Epsin 3 is a novel extracellular matrix-induced transcript specific to wounded epithelia.

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.

Cell-specific targeting of caveolin-1 to caveolae, secretory vesicles, cytoplasm or mitochondria.

In commonly used tissue culture cells, caveolin-1 is embedded in caveolae membranes. It appears to reach this location after being cotranslationally inserted into ER membranes, processed in the Golgi and shipped to the cell surface. We now report that caveolae are not the preferred location for caveolin-1 in all cell types. Skeletal muscle cells and keratinocytes target caveolin-1 to the cytosol while in exocrine and endocrine cells it accumulates in the secretory pathway. We also found that airway epithelial cells accumulate caveolin-1 in modified mitochondria. The cytosolic and the secreted forms appear to be incorporated into a soluble, lipid complex. We conclude that caveolin-1 can be targeted to a variety of intracellular destinations, which suggests a novel mechanism for the intracellular traffic of this protein.

Role of matrix metalloproteinases and their inhibition in cutaneous wound healing and allergic contact hypersensitivity.

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.

Keratinocyte collagenase-1 expression requires an epidermal growth factor receptor autocrine mechanism.

In response to cutaneous injury, expression of collagenase-1 is induced in keratinocytes via alpha2beta1 contact with native type I collagen, and enzyme activity is essential for cell migration over this substratum. However, the cellular mechanism(s) mediating integrin signaling remain poorly understood. We demonstrate here that treatment of keratinocytes cultured on type I collagen with epidermal growth factor receptor (EGFR) blocking antibodies or a specific receptor antagonist inhibited cell migration across type I collagen and the matrix-directed stimulation of collagenase-1 production. Additionally, stimulation of collagenase-1 expression by hepatocyte growth factor, transforming growth factor-beta1, and interferon-gamma was blocked by EGFR inhibitors, suggesting a required EGFR autocrine signaling step for enzyme expression. Collagenase-1 mRNA was not detectable in keratinocytes isolated immediately from normal skin, but increased progressively following 2 h of contact with collagen. In contrast, EGFR mRNA was expressed at high steady-state levels in keratinocytes isolated immediately from intact skin but was absent following 2 h cell contact with collagen, suggesting down-regulation following receptor activation. Indeed, tyrosine phosphorylation of the EGFR was evident as early as 10 min following cell contact with collagen. Treatment of keratinocytes cultured on collagen with EGFR antagonist or heparin-binding (HB)-EGF neutralizing antibodies dramatically inhibited the sustained expression (6-24 h) of collagenase-1 mRNA, whereas initial induction by collagen alone (2 h) was unaffected. Finally, expression of collagenase-1 in ex vivo wounded skin and re-epithelialization of partial thickness porcine burn wounds was blocked following treatment with EGFR inhibitors. These results demonstrate that keratinocyte contact with type I collagen is sufficient to induce collagenase-1 expression, whereas sustained enzyme production requires autocrine EGFR activation by HB-EGF as an obligatory intermediate step, thereby maintaining collagenase-1-dependent migration during the re-epithelialization of epidermal wounds.

Collagenase-1 and collagen in epidermal repair.

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.

Induction and repression of collagenase-1 by keratinocytes is controlled by distinct components of different extracellular matrix compartments.

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.

Cell type-specific inhibition of keratinocyte collagenase-1 expression by basic fibroblast growth factor and keratinocyte growth factor. A common receptor pathway.

Collagenase-1 is invariantly expressed by migrating basal keratinocytes in all forms of human skin wounds, and its expression is induced by contact with native type I collagen. However, net differences in enzyme production between acute and chronic wounds may be modulated by soluble factors present within the tissue environment. Basic fibroblast growth factor (bFGF, FGF-2) and keratinocyte growth factor (KGF, FGF-9), which are produced during wound healing, inhibited collagenase-1 expression by keratinocytes in a dose-dependent manner. However, KGF was >100-fold more effective than bFGF at inhibiting collagenase-1 expression, suggesting that this differential signaling is transduced via an FGF receptor that binds these ligands with different affinities. Reverse transcriptase-polymerase chain reaction analysis of human keratinocyte mRNA for fibroblast growth factor receptors (FGFRs) revealed expression of only FGFR-2 IIIb, the KGF-specific receptor, which also binds bFGF with low affinity, and FGFR-3 IIIb, which does not bind bFGF or KGF. FGFRs that bind bFGF with high affinity were not detected. Our results suggest that bFGF and KGF inhibit collagenase-1 expression through the KGF cell-surface receptor (FGFR-2 IIIb). Because bFGF induces collagenase-1 in most cell types, cell-specific expression of FGFR family members may dictate the regulation of matrix metalloproteinases in a tissue-specific manner.

The activity of collagenase-1 is required for keratinocyte migration on a type I collagen matrix.

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 alpha2 integrin subunit but not by antibodies against the alpha1 or alpha3 subunits. We propose that interaction of the alpha2beta1 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.

Modulation of intracellular calcium levels inhibits secretion of collagenase 1 by migrating keratinocytes.

Calcium concentration influences keratinocyte differentiation, and, following injury, keratinocytes move through an environment of changing calcium levels. Because these migrating cells in wounds invariably express collagenase 1, we assessed if modulation of calcium levels regulates collagenase 1 production by primary human keratinocytes. Accurately reflecting the confined spatial pattern of enzyme production seen in vivo, collagenase 1 mRNA was expressed only by keratinocytes migrating from foci of differentiated cells. Treatment with calcium ionophores A23187 or thapsigargin markedly inhibited the basal and phorbol 12-myristate 13-acetate-(PMA) stimulated accumulation of keratinocyte collagenase 1 in the medium but did not affect collagenase 1 production by control or PMA-treated fibroblasts. A23187-mediated inhibition of collagenase 1 protein was not associated with a decrease in mRNA levels but rather was controlled by a selective and reversible block of enzyme secretion. This block in secretion was likely not due to altered protein folding as the proenzyme within A23187-treated cells remained capable of autolytic activation upon treatment with p-aminophenylmercuric acetate. In contrast, 92-kDa gelatinase mRNA and secreted protein levels were coordinately reduced by A23187. Keratin 14 expression, a basal keratinocyte marker, was reduced with PMA treatment, but A23187 did not affect keratin 14 expression. In human wounds, both basal and suprabasal keratinocytes at the migrating front of epidermis stained for keratin 14, but only the basal cells expressed collagenase 1. These data suggest that collagenase 1 production is not necessarily linked with expression of basal cell markers and that modulation of intracellular calcium levels can block secretion of collagenase 1 by keratinocytes which have moved away from the stratum basalis and from their natural substrate.

Thrombin promotion of isometric contraction in fibroblasts: its extracellular mechanism of action.

Isometric force generated by fibroblasts plays an essential role in tissue contraction during normal wound healing and pathologic contractures. Thrombin, a serine protease present in all wounds, has been shown to promote wound healing. The purpose of this study was to determine the extracellular mechanism by which thrombin promotes isometric contraction by fibroblasts in an in vitro collagen lattice model of tissue contraction. The amount of isometric force generated by human fibroblasts can be measured directly with a stabilized collagen lattice attached to a force transducer. Thrombin promoted isometric contraction by human fibroblasts in a dose-dependent manner. In addition, thrombin-promoted isometric contraction is dependent on the enzymatic and anionic binding activity of thrombin, as demonstrated by inhibition with specific enzymatic and anionic binding inhibitors. These results suggest that thrombin may promote isometric contraction by fibroblasts through the enzymatic cleavage of its cell surface receptor, resulting in a new amino terminus that serves as a "tethered ligand" to activate the receptor directly. To test this mechanism of action, a synthetic peptide (SFLLRN) representing the "tethered ligand" region of the activated thrombin receptor was synthesized and examined for its ability to promote isometric contraction by fibroblasts. This peptide promoted fibroblast contraction in a dose-dependent manner. In contrast, a control isomer peptide (FSLLRN), in which the two amino-terminal amino acids were reversed, failed to promote this response. These findings demonstrate that human alpha-thrombin promotes isometric contraction by human fibroblasts and that binding to and cleavage of its cell surface receptor are integral to this response.

Thrombin stimulates fibroblast-mediated collagen lattice contraction by its proteolytically activated receptor.

Fibroblast contraction is proposed to play an important role in tissue contraction during events such as wound healing. Thrombin has been implicated to promote force generation in fibroblasts; however, its extracellular mode of action is unclear. The purpose of this study was to determine the role thrombin and the activation of its receptor plays in promoting the contraction of human fibroblasts in an in vitro collagen lattice contraction assay. Human alpha-thrombin promoted fibroblast contraction in a dose-dependent manner with maximal activity at 0.2 nM. In contrast, both hirudin-alpha-thrombin and D-phenylalanyl-L-propyl-L-arginyl chloromethyl ketone-alpha-thrombin, which lack enzymatic activity, failed to elicit fibroblast contraction. Thus, the enzymatic activity of thrombin appears to be necessary for promotion of fibroblast contraction. Northern analysis confirmed that these human fibroblasts expressed mRNA for the human alpha-thrombin receptor. Moreover, the synthetic peptide (SFLLRNPND-KYEPF) representing the "tethered ligand" portion of the activated alpha-thrombin receptor promoted fibroblast contraction, while a control isomer peptide, in which the first two amino acids were reversed, failed to elicit this response. These findings indicate that alpha-thrombin promotes the contraction of adult human fibroblasts and that cleavage of the human alpha-thrombin receptor is sufficient to produce this response.

Effects of MK-801 on rat primary afferent neurons and fibers.

Recent evidence suggests that glutamate and its N-methyl-D-aspartate (NMDA) receptor may participate in regulating neurite morphology and peptide expression. A previous study from this laboratory showed that treatment with the NMDA receptor antagonist, MK-801, induced an apparent increase in the density of calcitonin gene-related peptide (CGRP)-immunoreactive primary afferent fibers in the dorsal spinal cord of the rat. The present study was undertaken to extend this work by: 1) quantifying the MK-801-induced increase in CGRP immunostaining in the dorsal grey commissure/medial dorsal horn region and 2) examining the effect of MK-801 on the number of CGRP-immunoreactive primary afferent cell bodies in lumbar dorsal root ganglia. Following 7 days of MK-801 treatment, a significant increase (p less than 0.001) in CGRP immunostaining was observed in the dorsal grey commissure/medial dorsal horn. However, after MK-801 treatment, no significant difference was noted in the numbers of CGRP-immunoreactive primary afferent cell bodies in dorsal root ganglia. These data suggest that MK-801 produces significant alterations in the intraspinal projection of CGRP-immunoreactive fibers without inducing immunocytochemically detectable CGRP within a new population of primary afferent neurons.