Proteolytic Processing of the Laminin α3 G Domain Mediates Assembly of Hemidesmosomes but Has No Role on Keratinocyte Migration

Laminin-5 (Lm5), the major adhesion ligand of basal epithelial cells, undergoes complex extracellular proteolytic processing that influences cell adhesion and migration. In tumor cell lines, the proteolytic truncation of the C-terminal G domain of the Lm α3 chain induces assembly of hemidesmosomes and downregulates cell migration. To define the biological functions of the α3 G domain processing in physiological conditions, we have expressed a series of mutant α3 complementary DNA in human primary α3-null keratinocytes immortalized by human papillomavirus E6E7 (HKα3 cells). Using monolayer and organotypic cell cultures we show that: (1) the hinge region between subdomains G3 and G4 carries the proteolytic cleavage sites; (2) nucleation of the hemidesmosomal proteins is independent of the proteolytic maturation of the α3 G domain, whereas formation of mature hemidesmosomes relies on proteolytic cleavage of α3; and (3) the proteolytic processing plays no role in cell migration, which suggests that nucleation of hemidesmosomal structures in culture does not reflect the migratory potential of the epithelial cells. Our results also demonstrate that HKα3 cells are a unique model system, which will be useful to dissect the functions and molecular interactions of Lm5

Junctional Epidermolysis Bullosa in the German shorthaired pointer: a spontaneous model for Junctional Epidermolysis Bullosa in man

A Junctional Epidermolysis Bullosa (JEB), described in the German shorthaired pointer, is a remarkable spontaneous canine model for Junctional Epidermolysis Bullosa in man. In the German shorthaired pointer, JEB is caused by a homozygous substitution of the 1514C-T nucleotide. This variation in sequence results in a non-conservative change of 14 amino acids (505T –I) in domain I of laminin-5 α3. This non-conservative substitution modifies the hydrophobicity profile of the peptidic fragment, and therefore alters the stability of the binding between the α3 chain and the β3γ2 heterotrimer, which could result in a breakdown of the muted polypeptide. A fraction of muted α3 could be incorporated in laminin-5, modifying its function and hindering the extracellular cleavage of the γ2 chain. Genetic complementation studies were carried out. An MMLV retrovirus expressing the whole cDNA, muted and wild, of the α3 chain was developed to be transducted in the keratinocytes of JEB dogs (KJEB). This phenotypic transversion was successful and brought back a3 chain secretion, and thus the production of functional laminin-5 molecules. In addition, the adhesion capabilities of these transducted KJEB in cultures also returned. Finally, the reconstitution of canine JEB epithelia expressing a hybrid laminin-5 is now possible, and is currently used in studies on the fate of these ex vivo grafts in JEB dogs.Une Epidermolyse Bulleuse Jonctionnelle (EBJ) est décrite chez le Braque allemand et constitue un remarquable modèle canin de l'épidermolyse bulleuse jonctionnelle de l'Homme. Chez le Braque allemand, cette maladie est causée par une substitution homozygote du nucléotide 1514 C-T chez les chiens EBJ. Cette variation de séquence induit un changement non conservatif de 14 acides aminés (505T-I) dans le domaine I de la laminine a3. Cette substitution non conservative modifie le profil d'hydrophobicité du fragment peptidique et altère donc la stabilité d'association de la chaîne a3 avec l'hétérotrimère β3γ2. Ceci pourrait aboutir à une dégradation du polypeptide muté. Une fraction d'a3 mutée pourrait s'incorporer dans la laminine 5, en altérer le fonctionnement et entraver le clivage extra-cellulaire de la chaîne y2. Des expériences de complémentation génétique ont donc été réalisées. Pour cela, un rétrovirus MMLV exprimant l'ADNc entier muté et sauvage de la chaîne a3 a été construit pour être transduit dans les kératinocytes des chiens EBJ (KEBJ). Cette transversion phénotypique a été réussie et a permis aux KEBJ de sécréter de nouveau la chaîne a3 et ainsi, de produire des molécules de laminine 5 fonctionnelles. Par ailleurs, ces KEBJ transduits ont montré aussi la restauration de leur capacité d'adhésion en culture. Enfin, la reconstitution d'épithéliums canins EBJ exprimant une laminine 5 hybride est désormais possible et actuellement à l'origine d'études sur le devenir de ces greffes ex vivo chez des chiens atteints d'EBJ

Identification of a Lethal Form of Epidermolysis Bullosa Simplex Associated with a Homozygous Genetic Mutation in Plectin

Genetic mutations in plectin, a cytoskeleton linker protein expressed in a large variety of tissues including skin, muscle, and nerves, cause epidermolysis bullosa simplex with muscular dystrophy, a recessive inherited disease characterized by blistering of the skin and late onset of muscular dystrophy, and Ogna epidermolysis bullosa simplex, a rare dominant inherited form of epidermolysis bullosa simplex with no muscular involvement. Here we report a novel homozygous genetic mutation (2727del14) in the plectin gene (PLEC1) associated with a lethal form of recessive inherited epidermolysis bullosa in a consanguineous family with three affected offspring. This new clinical variant of epidermolysis bullosa is characterized by general skin blistering, aplasia cutis of the limbs, developmental complications, and rapid demise after birth. Mutation 2727del14 is the first genetic defect described in PLEC1 that disrupts the plakin domain of plectin. The severe phenotype of the patients may be linked to the role of the N-terminal domain in the function of plectin and develops the understanding of the genotype-phenotype correlations in the genodermatoses affecting the dermal-epidermal junction

Functional Correction of Type VII Collagen Expression in Dystrophic Epidermolysis Bullosa

Functional defects in type VII collagen, caused by premature termination codons on both alleles of the COL7A1 gene, are responsible for the severe autosomal recessive types of the skin blistering disease, recessive dystrophic epidermolysis bullosa (RDEB). The full-length COL7A1 complementary DNA (cDNA) is about 9kb, a size that is hardly accommodated by therapeutically used retroviral vectors. Although there have been successful attempts to produce functional type VII collagen protein in model systems of RDEB, the risk of genetic rearrangements of the large repetitive cDNA sequence may hamper the clinical application of full-length COL7A1 cDNA in the human system. Therefore, we used trans-splicing to reduce the size of the COL7A1 transcript. Retroviral transduction of RDEB keratinocytes with a 3′ pre-trans-splicing molecule resulted in correction of full-length type VII collagen expression. Unlike parental RDEB keratinocytes, transduced cells displayed normal morphology and reduced invasive capacity. Moreover, transduced cells showed normal localization of type VII collagen at the basement membrane zone in skin equivalents, where it assembled into anchoring fibril-like structures. Thus, using trans-splicing we achieved correction of an RDEB phenotype in vitro, which marks an important step toward its application in gene therapy in vivo.JID JOURNAL CLUB ARTICLE: For questions, answers, and open discussion about this article, please go to http://www.nature.com/jid/journalclu

LIF Mediates Proinvasive Activation of Stromal Fibroblasts in Cancer

SummarySignaling crosstalk between tumor cells and fibroblasts confers proinvasive properties to the tumor microenvironment. Here, we identify leukemia inhibitory factor (LIF) as a tumor promoter that mediates proinvasive activation of stromal fibroblasts independent of alpha-smooth muscle actin (α-SMA) expression. We demonstrate that a pulse of transforming growth factor β (TGF-β) establishes stable proinvasive fibroblast activation by inducing LIF production in both fibroblasts and tumor cells. In fibroblasts, LIF mediates TGF-β-dependent actomyosin contractility and extracellular matrix remodeling, which results in collective carcinoma cell invasion in vitro and in vivo. Accordingly, carcinomas from multiple origins and melanomas display strong LIF upregulation, which correlates with dense collagen fiber organization, cancer cell collective invasion, and poor clinical outcome. Blockade of JAK activity by Ruxolitinib (JAK inhibitor) counteracts fibroblast-dependent carcinoma cell invasion in vitro and in vivo. These findings establish LIF as a proinvasive fibroblast producer independent of α-SMA and may open novel therapeutic perspectives for patients with aggressive primary tumors

Compartmentalization of integrin α6β4 signaling in lipid rafts

Integrin α6β4 signaling proceeds through Src family kinase (SFK)–mediated phosphorylation of the cytoplasmic tail of β4, recruitment of Shc, and activation of Ras and phosphoinositide-3 kinase. Upon cessation of signaling, α6β4 mediates assembly of hemidesmosomes. Here, we report that part of α6β4 is incorporated in lipid rafts. Metabolic labeling in combination with mutagenesis indicates that one or more cysteine in the membrane-proximal segment of β4 tail is palmitoylated. Mutation of these cysteines suppresses incorporation of α6β4 in lipid rafts, but does not affect α6β4-mediated adhesion or assembly of hemidesmosomes. The fraction of α6β4 localized to rafts associates with a palmitoylated SFK, whereas the remainder does not. Ligation of palmitoylation-defective α6β4 does not activate SFK signaling to extracellular signal–regulated kinase and fails to promote keratinocyte proliferation in response to EGF. Thus, compartmentalization in lipid rafts is necessary to couple the α6β4 integrin to a palmitoylated SFK and promote EGF-dependent mitogenesis

Etablissement d'un modèle animal pour la thérapie génique des épidermolyses bulleuses jonctionnelles non létales

L'objectif de mon étude a été de mettre au point un modèle animal pour la thérapie génique cutanée. Nous avons identifié des chiens souffrant d'épidermolyse bulleuse jonctionnelle (EBJ), un groupe de génodermatoses autosomiques récessives causées par un défaut d'expression des composants des structures d'ancrage de l'épiderme au derme. Chez ces chiens, la maladie est causée par un défaut d'expression de la chaîne a3 de la laminine-5, le principal ligand d'adhésion des kératinocytes basaux. Une insertion intronique dans le gène codant pour la laminine a3 est à l'origine de la maladie, et aboutit à la production d'ARNm aberrants et à une diminution de la production de laminine-5 sauvage. La réversion phénotypique des kératinocytes canins a été réalisée par transduction rétrovirale de l'ADNc codant pour la chaîne a3 de type sauvage. Les kératinocytes transduits montrent une restauration de la capacité d'adhésion, de prolifération et de la clonogénicité. Des peaux artificielles générées in vitro à partir des kératinocytes transduits montrent une expression durable de la laminine-5 localisée à la jonction dermo-épidermique après la greffe sur des souris SCID. La greffe des épithéliums transgéniques autologues sur les chiens EBJ immunocompétents permettra d'évaluer l'éventuelle réponse immunitaire dirigée contre le produit du transgène. La laminine-5 et en particulier sa chaîne ?2 étant également impliquées dans la migration et l'invasion tumorale, nous avons procédé à la localisation chromosomique des gènes codant pour les trois chaînes de la laminine-5 chez le chien, et nous avons isolé et séquencé l'ADNc codant pour la chaîne ?2 canine.To prove the feasability and safety of gene therapy of mechanobullous diseases, we have characterized a breed of dogs suffering from a mild form of JEB, a devastating, untreatable inherited skin disease characterized by blistering and erosions of the skin. These dogs exhibit the clinical hallmarks of the human condition, associated with reduced expression and secretion of laminin-5, an heterotrimeric a3ß3?2 glycoprotein that is the principal adhesion ligand of basal keratinocytes. An intronic insertion was identified in the gene coding for the a3 chain of laminin-5 and lead to the synthesis of an aberrant messenger carrying a downstream PTC. Consequently, the JEB dog keratinocytes secrete low amounts of wild-type laminin-5. We achieved sustained expression of the transgene product by retroviral-mediated transduction of epidermal stem cells. The transduced keratinocytes displayed enhanced adhesion, proliferation and clonogenic potential, and deposited immunoreactive laminin-5 efficiently and permanently at the dermal-epidermal junction of in vitro reconstructed skin grafted into SCID mice. Our results set the basis for preclinical gene therapy on the first immunocompetent large animal model for an inherited skin disease. Expression of laminin-5 and particularly of its ?2 chain are also deregulated in number of human cancers. Because dog models of human cancers provide the opportunity to clarify the relationship between laminin-5 and tumor malignancy, we have isolated and characterized the cDNA of dog g2 chain. We have also determined the synthenic location of the dog laminin-5 loci on CFA7.NICE-BU Sciences (060882101) / SudocSudocFranceF