CSF1 GENE EXPRESSION IN OSTEOCLAST BIOLOGY

由成骨细胞和基质细胞释放的CSF-1刺激破骨细胞祖细胞的增殖和分化，提高破骨细胞的存活率。在op/op小鼠模型中，胸腺嘧啶插入编码骨质疏松。该提案的长期目标是确定可溶性(s)和膜结合(m) CSF-1亚型对破骨细胞发生的影响，并确定CSF-1启动子5'侧区域中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的合成方式不同，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。为了在体外解决这一问题，我们将用sCSF-1或mCSF-1 cDNA转染op/op基质细胞，并在靶细胞中检测稳定的转染物对CSF-1合成、生物活性、破骨细胞支持和c-fms酪氨酸激酶激活的影响。通过靶向同种异构体改善op/op小鼠的骨质疏松症，探索各同种异构体对体内破骨细胞形成的影响；通过sCSF-1与mCSF-1转基因小鼠与op/wt杂合小鼠的遗传杂交，建立表达各转基因的op/op突变体。小鼠将检测血清CSF-1骨生长、门牙爆发和骨质疏松的消退。将评估骨切片的破骨细胞活性；组织形态学分析将评估骨重塑的静态和动态指标。在op/op小鼠中，我们将使用腺病毒载体来限制成骨细胞中CSF-1的表达，从而评估将每种异构体靶向于骨的体内治疗效果。我们的第二个假设是，CSF-1启动子5'侧区域的特定调控元件在体外和体内指导小鼠发育过程中细胞特异性基因的表达。CSF-1在小鼠骨骼发育过程中的时空表达将通过原位杂交和免疫组织化学进行评估。为了确定控制CSF-1细胞特异性表达的潜在顺式作用元件，将测试CSF-1 5'侧翼区域产生的缺失构建体在成骨细胞、基质、肝脏、肌肉、上皮和B细胞系中直接转录的能力。相关的CSF-1细胞特异性启动子序列将通过产生含有与细菌lacZ报告基因相关的细胞特异性CSF-1启动子片段的转基因小鼠在体内进行评估。这些研究增加了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，旨在调节骨质疏松症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.