Regulation of Skeletal Growth by Nuclear Retinoid Receptors

描述（由申请方提供）：颅骨、躯干和四肢中软骨内骨骼结构的生长受生长板的活性控制，在生长板中软骨细胞增殖、成熟和肥大并被骨取代。生长板的功能反过来又取决于其独特的细胞外基质组成的聚集蛋白聚糖，胶原蛋白II和其他大分子，和异常的基质基因表达或结构引起生长板功能障碍和软骨发育不良，包括侏儒症。近年来，生长板和基质生物学和病理学取得了重大进展，但仍有许多有待理解和发现。 视黄酸受体a，和？(RARa、RAR？和RAR？）是调节许多基本过程并作为无配体转录抑制因子或配体依赖性转录激活因子的核激素受体。大约十年前进行的研究表明，缺乏RAR基因的小鼠具有骨骼缺陷，但此后一直缺乏对RAR作用的详细了解。为了研究肾脏特异性RAR功能，我们创建了条件RAR小鼠突变体。我们发现缺乏RAR α和RAR？(or RAR <$/RAR？）软骨中的基因表现出严重的生长迟缓。它们的生长板有缺陷，重要的是，聚集蛋白聚糖的表达和含量大幅下降。然而，缺乏RAR α和RAR β的小鼠是正常的，这表明RAR？运动规则可以称得上具有本质意义我们确实发现了RAR？是小鼠生长板中表达最强的RAR，其表达表征了聚集蛋白聚糖也强烈表达的增殖区和前肥大区。生化分析表明，这些区域缺乏活性内源性配体，表明RAR作为无配体因子起作用。小鼠软骨细胞培养的研究表明，（a）RAR？过表达增强聚集蛋白聚糖表达;（B）RAR？与转录辅阻遏物Zac 1合作发挥其功能;和（c）RAR对聚集蛋白聚糖表达的作用是间接的，并且涉及Sox蛋白。我们的中心假设是，RAR和RAR？特别是在生长板功能中发挥以前未被重视作用并促进聚集蛋白聚糖的表达和含量。配体较少的RAR阻遏物功能将导致Sox表达的刺激;这反过来将增加聚集蛋白聚糖的表达。我们的具体目标是：（i）确定RAR在生长板中的功能是否在很大程度上，如果不是唯一的话，是由于RAR？(ii)来定义RAR的分子机制通过Sox蛋白调节聚集蛋白聚糖基因表达;和（iii）表征共阻遏物Zac 1的作用。该项目的结果将在骨骼和生长板生物学方面产生根本性的新见解。RAR代表了骨骼生物学的一个关键但目前研究不足的领域，该项目将开始弥合这一明显的差距。由于RAR功能易受实验和药理学操作的影响，该项目还将导致设想未来的方法，其中这种治疗可以在治疗上用于纠正软骨细胞行为和基质稳态，并恢复骨骼生长缺陷和相关病理的功能。 公共卫生相关性：出生前和出生后的骨骼生长过程对于建立功能性骨骼至关重要，而这一过程易患先天性或后天性疾病，包括侏儒症。调节骨骼生长的机制仍然不清楚和理解。该项目的重点是在细胞核中发挥作用以调节基因表达的分子因子，并有望为未来可用于治疗的生长分子机制提供新的线索，以纠正和恢复受影响个体的骨骼生长和功能。

DESCRIPTION (provided by applicant): Growth of endochondral skeletal structures in skull, trunk and limbs is governed by the activity of the growth plates where chondrocytes proliferate, undergo maturation and hypertrophy and are replaced by bone. Functioning of the growth plates in turn depends on their unique extracellular matrix composed of aggrecan, collagen II and other macromolecules, and abnormalities in matrix gene expression or structure cause growth plate malfunction and chondrodysplasias, including dwarfism. There have been significant advances in growth plate and matrix biology and pathology in recent years, but much remains to be understood and discovered. The retinoic acid receptors a, ¿ and ? (RARa, RAR¿ and RAR?) are nuclear hormone receptors that regulate numerous fundamental processes and act as ligand-less transcriptional repressors or ligand-dependent transcriptional activators. Studies conducted a decade or so ago showed that mice lacking RAR genes have skeletal defects, but detailed understanding of RAR roles has been lacking since. To study skeletal-specific RAR function, we created conditional RAR mouse mutants. We find that mice lacking RARa and RAR? (or RAR¿/RAR?) genes in cartilage exhibit severe growth retardation. Their growth plates are defective and, importantly, display a major drop in aggrecan expression and content. Mice lacking RARa and RAR¿, however, are normal, suggesting that RAR? is essential. Indeed, we find that RAR? is the most strongly expressed RAR in mouse growth plates, and its expression characterizes the proliferative and pre-hypertrophic zones where aggrecan is strongly expressed also. Biochemical analysis shows that those zones are devoid of active endogenous ligands, signifying that the RARs operate as ligand-less factors. Studies with mouse chondrocyte cultures indicate that (a) RAR? over-expression enhances aggrecan expression; (b) RAR? exerts its function in cooperation with transcriptional co-repressor Zac1; and (c) RAR action on aggrecan expression is indirect and involves Sox proteins. Our central hypothesis is that RARs, and RAR? in particular, exert previously unappreciated roles in growth plate function and promote aggrecan expression and content. Ligand-less RAR repressor function would lead to stimulation of Sox expression; this, in turn, would increase aggrecan expression. Our specific goals are: (i) to determine whether RAR function in growth plates is largely, if not exclusively, due to RAR?; (ii) to define the molecular mechanisms by which RAR? regulates aggrecan gene expression via Sox proteins; and (iii) to characterize the roles of co-repressor Zac1. The results of the project will produce fundamentally new insights in skeletal and growth plate biology. The RARs represent a critical but currently understudied area of skeletal biology, and the project will begin to bridge this glaring gap. Because RAR function is susceptible to experimental and pharmacologic manipulations, the project will also lead to envision future ways in which such treatments could be exploited therapeutically to rectify chondrocyte behavior and matrix homeostasis and restore function in skeletal growth deficiencies and related pathologies. PUBLIC HEALTH RELEVANCE: The process of skeletal growth during prenatal and postnatal life is critical for establishment of a functioning skeleton, and this process is susceptible to congenital or acquired diseases including dwarfism. The mechanisms regulating skeletal growth remain ill defined and understood. This project focuses on molecular factors acting in the cell nucleus to regulate gene expression, and promises to shed new light into molecular mechanisms of growth that could be exploited therapeutically in the future to correct and restore skeletal growth and function in affected individuals.