E-cadherin Is a WT1 Target Gene

Seiyu Hosono,Isabelle Gross,Milton A English,Karen M Hajra,Eric R Fearon,Jonathan D Licht

doi:10.1074/jbc.275.15.10943

Abstract

The WT1 tumor suppressor gene encodes a transcription factor that can activate and repress gene expression. Transcriptional targets relevant for the growth suppression functions of WT1 are poorly understood. We found that mesenchymal NIH 3T3 fibroblasts stably expressing WT1 exhibit growth suppression and features of epithelial differentiation including up-regulation of E-cadherin mRNA. Acute expression of WT1 in NIH 3T3 fibroblasts after retroviral infection induced murine E-cadherin expression. In transient transfection experiments, the human and murine E-cadherin promoters were activated by co-expression of WT1. E-cadherin promoter activity was increased in cells overexpressing WT1 and was blocked by a dominant negative form of WT1. WT1 activated the murine E-cadherin promoter through a conserved GC-rich sequence similar to an EGR-1 binding site as well as through a CAAT box sequence. WT1 produced in vitro or derived from nuclear extracts bound to the WT1-response element within the murine E-cadherin promoter, but not the CAAT box. E-cadherin, a gene important in epithelial differentiation and neoplastic transformation, represents a downstream target gene that links the roles of the WT1 in differentiation and growth control.

Highlights

The WT1 tumor suppressor gene encodes a transcription factor that can activate and repress gene expression
We found that mesenchymal NIH 3T3 fibroblasts stably expressing WT1 exhibit growth suppression and features of epithelial differentiation including up-regulation of E-cadherin mRNA
Induction of E-cadherin Expression in NIH 3T3 Cells by Stable Transfection and Retroviral Infection—In previous studies, we created a number of normal (WR16 and WR35) and Ras-transformed (VW9) NIH 3T3 cell lines stably expressing WT1 isoform A (Ϫ17 amino acids, ϪKTS)

Summary

EXPERIMENTAL PROCEDURES

Cell Lines—The NIH 3T3 cells stably expressing WT1 (WR16 and WR35) and the Ras-transformed NIH 3T3 cells stably expressing WT1 (VW9) that were used in this study have been described previously [13, 26]. Binding reactions utilizing nuclear extracts were performed by preincubating 3 ␮g of extract in 20 mM HEPES (pH 7.5), 12% glycerol, 1 mg/ml bovine serum albumin, and 0.5 mM DTT with 2 ␮g of d(I-C) and 1 ␮g of DNase-free RNase A for 15 min at room temperature. The cell pellets were resuspended in 200 ␮l of PBS and solubilized in 200 ␮l of 2ϫ Laemmli buffer (60 mM Tris, pH 6.8, 2% SDS, 10% glycerol) for 1 h at 4 °C, The protein samples were incubated at 100 °C for 10 min and centrifuged in a microcentrifuge at 4 °C for 15 min, and the resulting supernatant was collected. After rinsing in Trisbuffered saline with Tween 20, the immunoblots were developed by chemiluminescence (ECL, Amersham Pharmacia Biotech)

RESULTS

Antibody supershift

DISCUSSION