Wilms’ tumor 1 inhibits aromatase P450 expression in human cells and is downregulated in endometriosis vs. endometrium-derived stromal cells

Abstract

Objective: Aberrant expression of aromatase P450 (P450arom) gives rise to increased estrogen in ectopic endometriotic tissue, but P450arom is absent in eutopic endometrium. In endometriosis, we previously showed that the stimulatory transcription factor, steroidogenic factor-1 (SF-1), was in part responsible for the activation of P450arom expression. Wilms’ Tumor 1 (WT1) is a tumor suppressor and functions as a transcription factor in a number of human tissues. Our current objective was to determine the cellular distribution and levels of WT1 in endometriotic vs. endometrial tissues and to define the role of WT1 in the regulation of P450arom expression in these tissues.Design: We used three model systems: 1)paired biopsies of eutopic endometrium and endometriosis performed simultaneously; 2)eutopic endometrial and endometriotic stromal cells maintained in primary culture; and 3)JEG-3,human placental choriocarcinoma cell line.Materials/Methods: We performed immunohistochemical analysis of simultaneously obtained paired samples (n = 6 patients) of eutopic endometrium and endometriosis using an antibody against WT1. We co-transfected serial deletion and site-directed mutants of the P450arom promoter II fused to the Luciferase reporter gene together with SF-1 and WT1 expression vectors into primary cultured eutopic endometrial and endometriotic stromal cells and JEG-3 cell lines. Confocal microscope was used for immunocolocalization of transcription factors in the nucleus.Results: Immunohistochemistry and H-scoring showed that WT1 was preferentially expressed in the nuclei of stromal and in significantly lower levels in epithelial cells. Moreover, WT1 levels in endometriotic stromal cells were significantly reduced compared to eutopic endometrial stromal cells. Thus, we hypothesized that in vivo downregulation of WT1 in endometriosis vs. eutopic endometrium may be responsible for aberrant P450arom expression in endometriosis. We determined in vitro whether WT1 modulated SF-1-dependent P450arom gene transcription. We showed that WT1 inhibited SF-1-induced activity of the human P450arom promoter II in a dose-dependent fashion in human endometriotic and endometrial stromal cells in primary culture. These data could also be reproduced in a reconstitutive fashion in JEG-3 choricarcinoma cells. Serial deletion constructs showed that WT1 could suppress SF-1-dependent activity of P450arom promoter region that contains two Nuclear Receptor Half Sites at -136/-124 bp and -263/-251 bp. Disruption of one these DNA motifs alone or in combination by site-directed mutagenesis suggested that the inhibition of SF-1-dependent activity of P450arom promoter is mediated by these two regulatory elements in endometriotic and endometrial stromal or JEG-3 cells. Moreover, coimmunolocalization of SF-1 and WT1 in the nuclei of JEG-3 cells suggested that WT1 exerts its inhibitory effect on P450arom expression by directly binding to SF-1.Conclusions: WT1 inhibits SF-1-dependent P450arom expression in primary human endometriotic and endometrial stromal cells. In vivo downregulation of WT1 in endometriotic stromal cells (vs. eutopic endometrial stromal cells) may in part be responsible for aberrantly increased P450arom expression and estrogen formation in this pathologic tissue.Supported by: NIH grant HD38691. Objective: Aberrant expression of aromatase P450 (P450arom) gives rise to increased estrogen in ectopic endometriotic tissue, but P450arom is absent in eutopic endometrium. In endometriosis, we previously showed that the stimulatory transcription factor, steroidogenic factor-1 (SF-1), was in part responsible for the activation of P450arom expression. Wilms’ Tumor 1 (WT1) is a tumor suppressor and functions as a transcription factor in a number of human tissues. Our current objective was to determine the cellular distribution and levels of WT1 in endometriotic vs. endometrial tissues and to define the role of WT1 in the regulation of P450arom expression in these tissues. Design: We used three model systems: 1)paired biopsies of eutopic endometrium and endometriosis performed simultaneously; 2)eutopic endometrial and endometriotic stromal cells maintained in primary culture; and 3)JEG-3,human placental choriocarcinoma cell line. Materials/Methods: We performed immunohistochemical analysis of simultaneously obtained paired samples (n = 6 patients) of eutopic endometrium and endometriosis using an antibody against WT1. We co-transfected serial deletion and site-directed mutants of the P450arom promoter II fused to the Luciferase reporter gene together with SF-1 and WT1 expression vectors into primary cultured eutopic endometrial and endometriotic stromal cells and JEG-3 cell lines. Confocal microscope was used for immunocolocalization of transcription factors in the nucleus. Results: Immunohistochemistry and H-scoring showed that WT1 was preferentially expressed in the nuclei of stromal and in significantly lower levels in epithelial cells. Moreover, WT1 levels in endometriotic stromal cells were significantly reduced compared to eutopic endometrial stromal cells. Thus, we hypothesized that in vivo downregulation of WT1 in endometriosis vs. eutopic endometrium may be responsible for aberrant P450arom expression in endometriosis. We determined in vitro whether WT1 modulated SF-1-dependent P450arom gene transcription. We showed that WT1 inhibited SF-1-induced activity of the human P450arom promoter II in a dose-dependent fashion in human endometriotic and endometrial stromal cells in primary culture. These data could also be reproduced in a reconstitutive fashion in JEG-3 choricarcinoma cells. Serial deletion constructs showed that WT1 could suppress SF-1-dependent activity of P450arom promoter region that contains two Nuclear Receptor Half Sites at -136/-124 bp and -263/-251 bp. Disruption of one these DNA motifs alone or in combination by site-directed mutagenesis suggested that the inhibition of SF-1-dependent activity of P450arom promoter is mediated by these two regulatory elements in endometriotic and endometrial stromal or JEG-3 cells. Moreover, coimmunolocalization of SF-1 and WT1 in the nuclei of JEG-3 cells suggested that WT1 exerts its inhibitory effect on P450arom expression by directly binding to SF-1. Conclusions: WT1 inhibits SF-1-dependent P450arom expression in primary human endometriotic and endometrial stromal cells. In vivo downregulation of WT1 in endometriotic stromal cells (vs. eutopic endometrial stromal cells) may in part be responsible for aberrantly increased P450arom expression and estrogen formation in this pathologic tissue. Supported by: NIH grant HD38691.