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

维生素 D 和雌激素在骨中的作用：C 家族中 hnRNP 的协同作用

• 批准号: 8325159
• 负责人: John S Adams
• 金额: $ 42.01万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-01-01 至 2014-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8325159
• 关键词: 1,25 (OH) vitamin D; Accident and Emergency department; Adolescent; Alternative Splicing; Attention; Beryllium; Binding; Binding Proteins; Biological Assay; Bone Diseases; Bone Growth; Cells; Chromatin; Coin; DNA Binding; Disease Outbreaks; Dominant-Negative Mutation; Drops; Estradiol; Estrogen Receptor 1; Estrogen Receptors; Estrogens; Event; Family; Female Adolescents; Gene Expression; Generations; Genes; Genetic Transcription; Health; Heterogeneous-Nuclear Ribonucleoprotein Group C; Heterogeneous-Nuclear Ribonucleoproteins; Hormone Receptor; Hormones; Human; Humulus; Immunoprecipitation; Individual; Investigation; Left; Ligands; Link; Los Angeles; Messenger RNA; Methodology; MicroRNAs; Modeling; Mus; Mutation; Nature; Osteoblasts; Osteogenesis; Participant; Phenotype; Precipitation; Primates; Process; Production; Proteins; Public Health; RNA; RNA Binding; RNA Splicing; Research; Resistance; Response Elements; Rickets; Role; Signal Pathway; Single-Stranded DNA; Site; Skeleton; Sterols; Technology; Testing; Therapeutic Intervention; Transcript; Transgenic Organisms; Translations; Untranslated RNA; Vehicle crash; Vitamin D; Vitamin D Response Element; Vitamin D3 Receptor; Work; bone; chromatin modification; chromatin remodeling; ds-DNA; expectation; hormone response element; human female; man; novel; nucleic acid binding protein; overexpression; receptor; receptor binding; research study; small molecule; steroid hormone; theories; young man

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.

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的佝偻病骨病爆发。我们对这个“自然实验”和一个具有相似表型的青春期人类女性的研究使我们发现了一种灵长类动物维生素D和雌激素抵抗的新方法，我们创造了这些引起耐药的蛋白质维生素D反应元件结合蛋白（VDRE-BP）和雌激素反应元件结合蛋白（ERE-BP）。因为它们能够与配体维生素D受体（VDR）和雌激素受体1（ER 1）反式竞争其同源反应元件，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hnRNP改变类固醇激素指导的基因表达的能力并不限于其在转录水平上的显性负作用。由于它们与单链DNA（ssDNA）、ssRNA以及双链DNA（dsDNA）相互作用的能力，这些hnRNP具有在细胞中的多个位点对基因表达施加控制的潜力。在这里，我们的理论，一个特定的hnRNP可以作为一个多位点的参与者，在一个单一的基因产物的同步表达的方式调节，连续，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）。因为我们研究的人类和次人类灵长类动物实验在hnRNP过表达的性质，导致一致的维生素D和雌激素拮抗作用的生长骨骼的青春期女性，在这里，我们将集中在生长骨和成骨细胞作为这些hnRNP的天然目标。本研究提出了三个具体目标：1）利用染色质修饰/沉淀、过表达/欠表达和新型细胞自由翻译技术，探讨hnRNP对负责1，25-二羟维生素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方法学研究这些hnRNPS的多功能性，以通过定向结合同一基因的RNA产物来控制转录本剪接和处理，所述同一基因的RNA产物的表达是同时进行的。在染色质重塑和转录水平上调节;和3）通过在小鼠骨中靶向转基因过表达VDRE-BP和ERE-BP hnRNP来确定这些hnRNP对生长的青春期骨骼的复合作用。我们期望，拟议的研究将为从机制上理解这些多功能hnRNP如何控制生长中骨骼中固醇/类固醇激素调节基因表达的过程铺平道路，并在该过程中描绘潜在治疗干预的新点。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春期雌性新世界灵长类动物的佝偻病爆发，确定了异质核核糖核蛋白C（hnRNPC）家族中的蛋白质，维生素D反应元件结合蛋白（VDRE-BP）和雌激素反应元件结合蛋白（ERE-BP），它们引起对维生素D和雌激素在骨骼上的作用的抵抗。由于它们与单链DNA（ssDNA）、ssRNA以及双链DNA（dsDNA）相互作用的能力，我们已经计划了实验来测试我们的理论，即VDRE-BP和ERE-BP可以在骨中作为多位点参与者，通过连续调节以下各项来同步表达单个基因产物：i）染色质重塑，ii）转录，iii）转录本剪接和iv）转录本处理。