Michel M. Sanders

Estrogen action: revitalization of the chick oviduct model

Despite decades of investigation, the molecular pathways triggered by estrogen that lead to tissue-specific cell proliferation, differentiation and survival are only superficially understood. If we are to modulate the actions of estrogen selectively in these processes, continued investigation using biologically relevant models is essential. The chick oviduct emerged as an early model for investigating the mechanism of action of steroid hormones because of its exquisite responsiveness to them. Unfortunately, because of experimental limitations, this model has been neglected in the past decade. Reviving this model has become intellectually attractive and technically feasible.

Identification of the Novel Player δEF1 in Estrogen Transcriptional Cascades

ABSTRACT 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 δEF1 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 δEF1. Characterization of this binding site revealed that δEF1 binds to and regulates the chick ovalbumin gene. Thus, a novel regulatory cascade that is triggered by estrogen has been defined.

Expression of the ZEB1 (δEF1) transcription factor in human: additional insights

The zinc finger E-box binding transcription factor ZEB1 (δEF1/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-β, estrogen, and progesterone. TGF-β 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.

Estrogen Opposes the Apoptotic Effects of Bone Morphogenetic Protein 7 on Tissue Remodeling

ABSTRACT 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 (TGFβ) in the uterus during the proliferative phase of the mammalian reproductive cycle. Bone morphogenetic protein 7 (BMP-7), a member of the TGFβ 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.

The ZEB1 transcription factor is a novel repressor of adiposity in female mice.

Background Four genome-wide association studies mapped an “obesity” gene to human chromosome 10p11–12. As the zinc finger E-box binding homeobox 1 (ZEB1) transcription factor is encoded by the TCF8 gene located in that region, and as it influences the differentiation of various mesodermal lineages, we hypothesized that ZEB1 might also modulate adiposity. The goal of these studies was to test that hypothesis in mice. Methodology/Principal Findings To ascertain whether fat accumulation affects ZEB1 expression, female C57BL/6 mice were fed a regular chow diet (RCD) ad libitum or a 25% calorie-restricted diet from 2.5 to 18.3 months of age. ZEB1 mRNA levels in parametrial fat were six to ten times higher in the obese mice. To determine directly whether ZEB1 affects adiposity, wild type (WT) mice and mice heterozygous for TCF8 (TCF8+/−) were fed an RCD or a high-fat diet (HFD) (60% calories from fat). By two months of age on an HFD and three months on an RCD, TCF8+/− mice were heavier than WT controls, which was attributed by Echo MRI to increased fat mass (at three months on an HFD: 0.517±0.081 total fat/lean mass versus 0.313±0.036; at three months on an RCD: 0.175±0.013 versus 0.124±0.012). No differences were observed in food uptake or physical activity, suggesting that the genotypes differ in some aspect of their metabolic activity. ZEB1 expression also increases during adipogenesis in cell culture. Conclusion/Significance These results show for the first time that the ZEB1 transcription factor regulates the accumulation of adipose tissue. Furthermore, they corroborate the genome-wide association studies that mapped an “obesity” gene at chromosome 10p11–12.

Regulation of the chicken ovalbumin gene by estrogen and corticosterone requires a novel DNA element that binds a labile protein, Chirp-1.

Because induction of the chicken ovalbumin (Ov) gene by steroid hormones requires concomitant protein synthesis, efforts have focused on defining the binding site in the Ov gene for a labile transcription factor. Previous gel mobility shift studies identified one such site in the steroid-dependent regulatory element (SDRE) between -900 and -853. To ascertain whether estrogen and glucocorticoid affect the binding of this labile protein, genomic footprinting of the Ov gene was done by treating primary oviduct cell cultures with dimethyl sulfate. Several alterations that include steroid-dependent protection of guanine residues -889 and -885 and hypersensitivity of adenine residues -892 and -865 were observed. Of particular importance, the in vivo footprinting data are corroborated by two functional studies, one with linker-scanning mutations and the other with point mutations. Ten-base-pair linker-scanning mutations between -900 and -878 severely reduced the induction by estrogen and glucocorticoid. Likewise, point mutations of the protected guanine residues profoundly attenuated the response to these steroid hormones. In addition, in vitro binding activity correlated with in vivo functional activity. For example, mutant A4e shows no transcriptional activity in response to steroid hormones, and a corresponding oligomer does not bind protein in vitro. In contrast, mutant A4c is fully active in both contexts. These data support the contention that the ovalbumin gene is regulated by a steroid hormone-induced transcriptional cascade that culminates in the binding of chicken ovalbumin induced regulatory protein or protein complex (Chirp-I) to a DNA element from -891 to -878 in the SDRE.

Comparison of the responsiveness of the pGL3 and pGL4 luciferase reporter vectors to steroid hormones

The ovalbumin gene (Ov) gene is responsive to estrogen, glucocorticoid, androgen, and progesterone. In our efforts to characterize the regulation of the Ov gene by steroid hormones, we have repeatedly observed that many vector backbones and promoters are responsive to steroids. In order to determine which vectors are most suitable for these types of analyses, vectors from Promegas pGL3 and newly engineered pGL4 Dual-Luciferase Reporter Assay System were tested with both estrogen and/or corticosterone. The results confirmed that both series are induced by glucocorticoids in transient transfections of primary oviduct tubular gland cells, which contain glucocorticoid receptors, but not in MCF-7 cells, which do not. Modest effects that were dependent upon backbone and promoter context were observed with both series of vectors with estrogen. Thus, use of these vectors for experiments analyzing the effects of steroid hormones, especially glucocorticoids, should be done with caution. However the new pGL4 series does have some advantages over the older series, and a comparison of transcription factor binding sites is reported.

Multiple promoter elements including a novel repressor site modulate expression of the chick ovalbumin gene.

As is the case with many eukaryotic genes, regulation of the chick ovalbumin (Ov) gene involves both positive and negative modulation. Recent studies indicate that positive regulation by steroids entails binding of several proteins to a hormone-response unit called the steroid-dependent regulatory element (SDRE; -892 to -780). In addition, gene activity is suppressed by factor(s) acting through the negative regulatory element (NRE; -308 to -88). Previous data suggested that the NRE is composed of multiple, independently acting negative elements. The goal of the present studies was to define more precisely the locations of these negative elements and to investigate their functional interactions. Transfection analyses of linker scanning mutants revealed a strong repressor site, designated the COUP-adjacent repressor (CAR) site, located between -119 and -111. Gel mobility shift analyses with the CAR element suggested that it may play a role in the developmental regulation of the Ov gene. A weaker repressor element was also identified at about -275. Surprisingly, two positive sites were found, one of which is the binding site for the estrogen-responsive transcription factor delta-EF1. These results demonstrate that the Ov NRE contains not only sites responsible for the repression of the gene but also a positive element that is required for responsiveness to steroid hormones.

Estrogen Modulates HNF-3 beta mRNA Levels in the Developing Chick Oviduct

Steroid hormones are involved in many physiological processes, including tissue-specific gene expression, homeostasis, and development. The chick oviduct represents an excellent system in which to study many of these events, as it is highly steroid responsive. Here, we report the cloning of chick HNF-3beta from an oviduct cDNA library and its expression pattern in adult tissues and in the developing oviduct in response to estrogen treatment. Overall, cHNF-3beta was expressed at high levels in the immature chick oviduct and lung and, to a lesser extent, in the liver, kidney, and muscle. This expression pattern is divergent from that of mammalian HNF-3beta, which is not expressed in kidney or muscle. Furthermore, several lengths of cHNF-3beta mRNA transcripts were detected that were expressed tissue specifically. Interestingly, cHNF-3beta mRNA levels were differentially influenced by estrogen as a result of a post-transcriptional effect on the cHNF-3beta message in some tissues. Finally, a role for cHNF-3beta is proposed in the estrogen-stimulated differentiation and development of the oviduct, as cHNF-3beta mRNA expression is induced in the early stages of oviduct development and declines as the animal becomes sexually mature.

Androgen Receptor Regulates Transcription of the ZEB1 Transcription Factor

The zinc finger E-box binding protein 1 (ZEB1) transcription factor belongs to a two-member family of zinc-finger homeodomain proteins involved in physiological and pathological events mostly relating to cell migration and epithelial to mesenchymal transitions (EMTs). ZEB1 (also known as δEF1, zfhx1a, TCF8, and Zfhep) plays a key role in regulating such diverse processes as T-cell development, skeletal patterning, reproduction, and cancer cell metastasis. However, the factors that regulate its expression and consequently the signaling pathways in which ZEB1 participates are poorly defined. Because it is induced by estrogen and progesterone and is high in prostate cancer, we investigated whether tcf8, which encodes ZEB1, is regulated by androgen. Data herein demonstrate that tcf8 is induced by dihydrotestosterone (DHT) in the human PC-3/AR prostate cancer cell line and that this induction is mediated by two androgen response elements (AREs). These results demonstrate that ZEB1 is an intermediary in androgen signaling pathways.