BMP'S IN SKELETAL GROWTH AND OSTEOGENIC DIFFERENTIATION

描述(由申请人提供)：BMP对于软骨形成和治疗骨关节炎的组织工程策略是必不可少的。在之前的资助期间，通过对缺乏BMP受体或下游转导R-SMADS的单、双和三个突变小鼠的广泛鉴定，我们发现体内生长板软骨形成的几乎每一个方面都需要BMPs，并且BMPs的大部分作用是通过R-SMADS转导的。出乎意料的是，Smad4，被认为是典型的BMP信号通路中形成复杂结构所必需的辅链Smad，在软骨形成中的作用要有限得多。这一发现挑战了被广泛接受的信条，即Smads1/5通过规范的途径传递它们的大部分影响。我们的中心假设是，BMP的大部分作用是通过R-Smad依赖但Smad4不依赖的机制来传递的，这一机制的重要性以前没有被认识到。我们将检验这一假设，并在四个具体目标中定义潜在的机制。在目标1中，我们研究了Smad1/5依赖、Smad4不依赖的机制如何转录调控软骨细胞中BMP途径的关键直接靶点IHH的表达。我们还将首次分析BMP对生长板软骨整体基因表达的影响。我们通过使用激光捕获显微解剖和体外器官培养来增加空间和时间分辨率。在目标2中，我们将测试是否需要Smads1/5作为一种新的机制来激活TAK1，这是一种依赖于Smad1/5、依赖于Smad4的信号转导机制，定义了R-Smads和非正则通路之间以前未知的相互作用。在目标3中，我们通过鉴定软骨细胞中BMPs诱导的miRNAs的转录和成熟来确定Smad1-DROSHA介导的miRNA成熟如何促进Smad1/5依赖的、Smad4不依赖的信号转导。在目标4中，我们通过在目标1-3中探索的机制来检验Smad1/5依赖的、Smad4独立的信号在成人关节软骨和生长板中同样重要的假设。这些实验有可能回答长期以来关于成人软骨中BMP功能的问题，并揭示BMP在软骨中传递信号的新机制。鉴于骨性关节炎的高患病率，BMP途径影响软骨形成和软骨维持的可能性，以及确定BMP下游最有可能促进BMP的软骨保护作用的途径的必要性，我们预计这些信息将具有临床意义。

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.