title: Inhibition of basement membrane formation by a nidogen-binding laminin gamma1-chain fragment in human skin-organotypic cocultures.
abstract: Basement membranes generally determine different tissue compartments in complex organs, such as skin, playing not only an important structural but also a regulatory role. We have previously demonstrated the formation of a regular basement membrane in organotypic three-dimensional (3D)-cocultures of human skin keratinocytes and fibroblasts by indirect immunofluorescence and transmission electron microscopy. In this assembly process, cross-linking of type IV collagen and the laminin gamma1 chain by nidogen is considered a crucial step. For a functional proof, we have now competitively inhibited nidogen binding to laminin in 3D-cocultures with a recombinant laminin gamma1 fragment (gamma1III3-5 module) spanning this binding site. Repeated treatment abolished the deposition of nidogen at the epithelial-matrix interface but also greatly perturbed the presence of other matrix constituents such as laminin and perlecan. This effect persisted over the entire observation period of 10 to 21 days. In contrast, some components of the basement membrane zone were only moderately affected, with the laminin-5 isoform (gamma2 chain), type IV collagen and integrin alpha6ss4 still showing a distinct staining at their regular position, when seen by light microscopy. Furthermore, epidermal morphology and differentiation remained largely normal as indicated by the regular location of keratins K1/K10 and also of late differentiation markers. Ultrastructural examination demonstrated that the gamma1 fragment completely suppressed any formation of basement membrane structures (lamina densa) and also of hemidesmosomal adhesion complexes. As a consequence of hemidesmosome deficiency, keratin filament bundles were not attached to the ventral basal cell aspect. These findings were further substantiated by immuno-electron microscopy, revealing either loss or drastic reduction and dislocation of basement membrane and hemidesmosomal components. Taken together, in this simplified human skin model (representing a 'closed system') a functional link has been demonstrated between compound structures of the extra- and intracellular space at the junctional zone providing a basis to interfere at distinct points and in a controlled fashion.
authors.affiliation: German Cancer Research Center, Division A080, Im Neuenheimer Feld 280, 69120, Heidelberg, Germany. d.breitkreutz@dkfz.de
authors.forename: Dirk
authors.initials: D
authors.lastname: Breitkreutz
authors.forename: Nicolae
authors.initials: N
authors.lastname: Mirancea
authors.forename: Cathrine
authors.initials: C
authors.lastname: Schmidt
authors.forename: Regina
authors.initials: R
authors.lastname: Beck
authors.forename: Ulrich
authors.initials: U
authors.lastname: Werner
authors.forename: Hans-Jürgen
authors.initials: HJ
authors.lastname: Stark
authors.forename: Martin
authors.initials: M
authors.lastname: Gerl
authors.forename: Norbert E
authors.initials: NE
authors.lastname: Fusenig
mesh_terms: D001485:Basement Membrane / Q000378:metabolism* / Q000648:ultrastructure; D001665:Binding Sites; D002352:Carrier Proteins / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure; D002454:Cell Differentiation; D002478:Cells, Cultured; D018920:Coculture Techniques; D024141:Collagen Type IV / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure; D003432:Cross-Linking Reagents / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D039642:Eukaryotic Initiation Factors; D005109:Extracellular Matrix / Q000737:chemistry; D019084:Fluorescent Antibody Technique, Indirect; D022002:Hemidesmosomes / Q000648:ultrastructure; D019812:Heparan Sulfate Proteoglycans / Q000378:metabolism; D006801:Humans; D039161:Integrin alpha6beta4 / Q000187:drug effects / Q000378:metabolism; D007381:Intermediate Filament Proteins / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure; D053556:Keratin-1; D053550:Keratin-10; D015603:Keratinocytes / Q000378:metabolism; D007633:Keratins / Q000378:metabolism / Q000648:ultrastructure; D007797:Laminin / Q000187:drug effects / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D008562:Membrane Glycoproteins / Q000378:metabolism*; D009924:Organ Culture Techniques; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012867:Skin / Q000166:cytology*; D013997:Time Factors
chemical_list: D002352:Carrier Proteins; D024141:Collagen Type IV; D003432:Cross-Linking Reagents; C109284:EIF6 protein, human; D039642:Eukaryotic Initiation Factors; D019812:Heparan Sulfate Proteoglycans; D039161:Integrin alpha6beta4; D007381:Intermediate Filament Proteins; C491616:KRT1 protein, human; C507493:KRT10 protein, human; D053556:Keratin-1; D007797:Laminin; D008562:Membrane Glycoproteins; D011994:Recombinant Proteins; C089141:laminin gamma 1; C029967:nidogen; C071927:perlecan; D053550:Keratin-10; D007633:Keratins