Regulation of Skeletal Growth by Nuclear Retinoid Receptors

描述（由申请人提供）：颅骨、躯干和四肢的软骨内骨骼结构的生长受生长板活动的支配，其中软骨细胞增殖、成熟和肥大，并被骨取代。生长板的功能反过来依赖于其独特的由聚集蛋白、胶原II和其他大分子组成的细胞外基质，基质基因表达或结构的异常导致生长板功能障碍和软骨发育不良，包括侏儒症。近年来，在生长板和基质生物学和病理学方面取得了重大进展，但仍有许多有待了解和发现。视黄酸受体a，¿和？（RARa， RAR¿和RAR?）是调节许多基本过程的核激素受体，并作为无配体转录抑制因子或依赖配体的转录激活因子。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，但对RAR作用的详细了解一直缺乏。为了研究骨骼特异性RAR功能，我们创建了条件RAR小鼠突变体。我们发现小鼠缺乏RARa和RAR？（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，它们的聚集蛋白表达和含量显著下降。然而，缺乏RARa和RAR¿的小鼠是正常的，这表明RAR？是至关重要的。事实上，我们发现RAR？是小鼠生长板中表达最强烈的RAR，其表达特征是增殖区和前肥厚区，聚集蛋白也在这些区域强烈表达。生化分析表明，这些区域缺乏活性内源性配体，表明RARs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aggrecan表达；(b) RAR吗?与转录共抑制因子Zac1协同发挥作用；(c) RAR对聚集蛋白表达的作用是间接的，涉及Sox蛋白。我们的中心假设是RAR， RAR？特别是在生长板功能中发挥以前未被认识到的作用，促进聚合蛋白的表达和含量。无配体RAR抑制因子的作用会刺激Sox的表达；这反过来又会增加聚合蛋白的表达。我们的具体目标是：(i)确定生长板中的RAR功能是否主要（如果不是完全）由RAR引起；（ii）界定RAR的分子机制？通过Sox蛋白调控聚集蛋白基因表达；（iii）表征共抑制因子Zac1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。RARs代表了骨骼生物学中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理学操作的影响，该项目也将导致设想未来这种治疗方法可以用于治疗性地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。

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.