In response to cutaneous injury, expression of collagenase-1 is induced in keratinocytes via α2β1 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-β1, and interferon-γ 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.

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-1 alpha and IL-1 beta, 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-1 alpha, but not against IL-1 beta, inhibited the induction of the mRNA for collagenase and of the mRNAs for both IL-1 alpha and IL-1 beta, Western analysis indicated that detectable levels of IL-1 alpha protein, but not that of IL-1 beta, are produced at the time of serotonin-dependent collagenase induction. In contrast, significant levels of IL-1 beta protein are detected only when bacterial lipopolysaccharide is added to the cells. Taken together, the results of our study indicate that IL-1 alpha, but not IL-1 beta, plays an obligatory role in multiple serotonin-mediated gene regulations in the myometrial smooth muscle cell. In addition, the data suggest that IL-1 beta production has the potential for modifying myometrial function in pathological settings, particularly that of uterine infection.