CUTANEOUS BIOLOGY OF KIT LIGAND

试剂盒配体的皮肤生物学

• 批准号: 2563923
• 负责人: BRUCE Jack LONGLEY
• 金额: $ 12.02万
• 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/2563923
• 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.

这项建议的目的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 其编码的受体酪氨酸激酶和KIT的配体。 皮肤肥大细胞增多症涉及肥大细胞黑色素细胞，两种细胞类型 其表达KIT并响应KIT配体（KL）。 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 一部分来源于肥大细胞的c-KIT cDNA显示出已知的突变， 在6名患者中的3名中导致KIT的组成性激活， 不同形式的肥大细胞增多症 KL，主要是膜结合的， 正常皮肤，在皮肤病变中以可溶性形式存在 肥大细胞增多症 还发现肥大细胞的糜酶 能特异性裂解KL产生具有生物活性的可溶性KL（sKL）。 的 拟议的研究将测试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，肥大细胞 糜蛋白酶或其它酶从基质细胞中切割膜结合KL（mKL 在皮肤肥大细胞增多症中，释放可激活黑素细胞的sKL， 进一步刺激肥大细胞增殖。 战略是确定 不同形式的活化c-KIT突变的频率 肥大细胞增多症，sKL在皮肤中产生的方式，以及 c-KIT突变导致转基因小鼠肥大细胞增多症的能力。 的 具体目标是： 目的1：确定活化c-KIT突变的频率， 不同形式的肥大细胞增多症 包含整个编码的cDNA c-KIT区域将来自皮肤肥大细胞增多症病变， 测序 具有新突变的cDNA将被亚克隆到哺乳动物中， 表达载体，并在体外表达，以确定其对KIT的影响 activation. 目的2：鉴定裂解KL并产生 生物活性sKL。 纯化的重组KL或mKL的制备 将与类胰蛋白酶、弹性蛋白酶和组织蛋白酶共孵育。 裂解 将通过蛋白质印迹和蛋白质测序鉴定产物， 通过黑素细胞-KIT磷酸化测定测定其生物活性。 目的3：在转基因小鼠中复制肥大细胞增多症。 为了检验这一假设 c-KIT突变可导致肥大细胞增多症，糜酶启动子将被 用于在转基因小鼠的肥大细胞中表达突变的KIT， 这些动物中肥大细胞增多症的发生率将表明c-KIT突变可导致 肥大细胞增多症