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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

基本信息

批准号：
7673282
负责人：
STEPHEN Eubank HARRIS
金额：
$ 30.76万
依托单位：
UNIVERSITY OF TEXAS HLTH SCIENCE CENTER
依托单位国家：
美国
项目类别：
财政年份：
2007
资助国家：
美国
起止时间：
2007-09-15 至 2012-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7673282
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

项目摘要

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）：人类BMP4和BMP2基因突变与骨质疏松症有关，这些BMP通过多种BMP受体发出信号。BMP2和BMP4在骨生物学中的作用是有限的，因为BMP4和BMP2的缺失是胚胎致命的。我们实验室的数据表明，使用Cre/loxP，在生成成骨细胞的基质中有条件地敲除BMP4，会导致出生后动物出现骨质减少表型。老年动物BMP4 cKO小鼠成骨细胞活性降低，破骨细胞增加。我们最近发现Shh应答基因Gli2通过Gli应答元件直接调控成骨细胞前体BMP2的表达和转录。我们的假设是早期间充质前体细胞需要BMP4和BMP2来驱动前体细胞进入成骨细胞阶段，然后再扩展和分化为成熟的成骨细胞。进一步提高BMP水平和通过BMP受体1A信号传导，则需要驱动成熟基质/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（早期成骨前阶段和后期基质生成成骨细胞阶段）选择性地去除BMP2和BMP4来验证这一假设。然后，我们将尝试通过选择性激活骨中重要的BMP受体之一BMPR1A来挽救这些骨表型。Specific Aim 1旨在确定BMP4和BMP2在成骨细胞生物学中的特异性和重叠作用，使用Osterix-CreERt2小鼠模型在成骨前阶段（特别是出生后的动物）暂时删除BMP4和/或BMP2。具体目标2将是确定BMP2和BMP4在后期成骨细胞中的作用，使用3.2 Col1a1-CreERtm模型诱导出生后缺失。具体目的3将是确定体外BMP2和BMP4的作用机制，使用骨髓间充质前体培养结合BMP2和BMP4缺失与腺病毒Cre体外。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。特异性目标4将旨在确定BMP2和BMP4的骨特异性缺失的哪些方面是由于通过BMP受体1A的信号传导引起的。这一假设将使用定量原位杂交、免疫细胞化学进行检验。西方对改变的信号通路的分析也将以定量的方式确定。b-catenin/TCF和BMP信号报告小鼠和谱系标记小鼠将用于确定BMP2和BMP4缺失小鼠中这些途径的水平和改变。体外原代成骨细胞培养，特异性基因缺失前后，将用于确定单个BMP2或BMP4以及BMP2和BMP4联合缺失的候选作用机制。