BETA3 INTEGRIN PROMOTER ACTIVATION IN OSTEOCLASTOGENESIS

整合素β3基因在破骨细胞和破骨细胞形成过程中高度表达，而在破骨细胞前体骨髓巨噬细胞中几乎没有表达。这种高水平的整合素β3基因表达是正常破骨细胞分化、形态和骨吸收所必需的。最近克隆并鉴定了小鼠整合素Beta3基因启动子，我们将确定哪些Beta3整合素基因启动子DNA序列直接在破骨细胞形成过程中表达。此外，我们还建议确定在破骨细胞形成过程中反式激活β3整合素基因的转录因子。最近，我们对指导破骨细胞发生的细胞因子的了解取得了革命性的进展，使得从永生的、更重要的是可转基因的巨噬细胞系诱导破骨细胞形成成为可能。因此，首次有可能通过将破骨细胞特异性基因启动子/报告结构导入巨噬细胞，然后诱导破骨细胞生成来研究破骨细胞的转录机制。启动子的缺失和突变，以及随后的生物活性分析，将有助于确定重要的启动子序列，而体外和体内DNA足迹技术将确定破骨细胞及其前体中与核蛋白相互作用的位置。以启动子寡核苷酸序列为基础的凝胶漂移分析(EMSA)将用于鉴定破骨细胞和前体核因子与β-3启动子DNA元件的体外结合作用。此外，已知转录因子的抗体将被用来“超级移动”核蛋白/DNA复合体，从而识别移动的复合体的成分。核因子结合位点将在整个启动子的背景下发生突变，并在破骨细胞形成过程中进行转录激活检测。虽然不是这项建议的具体目的，但上述实验在鉴定新的破骨细胞特异性转录因子方面具有令人兴奋的潜力。β-3基因的破骨转录机制可能与其他几个富含破骨细胞和破骨细胞特异的基因相同。因此，除了显著增加我们对引导破骨细胞分化的分子细胞事件的了解外，破骨细胞转录机制的特征可能有助于识别抗破骨细胞治疗的新靶点。

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.