Title: BMP/TGFbeta crosstalk in cartilage maintenance and osteoarthritis

项目摘要 在经典途径中，TGFβ分别与其I型和II型受体TGFβRII和ALK 5结合， 启动Smads 2和3。已经显示Smad 3对于关节软骨的维持是必需的，但是， Smad 2在体内的功能尚不清楚。研究表明，TGFβRII不是软骨形成所必需的 在发育过程中，但I型受体ALK 5的功能尚不清楚。这是一个重要的差距， 因为ALK 5可以独立于TGFβRII激活TGFβ通路。此外，ALK 5可以 与I型BMP受体ALK 1相互作用，使TGFβ能够激活BMP通路。我们发现， ALK 5导致致死性软骨发育不良，与缺乏TGFβRII的小鼠中的轻度存活表型相反， 这表明ALK 5在软骨中的作用独立于TGFβRII。此外，我们发现Smad 2/3 突变体是可行的，表明ALK 5并不主要作为典型TGFβ的转导子 软骨中的信号。出乎意料的是，ALK 5的缺失导致BMP输出的显著增加。生化 研究指出ALK 1是升高的BMP信号传导的来源。我们发现， 的Alk 1挽救了Alk 5突变体中的严重软骨发育不良。因此，我们的研究揭示了一种新的机制， ALK 5作为BMP输出调节剂的作用以及ALK 1以前未知的病理作用， 软骨我们将在目标1中研究ALK 1和ALK 5在生长板软骨中的作用。我们还消融了Alk 5， 成人关节软骨（AC）。突变体发展为早发性骨关节炎（OA）样病理。我们将解决 ALK 5是否作为典型TGFβ通路的转导子和/或作为BMP信号传导的抑制剂 通过Aim 2中AC中的ALK 1。最后，我们进行了交联，报告，邻近连接，和其他试验 研究ALK 1和ALK 5之间的遗传相互作用的基础。这些研究表明：（a） ALK 5的缺失触发ALK 1/ActRIIB复合物的从头形成，和（B）提供配体- 软骨中通过ALK 1的独立BMP信号传导。先前的体外研究表明，ALK 1是独特的， 在BMP受体中，其激活配体非依赖性信号传导的能力， 和生理上的相关性从未被研究过。我们在目标3中解决这些未知数。总的来说， 这些研究表明，ALK 5的主要作用不是抑制TGFβ信号，而是 通过软骨中的ALK 1阻止配体非依赖性BMP信号传导。完成这些研究可能会导致 我们对ALK 5的作用及其在调节 软骨中的TGFβ/BMP串扰。

PROJECT SUMMARY In the canonical pathway, TGFβs bind to their type I and II receptors TGFβRII and ALK5, respectively, to activate Smads 2 and 3. It has been shown that Smad3 is essential for articular cartilage maintenance, but the function of Smad2 in vivo is unknown. It has been shown that TGFβRII is not required for chondrogenesis during development, but the function of the type I receptor ALK5 is unknown. This is an important gap in knowledge because ALK5 can activate TGFβ pathways independently of TGFβRII. Moreover, ALK5 can interact with the type I BMP receptor ALK1, enabling TGFβs to activate BMP pathways. We found that loss of ALK5 leads to lethal chondrodysplasia, in contrast to the mild viable phenotype in mice lacking TGFβRII, demonstrating that ALK5 acts independently of TGFβRII in cartilage. Furthermore, we found that Smad2/3 mutants are viable, demonstrating that ALK5 does not act primarily as a transducer of canonical TGFβ signaling in cartilage. Unexpectedly, loss of ALK5 led to a significant increase in BMP output. Biochemical studies pointed to ALK1 as the source of elevated BMP signaling. This was confirmed by our finding that loss of Alk1 rescues the severe chondrodysplasia in Alk5 mutants. Our studies therefore uncover a new mechanism of action for ALK5 as a regulator of BMP output and a previously unknown pathological role for ALK1 in cartilage. We will investigate ALK1 and ALK5 action in growth plate cartilage in Aim 1. We also ablated Alk5 in adult articular cartilage (AC). Mutants develop early onset osteoarthritis (OA)-like pathologies. We will address whether ALK5 acts as a transducer of canonical TGFβ pathways and/or as an inhibitor of BMP signaling through ALK1 in AC in Aim 2. Finally, we performed crosslinking, reporter, proximity ligation, and other assays to investigate the basis for the genetic interaction between ALK1 and ALK5. These studies indicate that (a) loss of ALK5 triggers the de novo formation of ALK1/ActRIIB complexes, and (b) provide evidence for ligand- independent BMP signaling through ALK1 in cartilage. Prior in vitro studies have shown that ALK1 is unique among BMP receptors in its ability to activate ligand-independent signaling, but the underlying mechanisms and physiological relevance have never been investigated. We address these unknowns in Aim 3. In summary, these studies demonstrate that the primary role of ALK5 is not to transduce TGFβ signals, but rather is to prevent ligand-independent BMP signaling through ALK1 in cartilage. Completion of these studies could lead to a significant revision of our understanding of the role of ALK5 and its critical importance in regulating TGFβ/BMP crosstalk in cartilage.