Molecular Etiology of Enchondromatosis: Hedgehog Regulation in Chondrocytes

描述（由申请人提供）：内生软骨瘤（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，并可进展为软骨肉瘤。圆线虫病是一种有多个ECA的疾病。目前没有有效的药物治疗这种情况。我们鉴定了PTHR 1中导致内生软骨瘤病中Hedgehog（Hh）信号传导组成性激活的突变，并发现组成性Hh信号传导在ECA中常见。我们产生了一种小鼠，其中组成型Hh转录激活导致ECA，并使用它来研究介质在正常和ECA软骨细胞中的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（Suppressor of fused，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。我们在内生软骨瘤病中发现的突变存在于约5%的病例中。最近，突变异柠檬酸脱氢酶1和2（IDH 1和IDH 2），被发现在内生软骨瘤病的情况下，更大的比例。我们发现小鼠外植体培养物中的Idh突变抑制软骨细胞分化并激活Hh配体激活上游的Hh信号传导。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信号转导的机制，使用我们产生的小鼠，在软骨细胞中表达突变体Idh 1，并开发ECA。突变体Idh和Hh如何相互作用在开发用于ECA的新靶向疗法中是重要的。我们以前的工作表明，腐乳与其他蛋白质相互作用，以调节Gli信号。我们发现斑点型Poz蛋白（Spop），一种基于cullin 3的泛素连接酶，与腐乳以重叠的方式表达。在我们的细胞培养研究中，Spop在Gli转录因子的调节中对抗Sufu的作用。我们建议使用Spop条件性基因敲除小鼠通过北美条件性小鼠诱变项目产生的研究其与腐乳和生长板中软骨细胞分化的其他调节剂的相互作用。我们使用Hh激活的Gli转录因子的抗体通过ChIP-Seq鉴定了Hh靶基因。利用这项技术，我们发现 在软骨细胞和ECA中作为Hh靶基因的胆固醇代谢介导物，并且Hh活化导致软骨细胞中胆固醇的细胞内积累。这些基因在软骨细胞中的功能作用将在生长板和ECA中使用转基因小鼠进行研究。我们的最终目标是利用这些信息来开发新的疗法来治疗ECA和内生软骨瘤病。为此，我们还建议测试基于调节Hh、Idh和/或胆固醇的新的治疗方法。

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.