Integrin-linked kinase is required for epidermal and hair follicle morphogenesis

Integrin-linked kinase (ILK) links integrins to the actin cytoskeleton and is believed to phosphorylate several target proteins. We report that a keratinocyte-restricted deletion of the ILK gene leads to epidermal defects and hair loss. ILK-deficient epidermal keratinocytes exhibited a pronounced integrin-mediated adhesion defect leading to epidermal detachment and blister formation, disruption of the epidermal–dermal basement membrane, and the translocation of proliferating, integrin-expressing keratinocytes to suprabasal epidermal cell layers

Laminin 332 Is Indispensable for Homeostatic Epidermal Differentiation Programs

The skin epidermis is attached to the underlying dermis by a laminin 332 (Lm332)-rich basement membrane. Consequently, loss of Lm332 leads to the severe blistering disorder epidermolysis bullosa junctionalis in humans and animals. Owing to the indispensable role of Lm332 in keratinocyte adhesion in vivo, the severity of the disease has limited research into other functions of the protein. We have conditionally disrupted Lm332 expression in basal keratinocytes of adult mice. Although blisters develop along the interfollicular epidermis, hair follicle basal cells provide sufficient anchorage of the epidermis to the dermis, making inducible deletion of the Lama3 gene compatible with life. Loss of Lm332 promoted the thickening of the epidermis and exaggerated desquamation. Global RNA expression analysis revealed major changes in the expression of keratins, cornified envelope proteins, and cellular stress markers. These modifications of the keratinocyte genetic program are accompanied by changes in cell shape and disorganization of the actin cytoskeleton. These data indicate that loss of Lm332-mediated progenitor cell adhesion alters cell fate and disturbs epidermal homeostasis.Peer reviewe

Kindlin-1 is a phosphoprotein involved in regulation of polarity, proliferation, and motility of epidermal keratinocytes

A novel family of focal adhesion proteins, the kindlins, is involved in attachment of the actin cytoskeleton to the plasma membrane and in integrin-mediated cellular processes. Deficiency of kindlin-1, as a result of loss-of-function mutations in the KIND1 gene, causes Kindler syndrome, an autosomal recessive genodermatosis characterized by skin blistering, progressive skin atrophy, photosensitivity and, occasionally, carcinogenesis. Here we characterized authentic and recombinantly expressed kindlin-1 and show that it is localized in basal epidermal keratinocytes in a polar fashion, close to the cell surface facing the basement membrane, in the areas between the hemidesmosomes. We identified two forms of kindlin-1 in keratinocytes, with apparent molecular masses of 78 and 74 kDa, corresponding to phosphorylated and desphosphorylated forms of the protein. In kindlin-1-deficient skin, basal keratinocytes show multiple abnormalities: cell polarity is lost, proliferation is strongly reduced, and several cells undergo apoptosis. In vitro, deficiency of kindlin-1 in keratinocytes leads to strongly reduced cell proliferation, decreased adhesion, undirected motility, and intense protrusion activity of the plasma membrane. Taken together, these results show that kindlin-1 plays a role in keratinocyte adhesion, polarization, proliferation, and migration. It is involved in organization and anchorage of the actin cytoskeleton to integrin-associated signaling platforms

C-terminal truncation impairs glycosylation of transmembrane collagen XVII and leads to intracellular accumulation

Collagen XVII, a type II transmembrane protein in hemidesmosomes, is involved in the anchorage of stratified epithelia to the underlying mesenchyme. Its functions are regulated by ectodomain shedding, and its genetic defects lead to epidermal detachment in junctional epidermolysis bullosa (JEB), a heritable skin fragility syndrome, but the molecular disease mechanisms remain elusive. Here we used a spontaneously occurring homozygous COL17A1 deletion mutant in JEB to discern glycosylation of collagen XVII. The mutation truncated the distal ectodomain and positioned the only N-glycosylation site 34 amino acids from the newly formed C terminus, which impaired efficient N-glycosylation. Immunofluorescence staining of authentic JEB keratinocytes and of COS-7 cells transfected with the mutant indicated intracellular accumulation of collagen XVII precursor molecules. Cell surface biotinylation and quantification of ectodomain shedding demonstrated that only about 15% of the truncated collagen XVII reached the cell surface. The cell surface-associated molecules were N-glycosylated in a normal manner, in contrast to the molecules retained within the cells, indicating that N-glycosylation of the ectodomain is required for targeting of collagen XVII to the plasma membrane and that reduced accessibility of the N-glycosylation site negatively regulates this process. Functional consequences of the strong reduction of collagen XVII on the cell surface included scattered deposition of cell adhesion molecule laminin 5 into the extracellular environment and, as a consequence of faulty collagen XVII-laminin ligand interactions, aberrant motility of the mutant cells

Regulation of B cell homeostasis and activation by the tumor suppressor gene CYLD

B cell homeostasis is regulated by multiple signaling processes, including nuclear factor-κB (NF-κB), BAFF-, and B cell receptor signaling. Conditional disruption of genes involved in these pathways has shed light on the mechanisms governing signaling from the cell surface to the nucleus. We describe a novel mouse strain that expresses solely and excessively a naturally occurring splice variant of CYLD (CYLDex7/8 mice), which is a deubiquitinating enzyme that is integral to NF-κB signaling. This shorter CYLD protein lacks the TRAF2 and NEMO binding sites present in full-length CYLD. A dramatic expansion of mature B lymphocyte populations in all peripheral lymphoid organs occurs in this strain. The B lymphocytes themselves exhibit prolonged survival and manifest a variety of signaling disarrangements that do not occur in mice with a complete deletion of CYLD. Although both the full-length and the mutant CYLD are able to interact with Bcl-3, a predominant nuclear accumulation of Bcl-3 occurs in the CYLD mutant B cells. More dramatic, however, is the accumulation of the NF-κB proteins p100 and RelB in CYLDex7/8 B cells, which, presumably in combination with nuclear Bcl-3, results in increased levels of Bcl-2 expression. These findings suggest that CYLD can both positively and negatively regulate signal transduction and homeostasis of B cells in vivo, depending on the expression of CYLD splice variants

Laminins and interaction partners in the architecture of the basement membrane at the dermal-epidermal junction

The basement membrane at the dermal-epidermal junction keeps the epidermis attached to the dermis. This anatomical barrier is made up of four categories of extracellular matrix proteins: collagen IV, laminin, nidogen and perlecan. These proteins are precisely arranged in a well-defined architecture through specific interactions between the structural domains of the individual components. Some of the molecular constituents are provided by both fibroblasts and keratinocytes, while others are synthesized exclusively by fibroblasts or keratinocytes. It remains to be determined how the components from the fibroblasts are targeted to the dermal-epidermal junction and correctly organized and integrated with the proteins from the adjacent keratinocytes to form the basement membrane

The role of laminins in basement membrane function

Laminins are a family of multifunctional macromolecules, ubiquitous in basement membranes, and represent the most abundant structural noncollagenous glycoproteins of these highly specialised extracellular matrices. Their discovery started with the difficult task of isolating molecules produced by cultivated cells or extracted from tissues. The development of molecular biology techniques has facilitated and accelerated the identification and the characterisation of new laminin variants making it feasible to identify full-length polypeptides which have not been purified. Further, genetically engineered laminin fragments can be generated for studies of their structure-function relationship, permitting the demonstration that laminins are involved in multiple interactions with themselves, with other components of the basal lamina, and with cells. It endows laminins with a central role in the formation, the architecture, and the stability of basement membranes. In addition, laminins may both separate and connect different tissues, i.e. the parenchymal and the interstitial connective tissues. Laminins also provide adjacent cells with a mechanical scaffold and biological information either directly by interacting with cell surface components, or indirectly by trapping growth factors. In doing so they trigger and control cellular functions. Recently, the structural and biological diversity of the laminins has started to be elucidated by gene targeting and by the identification of laminin defects in acquired or inherited human diseases. The consequent phenotypes highlight the pivotal role of laminins in determining heterogeneity in basement membrane functions

Rôle de l'interaction mésenchyme-épithélium dans l'expression, la maturation protéolytique et la déposition de la laminine 5

La lamine 5 (a3b3g2) est une glycoprotéine composant la lame basale de la peau et elle est importante pour la cohésion de la jonction dermo-épidermique. Cette protéine est synthétisée et sécrétée par les cellules épithéliales sous forme d'un précurseur de 490 kDa qui est converti en formes matures de 440 et 440 kDa, par clivage des domaines N-terminaux des chaînes a3 et g2. Ce clivage est important pour la maturation de la lamine 5 et l'objectif de ce travail a été de mettre en évidence le rôle des interactions épithélio-mésenchymateuses physiologiques et pathologiques sur le clivage et la déposition de la laminine 5. Les effets de l'interaction épithélio-mésenchymateuse sur la laminine 5 ont été reproduits en co-cultivant ou pas des cellules éphitéliales normales ou tumorales avec des fibroblastes...Laminin 5 (a3b3g2), a glycoprotein, is a component of basement membranes and is important for the dermal-epidermal stability. This protein is synthesised and secreted by epithelial cells under a precursor from of 490 kDa which is converted into a mature from of 440 kDa and 400 kDa, after cleavage of the a3 and g2 N-terminal domains. Cleavage is important for the maturation of laminin 5. The aim of this study was to find out the phyiological and pathological role of the epithelial-mesenchymal interaction as a result of the cleavage and deposition of laminin 5....PARIS5-BU Saints-Pères (751062109) / SudocSudocFranceF

INTERACTIONS AU NIVEAU DES DOMAINES INTRACELLULAIRES DES SOUS-UNITES ALPHA3A, ALPHA6A ET BETA1A DES INTEGRINES

LES ADHESIONS FOCALES (AF) CONSTITUENT UN LIEN ESSENTIEL ENTRE LA MATRICE EXTRACELLULAIRE ET LE CYTOSQUELETTE, AINSI QUE LE SITE DE TRANSMISSION DE SIGNAUX PAR LES INTEGRINES. LES INTEGRINES 31 ET 61, RECEPTEURS DES LAMININES, DIFFERENT DANS LEUR SPECIFICITE DE RECONNAISSANCE : LA LAMININE 1 SE LIE A L'INTEGRINE 61 SEULEMENT ET L'ADHERENCE DES CELLULES A CE SUBSTRAT ENTRAINE LA FORMATION D'AF DISTINCTES DE CELLES FORMEES SUR D'AUTRES ISOFORMES DE LAMININE LIANT A LA FOIS 31 ET 61. L'INTEGRINE 31 POURRAIT DONC REGULER DE MANIERE TRANSDOMINANTE L'INTEGRINE 61. AFIN DE TESTER CETTE HYPOTHESE, DES PEPTIDES REPRESENTANT LE DOMAINE INTRACELLULAIRE DES SOUS-UNITES 3A OU 6A D'INTEGRINES ONT ETE INJECTES DANS DES FIBROBLASTES VIVANTS ADHERES A LA LAMININE 1. SEULE LA MICROINJECTION DU PEPTIDE 3A ENTRAINE UNE REORGANISATION SPECIFIQUE DES AF, RAPPELANT CELLES FORMEES LORS DE L'ACTIVATION DU RECEPTEUR 31 PAR SES LIGANDS. DE PLUS, DES ETUDES PAR RESONANCE PLASMONIQUE DE SURFACE (SPR) PERMETTENT DE DETECTER UNE INTERACTION DIRECTE ENTRE LES PEPTIDES CORRESPONDANT AUX DOMAINES INTRACELLULAIRES DES SOUS-UNITES 3A ET 1A D'INTEGRINE. ENFIN, UN CRIBLAGE DE BANQUE D'ADNC AVEC LE DOMAINE INTRACELLULAIRE DE LA SOUS-UNITE 3A COMME AMORCE DANS LE SYSTEME DOUBLE HYBRIDE CHEZ LA LEVURE, A PERMIS L'IDENTIFICATION DE PLUSIEURS PARTENAIRES D'INTERACTION. PARMI CEUX-CI, LA PROTEINE DRAL/FHL2 INTERAGIT AVEC L'INTEGRINE 31 IN VIVO. EN CONCLUSION, DIFFERENTES INTERACTIONS SPECIFIQUES AU DOMAINE INTRACELLULAIRE DE LA SOUS-UNITE 3A, CONTRIBUENT A LA REGULATION DE L'ORGANISATION DES AF PAR L'INTEGRINE 31. PAR AILLEURS, NOUS AVONS EXAMINE LA CONTRIBUTION DU DOMAINE INTRACELLULAIRE DE LA SOUS-UNITE 1A DANS LE PHENOMENE DE CLUSTERING DES INTEGRINES. DES ANALYSES EN SPR REVELENT UNE AFFINITE DU DOMAINE INTRACELLULAIRE DE 1A POUR LUI-MEME. DES EXPERIENCES EN GEL FILTRATION ET EN SDS-PAGE MONTRENT QUE CE DOMAINE FORME DES MULTIMERES QUI, D'APRES DES MESURES EN DICHROISME CIRCULAIRE, ADOPTENT UNE STRUCTURE EN HELICE . L'OLIGOMERISATION ET LE CHANGEMENT DE STRUCTURE SECONDAIRE DU DOMAINE INTRACELLULAIRE DE LA SOUS-UNITE 1A POURRAIENT INTERVENIR DE MANIERE ACTIVE DANS L'AGREGATION DES INTEGRINES ET LA FORMATION DES AF.PARIS-BIUSJ-Thèses (751052125) / SudocCentre Technique Livre Ens. Sup. (774682301) / SudocPARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF