CONTROL OF COLLAGENASE IN HUMAN FIBROBLAST CULTURES

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Collagenase is critical in initiating the degradation of interstitial collagens in a variety of morphogenetically important events. We shall use cultured skin fibroblasts to examine the effects of collagenase-stimulatory proteins, including a 20 kDa epidermal cytokine and platelet-derived growth factor, on the expression of collagenase by these target cells. We shall assess whether the response to these peptide agonists is developmentally modulated by examining (i) collagenase expression as reflected by enzyme activity, synthesis of immunoreactive enzyme protein and alterations in hybridizable mRNA and (ii) the mitogenic response of the target fibroblasts to the cytokines. We shall use cultured fibroblasts from various stages of development including from the genetic "aging syndromes" to address whether these two responses are reflective of separate mechanistic pathways. These investigations of the mechanisms for the effects of cytokines on collagenase expression will be complemented by defining if the gene product itself, collagenase, can in any way affect its own synthesis, by probing the effects of other agonists on normal cells and cells from various types of epidermolysis bullosa, and by examining the effects at a nucleic acid level of agents such as retinoids and glucocorticoids that inhibit collagenase synthesis. Since there is evidence for a structurally mutant form of collagenase in recessive dystrophic epidermolysis bullosa (RDEB), we shall employ cultured fibroblasts from this disease as a source for collagenase mRNA to clone the putatively mutant collagenase gene. As a probe for the isolation and initial characterization of the RDEB cDNa clones we shall employ a full-length cDNA clone for normal human skin collagenase pCol 185.2. Our goal is to focus intensely on defining the site(s) of mutation leading to the structurally altered protein, so that we can better understand regulatory mechanisms, provide improved diagnosis and genetic counseling, and devise rational therapeutic modalities in this devastating genodermatosis.