The Wilms tumour suppressor gene WT1 is mutated in 10-15% of Wilms tumours and mutations in WT1 are also associated with Denys Drash Syndrome (DDS). DDS is characterised by childhood nephrotic syndrome, glomerulosclerosis, end-stage renal failure and Wilms tumours. WT1 encodes a zinc finger transcription factor that also regulates gene expression at the mRNA level. The search for WT1’s DNA and mRNA targets is ongoing – a recent report has suggested that WT1 regulates the expression of a key growth factor, VEGF (Vascular Endothelial Growth Factor). There are two families of VEGF isoforms; an angiogenic family (VEGFxxx); and a sister family of anti-angiogenic isoforms (VEGFxxxb) that result from a distal alternative 3’ splice site in exon 8 (Bates et al 2002). Although WT1 regulates VEGF expression transcriptionally, the extent to which WT1 affects VEGF isoform levels is not known, but splicing is altered in Denys Drash patients (Schumacher et al 2007). We measured VEGF isoform expression in differentiated wild-type and DDS patient-derived podocytes with a mutation in WT1 (R366C). The differentiated podocytes represents the closest in vivo model of podocytes in the kidney within the constraints of a cell culture model. Total VEGF and VEGFxxxb levels were determined by ELISA and RT-PCR. In the differentiated DDS podocytes there was a 0.13 ±0.002 fold reduction in the amount of VEGFxxxb compared to wildtype differentiated podocytes while DDS stably transfected with wildtype WT1 restored the expression of VEGFxxxb to 0.73 ±0.08. In the differentiated DDS podocytes there was a 2.32 ±0.483 fold increase in the amount of panVEGF compared to wildtype differentiated podocytes. Transfection of the wildtype WT1 into the differentiated DDS podocytes manages to rescue the DDS phenotype by decreasing the amount of panVEGF to 0.618 ±0.114 fold compare to wildtype podocytes. These results suggest that WT1 plays a role in the regulation of VEGF isoform expression; either by direct binding to VEGF pre-mRNA or by modulating splice factor activities
University of Bristol (2008) Proc Physiol Soc 9, C9
Oral Communications: Role of WT1 in the regulation of expression of pro- and anti-angiogenic isoforms of VEGF
E. Amin1, 2, D. G. Nowak2, 1, S. J. Harper2, D. O. Bates2, M. R. Ladomery1
1. Centre for Research in Biomedicine, University of the West of England, Bristol, United Kingdom. 2. Microvascular Research Laboratories, University of Bristol, Bristol, United Kingdom.
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