FIG. 6.

Correlation between fibronectin promoter activity and presence of LEF-1-related proteins in kidney epithelial cells (A6). (A) LEF-1 immunoblot and RT-PCR of fibroblasts, cadherin-transfected fibroblasts, and epithelial cells. In transfected and untransfected fibroblasts a protein band of approximately 55 kDa which corresponds to the size of murine LEF-1 was detectable, while lysates of epithelial cells gave no signal. The protein of 90 kDa might represent a LEF-1-related HMG box-containing protein. The RT-PCR showed XTcf-3 and XTcf-4 expression in both fibroblasts and epithelial cells, whereas XLEF-1 was detected only in the fibroblasts. Both cell lines also express XGrg4 and XGrg5. As an internal standard, histone H4 was used. (B) Subcellular localization of LEF-TCF-related proteins in immunostaining with LEF-1 antiserum. In the fibroblasts the signal was found concentrated in the nuclei, while epithelial cells were negative in staining. (C) The fibronectin promoter was inactive in epithelial cells compared to fibroblasts. The activity of the promoter is shown in relation to that of the CMV promoter. The reporter gene constructs used are indicated. n, number of independent transfections. (D) The fibronectin promoter containing the Wnt/Wg response element was activated by LEF-1 alone or by the combination of LEF-1 and β-catenin, while β-catenin alone was not able to enhance promoter activity. Wild-type XTcf-3 does not influence FN promoter activity, either alone or in combination with β-catenin, while the mutant XTcf-3Δgrg upregulates the promoter activity. n, number of independent transfections; asterisks, significant differences by the Student t test (P < 0.005). Activity of the corresponding promoter construct was set at 100%.