ROLE OF SRC TYROSINE KINASE IN OSTEOCLAST FUNCTION

虽然骨吸收增加是其机制 在绝经后时期，在恶性 涉及骨骼的疾病和佩吉特病， 骨吸收的机制尚不清楚。 最近，人们对这一进程有了新的认识， src原癌基因（pp 6 Ocsrc）的表达是必需的（和必要的） 正常的骨吸收。 这一点出乎意料地表现在 SRC表达缺陷的转基因小鼠和通过该技术产生的转基因小鼠 通过同源重组将无效突变引入基因。 我们在本申请中的计划是检查负责 使用体外和体内技术， src缺陷型（src-）和正常野生型小鼠。 我们计划确定 src表达是否是破骨细胞形成或活化所必需的 已经形成的细胞，或者两者都有。 为了评估这个问题，我们计划 培养纯合子src缺陷的骨髓细胞和脾细胞 变种人 我们还计划确定石骨症是否是由于 微环境缺陷或破骨细胞中的细胞缺陷 脉 在这些实验中将使用两种方法。在一个 方法，实验目前正在进行的博士Soriano使用 将胎儿肝细胞移植给受致命辐射的受体。 中 第二种方法，我们将使用脾细胞或 骨髓细胞（作为破骨细胞前体的来源）与 基质细胞从骨髓培养物中获得 腔或来自新鲜分离的颅骨细胞。 这些实验应该 允许我们确定src表达是否需要在细胞中， 破骨细胞系或参与骨吸收的辅助细胞。 我们 还计划研究src酪氨酸激酶抑制剂对 在体外和体内的骨吸收。 我们有许多 其中一种优先抑制src 酪氨酸激酶 此外，我们计划研究 甲状旁腺激素和其他骨吸收刺激物 酪氨酸激酶活性的表达， 酪氨酸激酶和src在分离的破骨细胞，成骨细胞， 以及与骨吸收相关的辅助细胞。 这些实验 应该使我们能够确定a）是否需要src表达式 是在破骨细胞谱系中还是在辅助细胞中; B）src是否 表达是破骨细胞形成所必需的，或者是破骨细胞形成所必需的。 活化; c）PTH是否对骨吸收发挥作用以增强骨吸收; src酪氨酸激酶的表达;和d）探索 在体外和体内抑制骨吸收的新方法。

Although increased osteoclastic bone resorption is the mechanism responsible for bone loss in the postmenopausal period, in malignant diseases which involve the skeleton and Paget's disease, the molecular mechanisms responsible for osteoclastic bone resorption remain unclear. Recently, new light has been shed on this process with the observation that expression of the src proto-oncogene (pp6Ocsrc) is required (and essential) for normal osteoclastic bone resorption. This was shown unexpectedly in transgenic mice deficient in src expression and produced by the technique of introducing the null mutation into the gene by homologous recombination. Our plan In this application is to examine the mechanisms responsible for this impaired osteoclast function using in vitro and in vivo techniques in src deficient (src-) and normal wild type mice. We plan to determine whether src expression is required for osteoclast formation, or activation of cells already formed, or both. To evaluate this question, we plan to culture bone marrow cells and spleen cells from homozygous src deficient mutants. We also plan to determine whether the osteopetrosis is due to a microenvironmental defect or to a defect in cells in the osteoclast lineage. Two approaches will be utilized in these experiments. In one approach, experiments are currently being performed by Dr. Soriano using transplants of fetal liver cells to lethally irradiated recipients. In a second approach, we will perform experiments in vitro using spleen cells or bone marrow cells (as a source of osteoclast precursors) co-cultured with stromal cells which are obtained either from cultures from the marrow cavity or from freshly isolated calvarial cells. These experiments should allow us to determine whether src expression is required in cells in the osteoclast lineage or the accessory cells involved in bone resorption. We also plan to examine the effects of inhibitors of src tyrosine kinase on osteoclastic bone resorption both in vitro and in vivo. We have a number of inhibitors available to us, one of which preferentially inhibits src tyrosine kinase. In addition, we plan to examine the effects of parathyroid hormone and other stimulators of bone resorption on the expression of tyrosine kinase activity, the phosphorylated substrate of tyrosine kinase, and src expression in isolated osteoclasts, osteoblasts, and accessory cells associated with bone resorption. These experiments should enable us to determine a) whether the requirement for src expression is in the osteoclast lineage or in accessory cells; b) whether src expression is required for formation of osteoclasts, or for their activation; c) whether PTH exerts its effects on bone resorption to enhance expression of src tyrosine kinase; and d) exploration of the potential of novel approaches of inhibiting bone resorption both in vitro and in vivo.