of pre-miR-34b. This sequence is indeed very different from miR-34a and miR-34c. The sequence called miR-34bn in miRBase derives from the 5p arm of the pre-miR-34b. We observed in human cell lines that miR-34bn is expressed (unpublished observations), which means that miR-34bn is a mature miRNA produced from the pre-miR-34b. In Rattus norvegicus and in Mus musculus, the mature miRNAs derived from the 5p arm of the precursors are called miR-34b and miR34b-5p, respectively. For the sake of clarity, we use in the rest of the manuscript the nomenclature: miR-34b-5p. By looking at sequences showing exact matches with nucleotides (nts) 2–7 of the three members of the miR-34 family (as implemented by the MicroCible function available at http://www.microarray.fr/microRNA), one can predict that TCL1A is targeted by miR-34b-5p at four different positions but not by miR-34a nor by miR-34c. Three of these are contained within a 28-nt sequence repeated three times in the 30-UTR (Figure 2b). A fragment of TCL1A 30-UTR containing the 28-nt repeats (nts 437–589 from the 30-UTR of transcript NM_021966) was inserted into a psiCheck2 plasmid (Promega, Charbonnières-les-Bains, France) downstream to a luciferase coding sequence. HEK293T cells were co-transfected with this plasmid and with each of the three miR-34s or with a negative control. MiR-34b-5p, but not miR-34a or miR-34c, was able to downregulate the luciferase activity, suggesting that TCL1A is indeed targeted by this miRNA (N1⁄4 3 independent experiments, Figure 2d). Having almost identical sequences at their 50 extremities, and being identically induced by p53, one could argue that miR-34a and miR-34b/c are functionally redundant, and thus that the deletion of the miR-34b/c gene in 11q CLL could be compensated by the expression of miR-34a. The inhibitory role that miR-34b-5p specifically exerts on TCL1 expression is an argument against this hypothesis. To reinforce the concept that some of the targets of miR-34b-5p are not modulated by miR34a/c, we noticed that miR-34b-5p differs from mir-34a and Letters to the Editor

BMP signalling controls the construction of vertebrate mucociliary epithelia

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. Summary: The BMP pathway controls both morphognenesis of and cell type specification in mucociliary epithelia: the Xenopus embryonic epidermis and regenerating human airways.

miR-34/449 control apical actin network formation during multiciliogenesis through small GTPase pathways

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.

Impact of MicroRNA in Normal and Pathological Respiratory Epithelia

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.

MicroRNAs as key regulators of GTPase-mediated apical actin reorganization in multiciliated epithelia.

ABSTRACT 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.

MicroRNAs pull the strings of motile cilia

> Le cil, organite cellulaire conserve chez de nombreux eucaryotes, est un prolongement membranaire constitue de microtubules et dote de fonctions sensorielles et/ou locomotrices particulieres [1]. On distingue le cil primaire, non-motile et present dans la plupart des types cellulaires, du cil motile qui est present en un unique exemplaire dans des cellules « monociliees », et jusqu’a plusieurs centaines dans des cellules dites « multiciliees » (➜). Chez les vertebres, ces cellules multiciliees bordent la surface de certains tissus comme les voies respiratoires, les ventricules cerebraux, le tractus genital feminin, les placodes1 olfactives du poisson zebre ou encore l’epiderme d’embryon de grenouille [2, 3]. Grâce au battement coordonne des cils motiles, les cellules multiciliees jouent un role important dans divers processus physiologiques comme l’evacuation, par l’epithelium respiratoire, des particules inhalees piegees dans le mucus, la circulation du liquide cephalo-rachidien ou la progression de l’embryon dans le tractus genital [2]. Un dysfonctionnement ou une diminution du nombre de ces cils motiles peut etre la cause ou aggraver les symptomes de nombreuses pathologies telles que des ciliopathies (comme les dyskinesies ciliaires touchant le systeme respiratoire) ou des maladies respiratoires chroniques (comme la mucoviscidose,