Integration of Signaling Pathways Regulating Chondrocyte Differentiation

描述（由申请人提供）：本资助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。在这个过程中，软骨细胞经历了一个以肥大为特征的分化过程，并产生了一种独特的蛋白质，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的主要活性代谢物）在这一过程中发挥着深远的作用。这些信号通路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；任何一种途径（如甲状腺功能减退症，它阻断T3信号）的消失都会阻止分化，导致骨骼缩短。Col10基因的调控为研究T3、RA和bmp控制软骨细胞成熟的分子机制提供了一个窗口。在目前的资助期内，我们已经证明T3、RA和BMP信号通路集中在鸡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AP1。所有这些位点都在基础和RA/ bmp刺激的转录活性中发挥重要作用；因此，该增强子可以作为激活Col10基因转录的各种信号通路的整合子。我们已经证明了这些效应物的分层网络，其中T3通过刺激类视黄醛和BMP信号间接作用于Col10基因表达。此外，我们已经证明RA具有直接和间接作用，可以直接刺激Col10转录，还可以进一步刺激BMP信号传导。在这一更新申请中，我们提出以下具体目标：1)我们将验证以下假设：在体内软骨细胞成熟过程中，以及在培养中对T3、bmp和RA的反应中，改变启动子和染色质结构的转录因子和调节因子的结合变化是Col10急剧增加的原因；2)我们将验证T3通过刺激BMP和RA信号激活Col10转录的假设，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靶向甲状腺激素受体失活的小鼠模型。一种表现为骨性甲状腺功能减退，导致软骨内成骨延迟，而另一种表现为骨性甲状腺毒性炎，导致软骨内成骨加速；两者都会导致身材矮小。这些实验将为研究由于生长板分化失败或生长板过早闭合导致的矮小问题提供重要的机制见解。公共卫生相关性：骨骼生长深受甲状腺激素、维生素A和骨形态发生蛋白（BMPs）的影响。这个国家有数百万人患有未确诊的甲状腺疾病全世界有更多的人缺乏维生素A；此外，维生素A衍生物和bmp在临床广泛使用，其使用对骨骼生长的影响尚不清楚。拟议的实验将为这些生长因子和激素在正常骨骼生长失败中所起的作用提供重要的见解，从而导致身材矮小。

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.