CUTANEOUS BIOLOGY OF KIT LIGAND

试剂盒配体的皮肤生物学

• 批准号: 2633661
• 负责人: BRUCE Jack LONGLEY
• 金额: $ 23.37万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-01-01 至 2000-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2633661
• 关键词: biopsy; cell proliferation; chymase; clinical research; gene frequency; gene mutation; genetic promoter element; genetically modified animals; human subject; laboratory mouse; mastocytosis; molecular cloning; neoplastic transformation; nucleic acid sequence; pathologic process; protein tyrosine kinase; protooncogene

The goal of this proposal is to determine the role in the pathogenesis of the disease mastocytosis played by the c-KIT proto-oncogene, the KIT receptor tyrosine kinase that it encodes, and the ligand for KIT. Cutaneous mastocytosis involves mast cells melanocytes, two cell types which express KIT and respond to the KIT ligand(KL). Because activation of KIT can cause neoplastic transformation, abnormalities of KIT and KL were sought in patients with mastocytosis. Targeted sequencing a short portion of c-KIT cDNAs derived from mast cells showed a mutation know to result in constitutive activation of KIT in three of six patients with different forms of mastocytosis. KL, which is mostly membrane bound in normal skin, was found in a soluble form in lesions of cutaneous mastocytosis. It was also discovered that the mast cell enzyme chymase could specifically cleave KL to produce bioactive soluble KL (sKL). The proposed research will test the hypothesis that mast cell proliferation in mastocytosis is initiated by activating c-KIT mutations, and that mast cell chymase or other enzyme cleave membrane bound KL (mKL) from stromal cells in cutaneous mastocytosis, releasing sKL which may activate melanocytes and stimulate further mast cell proliferations. The strategy is to determine the frequency of activating c-KIT mutations in different forms of mastocytosis, the ways in which sKL is generated in the skin, and the ability of c-KIT mutations to cause mastocytosis in transgenic mice. The specific aims are: Aim 1: To determine the frequency of activating c-KIT mutations in different forms of mastocytosis. cDNAs that include the entire coding region of c-KIT will be derived from lesions of cutaneous mastocytosis and sequenced. cDNAs with novels mutations will be subcloned into mammalian expression vectors and expressed in vitro to determine their effects on KIT activation. Aim 2: To identify inflammatory cell enzymes than cleave KL and produce biologically active sKL. Preparations of purified recombinant KL or mKL will be co-incubated with tryptase, elastase, and cathepsin. Cleavage products will be identified by western blotting and protein sequencing, and their bioactivity determined by melanocyte-KIT phosphorylation assays. Aim 3: To reproduce mastocytosis intransgenic mice. To test the hypothesis that c-KIT mutations can cause mastocytosis, the chymase promoter will be used to express mutated KIT in mast cells of transgenic mice the occurrence of mastocytosis in these animals will show that c-KIT mutation can cause mastocytosis.

该提案的目标是确定在发病机制中的作用 由 c-KIT 原癌基因 KIT 引起的肥大细胞增多症 它编码的受体酪氨酸激酶以及 KIT 的配体。 皮肤肥大细胞增多症涉及肥大细胞黑素细胞，两种细胞类型 表达 KIT 并对 KIT 配体 (KL) 做出反应。 因为激活 KIT可引起肿瘤转化、KIT和KL异常 在肥大细胞增多症患者中寻找。 靶向测序短 来自肥大细胞的 c-KIT cDNA 部分显示出已知的突变 导致六名患者中的三名患者的 KIT 组成型激活 不同形式的肥大细胞增多症。 KL，主要是膜结合 正常皮肤，在皮肤损伤处发现可溶形式 肥大细胞增多症。 还发现肥大细胞食糜酶 可以特异性裂解 KL 产生具有生物活性的可溶性 KL (sKL)。 这 拟议的研究将检验肥大细胞增殖的假设 肥大细胞增多症是通过激活 c-KIT 突变启动的，并且肥大细胞 食糜酶或其他酶从基质细胞中裂解膜结合 KL (mKL) 在皮肤肥大细胞增多症中，释放 sKL 可能会激活黑素细胞并 刺激肥大细胞进一步增殖。 策略是确定 不同形式的 c-KIT 突变激活频率 肥大细胞增多症、皮肤中 sKL 的产生方式以及 c-KIT 突变导致转基因小鼠肥大细胞增多的能力。 这 具体目标是： 目标 1：确定 c-KIT 突变的激活频率 不同形式的肥大细胞增多症。 包含完整编码的 cDNA c-KIT区域将源自皮肤肥大细胞增多症的病变和 已测序。 具有新突变的 cDNA 将被亚克隆到哺乳动物体内 表达载体并体外表达以确定其对 KIT 的影响 激活。 目标 2：鉴定炎症细胞酶，然后裂解 KL 并产生 具有生物活性的 sKL。 纯化的重组KL或mKL的制剂 将与类胰蛋白酶、弹性蛋白酶和组织蛋白酶共同孵育。 乳沟 产品将通过蛋白质印迹和蛋白质测序进行鉴定，并且 它们的生物活性通过黑素细胞-KIT 磷酸化测定确定。 目标 3：在转基因小鼠中重现肥大细胞增多症。 检验假设 c-KIT 突变可导致肥大细胞增多症，食糜酶启动子将 用于在转基因小鼠的肥大细胞中表达突变KIT的发生 这些动物的肥大细胞增多症将表明 c-KIT 突变可导致 肥大细胞增多症。