The overall goal of this project is to improve the prevention and treatment of inflammatory disease through targeting a novel pathway by which the estrogen receptor (ER) reduces the action of a key transcription factor, NF-B. ER suppression of NFB is critical for protective effects of estrogens in inflammatory bowel disease, sepsis, arthritis, atherosclerosis, and lipid metabolism, highlighting the broad importance of this pathway. Our hypothesis is that ER ligands that modulate NF-B induce a unique conformation of ER, facilitating association with gene-specific protein complexes, inducing displacement of gene-selective coregulators and transcription factors. This hypothesis will be pursued in the following Specific Aims:
SPECIFIC AIM 1. To investigate the importance of CBP and Grip1 in the molecular interactions required for ER-mediated suppression of NF-B, we will measure their recruitment to the MCP-1, IL-6, IL-8, MIP-1 and TNF-a genes by chromatin immunoprecipitation, and assay the effects of RNA interference targeting CBP and Grip1 on expression of the same genes. We will also examine these interactions in ER target tissues, including breast, uterine, bone, and macrophage cells. We propose that coactivator/transcription factor displacement is a gene specific phenomenon, and that other contributing transcription factors, such as c-jun, determine whether ER competes with CBP or Grip1. We also hypothesize that there are ligand pharmacophores that are selective for displacement of CBP versus Grip1 to specific inflammatory genes, which we propose to test with our large sets of NF-B selective ER ligands.
SPECIFIC AIM 2. To define the structural and chemical features of ER-ligand complexes that mediate selectivity for the NF-B pathway, we will characterize the structure of the receptor with pathway-selective ligands, using x-ray crystallography. We propose that pathway-selective ligands induce a unique conformation of the receptor that is distinct from the agonist conformation. We recently developed a novel technique that accelerates the crystallization of the nuclear receptor ligand-binding domain by several orders of magnitude, allowing us to characterize entire classes of receptor ligand complexes, as we propose here. Using our robotic cell based screening core, we have identified clusters of cofactors that interact with ER bound to compounds that are selective for NF-B or estrogen response element signaling. We also propose a genome scale siRNA screen for genes required for ER cross-talk with NF-B, providing an unbiased approach to define components of the repressive complex. The goal of these experiments is to understand how subtle changes in ligand chemistry alter receptor structure, and associated protein complexes, to effect signaling specificity through the estrogen receptor. Advances in each of these areas will significantly improve our understanding of how nuclear receptors act as molecular machines to achieve signaling specificity. Due to the critical importance of this pathway in inflammatory disease, this work also has great potential to directly impact the development of improved therapeutics. The overall goal of this project is to improve the prevention and treatment of inflammatory disease through targeting a novel pathway by which the estrogen hormones reduces the action of a key cellular protein that is a mediator of inflammation, NF-kappaB. We have generated a number of synthetic estrogens that inhibit the NFkappaB inflammatory pathway without stimulating other estrogenic responses in the cell. We propose to use these synthetic compounds to understand the molecular details of this important anti-inflammatory pathway.
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