TGF-beta Signaling and Degenerative Joint Diseases

描述(申请人提供)：人类骨关节炎(OA)是一种以关节软骨退化为特征的进行性关节疾病。在骨性关节炎发育过程中通常出现的其他主要特征包括软骨下骨增厚、骨赘的形成、不同程度的滑膜炎症、韧带和半月板的退化以及关节囊肥大。然而，这些事件的后果和之间的关系目前尚不清楚。转化生长因子β信号在控制软骨细胞分化中起关键作用。据报道，抑制转化生长因子β信号转导可导致小鼠严重的骨关节炎样表型。最近的遗传学研究也表明，SMAD3基因突变与患者髋关节和膝关节骨性关节炎的高发病率有关。然而，关节软骨细胞转化生长因子β信号转导下游的关键靶基因仍有待确定。我们的初步研究表明，软骨细胞特异性缺失的II型转化生长因子β受体基因(TGFBR2)在出生后/成年阶段会导致严重的OA样表型的发展。TGFBR2条件性基因敲除(Tgfbr2Col2ER)小鼠表现出骨性关节炎最重要的特征。在这些小鼠中，Runx2和ATF4的表达显着增加。我们进一步证明，抑制转化生长因子β信号转导上调MMP13和Adamts5的表达。我们建立了TGFBR2/Mmp13和TGFBR2/Adamts5双基因敲除小鼠，并证明了Tgfbr2Col2ER背景中Mmp13或Adamts5基因的缺失显著逆转了Tgfbr2Col2ER小鼠观察到的大部分OA特征。基于这些观察，我们推测Runx2和ATF4在激活Mmp13和Adamts5表达(MMP13和ADAMTS5是软骨基质降解的两个关键酶)中发挥核心作用，从而导致Tgfbr2Col2ER小鼠发生骨性关节炎。在拟议的研究中，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转化生长因子β抑制--Runx2/ATF4-&Gt；MMP13/Adamts5通路的信号机制。我们提出了三个具体目标来检验我们的假设。具体目标1：我们将确定转化生长因子β信号在成年小鼠骨性关节炎发生中的作用。我们的假设是，成年小鼠关节软骨细胞中TGFBR2基因的缺失会导致成年Tgfbr2Col2ER小鼠出现类似骨关节炎的形态变化和疼痛相关的行为变化。在特定目标2：我们将确定Mmp13在Tgfbr2Col2ER诱导的OA表型中的作用。我们的假设是，转化生长因子β信号抑制诱导MMP13的表达是由转录因子Runx2和ATF4介导的。具体目标3：我们将确定Adamts5在Tgfbr2Col2ER诱导的OA表型中的作用。我们的假设是，在Tgfbr2Col2ER小鼠中缺失Adamts5/Mmp13基因将比单独缺失Mmp13和Adamts5对Tgfbr2Col2ER诱导的关节软骨退化具有更好的保护作用。我们提出的研究将为了解骨性关节炎发生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.