We have shown in a variety of human wounds that collagenase-1 (MMP-1), a matrix metalloproteinase that cleaves fibrillar type I collagen, is invariably expressed by basal keratinocytes migrating across the dermal matrix. Furthermore, we have demonstrated that MMP-1 expression is induced in primary keratinocytes by contact with native type I collagen and not by basement membrane proteins or by other components of the dermal or provisional (wound) matrix. Based on these observations, we hypothesized that the catalytic activity of MMP-1 is necessary for keratinocyte migration on type I collagen. To test this idea, we assessed keratinocyte motility on type I collagen using colony dispersion and colloidal gold migration assays. In both assays, primary human keratinocytes migrated efficiently on collagen. The specificity of MMP-1 in promoting cell movement was demonstrated in four distinct experiments. One, keratinocyte migration was completely blocked by peptide hydroxymates, which are potent inhibitors of the catalytic activity of MMPs. Two, HaCaTs, a line of human keratinocytes that do not express MMP-1 in response to collagen, did not migrate on a type I collagen matrix but moved efficiently on denatured type I collagen (gelatin). EGF, which induces MMP-I production by HaCaT cells, resulted in the ability of these cells to migrate across a type I collagen matrix. Three, keratinocytes did not migrate on mutant type I collagen lacking the collagenase cleavage site, even though this substrate induced MMP-1 expression. Four, cell migration on collagen was completely blocked by recombinant tissue inhibitor of metalloproteinase-1 (TIMP-1) and by affinity-purified anti–MMP-1 antiserum. In addition, the collagen-mediated induction of collagenase-1 and migration of primary keratinocytes on collagen was blocked by antibodies against the α2 integrin subunit but not by antibodies against the α1 or α3 subunits. We propose that interaction of the α2β1 integrin with dermal collagen mediates induction of collagenase-1 in keratinocytes at the onset of healing and that the activity of collagenase-1 is needed to initiate cell movement. Furthermore, we propose that cleavage of dermal collagen provides keratinocytes with a mechanism to maintain their directionality during reepithelialization.

Normal wounds can heal by secondary intention (epidermal migration to cover a denuded surface) or by approximation of the wound edges (e.g., suturing). In healing by secondary intention, epidermis-derived MMPs are important. Keratinocyte migration begins within 3-6 hr post injury, as basal cells detach from underlying basal lamina and encounter a dermal substratum rich in type I collagen. Cell contact with type I collagen in vitro stimulates collagenase-1 expression, which is mediated by the alpha 2 beta 1 integrin, the major keratinocyte collagen-binding receptor. Collagenase-1 activity alone is necessary and sufficient for keratinocyte migration over a collagen subsurface. Stromelysins-1 and -2 are also found in the epidermis of normal acute wounds. Stromelysin-2 co-localizes with collagenase-1 and may facilitate cell migration over non-collagenous matrices of the dermis. In contrast, stromelysin-1 is expressed by keratinocytes behind the migrating front and which remain on basal lamina, i.e., the proliferative cell population. Studies with stromelysin-1-deficient mice that suggest this MMP plays a role in keratinocyte detachment from underlying basement membrane to initiate cell migration. In chronic ulcers, MMP levels are markedly elevated, in contrast to their precise temporal and spatial expression in acute wounds. Both collagenase-1 and stromelysin-1 are found in fibroblasts underlying the nonhealing epithelium, and stromelysin-1 expression is especially prominent. Two key questions underlie the use of MMP inhibitors and wound healing: (1) will these agents impair normal reepithelialization in wounds that heal by secondary intention; and (2) can MMP inhibitors be effective therapy for chronic ulcers? The answer to neither is known. Batimastat and marimastat appear not to interfere with normal wound healing, but only in sutured surgical wounds, a situation in which MMP expression has practically no role. We also show the first example of an in vivo immune response, contact hypersensitivity, which is dependent upon MMP activity. Using gene-deficient mice, we demonstrate that stromylysin-1 (MMP-3) is required for sensitization, whereas gelatinase B (MMP-9) is required for timely resolution of the reaction to antigenic challenge.

In all forms of cutaneous wounds, collagenase-1 (matrix metalloproteinase-1 (MMP-1)) is invariably expressed by basal keratinocytes migrating over the dermal matrix. We report that native type I collagen mediates induction of MMP-1 by primary human keratinocytes. Collagen-mediated induction of MMP-1 was rapid, being detected 2 h after plating, and was transcriptionally regulated. As demonstrated by in situ hybridization, only migrating keratinocytes expressed MMP-1, suggesting that contact with collagen is not sufficient to induce MMP-1 expression in keratinocytes; the cells must also be migrating. Upon denaturation, type I collagen lost its ability to induce MMP-1 expression but still supported cell adhesion. Other dermal or wound matrix proteins, such as type III collagen, fibrin, and fibronectin, and a mixture of basement membrane proteins did not induce MMP-1 production. In the presence of collagen, laminin-1 inhibited induction of MMP-1 but laminin-5 did not. Taken together, these observations suggest that as basal keratinocytes migrate from the basal lamina onto the dermal matrix contact with native type I collagen induces MMP-1 expression. In addition, our findings suggest that re-establishment of the basement membrane and, in particular, contact with laminin-1 provides a potent signal to down-regulate MMP-1 production as the epithelium is repaired.Normal cutaneous wound healing, as well as healing in essentially all tissues, involves an orderly progression of events to re-establish the integrity of the injured tissue. The initial injury starts a programmed series of interdependent yet functionally separate responses, such as re-epithelialization and epithelial proliferation, inflammation, angiogenesis, fibroplasia, matrix accumulation, and eventually resolution. During each stage in this process, proteinases are needed to remove or remodel extracellular matrix components in both the epithelial and interstitial compartments to accommodate cell migration and tissue repair (1).In a thorough examination of normally healing wounds and of a variety of chronic ulcers, we found that collagenase-1, a member of the matrix metalloproteinase (MMP) 1 family with the ability to cleave fibrillar collagens type I, II, and III at a specific locus in their triple helical domain, is invariably expressed by basal keratinocytes at the edge of repairing tissue (2-4). In all, we have examined Ͼ100 different human skin specimens, representing a variety of chronic ulcers, blisters, and normally healing wounds, and in each sample with injury that breached the basement membrane, collagenase-1 was prominently and invariantly expressed by basal keratinocytes migrating over the dermal wound bed. The invariable expression of collagenase-1 in all forms of wounds and the confinement of its expression to periods of active re-epithelialization suggest that this enzyme plays a critical role in keratinocyte function during healing. Indeed, we recently demonstrated that keratinocyte migration on a native collagen matrix requires the catalyti...

An invariable feature of wounded skin, whether a normally healing or chronic lesion, is the expression of collagenase-1 by migrating basal keratinocytes. Collagenase-1 is a member of the matrix metalloproteinase family of enzymes and is the principal human enzyme which cleaves native fibrillar collagen. Following injury, basal keratinocytes move from the basement membrane and interact with new connective tissue proteins in the dermis and wound bed. Contact with native type I collagen, the most abundant protein in the dermis, induces expression of collagenase-1. This metalloproteinase cleaves collagen, thereby altering its structure and, hence, the affinity to which cells bind it. Thus, collagenase-1 serves a beneficial role in wound healing by facilitating the movement of keratinocytes over the collagen-rich dermis during reepithelialization.

Using an in vitro model of keratinocyte activation by the extracellular matrix following injury, we have identified epsin 3, a novel protein closely related to, but distinct from previously described epsins. Epsin 3 contains a domain structure common to this gene family, yet demonstrates novel differences in its regulation and pattern of expression. Epsin 3 mRNA and protein were undetectable in keratinocytes isolated from unwounded skin, but induced in cells following contact with fibrillar type I collagen. The native triple helical structure of collagen was required to mediate this response as cells failed to express epsin 3 when plated on gelatin. Consistent with the reported function of other epsins, epsin 3 was evident in keratinocytes as punctate vesicles throughout the cytoplasm that partially co-localized with clathrin. In addition, epsin 3 exhibited nuclear accumulation when nuclear export was inhibited. In contrast to other known epsins, epsin 3 was restricted to keratinocytes migrating across collagen and down-regulated following cell differentiation, suggesting that expression was spatially and temporally regulated. Indeed, epsin 3 was localized specifically to migrating keratinocytes in cutaneous wounds, but not found in intact skin. Intriguingly, Northern hybridization and reverse transcriptase-polymerase chain reaction experiments indicated that epsin 3 expression was restricted to epithelial wounds or pathologies exhibiting altered cell-extracellular matrix interactions. Thus, we have identified a novel type I collagen-induced epsin that demonstrates structural and behavioral similarity to this gene family, yet exhibits restricted and regulated expression, suggesting that epsin 3 may serve an important function in activated epithelial cells during tissue morphogenesis.The epidermis consists of a multilayered epithelial sheet that provides a physical barrier against the outside environment and heals in response to injury. In unwounded skin, basal keratinocytes reside on a basement membrane that physically separates these cells from the underlying dermal connective tissue rich in type I collagen. While in contact with this extracellular matrix (ECM), 1 keratinocytes express a programmed subset of genes that promotes proliferation and differentiation. Following injury, however, keratinocytes from the surrounding tissue are activated by exposure to ligands released into the wound site and by contact with ECM macromolecules (1-3). Keratinocyte activation, which typically begins 18 -24 h prior to the onset of migration, occurs as cells at the wound edge enhance the expression of genes that support repair of the tissue defect (4, 5). Many of the genes up-regulated in keratinocytes during healing, including secreted proteinases and integrin receptors, enable a fundamental shift in cell behavior that supports sustained and directed migration to re-establish the normal cytoarchitecture of the skin (3). The preponderance of studies to date attempting to identify signals that stimulate keratinocyte activation...