CSF1 GENE EXPRESSION IN OSTEOCLAST BIOLOGY

由成骨细胞和基质细胞释放的CSF-1刺激破骨细胞前体细胞的增殖和分化，提高破骨细胞的存活率。在OP/OP小鼠模型中，胸腺嘧啶核苷插入编码骨化病。这一建议的长期目标是确定可溶性(S)和膜结合型(M)脑脊液-1亚型对破骨细胞形成的影响，并确定在脑脊液-1启动子5‘侧翼区域调节其表达的细胞特异性顺式作用元件。我们的第一个假设是，sCSF-1和MCSF-1是不同的合成，并刺激破骨细胞的形成，部分原因是它们与c-FMS受体的相互作用。为了解决这一问题，在体外，将sCSF-1或MCSF-1基因导入OP/OP基质细胞，并在靶细胞中检测CSF-1合成、生物活性、破骨细胞支持和c-FMS酪氨酸激酶激活情况。通过靶向异构体改善OP/OP小鼠的骨化，sCSF-1和MCSF-1转基因小鼠与杂合OP/wt小鼠之间的遗传杂交，sCSF-1和MCSF-1转基因小鼠之间的遗传杂交和杂合OP/wt小鼠之间的遗传杂交，以建立表达每个转基因的OP/OP突变体，以探讨每种亚型对体内破骨细胞形成的影响。将对小鼠进行血清CSF-1骨生长、门牙萌出和骨化症消退的检查。骨切片将评估破骨细胞活性；组织形态计量学分析将评估骨重建的静态和动态指标。通过腺病毒载体限制成骨细胞中CSF-1的表达，将评估在OP/OP小鼠体内靶向每种异构体的骨的治疗效果。我们的第二个假设是，在小鼠发育过程中，CSF-1启动子5‘侧翼区的特定调控元件在体外和体内指导细胞特异性基因的表达。通过原位杂交和免疫组织化学方法研究小鼠骨骼发育过程中脑脊液-1的时空表达。为了确定控制细胞特异性表达CSF-1的潜在顺式作用元件，将测试从CSF-1的5‘侧翼区产生的缺失构建体在成骨细胞、基质、肝脏、肌肉、上皮和B细胞系中指导转录的能力。相关的脑脊液-1细胞特异性启动子序列将通过产生含有与细菌LacZ报告基因相连的细胞特异性脑脊液-1启动子片段(S)的转基因小鼠在体内进行评估。这些研究将增加我们对发育过程中激活CSF-1的分子机制的理解，并可能提出新的治疗策略，旨在调节骨质疏松和骨折等各种骨骼疾病中破骨细胞的形成。

CSF-1, released by osteoblasts and stromal cells, stimulates the proliferation and differentiation of osteoclast progenitors and enhances osteoclast survival. In the op/op mouse model, a thymidine insertion in the coding osteopetrosis. The long-term goal of this proposal is to determine the effect of soluble(s) and membrane-bound (m) CSF-1 isoforms on osteoclastogenesis and define cell-specific cis-acting elements in the 5' flanking region of the CSF-1 promoter that regulate their expression. Our first hypothesis is that sCSF-1 and mCSF-1 are differentially synthesized and stimulate osteoclast formation due, in part, to their interaction with the c-fms receptor. To address this issue in vitro, op/op stromal cells will be transfected with sCSF-1 or mCSF-1 cDNA and stable transfectants examined for CSF-1 synthesis, bioactivity, osteoclast support and c-fms tyrosine kinase activation in target cells. The effect of each isoform on osteoclast formation in vivo will be explored by targeting isoform to ameliorate osteopetrosis in op/op mice, genetic crosses between sCSF-1 and mCSF-1 transgenic mice and heterozygous op/wt mice, genetic crosses between sCSF-1 and mCSF-1 transgenic mice and heterozygous op/wt mice will be carried out to establish op/op mutants expressing each transgene. Mice will be examined for serum CSF-1 bone growth, incisor eruption and resolution of osteopetrosis. Bone sections will be assessed for osteoclast activity; histomorphometric analysis will evaluate both static and dynamic indices of bone remodeling. The in vivo therapeutic effect of targeting each isoform to the bone in op/op mice will be assessed using adenoviral vectors designed to limit CSF-1 expression in osteoblasts. Our second hypothesis is that specific regulatory elements in the 5' flanking region of the CSF-1 promoter direct cell-specific gene expression in vitro and in vivo during murine development. The temporal and spacial expression of CSF-1 during murine skeletal development will be assessed by in situ hybridization and immunohistochemistry. To determine potential cis-acting elements that control cell-specific expression of CSF-1, deletion constructs generated from the 5' flanking region of CSF- 1 will be tested for their ability to direct transcription in osteoblast, stromal, liver, muscle, epithelial and B cell lines. Relevant CSF-1 cell specific promoter sequences will be assessed in vivo by generating transgenic mice harboring cell-specific CSF-1 promoter segment(s) linked to the bacterial lacZ reporter gene. These studies should increase our understanding of the molecular mechanisms that activate CSF-1 during development and may suggest novel therapeutic strategies designed to regulate osteoclast formation in a variety of bone disorders such as osteoporosis and bone fracture.