The long term objective of this application is to characterize the molecular mechanisms by which IL-1 enhances the stability and function of cytokine mRNA in stromal cells of the human hematopoietic microenvironment. During the past funding period, we have determined that IL-1 governs the expression of multiple hematopoietic growth factor genes in endothelial cells and fibroblasts, in large part, by stabilizing gene transcripts. Specifically, we have found that GM-CSF, G-CSF, IL-1beta, and IL-6 mRNAs are stabilized in IL-1 stimulated diploid fibroblasts and human vascular endothelial cells. Using a cell-free method that faithfully reproduces the effect of IL-1 on mRNA stability, we have noted that an IL-1-induced RNase inhibitory factor can be reconstituted by reaction mixtures containing polysome-bound proteins and cytosolic co-factors. Using these same mixtures, we have also demonstrated that IL-1 induces mRNA binding proteins to bind to specific regions of the GM-CSF mRNA molecule known to destabilize the transcript. These methods, combined with promoter/reporter analysis in intact cells provide us with the tools necessary to achieve our specific aims; (1) to purify and characterize the biological activity of cellular factors activated by IL-1 that stabilize cytokine mRNA and (2) to identify specific attributes (sequences and/or secondary structural features) of cytokine mRNA chains that permit them to be stabilized in response to IL-1.
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