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

描述(申请人提供)：糖皮质激素(GC)是一种经常用于治疗自身免疫性和炎症性疾病的类固醇激素。这些药物可能会导致严重的不良反应，如骨质丢失和GC诱发的骨质疏松症(GIOP)。胰岛素样生长因子(IGF)系统是骨内GC抑制的主要靶点。我们发现GCs抑制IGF结合蛋白-5(IGFBP-5)的表达，IGFBP-5结合IGF并通过IGF依赖和独立的机制刺激成骨细胞。GC对IGFPB-5启动子活性的抑制是由一个与转录因子激活蛋白-2(AP-2)和核因子I(NFI)结合的复合反应元件介导的。我们的长期目标是确定IGFBP-5在人类成骨细胞中的调控机制。这笔赠款的重点是NFI-B基因家族成员NFI-B对IGFBP-5基因表达的调控。为了明确IGFBP-5基因调控的潜在机制，我们将检验两个假设：1)NFI通过激活IGFBP-5基因转录来调节成骨细胞的增殖和分化；2)GR与NFI的配体依赖结合介导GC抑制IGFBP-5的表达。这些假说的具体目的如下：1)研究在成骨细胞中参与IGFBP-5转录调控的NFI-B、GR和AP-2之间的相互作用；2)确定NFI-B基因敲除/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改变分析来研究参与调控IGFBP-5启动子激活的NFI-B、GR、AP-2之间的相互作用。NFI-B基因敲除对IGFBP-5表达的影响将通过合成的siRNA寡核苷酸瞬时转染来评估。通过瞬时转染NFI-B表达载体实现NFI-B的过表达。基因的表达水平将通过定量实时聚合酶链式反应(qRT-PCR)和蛋白质的蛋白质印迹来确定。成骨细胞的增殖和分化将通过基于细胞数量的分析和成骨细胞特异性基因的表达来进行。拟议的工作不仅将为NFI-B转录因子在成骨细胞调控中的作用以及GC诱导的IGFBP-5表达抑制提供新的见解。此外，这些研究将发现可针对骨质疏松症和GIOP的药物预防和治疗的机制，这两种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.