Cutaneous Biology of KIT Ligand

KIT 配体的皮肤生物学

• 批准号: 6399913
• 负责人: BRUCE Jack LONGLEY
• 金额: $ 13.38万
• 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 2002-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6399913
• 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 细胞生长因子），是 KIT（一种受体酪氨酸激酶）的配体。这 该提案的目标是确定 SCF-KIT 信号通路的作用 肥大细胞增多症和皮肤炎症。 成人中偶发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 PI3-K，引起磷酸化 AKT。家族性和大多数儿童肥大细胞增多症病例显示正常 KIT 蛋白质编码序列，但在病变肥大细胞中磷酸化 AKT。我们的 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 信号通路或通路的突变引起 在 AKT 或以上时与其收敛。 人表皮角质细胞产生SCF，真皮注射SCF引起 炎。与人类一样，表达表皮 SCF 的转基因小鼠表现出 对过敏和刺激性接触物的过度肿胀反应。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肿瘤发生机制， 来自病变肥大细胞的 mRNA 将被 RT-PCR 扩增并测序以检测 可能影响 AKT 磷酸化的编码分子基因突变 包括 AKT、PTEN、Lyn 和 PI3-K。由于 PTEN 的丢失可能会导致 PI3-K 信号传导增加，病变肥大细胞 DNAk 也将被检测是否丢失 通过微卫星分析确定 10q23 区域的杂合性。功能性的 突变或基因丢失的影响将在培养的骨髓中确定 通过突变体的逆转录病毒表达衍生的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，两个不同的亲属 具有显性遗传性肥大细胞增多症的患者将接受基因连锁测试 通过微卫星分析已知其影响 KIT-P13-K 信号通路。 如有必要，对受影响的和遗传相关的进行全基因组筛查 未受影响的个体将使用基因座以 10 cm 的间隔进行检测，然后 通过定位克隆和基因鉴定。 3. 检验假设 SCF-KIT 信号传导积极参与传入臂、传出臂或双臂 皮肤免疫反应，我们将使用免疫的过继转移 淋巴细胞、KIT 阻断抗体和 KIT 小分子抑制剂 在正常小鼠和经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。这些研究将决定 SCF-KIT 信号传导对接触性皮炎的具体贡献，提供 支持 KIT 抑制剂可能是新型治疗药物的假设 人类皮肤炎症剂。