The Major Podocyte Protein Nephrin Is Transcriptionally Activated by the Wilms’ Tumor Suppressor WT1

Abstract

Abstract. NPHS1 encodes the structural protein nephrin, which has a crucial role in the filtration barrier of the glomerular podocyte. Mutations or deregulation of NPHS1 are associated with a variety of renal diseases, including the Finnish type congenital nephrotic syndrome. This study analyzed a potential regulation of nephrin by the Wilms' tumor protein, Wt1. Using an inducible U2OS osteosarcoma cell line, it is shown that upon Wt1 induction, endogenous nephrin mRNA becomes highly upregulated. Co-transfection studies demonstrate that Wt1 can activate the nephrin promoter Ͼ10-fold. DNase footprinting and mutation analysis identify a Wt1 responsive element in the nephrin promoter, which is required for the binding of Wt1 protein. Mutations or deletion of this Wt1 responsive element completely abolished transactivation of the nephrin promoter by Wt1. Moreover, transgenic analysis demonstrates the requirement of the identified binding site to direct podocyte-specific expression of a reporter gene in transgenic mice, thus confirming the importance of this site for the regulation of nephrin in vivo. Finally, it is shown that nephrin expression is lowest in kidneys of mice that lack specifically the Wt1(ϪKTS) splice variant, but in comparison with wild-type littermates, it is also reduced in animals with disruption of the Wt1(ϩKTS) splice variant. Taken together, these data identify nephrin as a direct transcriptional target for Wt1 and underline the importance of Wt1 as a key regulator in podocyte function.

The Wilms' tumour suppressor WT1 is involved in endothelial cell proliferation and migration: expression in tumour vessels in vivo

“…In chromatin immunoprecipitation experiments using HUVECs, we could identify a binding region for WT1 protein, which contained the sequence À387 to À27 bp relative to the ATG of ETS-1, whereas a sequence from the 3 0 -untranslated region (UTR) of ETS-1 did not bind to WT1 (Figure 7b). Using multiple alignments between mouse and human ETS-1 promoters and our identified WT1-binding site in the nephrin promoter (Wagner et al, 2004), we predicted a binding site for WT1 in the ETS-1 promoter. Physical interaction of WT1(ÀKTS), but not WT1( þ KTS) with this element was confirmed by electrophoretic mobility shift assays.…”

“…A 4 kb VEGFR2 promoter construct in the luciferase expression vector was a gift from C Patterson (Labrecque et al, 2003). Deletion of the WT1-binding site in the ETS-1 promoter was performed using the Quik Change II site-directed mutagenesis kit (Stratagene, Amsterdam, Netherlands) as described by Wagner et al (2004) using the following primers: 5 0 -CCCTGGGCCTGGGCTCGGTCTGC ACTGCGCCAGCGCCG-3 0 (forward, reverse primer in the corresponding antisense orientation). HEK 293 cells at WT1 activates ETS-1 in endothelial cells N Wagner et al approximately 60% confluence were transfected in 60 mm tissue culture plates using Fugene 6 reagent (Roche Diagnostics, Meylan, France).…”

“…HEK 293 cells at WT1 activates ETS-1 in endothelial cells N Wagner et al approximately 60% confluence were transfected in 60 mm tissue culture plates using Fugene 6 reagent (Roche Diagnostics, Meylan, France). About 0.3 mg of the reporter constructs together with 0.1 mg of a cytomegalovirus (CMV)-driven b-galactosidase plasmid, and 1.6 mg of the expression constructs encoding different WT1 forms were transiently co-transfected and assayed for luciferase-and b-galactosidase activity as described in detail elsewhere (Wagner et al, 2004).…”

“…Electrophoretic mobility shift assay Electrophoretic mobility shift assays using recombinant WT1 protein were performed as described elsewhere (Wagner et al, 2004). The 26-bp double stranded oligonucleotide (5 0 -GAG GAACGCCCTAAAGCGGAGGCGAG-3 0 ) represented the WT1 responsive element from the ETS-1 promoter.…”

hnRNP-U directly interacts with WT1 and modulates WT1 transcriptional activation

“…The majority of studies to date have focused on identifying transcriptional targets of WT1, although as WT1-transfected cell lines are not a physiologically relevant system, very few bona fide WT1 target genes have been identified. Those that have include amphiregulin, SF-1, Wnt-4, nephrin and TrkB (Lee et al, 1999;Wilhelm and Englert, 2002;Sim et al, 2002;Wagner et al, 2004Wagner et al, , 2005) and the situation is further complicated by the fact that WT1 activity is likely to be modulated by its interactions with other proteins. For example, WT1 interacts with steroidogenic factor 1 (SF-1) to activate mullerian inhibitory substance (MIS) expression in the male gonadal ridge, but this interaction is antagonized by the X-linked sex reversal protein, Dax1, which competes WT1 off SF-1, thereby inactivating the complex (Nachtigal et al, 1998).…”