Structural analysis of the alpha(2) integrin I domain/procollagenase-1 (matrix metalloproteinase-1) interaction.

Previous studies have established that ligation of keratinocyte alpha(2)beta(1) integrin by type I collagen induces expression of matrix metalloproteinase-1 (MMP-1) and that MMP-1 activity is required for the alpha(2)beta(1) integrin-dependent migration of primary keratinocytes across collagenous matrices. We now present evidence that MMP-1 binds the alpha(2)beta(1) integrin via the I domain of the alpha(2) integrin subunit. Using an enzyme-linked immunosorbent assay with purified human MMP-1 and recombinant alpha(2) integrin I domain, we showed that the alpha(2) integrin I domain specifically bound in a divalent cation-dependent manner to both the pro and active forms of MMP-1, but not to MMP-3 or MMP-13. Although both the I domain and MMP-1 bind divalent cations, MMP-1 bound, in a divalent cation-dependent manner, to alpha(2) integrin I domains containing metal ion-dependent adhesion sites motif mutations that prevent divalent cation binding to the I domain, demonstrating that the metal ion dependence is a function of MMP-1. Using a series of MMP-1-MMP-3 and MMP-1-MMP-13 chimeras, we determined that both the linker domain and the hemopexin-like domain of MMP-1 were required for optimal binding to the I domain. The alpha(2) integrin/MMP-1 interaction described here extends an emerging paradigm in matrix biology involving anchoring of proteinases to the cell surface to regulate their biological activities.

Pro-collagenase-1 (matrix metalloproteinase-1) binds the alpha(2)beta(1) integrin upon release from keratinocytes migrating on type I collagen.

In injured skin, collagenase-1 (matrix metalloproteinase-1 (MMP-1)) is induced in migrating keratinocytes. This site-specific expression is regulated by binding of the alpha(2)beta(1) integrin with dermal type I collagen, and the catalytic activity of MMP-1 is required for keratinocyte migration. Because of this functional association among substrate/ligand, receptor, and proteinase, we assessed whether the integrin also directs the compartmentalization of MMP-1 to its matrix target. Indeed, pro-MMP-1 co-localized to sites of alpha(2)beta(1) contacts in migrating keratinocytes. Furthermore, pro-MMP-1 co-immunoprecipitated with alpha(2)beta(1) from keratinocytes, and alpha(2)beta(1) co-immunoprecipitated with pro-MMP-1. No other MMPs bound alpha(2)beta(1), and no other integrins interacted with MMP-1. Pro-MMP-1 also provided a substrate for alpha(2)beta(1)-dependent adhesion of platelets. Complex formation on keratinocytes was most efficient on native type I collagen and reduced or ablated on denatured or cleaved collagen. Competition studies suggested that the alpha(2) I domain interacts with the linker and hemopexin domains of pro-MMP-1, not with the pro-domain. These data indicate that the interaction of pro-MMP-1 with alpha(2)beta(1) confines this proteinase to points of cell contact with collagen and that the ternary complex of integrin, enzyme, and substrate function together to drive and regulate keratinocyte migration.

Serotonin-dependent collagenase transcription in myometrial cells requires extended AP-1 site.

Primary cultures of uterine smooth muscle cells from post-partum rats express interstitial collagenase in response to serotonin and the serotonin-dependent production of interleukin-1 (IL-1) [Wilcox, B.D., Dumin, J.A. and Jeffrey, J.J. Serotonin regulation of interleukin-1 messenger RNA in rat uterine smooth muscle cells. Relationship to the production of interstitial collagenase J. Biol. Chem., 269, (1994a), 29658]. Transient transfections of these cells indicate that rat collagenase transcription is regulated via a proximal consensus AP-1 site within an extended palindrome. Mutation of either the AP-1 site or extended palindrome (EP) decreases promoter activity to approximately 30% of the wild-type. Electrophoretic mobility shift assays reveal the binding of smooth muscle cell nuclear proteins to the AP-1 EP. This binding is barely detectable after mutation of the EP and is completely eliminated by mutation of the AP-1 heptamer. Competition experiments demonstrate that binding to the AP-1 EP is specific and of higher affinity than binding to oligonucleotides containing a mutated EP. Binding to the AP-1 EP is higher when smooth muscle cells are cultured in the presence of serotonin than in its absence. Although IL-1 is required for collagenase transcription, binding to the AP-1 EP appears to be IL-1-independent. FosB, Fra-2, c-Jun, JunB and, most abundantly, JunD bind the AP-1 EP in the absence and presence of serotonin. In contrast, Fra-1 expression and binding are serotonin-dependent suggesting that the activation of Fra-1 may be a key component of collagenase transcriptional activation.

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.

Serotonin-mediated production of interstitial collagenase by uterine smooth muscle cells requires interleukin-1alpha, but not interleukin-1beta.

The activation of the gene for interstitial collagenase in myometrial smooth muscle cells is absolutely dependent upon the presence of serotonin. Our previous studies investigating the mechanisms of this induction demonstrated that the mRNAs of both interleukin-1 (IL-1) isoforms, IL-1alpha and IL-1beta, are induced by serotonin and that the induction of IL-1 is required for the subsequent induction of collagenase. These data provided compelling evidence that serotonin-induced IL-1 acts via an autocrine loop in activating the collagenase gene. The experiments described here were designed to examine the potential role of each IL-1 isoform in collagenase production by using neutralizing antisera specific to each isoform of the cytokine. The antisera were examined for their ability to inhibit the serotonin-dependent production of the mRNA for collagenase and of the cytokines themselves. Neutralizing antiserum against IL-1alpha, but not against IL-1beta, inhibited the induction of the mRNA for collagenase and of the mRNAs for both IL-1alpha and IL-1beta. Western analysis indicated that detectable levels of IL-1alpha protein, but not that of IL-1beta, are produced at the time of serotonin-dependent collagenase induction. In contrast, significant levels of IL-1beta protein are detected only when bacterial lipopolysaccharide is added to the cells. Taken together, the results of our study indicate that IL-1alpha, but not IL-1beta, plays an obligatory role in multiple serotonin-mediated gene regulations in the myometrial smooth muscle cell. In addition, the data suggest that IL-1beta production has the potential for modifying myometrial function in pathological settings, particularly that of uterine infection.

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.

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.

Serotonin regulation of interleukin-1 messenger RNA in rat uterine smooth muscle cells. Relationship to the production of interstitial collagenase.

Previous studies have shown that the production of interstitial collagenase by rat myometrial smooth muscle cells is dependent on serotonin. These cells fail to produce collagenase early in culture, however, and produce the enzyme only 8-12 days after confluence. During the early quiescent period, collagenase production can be induced by low concentrations of bacterial endotoxin. Under these conditions, interleukin (IL)-1 alpha and IL-1 beta mRNAs increase coincident with collagenase and collagenase mRNA. Serotonin removal decreases IL-1 alpha and IL-1 beta mRNAs, and effect that is rapidly reversed upon readdition of serotonin. Conversely, serotonin-dependent increases in IL-1 mRNAs are blocked by progesterone. Experiments with 5-HT2 receptor agonists and antagonists indicate that induction is mediated by the 5-HT2 receptor subtype. In serotonin-treated cells late in culture, IL-1 mRNAs increase coincident with the production of collagenase. Similarly, exogenous IL-1 fully substitutes for lipopolysaccharide in stimulating myometrial cells to produce collagenase early in culture. Cells treated with IL-1 receptor antagonist fail to make IL-1 mRNAs or collagenase but produce collagenase and IL-1 mRNAs following antagonist removal. These results indicate that serotonin-dependent IL-1 production by the myometrial cell is required for collagenase production and that IL-1 participates in its own production via an autocrine mechanism.