Requirement of Ras-Dependent Pathways for Activation of the Transforming Growth Factor β3 Promoter by Estradiol**This work was supported by the Canadian Institutes of Health Research and the National Cancer Institute of Canada through the Canadian Breast Cancer Research Initiative program.

It has been previously observed that the transforming growth factor beta3 (TGFbeta3) gene can be activated by both estradiol (E(2)) and selective estrogen receptor modulators (SERMs) in vivo but that only SERMs have a potent stimulatory effect on the TGFbeta3 promoter in cultured cells. We demonstrate in this report that E(2) can act also as a potent inducer of the TGFbeta3 promoter via a novel and specific estrogen receptor (ER)alpha-mediated mechanism. Our results show that treatment with epidermal growth factor or transfection of a constitutively active Ras mutant allows E(2) to induce the TGFbeta3 promoter via ERalpha in cotransfected HeLa and osteosarcoma MG63 cells. Both protein kinase C (PKC) and mitogen-activated protein kinase (MAPK) inhibitors can block the combined stimulatory effect of E(2) and epidermal growth factor/Ras. However, E(2) induction of the TGFbeta3 promoter was found to be unaffected by mutation of ERalpha serine 118, a well-characterized target of MAPK. Progressive deletion analysis of the ERalpha amino-terminal region delineated three separate domains modulating the E(2)/activated Ras response, revealing a complex functional organization of the ERalpha A/B domain required for regulation of the TGFbeta3 promoter. In addition, PKC and MAPK inhibitors had no effect on the induction of TGFbeta3 promoter activity by the SERM EM-652. These results indicate that induction of the TGFbeta3 promoter by the E(2)/ERalpha complex requires the concomitant activation of PKC and MAPK signaling and provide a novel framework for the design of more effective estrogen-based therapeutic strategies.