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.

该提案的目的是确定 C-kit原始癌基因（套件）发挥的疾病肥大症 它编码的受体酪氨酸激酶和套件的配体。 皮肤肥大性涉及肥大细胞黑素细胞，两种细胞类型 表达套件并响应套件配体（KL）。 因为激活 试剂盒的肿瘤转化，套件和KL的异常 在肥大症患者中寻求寻求。 有针对性的排序 从肥大细胞衍生的C-KIT cDNA的一部分显示出一种突变。 导致六名患者中三名的构型激活 不同形式的肥大症。 kl，大部分是膜绑定的 正常皮肤，在皮肤病变中以可溶性形式发现 肥大症。 还发现肥大细胞酶Chymase 可以特异性裂解KL以产生生物活性可溶性KL（SKL）。 这 拟议的研究将检验以下假设 肥大细胞增多是通过激活C-Kit突变引发的，该突变是肥大细胞 来自基质细胞的Chymase或其他酶裂解膜结合Kl（MKL） 在皮肤肥大性中，释放SKL可能会激活黑素细胞和 刺激进一步的肥大细胞增殖。 策略是确定 以不同形式激活C-kit突变的频率 肥大细胞增多症，皮肤中SKL产生的方式，以及 C-KIT突变引起转基因小鼠肥大的能力。 这 具体目的是： 目标1：确定激活C-KIT突变的频率 不同形式的肥大症。 包括整个编码的cDNA C-KIT区域将来自皮肤肥大性的病变和 测序。 带有小说突变的cDNA将被亚克隆到哺乳动物中 表达向量并在体外表达以确定其对套件的影响 激活。 目标2：鉴定炎症细胞酶比裂解KL并产生 生物活性SKL。 纯化的重组KL或MKL的准备 将与胰蛋白酶，弹性酶和组织蛋白酶共结合。 乳沟 产品将通过蛋白质印迹和蛋白质测序来识别，并且 它们由黑素蛋白酶磷酸化测定法确定的生物活性。 目标3：再现肥大性肌细胞骨质骨质小鼠。 检验假设 C-KIT突变会引起肥大症，Chymase启动子将是 用于在转基因小鼠的肥大细胞中表达突变试剂盒的发生 这些动物中肥大细胞增多症会表明C-kit突变会导致 肥大症。