Molecular Mechanisms of RANKL Activation in Osteoblasts

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DESCRIPTION (provided by applicant): Osteoclasts are large, multinucleated cells that play a central role in bone resorption. They are derived from hematopoietic precursors in response to RANKL. RANKL is both necessary and sufficient for osteoclastogenesis, although factors such as M-CSF, TGFb, inflammatory cytokines and prostaglandins are also important to this process. RANKL is synthesized in a number of cell types, including those of the osteoblast lineage and the activated T cell. Studies both in vitro and in vivo indicate that whereas RANKL mRNA expression is regulated physiologically by key bone remodeling hormones such as PTH and 1,25(OH)2D3 as well as by cytokines such as IL-1, TNFa, IL-6 and the prostaglandin PGE2, its expression in T cell subsets is largely due to T cell activation. Aberrant production and/or expression of any one these modulators or the cell types has been implicated in RANKL overexpression, enhanced bone resorption and osteolytic or osteoporotic disease. Despite a recent increase in our understanding of the molecular mechanisms of Rankl expression in osteoblast lineage cells, almost nothing is known of these mechanisms relative to T cells. In view of the critical impact of RANKL on bone resorption associated with activation of T cells in chronic inflammatory disease, autoimmune disease and metastatic cancer, we propose to define key mechanisms instrumental to RANKL gene expression at the molecular level in T cells. Aim 1: To investigate the expression of RankL in both mouse and human T cell models and in mouse and human primary T Cells. RankL is absent in naive T cells, but strikingly induced in activated CD4+ and CD8+ T cell populations. We plan to establish both mouse and human T cell models to explore the activation process and to study the induction of RankL at the mRNA and protein levels. Aim 2: To characterize the molecular mechanisms of RankL induction in mouse and human T cells using ChIP and ChIP-chip analysis. We plan to utilize an unbiased approach using ChIP-chip analysis to identify T cell specific enhancers in the RankL intergenic region analysis to identify T cell specific enhancers in this intergenic region using T cell activation-induced RNA pol II recruitment and T cell activation-induced histone 4 (H4) acetylation (H4ac) and then identify and characterize the transcription factors that are involved in the activation process. Aim 3: To identify key components of the Rankl locus essential for T cell expression of RankL in vivo. We will prepare a mouse BAC clone containing the RankL locus bounded by CTCF insulator sites, introduce a luciferase reporter into the 3' UTR, and then evaluate the expression of this clone in transgenic mouse strains. These studies will provide important detail into the mechanisms through which RANKL expression is regulated in T cells in vitro and in vivo. PUBLIC HEALTH RELEVANCE: RANKL is a molecule which plays a significant role in both normal bone turnover and the bone loss associated with ageing, menopause, autoimmune disease and cancer. The studies herein seek to identify the mechanisms that underlie the expression of this factor such that better and more selective medicines can be created.