BMP'S IN SKELETAL GROWTH AND OSTEOGENIC DIFFERENTIATION

描述（由申请人提供）：BMP对于治疗骨关节炎的软骨形成和组织工程策略至关重要。在先前的授权期间，通过对缺乏BMP受体或下游换能器R-Smads的单突变、双突变和三突变小鼠的广泛表征，我们发现BMP几乎是体内生长板软骨形成的各个方面所需的，并且BMP的大部分作用都是通过R-Smads转导的。出乎意料的是，Smad 4，被认为是在典型的BMP信号通路中形成复合物所需的co-Smad，在软骨形成中的作用要有限得多。这一发现挑战了广泛接受的原则，即Smads 1/5通过经典途径抑制其大部分作用。我们的中心假设是，骨形态发生蛋白通过R-Smad依赖性，但Smad 4独立的机制，其重要性已被以前认识到的影响。我们将测试这一假设，并在四个具体目标中定义潜在的机制。 在目的1中，我们研究了Smad 1/5依赖性，Smad 4独立性机制如何转录调节Ihh的表达，Ihh是软骨细胞中BMP通路的关键直接靶点。我们还将进行第一次分析BMP对生长板软骨中全局基因表达的影响。我们增加了空间和时间分辨率，采用激光捕获显微切割和离体器官培养。在目标2中，我们将测试是否Smads 1/5所需的TAK 1作为一种新的机制，潜在的Smad 1/5依赖，Smad 4独立的信号转导，定义一个以前未知的R-Smads和非经典途径之间的相互作用。在目标3中，我们确定Smad 1-Drosha介导的miRNA成熟如何通过鉴定软骨细胞中BMP诱导转录和成熟的miRNA来促进Smad 1/5依赖性，Smad 4非依赖性信号传导。在目的4中，我们通过目的1-3中探索的机制检验了Smad 1/5依赖性、Smad 4独立性信号传导在成人关节软骨中与在生长板中一样重要的假设。这些实验有可能回答长期存在的关于BMP在成人软骨中功能的问题，并揭示BMP在软骨中传递信号的新机制。我们预计，这些信息将具有临床相关性，因为OA的患病率很高，BMP途径可能影响软骨形成和软骨维持，并且需要确定最有可能促进BMP软骨保护作用的BMP下游途径。

DESCRIPTION (provided by applicant): BMPs are essential for chondrogenesis and tissue engineering strategies for the treatment of osteoarthritis. In the prior grant period, through an extensive characterization of single, double, and triple mutant mice lacking BMP receptors or the downstream transducers R-Smads, we found that BMPs are required for nearly every aspect of growth plate chondrogenesis in vivo, and that most of the effects of BMPs are transduced through R-Smads. Unexpectedly, Smad4, the co-Smad that is thought to be required for complex formation in the canonical BMP signaling pathway, has a much more limited role in chondrogenesis. This finding challenges the widely accepted tenet that Smads1/5 transduce the majority of their effects through canonical pathways. Our central hypothesis is that BMPs transduce the majority of their effects through R-Smad-dependent but Smad4-independent mechanisms, whose importance has been previously unrecognized. We will test this hypothesis and define the underlying mechanisms in four Specific Aims. In Aim 1, we investigate how Smad1/5-dependent, Smad4-independent mechanisms transcriptionally regulate the expression of Ihh, a key direct target of BMP pathways in chondrocytes. We will also perform the first analysis of BMP effects on global gene expression in growth plate cartilage. We add spatial and temporal resolution by employing laser capture microdissection and ex vivo organ culture. In Aim 2, we will test whether Smads1/5 are required for activation of TAK1 as a novel mechanism underlying Smad1/5-dependent, Smad4-independent signaling, defining a previously unknown interaction between R-Smads and non-canonical pathways. In Aim 3, we determine how Smad1-Drosha mediated miRNA maturation contributes to Smad1/5-dependent, Smad4-independent signaling through identification of miRNAs whose transcription and maturation are induced by BMPs in chondrocytes. In Aim 4, we test the hypothesis that Smad1/5-dependent, Smad4 independent signaling through the mechanisms explored in Aims 1-3 is as important in adult articular cartilage as it is in the growth plate. These experiments hod the potential to answer longstanding questions about BMP function in adult cartilage, and to uncover novel mechanisms through which BMPs transduce their signals in cartilage. We anticipate that this information will have clinical relevance given the high prevalence of OA, the potential for BMP pathways to impact chondrogenesis and cartilage maintenance, and the need to identify the pathways downstream of BMPs that are most likely to promote the chondroprotective effects of BMPs.