During the previous grant period, we developed an immortalized OCL precursor cell line by targeting bcl-XL and SV40 large T antigen to cells in the OCL lineage (B/T cells), using the tartrate-resistant acid phosphatase (TRAP) promoter. We used subtractive hybridization techniques with OCL formed by B/T cells and untreated B/T cells to identify four genes that regulate OCL formation and activity. Three of these genes were novel regulators of OCL formation. One of these genes, ADAM8 (A Disintegrin And Metalloproteinase) and its receptor alpha9beta1 integrin, act as adhesion molecules that induce OCL differentiation and regulate the OCL cytoskeleton. ADAM8 is upregulated by inflammatory cytokines. It is our hypothesis that characterizing the molecular mechanisms involved in alpha9beta1-induced signaling in OCL precursors and mature OCL will lead to the identification of novel therapeutic targets for inhibiting the bone destruction associated with inflammatory arthritis such as rheumatoid arthritis (RA). In support of an important role for ADAM8/alpha9 in regulation of OCL differentiation and activity are our preliminary data and recent publications which demonstrate: a) The disintegrin domain rather than the metalloproteinase domain of ADAM-8 regulates OCL differentiation via alpha9beta1 integrin, a previously unidentified integrin on OCL precursors; b) blocking ADAM-8 or its receptor alpha9beta1 inhibits OCL formation; c) treatment of beta3-/- OCL precursors with ADAMS rescues the abnormal cytoskeletal phenotype of beta3 -/- OCL; and d) our preliminary studies which suggest that a9 -/-mice have a bone phenotype. Thus, alpha9beta1 signaling induced by ADAM8 regulates both OCL precursor differentiation and the OCL cytoskeleton. Characterization of the mechanisms responsible for ADAM8/alpha9beta1's regulation of OCL formation and the OCL cytoskeleton is the focus of this application. The following specific aims will be pursued: I.Characterize the mechanisms responsible for regulation of OCL differentiation and the OCL cytoskeleton by ADAM8/alpha9beta1. A) Confirm that alpha9beta1 is the primary receptor for signaling ADAM8's regulation of OCL differentiation using GST-ADAM8 chimeric protein pulldown experiments with WT and alpha9 -/- OCL precursors. B) Determine the signaling pathways utilized by alpha9beta1, to regulate OCL differentiation and the OCL cytoskeleton, and their relative contribution to these processes using specific inhibitors of these pathways. These experiments, will utilize OCL precursors from wild-type (WT) mice, alpha9 -/- OCL precursors obtained from lethally irradiated WT mice rescued with alpha9 -/- marrow, and beta3 -/- mice treated with ADAM8 and low dose RANKL/M-CSF to distinguish the activation of alpha9beta1, from alphavbeta3, a major integrin involved in the regulation of OCL differentiation and the OCL cytoskeleton. C) Perform structure-function experiments to assess the critical amino acids present in the cytoplasmic tail of alpha9 that signal alpha9's regulation of OCL differentiation and the OCL cytoskeleton. We will treat highly purified alpha9 -/- OCL precursors transfected with full-length a9 and deletion constructs of the cytoplasmic tail of alpha9 with ADAM8 and low dose RANKL/M-CSF, and test the capacity of these cells to form OCL with a normal cytoskeleton. D) Use confocal microscopy, to determine the cellular location of a9 and av in alpha9 -/-, WT, and beta3 -/- OCL treated with ADAM8, and the effects of inhibiting the a9b1 signaling pathways identified above on the OCL cytoskeleton and actin ring formation. II. Characterize ADAM8/alpha9beta1's regulation of OCL formation and activity in vivo,by assessing the bone phenotype of mice in which ADAM8 is either overexpressed (TRAP-ADAM8) or deleted (TRAP-cre x floxed ADAM8 mice) in cells of the OCL lineage, alpha9 -/- mice and radiation chimeras transplanted with alpha9 -/- marrow. OCL formation, bone histomorphometry, bone architecture by microqCT and bone resorption capacity of OCL formed in marrow cultures from these animals will be compared to syngeneic age-matched control mice. We will also breed WT, TRAP-ADAM8 or TRAP-cre x floxed ADAM8 mice to beta3 -/- mice to determine if overexpression of ADAM8 in OCL precursors in vivo can prevent the osteosclerosis that occurs in b3 -/- mice. Our long-term goal will be to characterize the role of ADAM8/a9b1 in the bone destruction which accompanies collagen induced arthritis, which will be started in grant year 5.
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