FUNCTIONAL ROLE OF IL 6 RECEPTOR SUBUNITS IN RA

IL 6 受体亚基在 RA 中的功能作用

• 批准号: 3727961
• 负责人: GERALD M FULLER
• 金额: --
• 依托单位: UNIVERSITY OF ALABAMA AT BIRMINGHAM
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3727961
• 关键词: RNase protection assay; cellular pathology; cytokine receptors; endopeptidases; enzyme linked immunosorbent assay; glycoprotein structure; in situ hybridization; interleukin 6; laboratory mouse; laboratory rat; membrane proteins; messenger RNA; mixed tissue /cell culture; molecular pathology; monoclonal antibody; osteoclasts; polymerase chain reaction; protein signal sequence; protein structure function; receptor expression; rheumatoid arthritis; synovial fluid; western blottings

The central focus of this proposal is to determine the functional role(s) of IL-6 and its receptors in synoviocytes found in normal and rheumatoid arthritis (RA) joints. Interleukin-6 is the most prominently expressed cytokine in synovial fluids from patients with RA. Recruited inflammatory cells (monocytes, neutrophils and T and B lymphocytes) as well as fibroblasts may contribute to the high levels of IL-6. For cells to respond to IL-6 they must possess a cognant IL-6 receptor subunit (IL- 6R) and its signal transducing subunit (gp130). It is not currently known what cells in the RA lesion possess the IL-6 receptors nor if expression of the receptor is altered in the synoviocytes as part of the etiology of this disorder. Not all cell types express IL-6R however, the signal transducing subunit (gp130) is found in nearly all cell types. Induction of IL-6R during an arthritic crisis could make quiescent cells responsive to the cytokine. Experiments are planned using sensitive ribonuclease protection assays (RPA) and reverse transcriptase polymerase chain reaction (RT-PCR) methodologies to quantitate IL-6R and gp 130 mRNAs in synoviocytes from normal and induced arthritic joints of an animal model. Additionally, the IL-6R mRNA and protein from human synoviocyte cell lines will be measured. Of considerable interest is the finding that soluble forms of the two subunits of the IL-6 receptor complex have been identified both in serum and urine. Unlike soluble forms of receptors from other cytokines, sIL-6R can bind with IL-6 and this complex activate membrane bound gp130. This feature makes the sIL- 6R an agonist, and renders cells IL-6 responsive, which by themselves cannot bind IL-6. Another experimental aim of this project will be determined the concentration of sIL-6R in synovial fluid and from conditioned medium of synoviocytes exposed to factors known to be present in the arthritic synovial fluids. sIL-6R is expressed by being proteolytically cleaved by a yet to be identified cell surface protease. Experiments are described in which this protease will be identified using a genetically constructed form of the IL-6R for a functional assay system. It has recently been reported that when IL-6 and sIL-6R are added to a co-culture of osteoblasts and bone marrow stromal cells a dramatic increase in the number of osteoclast-like cells are formed. Since bone dissolution is a part of the pathology of rheumatoid arthritis an understanding of how these cells are formed from the IL-6 signal will be investigated. Our specific aims will be to identify the cells responsible for producing sIL-R, determine what factors alter the expression of the receptor, identify the protease that cleaves the receptor from the cell surface and to determine some of the events that lead to osteoclast formation. We believe results from this investigation will provide new information on the cellular and molecular events that take place in the RA lesion.

本建议的中心重点是确定职能作用 IL-6及其受体在正常和类风湿关节炎滑膜细胞中的表达 关节炎（RA）关节。 白细胞介素-6是最突出的表达 RA患者滑液中的细胞因子。 招募 炎性细胞（单核细胞、嗜中性粒细胞以及T和B淋巴细胞）， 以及成纤维细胞可能有助于高水平的IL-6。 的细胞 为了响应IL-6，它们必须具有同源的IL-6受体亚单位（IL- 6 R）及其信号转导亚基gp 130。 它当前未 已知RA病变中的哪些细胞具有IL-6受体， 受体的表达在滑膜细胞中发生改变， 这种疾病的病因。 然而，并非所有细胞类型都表达IL-6 R， 信号转导亚基（gp 130）存在于几乎所有细胞类型中。 在关节炎危象期间诱导IL-6 R可以使静止细胞 对细胞因子有反应。 计划使用敏感的 核糖核酸酶保护试验（RPA）和逆转录酶聚合酶 定量IL-6 R和gp 130的链反应（RT-PCR）方法 正常和诱导性关节炎关节滑膜细胞中的mRNA 动物模型 此外，人IL-6 R mRNA和蛋白质表达水平与人IL-6 R mRNA和蛋白质表达水平呈负相关。 将测量滑膜细胞系。 相当有趣的是， 发现IL-6受体的两个亚单位的可溶形式 在血清和尿液中均发现了这种复合物。 与可溶性 作为其他细胞因子受体的一种形式，sIL-6 R可与IL-6结合， 该复合物激活膜结合的GP 130。 这一特点使SIL- 6 R是一种激动剂，并使细胞对IL-6产生反应， 不能与IL-6结合。 该项目的另一个实验目标是 测定滑膜液中sIL-6 R的浓度， 滑膜细胞的条件培养基暴露于已知存在的因子 关节炎的滑液中 sIL-6 R表达为 由尚待鉴定的细胞表面蛋白酶进行蛋白水解切割。 描述了实验，其中该蛋白酶将使用 用于功能测定的IL-6 R的遗传构建形式 系统 最近有报道称，当IL-6和sIL-6 R被抑制时， 加入到成骨细胞和骨髓基质细胞的共培养物中， 破骨细胞样细胞的数量急剧增加。 由于骨溶解是类风湿关节炎病理学的一部分， 了解这些细胞是如何从IL-6信号中形成的， 追究 我们的具体目标将是识别细胞 负责产生sIL-R，确定哪些因素改变了 受体的表达，鉴定切割受体的蛋白酶。 从细胞表面的受体，并确定一些事件， 导致破骨细胞形成。 我们相信这次调查的结果 将提供关于细胞和分子事件的新信息， 发生在RA病变中。