Elastin is the resilient protein of connective tissue, formed from a soluble, hydrophobic precursor and extensively crosslinked to provide remarkable durability. A number of pathological processes are linked to elastic fiber abnormalities. In this renewal application, experimental approaches focus on the normal and abnormal regulation of elastin abundance in human and animal tissues. Human genetic diseases involving elastin are phenotypically stable to analysis in fibroblast culture, and several strains show altered elastin production, suggesting the cause some elastic tissue pathology is linked to regulation of elastin production. Recent data from this laboratory at both protein production and mRNA levels estimated by blotting and in situ hybridization reveal the complex influence of cytokines, glucocorticoids, and ascorbic acid on elastin expression. Cytokines, in particular, are likely to influence behavior of elastin-producing cells during development, growth, and response to injury. Transforming growth factor-beta (TGF-beta) strongly stimulates elastin in many cell types, while basic fibroblast growth factor (bFGF) and transforming growth factor-alpha reduce elastin expression and antagonize the TGF-beta effect. Glucocorticoids suppress elastin expression in normal, adult cells but have no or weak positive effects in keloid and fetal skin cells. Ascorbate reduces elastin mRNA levels rapidly. Differences in regulatory responses are found in abnormal fibroblast strains. Experimental protocols will focus on the evaluation of cytokine and hormonal regulation in normal and diseased skin cells. Studies of elastin gene function will concentrate on the role of mRNA stability in regulation of elastin expression, with a hypothesis that untranslated sequences in elastin mRNA affect transcript stability and are responsible for differences in production evoked by cytokine treatment. Segments of the human elastin gene containing candidate sequences isolated during the previous grant period will be introduced into mammalian expression plasmids and tested for their ability to modulate transcript abundance and reporter gene expression in fibroblasts transiently transfected by liposome-mediated transfer. Experiments are proposed to discern the molecular basis of abnormal regulation, whether it be in cis- or trans-acting elements. A second gene expressed at high levels in elastic tissue will be characterized with respect to structure and expression patterns. Regulation of elastin gene expression is critical during development and repair of skin, lung, and vascular tissue, given that the structural gene is now isolated, these proposed studies hopes to define the cellular mechanisms which impinge upon elastin gene activity.
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