Department of Pathology, Haartman Institute, University of Helsinki, Helsinki, Finland.
Nephrol Dial Transplant. 2012 May;27(5):1737-45. doi: 10.1093/ndt/gfr576. Epub 2011 Oct 6.
Nephrin and Neph3 are homologous molecules expressed in the podocyte slit diaphragms that are essential for normal glomerular ultrafiltration. Nephrin and Neph3 genes form a bidirectional gene pair suggesting that they may share key features in their regulation. We investigated if nephrin and Neph3 genes have similar mechanisms in their transcriptional regulation focussing on transcription factor Wilms' tumour 1 (WT1) and nuclear factor-κB (NF-κB) and DNA methylation.
Transcriptional regulation of nephrin and Neph3 by WT1 and NF-κB was analysed by overexpression studies, reporter gene assay and chromatin immunoprecipitation using A293 cells and cultured podocytes. The interaction between WT1 and NF-κB was studied by co-immunoprecipitation. The effect of NF-κB activator tumour necrosis factor-α (TNF-α) with or without NF-κB pathway inhibitor (BAY 11-7082) on nephrin and Neph3 messenger RNA (mRNA) expression and on cellular distribution of NF-κB was determined by quantitative polymerase chain reaction (PCR) and immunostaining, respectively. The role of DNA methylation in regulating nephrin and Neph3 genes was studied by demethylating agent (5-aza-2'-deoxycytidine) treatment and quantitative PCR.
WT1 and NF-κB interact with nephrin and Neph3 promoter and cooperatively regulate nephrin and Neph3. The cooperation was further supported by the physical interaction between WT1 and NF-κB. TNF-α increased nephrin and Neph3 mRNA expression and this effect was mediated by NF-κB. Furthermore, DNA methylation played a role in silencing nephrin and Neph3 expression in a cell-type and differentiation stage-dependent manner.
These results provide novel insights into the transcriptional regulation of nephrin and Neph3 genes and indicate that nephrin and Neph3 share the same mechanisms in their regulation.
nephrin 和 Neph3 是在足细胞裂孔隔膜中表达的同源分子，对于正常肾小球超滤是必不可少的。nephrin 和 Neph3 基因形成一个双向基因对，表明它们可能在调节方面具有关键特征。我们研究了 nephrin 和 Neph3 基因在转录调节中是否具有相似的机制，重点关注转录因子 Wilms 肿瘤 1（WT1）和核因子-κB（NF-κB）和 DNA 甲基化。
通过过表达研究、报告基因测定和染色质免疫沉淀，使用 A293 细胞和培养的足细胞分析 WT1 和 NF-κB 对 nephrin 和 Neph3 的转录调节。通过共免疫沉淀研究 WT1 和 NF-κB 之间的相互作用。用肿瘤坏死因子-α（TNF-α）（NF-κB 途径抑制剂 BAY 11-7082）刺激或不刺激，分别通过定量聚合酶链反应（PCR）和免疫染色，研究 NF-κB 激活剂 TNF-α对 nephrin 和 Neph3 信使 RNA（mRNA）表达和 NF-κB 细胞分布的影响。通过去甲基化剂（5-氮杂-2'-脱氧胞苷）处理和定量 PCR 研究 DNA 甲基化在调节 nephrin 和 Neph3 基因中的作用。
WT1 和 NF-κB 与 nephrin 和 Neph3 启动子相互作用，并协同调节 nephrin 和 Neph3。WT1 和 NF-κB 之间的物理相互作用进一步支持了这种合作。TNF-α增加了 nephrin 和 Neph3 mRNA 的表达，这种作用是由 NF-κB 介导的。此外，DNA 甲基化在细胞类型和分化阶段依赖性沉默 nephrin 和 Neph3 表达方面发挥作用。
这些结果为 nephrin 和 Neph3 基因的转录调节提供了新的见解，并表明 nephrin 和 Neph3 在调节方面具有相同的机制。
