RECEPTOR FOR ADVANCED GLYCATION END-PRODUCTS: EXPRESSION AND SIGNALING

The NF-B transcription factor family plays a central role in many aspects of the immune response, and activation of this family of transcription factors has been shown to trigger many disease processes. Thus, the ability to modulate NF-kB activity may be an attractive way to treat these diseases. We used an in vitro cell-based assay to test potential NF-kB inhibitors by measuring their effect on IL-1-induced expression of the NF-B dependent intracellular adhesion molecule-1 (ICAM-1, CD54). To develop the cell-based system we sorted IL-1b-responsive U373 human astrocytoma cells to obtain a population of cells with minimum background expression and maximum induced expression of CD54 following stimulation with IL-1. We tested ethyl pyruvate, a novel anti-inflammatory drug candidate, and the ability of related compounds to block activation of NF-kB activity by measuring the expression of CD54 on U373 cells exposed to IL-1. 4-hydroxyphenylpyruvic acid was the best inhibitor of CD54 upregulation. We further tested the compounds using the mouse macrophage-like RAW 264.7 cell line which produce a variety of cytokines and nitric oxide (NO•) following exposure to lipopolysaccharide (LPS) in an NF-kB-dependent manner. The drugs downregulated LPS-induced IL-6 production, iNOS upregulation, and NO• production following the same efficacy trend observed in the primary screening using CD54 expression in U373 cells. These studies show the ease of using an endogenous reporter gene (i.e., CD54) and FACS analysis to rapidly characterize the relative efficacy of pharmacologic inhibitors. A second completely unrelated topic of the dissertation dealt with the receptor for advanced glycation end-products (RAGE). RAGE is thought to be important in a variety of pathological conditions, including diabetes, sepsis, atherosclerosis, renal diseases, hypertension and Alzheimer's disease. However, RAGE proximal signaling events are still unclear. We were able to establish that original RAGE, sequenced from bovine lung, is only present in the lung. This observation was based on antibody specificity, Northern blotting and N-glycosylation analysis. One of the antibodies that we used (H-300, Santa Cruz, CA) was very selective for lung RAGE and not cross-react with other RAGE isoforms. Only lung RAGE had a transcript size of 1.4 kb as determined by Northern blot and only lung RAGE was N-glycosylated. Non-lung tissues and cell lines appeared to express their own unique RAGE isoforms. Non-lung derived cell lines were permissive for lung RAGE isoform expression but lung derived cell lines were not. Interestingly, all transfected cell lines (of lung and non-lung origin) expressed RAGE mRNA transcripts. In addition, we established that previously described endogenous soluble RAGE (esRAGE) does not contain any of the canonical RAGE epitopes, but includes sequence encoded in intron 9. RAGE knockout mice lose esRAGE isoform along with the canonical one confirming that esRAGE originates from the RAGE gene. Signaling studies with pro-inflammatory stimuli in mouse lung slices of wild-type and knockout mice revealed the importance of RAGE in LPS and IL-1-induced inflammatory response, but not when reported RAGE ligands, including AGEs, HMGB1 and S100B, were applied