In many heritable skin diseases the characteristic feature is loss of the epidermal-dermal junction. An example is the skin disorder known as recessive dystrophic epidermolysis bullosa (RDEB), where the loss is associated with increased expression of collagenase by the skin cells. However, skin fibroblast collagenase cleaves native fibrillar collagens I, II and III at a single point only. Therefore, collagenase alone cannot be the only causative component in RDEB. Accordingly, other proteolytic enzymes, specifically gelatinase and stromelysin, are probably involved as well. Of note is that gelatinase degrades denatured collagens, such as those released after collagenase digestion, and that stromelysin is thought to be the physiologically relevant enzyme which activates latent collagenase. Thus, there are several reasons to carry out detailed studies on the expression of gelatinase, stromelysin, and collagenase in cultured RDEB skin fibroblasts. To this end, we have recently developed cloned complementary DNA (cDNA) probes for human collagenase and stromelysin. In addition, work in in progress to develop similar cDNA probes for human gelatinase. In the first phase of the proposed research, we will use these cDNA probes to monitor and quantitate the messenger RNA levels and study the expression of these enzymes in cultured skin fibroblasts from normal and RDEB patients. In the second phase, we will determine the structure for RDEB collagenase and examine the properties of the gene. It is hoped that through this work we will be able to define additional biochemical markers useful in the diagnosis and treatment of the RDEB skin disorder. In the third phase of this research, we will study the mechanisms involved in the regulation of collagenase, gelatinase and stromelysin genes using appropriate gene elements linked to reporter genes and transfected into cultured human skin fibroblasts.
The aim of these experiments is to provide a detailed picture of the gene structures and regulatory regions that are important in their controlled expression. For this purpose, we have recenlty isolated human genes for collagenase and stromelysin. We are convinced that studies which first characterize the regulation of these genes in normal circumstances will provide a solid basis for understanding abnormal regulation of proteolytic enzymes that may operate in skin disorders such as RDEB.
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