RABBIT COLLAGENASE GENE REGULATION BY IL-1

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The long term goal of this research is to gain a detailed understanding of the molecular mechanisms that regulate collagenase (matrix metalloproteinase 1, MMP-1) expression in response to IL- 1. IL-1 is a potent physiological inducer of collagenase mRNA, and under pathological conditions collagenase, the only enzyme capable to degrade interstitial collagens at neutral pH, is instrumental in cartilage destruction in rheumatoid arthritis. Because of the central role IL-1 plays in the pathophysiology of connective tissue breakdown it is an important area of investigation. An understanding of the transcriptional and post-transcriptional mechanisms may influence the development of therapies designed to abrogate IL-1 induced collagenase gene expression. Therefore, the transcriptional and post- transcriptional mechanisms that regulate collagenase gene expression in response to IL-1 will be investigated using a rabbit model of synovial fibroblast cultures. Both experimentally and on the nucleotide sequence level this model mimics primary cultures of human cells from patients with rheumatoid arthritis. IL-1 responsive elements in the rabbit collagenase gene will be identified by transient transfection reporter constructs and deletional/mutational analysis. The dependency of these fragments on sequences in the proximal promoter, including AP-1 sites will be investigated. Specific DNA/protein interactions occurring on these elements will be investigated using electrophoretic mobility shift analysis, antibodies for specific transcription factors and UV crosslinking. RNA/protein interaction occurring at the 3' untranslated region of the collagenase mRNA will be determined by the ability of native or mutated AUUUA sequences to mediate binding of proteins from IL-1 treated and untreated cells.