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

描述(申请人提供)：人类BMP4和BMP2基因的突变与骨质疏松症有关，这些BMPs通过各种BMP受体传递信号。BMP2和BMP4在骨生物学中的作用是有限的，因为BMP4和BMP2的缺失是胚胎死亡。我们实验室的数据显示，使用Cre/loxP有条件地敲除产生基质的成骨细胞中的BMP4，会导致出生后的动物出现骨量减少的表型。BMP4 CKO小鼠在老年动物中成骨细胞活性降低，破骨细胞增加。我们最近发现Shh反应基因Gli2通过Gli反应元件直接调节成骨细胞前体细胞中BMP2的表达和转录。我们的假设是，BMP4和BMP2在早期间充质前体细胞中是必需的，以驱动前体细胞在扩增和分化为成熟成骨细胞之前进入成骨细胞承诺期。通过BMP受体1A进一步提高BMP水平和信号，然后需要驱动成熟的基质/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选择性地去除BMP2和BMP4来检验这一假说，这两个阶段是早期成骨细胞阶段和晚期基质形成成骨细胞阶段。然后，我们将尝试通过选择性地激活骨骼中重要的BMP受体之一BMPR1A来挽救其中一些骨骼表型。具体目标1旨在确定BMP4和BMP2在成骨细胞生物学中的特定和重叠的作用，使用Osterix-CreERt2小鼠模型在成骨前阶段暂时删除BMP4和/或BMP2，特别是在出生后的动物中。具体目标2将确定BMP2和BMP4在晚期成骨细胞中的作用，使用3.2Col1a1-Creertm模型在出生后诱导缺失。具体目的3是利用骨髓间充质前体细胞培养结合BMP2和BMP4基因缺失的腺病毒Cre体外培养方法，研究BMP2和BMP4的体外作用机制。将利用微阵列分析来确定生长、凋亡、分化和改变的途径，以及基因表达模式。具体目标4将旨在确定BMP2和BMP4的骨骼特异性缺失的哪些方面是由于通过BMP受体1A的信号所致。这一假说将通过定量原位杂交、免疫细胞化学进行验证。对改变的信号通路的蛋白质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被用来确定这些通路的水平和BMP2和BMP4缺失小鼠的变化。在体外原代培养的成骨细胞中，特定基因缺失前后将被用来确定单一BMP2或BMP4以及BMP2和BMP4联合缺失的候选作用机制。

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.