MICROPHTHALMIA IN OSTEOCLAST DEVELOPMENT

小眼球(Mi)基因是破骨细胞发育的关键基因 基于mi/mi突变小鼠严重的破骨细胞缺陷。这些老鼠 代表了继发性骨化病的基因定义动物模型 尽管破骨细胞正常，但破骨细胞功能严重受损 数字。破骨细胞在骨质疏松症的发病机制中起重要作用 骨质疏松症、骨质疏松症及多种病理特征 转移性癌症，如骨侵犯、病理性骨折和骨 由许多人类肿瘤引起的疼痛。因此，破骨细胞的功能在于 许多与骨骼相关的人类疾病的十字路口 发育和衰老。此外，破骨细胞提供了一种诱人的 转录因子小眼炎的功能研究系统 (Mi)。 Mi基因编码一个基本/螺旋-环-螺旋/亮氨酸拉链(bhlh-zip)。 与癌蛋白Myc相关的转录因子。Mi突变体 小鼠的表型包括骨化症，缺乏色素，体积小 眼睛和肥大细胞缺陷。我们实验室的生物化学 表征DNA结合、转录活性，并鉴定 Mi.的三个二聚体伙伴。此外，我们最近还 发现Mi蛋白在c-kit的作用下被磷酸化 (干细胞因子受体)通过信号通路激活，包括 MAP激酶和这种磷酸化增强Mi的转录 激活。这一观察是由色素的相似性引起的 Mi/mi和kit突变小鼠的细胞缺陷。M-CSF基因突变的小鼠 出现骨化症。M-CSF受体与c-kit密切相关， 在目前的初步证据中，当初级破骨细胞样时 培养物用M-CSF刺激，Mi蛋白经历后 翻译修饰(可能是磷酸化)。鉴于两者都是 M-CSF和Mi对破骨细胞至关重要，我们的观察可能与 这些因子参与破骨细胞的信号传递和转录。整体而言 本项目的目标将是阐明 MI转录因子与破骨细胞发育具体目标 1)研究Mi在破骨细胞中的表达和功能 发展，2)研究M-CSF对Mi的可能调节 受体信号转导和3)转录分析潜在基因 在破骨细胞中受Mi调控。MI表达与二聚体配对 将通过免疫组织化学、免疫沉淀和 用单抗和多克隆抗体进行免疫印迹分析 已经发展起来并具有特征。MI在M-CSF中的潜在作用 信号将通过分析MAP激酶通路和In 以Mi或特定突变体为底物的体外激酶测定。品种 已经对Mi表达结构进行了改造，包括显性 阴性突变体，将被用来帮助识别目标基因 在破骨细胞中受Mi转录调控。 David Fisher博士将监督该项目并领导一个顾问委员会 信号、骨生物学、细胞生理学方面的专家组成 提供其他指导和帮助，帮助我过渡到独立的 调查员。

The microphthalmia (mi) gene is critical for osteoclast development based on severe osteoclast defects in mi/mi mutant mice. These mice represent a genetically defined animal model of osteopetrosis secondary to profound failure of osteoclast function, despite normal osteoclast numbers. Osteoclasts play an important role in the pathogenesis of osteoporosis, osteopetrosis, and a variety of pathological features of metastatic cancer, such as bony invasion, pathologic fractures, and bone pain caused by many human tumors. Thus, osteoclast function lies at the crossroads of many human diseases of particular relevance in bone development and aging. In addition, osteoclasts provide an attractive system to study the function of the transcription factor microphthalmia (Mi). The mi gene encodes a basic/helix-loop-helix/leucine zipper (bHLH-ZIP) transcription factor related to the oncoprotein, Myc. The mi mutant phenotype in mice includes osteopetrosis, a lack of pigmentation, small eyes, and a mast cell defects. Our laboratory has biochemically characterized DNA binding, transcriptional activity, and identified three dimerization partners of Mi. In addition we have recently discovered that the Mi protein is phosphorylated in response to c-kit (stem cell factor receptor) activation via a signaling pathway involving MAP kinase and that this phosphorylation enhances Mi transcriptional activation. This observation was sparked by the similarity of pigment cell defects in mi/mi and kit mutant mice. Mice with mutations in M-CSF develop osteopetrosis. M-CSF receptor is closely related to c-kit, and in present preliminary evidence that when primary osteoclast-like cultures are stimulated with M-CSF, the Mi protein undergoes a post- translational modification (likely phosphorylation). Given that both M-CSF and Mi are critical for osteoclasts, our observation may relate these factors in osteoclast signaling and transcription. The overall goal of this project will be to elucidate the critical function of the Mi transcription factor in osteoclast development. The specific aims are 1) To Characterize Mi's expression and function during osteoclast development, 2) To examine the possible regulation of Mi via M-CSF receptor signaling and 3) To analyze potential genes transcriptionally regulated by Mi in osteoclasts. Mi expression and dimerization partners will be analyzed by immunohistochemistry, immunoprecipitation, and Western blot analysis with monoclonal and polyclonal antibodies that I have developed and characterized. Mi's potential role in M-CSF signaling will be assessed by analyzing the MAP kinase pathway and in vitro kinase assays using Mi or specific mutants as substrate. Variety of Mi expression constructs have been engineered including dominant negative mutants and will be employed to help identify target genes transcriptionally regulated by Mi in osteoclasts. Dr. David Fisher will supervise the project and head an advisory board of experts in signalling, bone biology, cellular physiology formed to provide additional guidance and aid in my transition to an independent investigator.