INHIBITION OF OSTEOCLAST ACTIVITY BY OIP-1/HSCAL

OIP-1/HSCAL 对破骨细胞活性的抑制

• 批准号: 6951560
• 负责人: Sakamuri V. Reddy
• 金额: $ 20.76万
• 依托单位: MEDICAL UNIVERSITY OF SOUTH CAROLINA
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-04-10 至 2009-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6951560
• 关键词: apoptosis; biological signal transduction; cell differentiation; cell growth regulation; gene expression; gene targeting; genetic transcription; genetically modified animals; inhibitor /antagonist; interleukin 1; laboratory mouse; membrane proteins; osteoclasts; osteoprotegerin; pathologic bone resorption; physiologic bone resorption; protein protein interaction; recombinant proteins; tissue /cell culture; transfection; yeast two hybrid system

DESCRIPTION (provided by applicant): The present application is an ongoing project that will identify the molecular mechanisms responsible for osteoclast (OCL) inhibition by OCL inhibitory peptide-1 (OIP-1/hSca 1). During the previous proposal period, we have identified the OCL Inhibitory Peptide-1 (OIP-1/hSca), a local factor produced in the bone microenvironment as a novel inhibitor of OCL formation and bone resorption that is produced by OCLs. OIP-1 is a glycosylphosphatidyl-inositol (GPI) linked membrane protein (17 kDa) related to the Ly-6 family of hematopoietic proteins. We have determined that the OIP-1 c-peptide region (32 amino acids) of OIP-1 is critical for OIP-1's inhibition of OCL formation. Synthetic OIP-1 c-peptide inhibits OCL formation through suppression of TRAF-2 and p-c-Jun kinase activity, but has no effect on TRAF-6 and NF-kappaB activation. We also found that cytokines such as IFN-gamma, and IL-1beta significantly enhance OIP-1 mRNA expression in OCL precursors, CFU-GM. It is our hypothesis that OIP-1 plays a critical role in the physiologic regulation of OCL development by cellular signaling through unique transmembrane protein interactions and that OIP-1 expression in OCL precursors is controlled by specific transcriptional regulatory mechanisms. To test this hypothesis, we will pursue the following Specific Aims: Aim (1) Identify and clone the osteoclast transmembrane proteins which interacts with the OIP-1 c-peptide: (i) Clone and characterize the osteoclast membrane receptor for OIP-1; (ii) Identify OIP-1 interacting proteins using yeast two-hybrid system; (iii) Determine the effects of over-expression or blocking the expression of OIP-1 interacting proteins on OIP-1 capacity to inhibit OCL formation (iv) Determine the participation of OIP-1 interacting proteins in OIP-1 signaling to inhibit OCL formation; Aim (2) Determine if over-expression of OIP-1 in cells of OCL lineage inhibit osteoclast formation: (a) Develop TRAP-OIP-1 transgenic mice targeting OIP-1 expression in cells of OCL lineage to assess OCL development in vivo; (b) Determine if overexpression of OIP-1 in OCL precursor cells affects OCL differentiation and the status of RANKL signaling molecules during OCL differentiation in vitro; Aim (3) Isolate and characterize the OIP-1 gene promoter sequence for cytokine regulatory regions and potential transcription factors that module OIP-1 gene expression in osteoclasts. These studies should provide important insights into the regulatory mechanisms responsible for OIP-1 inhibition of OCL formation in the bone microenvironment and allow development of novel rational approaches and therapeutic agents to control enhanced osteoclast activity in Paget's disease and other bone diseases such as osteoporosis and multiple myeloma.

描述（由申请人提供）：本申请是一个正在进行的项目，将确定 OCL 抑制肽-1 (OIP-1/hSca 1) 抑制破骨细胞 (OCL) 的分子机制。在之前的提案期间，我们已经确定了 OCL 抑制肽-1 (OIP-1/hSca)，这是一种在骨微环境中产生的局部因子，作为 OCL 形成和 OCL 产​​生的骨吸收的新型抑制剂。 OIP-1 是一种糖基磷脂酰肌醇 (GPI) 连接膜蛋白 (17 kDa)，与造血蛋白 Ly-6 家族相关。我们已经确定，OIP-1 的 OIP-1 c 肽区域（32 个氨基酸）对于 OIP-1 抑制 OCL 形成至关重要。合成的 OIP-1 c 肽通过抑制 TRAF-2 和 p-c-Jun 激酶活性来抑制 OCL 形成，但对 TRAF-6 和 NF-kappaB 激活没有影响。我们还发现，IFN-gamma 和 IL-1beta 等细胞因子显着增强 OCL 前体 CFU-GM 中 OIP-1 mRNA 的表达。我们假设 OIP-1 通过独特的跨膜蛋白相互作用通过细胞信号传导在 OCL 发育的生理调节中发挥关键作用，并且 OIP-1 在 OCL 前体中的表达受到特定转录调节机制的控制。为了检验这一假设，我们将追求以下具体目标： 目标 (1) 鉴定并克隆与 OIP-1 c 肽相互作用的破骨细胞跨膜蛋白： (i) 克隆并表征 OIP-1 的破骨细胞膜受体； (ii) 使用酵母双杂交系统鉴定 OIP-1 相互作用蛋白； (iii) 确定 OIP-1 相互作用蛋白的过度表达或阻断表达对 OIP-1 抑制 OCL 形成的能力的影响 (iv) 确定 OIP-1 相互作用蛋白在 OIP-1 信号传导中的参与，以抑制 OCL 形成;目的 (2) 确定 OCL 谱系细胞中 OIP-1 的过度表达是否抑制破骨细胞形成： (a) 开发针对 OCL 谱系细胞中 OIP-1 表达的 TRAP-OIP-1 转基因小鼠，以评估 OCL 体内发育； (b) 体外确定OCL前体细胞中OIP-1的过表达是否影响OCL分化以及OCL分化过程中RANKL信号分子的状态；目标 (3) 分离并表征破骨细胞中调节 OIP-1 基因表达的细胞因子调控区和潜在转录因子的 OIP-1 基因启动子序列。这些研究应该为骨微环境中 OIP-1 抑制 OCL 形成的调节机制提供重要见解，并允许开发新的合理方法和治疗药物来控制佩吉特病和其他骨疾病（如骨质疏松症和多发性骨病）中破骨细胞活性的增强。骨髓瘤。