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并增强破骨细胞存活。在op/op小鼠模型中，编码骨硬化的胸苷插入。该建议的长期目标是确定可溶性和膜结合CSF-1同种型对破骨细胞生成的影响，并确定CSF-1启动子5'侧翼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的差异合成和刺激破骨细胞的形成，部分是由于它们与c-fms受体的相互作用。为了在体外解决这个问题，将用sCSF-1或mCSF-1 cDNA转染op/op基质细胞，并检查稳定转染子在靶细胞中的CSF-1合成、生物活性、破骨细胞支持和c-fms酪氨酸激酶活化。将通过靶向同种型以改善op/op小鼠中的骨石化病来探索每种同种型对体内破骨细胞形成的影响，将进行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转基因小鼠与杂合op/wt小鼠之间的遗传杂交，以建立表达每种转基因的op/op突变体。将检查小鼠的血清CSF-1骨生长、切牙萌出和石骨症消退。将评估骨切片的破骨细胞活性;组织形态计量学分析将评价骨重建的静态和动态指标。将使用设计用于限制成骨细胞中CSF-1表达的腺病毒载体来评估将每种同种型靶向至op/op小鼠中的骨的体内治疗效果。我们的第二个假设是，在CSF-1启动子的5'侧翼区的特异性调控元件指导细胞特异性基因表达在体外和体内在小鼠发育过程中。将通过原位杂交和免疫组织化学评估CSF-1在小鼠骨骼发育期间的时间和空间表达。为了确定控制CSF-1的细胞特异性表达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、基质细胞、肝细胞、肌肉细胞、上皮细胞和B细胞系中指导转录的能力。将通过产生携带与细菌lacZ报告基因连接的细胞特异性CSF-1启动子片段的转基因小鼠，在体内评估相关CSF-1细胞特异性启动子序列。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，旨在调节破骨细胞形成的各种骨骼疾病，如骨质疏松症和骨折。

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.