Transforming growth factor-beta promotes deposition of collagen VII in a modified organotypic skin model.

BACKGROUND

Anchoring fibrils, which consist mainly of collagen VII, form a three-dimensional network below stratified squamous epithelia thus attaching the epithelial basement membrane to the underlying connective tissue. In skin, collagen VII is mainly produced by keratinocytes, and in vitro findings of keratinocyte-fibroblast cocultures suggest that mesenchymal-epithelial signaling plays a major role in the regulation of its expression. The matrix cytokine transforming growth factor-beta (TGF-beta) is a candidate mediator of such interactions, and exogenous TGF-beta has been shown to stimulate collagen VII expression in vitro. In the present study, the role of TGF-beta and other growth factors as mediators of mesenchymal-epithelial interactions in the regulation of collagen VII synthesis and deposition was investigated.

EXPERIMENTAL DESIGN

The synthesis of collagen VII in monolayer cultures of normal human skin cells and in an organotypic skin equivalent system was investigated by indirect immunofluorescence staining. The role of soluble factors in collagen VII regulation was examined in a two-compartment coculture system, and the effect of medium conditioned by cocultures was tested on monocultures. Neutralizing antibodies to TGF-beta 2 were used to establish the relevance of this growth factor in the regulation of collagen VII synthesis. Modified skin equivalent cultures were constructed by cultivating an epithelium on top of devitalized human dermis deprived of the basement membrane.

RESULTS

TGF-beta 2 was found to be a major autocrine or paracrine stimulator of collagen VII synthesis in cocultures. Incubation of the cultures with neutralizing antibodies to this growth factor abolished expression of collagen VII almost completely. Among other putative regulators of extracellular matrix metabolism, such as insulin-like growth factor, basic fibroblast growth factor, platelet-derived growth factor, epidermal growth factor, and calcium, insulin-like growth factor and calcium stimulated collagen VII synthesis by keratinocytes, but the extent was clearly less than by TGF-beta. Fibroblasts did not respond to either growth factor. However, experiments with fibroblasts grown on a keratinocyte-derived extracellular matrix suggested that collagen VII synthesis by fibroblasts is enhanced by matrix-mediated stimuli. A modified organotypic skin equivalent proved to be a suitable model to study the expression and deposition of basement membrane components. Collagen VII was expressed by basal keratinocytes after 3 weeks in culture under serum-free conditions. Its deposition at the dermoepidermal junction, however, depended on the addition of TGF-beta.

CONCLUSIONS

TGF-beta is an important mediator of mesenchymal-epithelial interactions regulating collagen VII expression, and the deposition of collagen VII at the dermoepidermal interface is promoted by this growth factor. TGF-beta is a candidate factor for stabilization of the dermoepidermal junction in vivo during wound healing and in conditions where the junction zone is pathologically altered, e.g., dystrophic epidermolysis bullosa.