No abstract

Steroid hormones regulate the transcriptional activity of the chicken ovalbumin gene both in vivo and in cell culture. To identify the regulatory elements involved, primary oviduct cell cultures were transfected with constructs containing the promoter and 5'-flanking region of the ovalbumin gene fused to the bacterial chloramphenicol acetyltransferase (CAT) gene. Induction of the OvCAT genes by estrogen, progesterone, or corticosterone mimics that of the endogenous ovalbumin gene, indicating that the transfected DNA is accurately regulated. Deletion analysis revealed that a steroid response element (SRE) resides between nucleotide coordinates -880 and -585 and that a negative regulatory element (NRE) resides between -350 and -248 in the ovalbumin gene. Thus, an NRE represses expression of the ovalbumin gene unless steroid hormones relieve this negative control through interactions involving a more distal SRE. Neither the SRE nor the NRE alone regulates the heterologous thymidine kinase promoter, suggesting either that they function as a single entity or that they are conditional regulatory elements. The NRE is functional in MCF-7 cells, but the SRE cannot be activated by steroids in this heterologous estrogen-responsive cell line. These data indicate that the steroid-receptor complex induces the ovalbumin gene through direct or indirect actions at an SRE to relieve represssion at an NRE.

Although many genes are regulated by estrogen, very few have been shown to directly bind the estrogen receptor complex. Therefore, transcriptional cascades probably occur in which the estrogen receptor directly binds to a target gene that encodes another transcription factor that subsequently regulates additional genes. Through the use of a differential display assay, a transcription factor has been identified that may be involved in estrogen transcriptional cascades. This report demonstrates that transcription factor ␦EF1 is induced eightfold by estrogen in the chick oviduct. Furthermore, the regulation by estrogen occurs at the transcriptional level and is likely to be a direct effect of the estrogen receptor complex, as it does not require concomitant protein synthesis. A putative binding site was identified in the 5-flanking region of the chick ovalbumin gene identifying it as a possible target gene for regulation by ␦EF1. Characterization of this binding site revealed that ␦EF1 binds to and regulates the chick ovalbumin gene. Thus, a novel regulatory cascade that is triggered by estrogen has been defined.

The zinc finger E-box binding transcription factor ZEB1 (deltaEF1/Nil-2-a/AREB6/zfhx1a/TCF8/zfhep/BZP) is emerging as an important regulator of the epithelial to mesenchymal transitions (EMT) required for development and cancer metastasis. ZEB1 promotes EMT by repressing genes contributing to the epithelial phenotype while activating those associated with the mesenchymal phenotype. TCF8 (zfhx1a), the gene encoding ZEB1, is induced by several potentially oncogenic ligands including TGF-beta, estrogen, and progesterone. TGF-beta appears to activate EMT, at least in part, by inducing ZEB1. However, our understanding of how ZEB1 contributes to signaling pathways elicited by estrogen and progesterone is quite limited, as is our understanding of its functional roles in normal adult tissues. To begin to address these questions, a human tissue mRNA array analysis was done. In adults, the highest ZEB1 mRNA expression is in bladder and uterus, whereas in the fetus highest expression is in lung, thymus, and heart. To further investigate the regulation of TCF8 by estrogen, ZEB1 mRNA was measured in ten estrogen-responsive cell lines, but it is only induced in the OV266 ovarian carcinoma line. Although high expression of ZEB1 mRNA is estrogen-dependent in normal human ovarian and endometrial biopsies, high expression is estrogen-independent in late stage ovarian and endometrial carcinomas, raising the possibility that deregulated expression promotes cancer progression. In contrast, TCF8 is at least partially deleted in 4 of 5 well-differentiated, grade I endometrial carcinomas, which may contribute to their non-aggressive phenotype. These data support the contention that high ZEB1 encourages gynecologic carcinoma progression.

Interactions between estrogen and growth factor signaling pathways at the level of gene expression play important roles in the function of reproductive tissues. For example, estrogen regulates transforming growth factor beta (TGF␤) in the uterus during the proliferative phase of the mammalian reproductive cycle. Bone morphogenetic protein 7 (BMP-7), a member of the TGF␤ superfamily, is also involved in the development and function of reproductive tissues. However, relatively few studies have addressed the expression of BMP-7 in reproductive tissues, and the role of BMP-7 remains unclear. As part of an ongoing effort to understand how estrogen represses gene expression and to study its interactions with other signaling pathways, chick BMP-7 (cBMP-7) was cloned. cBMP-7 mRNA levels are repressed threefold within 8 h following estrogen treatment in the chick oviduct, an extremely estrogen-responsive reproductive tissue. This regulation occurs at the transcriptional level. Estrogen has a protective role in many tissues, and withdrawal from estrogen often leads to tissue regression; however, the mechanisms mediating regression of the oviduct remain unknown. Terminal transferase-mediated end-labeling and DNA laddering assays demonstrated that regression of the oviduct during estrogen withdrawal involves apoptosis, which is a novel observation. cBMP-7 mRNA levels during estrogen withdrawal increase concurrently with the apoptotic index of the oviduct. Furthermore, addition of purified BMP-7 induces apoptosis in primary oviduct cells. This report demonstrates that the function of BMP-7 in the oviduct involves the induction of apoptosis and that estrogen plays an important role in opposing this function.