Vitamin D and Estrogen Action in Bone: Concerted Role for hnRNPs in the C Family

描述（由申请人提供）：25年前，我们开始调查居住在洛杉矶动物园的青春期雌性新世界灵长类动物中爆发的脊骨病。我们对这一“自然实验”的研究，以及对一名具有相似表型的青春期人类女性的研究，使我们发现了灵长类动物（包括人类）对维生素D和雌激素抗性的一种新方法。我们将这些耐药蛋白命名为维生素D反应元件结合蛋白（VDR - bp）和雌激素反应元件结合蛋白（re - bp），因为它们能够与配体维生素D受体（VDR）和雌激素受体1 （ER1）反式竞争其同源反应元件，并将它们鉴定为异质核核糖核蛋白C （hnRNPC）家族中的核酸结合蛋白。最近的工作使我们认识到，这些hnRNPs改变类固醇激素导向的基因表达的能力并不局限于它们在转录水平上的显性负作用。凭借其与单链DNA （ssDNA）、ssRNA以及双链DNA （dsDNA）相互作用的能力，这些hnRNPs具有在细胞中多个位点控制基因表达的潜力。在这里，我们推测一个特定的hnRNP可以通过依次调节染色质重塑（ssDNA结合）、转录（dsDNA结合）、剪接（ssDNA和ssRNA结合）和microRNA （miRNA； ssRNA结合），在单个基因产物的同步表达中充当多位点参与者。由于我们研究的hnRNP过表达的人类和亚人类灵长类动物实验都导致了青春期女性骨骼生长中维生素D和雌激素的一致拮抗作用，因此在这里，我们将集中研究作为这些hnRNP天然靶点的生长骨骼和成骨细胞。提出了三个具体目标：1)利用染色质修饰/沉淀，过/过表达和新型细胞自由翻译技术，探索hnRNPs对负责1,25-二羟基维生素D和雌二醇驱动的骨生长的细胞的影响；2)采用新的RNA免疫沉淀和微RNA方法研究这些hnRNPS通过直接结合同一基因的RNA产物来控制转录物剪接和处理的多功能性，该基因的表达在染色质重塑和转录水平上同时受到调节；3)通过在小鼠骨骼中靶向转基因过表达vre - bp和re - bp hnRNPs，确定这些hnRNPs对生长中的青少年骨骼的综合作用。我们期望所提出的研究将为了解这些多功能hnRNPs如何控制生长骨骼中固醇/类固醇激素调节的基因表达过程铺平道路，并描绘该过程中潜在治疗干预的新观点。公共卫生相关性：维生素D和雌激素是两种类固醇激素，长期以来被认为具有影响骨骼生长的能力。25年前，我们开始调查居住在洛杉矶动物园的青少年雌性新世界灵长类动物的佝偻病暴发，鉴定了异质核糖核蛋白C （hnRNPC）家族中的蛋白质、维生素D反应元件结合蛋白（vdrep）和雌激素反应元件结合蛋白（re - bp），这些蛋白质导致骨骼对维生素D和雌激素激素的作用产生抗性。由于它们能够与单链DNA （ssDNA）、ssRNA和双链DNA （dsDNA）相互作用，我们已经计划了实验来验证我们的理论，即vdrer - bp和ERE-BP可以通过连续调节i)染色质重塑，ii)转录，iii)转录物剪接和iv)转录物处理，在骨中作为单个基因产物同步表达的多位点参与者。

DESCRIPTION (provided by applicant): Twenty-five years ago we began to investigate an outbreak of rachitic bone disease in adolescent, female New World primates residing at the Los Angles Zoo. Our investigation of this "experiment of nature" and that of an adolescent human female with a similar phenotype led us to the discovery of a novel means for vitamin D and estrogen resistance in primates, including man. We coined these resistance-causing proteins the vitamin D response element binding protein (VDRE-BP) and estrogen response element binding (ERE-BP) for their ability to compete in trans with the liganded vitamin D receptor (VDR) and estrogen receptor 1 (ER1) for their cognate response elements, identifying them as nucleic acid binding proteins in the heterogeneous nuclear ribonucleoprotein C (hnRNPC) family. Recent work has led us to realize that the ability of these hnRNPs to alter steroid hormone-directed gene expression is not limited to their dominant-negative actions at the level of transcription. By virtue of their capacity to interact with single-strand DNA (ssDNA), ssRNA as well as double- strand DNA (dsDNA), these hnRNPs have the potential to exert control over gene expression at multiple sites in the cell. Here we theorize that a specific hnRNP can act as a multi-site participant in the synchronized expression of a single gene product by way of regulating, in succession, chromatin remodeling (ssDNA binding), transcription (dsDNA binding), splicing (ssDNA and ssRNA binding) and microRNA (miRNA; ssRNA binding). Because both the human and subhuman primate experiments of nature in hnRNP overexpression we have studied resulted in coincident vitamin D- and estrogen-antagonism on the growing skeleton of adolescent females, here we will concentrate on growing bone and the osteoblast as natural targets for these hnRNPs. Three specific aims are proposed: 1) using chromatin modification/precipitation, over/under expression and novel cell free translation technologies, explore the impact of hnRNPs on the cells responsible for 1,25- dihydroxyvitamin D and estradiol-driven bone growth; 2) employ new RNA immunoprecipitation and micro- RNA methodology to study the versatility of these hnRNPS to control transcript splicing and handling by directed binding to RNA products of the same gene whose expression is simultaneously regulated at the level of chromatin remodeling and transcription; and 3) ascertain the composite effect of these hnRNPs on the growing, adolescent skeleton by targeted transgenic overexpression of the VDRE-BP and ERE-BP hnRNPs in mouse bone. It is our expectation that the proposed research will pave the way to a mechanistic understanding of how these multi-functional hnRNPs control the process of sterol/steroid hormone-regulated gene expression in growing bone and delineate novel points of potential therapeutic intervention in that process. PUBLIC HEALTH RELEVANCE: Vitamin D and estrogen are two steroid hormones long recognized for their ability to influence the growing skeleton. Twenty five years ago we began to investigate an outbreak of rickets in adolescent, female New World primates residing at the Los Angeles Zoo, identifying proteins in the heterogeneous nuclear ribonucleoprotein C (hnRNPC) family, the vitamin D response element binding protein (VDRE-BP) and estrogen response element binding (ERE-BP) which caused resistance to the actions vitamin D and estrogen hormones on the skeleton. By virtue of their capacity to interact with single-strand DNA (ssDNA), ssRNA as well as double- strand DNA (dsDNA), we have planned experiments to test our theory that the VDRE-BP and ERE-BP can act in bone as a multi-site participant in the synchronized expression of a single gene product by way of regulating, in succession, i) chromatin remodeling, ii) transcription, iii) transcript splicing and iv) transcript handling.