INHIBITION OF OSTEOCLAST ACTIVITY BY OIP1/HSCAL

OIP1/HSCAL 对破骨细胞活性的抑制

基本信息

批准号：
6540882
负责人：
Sakamuri V. Reddy
金额：
$ 13.8万
依托单位：
UNIVERSITY OF PITTSBURGH AT PITTSBURGH
依托单位国家：
美国
项目类别：
财政年份：
1999
资助国家：
美国
起止时间：
1999-04-10 至 2003-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/6540882
关键词：
CHO cells Escherichia coli apoptosis cell differentiation cell growth regulation expression cloning gene expression inhibitor /antagonist interleukin 1 laboratory mouse osteoclasts pathologic bone resorption physiologic bone resorption recombinant proteins tissue /cell culture transfection

项目摘要

Enhanced osteoclast (OCL) activity plays a major role in the pathogenesis of osteoporosis, bone metastasis, and hypercalcemia of malignancy. Thus, identification and characterization of inhibitors of OCL activity should be clinically important. We have used an expression cloning approach to identify novel inhibitors of OCL activity that are produced by OCL and have recently identified, cloned, and produced in E. coli, osteoclast inhibitory peptide 1 (OIP-1), a factor that blocks both OCL formation and bone resorption. The factor is the human homologue of murine Sca1, a member of the Ly6 family of proteins that is expressed on hematopoietic precursors and also expressed by osteoblasts, but heretofore had no known effect on OCL activity. This factor represents a novel class of factors which inhibit OCL activity and that is both membrane-bound and also released from the cell. In this proposal, we will determine the mechanism of OCL inhibition by OIP-1/hSca1 by: (1) Assessing if the membrane-bound form as well as the extracellular form of OIP-1/hSca1 inhibits OCL formation. We will express a c-terminal truncation form of OIP-1/hSca1, label the soluble recombinant protein and determine if there is a target receptor by Scatchard binding analysis and cross-linking studies; (2) Examining the effects of E. coli-derived recombinant OIP-1/hSca1 on the growth and differentiation of the different stages of OCL development and determining the effect of OIP-1/hSca1 on OCL apoptosis; and (3) Testing OIP-1/hSca1 on OCL formation in vivo. As part of these studies, we will stably transfect Chinese Hamster Ovary (CHO) cells with an OIP-1/hSca1 cDNA expression construct and implant them into nude mice to assess the effects of continuous exposure of mammalian OIP-1/hSca1 on basal and IL-1 stimulated bone remodeling.

增强的破骨细胞（OCL）活性在骨质疏松、骨转移和高钙血症的发病机制恶性肿瘤。因此，抑制剂的鉴定和表征 OCL 活动应该具有临床重要意义。我们使用了一个表达方式克隆方法来鉴定新型 OCL 活性抑制剂由 OCL 生产，最近已在大肠杆菌，破骨细胞抑制肽 1 (OIP-1)，一种阻断因子 OCL 形成和骨吸收。因素是人鼠 Sca1 的同源物，Ly6 蛋白家族的成员，在造血前体细胞上表达，也由成骨细胞，但迄今为止对 OCL 活性尚无已知影响。这该因子代表一类抑制 OCL 活性的新型因子它既是膜结合的，也是从细胞中释放的。在在这个提案中，我们将通过以下方式确定 OCL 抑制机制： OIP-1/hSca1 通过： (1) 评估膜结合形式以及 OIP-1/hSca1 的细胞外形式抑制 OCL 形成。我们将表达 OIP-1/hSca1 的 C 端截断形式，标记可溶性重组蛋白并确定是否存在目标受体 Scatchard 结合分析和交联研究； (2) 检查大肠杆菌来源的重组 OIP-1/hSca1 对生长和生长的影响 OCL发展不同阶段的分化测定OIP-1/hSca1对OCL凋亡的影响； (3) 测试 OIP-1/hSca1 对体内 OCL 形成的影响。作为这些研究的一部分，我们将用 OIP-1/hSca1 稳定转染中国仓鼠卵巢 (CHO) 细胞 cDNA表达构建体并将其植入裸鼠以评估持续暴露于哺乳动物 OIP-1/hSca1 对基础和 IL-1刺激骨重塑。