Integration of Signaling Pathways Regulating Chondrocyte Differentiation

描述（由申请人提供）：该基金的主要重点是确定在软骨内骨形成过程中调节软骨细胞成熟的分子机制。在此过程中，软骨细胞经历分化过程，其特征在于肥大和产生独特的蛋白质胶原X（Col10）。甲状腺激素（T3）、骨形态发生蛋白（BMPs）和维甲酸（RA，维生素A的主要活性代谢产物）对这一过程产生深远的影响。这些信号传导途径中的每一个都是软骨内骨形成所必需的，并且不能补偿其他的;废除任何一个途径（例如甲状腺功能减退症，其阻断T3信号传导）会阻止分化，导致骨骼缩短。Col10基因的调控为T3、RA和BMP控制软骨细胞成熟的分子机制提供了一个窗口。在目前的资助期间，我们已经表明，T3，RA和BMP信号通路会聚在鸡Col10启动子的一个小的远端区域，该区域包含多个转录因子的结合位点，包括RA受体，Runx2，Ets家族成员和两个DNA弯曲因子，Lef1和AP 1。所有这些位点在基础和RA/BMP刺激的转录活性中发挥重要作用;因此，该增强子可能作为激活Col10基因转录的各种信号通路的整合剂发挥作用。我们已经证明了这些效应器的分层网络，其中T3通过刺激类维生素A和BMP信号传导间接作用于Col10基因表达。此外，我们已经表明，RA有直接和间接的影响，刺激Col10的转录直接，但也进一步刺激BMP信号。在本更新申请中，我们提出了以下具体目标：1）我们将检验这样的假设，即改变启动子和染色质结构的转录因子和共调节剂的结合的改变是体内软骨细胞成熟期间以及培养物中响应于T3、BMP和RA而发生的Col10急剧增加的原因; 2）我们将检验T3通过刺激BMP和RA信号而激活Col10转录的假设，并且这种刺激对于T3的作用是必不可少的。体内实验将利用两种独特的甲状腺激素受体靶向失活小鼠模型。一种菌株显示骨骼甲状腺功能减退，导致软骨内骨形成延迟，而另一种菌株显示骨骼甲状腺毒症，导致软骨内骨化加速;两者均导致身材矮小。这些实验将为由于生长板分化失败或生长板过早闭合而导致的身材矮小问题提供重要的机理性见解。公共卫生相关性：骨骼生长受到甲状腺激素、维生素A和骨形态发生蛋白（BMP）的深刻影响。美国有数百万人患有未确诊的甲状腺疾病，全世界有数百万人患有维生素A缺乏症;此外，维生素A衍生物和BMP在临床上广泛使用，其使用对骨骼生长的影响尚不清楚。拟议的实验将提供重要的见解，这些生长因子和激素在正常骨骼生长失败中发挥的作用，导致身材矮小。

DESCRIPTION (provided by applicant): The major focus of this grant is to identify the molecular mechanisms that regulate chondrocyte maturation during endochondral bone formation. During this process, chondrocytes undergo a differentiation process characterized by hypertrophy and production of a unique protein, collagen X (Col10). Thyroid hormone (T3), bone morphogenetic proteins (BMPs) and retinoic acid (RA, the major active metabolite of vitamin A) exert profound effects on this process. Each of these signaling pathways is essential for endochondral bone formation and cannot compensate for the others; abrogation of any one pathway (e.g. hypothyroidism, which blocks T3 signaling) prevents differentiation, resulting in shortened bones. Regulation of the Col10 gene provides a window into the molecular mechanisms by which T3, RA and BMPs control chondrocyte maturation. During the current funding period, we have shown that the T3, RA and BMP signaling pathways converge on a small distal region of the chick Col10 promoter that contains binding sites for multiple transcription factors, including RA receptors, Runx2, an Ets family member and two DNA-bending factors, Lef1 and AP1. All of these sites play a important roles in both basal and RA/BMP-stimulated transcriptional activity; thus this enhancer may function as an integrator of the various signaling pathways that activate Col10 gene transcription. We have demonstrated a hierarchical network of these effectors, in which T3 acts indirectly on Col10 gene expression by stimulating both retinoid and BMP signaling. In addition, we have shown that RA has both direct and indirect effects, stimulating Col10 transcription directly, but also further stimulating BMP signaling. In this renewal application, we propose the following specific aims: 1) We will test the hypothesis that alterations in binding of transcription factors and comodulators that alter promoter and chromatin architecture are responsible for the dramatic increase in Col10 that occur during chondrocyte maturation in vivo and in response to T3, BMPs and RA in culture; and 2) We will test the hypothesis that T3 activates Col10 transcription through stimulation of BMP and RA signaling and this stimulation is essential for T3 effects. The in vivo experiments will utilize two unique mouse models with targeted inactivation of thyroid hormone receptors. One strain displays skeletal hypothyroidism, resulting in delayed endochondral bone formation, while the other displays skeletal thyrotoxicotis, resulting in accelered endochondral ossification; both result in short stature. These experiments will provide significant mechanistic insights into the problems of short stature due to failure of growth plate differentiation or premature growth plate closure. PUBLIC HEALTH RELEVANCE: Skeletal growth is profoundly affected by thyroid hormones, vitamin A and bone morphogenetic proteins (BMPs). There are millions of people in this country with undiagnosed thyroid disease and many more millions worldwide with vitamin A deficiency; furthermore, vitamin A derivatives and BMPs are in widespread clinical use, and the impact of their use on skeletal growth is not well understood. The proposed experiments will provide significant insights into the roles these growth factors and hormones play in failure of normal skeletal growth, resulting in short stature.