The role of B-cells in regulating osteoclastogenesis is contentious and remains poorly understood. We have recently evaluated the role of B-cells in osteoclast formation in a human model system and demonstrated that contrary to some previous reports, B-cells are inhibitory to osteoclastogenesis in vitro. We find that B-cell depleted cultures generate higher numbers of osteoclasts than B-cell replete cultures. This inhibitory factor is resident in B-cell conditioned medium and induces apoptosis of osteoclast precursors and multinucleated mature osteoclasts. We have determined that these apoptotic effects of B-cells are the result of TGFbeta secretion. Secretion of TGFbeta by B-cells thus represents a new and important mechanism of bone regulation. Our data also shows that IL-7 downregulates the production of TGFbeta in vitro. We thus hypothesize that stromal cell secretion of IL-7 in the bone marrow may constitute a key mechanism of regulating the B-cell production of TGFbeta, thus playing a pivotal role in regulating bone homeostasis. We now propose to investigate the mechanism by which IL-7 regulates TGFbeta production, by making a detailed examination of the human TGFbeta promoter. We plan to utilize an IL-7 promoter-luciferase reporter construct to investigate which transcription factor motifs for IL-7 responsive. We plan to initially explore the relative contributions of different transcription factor binding motifs such as SP-1, TRE, and NF1, that are known to be present in the TGFbeta promoter. Using site-directed mutagenesis we will mutate these sites to render them inoperable and then assess the ability of IL-7 to inhibit transcription. If these sites are not found to explain TGFbeta responsiveness we will generate systematic 5' deletions of the promoter to identify the relevant IL-7 responsive sequences.
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