MICROPHTHALMIA IN OSTEOCLAST DEVELOPMENT

小眼畸形（mi）基因对破骨细胞的发育至关重要 基于MI/MI突变小鼠中严重的破骨细胞缺陷。 这些小鼠 代表继发性石骨症的遗传定义的动物模型 尽管破骨细胞正常， 号码 破骨细胞在骨质疏松症的发病机制中起着重要的作用， 骨质疏松症，骨硬化症，和各种病理特征， 转移性癌症，如骨侵袭、病理性骨折和骨 许多人类肿瘤引起的疼痛。 因此，破骨细胞的功能在于 许多人类疾病的十字路口，特别是与骨骼相关的疾病 发育和衰老。 此外，破骨细胞提供了一个有吸引力的 系统研究转录因子小眼症的功能 （完） mi基因编码碱性/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变种人 小鼠中的表型包括骨硬化症、缺乏色素沉着、小 眼睛和肥大细胞缺陷。 我们的实验室从生物化学的角度 表征DNA结合、转录活性，并鉴定 Mi的三个二聚化伙伴。 此外，我们最近 发现Mi蛋白在对c-kit的反应中被磷酸化 (stem细胞因子受体）活化 MAP激酶，并且这种磷酸化增强Mi转录 activation. 这一发现的灵感来自于 MI/MI和Kit突变小鼠中的细胞缺陷。 M-CSF突变小鼠 发展成骨硬化症。 M-CSF受体与c-kit密切相关， 目前初步证据表明，当原发性破骨细胞样 用M-CSF刺激培养物，Mi蛋白经历后 翻译修饰（可能是磷酸化）。 鉴于双方 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导和转录。 整体 本项目的目标将是阐明的关键功能 Mi转录因子在破骨细胞发育中的作用。 具体目标 目的：1）研究破骨细胞分化过程中Mi的表达和功能 2）研究M-CSF对Mi的可能调控作用 受体信号传导和3）分析潜在的基因转录 在破骨细胞中由Mi调节。Mi表达和二聚化伴侣 将通过免疫组织化学、免疫沉淀和 用单克隆和多克隆抗体进行Western印迹分析， 已经发展和特征化了。 Mi在M-CSF中的潜在作用 将通过分析MAP激酶途径和在细胞内的表达来评估信号传导。 使用Mi或特定突变体作为底物的体外激酶测定。 各种 的Mi表达构建体已经被工程化，包括显性的 阴性突变体，并将用于帮助确定靶基因 在破骨细胞中受Mi的转录调节。 博士大卫·费舍尔将监督该项目并领导一个咨询委员会 信号传导、骨生物学、细胞生理学方面的专家组成， 在我过渡到独立的过程中提供额外的指导和帮助 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.