Ectopic expression of mouse Sry interferes with Wnt/β-catenin signaling in mouse embryonal carcinoma cell lines

Abstract

In mammals, Sry is the master regulator of male sex determination, although how it functions is still unclear. By contrast, female sex determination depends on the action of Rspo1 and Wnt4, the regulators of Wnt/β-catenin signaling. To seek a possible interaction between male and female sex determination mechanisms, we examined whether Sry affects Wnt/β-catenin signaling. Using the TOPFLASH reporter system to measure Lef/Tcf-dependent transcriptional activity, we showed that ectopic expression of mouse Sry strongly suppressed Wnt/β-catenin signaling in mouse embryonal carcinoma and human embryonic kidney cell lines. This inhibition occurred downstream of β-catenin but upstream of Lef/Tcf, and depended on both the HMG-box and the C-terminal transcriptional activation domain. By contrast, TOPFLASH was not inhibited by human SRY, which apparently lacks a transcriptional activation domain. However, a fusion construct consisting of human SRY attached to the C-terminal domain of mouse Sry was able to inhibit TOPFLASH effectively. Furthermore, Sry constructs carrying point mutations equivalent to those in human sex reversal mutations were less effective in inhibiting Wnt/β-catenin signaling. Also, we showed that the action of Sry as a transcriptional activator was both necessary and sufficient to inhibit Wnt/β-catenin signaling, suggesting that the transcriptional targets of Sry are responsible for the inhibition of signaling. Sox9 is a potential transcriptional target of Sry, although quantitative RT-PCR analysis indicates that the expression of Sox9 was not up-regulated by the ectopic expression of mouse Sry in mouse embryonal carcinoma cells. While the present study demonstrates an impact of mouse Sry on Wnt/β-catenin signaling at an in vitro level, it requires further investigations to assess whether such action also takes place in vivo to regulate male sex determination.