Integration of Signaling Pathways Regulating Chondrocyte Differentiation

描述(申请人提供)：这项资助的主要重点是确定在软骨内骨形成过程中调控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分化过程，其特征是肥大并产生一种独特的蛋白质，即X型胶原(Col10)。甲状腺激素(T3)、骨形态发生蛋白(BMPs)和维生素A的主要活性代谢物维甲酸(RA)对这一过程有深远的影响。这些信号通路中的每一个都是软骨内骨形成所必需的，不能补偿其他的信号通路；取消任何一个信号通路(例如，甲状腺功能减退症，阻断T3信号)会阻止分化，导致骨骼缩短。COL10基因的调控为了解T3、RA和BMPs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，我们已经发现T3、RA和BMP信号通路聚集在鸡COL10启动子的一个小的末端区域，该区域包含多种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。所有这些位点在基础转录和RA/BMP刺激的转录活动中都发挥着重要作用；因此，该增强子可能作为激活CO10基因转录的各种信号通路的整合子。我们已经证明了一个由这些效应器组成的分层网络，其中T3通过刺激维甲酸和BMP信号间接作用于Col10基因的表达。此外，我们还发现RA具有直接和间接两种作用，直接刺激COL10转录，但也进一步刺激BMP信号转导。在这一更新应用中，我们提出了以下具体目标：1)我们将测试一种假设，即在体内软骨细胞成熟过程中，以及在对T3、BMPs和RA的响应中，转录因子和共调节器结合的变化改变了启动子和染色质的结构，从而导致Col10的急剧增加；以及2)我们将测试T3通过刺激BMP和RA信号来激活Col10转录的假设，这种刺激是T3效应的关键。体内实验将利用两种独特的小鼠模型，定向灭活甲状腺激素受体。一个菌株表现为骨骼甲状腺功能减退，导致软骨内骨形成延迟，而另一个菌株表现为骨骼甲亢性炎症，导致软骨内骨化加速；两者都导致身材矮小。这些实验将对由于生长板分化失败或生长板过早关闭而导致的身材矮小问题提供重要的机械见解。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。在这个国家，有数百万人患有未确诊的甲状腺疾病，全球有数百万人患有维生素A缺乏症；此外，维生素A衍生物和骨形态发生蛋白在临床上得到广泛使用，它们对骨骼生长的影响尚不清楚。拟议中的实验将对这些生长因子和激素在导致身材矮小的正常骨骼生长失败中所起的作用提供重要的见解。

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.