Septic shock is the thirteenth leading cause of death in the United States and sepsis due to gram negative bacteria accounts for over 50% of those cases. Bacterial lipopolysaccharide from gram negative bacteria stimulates monocytes and macrophages to release a variety of soluble immune system mediators and when introduced systemically, to trigger the pathological events observed during endotoxic shock. The long-term objective of the proposed research is to define the biochemical mechanisms through which bacterial lipopolysaccharide (LPS) acts to modulate monocyte and macrophage functions. Specifically, this project proposes to define the LPS-induced nuclearsignalling events which lead to the induction of the interleukin-1 receptor antagonist (IL-1ra) gene. Previously, we have identified regions of the human secretory IL-1ra promoter which contain three positive acting and one inhibitory LPS responsive elements. The proposed studies will expand this work to conclusively identified the cis-acting DNA sequences and the trans- acting factors which regulate the LPS-induced expression of the IL-1ra gene.
Specific aim 1 will define the LPS-responsive cis-acting DNA elements in the human IL-1ra promoter. An IL-1ra promoter/luciferase reporter chimeric gene construct will be used in transient transfection studies. The sequences of important transcriptional elements in the promoter will be determined through the use of heterologous promoter constructs, DNase I protection assays, and electrophoretic mobility shift assays (EMSA). Confirmation of the importance of an element in the LPS-induced activation will be made through site-directed mutagenesis of each element alone and in combination with other identified elements.
Specific aim 2 will characterize the factors which bind to the LPS responsive elements defined in specific aim 1. We will determine the specific translational and posttranslational requirements for factor binding and activation as well as its molecular weight. The goal of these studies is to determine if the LPS responsive factors are newly identified transcription factor or previously characterized ones. It is expected that these studies will provide significant new insights into the mechanisms through which LPS acts to regulate gene expression in monocytes and macrophages.
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