HORMONAL REGULATION OF BONE COLLAGENASE MRNA

描述（改编自申请人的摘要）：长期目标 本更新申请的目的是了解 间质胶原酶（MMP-1）在骨重建过程中的作用。 骨 重塑是生长和骨折愈合所必需的。 此外，本发明还提供了一种方法， 该过程用于维持体内钙的稳态。 MMP-1， 一种由成骨细胞产生的基质金属蛋白酶， 引发骨骼的分解 有越来越多的证据表明 MMP-1蛋白与骨重建区域相关 正在发生。 利用大鼠克隆细胞系UMR 106-01作为 成骨细胞模型，已经确定MMP-1的分泌 蛋白质是响应于骨重塑剂而启动的， 甲状旁腺激素（PTH）。 甲状旁腺素诱导子宫内膜异位症MMP-1分泌的研究 106-01细胞主要由cAMP介导。 这种归纳在某种程度上 转录调控。 MMP-1转录的调控 通过因子与上游区域的序列结合而发生 的基因。 CREB蛋白通过相互作用激活转录 具有对cAMP有反应的基因的调节区中的序列。 因此，CREB与调控蛋白中的序列的相互作用 MMP-1区域是PTH刺激转录所必需的。 因此，为了了解MMP-1在骨重建中的调节作用， 本项目的目标是：1）确定CREB的作用 在MMP-1 mRNA的PTH诱导中，使用（a）凝胶超位移，（B）位移- 蛋白质印迹法和（c）磷蛋白的免疫沉淀; 2） 表征与CREB相互作用以诱导MMP-1 mRNA的蛋白质 通过PTH，通过（a）筛选表达文库，（B）凝胶迁移和DNA酶 I足迹法，和（c）DNA测序; 3）证实PTH对 培养的原代大鼠成骨细胞中MMP-1 mRNA的表达，通过（a） 北方印迹分析，和（B）DNA酶I足迹法和凝胶位移;和 4)破译与AP-2结合的转录因子的作用， MMP-1的5'调控区中的PEA-3共有序列位点， 通过（a）凝胶超位移，（B）位移-蛋白质印迹法测定PTH对MMP-1 mRNA的活化 印迹，和（c）蛋白质的表征。 这些研究 旨在帮助我们了解 动物骨吸收和骨形成之间的临界平衡 因此，在未来， 疾病、肌肉骨骼肿瘤和骨折愈合延迟。

DESCRIPTION (Adapted from the Applicant's Abstract): The long range goal of this renewal application is to understand the regulation of interstitial collagenase (MMP-1) in the bone remodeling process. Bone remodeling is necessary for growth and fracture healing. In addition, this process serves to maintain calcium homeostasis in the body. MMP-1, a matrix metalloprotease produced by the osteoblast, is thought to initiate the breakdown of bone. Strong evidence has been accumulating that MMP-1 protein is associated with areas in which bone remodeling is occurring. Utilizing the rat clonal cell line UMR 106-01 as an osteoblastic model, it has been determined that secretion of MMP-1 protein is initiated in response to bone remodeling agents such as parathyroid hormone (PTH). PTH induction of MMP-1 secretion in UMR 106-01 cells is mediated mainly by cAMP. This induction is in part transcriptionally regulated. The regulation of MMP-1 transcription occurs through binding of factors to sequences in the upstream region of the gene. CREB protein activates transcription through interaction with sequences in the regulatory region of genes responsive to cAMP. Thus it follows that interaction of CREB with sequences in the regulatory region of MMP-1 is necessary for PTH stimulation of transcription. Therefore, to understand the regulation of MMP-1 by bone remodeling agents, the goals of this project are to: 1) determine the role of CREB in the PTH induction of MMP-1 mRNA, using (a) gel supershift, (b) shift- Western blotting, and (c) immunoprecipitation of phosphoproteins; 2) characterize protein(s) interacting with CREB for induction of MMP-1 mRNA by PTH, by (a) screening an expression library, (b) gel shift and DNase I footprinting, and (c) DNA sequencing; 3) affirm the effects of PTH on MMP-1 mRNA expression in primary rat osteoblasts in culture, through (a) Northern blot analysis, and (b) DNase I footprinting and gel shift; and 4) decipher the role of transcription factors that bind to the AP-2 and PEA-3 consensus sequence sites in the 5' regulatory region of MMP-1 for PTH activation of MMP-1 mRNA by (a) gel supershift, (b) shift-Western blotting, and (c) characterization of proteins. These studies are intended to contribute to our understanding of the regulation of the critical balance between bone resorption and formation in the animal and, thus, lead to therapies in the future to treat metabolic bone disorders, musculoskeletal neoplasms and delays in fracture healing.