Abstract. The molecular mechanism underlying the promotion of wound healing by TGF-/31 is incompletely understood. We report that TGF-/5'I regulates the regenerative/migratory phenotype of normal human keratinocytes by modulating their integrin receptor repertoire. In growing keratinocyte colonies but not in fully stratified cultured epidermis, TGF-~I: (a) strongly upregulates the expression of the fibronectin receptor c~5~1, the vitronectin receptor t~v/~5, and the collagen receptor t~2/~l by differentially modulating the synthesis of their ot and/~ subunits; (b) downregulates the multifunctional oL3/~l heterodimer; (c) induces the de novo expression and surface exposure of the av#6 fibronectin receptor; (d) stimulates keratinocyte migration toward fibronectin and vitronectin; (e) induces a marked perturbation of the general mechanism of polarized domain sorting of both #1 and ~4 dimers; and (f) causes a pericellular redistribution of ~v/~5. These data suggest that ot5~l, ave6, and av/~5, not routinely used by keratinocytes resting on an intact basement membrane, act as "emergency" receptors, and uncover at least one of the molecular mechanisms responsible for the peculiar integrin expression in healing human wounds. Indeed, TGF-~I reproduces the integrin expression pattern of keratinocytes located at the injury site, particularly of cells in the migrating epithelial tongue at the leading edge of the wound. Since these keratinocytes are inhibited in their proliferative capacity, these data might account for the apparent paradox of a TGF-/5'l-dependent stimulation of epidermal wound healing associated with a growth inhibitory effect on epithelial cells. HUMAr~ epidermis, the outermost layer of skin, is a stratified squamous epithelium mainly composed of a single cell type, the keratinocyte. The epidermis survives through a self-renewal process (Green, 1980). Small progenitor keratinocytes (Barrandon and Green, 1987b), forming the innermost epidermal basal layer, regularly undergo mitosis, differentiation, and upward migration to replace terminally differentiated cornified cells that are continuously shed into the environment (Green, 1980;Watt, 1989;Fuchs, 1990). Basal epidermal keratinocytes rest on a basement membrane composed of a specific subset of extracellular matrix proteins such as laminin, type IV collagen, kalinin, nidogen, and heparan sulfate proteoglycan. The firm adhesion of basal keratinocytes, hence of the whole epidermis, to the basal lamina is mediated by hemidesmoAddress all correspondence to Dr. Michele De Luca, Unit of Epithelial Biology and Biotechnology, CBA, Centro di Biotecnologie Avanzate, Viale Benedetto XV no. 10, 16132, Genoa, Italy. Ph.: (39) (10) 5737423. Fax: (39) (10) 5737405.somes. These structures link the epithelial intermediate filament network to the dermal anchoring fibrils, which are mainly composed of type VII collagen and extend from the basement membrane to anchoring plaques in the papillary dermis (Jones et al., 1994). The keratinocyte behavior changes dramatically when ...

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The 72-kd type IV collagenase is a member of the collagenase enzyme family that has been closely linked with the invasive phenotype of cancer cells. Previous studies have shown that both normal cells and highly invasive tumor cells produce the 72-kd type IV procollagenase enzyme in a complexed form consisting of the proenzyme and a novel tissue inhibitor of metalloproteinases, TIMP-2. The balance between activated enzyme and available inhibitor is thought to be a critical determinant of the matrix proteolysis associated with a variety of pathologic processes, including tumor cell invasion. In the present study, we demonstrate that alteration of the metalloproteinase-metalloproteinase-inhibitor balance in favor of excess inhibitor blocks human fibrosarcoma HT-1080 tumor cell invasion of a reconstituted basement membrane. The HT-1080 cell line produces both the 72-kd and the 92-kd type IV collagenases. Alteration of the type IV collagenase-inhibitor balance was achieved by addition of free TIMP-2 or antibodies to 72-kd type IV collagenase. Native, purified TIMP-2 was inhibitory in the range of 1-25 micrograms/mL. Addition of specific antiserum against the 72-kd type IV collagenase, which did not cross-react with the 92-kd type IV collagenase, inhibited HT-1080 cell invasion to the same extent. These results suggest that metalloproteinases, in particular the 72-kd type IV collagenase, are critical for tumor cell invasion of the reconstituted basement membrane. Our findings demonstrate that addition of the endogenous inhibitor TIMP-2 is able to block invasion. Thus, we recommend initiation of in vivo studies of the therapeutic potential of TIMP-2 to block tumor cell invasion and intravasation into the circulation.

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