Regulation of Functional Activity of Osteoclasts

破骨细胞功能活动的调节

• 批准号: 9278960
• 负责人: Kin-Hing William Lau
• 金额: --
• 依托单位: VA LOMA LINDA HEALTHCARE SYSTEM
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-07-01 至 2020-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9278960
• 关键词: Address; Aging; Bone Resorption; Calcium; Cell Nucleus; Cell Size; Cells; ChIP-seq; Coupling; DNA cassette; Development; Dinoprostone; Disease; Etiology; Female; Formulation; Foundations; Functional disorder; Future; Gene Cluster; Gene Expression Profiling; Genetic Transcription; Hip Fractures; Human; Impairment; In Vitro; Incidence; Interleukin-1; Knockout Mice; Knowledge; Medical; Messenger RNA; MicroRNAs; Mission; Molecular; Morbidity - disease rate; Mus; Mutate; Osteoblasts; Osteoclasts; Osteoporosis; Ovariectomy; Pathologic; Pathway Analysis; Patient Care; Phenotype; Physiological; Post-Transcriptional Regulation; Production; Protein Tyrosine Phosphatase; Quality of life; Regulation; Research; Response Elements; Role; Signal Transduction; Testing; Therapeutic; Transcriptional Regulation; Transgenes; Transgenic Mice; Up-Regulation; Veterans; Wasting Syndrome; Work; bone; bone cell; bone loss; cost; cytokine; design; disabling symptom; effective therapy; improved; in vivo; innovation; insight; novel; overexpression; patient population; prevent; promoter; protein expression; public health relevance; screening; transcription factor; transcriptome sequencing; working group

 DESCRIPTION (provided by applicant): Past work of this group has demonstrated that an osteoclastic protein-tyrosine phosphatase (PTP-oc) is a positive activator of the osteoclast activity. Recent preliminary studies have disclosed four exciting discoveries relevant to the mechanism of how PTP-oc expression in osteoclasts is regulated: First, PTP-oc expression is controlled primarily by post-transcriptional mechanisms. Second, the post-transcriptional regulation of PTP-oc expression in osteoclasts is regulated in part by miR17. Third, the miR-17 expression in mature osteoclasts is controlled by certain resorption regulators that also regulate cellular PTP-oc levels in osteoclasts; and fourth, conditional deletion of the miR17~92 gene cluster in osteoclastic cells resulted in not only an increase in PTP- oc mRNA level but also increases in the cell size, number of nuclei, fusion, and bone resorption activity of osteoclasts. These ground-breaking discoveries led to the formulation of three novel hypotheses: 1) Certain resorption cytokines, such as PTH, IL-1, 1,25(OH)2D3, PGE2, activates osteoclasts by reducing miR17 transcription through suppression of its promoter activity; 2) conditional deletion of miR17 in osteoclasts increases expression of PTP-oc, which in turn stimulates osteoclast activity via up-regulation of the PTP-oc signaling; and 3) conditional overexpression of miR17 in osteoclasts suppresses PTP-oc expression and inhibits physiologically and pathologically-induced bone resorption. The first Aim tests the first hypothesis by a) determining if treatment with selected resorption modulators regulate the promoter activity of miR17 in osteoclasts in vitro; b) identifying and mutating essential response elements to demonstrate direct effects of these resorption modulators on miR17~92 gene transcription; and c) performing ChIP-seq analysis to identify essential transcription factors that are involved in the transcriptional regulation of the expression of miR1 in osteoclasts. Aim 2 tests the second hypothesis by characterizing the in vivo and in vitro bone and osteoclast phenotypes of osteoclast miR17~92 conditional knockout mice, determining effects of miR17~92 deficiency on PTP-oc mRNA level, the PTP-oc signaling, and resorption activity of osteoclasts in vitro, and determining whether re-introduction of miR17 into miR17~92 deficient osteoclasts would "restore" the osteoclast phenotype in vitro; b) evaluating effects of miR17~92 deficiency on osteoclast differentiation by performing RNA-seq gene expression profiling and pathways analyses and by determining the role of Rac1/Rac2 in the miR17-deficiency- induced osteoclast fusion; and c) showing that the observed defective bone coupling in conditional knockout mice is not due to deletion of miR17 in osteoblasts, reduced osteoblast activity, but instead to an impaired bone cell production of "coupling factors". Aim 3 tests the third hypothesis by generating transgenic mice with conditional overexpression of miR17~92 in osteoclasts by crossing LysM-Cre mice with mice harboring the miR17~92 transgene downstream to a loxP-flanked Neo-STOP cassette at the ROSA locus, determining the effects of conditional overexpression of miR17~92 in osteoclasts on bone and osteoclast phenotypes in vivo and in vitro, and determining if overexpression of miR17~92 in osteoclasts would blunt the ovariectomy-and calcium depletion-induced bone resorption and bone loss. This research addresses an innovative concept that a miRNA (i.e., miR17) is a key regulator of the functional activity of mature osteoclasts through modulation of expression of PTP-oc in osteoclasts. If successful as expected, this work will yield important insights into how PTP-oc expression is regulated, which is critically important not only with respect to the overall understanding of the role of PTP-oc and its signaling mechanisms in the pathophysiology in various types of bone-wasting diseases, including osteoporosis. It could also provide novel targets (i.e., miR17) for future screening of small molecular therapeutic compounds as a rational design for an effective means to modulate PTP-oc expression and the functional activity of osteoclasts, which can then be used to develop novel and effective anti-resorption therapies for certain subtypes of osteoporosis and bone-wasting diseases.

 描述（由申请人提供）： 该小组过去的工作已经证明，破骨细胞蛋白酪氨酸磷酸酶（PTP-oc）是破骨细胞活性的正激活剂。最近的初步研究已经公开了四个令人兴奋的发现，相关的机制，如何在破骨细胞中的PTP-OC表达的调节：第一，PTP-OC的表达主要是由转录后机制控制。其次，破骨细胞中PTP-oc表达的转录后调节部分由miR 17调节。第三，成熟破骨细胞中的miR-17表达受某些再吸收调节剂控制，所述再吸收调节剂也调节破骨细胞中的细胞PTP-α水平;以及第四， 破骨细胞中miR 17 ~92基因簇的条件性缺失不仅导致PTP- oc mRNA水平增加，而且还导致破骨细胞的细胞大小、细胞核数量、融合和骨吸收活性增加。这些突破性的发现导致了三个新的假说的形成：1）某些吸收细胞因子，如PTH、IL-1、1，25（OH）2D 3、PGE 2，通过抑制miR 17的启动子活性来减少其转录从而激活破骨细胞; 2）破骨细胞中miR 17的条件性缺失增加了PTP-oc的表达，其又通过上调PTP-α信号传导刺激破骨细胞活性;和3）破骨细胞中miR 17的条件性过表达抑制PTP-α表达并抑制生理学和病理学诱导的骨吸收。第一个目的通过以下步骤检验第一个假设：a）确定用选定的再吸收调节剂处理是否在体外调节破骨细胞中miR 17的启动子活性; B）鉴定和突变必需的应答元件以证明这些再吸收调节剂对miR 17 ~92基因转录的直接作用;和c）进行ChIP-seq分析以鉴定参与破骨细胞中miR 1表达的转录调节的必需转录因子。目的2通过表征破骨细胞miR 17 ~92条件性基因敲除小鼠的体内和体外骨和破骨细胞表型，确定miR 17 ~92缺陷对体外破骨细胞的PTP-oc mRNA水平、PTP-oc信号传导和再吸收活性的影响，并确定将miR 17重新引入miR 17 ~92缺陷型破骨细胞是否会在体外“恢复”破骨细胞表型; B）通过进行RNA-seq基因表达谱分析和途径分析以及通过确定Rac 1/Rac 2在miR 17缺陷诱导的破骨细胞融合中的作用来评估miR 17 ~92缺陷对破骨细胞分化的影响;和c）表明在条件性敲除小鼠中观察到的有缺陷的骨偶联不是由于成骨细胞中miR 17的缺失、成骨细胞活性的降低，而是由于骨细胞产生的“偶联因子”受损。目的3通过将LysM-Cre小鼠与在ROSA基因座处的loxP侧翼的Neo-STOP盒下游携带miR 17 ~ 92转基因的小鼠杂交来产生在破骨细胞中具有miR 17 ~ 92的条件性过表达的转基因小鼠，来测试第三种假设，确定在破骨细胞中miR 17 ~92的条件性过表达对骨和破骨细胞表型的体内和体外影响，并确定在破骨细胞中过表达miR 17 ~92是否会减弱卵巢切除和钙缺乏诱导的骨吸收和骨丢失。这项研究提出了一个创新的概念，即miRNA（即，miR 17）是通过调节破骨细胞中PTP-α的表达来调节成熟破骨细胞的功能活性的关键调节剂。如果成功的预期，这项工作将产生重要的见解如何PTP-OC表达的调节，这是至关重要的，不仅是相对于PTP-OC的作用和其信号传导机制在各种类型的骨消耗性疾病，包括骨质疏松症的病理生理学的整体理解。它还可以提供新的靶点（即，miR 17）用于未来筛选小分子治疗化合物作为调节PTP-a表达和破骨细胞功能活性的有效手段的合理设计，其然后可用于开发用于某些亚型的骨质疏松症和骨消耗疾病的新颖且有效的抗吸收疗法。