The integrin beta3 gene is highly expressed in osteoclasts and during osteoclastogenesis yet the beta3 promoter is nearly silent in bone marrow macrophage, the osteoclast precursor. This high level of integrin beta3 gene expression is required for normal osteoclast differentiation, morphology, and bone resorption. Having recently cloned and characterized the mouse integrin beta3 gene promoter, we will determine which beta3 integrin gene promoter DNA sequences direct expression during osteoclastogenesis. In addition we propose to identify the transcription factors which trans-activate the beta3 integrin gene in osteoclastogenesis. Recent revolutionary advances in our understanding of the cytokine factors directing osteoclastogenesis have made it possible to induce osteoclast formation from immortal, and importantly, transfectable macrophage cell lines. It is therefore possible, for the first time, to study the transcription machinery of osteoclasts by transfection of macrophage with an osteoclast-specific gene promoter/reporter construct followed by induction of osteoclastogenesis. Promoter deletions and mutagenesis, followed by assays for biological activity, will help identify important promoter sequences, while in-vitro and in-vivo DNA footprinting techniques will identify sites of interaction with nuclear proteins in osteoclasts and their precursors. Gel-shift assays (EMSA), with promoter oligonucleotide sequences, compared with control consensus transcription factor binding site oligonucleotides, will be employed to identify in-vitro binding interactions between osteoclast and precursor nuclear factors and beta-3 promoter DNA elements. In addition, antibodies to known transcription factors will be used to """"""""super-shift"""""""" the nuclear protein/DNA complexes, thereby identifying components of shifted complexes. Nuclear factor binding sites will then be mutated in the context of the entire promoter and assayed for transcriptional activation during osteoclastogenesis. While not the specific aim of this proposal, the experiments described above hold the exciting potential for the identification of novel osteoclast-specific transcription factors. The osteoclastic transcriptional machinery for the beta-3 gene is likely shared by several other osteoclast-enriched and osteoclast-specific genes. Therefore, in addition to significantly increasing our understanding of the molecular cellular events directing osteoclast differentiation, the characterization of osteoclast transcriptional machinery may help identify novel targets for anti-osteoclastogenic therapies.

National Institute of Health (NIH)
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Research Project (R01)
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Orthopedics and Musculoskeletal Study Section (ORTH)
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Sharrock, William J
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Children's Hospital Boston
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Crotti, Tania N; O'Sullivan, Regina P; Shen, Zhenxin et al. (2011) Bone matrix regulates osteoclast differentiation and annexin A8 gene expression. J Cell Physiol 226:3413-21
Crotti, Tania N; Sharma, Sudarshana M; Fleming, Joseph D et al. (2008) PU.1 and NFATc1 mediate osteoclastic induction of the mouse beta3 integrin promoter. J Cell Physiol 215:636-44
McHugh, Kevin P (2008) Osteoimmunology in skeletal cell biology and disease. Autoimmunity 41:181-2
Shen, Zhenxin; Crotti, Tania N; Flannery, Merrilee R et al. (2007) A novel promoter regulates calcitonin receptor gene expression in human osteoclasts. Biochim Biophys Acta 1769:659-67
Cuetara, Bethany L V; Crotti, Tania N; O'Donoghue, Anthony J et al. (2006) Cloning and characterization of osteoclast precursors from the RAW264.7 cell line. In Vitro Cell Dev Biol Anim 42:182-8
Crotti, Tania N; Flannery, Merrilee; Walsh, Nicole C et al. (2006) NFATc1 regulation of the human beta3 integrin promoter in osteoclast differentiation. Gene 372:92-102
Shen, Zhenxin; Crotti, Tania N; McHugh, Kevin P et al. (2006) The role played by cell-substrate interactions in the pathogenesis of osteoclast-mediated peri-implant osteolysis. Arthritis Res Ther 8:R70
Crotti, T N; Flannery, M; Walsh, N C et al. (2005) NFATc1 directly induces the human beta3 integrin gene in osteoclast differentiation. J Musculoskelet Neuronal Interact 5:335-7