Non-invasive study of gelatinases in sun-exposed and unexposed healthy human skin based on measurements in stratum corneum.

Gelatinases, which belong to the family of matrix metalloproteinases, degrade various components of skin, and may be involved in photoaging, since they are upregulated by low-dose UV exposure to the skin. However, their behavior in healthy human skin is still unclear. In the present study, gelatinases was specifically detected in stratum corneum (SC) of skin from sun-exposed sites, including the face, in healthy humans, but not in SC of skin from unexposed sites. Following experimental UVB irradiation of the abdomen in volunteers, gelatinases were detected in tape-stripped SC from the site for several weeks, and subsequently disappeared. The appearance of gelatinase in SC after a lag time consistent with SC turnover is considered to reflect upregulation of gelatinase expression in keratinocytes in response to UVB-exposure of the skin. A survey of gelatinases in facial SC samples collected by tape-stripping from the cheeks of 100 healthy women revealed that the enzyme was present in 90% of subjects. These results, taken together, suggest that gelatinase is constantly upregulated by sunlight in the facial epidermis of most women during their daily lives, and may be an etiological factor in photoaging, e.g., by promoting wrinkle formation.

Possible involvement of gelatinases in basement membrane damage and wrinkle formation in chronically ultraviolet B-exposed hairless mouse.

A number of studies indicate that matrix metalloproteinase might be involved in photoaging, but little is known about their direct contribution to ultraviolet-induced histologic and morphologic changes in the skin in vivo. This study reports the relationship between changes of matrix metalloproteinase activities and ultraviolet B-induced skin changes in hairless mouse. The role of matrix metalloproteinase in the skin changes was studied by topical application of a specific matrix metalloproteinase inhibitor. The backs of mice were exposed to ultraviolet B three times a week for 10 wk. Histologic studies showed that the basement membrane structure was damaged, with epidermal hyperplasia, in the first 2 wk of ultraviolet B irradiation, followed by the appearance of wrinkles, which gradually extended in the latter half of the ultraviolet B irradiation period. We observed enhancement of type IV collagen degradation activity, but not collagenase or matrix metalloproteinase-3 activity, in extracts of ultraviolet B-irradiated, wrinkle-bearing skin. Gelatin zymographic analysis revealed that gelatinases, matrix metalloproteinase-9 and matrix metalloproteinase-2, were significantly increased in the extract. In situ zymographic study clarified that the activity was specifically localized in whole epidermis of ultraviolet B-irradiated, wrinkled skin in comparison with normal skin. The activity was induced around the basal layer of the epidermis by a single ultraviolet exposure of at least one minimal erythema dose. Furthermore, topical application of a specific matrix metalloproteinase inhibitor, CGS27023A, inhibited ultraviolet B-induced gelatinase activity in the epidermis, and its repeated application prevented ultraviolet B-induced damage to the basement membrane, as well as epidermal hyperplasia and dermal collagen degradation. Ultraviolet B-induced wrinkles were also prevented by administration of the inhibitor. These results, taken together, suggest that ultraviolet B-induced enhancement of gelatinase activity in the skin contributes to wrinkle formation through the destruction of basement membrane structure and dermal collagen in chronically ultraviolet B-exposed hairless mouse, and thus topical application of matrix metalloproteinase inhibitors may be an effective way to prevent ultraviolet B-induced wrinkle formation.

Urokinase-type plasminogen activator is activated in stratum corneum after barrier disruption.

The plasminogen/plasmin system in epidermis is thought to be the major protease involved in the delay of barrier recovery. However, little is known about the mechanism through which this system is activated. In order to clarify this mechanism, we first determined the distribution of proteolytic activity by using in situ zymography. As a result, plasminogen-activator activity was found to be present in the stratum corneum (SC) after barrier disruption. Next, SC subjected to repeated barrier disruption was collected to identify the protease. The protease was identified as urokinase-type plasminogen activator, because flybrinolytic activity of the collected SC was abolished by addition of anti-urokinase antibody. Urokinase activation in SC was confirmed by means of an in vitro assay, in which the precursor of urokinase (pro-uPA) became active after incubation with the insoluble component of SC homogenate. These findings indicated that urokinase-type plasminogen activator is activated in SC after barrier disruption and this activation might trigger the plasminogen/plasmin system in the epidermis.