. Basal keratinocytes attach to the underlying dermal stroma through an ultrastructurally unique and complex basement membrane zone. Electron-dense plaques along the basal surface plasma membrane, termed hemidesmosomes, appear to attach directly to the lamina densa of the basement membrane through fine strands, called anchoring filaments . The lamina densa is secured to the stroma through a complex of type VII collagen containing anchoring fibrils and anchoring plaques . We have identified what we believe is a novel antigen unique to this tissue region . The mAbs to this antigen localize to the anchoring filaments, just below the basal-dense plate of the hemidesmosomes . In cell culture, the antigen is deposited upon the culture substate by growing and migrating human keratinocytes. Addition of mAb to the cultures causes the cells to round and detach, but does not impair them metabolically. Skin fragments incubated with antibody exten-HE basement membrane zones at epithelial-stromml interfaces of external tissues are unique and complex . The dermal-epidermal junction is one ofthe best stud ied (Palade and Farquhar, 1965 ;Rowlatt, 1969;Susi, 1967;Bruns, 1969;Briggaman and Wheeler, 1975a ;Kawanami et al., 1978) . When visualized by EM following standard conditions of fixation, dehydration, and staining, a typical basal lamina is seen, containing a lamina lucida and lamina densa extending -100 nm from the epithelial basal surface (see Fig . 2 a). In addition, electron-dense thickenings, termed hemidesmosomes (Weiss and Ferris, 1954;Kelly, 1966), are seen along the basal keratinocyte surface. Thin filaments, termed anchoring filaments (Ellison and Garrod, 1984), appear to transverse the lamina lucida, bridging the hemidesmosome and the lamina densa . Along the dermal margin ofthe lamina densa, anchoring fibrils extend fromthe basement membrane. The anchoring fibrils either loop back and reinsert into the lamina densa, or extend perpendicularly from the basement membrane and insert into anchoring plaques, which are electron-dense condensations of the ends of anchoring fibrils toDr. Rousselle'spresent address is Laboratoire desSubstituts Cutan6s, H6pi-tal Fdouard Herriot, Lyon, France .C The Rockefeller University Press, 0021-9525/91/08/567/10 $2 .00 The Journal of Cell Biology, Volume 114, Number 3, August 1991567-576 sively de-epithelialize. These findings strongly suggest that this antigen is intimately involved in attachment of keratinocytes to the basement membrane.This antigen was isolated from keratinocyte cultures by immunoaffinity chromatography. Two molecules are observed. The most intact species contains three nonidentical chains, 165, 155, and 140 kD linked by interchain disulfide bonds. The second and more abundant species contains the 165-and 140-kD chains, but the 155-kD chain has been proteolytically cleaved to 105 kD. Likewise, two rotary-shadowed images are observed . The larger ofthe two, presumably corresponding to the most intact form, appears as an asymmetric 107-run-long rod, with a si...

Cutaneous wound healing in adult mammals is a complex multi-step process involving overlapping stages of blood clot formation, inflammation, re-epithelialization, granulation tissue formation, neovascularization, and remodelling. Re-epithelialization describes the resurfacing of a wound with new epithelium. The cellular and molecular processes involved in the initiation, maintenance, and completion of epithelialization are essential for successful wound closure. A variety of modulators are involved, including growth factors, cytokines, matrix metalloproteinases, cellular receptors, and extracellular matrix components. Here, we focus on cellular mechanisms underlying keratinocyte migration and proliferation during epidermal closure. Inability to re-epithelialize is a clear indicator of chronic non-healing wounds, which fail to proceed through the normal phases of wound healing in an orderly and timely manner. This review summarizes the current knowledge regarding the management and treatment of acute and chronic wounds, with a focus on re-epithelialization, offering some insights into novel future therapies.

Mutational analyses of genes that encode components of the anchoring complex underlying the basolateral surface of external epithelia indicate that this structure is the major element providing for resistance to external friction. Ultrastructurally, laminin 5 (α3β3γ2; a component of the anchoring filament) appears as a thin filament bridging the hemidesmosome with the anchoring fibrils. Laminin 5 binds the cell surface through hemidesmosomal integrin α6β4. However, the interaction of laminin 5 with the anchoring fibril (type VII collagen) has not been elucidated. In this study we demonstrate that monomeric laminin 5 binds the NH2-terminal NC-1 domain of type VII collagen. The binding is dependent upon the native conformation of both laminin 5 and type VII collagen NC-1. Laminin 6 (α3β1γ1) has no detectable affinity for type VII collagen NC-1, indicating that the binding is mediated by the β3 and/or γ2 chains of laminin 5. Approximately half of the laminin 5 solubilized from human amnion or skin is covalently complexed with laminins 6 or 7 (α3β2γ1). The adduction occurs between the NH2 terminus of laminin 5 and the branch point of the short arms of laminins 6 or 7. The results are consistent with the presumed orientation of laminin 5, having the COOH-terminal G domain apposed to the hemidesmosomal integrin, and the NH2-terminal domains within the lamina densa. The results also support a model predicting that monomeric laminin 5 constitutes the anchoring filaments and bridges integrin α6β4 with type VII collagen, and the laminin 5–6/7 complexes are present within the interhemidesmosomal spaces bound at least by integrin α3β1 where they may mediate basement membrane assembly or stability, but contribute less significantly to epithelial friction resistance.

The ability of skin to act as a barrier is primarily determined by cells that maintain the continuity and integrity of skin and restore it after injury. Cutaneous wound healing in adult mammals is a complex multi-step process that involves overlapping stages of blood clot formation, inflammation, re-epithelialization, granulation tissue formation, neovascularization, and remodeling. Under favorable conditions, epidermal regeneration begins within hours after injury and takes several days until the epithelial surface is intact due to reorganization of the basement membrane. Regeneration relies on numerous signaling cues and on multiple cellular processes that take place both within the epidermis and in other participating tissues. A variety of modulators are involved, including growth factors, cytokines, matrix metalloproteinases, cellular receptors, and extracellular matrix components. Here we focus on the involvement of the extracellular matrix proteins that impact epidermal regeneration during wound healing.

Abstract. Stable attachment of external epithelia to the basement membrane and underlying stroma is mediated by transmembrane proteins such as the integrin et6134 and buUous pemphigoid antigen 2 within the hemidesmosomes along the basolateral surface of the epithelial cell and their ligands that include a specialized subfamily of laminins. The laminin 5 molecule (previously termed kalinin/nicein/epiligrin) is a member of this epithelial-specific subfamily. Laminin 5 chains are not only considerably truncated within domains III-VI, but are also extensively proteolytically processed in vitro and in vivo. As a result, the domains expected to be required for the association of laminins with other basement membrane components are lacking in the mature laminin 5 molecule. Therefore, the tight binding of laminin 5 to the basement membrane may occur by a unique mechanism. To examine laminin 5 in tissue, we chose human amnion as the source, because of its availability and the similarity of the amniotic epithelial basement membrane with that of skin. We isolated the laminin 5 contained within the basement membrane of human amnion. In addition to monomeric laminin 5, we find that much of the laminin 5 isolated is covalently adducted with laminin 6 (o~31M~/1) and a novel laminin isotype we have termed laminin 7 (or3 1~2 ~/1). We propose that the association between laminin 5 and laminins 6 and 7 is a mechanism used in amnion to allow stable association of laminin 5 with the basement membrane. The 1~2 chain is seen at the human amniotic epithelial-stromal interface and at the dermal-epidermal junction of fetal and adult bovine skin by immunofluorescence, but is not present, or only weakly present, in neonatal human skin.T hE attachment of external epithelia to the underlying stroma is mediated by a unique set of ultrastructural entities within the basement membrane zone called the attachment complex (Gipson et al., 1987). This complex includes hemidesmosomes on the basolateral surface of the epithelium (Kelly, 1966), anchoring filaments that bridge the hemidesmosomes with the lamina densa (Komura, 1973), and anchoring fibrils that form an extended network surrounding stromal fibrous elements and insert into the basement membrane (Bruns, 1969). Characterization of the components of the complex show that the hemidesmosomes contain an intracellular protein, bullous pemphigoid antigen (BPAG)X-1, with homology to desmoplakin that is assumed to mediate the interaction of Address all correspondence to Robert E. Burgeson,

No abstract

Abstract. Kalinin was purified from squamous cell carcinoma (SCC25) spent culture media using an immunoaffinity column prepared from the mAb BM165. The affinity-purified material was separated by SDS-PAGE into three bands of 140, and 105 kD identical to those obtained from normal human keratinocyte cultures and previously identified as kalinin. Kalinin promoted adhesion of a large number of normal cells and established cell lines with an activity similar to other adhesion molecules such as the laminin-nidogen complex, fibronectin, or collagen IV. However, kalinin was a much better substrate than laminin-nidogen complex for adhesion of cells of epithelial origin including primary human keratinocytes. Adhesion to kalinin was followed by cell shape changes ranging from rounded to fully spread cells depending on the cell types. The adhesion-promoting activity of kalinin was conformation dependent and was abolished by heat denaturation. mAb BM165 prevented cell adhesion to kalinin but not to other extracellular matrix substrates. However, either complete or partial inhibition was observed with different cells suggesting the existence of at least two cell-binding sites on the kalinin molecule. Experiments inhibiting cell adhesion with function-blocking antiintegrin subunit antibodies indicated that both a3/~l and ot6/~l integrins are involved in the cellular interactions with kalinin, while for cell adhesion to classical mouse Engelbreth-Holm-Swarm laminin only ot6/31 integrins, and not a3/~l, appeared to be functional. Altogether, these results suggest that kalinin may fulfill additional functions than laminin, particularly for epithelial cells.

Laminin 332, composed of the α3, β3 and γ2 chains, is an epithelial-basement membrane specific laminin variant. Its main role in normal tissues is the maintenance of epithelial-mesenchymal cohesion in tissues exposed to external forces, including skin and stratified squamous mucosa. After being secreted and deposited in the extracellular matrix, laminin 332 undergoes physiological maturation processes consisting in the proteolytic processing of domains located within the α3 and the γ2 chains. These maturation events are essential for laminin 332 integration into the basement membrane where it plays an important function in the nucleation and maintenance of anchoring structures. Studies in normal and pathological situations have revealed that laminin 332 can trigger distinct cellular events depending on the level of its proteolytic cleavages. In this review, the biological and structural characteristics of laminin 332 domains are presented and we discuss whether they trigger specific functions.