CUTANEOUS BIOLOGY OF KIT LIGAND

试剂盒配体的皮肤生物学

• 批准号: 2856148
• 负责人: BRUCE Jack LONGLEY
• 金额: $ 23.9万
• 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/2856148
• 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.

这项建议的目标是确定在致病机制中的作用。 C-kit原癌基因所引起的肥大细胞增多症 它编码的受体酪氨酸激酶，以及试剂盒的配体。 皮肤肥大细胞增多症涉及肥大细胞和黑素细胞，两种细胞类型 它们表达KIT并与KIT配体(KL)反应。因为激活 KIT可引起肿瘤转化，KIT和KL异常 适用于肥大细胞增多症患者。有针对性的测序一小段 来自肥大细胞的部分c-kit cDNA显示出已知的突变 6例患者中有3例导致试剂盒的结构性激活 不同形式的肥大细胞增多症。KL，主要由膜结合在一起 正常皮肤，在皮损中以可溶性形式存在 肥大细胞增多症。还发现肥大细胞酶类的乳糜酶 能特异性裂解KL，产生具有生物活性的可溶性KL(SKL)。这个 拟议的研究将检验肥大细胞在体内增殖的假设 肥大细胞增多症是通过激活c-kit突变启动的，而肥大细胞 凝乳酶或其他酶裂解基质细胞膜结合的KL(MKL) 在皮肤肥大细胞增多症中，释放SKL，它可能激活黑素细胞和 刺激肥大细胞进一步增殖。战略是确定 不同形式c-kit基因突变的激活频率 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。这个 具体目标是： 目的1：确定c-kit基因突变的激活频率 不同形式的肥大细胞增多症。包含完整编码的cDNA C-kit区域将来自皮肤肥大细胞增多症和 已排序。具有小说突变的cDNA将被亚克隆到哺乳动物体内 表达载体和体外表达，以确定其对KIT的影响 激活。 目的2：鉴定炎性细胞酶并裂解KL并产生 具有生物活性的SKL。纯化重组KL或MKL的制备 将与类胰蛋白酶、弹力酶和组织蛋白酶共同孵育。卵裂 产品将通过蛋白质印迹和蛋白质测序进行鉴定，以及 用黑素细胞试剂盒磷酸化试验测定其生物活性。 目的3：在转基因小鼠体内复制肥大细胞增多症。来检验这一假设 C-kit突变会导致肥大细胞增多症，乳糜酶启动子将 用于在转基因小鼠的肥大细胞中表达突变的KIT 这些动物的肥大细胞增多症的研究将表明c-kit突变可以导致 肥大细胞增多症。