Cutaneous Biology of KIT Ligand

KIT 配体的皮肤生物学

• 批准号: 6667090
• 负责人: B Jack Longley
• 金额: $ 31.1万
• 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 2006-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6667090
• 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(申请人提供)：干细胞因子(SCF，又称MAST 细胞生长因子)是KIT的配体，KIT是一种受体酪氨酸激酶。这个 这项建议的目的是确定SCF-KIT信号通路的作用 肥大细胞增多症和皮肤炎。 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 影响试剂盒的初级序列，并导致组成性激活 Kit及其下游导致磷酸化的转导分子PI3-K AKT的。家族性和大多数儿童肥大细胞增多症病例显示正常试剂盒 蛋白质编码序列，但在皮损肥大细胞中已磷酸化AKT。我们的 第一种假设是家族性和散发性的儿童肥大细胞增多症 由影响SCF-KIT信号通路的突变引起 在AKT或AKT以上与之收敛。 人表皮角质形成细胞产生干细胞因子，皮肤注射干细胞因子引起 发炎。像人类一样，表达表皮干细胞因子的转基因小鼠表现出 对过敏性和刺激性接触物的耳肿胀反应夸张。我们的 第二个假设是，SCF-KIT信号在 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致癌机制(S)， 来自皮损肥大细胞的mRNA将被RT-PCR扩增并测序以检测 编码可能影响AKT磷酸化的分子的基因突变 包括AKT、PTEN、LYN和PI3-K。因为PTEN的缺失可能会导致 PI3-K信号增强，皮损肥大细胞DNAk也将接受丢失测试 微卫星分析10q23区域杂合性。功能界别 突变或基因缺失的影响将在培养的骨髓中确定 逆转录病毒表达突变体诱导的肥大细胞系 激活或显性负性蛋白，或通过反义抑制。2.至 确定两个不同家族的家族性肥大细胞增多症的遗传基础 患有显性遗传性肥大细胞增多症的患者将接受基因连锁检测 利用微卫星分析已知影响KIT-P13-K信号通路。 如有必要，对受影响和基因相关的全基因组进行筛查 未受影响的个体将每隔10厘米使用基因座进行检查 通过定位克隆和基因鉴定。3.检验假设 SCF-KIT信号积极参与传入、传出或同时参与传入和传出 对皮肤的免疫反应，我们将采用过继转移免疫 淋巴细胞、KIT阻断抗体和KIT的小分子抑制剂 在正常小鼠和已证实的转基因小鼠中进行的一系列DNFGB敏感性研究 SCF-KIT介导的皮肤炎症模型。这些研究将确定 SCF-KIT信号在接触性皮炎中的特殊作用 支持KIT抑制剂可能是新的治疗药物的假设 治疗人体皮肤炎的药物。