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First published online May 24, 2004
doi: 10.1242/10.1242/jcs.01127

Journal of Cell Science 117, 2611-2622 (2004)
Published by The Company of Biologists 2004
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Research Article

Inhibition of basement membrane formation by a nidogen-binding laminin {gamma}1-chain fragment in human skin-organotypic cocultures

Dirk Breitkreutz1,*, Nicolae Mirancea2, Cathrine Schmidt1, Regina Beck1, Ulrich Werner3, Hans-Jürgen Stark1, Martin Gerl3 and Norbert E. Fusenig1

1 German Cancer Research Center, Division A080, Im Neuenheimer Feld 280, 69120, Heidelberg, Germany
2 Romanian Academy of Sciences, Spaiul Independentei 296, 77703, Bucharest, Romania
3 Aventis Pharma Deutschland GmbH, Industriepark Hoechst H811/815, 65926, Frankfurt am Main, Germany

* Author for correspondence (e-mail: d.breitkreutz{at}dkfz.de)

Accepted 28 January 2004

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 {gamma}1 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 {gamma}1 fragment ({gamma}1III3-5 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 ({gamma}2 chain), type IV collagen and integrin {alpha}6ß4 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 {gamma}1 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.

Key words: Nidogen, Basement membrane, Human skin-organotypic coculture


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© The Company of Biologists Ltd 2004