Regulation of Skeletal Growth by Nuclear Retinoid Receptors

描述(申请人提供)：颅骨、躯干和四肢的软骨内骨骼结构的生长由生长板的活动控制，在生长板上软骨细胞增殖、成熟和肥大，并被骨取代。生长板的功能反过来又依赖于它们独特的细胞外基质，包括聚集素、II型胶原和其他大分子，基质基因表达或结构的异常会导致生长板功能障碍和软骨发育不良，包括侏儒症。近年来，生长板和基质的生物学和病理学研究取得了重大进展，但仍有许多有待了解和发现的地方。视黄酸受体a、？和？(RARA、RAR？和RAR？)核激素受体是调节许多基本过程的核激素受体，充当非配体转录抑制因子或配体依赖转录激活因子。大约十年前进行的研究表明，缺乏RAR基因的小鼠存在骨骼缺陷，但自那以来一直缺乏对RAR作用的详细了解。为了研究骨骼特异的RAR功能，我们创造了条件RAR小鼠突变体。我们发现缺乏RARA和RAR？的小鼠(或RAR？/RAR？)软骨中的基因表现出严重的生长迟缓。它们的生长板是有缺陷的，重要的是，显示出凝集素表达和含量的大幅下降。然而，缺乏RARA和RAR的小鼠是正常的，这表明RAR？是必不可少的。的确，我们发现RAR？是在小鼠生长板中表达最强的RAR，它的表达特征是增殖区和肥大前期区，在那里aggrecan也强烈表达。生化分析表明，这些区域缺乏活跃的内源性配体，这意味着RARs作为非配体因子发挥作用。对小鼠软骨细胞培养的研究表明：(A)RAR？过度表达增强了集聚蛋白的表达；(B)RAR？与转录共抑制因子Zac1协同发挥作用；以及(C)RAR对aggrecan表达的作用是间接的，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认识的作用，促进聚集素的表达和含量。缺乏配体的RAR抑制功能会刺激SOX的表达，这反过来又会增加aggrecan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主要(如果不是完全)是由于RAR？；(Ii)确定RAR？通过Sox蛋白调节aggrecan基因的表达；以及(Iii)研究共抑制因子Zac1的作用。该项目的结果将从根本上对骨骼和生长板生物学产生新的见解。RARs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功能容易受到实验和药物操作的影响，该项目还将展望未来此类治疗方法可以用于治疗纠正软骨细胞行为和基质动态平衡，并恢复骨骼生长缺陷和相关病理的功能。 公共卫生相关性：出生前和出生后骨骼的生长过程对于建立正常的骨骼至关重要，而且这一过程容易受到包括侏儒症在内的先天性或后天疾病的影响。调节骨骼生长的机制仍然定义不清，也不清楚。该项目专注于作用于细胞核中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.