Identification of novel genes regulating metalloproteinase activity

调控金属蛋白酶活性的新基因的鉴定

• 批准号: 8136114
• 负责人: Andreas Herrlich
• 金额: $ 24.65万
• 依托单位: BRIGHAM AND WOMEN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-01 至 2013-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8136114
• 关键词: Acute; Adverse effects; Alzheimer' s Disease; Amyloid beta-Protein Precursor; Angiotensin II; Antibodies; Biological Assay; C-terminal; Candidate Disease Gene; Cell Count; Cell physiology; Cells; Chronic Kidney Failure; Cleaved cell; Complementary DNA; Coupled; DTR gene; Development; Disease; EGF gene; Enhancers; Epithelial Cells; Epitopes; Evaluation; Fluorescence; Fluorochrome; Genes; Goals; Grant; Heart Diseases; Heart failure; Human; Infection; Inflammation; Injury; Instruction; Jurkat Cells; Kidney; Kidney Diseases; Knockout Mice; LacZ Genes; Length; Libraries; Life; Ligands; Lung; Malignant Neoplasms; Measures; Metalloproteases; Modeling; Mus; Phase; Phorbol Esters; Phosphoric Monoester Hydrolases; Phosphotransferases; Physiological; Polybrene; Progress Reports; Protein Isoforms; Puromycin; Regulation; Resistance; Signal Transduction Pathway; Staining method; Stains; Transforming Growth Factor alpha; Tubular formation; Virus Diseases; atypical protein kinase C; base; cDNA Library; high throughput screening; inhibitor/antagonist; killings; knock-down; novel; overexpression; pro-EGF; repaired; response; small hairpin RNA

Ectodomain (ECD) cleavage by metalloproteinases is involved in many diseases including kidney and cardiac disease, Alzheimer's disease, cancer and inflammation. EGF pro-ligands mature by metalloproteinase-cleavage of the ECD that represents the active ligand. They are important in a broad range of physiological and disease states. In the kidney, as examples, the EGF ligand TGFalpha is cleaved in response to Angiotensin II (Angll). TGFalpha knock-out mice or mice treated with a metalloprotease inhibitor are protected against Angll-induced chronic kidney disease. HB-EGF, another EGF ligand, is involved in tubular branching in the developing kidney and in tubular repair after kidney injury. Metalloproteinase-directed therapies in humans are limited by side effects, or non-existent. The signal transduction pathways regulating ECD cleavage are essentially unknown. The goal of our study is to identify novel genes that regulate ECD cleavage using a lentiviral shRNA gene knock-down approach. We have developed a high-throughput assay that detects cleavage of EGF-ligands in a FACS-based assay using cells stably expressing a ligand with an ECD epitope tag and a C-terminal GFP-fusion. The ECD can be tracked by staining with a fluorochrome-coupled antibody ("red"). In the uncleaved state any given cell has a 1:1 ratio of outside (ECD, "red") to inside (GFP, "green") fluorescence, as measured by FACS on life single cells. Stimulation of EGF ligand cleavage decreases, while inhibition increases this ratio. During the K99 phase of this grant we began examining the effect of knock-down of human kinases and phosphatases on EGF ligand cleavage. Here we present up-to-date results on the initial screen that identified several inhibitors and activators of phorbol ester-induced TGFalpha cleavage in Jurkat cells. Knock-down of PKCzeta, an atypical protein kinase C isoform not activated by phorbol ester is identified as an important inhibitor of cleavage in Jurkat cells and also in mouse lung epithelial cells and partipates in differential regulation of ECD cleavage of three different EGF ligands examined. We are outlining our plans for completion of the high-throughput screen and for further evaluation of candidate genes, including PKCzeta.

金属蛋白酶的胞外结构域（ECD）裂解涉及许多疾病，包括肾脏和 心脏病、阿尔茨海默病、癌症和炎症。EGF的成熟 代表活性配体的ECD的金属蛋白酶裂解。它们在广泛的 生理和疾病状态的范围。例如，在肾脏中，EGF配体TGF α被切割， 血管紧张素II（AngII）。TGF α敲除小鼠或用金属蛋白酶处理的小鼠 抑制剂被保护免于AngII诱导慢性肾病。HB-EGF是EGF的另一种配体， 参与发育中的肾的肾小管分支和肾损伤后的肾小管修复。 金属蛋白酶定向疗法在人类中受到副作用的限制，或不存在。信号 调节ECD裂解的转导途径基本上是未知的。我们研究的目的是 使用慢病毒shRNA基因敲低方法调节ECD切割的新基因。我们有 开发了一种高通量测定法，该测定法在基于FACS的测定法中检测EGF配体的裂解， 稳定表达具有ECD表位标签和C-末端GFP融合体的配体的细胞。ECD可以是 通过用荧光染料偶联抗体（“红色”）染色来追踪。在未分裂状态下，任何给定的细胞都具有 外部（ECD，“红色”）与内部（GFP，“绿色”）荧光的比例为1：1，如通过FACS对寿命所测量的 单细胞刺激EGF配体裂解减少，而抑制增加这一比例。期间 在K99阶段，我们开始研究敲低人类激酶和磷酸酶的效果 表皮生长因子配体裂解。在这里，我们在初始屏幕上展示了最新的结果，其中识别了几个 在Jurkat细胞中佛波酯诱导的TGF α裂解的抑制剂和激活剂。敲低 PKCzeta是一种不被佛波酯激活的非典型蛋白激酶C亚型，被鉴定为重要的 在Jurkat细胞和小鼠肺上皮细胞中的切割抑制剂，以及在分化 检测三种不同EGF配体的ECD切割的调节。我们正在计划 完成高通量筛选和进一步评估候选基因，包括PKCzeta。