LAMININ GENE EXPRESSION IN GLOMERULAR CELLS

进行性肾病最严重的后遗症是肾小球性 疤痕和硬化 肾小球硬化是一种异常的 正常肾小球基底膜（GBM）成分的积累。 层粘连蛋白是GBM的正常成分之一，其在GBM内积累。 免疫介导的肾小球疾病进展过程中的肾小球 疾病或糖尿病。 白细胞介素-1（IL-1）是一种普遍存在的细胞因子 由侵入的巨噬细胞和驻留的巨噬细胞在肾小球中产生 系膜细胞 IL-1或其他生长因子的过度产生 被认为激活肾小球细胞，导致细胞增殖 和细胞外基质成分的异常积累。 虽然 我们对肾小球疾病生物学的理解已经取得了进展， 病变，对细胞内分子事件知之甚少， 介导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链的积累， mRNA和刺激层粘连蛋白B2链基因启动子提供了一种新的免疫抑制剂。 有机会研究参与细胞内分子机制， 正常和异常生产的一个组成部分GBM的一个 肾小球细胞 本建议的目的是确定 控制层粘连蛋白B2基因表达的转录因子 肾小球细胞 通过在大鼠和人中使用比较缺失分析， 肾小球细胞，我们将首先尝试识别增强子元件 层粘连蛋白的组成性和IL-1诱导活性所需 B2链基因启动子。 首先，大鼠肾小球上皮和系膜 细胞将用含有野生型 或与报道基因连接的突变型大鼠层粘连蛋白B2启动子。 大鼠层粘连蛋白B2启动子中的转录元件 将通过比较大鼠中报告基因的活性来鉴定 上皮细胞和系膜细胞转染这些重组 质粒。 第二，我们将在人类系膜细胞中测试人类层粘连蛋白， 含有缺失同源区的B2启动子构建体， 在大鼠启动子中具有转录活性。 删除比较 大鼠和人类系统中的突变体应有助于 在启动子中具有转录活性和同源性的序列， 两个物种。 根据比较删除的信息， 分析人和大鼠层粘连蛋白B2启动子，我们将使用特异性 电泳凝胶位移分析（EMSA）中的DNA探针，西南印迹 和甲基化干扰，以鉴定组成型和IL-1诱导型 调节层粘连蛋白B2活性的转录因子 启动子 我们鉴定为新的和/或特异性的蛋白质 将使用连续阴离子交换纯化肾小球细胞， 串联DNA亲和层析。 其部分氨基酸序列 蛋白质将用于合成简并寡核苷酸引物 聚合酶链反应（PCR）。 cDNA文库来源于 将用PCR扩增的探针筛选肾小球细胞以克隆 层粘连蛋白B2启动子特异性转录因子。 大鼠和人肿瘤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 完整肾小球层粘连蛋白产生的转录过程 在肾小球疾病的发展过程中。

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.