Regulation of Hepatic Glutathione Synthesis

肝脏谷胱甘肽合成的调节

基本信息

批准号：
7221846
负责人：
Shelly Chi-Loo Lu
金额：
$ 27.2万
依托单位：
UNIVERSITY OF SOUTHERN CALIFORNIA
依托单位国家：
美国
项目类别：
财政年份：
1992
资助国家：
美国
起止时间：
1992-09-30 至 2009-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7221846
关键词：
1-Phosphatidylinositol 3-Kinase 2-tert-butylhydroquinone 5&apos Flanking Region Acetaldehyde Address Anabolism Antioxidants Binding Binding Sites Biological Assay Biological Testing Buthionine Sulfoximine CXCL9 gene Ca(2+)-Calmodulin Dependent Protein Kinase Cancer Cell Growth Catalytic Domain Cell Line Cell physiology Cells Comparative Study Consensus Cyclic AMP Cyclic AMP-Dependent Protein Kinases DNase-I Footprinting Data Dithiothreitol Electrophoretic Mobility Shift Assay Elements Enhancers Enzymes Ethanol Exhibits Fibroblasts GCLC gene GCLM gene Gamma-glutamyl transferase Genes Glucocorticoids Glucose Glutamate-Cysteine Ligase Glutamates Glutathione Glutathione Disulfide Goals Growth Hepatic Hepatocyte Holoenzymes Homeostasis Hormones Human Hydrocortisone Hydroquinones In Vitro Insulin Laboratories Ligase Liver Liver Regeneration Luciferases Malignant Neoplasms Malignant neoplasm of liver Mediating Metals Methods Molecular NF-kappa B Natural regeneration Nuclear Oxidative Stress Partial Hepatectomy Phosphoinositide-3-Kinase, Catalytic, Gamma Polypeptide Play Primary carcinoma of the liver cells Protein Kinase C Rate Rattus Recombinants Reduced Glutathione Regulation Regulatory Element Reporter Reporting Research Design Role S-ethyl glutathione Site-Directed Mutagenesis TNF gene Techniques Thioacetamide Transcription Factor AP-1 Transcriptional Activation Transcriptional Regulation Transfection Transgenic Organisms Tumor Necrosis Factor-alpha Tumor Necrosis Factors Up-Regulation Work cell growth cis acting element diethyl maleate hepatoma cell human TNF protein hydroquinone improved in vitro Model in vivo in vivo Model nuclear factor 1 prevent promoter response synthetic enzyme transcription factor

项目摘要

DESCRIPTION (provided by applicant): Glutathione (GSH) plays a vital defensive role and modulates critical cellular processes. One important factor that determines the rate of GSH synthesis is the activity of glutamate-cysteine ligase (GCL, also known as glutamylcysteine synthetase). GCL is made up of a catalytic and a modifier subunit (GCLC and GCLM), the former exhibits all of the catalytic activity of the holoenzyme but the latter makes the enzyme function more efficiently. During the reporting period we showed that the two subunits are differentially regulated. While hormones and rapid liver growth transcriptionally activate GCLC, some but not all inducers of oxidative stress activate both GCL subunits. Although much emphasis has been placed on GCL, our results indicate that the second enzyme in GSH synthesis, GSH synthetase (GS) may be just as important in certain cells and has been overlooked. Treatments that induce both GCL subunits also induced GS. Importantly, we found GS up-regulation can further enhance the cell's GSH synthetic capacity. In order to study transcriptional regulation of the GSH synthetic enzymes using our well-defined in vivo and in vitro models, we have cloned the 5'-flanking regions of both GCL subunits and GS from the rat and identified candidate transcription factors that may regulate these genes. We found AP-1 is required for the basal expression and the tert-butylhydroquinone-mediated increase in expression of both GCL subunits and GS. The advantage of studying the rat promoters is the ability to perform comparative studies using in vitro and in vivo models, which would be difficult to achieve with human promoters. Finally, we characterized GSH homeostasis in regenerating rat liver and human hepatocellular carcinoma (HCC) and found that both GCLC and GS are transcriptionally up-regulated in HCC and increased GSH can facilitate liver cancer cell growth. The following aims are direct extensions of these observations which will: 1) examine transcriptional regulation of rat GCLC - define the molecular mechanism of transcriptional regulation by various agents we have identified using in vitro and in vivo models; 2) examine transcriptional regulation of rat GCLM - similar types of studies will be performed here as for GCLC; and 3) examine transcriptional regulation of rat GS - identify cis-acting elements and transcription factors important for basal expression and in response to various treatments. These studies should improve our understanding of transcriptional regulation of the GSH synthetic enzymes. Our ultimate goal is to utilize this information to improve the treatment and prevent complications that may result from altered hepatic GSH synthesis.

描述（由申请人提供）：谷胱甘肽（GSH）起着至关重要的防御作用，并调节关键细胞过程。确定GSH合成速率的一个重要因素是谷氨酸 - 半胱氨酸连接酶（GCL，也称为谷氨酰胺半胱氨酸合成酶）的活性。 GCL由催化和修饰剂亚基（GCLC和GCLM）组成，前者表现出全酶的所有催化活性，但后者使酶的功能更有效。在报告期间，我们表明这两个亚基受到差异调节。尽管激素和肝脏生长转录会激活GCLC，但有些氧化应激的诱导剂激活了两个GCL亚基。尽管已在GCL上强调了很多重点，但我们的结果表明，GSH合成中的第二种酶，GSH合成酶（GS）在某些细胞中可能同样重要，并且被忽略了。诱导两个GCL亚基的处理也诱导GS。重要的是，我们发现GS上调可以进一步增强细胞的GSH合成能力。为了使用我们定义明确的体内和体外模型研究GSH合成酶的转录调节，我们将大鼠和GCL亚基和GS的5 f频型区域克隆起来，并确定了可能调节这些基因的候选转录因子。我们发现AP-1是基础表达和TERT叔丁基氢醌介导的GCL亚基和GS表达的增加所必需的。研究大鼠启动子的优点是能够使用体外和体内模型进行比较研究，这很难用人类启动子来实现。最后，我们表征了在再生大鼠肝脏和人肝细胞癌（HCC）中的GSH稳态，并发现GCLC和GS在HCC中都在转录上调，而GSH的增加可以促进肝癌细胞的生长。以下目的是这些观察值的直接扩展：1）检查大鼠GCLC的转录调控 - 定义了我们使用体外和体内模型确定的各种药物的转录调控的分子机制； 2）检查大鼠GCLM的转录调控 - 对于GCLC，将进行类似的研究类型； 3）检查大鼠GS的转录调控 - 确定对基础表达和对各种治疗的反应重要的顺式作用元件和转录因子。这些研究应提高我们对GSH合成酶的转录调节的理解。我们的最终目标是利用这些信息来改善治疗方法，并防止肝GSH合成改变可能导致的并发症。