Molecular Etiology of Enchondromatosis: Hedgehog Regulation in Chondrocytes

描述（由申请人提供）：内生性软骨瘤（ECA）是一种软骨肿瘤，由生长板软骨细胞未能进行终末分化引起，存在于超过3%的人群中，引起疼痛、畸形，并可发展为软骨肉瘤。内啡肽瘤病是一种有多个eca的疾病。目前还没有有效的药物治疗这种情况。我们发现PTHR1突变导致内生软骨瘤病中Hedgehog （Hh）信号的组成性激活，并发现组成性Hh信号在eca中很常见。我们制造了一只小鼠，其中组成性Hh转录激活导致ECA，并利用它来研究介质在正常和ECA软骨细胞中Hh信号级联中的作用。我们研究的一种介质是融合抑制因子（Sufu），我们揭示了其作为Hh信号负调节因子的重要作用，但发现它需要其他尚未确定的相互作用伙伴来调节Hh信号。我们在软骨瘤病中发现的突变存在于约5%的病例中。最近，异柠檬酸脱氢酶1和2 （IDH1和IDH2）的突变在较大比例的内生软骨瘤病中被发现。我们发现小鼠外植体培养中的Idh突变抑制软骨细胞分化并激活Hh配体激活上游的Hh信号。在这里，我们将扩展这项工作，以确定突变体Idh调节软骨细胞分化和Hh信号的机制，使用我们产生的在软骨细胞中表达突变体Idh1并产生ECA的小鼠。突变体Idh和Hh如何相互作用对于开发针对ECA的新型靶向治疗非常重要。我们之前的工作表明，Sufu与其他蛋白质相互作用，调节Gli信号传导。我们发现斑点型Poz蛋白（Spop）是一种基于cullin 3的泛素连接酶，与Sufu以重叠方式表达。在我们的细胞定制研究中，Spop反对Sufu在Gli转录因子调控中的作用。我们拟利用North American conditional mouse Mutagenesis项目产生的Spop条件敲除小鼠，研究其在生长板中与Sufu等软骨细胞分化调控因子的相互作用。我们使用Hh激活Gli转录因子抗体，通过ChIP-Seq鉴定Hh靶基因。使用这种技术，我们确定

DESCRIPTION (provided by applicant): Enchondromas (ECA) are a cartilage tumor, arising from growth plate chondrocytes that fail to undergo terminal differentiation, present in greater than 3% of the population causing pain, deformity, and can progress to chondrosarcoma. Enchodnromatosis is a condition in which there are multiple ECAs. There are currently no effective drug therapies for this condition. We identified a mutation in PTHR1 that results in constitutive activation of Hedgehog (Hh) signaling in Enchondromatosis, and found that constitutive Hh signaling is a common occurrence in ECAs. We generated a mouse in which constitutive Hh transcriptional activation causes ECAs, and used this to investigate the role of mediators in the Hh signaling cascade in normal and ECA chondrocytes. One mediator we investigated was Suppressor of fused (Sufu), and we unraveled important insight about its role as a negative regulator of Hh signaling, but found that it requires other, yet to be identified, interacting partners in its regulation of Hh signaling. The mutation we identified in enchondromatosis is present in about 5% of cases. Recently, mutations in isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2), were found in a larger proportion of cases of enchondromatosis. We found that Idh mutations in mouse explant cultures inhibit chondrocyte differentiation and activate Hh signaling upstream of Hh ligand activation. Here we will expand on this work to identify the mechanism by which the mutant Idh regulates chondrocyte differentiation and Hh signaling using a mouse we generated that expresses a mutant Idh1 in chondrocytes and develops ECA. How the mutant Idh and Hh interact is important in developing novel targeted therapies for ECA. Our previous work shows that Sufu interacts with other proteins to regulate Gli signaling. We found that speckle-type Poz protein (Spop), a cullin 3 - based ubiquitin ligase, is expressed in an overlapping manner with Sufu. In our cell couture studies, Spop opposes the action of Sufu in the regulation of Gli transcription factors. We propose to use a Spop conditional knockout mouse generated though the North American Conditional Mouse Mutagenesis project to study its interaction with Sufu and other regulators of chondrocyte differentiation in the growth plate. We identified Hh target genes by ChIP-Seq using antibodies to the Hh activated Gli transcription factors. Using this technique, we identified mediators of cholesterol metabolism as Hh target genes in chondrocytes and in ECAs, and that Hh activation causes intracellular accumulation of cholesterol in chondrocytes. The functional role of these genes in chondrocytes will be studies in the growth plate and in ECAs, using genetically modified mice. Our ultimate goal is to use this information to develop novel therapies to treat ECAs and enchondromatosis. To this end, we also propose to test novel therapeutic approaches based on modulating Hh, Idh, and/or cholesterol.