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In normal human epidermal keratinocytes (NHEK) proteolytic detachment from the substrate induces a complex activation cascade including expression of new proteins, morphological alterations, and the onset of migration for epidermal regeneration. By subtractive cloning we have shown that L6, a four-transmembrane protein, is newly expressed after proteolytic keratinocyte detachment. In this study, we have generated a novel anti-L6 antibody (clone HD-pKe#104-1.1) and investigated L6 expression regulation in vitro and in vivo as well as L6 function in keratinocyte migration. Dispase-mediated detachment induced L6 expression in NHEK at the mRNA and protein level. Immunohistology of skin biopsies displayed a strong expression of L6 in follicular epidermis and epidermolytic lesions of autoimmune bullous dermatoses (bullous pemphigoid, pemphigus vulgaris), but not in normal interfollicular epidermis. In contrast to normal keratinocytes, HaCaT cells showed constitutive L6 expression, indicating a constitutively active phenotype. After artificial wounding of confluent HaCaT cultures, anti-L6 antibody strongly impaired cell migration velocity and migratory reepithelization of the defect, indicating L6 involvement in keratinocyte migration. These findings suggest that L6 is an important activation-dependent regulator of keratinocyte function and epidermal tissue regeneration., (Copyright 2001 Academic Press.)

To analyze the inhibitor of DNA-binding type 1 (ID1) in the human epidermis and in cultured keratinocytes we generated and characterized ID1-specific monoclonal antibodies. Immunohistological studies on human skin biopsies revealed that ID1 is not detectable in normal human epidermis but in lesional epidermis of bullous pemphigoid. In the latter case we found ID1 in the cytoplasm of basal and proximal suprabasal keratinocytes. Cultured normal human epidermal keratinocytes displayed ID1 in the cytoplasm; upon differentiation into a multilayered keratinocyte sheet, ID1 was no longer detectable. It was reexpressed after dispase-mediated detachment of the keratinocyte cultures from the growth substratum. In this case ID1 was localized to the cytoplasm and the nucleus. Our data indicate that after epidermal injury-in our case loss of cell-matrix contact-ID1 is upregulated in affected keratinocytes. In view of the ID1 function in other cell types, we speculate that ID1 facilitates the transition from the resting to the migrating and proliferating keratinocyte required for efficient repair of epidermal lesions by reepithelialization. Taken together we suggest that ID1 is an important player in epidermal (patho-)physiology., (Copyright 2001 Academic Press.)

Keratinocytes synthesize and secrete urokinase-type plasminogen activator (uPA), which is bound in an autocrine manner to a specific receptor (uPA-R, CD87) at their surface. Plasminogen, which is also bound to membrane binding sites, is readily activated by uPA-R-bound uPA. Thus, plasmin for proteolysis of pericullular glycoproteins is provided. While uPA-R and uPA are at low to undetectable levels in keratinocytes of the normal epidermis, both compounds are upregulated in migrating keratinocytes during reepithelialization of epidermal defects and in affected keratinocytes of various epidermal disorders, including bullous dermatoses. We have hypothesized that the disturbance of cell/matrix interactions--a common feature of these diverse pathological situations--induces uPA/uPA-R. Accordingly, we explored whether the dispase-mediated detachment of cultured keratinocytes, which have formed a multilayered epidermis-like structure in vitro, induced uPA and uPA-R. We found increases in uPA secretion, cell-associated uPA activity, and uPA- and uPA-R-antigen in keratinocytes upon dispase-mediated detachment from their growth substratum. The increase was preceded by an increase in uPA-R- and uPA-specific mRNA, which was not observed when the proteinase inhibitor phosphoramidon was added together with dispase. In conclusion, we present evidence that experimental detachment with dispase provides signals for the concomitant upregulation of uPA-R and uPA. The findings support the hypothesis that cell/matrix interactions may influence the expression of the cell surface-associated PA system in human keratinocytes.

Keratinocytes synthesize and secrete urokinase-type plasminogen activator (uPA) which is bound in an autocrine manner to a specific receptor (uPA-R) at the keratinocyte surface. Plasminogen that is also bound to specific membrane binding sites is readily activated by uPA-R-bound uPA. Thus, plasmin is provided for proteolysis of pericellular glycoproteins. The expression of uPA and the uPA-R is confined to migrating keratinocytes during epidermal wound healing, rather than to keratinocytes of the normal epidermis. The regulatory factors of uPA/uPA-R expression in keratinocytes remained largely elusive. Proinflammatory cytokines, such as tumor necrosis factor-alpha (TNF-alpha) or interleukin-1 beta (IL-1 beta), are present in epidermal wounds. We have therefore tested IL-1 beta and TNF-alpha for their influence on surface-associated plasminogen activation in a human keratinocyte cell line (HaCaT) as well as in primary cultures of normal human epidermal keratinocytes. Both cytokines induced the secretion of uPA into the culture supernatants and a concomitant increase in uPA activity as well as in uPA and uPA-R antigen at the cell surface. The increase was preceded by an increase in specific mRNA. The induction was accompanied by an accelerated uPA-dependent and plasmin-mediated detachment of HaCaT cells from the culture substratum. Taken together, the proinflammatory cytokines IL-1 beta and TNF-alpha induced a coordinated increase in uPA and uPA-R as well as increased pericellular plasmin-mediated proteolysis in human epidermal keratinocytes. This function might be an element of the molecular cell biological events during epidermal wound healing.

Cultured keratinocytes resemble migrating keratinocytes under conditions of reepithelialization during wound healing. Such keratinocytes express urokinase-type plasminogen activator (uPA) and its specific receptor (uPA receptor). Receptor-bound uPA activates plasminogen, thus providing plasmin for pericellular proteolysis. uPA is regulated by the plasminogen activator inhibitors PAI-1 and PAI-2. As indicated by immunohistology, neither uPA nor uPA receptor is expressed in normal epidermis. Thus, the down-regulation of uPA and uPA-receptor expression in keratinocytes appears to be an important event in epidermal healing and restoration of a normal epidermal tissue architecture. We have addressed this matter by using a culture and differentiation system for keratinocytes in vitro. Keratinocytes were grown in organotypic cocultures for 4, 7, and 14 days. Frozen sections were analyzed with indirect immunofluorescence staining and overlay zymography, the latter detecting activity of plasminogen activators. While tPA and PAI-1 stainings were consistently negative over the entire observation period, uPA and uPA receptor were expressed by basal keratinocytes at Days 4 and 7, but not at Day 14. Accordingly, overlay zymography revealed uPA activity at Days 4 and 7. PAI-2 was found throughout the entire observation period, but with varying distribution: at Days 4 and 7 all suprabasal keratinocytes stained positive for PAI-2. At Day 14, PAI-2-specific stainings were confined to the uppermost cells of the stratum spinosum. Our data demonstrate that uPA and uPA receptor, which are up-regulated in cultured keratinocytes, are down-regulated upon restoration of an epidermis-like structure. The distribution of PAI-2 varied over the observation period and at Day 14 resembled the distribution of PAI-2 in normal epidermis. Taken together, keratinocytes in organotypic coculture behave like keratinocytes in healing wounds in vivo with respect to the expression of the plasminogen activator system.