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 ...

Abstract. This paper shows that, in confluent human umbilical vein endothelial cell (EC) monolayers, the integrin heterodimers c~2/3~ and ot5/3~, but not other members of the/3~ subfamily, are located at cell-cell contact borders and not at cellular free edges. Also the cry chain, but not its most common partner/33, that is widely expressed in EC cell-matrix junctions, is found at cell-cell borders. In EC monolayers, the putative ligands of oe2/3~ and o~5/3t receptors, i.e., laminin, collagen type IV, and fibronectin, are also organized in strands corresponding to cell-cell borders. The location of the above integrin receptors is not an artifact of in vitro culture since it has been noted also in explanted islets of the native umbilical vein endothelium. The integrins tx2~ and ot5/3~ play a role in the maintenance of endothelial monolayer continuity in vitro. Indeed, specific antibodies to ot2#~, ot5/3~, and the synthetic peptide GRGDSP alter its continuity without any initial cell detachment. Moreover, antibodies to oLs/3~ increase the permeation of macromolecules across confluent EC monolayers. In contrast/33 antibodies were ineffective. It is suggested that the relocation of integrins to cell-cell borders is a feature of cells programmed to form polarized monolayers since integrins have a different distribution in nonpolar confluent dermal fibroblasts. The conclusion is that some members of the integrin superfamily collaborate with other intercellular molecules to form lateral junctions and to control both the monolayer integrity and the permeability properties of the vascular endothelial lining. This also suggests that integrins are adhesion molecules provided with a unique biochemical adaptability to different biological functions.

Abstract. Human umbilical vein endothelial cells (ECs) adhere in vitro to proteins of the extracellular matrix including fibronectin (fn) and vitronectin (vn). Specific receptors for fn and vn have been previously characterized. These receptors belong to a family of membrane glycoproteins characterized (a) by being a transmembrane complex of two noncovalently linked subunits and (b) by recognizing the tripeptide ArgGly-Asp on their respective ligands. In this paper we investigated how vn and fn control the organization of their respective receptors over the surface of ECs. It was found that the clustering of individual receptors and the organization thereafter of focal contacts occurred only when ECs were exposed to the specific ligand and did not occur on the opposite ligand. The shape of receptor clusters was slightly different and a colocalization of the two receptors was found when ECs were cultured on a mixed matrix of fn plus vn. Adhesion was selectively inhibited by vn or fn receptor antibodies on their respective substrates. The clustering of both receptors preceded the association of vinculin with focal contacts and stress fiber formation. Also, the vn receptor, in the absence of associated fn receptor, was capable of inducing the organization of the membrane-microfilament interaction complex. Overall, these results indicate that individual matrix ligands induce only the clustering of their respective membrane receptors. The clustering of only one receptor is capable of supporting the subsequent formation of focal contacts and the local assembly of related cytoskeletal proteins.H UMAN endothelial cells (ECs) ~ adhere, spread, and organize their cytoskeleton on different molecules of the extracellular matrix such as fibronectin (fn), vitronectin (vn), and collagen (for review see references 8, 32). The reasons for such multiple recognition may be found in the fact that ECs express and expose on their surface several receptor molecules that, on the outer side of the membrane, specifically recognize and bind different components of the extracellular matrix and, on the cytoplasmic side, link a chain of proteins of the membrane-microfilament interaction complex involved in the mechanism of adhesion and cytoskeletal organization (for review see reference 9).Recently, a family of cell adhesion receptors that recognizes a number of extracellular matrix components has been described (15, 27). These receptors have several structural and functional homologies. They consist of two noncovalently linked subunits (denominated a and 13 chains) and are capable of recognizing the sequence Arg-Gly-Asp (RGD) which is present in many extracelllar matrix proteins and is : EC, endothelial cell; fn, fibronectin; vn, vitronectin. believed to play a key role in cell adhesion. Within this family fn-and vn-specific receptors have been isolated (25,26). Although they similarly recognize the RGD sequence on their targets, fn and vn receptors have mutually exclusive specificities. Indeed, liposomes containing these receptors do s...

Epithelial cell interactions with matrices are critical to tissue organization. Indirect immunofluorescence and immunoprecipitations of cell lysates prepared from stratified cultures of human epidermal cells showed that the major integrins expressed by keratinocytes are ad3,4 (also called a644) and a2pl/a3.l1. The adP4 integrin is localized at the surface of basal cells in contact with the basement membrane, whereas a2fil/a3fl1 integrins are absent from the basal surface and are localized only on the lateral surface of basal and spinous keratinocytes. Anti-fl4 antibodies potently inhibited keratinocyte adhesion to matrigel or purified laiinin, whereas anti-fl1 antibodies were ineffective. Only anti-fl4 antibodies were able to detach established keratinocyte colonies. These data suggest that aEP4 mediates keratinocyte adhesion to basal lamina, whereas the ,BI subfamily is involved in cell-cell adhesion of keratinocytes.

The adhesion of osteoclasts to the bone matrix is mandatory for bone resorption. Contact of the osteoclast with bone surface induces, in fact, cell polarization and organization of the resorbing apparatus, the so-called "ruffled border." Cell-matrix interaction in osteoclasts is a complex phenomenon resulting from formation of the "clear zone," a cytoplasmic area presenting the adhering plasma membrane, or "sealing membrane." The sealing membrane surrounds the ruffled border and seals the resorbing compartment, namely the extracellular space in which bone resorption takes place. Adhesion at this level occurs via specialized discrete structures, the "podosomes." Podosomes present most of the protein commonly found in focal adhesions, but with a peculiar organization. They are dynamic elements suitable for regulation, according with the functional demand of the cell. Their assembly increases during bone resorption and is regulated by the cytosolic free calcium concentration and the activity of protein kinase C.