B-CELLS REGULATE OSTEOCLASTOGENESIS BY TGF-BETA SECRETIO

b细胞在调节破骨细胞发生中的作用是有争议的，并且仍然知之甚少。我们最近在人体模型系统中评估了b细胞在破骨细胞形成中的作用，并证明与之前的一些报道相反，b细胞在体外抑制破骨细胞的形成。我们发现b细胞缺失的培养比b细胞充满的培养产生更多的破骨细胞。该抑制因子驻留在b细胞条件培养基中，诱导破骨细胞前体和多核成熟破骨细胞凋亡。我们已经确定b细胞的这些凋亡效应是tgfβ分泌的结果。因此，b细胞分泌tgf - β代表了一种新的重要骨调节机制。我们的数据还表明，IL-7在体外下调tgf - β的产生。因此，我们假设骨髓基质细胞分泌IL-7可能是调节b细胞tgf β产生的关键机制，从而在调节骨稳态中起关键作用。我们现在建议通过对人类TGFbeta启动子进行详细检查来研究IL-7调节TGFbeta产生的机制。我们计划利用IL-7启动子-荧光素酶报告结构来研究哪些转录因子基序对IL-7有响应。我们计划初步探索不同转录因子结合基序（如SP-1、TRE和NF1）的相对贡献，这些基序已知存在于TGFbeta启动子中。利用位点定向诱变，我们将使这些位点发生突变，使它们无法工作，然后评估IL-7抑制转录的能力。如果没有发现这些位点能够解释TGFbeta的响应性，我们将对启动子进行系统的5'删除，以确定相关的IL-7响应序列。

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.