LAMININ GENE EXPRESSION IN GLOMERULAR CELLS

进行性肾脏疾病最严重的后遗症是肾小球 伤疤和硬化症。肾小球硬化症代表一种异常 正常肾小球基底膜(GBM)成分积聚。 层粘连蛋白是基底膜的正常成分之一，它积累在 免疫介导性肾小球进展过程中的肾小球 疾病或糖尿病。白介素1是一种普遍存在的细胞因子 由入侵的巨噬细胞和常驻的巨噬细胞在肾小球中产生 系膜细胞。过度产生IL-1或其他生长因子 被认为能激活肾小球细胞，导致细胞增殖 细胞外基质成分异常积聚。虽然 我们对肾小球生物学的了解已经取得了进展 损伤，对细胞内分子事件知之甚少 调节细胞外基质成分的产生。我们的观察 在肾小球细胞中IL-1增加层粘连蛋白B2链的积聚 MRNA和刺激层粘连蛋白B2链基因启动子提供了 有机会研究细胞内涉及的分子机制 一种GBM组分的正常和异常产生 肾小球细胞。这项提案的目标是确定 控制层粘连蛋白B2基因表达的转录因子 肾小球细胞。 通过对大鼠和人进行比较缺失分析 肾小球细胞，我们将首先尝试鉴定增强子元件 层粘连蛋白的构成活性和IL-1诱导活性所必需的 B2链基因启动子。第一，大鼠肾小球上皮和系膜 细胞将被含有野生型或野生型的载体 或突变的大鼠层粘连蛋白B2启动子与报告基因连锁。 大鼠层粘连蛋白B2启动子中的转录元件 将通过比较报告基因在大鼠体内的活性来鉴定 重组人肾小管上皮细胞和系膜细胞 质粒。第二，我们将在人肾小球系膜细胞中测试人层粘连蛋白 具有缺失同源区域的B2启动子的构建 在大鼠启动子中具有转录活性。删除的比较 大鼠和人类系统中的突变应该有助于定位 启动子中转录活性和同源性的序列 这两个物种。基于来自比较删除的信息 分析人和大鼠层粘连蛋白B2启动子，我们将使用特异的 凝胶电泳法(EMSA)中的DNA探针，西南斑点 和甲基化干扰以识别组成性和IL-1诱导性 调节层粘连蛋白B2活性的转录因子 推动者。蛋白质(S)，我们确定为新的和/或特定于 肾小球细胞将使用顺序阴离子交换和 串联DNA亲和层析。它的部分氨基酸序列 蛋白质(S)将用于合成简并寡核苷酸引物 用于聚合酶链式反应(PCR)。来源于 用聚合酶链式反应扩增的探针筛选肾小球细胞克隆 层粘连蛋白B2启动子特异性转录因子(S)。 大鼠和人相关转录因子的鉴定 层粘连蛋白B2基因的表达将为未来的研究奠定基础 完整肾小球中层粘连蛋白的转录过程 在发育过程中和在肾小球疾病过程中。

The most serious sequela of progressive renal disease is glomerular scarring and sclerosis. Glomerular sclerosis represents an abnormal accumulation of normal glomerular basement membrane (GBM) components. Laminin is one of the normal components of GBM that accumulates within glomeruli during the progression of immunologically-mediated glomerular diseases or diabetes. Interleukin-1 (IL-1) is a ubiquitous cytokine produced in the glomeruli by both invading macrophages and by resident mesangial cells. Excessive production of IL-1 or other growth factors is thought to activate glomerular cells resulting in cell proliferation and abnormal accumulation of extracellular matrix components. although progress has been made in our understanding of the biology of glomerular lesions, little is known about intracellular molecular events that mediate production of extracellular matrix components. Our observation that in glomerular cells IL-1 increases accumulation of laminin B2 chain mRNA and stimulates the laminin B2 chain gene promoter provides an opportunity to study the intracellular molecular mechanisms involved in the normal and abnormal production of one of the components of GBM by a glomerular cell. The objective of this proposal is to identify transcription factors that control laminin B2 gene expression in glomerular cells. Through the use of comparative deletion analysis in rat and human glomerular cells, we will first attempt to identify enhancer elements required for the constitutive and IL-1-inducible activity of the laminin B2 chain gene promoter. First, rat glomerular epithelial and mesangial cells will be transfected with plasmids containing either the wild type or mutant rat laminin B2 promoter linked to a reporter gene. Transcriptional elements contained within the rat laminin B2 promoter will be identified by comparing activity of the reporter gene in rat epithelial and mesangial cells transfected in with these recombinant plasmids. Second, we will test in human mesangial cells human laminin B2 promoter constructs with deletion of homologous regions contained and transcriptionally active in the rat promoter. Comparison of deletion mutants in the rat and human systems should facilitate localization of sequences transcriptionally active and homologous in the promoters from the two species. Based on the information from the comparative deletion analysis of the human and rat laminin B2 promoter, we will use specific DNA probes in electrophoretic gel shift assays (EMSA), Southwestern blots and methylation interference to identify constitutive and IL-1-inducible transcription factors that regulate the activity of the laminin B2 promoter. Protein(s) that we identify as novel and/or specific for glomerular cells will be purified using sequential anion exchange and tandem DNA-affinity chromatography. Partial amino acid sequences of this protein(s) will be used to synthesize degenerate oligonucleotide primers for polymerase chain reaction (PCR). cDNA libraries derived from glomerular cells will be screened with the PCR-amplified probes to clone the laminin B2 promoter-specific transcription factor(s). Identification of transcription factors involved in the rat and human laminin B2 gene expression would set the ground work for future studies on transcriptional processes of laminin production in intact glomeruli during development and in the course of glomerular disease.