Mitf-Signal Responsive Transcription in Osteoclasts

描述(申请人提供)：MITF是一种螺旋-环-螺旋转录因子，其突变会在多种物种中导致严重的骨化病。来自MITF突变小鼠的破骨细胞谱系细胞发育，但似乎停滞在大约融合阶段，并表现出严重的骨吸收缺陷。这种晚期发育缺陷表明，MITF转录因子调节晚期破骨细胞分化基因，而不是谱系决定的全局调节者。这些基因的识别和调节其表达的转录调控将为人类骨骼疾病的治疗提供重要的新靶点。我们观察到，MITF的显性负性等位基因会导致严重的骨化病，而空等位基因则不会。在此基础上，我们确定了TFE3，一个与HLH相关的因子，作为一个二聚体伙伴，在破骨细胞谱系中与MITF分享转录活性。MITF和TFE3的这种重叠作用已经在双零小鼠模型中得到验证。我们还证明，M-CSF是破骨细胞早期和晚期发育的重要细胞因子，它可以触发MITF和TFE3在保守的丝氨酸上的MAPK磷酸化，刺激它们重新招募辅助激活因子p300。这种磷酸受体丝氨酸的突变破坏了后期的破骨细胞分化。除了这种翻译后调控外，我们还发现了几个MITF/TFE3转录靶基因，包括分泌的组织蛋白K和生存基因Bcl2。体内这些基因的破坏会导致骨化症。MITF/TFE3在破骨细胞晚期成熟过程中所起的重要作用为通过分析其生化调控和靶基因鉴定发现骨吸收过程中的关键途径和介体提供了独特的机会。我们建议在两个具体目标下进行这一分析。第一个重点是阐明调控MITF和TFE3表达和活性的转录和翻译后机制，它们反过来调节后期破骨细胞的发育和骨吸收。第二种方法代表了一种系统的方法来识别MITF/TFE3的转录靶标，使用将它们的生化活性与它们所在的信号通路相耦合的屏幕。

DESCRIPTION (provided by applicant): Mitf is a helix-loop-helix transcription factor whose mutation produces severe osteopetrosis in multiple species. Osteoclast lineage cells from Mitf-mutant mice develop, but appear to arrest at approximately the fusion stage and exhibit profoundly defective bone resorption. This late developmental defect suggests that the Mitf transcription factor regulates late osteoclast differentiation genes, rather than global regulators of lineage determination. The identification of those genes and the transcriptional controls which modulate their expression stand to provide important new targets for the treatment of bone disorders in man. We observed that dominant negative alleles of Mitf produce severe osteopetrosis whereas null alleles do not. On this basis we identified TFE3, a related HLH factor, as a dimerization partner which shares transcriptional activities with Mitf in the osteoclast lineage. This overlapping role for Mitf and TFE3 has been verified in a double-null mouse model. We have also demonstrated that M-CSF, a vital cytokine for both early and late osteoclast development, triggers MAPK phosphorylation of both Mitf and TFE3 on a conserved serine, stimulating their recruitment of the coactivator p300. Mutation of this phosphoacceptor serine disrupts late osteoclastic differentiation. In addition to this post-translational regulation, we have identified several Mitf/TFE3 transcriptional target genes including the secreted protease cathepsin K, and the survival gene Bcl2. In vivo disruption of these genes results in osteopetrosis. The essential roles for Mitf/TFE3 specifically for late osteoclast maturation affords a unique opportunity to discover key pathways and mediators of the bone resorption process through the analysis of their biochemical regulation and target gene identification. We propose to carry out this analysis in two Specific Aims. The first focuses on elucidating transcriptional and post-translational mechanisms which regulate expression and activity of Mitf and TFE3, which in turn modulate late osteoclast development & bone resorption. The second represents a systematic approach to the identification of transcriptional targets of Mitf/TFE3 using screens which couple their biochemical activities to the signaling pathways in which they reside.