Recent understanding of factors regulating collagenase production facilitates new approaches to the study of recessive dystrophic epidermolysis bullosa (RDEB), in which collagenase production is elevated, and other forms of epidermolysis bullosa (EB). 1) Following leads suggested by this new information, we wish to determine whether RDER fibroblasts have a defect in regulation of production of collagenase such that they appear constitutively stimulated and therefore not only secrete high levels of collagenase but fail to respond to stimulation. In order to evaluate this hypothesis, we will determine whether RDEB fibroblasts show reduced sensitivity compared to normal fibroblasts on exposure to interleukin-1 and other stimulators of collagenase production, including such agents produced by keratinocyte cultures. We will examine culture supernatants for excessive production of factors capable of stimulating production of collagenase. Since extracellular matrix, especially collagen, can increase collagenase production, we will assess collagenase modulation in normal and affected cells plated with or without collagen or fibronectin and on collagen gels. We have recently documented production of collagenase by cultures of human keratinocytes exposed to the phorbol ester, TPA. We will compare production of collagenase in keratinocytes from controls and patients with various types of EB. We will search for physiologic stimuli for the production of collagenase by keratinocytes and will characterize the collagenase. We will examine supernatants from EB keratinocytes in an assay designed to detect activity dissociating keratinocytes, as occurs in vivo. 2) Since abnormal cytoskeletal regulation may underly elevated collagenase in RDEB fibroblasts, we will use antibodies to vinculin, actin, talin, spectrin, and desmoplakin to examine the cytoskeleton of normal and affected cells by immunofluorescence, immunoblotting, and immunoprecipitation. We have recently shown alterations in distribution of vinculin in keratinocytes after an increase in Ca+ in the medium from 0.1 to 1.0 mM and will similarly examine the keratinocyte cytoskeleton under these two conditions in cells from patients with other types of EB. 3) Finally, we will explore collagenase production by an in vitro model of skin made from a fibroblast-populated. Collagen lattice covered by added cultured keratinocytes. We will attempt to produce basement membrane antigens and lamina densa in vitro with this model. These studies may help elucidate the abnormal regulation of collagenase in EB.