Regulation of IGFBP-5 and osteoblast functions by Nuclear Factor I

核因子 I 对 IGFBP-5 和成骨细胞功能的调节

• 批准号: 7322181
• 负责人: Laura A Perez-Casellas
• 金额: $ 2.9万
• 依托单位: LOMA LINDA UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-08-01 至 2010-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7322181
• 关键词: Address; Adverse effects; Affect; Age-Years; AlamarBlue; Alkaline Phosphatase; American; Binding; Binding Sites; Biological Assay; Cell Count; Cells; Co-Immunoprecipitations; Condition; Development; Dexamethasone; Differentiation Antigens; Disease; Distress; EMSA; Electrophoretic Mobility Shift Assay; Family; Foundations; Gene Expression; Gene Expression Regulation; Gene Family; Genes; Genetic Transcription; Glucocorticoid Receptor; Glucocorticoids; Goals; Grant; Growth Factor; Human; Immune; Inflammatory; Insulin-Like Growth Factor Binding Protein 5; Insulin-Like Growth-Factor-Binding Proteins; Ligands; Measures; Mediating; Messenger RNA; Molecular Target; Osteoblasts; Osteoporosis; Pharmaceutical Preparations; Polymerase Chain Reaction; Prevention; Protein Overexpression; Proteins; Public Health; RNA Interference; Regulation; Research; Response Elements; Risk Factors; Role; Small Interfering RNA; Somatomedins; System; Testing; Thymidine; Time; Transcription Factor AP-2 Alpha; Transfection; Western Blotting; Work; base; bone; bone loss; chromatin immunoprecipitation; design; expression vector; human NFIB protein; innovation; insight; mRNA Expression; member; nuclear factor 1; osteosarcoma; promoter; steroid hormone; transcription factor

DESCRIPTION (provided by applicant): Glucocorticoids (GCs) are steroid hormones that are frequently used in therapy for auto-immune and inflammatory diseases. These drugs can cause severe adverse effects such as bone loss and GC-induced osteoporosis (GIOP). The Insulin-like Growth Factor (IGF) system is a major target of GC inhibition in bone. We found that GCs inhibit expression of IGF binding protein-5 (IGFBP-5) which binds IGFs and stimulates osteoblast by IGF dependent and independent mechanisms. GC-induced inhibition of IGFPB-5 promoter activity was mediated by a composite response element that has binding sites for the transcription factor activator protein-2 (AP-2) and nuclear factor I (NFI). Our long term goal is to determine the mechanism by which IGFBP-5 is regulated in human osteoblasts. The focus of this grant is on the regulation of IGFBP-5 gene expression by NFI-B, a member of the NFI gene family. To define underlying mechanisms of IGFBP-5 gene regulation we will test two hypotheses: 1) NFI functions to regulate osteoblast proliferation and differentiation through activation of IGFBP-5 gene transcription and 2) Ligand dependent binding of GR to NFI mediates GC inhibition of IGFBP-5 expression. These hypotheses will be addressed with the following specific aims: 1) Characterize interactions between NFI-B, GR and AP-2 that are involved in regulation of IGFBP-5 transcription in osteoblasts and 2) Determine effects of NFI-B knockdown/ overexpression in IGFBP-5 expression, osteoblast proliferation and differentiation. We will investigate interactions between NFI-B, GR, AP-2 involved in the regulation of IGFBP-5 promoter activation using chromatin immunoprecipitation and electrophoretic mobility shift assays. Effects of NFI-B gene knockdown on IGFBP-5 expression will be assessed by transient transfection with synthetic siRNA oligos. Overexpression of NFI-B will be performed by transient transfection of NFI-B expression vector. Gene expression levels will be determined by quantitative Real Time PCR (qRT-PCR) for mRNA levels and Western blot for proteins. Osteoblast proliferation and differentiation will be performed by cell number based assays and by expression of osteoblast specific genes using qRT-PCR. Proposed work will not only provide new insights regarding the role of NFI-B transcription factor in osteoblast regulation, and GC-induced inhibition of IGFBP- 5 expression. Additionally, these studies will discover mechanisms that can be targeted for pharmacological prevention and treatment of osteoporosis and GIOP, conditions that significantly distress Americans.

描述（由申请人提供）：糖皮质激素（GC）是类固醇激素，常用于治疗自身免疫性疾病和炎症性疾病。这些药物可引起严重的副作用，例如骨质流失和GC诱导的骨质疏松症（GIOP）。胰岛素样生长因子 (IGF) 系统是骨中 GC 抑制的主要目标。我们发现 GC 抑制 IGF 结合蛋白 5 (IGFBP-5) 的表达，IGFBP-5 与 IGF 结合并通过 IGF 依赖和独立机制刺激成骨细胞。 GC 诱导的 IGFPB-5 启动子活性抑制是由复合反应元件介导的，该元件具有转录因子激活蛋白 2 (AP-2) 和核因子 I (NFI) 的结合位点。我们的长期目标是确定 IGFBP-5 在人类成骨细胞中的调节机制。本次资助的重点是 NFI 基因家族成员 NFI-B 对 IGFBP-5 基因表达的调节。为了定义 IGFBP-5 基因调控的潜在机制，我们将测试两个假设：1) NFI 通过激活 IGFBP-5 基因转录来调节成骨细胞增殖和分化，2) GR 与 NFI 的配体依赖性结合介导 GC 对 IGFBP-的抑制。 5 表达。这些假设将通过以下具体目标得到解决：1) 表征参与成骨细胞中 IGFBP-5 转录调节的 NFI-B、GR 和 AP-2 之间的相互作用；2) 确定 NFI-B 敲低/过表达对成骨细胞的影响IGFBP-5 表达、成骨细胞增殖和分化。我们将使用染色质免疫沉淀和电泳迁移率变动分析来研究参与 IGFBP-5 启动子激活调节的 NFI-B、GR、AP-2 之间的相互作用。将通过用合成 siRNA 寡核苷酸瞬时转染来评估 NFI-B 基因敲低对 IGFBP-5 表达的影响。 NFI-B的过表达将通过NFI-B表达载体的瞬时转染来进行。基因表达水平将通过定量实时 PCR (qRT-PCR) 确定 mRNA 水平，并通过蛋白质印迹确定。成骨细胞增殖和分化将通过基于细胞数量的测定和使用 qRT-PCR 表达成骨细胞特异性基因来进行。拟议的工作不仅将提供关于 NFI-B 转录因子在成骨细胞调节中的作用以及 GC 诱导的 IGFBP-5 表达抑制的新见解。此外，这些研究还将发现可针对骨质疏松症和 GIOP 进行药物预防和治疗的机制，这些疾病使美国人深受困扰。