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

描述（由申请人提供）：糖皮质激素（GC）是常用于治疗自身免疫性和炎症性疾病的类固醇激素。这些药物可引起严重的不良反应，如骨丢失和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抑制的主要靶点。我们发现GCs通过IGF依赖和非依赖机制抑制IGF结合蛋白-5（IGFBP-5）的表达，IGFBP-5结合IGF并刺激成骨细胞。GC诱导的IGFPB-5启动子活性抑制由复合反应元件介导，该复合反应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在人成骨细胞中的调节机制。这项资助的重点是通过NFI基因家族的成员NFI-B调节IGFBP-5基因表达。为了确定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，以及2）GR与NFI的配体依赖性结合介导GC对IGFBP-5表达的抑制。这些假设将通过以下具体目的来解决：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，和2）确定NFI-B敲低/过表达对IGFBP-5表达、成骨细胞增殖和分化的影响。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调节IGFBP-5启动子激活的NFI-B、GR、AP-2之间的相互作用。NFI-B基因敲低对IGFBP-5表达的影响将通过用合成siRNA寡核苷酸瞬时转染来评估。通过瞬时转染NFI-B表达载体进行NFI-B的过表达。将通过定量真实的时间PCR（qRT-PCR）测定mRNA水平和蛋白质印迹法测定蛋白质表达水平。将通过基于细胞数量的测定和通过使用qRT-PCR表达成骨细胞特异性基因来进行成骨细胞增殖和分化。拟议的工作不仅将提供新的见解，在成骨细胞的调节，和GC诱导的IGFBP- 5的表达抑制NFI-B转录因子的作用。此外，这些研究将发现可以用于骨质疏松症和GIOP的药物预防和治疗的机制，这些疾病严重困扰着美国人。

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.