TGF-beta Signaling and Degenerative Joint Diseases

描述（由申请人提供）：人骨关节炎（OA）是一种进行性关节疾病，其特征为关节软骨退化。在OA发展过程中通常发生的其他关键特征包括软骨下骨增厚、骨赘形成、不同程度的滑膜炎症、韧带和滑膜变性以及关节囊肥大。然而，这些事件之间的后果和关系目前尚不清楚。TGFβ信号传导在控制软骨细胞分化中起关键作用。据报道，抑制TGFβ信号传导导致小鼠中严重的OA样表型。最近的遗传学研究也表明，Smad 3基因的突变与患者髋关节和膝关节OA的高发病率相关。然而，在OA发展过程中关节软骨细胞中TGFβ信号传导的关键下游靶基因仍有待确定。我们的初步研究表明，软骨细胞特异性缺失II型TGFβ受体基因（Tgfbr 2）在出生后/成人阶段导致严重的OA样表型的发展。Tgfbr 2条件性基因敲除（Tgfbr 2Col 2 ER）小鼠显示出OA的最重要特征。在这些小鼠中，Runx 2和Atf 4表达显著增加。我们进一步证明，抑制TGFβ信号传导上调Mmp 13和Adamts 5表达。我们产生了Tgfbr 2/Mmp 13和Tgfbr 2/Adamts 5双敲除小鼠，并证明在Tgfbr 2Col 2 ER背景中缺失Mmp 13或Adamts 5基因显著逆转了在Tgfbr 2Col 2 ER小鼠中观察到的大多数OA特征。基于这些观察结果，我们假设Runx 2与ATF 4在激活Mmp 13和ADAMTS 5表达（MMP 13和ADAMTS 5是软骨基质降解中的两种关键酶）中起核心作用，从而导致Tgfbr 2Col 2 ER小鼠中OA的发展。在本研究中，我们将使用综合的分子和遗传学方法来确定关节软骨细胞中TGFβ抑制-> Runx 2/ATF 4-> Mmp 13/Adamts 5通路的信号传导机制。我们提出了三个具体目标来检验我们的假设。在具体目标1中：我们将确定TGFβ信号在成年小鼠OA发展中的作用。我们的假设是，成年小鼠关节软骨细胞中Tgfbr 2基因的缺失导致成年Tgfbr 2Col 2 ER小鼠OA样形态学变化和疼痛相关的行为变化。在具体目标2中：我们将确定Mmp 13在Tgfbr 2Col 2 ER诱导的OA表型中的作用。我们的假设是，TGFβ信号转导抑制诱导Mmp 13表达由转录因子Runx 2和ATF 4介导。在具体目标3中：我们将确定Adamts 5在Tgfbr 2Col 2 ER诱导的OA表型中的作用。我们的假设是，在Tgfbr 2Col 2 ER小鼠中，Adamts 5/Mmp 13基因的缺失将比单独缺失Mmp 13和Adamts 5对Tgfbr 2Col 2 ER诱导的关节软骨降解具有更好的保护作用。我们提出的研究将为OA发展的分子机制提供新的见解。

DESCRIPTION (provided by applicant): Human osteoarthritis (OA) is a progressive joint disease characterized by degradation of articular cartilage. Other key features normally occurring during OA development include thickening of the subchondral bone, formation of osteophyte, variable degrees of inflammation of the synovium, degeneration of ligaments and the menisci, and hypertrophy of the joint capsule. However, the consequence and relationship among these events are currently unknown. TGFβ signaling plays a critical role in controlling chondrocyte differentiation. It has been reported that inhibition of TGFβ signaling causes severe OA-like phenotype in mice. Recent genetic studies also demonstrated that mutations of the Smad3 gene are associated with high incidence of hip and knee OA in patients. However, the key downstream target genes of TGFβ signaling in articular chondrocytes during OA development remains to be determined. Our preliminary studies demonstrated that chondrocyte-specific deletion of the type II TGFβ receptor gene (Tgfbr2) at postnatal/adult stages lead to development of a severe OA-like phenotype. Tgfbr2 conditional knockout (Tgfbr2Col2ER) mice display the most important features of OA. In these mice Runx2 and Atf4 expression was significantly increased. We further demonstrated that inhibition of TGFβ signaling up regulates Mmp13 and Adamts5 expression. We generated Tgfbr2/Mmp13 and Tgfbr2/Adamts5 double knockout mice and demonstrated that deletion of either the Mmp13 or Adamts5 gene in the Tgfbr2Col2ER background significantly reversed the majority of the OA features observed in Tgfbr2Col2ER mice. Based on these observations, we hypothesize that Runx2, with ATF4, plays a central role in activation of Mmp13 and Adamts5 expression (MMP13 and ADAMTS5 are two key enzymes in cartilage matrix degradation) and consequently leading to OA development in Tgfbr2Col2ER mice. In the proposed studies we will determine the signaling mechanism of the TGFβ inhibition->Runx2/ATF4->Mmp13/Adamts5 pathway in articular chondrocytes using comprehensive molecular and genetic approaches. We have proposed three specific aims to test our hypothesis. In Specific Aim 1: we will determine the role of TGFβ signaling in OA development in adult mice. Our hypothesis is that deletion of the Tgfbr2 gene in articular chondrocytes of adult mice leads to OA-like morphological changes and pain-related behavioral changes in adult Tgfbr2Col2ER mice. In Specific Aim 2: we will determine the role of Mmp13 in Tgfbr2Col2ER-induced OA phenotype. Our hypothesis is that TGFβ signaling inhibition-induced Mmp13 expression is mediated by transcription factors Runx2 and ATF4. In Specific Aim 3: we will determine the role of Adamts5 in Tgfbr2Col2ER-induced OA phenotype. Our hypothesis is that deletion of Adamts5/Mmp13 genes in Tgfbr2Col2ER mice will have better protective effect than deletion of Mmp13 and Adamts5 alone on Tgfbr2Col2ER-induced articular cartilage degradation. Our proposed studies will provide novel insights into the molecular mechanisms of OA development.