TGF BETA SIGNALING IN CARTILAGE REPAIR

描述：（改编自研究者摘要）长期目标 拟议的研究的一部分是设计治疗方式， 退行性软骨疾病的软骨修复。 申请人 预期由转化生长启动的信号操纵 因子-β（TGF-β）将允许他们选择性地激活其强大的 刺激软骨基质产生和软骨细胞的能力 差异化的状态，并规避通常 限制或调节这些重要活动。 这种分子 这种方法可以增强替换局部受损软骨的能力， 用来自体外细胞培养的活软骨， 基因表达和细胞代谢在早期临床病变， 减缓或防止退化。 该研究将提供 关于TGF-b信号传导中潜在靶点的必要细节， 通过扩展TGF-β结合的新兴模型， 诱导异型受体复合物的形成， I和II受体固有激酶活性和激酶的起始 级联。 新开发和表征的抗肽抗体 II型受体的N-和C-末端结构域将用于 评价内源二元和三元异型的形成 受体复合物，并决定其信号传导能力 选择性地用于基质合成、分化状态和 增殖 这种方法是可能的，因为独特的属性 以阻断I型抗体的亲和标记 受体的 受体免疫沉淀和二维蛋白质图谱 将用于表征内源性磷酸化状态， II型受体及其相关蛋白的研究和需求 用于启动假定的激酶级联反应。 调查人员将 将这些磷酸化事件与早期激酶级联反应联系起来 他们已经确定并表征了其细胞基质 定位，生物化学，与选择性TGF-β依赖性 信号通路和激酶活性的潜力。 所获取的 因此，信息将包括TGF-β中三个重要靶域 信号传导：细胞表面受体复合物，受体相关 蛋白质/效应物和受体后激酶信号传导。每一个都将是 评估对蛋白质的TGF-β增强作用的敏感性 激酶抑制剂HA 1004和微丝修饰剂， 二氢细胞松弛素B，以确定它们与此相互作用的位点 信号系统，并提供其操纵的初步模型。

DESCRIPTION: (Adapted From Investigator's Abstract) The long-term goal of the proposed research is to devise therapeutic modalities to enhance cartilage repair in degenerative cartilage disease. The applicants expect that manipulation of signaling initiated by transforming growth factor-beta (TGF-b) will allow them to selectively activate its potent capacity to stimulate cartilage matrix production and the chondrocyte differentiated state, and circumvent the mechanisms that normally restrict or modulate these important activities. This molecular approach could enhance the capacity to replace local damaged cartilage with living cartilage derived from in vitro cell culture, and manipulate gene expression and cellular metabolism in early clinical lesions to slow or prevent degeneration. The proposed research will provide necessary details concerning potential targets in TGF-b signaling in articular chondrocytes by extending the emerging model that TGF-b binding induces formation of heterotypic receptor complexes, activation of type I and II receptor intrinsic kinase activity and initiation of a kinase cascade. Newly developed and characterized antipeptide antibodies to the N- and C-terminal domains of the type II receptor will be used to evaluate the formation of endogenous binary and ternary heterotypic receptor complexes and determine their capacity for signaling selectively for changes in matrix synthesis, differentiated state, and proliferation. This approach is possible because of the unique property of the N-terminal antibody to block affinity labeling of the type I receptor. Receptor immunoprecipitation and 2-dimensional protein mapping will be used to characterize the phosphorylation state of the endogenous type II receptor and its associated proteins and study the requirements for initiation of the putative kinase cascade. The investigators will link these phosphorylation events with the early kinase cascade substrate that they have identified and characterize its cellular localization, biochemistry, affiliation with selective TGF-b-dependent signaling pathways, and potential for kinase activity. The acquired information will thus encompass three important target domains in TGF-b signaling: cell surface receptor complexes, receptor associated proteins/effectors, and post-receptor kinase signaling.Each will be evaluated for sensitivity to the TGF-b -enhancing effects of the protein kinase inhibitor, HA1004, and the microfilament modifier, dihydrocytochalasin B, to identify their sites of interaction with this signaling system and to provide preliminary models for its manipulation.