The importance of laminin 5 in the dermal–epidermal basement membrane
Introduction
The basement membrane at the dermal–epidermal junction has been proposed to have many functions. As described later, its most obvious function is to tightly link the epidermis to the dermis. Another obvious function of the basement membrane is to determine the polarity of the epidermis and to provide a barrier to epidermal migration. Once the basement membrane has been assembled, the epidermal cells recognize the surface adjacent to the basement membrane as the basal surface. Stratification of the epidermis proceeds, with the proliferating cells remaining attached to the basement membrane and the daughter cells migrating into the upper layers [1], [2], [3]. It is assumed that the basement membrane influences the epidermal differentiation, to maintain the proliferative state of the basal layer. Under normal circumstances, basement membrane prevents the direct contact of epidermal cells with the dermis. However, following injury that penetrates or disrupts the basement membrane, the epidermal cells lose their contact with the basement membrane, and come in contact with naked dermis. Under these conditions, the epithelial cells modify their behavior to cover and close the wound. These behavioral changes include the upregulation of proteolytic enzymes and other changes that accompany conversion to a migratory phenotype.
Other functions of the basement membrane derive from the positioning of the structure between the epidermal cells and the dermal cells. The epidermis and the dermis never function independently [4], [5], [6]. Instead, normal skin homeostasis requires the constant passage of signals back and forth between the two cell types. In general, these signals are small molecules made in one compartment that are thought to diffuse to the opposite compartment. These signals must cross the basement membrane. Components of the basement membrane can selectively facilitate or prevent the passage of these signals. In some cases, the signaling molecules are stored by the basement membrane and only released if the basement membrane is destroyed.
The basement membrane between the epidermis and the dermis contains unique structures that maintain the attachment of the epidermis. The components of the attachment complex provide links to the intracellular intermediate filament network of basal keratinocytes and to the extracellular matrix of the papillary dermis [7]. One of the key components of the anchoring complex is laminin 5 [8]. Past studies have shown that laminin 5 is essential to epidermal attachment, as mutations in the genes encoding the laminin 5 chains underlie the severe blistering phenotype of Herlitz’ junctional epidermolysis bullosa [9], [10]. It is clear that laminin 5 constitutes the anchoring filaments and binds the transmembrane hemidesmosomal integrin α6β4, which is known to be the receptor of laminin 5. With regard to binding of laminin 5 with either components of the basement membrane or of the papillary dermis, it has recently been elucidated that: (1) laminin 5 directly binds type VII collagen which forms the anchoring fibrils that insert into the papillary dermis [11]; and (2) laminin 5 forms a covalent complex with laminin 6 or 7 and this laminin 5–6/7 complex interacts with type IV collagen in the basement membrane through nidogen [12].
It was hypothesized that laminin 5 is a major contributor to epidermal–dermal stability. One corollary to this hypothesis is that laminin 5 might improve epidermal attachment in a variety of healing processes or clinical situations where epidermal–dermal attachment and basement membrane formation might be compromised. Therefore, the effect of laminin 5 during the cell culture of a skin equivalent model, and during keratinocyte sheet grafting has been investigated. The results strongly suggest that laminin 5 promotes epidermal attachment by increasing the rate of basement membrane formation.
Section snippets
Purification of laminin 5
Human keratinocytes were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS). The spent medium was filtered to remove cells. EDTA, PMSF, and N-ethylmaleimide were then added to final concentrations of 5 mM, 50, and 50 μM, respectively, to inhibit endogenous protease activity. To remove non-specific binding by fibronectin, the medium was first passed through gelatin-Sepharose (Pharmacia Biotech AB, Uppsala, Sweden) and then applied to an
Deposition of basement membrane components in cultured skin equivalents
Keratinocytes were cultured on top of dermal equivalents formed from type I collagen and fibroblasts raised to an air–liquid interface. As observed by light microscopy, the keratinocytes formed a stratified squamous epithelium within 7 days with the presence of basal, spinous, granular, and corneal cell layers. Immunohistochemical analysis showed that type IV collagen and the α3 chain of laminin (contained in laminins 5, 6 and 7) were deposited on the dermal–epidermal interface area in 14-day
Discussion
The present study demonstrates that laminin 5 has the potential to accelerate formation of basement membrane in cultured skin equivalents. Addition of exogenous laminin 5 increased the length of lamina densa, which was recognized by an anti-type IV collagen antibody, in a concentration-dependent manner [13]. It was also found that laminin 5 increased the frequency of hemidesmosomal structures at the dermal–epidermal junction and that most laminae densae were associated with the hemidesmosomal
Conclusion
In vitro and in vivo evidence indicates that laminin 5 participates in several essential functions in skin biology. From published reports [8], [11], [12] and the present studies, it was concluded that the function of laminin 5 is as follows. Laminin 5 has two functional domains which can bind integrins of basal keratinocytes and type VII collagen, forming anchoring fibrils, and the anchoring complex provides an essential link between the epidermal basal cells and the papillary dermis. This
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