Cutaneous Biology of KIT Ligand

KIT 配体的皮肤生物学

• 批准号: 6583888
• 负责人: BRUCE Jack LONGLEY
• 金额: $ 19.19万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 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 2005-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6583888
• 关键词: biopsy; cell proliferation; contact dermatitis; family genetics; gene mutation; genetic mapping; genetic promoter element; genetically modified animals; human subject; inflammation; laboratory mouse; loss of heterozygosity; mastocytosis; neoplastic transformation; nucleic acid sequence; pathologic process; patient oriented research; phosphatidylinositol 3 kinase; protein tyrosine kinase; skin disorder; stem cell factor; tissue /cell culture

DESCRIPTION (provided by applicant): Stem Cell Factor (SCF, also known as mast cell growth factor), is the ligand for KIT, a receptor tyrosine kinase. The goal of this proposal is to determine the role of the SCF-KIT signaling pathway in mastocytosis and cutaneous inflammation. Mastocytosis occurring sporadically in adults is caused by somatic mutations affecting the primary sequence of KIT and causing constitutive activation of KIT and its downstream transducing molecule PI3-K, which causes phosphorylation of AKT. Familial and most pediatric cases of mastocytosis cases show normal KIT protein coding sequence but have phosphorylated AKT in lesional mast cells. Our first hypothesis is that familial and sporadic pediatric mastocytosis are caused by mutations affecting the SCF-KIT signaling pathway, or pathways convergent with it at or above AKT. Human epidermal keratincytes produce SCF, and dermal injection of SCF causes inflammation. Trangenic mice which express epidermal SCF, like humans, show an exaggerated ear swelling response to allergic and irritant contactants. Our second hypothesis is that SCF-KIT signaling plays an active role in the cutaneous inflammatory response. Our specific aims are: 1. To determine the mechanism(s) of oncogenesis in c-KIT mutation negative pediatric mastocytosis, mRNA from lesional mast cells will be RT-PCR amplified and sequenced to detect mutations in genes encoding molecules which may affect AKT phosphorylation including AKT, PTEN, Lyn and PI3-K. Since loss of PTEN could result in increased PI3-K signaling, lesional mast cell DNAk will also be tested for loss of heterozygosity in region 10q23 by microsatellite analysis. The functional effects of mutations or gene loss will be determined in cultured bone marrow derived mast cells an mast cell lines by retroviral expression of mutant activating or dominant negative proteins, or by anti-sense suppression. 2. To determine the genetic basis of familial mastocytosis, two separate kindreds with dominantly inherited mastocytosis will be tested for linkage to genes known to affect the KIT-P13-K signaling pathway using microsatellite analysis. If necessary, a genome-wide screen of affected and genetically relevant unaffected individuals will be performed using loci at 10 cM intervals followed by positional cloning and gene identification. 3. To test the hypothesis that SCF-KIT signaling is actively involved in the afferent, efferent, or both arms of the cutaneous immune response, we will use adoptive transfer of immune lymphocytes, KIT blocking antibodies, and small molecule inhibitors of KIT in a series of DNFGB sensitivity studies in normal mice, and in a proven transgenic model of SCF-KIT mediated cutaneous inflammation. These studies will determine specific contributions of SCF-KIT signaling to contact dermatitis, an provide support for the hypothesis that inhibitors of KIT may be novel therapeutic agents for human cutaneous inflammation.

描述（由申请人提供）：干细胞因子（SCF，也称为肥大细胞） 细胞生长因子）是KIT（一种受体酪氨酸激酶）的配体。的 本提案的目标是确定SCF-KIT信号通路的作用 肥大细胞增多症和皮肤炎症 成人中零星发生的肥大细胞增多症是由体细胞突变引起的 影响KIT的一级序列，并引起 KIT及其下游转导分子PI 3-K，其引起磷酸化 的AKT。家族性和大多数儿童肥大细胞增多症病例显示KIT正常 蛋白编码序列，但在病变肥大细胞中有磷酸化的AKT。我们 第一个假设是家族性和散发性儿童肥大细胞增多症是 由影响SCF-KIT信号通路的突变引起， 在AKT或AKT以上收敛。 人表皮角化细胞产生SCF，真皮注射SCF引起 炎症与人类一样，表达表皮SCF的转基因小鼠表现出与人类相似的免疫反应。 对过敏性和刺激性接触物的过度耳肿胀反应。我们 第二个假设是，SCF-KIT信号转导在细胞凋亡中起着积极的作用。 皮肤炎症反应。我们的具体目标是：1.确定 c-KIT突变阴性儿童肥大细胞增多症的肿瘤发生机制， 将来自病变肥大细胞的mRNA进行RT-PCR扩增和测序，以检测 编码可能影响AKT磷酸化的分子的基因突变 包括AKT、PTEN、林恩和PI 3-K。由于PTEN的缺失可能导致 增加的PI 3-K信号传导，也将测试病变肥大细胞DNAk的损失 10 q23区域的微卫星分析表明，功能 将在培养的骨髓中确定突变或基因丢失的影响 通过逆转录病毒表达突变体衍生肥大细胞肥大细胞系 激活或显性失活蛋白，或通过反义抑制。2.到 确定家族性肥大细胞增多症的遗传基础， 与显性遗传性肥大细胞增多症将测试连锁基因 使用微卫星分析已知影响KIT-P13-K信号通路。 如有必要，对受影响的和遗传相关的基因组进行全基因组筛查。 将使用10 cM间隔的基因座对未受影响的个体进行检测， 通过定位克隆和基因鉴定。3.为了验证这个假设， SCF-KIT信号传导活跃地参与传入臂、传出臂或两臂 皮肤免疫反应，我们将使用过继转移免疫 淋巴细胞，KIT阻断抗体和KIT的小分子抑制剂， 一系列DNFGB敏感性研究在正常小鼠，并在一个证明转基因 SCF-KIT介导的皮肤炎症模型。这些研究将决定 SCF-KIT信号传导对接触性皮炎的特异性贡献， 支持KIT抑制剂可能是新型治疗药物的假设 用于人类皮肤炎症的药剂。