The studies outlined in this proposal will pursue the compelling finding that UVA irradiation stimulates collagenase production in cultured human skin fibroblasts and further elucidate the biochemical and molecular mechanisms of connective tissue damage in human skin caused by ultraviolet radiation (UVR). Photodamage is seen in most fair-skinned individuals by the fifth decade of life and is manifest clinically by wrinkled, lax and fragile skin and histologically by accumulation of elastotic material and collagen degradation. The biochemical mechanisms responsible for these connective tissue changes are poorly understood. Degradation of connective tissue is mediated, in part, by enzymes termed matrix metalloproteinases (MMPs), which include interstitial collagenase and type IV collagenases. Preliminary studies have demonstrated a dose dependent increase in interstitial collagenase and collagenase mRNA in cultured human skin fibroblasts irradiated with ultraviolet A. These preliminary data support the hypothesis that UVR induces expression of MMPs, resulting in connective tissue damage. This study will examine the effect of UVR on the synthesis of MMPs in vitro and in vivo. The effect of long- and short-wave UVR, as well as solar- simulating radiation, on the production and expression of MMPs, and their common inhibitor, tissue inhibitor of metalloproteinase (TIMP), by human fibroblasts and keratinocytes in culture will be investigated. A comprehensive examination of interstitial collagenase, 72-kD and 92-kD type IV collagenase and TIMP regulation in vitro will be undertaken using functional, immunological and molecular biological methods. Specific experiments will address these questions: a) Is cellular response dependent on cellular age or on past UVR exposure? b) Do extracellular matrix molecules alter the cellular response to UVR of MMPs and TIMP? c) Will inhibitors of collagenase production modify cellular responses to UVR? d) Does UVR indirectly regulate MMP and TIMP expression via cytokines? The effect of UVR on MMP and TIMP synthesis and gene expression in vivo will be studied in human skin irradiated with UVR using immunohistochemical and in situ hybridization techniques, as well as in human skin grafted onto athymic mice. The effect of topically applied inhibitors of collagenase and sunscreens on UV-induced MMP and TIMP expression will be studied in human subjects and the effect of orally administered inhibitors will be studied using the athymic mouse model.
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