Role and Mechanism of BMP4 and BMP2 Action and Signaling in Bone

BMP4 和 BMP2 作用和信号传导在骨中的作用和机制

• 批准号: 8120408
• 负责人: STEPHEN Eubank HARRIS
• 金额: $ 29.24万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCIENCE CENTER
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-09-15 至 2013-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8120408
• 关键词: Adenoviruses; Age; Age-Months; Animals; Antibodies; Apoptosis; BMP2 gene; BMP4; BMPR1A gene; Biology; Birth; Bone Density; Bone Marrow; Calvaria; Cell Culture Techniques; Cells; Collagen; Collagen Type I; Data; Defect; Development; Differentiation Antigens; Differentiation and Growth; Embryo; Erinaceidae; Failure; Feedback; Gene Deletion; Gene Expression; Gene Expression Microarray Analysis; Gene Expression Profile; Genes; Genetic Transcription; Growth; Human; In Situ Hybridization; In Vitro; Knock-out; Knockout Mice; Ligands; MAPK14 gene; Maps; Mesenchymal; Mesenchyme; Microarray Analysis; Minor; Modeling; Mus; Mutation; Osteoblasts; Osteoclasts; Osteocytes; Osteogenesis; Osteoporosis; Pathway Analysis; Pathway interactions; Pattern; Phenotype; Phospho-Specific Antibodies; Play; Population; Population Decreases; Production; Reporter; Response Elements; Role; Signal Pathway; Signal Transduction; Staging; System; TNFSF11 gene; Testing; Time; Vertebral column; Virus; Work; bone; bone loss; bone mass; bone morphogenetic protein receptors; experience; immunocytochemistry; in vivo; long bone; mouse model; mutant; osteoblast differentiation; osteoclastogenesis; postnatal; precursor cell; progenitor; recombinase; smoothened signaling pathway; stem cell population; substantia spongiosa

DESCRIPTION (provided by applicant): Mutations in the human BMP4 and BMP2 genes have been associated with osteoporosis and these BMPs signal through a variety of BMP receptors. The role for BMP2 and BMP4 in bone biology is limited since deletions of BMP4 and BMP2 are embryonic lethals. Data from our lab has shown that conditional knock-out of BMP4 in matrix producing osteoblasts, using Cre/loxP, results in postnatal animals with an osteopenic phenotype. BMP4 cKO mice have decreased osteoblast activity and increased osteoclasts in older animals. We have recently shown that the Shh responsive gene, Gli2, directly regulates BMP2 expression and transcription in osteoblast precursors through Gli response elements. Our hypothesis is that BMP4 and BMP2 are required at early mesenchymal precursor cells to drive the precursors to a commitment osteoblast stage before expansion and differentiation to mature osteoblasts. Further elevated BMP levels and signaling through BMP receptor 1A, is then required to drive the development of the mature matrix/mineralizing osteoblasts and osteocytes. We will test this hypothesis by selectively removing BMP2 and BMP4 at two different osteoblast stages, the early preosteoblast stage and at later matrix producing osteoblast stage. We will then attempt to rescue some of these bone phenotypes by selectively activating one of the important BMP receptors in bone, BMPR1A. Specific Aim 1 will be directed at determining the specific and overlapping roles of BMP4 and BMP2 in osteoblast biology, using the Osterix-CreERt2 mouse model to temporally delete BMP4 and/or BMP2 in preosteoblast stage, specifically in animals after birth. Specific Aim 2 will be to determine the role of BMP2 and BMP4 in later stage osteoblasts, using the 3.2 Col1a1-CreERtm model to induce deletions after birth. Specific Aim 3 will be to determine the mechanism of action of BMP2 and BMP4 in vitro, using bone marrow mesenchymal precursor cultures in combination with BMP2 and BMP4 deletions in vitro with Adenovirus Cre. Growth, apoptosis, differentiation, and altered pathways will be determined, as well as gene expression patterns using microarray analysis. Specific Aim 4 will be directed at determining what aspects of the bone specific deletions of BMP2 and BMP4 are due to signaling through the BMP receptor 1A. This hypothesis will be tested using quantitative in situ hybridization, immunocytochemistry. Western analysis of altered signaling pathways will also be determined in a quantitative manner. b-catenin/TCF and BMP signaling reporter mice and lineage marker mice will be used to determine levels of these pathways and alterations in the BMP2 and BMP4 deletion mice. In vitro primary osteoblast cell cultures, before and after specific gene deletion will be used to determine candidate mechanism of action of the single BMP2 or BMP4 and the combined deletion of both BMP2 and BMP4.

描述（由申请人提供）：人BMP 4和BMP 2基因突变与骨质疏松症相关，这些BMP通过多种BMP受体发出信号。BMP 2和BMP 4在骨生物学中的作用是有限的，因为BMP 4和BMP 2的缺失是胚胎致死的。来自我们实验室的数据表明，使用Cre/loxP在基质产生成骨细胞中条件性敲除BMP 4，导致出生后动物具有骨质减少表型。BMP 4 cKO小鼠在老年动物中具有降低的成骨细胞活性和增加的破骨细胞。我们最近发现Shh反应基因Gli 2通过Gli反应元件直接调节成骨细胞前体中BMP 2的表达和转录。我们的假设是，BMP 4和BMP 2需要在早期间充质前体细胞，以驱动前体细胞的承诺成骨细胞阶段之前，扩展和分化为成熟的成骨细胞。然后需要进一步升高的BMP水平和通过BMP受体1A的信号传导来驱动成熟基质/矿化成骨细胞和骨细胞的发育。我们将通过在两个不同的成骨细胞阶段，即早期前成骨细胞阶段和后期基质生成成骨细胞阶段选择性地去除BMP 2和BMP 4来测试这一假设。然后，我们将尝试通过选择性激活骨中重要的BMP受体之一BMPR 1A来拯救这些骨表型。具体目标1旨在确定BMP 4和BMP 2在成骨细胞生物学中的特定和重叠作用，使用Osterix-CreERt 2小鼠模型暂时删除前成骨细胞阶段的BMP 4和/或BMP 2，特别是在出生后的动物中。具体目标2将确定BMP 2和BMP 4在后期成骨细胞中的作用，使用3.2 Col 1a 1-CreERtm模型在出生后诱导缺失。具体目标3将是使用骨髓间充质前体培养物与体外BMP 2和BMP 4缺失以及腺病毒Cre来确定BMP 2和BMP 4的体外作用机制。生长，凋亡，分化和改变的途径将被确定，以及基因表达模式，使用微阵列分析。具体目标4将针对确定BMP 2和BMP 4的骨特异性缺失的哪些方面是由于通过BMP受体1A的信号传导。这一假设将使用定量原位杂交、免疫细胞化学进行检验。改变的信号通路的Western分析也将以定量方式确定。将使用β-连环蛋白/TCF和BMP信号转导报告小鼠和谱系标记小鼠来确定BMP 2和BMP 4缺失小鼠中这些途径和改变的水平。在特定基因缺失之前和之后的体外原代成骨细胞培养物将用于确定单一BMP 2或BMP 4以及BMP 2和BMP 4两者的组合缺失的候选作用机制。

项目成果

Role of bone morphogenetic proteins on cochlear hair cell formation: analyses of Noggin and Bmp2 mutant mice.

• DOI: 10.1002/dvdy.22200
• 发表时间: 2010-03
• 期刊: Developmental dynamics : an official publication of the American Association of Anatomists
• 作者: Hwang CH;Guo D;Harris MA;Howard O;Mishina Y;Gan L;Harris SE;Wu DK
• 通讯作者: Wu DK