Transcription of nephrin-Neph3 gene pair is synergistically activated by WT1 and NF- B and silenced by DNA methylation

Abstract

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.