Regulation of Neph3 gene in podocytes--key roles of transcription factors NF-kappaB and Sp1.
Regulation of Neph3 gene in podocytes--key roles of transcription factors NF-kappaB and Sp1.
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DOI：
10.1186/1471-2199-10-83
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发表时间：
2009-08-24
影响因子：
--
通讯作者：
Holthöfer H
中科院分区：
文献类型：
--
作者：
Ristola M;Arpiainen S;Saleem MA;Mathieson PW;Welsh GI;Lehtonen S;Holthöfer H
Neph3(滤蛋白)表达于肾小球足细胞，定位于足突的特殊细胞黏附结构，称为裂隙隔膜，构成肾小球滤过屏障的最外层。Neph3蛋白与Neph1、Neph2和newitin具有同源性和结构相似性，它们都是维持正常肾小球超滤功能的关键。Neph3在肾脏中的确切功能尚不清楚，但我们之前已经证明，在蛋白尿疾病中，Neph3的mRNA水平降低。这提示Neph3可能在肾脏损伤的发病机制中起作用，并强调了分析Neph3基因的调控机制的必要性。在这项研究中，我们通过寻找控制Neph3表达的转录因子来研究Neph3基因的转录调控。我们克隆并鉴定了Neph3基因上游约5kb的片段。转录起始点附近的Neph3启动子没有TATA和CAAT盒，但含有一个高度富含GC的区域。利用启动子报告基因构建，我们将Neph3基因的主要激活调控区定位在其近端的启动子区域-105至-57。通过计算分析，在该区域内发现了与NF-κB和Sp1可能的转录因子结合位点。突变筛选表明，NF-κB和Sp1反应元件是Neph3启动子基本转录活性所必需的。共转染研究进一步表明，NF-κB和Sp1调节Neph3启动子的活性。此外，NF-κB的过表达增加了内源性Neph3基因的表达。用培养的人足细胞进行染色质免疫沉淀分析表明，NF-κB和Sp1都与Neph3启动子相互作用。我们的结果表明，NF-κB和Sp1在足细胞的基础水平上是Neph3表达的关键调节因子，从而为了解促进Neph3基因表达的分子机制提供了新的视角。
Neph3 (filtrin) is expressed in the glomerular podocytes where it localizes at the specialized cell adhesion structures of the foot processes called slit diaphragms which form the outermost layer of the glomerular filtration barrier. Neph3 protein shows homology and structural similarity to Neph1, Neph2 and nephrin, which all are crucial for maintaining the normal glomerular ultrafiltration function. The exact function of Neph3 in the kidney is not known but we have previously shown that the level of Neph3 mRNA is decreased in proteinuric diseases. This suggests that Neph3 may play a role in the pathogenesis of kidney damage, and emphasizes the need to analyze the regulatory mechanisms of Neph3 gene. In this study we investigated the transcriptional regulation of Neph3 gene by identifying transcription factors that control Neph3 expression. We cloned and characterized approximately 5 kb fragment upstream of the Neph3 gene. Neph3 proximal promoter near the transcription start site was found to be devoid of TATA and CAAT boxes, but to contain a highly GC-rich area. Using promoter reporter gene constructs, we localized the main activating regulatory region of Neph3 gene in its proximal promoter region from -105 to -57. Within this region, putative transcription factor binding sites for NF-κB and Sp1 were found by computational analysis. Mutational screening indicated that NF-κB and Sp1 response elements are essential for the basal transcriptional activity of the Neph3 promoter. Co-transfection studies further showed that NF-κB and Sp1 regulate Neph3 promoter activity. In addition, overexpression of NF-κB increased endogenous Neph3 gene expression. Chromatin immunoprecipitation assay using cultured human podocytes demonstrated that both NF-κB and Sp1 interact with the Neph3 promoter. Our results show that NF-κB and Sp1 are key regulators of Neph3 expression at the basal level in podocytes, therefore providing new insight into the molecular mechanisms that contribute to the expression of Neph3 gene.
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影响因子：
5
作者：
Matsui, Isao;Ito, Takahito;Hori, Masatsugu
通讯作者：
Hori, Masatsugu
影响因子：
2.9
作者：
Minaki, Y;Mizuhara, E;Ono, Y
通讯作者：
Ono, Y
影响因子：
64.5
作者：
COUREY, AJ;TJIAN, R
通讯作者：
TJIAN, R
影响因子：
5.3
作者：
Donoviel, DB;Freed, DD;Powell, DR
通讯作者：
Powell, DR
DOI：
10.1073/pnas.98.2.646
发表时间：
2001-01-16
影响因子：
11.1
作者：
O'Hare, MJ;Bond, J;Jat, PS
通讯作者：
Jat, PS