Our preliminary data that collagenase (MMP-1) expression in scleroderma (SSc) fibroblasts is inhibited even after stimulation with TNFalpha, IL-1 Beta and PMA is extremely relevant for the accumulation of collagen in this disease. Both rnRNA and protein are reduced in stimulated cells and that these phenomena are seen in the, lesion but not the non-lesion fibroblasts. We will focus on the phenotype of the scleroderma fibroblast in relation to the level and degree of collagenase inhibition with regard to transcriptional or translational control in SSc fibroblasts stimulated with TNF-alpha or PMA as compared to collagenase induction in normal fibroblasts, Our study of transcriptional regulation of MMP- 1 levels will investigate the level and function of AP- 1 complexes as these represent the predominant intersection of the signaling pathways of the different stimuli. We will quantitate the amount and activity of different Fos and Jun family members which have been identified in these complexes in extracts of basal and stimulated SSc and normal fibroblasts and determine their ability to bind to the two AP-1 sequences which are active in the MMP-1 promoter. We will also examine levels of the many other genes known to affect collagenase expression using DNA microarray analyses of mRNA from stimulated and unstimulated cultures of SSc and normal fibroblasts. Finally, we will address the relevancy of the observation that icIRAP is elevated in Sscby using a retroviral vector to transfect antisense for icIRAP into SSc cells. The cells will then be stimulated with TNF and PMA to determine if collagenase responsiveness is recovered (or possibly exaggerated). The mRNA for collagenase and the collagenase protein synthesized will be correlated with the amounts of icIRAP protein. Modeling of observed effects in SSc collagenase pathways will be done using normal fibroblasts transfected with retrovirus containing cDNA for icIRAP or stimulated with peptide fragments of icIRAP. The experiments should provide information for the control of collagenase expression in this disease as well as to provide information for the association of icIRAP with collagenase inhibition.
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