This proposal seeks to understand the molecular mechanisms controlling expression of the gene for collagenase (matrix metalloproteinase-1; MMP- 1), the only enzyme active at neutral pH that can degrade the interstitial collagens (types I, II and III). Collagenase can be induced by phorbol esters (PMA) and by cytokines such as interleukin-1 (IL-1), and suppressed by vitamin A analogues (retinoids). Collagenase is over- expressed in the joints of patients with rheumatoid arthritis, and this over-expression contributes significantly to the pathophysiology of this disease. To study mechanisms of transcriptional activation and suppression of the collagenase gene, we made chimeric constructs containing fragments of the collagenase promoter linked to a reporter gene, and we transfected these chimeric constructs into fibroblasts, cells that normally express the collagenase gene endogenously. We found that although the AP-1 (activator protein) motif, 5'-TGAGTCAC-3', which binds the transcription factors Fos and Jun is important, additional sequences are essential for both activation and suppression. We also found that upstream sequences compensate for mutations in the AP-1 site, and that repression by retinoic acid involves differential activity of several Retinoic Acid Receptors (RARs and RXRs), at multiple sites along the promoter in a RAR type-specific manner. Furthermore, the mechanisms mediating IL-1 induction of collagenase mRNA may not be identical to those mediating induction by PMA. Different cis-acting elements in the promoter may respond transcriptionally, and post-transcriptional mechanisms, i.e. mRNA stability, may also contribute. Since the 3' untranslated region of collagenase mRNA contains 3 copies of the sequence AUUUA, which is associated with mRNA instability, these sequences may be important in regulating IL-1 induced collagenase gene expression. Thus, the specific aims of this proposal are to: (1) Continue our studies to (a) identify cis-acting regulatory elements in the collagenase promoter involved in basal/constitutive transcription and in the response to PMA (b) determine their dependency on a functional AP-1 site, and (c) characterize the protein-DNA interactions involved; (2) Identify retinoic acid responsive elements within the collagenase promoter and characterize the specific role of RARs (alpha, Beta and gamma) and RXR alpha in modulating basal and phorbol induced transcription; and (3) Characterize the transcriptional and post-transcriptional mechanisms (i.e., mRNA stability and the role of the AUUUA sequences) that regulate collagenase gene expression in response to IL-1. Knowledge of mechanisms regulating expression of the collagenase gene will enhance our understanding of the pathophysiology of arthritic disease and may form the basis for new and novel therapies designed to reduce joint destruction.
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