April K. Binder

Welcoming β-Catenin to the Gonadotropin-Releasing Hormone Transcriptional Network in Gonadotropes

GnRH binds its G-coupled protein receptor, GnRHR, on pituitary gonadotropes and stimulates transcription of Cga, Lhb, and Fshb. These three genes encode two heterodimeric glycoprotein hormones, LH and FSH, that act as gonadotropins by regulating gametogenesis and steroidogenesis in both the testes and ovary. GnRH also regulates transcription of Gnrhr. Thus, regulated expression of Cga, Lhb, Fshb, and Gnrhr provides a genomic signature unique to functional gonadotropes. Steadily increasing evidence now indicates that GnRH regulates transcription of its four signature genes indirectly through a hierarchical transcriptional network that includes distinct subclasses of DNA-binding proteins that comprise the immediate early gene (IEG) family. These IEGs, in turn, confer hormonal responsiveness to the four signature genes. Although the IEGs confer responsiveness to GnRH, they cannot act alone. Instead, additional DNA-binding proteins, including the orphan nuclear receptor steroidogenic factor 1, act permissively to allow the four signature genes to respond to GnRH-induced changes in IEG levels. Emerging new findings now indicate that beta-catenin, a transcriptional coactivator and member of the canonical WNT signaling pathway, also plays an essential role in transducing the GnRH signal by interacting with multiple DNA-binding proteins in gonadotropes. Herein we propose that these interactions with beta-catenin define a multicomponent transcriptional network required for regulated expression of the four signature genes of the gonadotrope, Cga, Lhb, Fshb, and Gnrhr.

The absence of ER-β results in altered gene expression in ovarian granulosa cells isolated from in vivo preovulatory follicles.

Determining the spatial and temporal expression of genes involved in the ovulatory pathway is critical for the understanding of the role of each estrogen receptor in the modulation of folliculogenesis and ovulation. Estrogen receptor (ER)-β is highly expressed in ovarian granulosa cells, and mice lacking ER-β are subfertile due to inefficient ovulation. Previous work has focused on isolated granulosa cells or cultured follicles and, although informative, provides confounding results due to the heterogeneous cell types present including granulosa and theca cells and oocytes and exposure to in vitro conditions. Herein we isolated preovulatory granulosa cells from wild-type (WT) and ERβ-null mice using laser capture microdissection to examine the genomic transcriptional response downstream of pregnant mare serum gonadotropin (mimicking FSH) and pregnant mare serum gonadotropin/human chorionic gonadotropin (mimicking LH) stimulation. This allows for a direct comparison of in vivo granulosa cells at the same stage of development from both WT and ERβ-null ovaries. ERβ-null granulosa cells showed altered expression of genes known to be regulated by FSH (Akap12 and Runx2) as well as not previously reported (Arnt2 and Pou5f1) in WT granulosa cells. Our analysis also identified 304 genes not previously associated with ERβ in granulosa cells. LH-responsive genes including Abcb1b and Fam110c show reduced expression in ERβ-null granulosa cells; however, novel genes including Rassf2 and Megf10 were also identified as being downstream of LH signaling in granulosa cells. Collectively, our data suggest that granulosa cells from ERβ-null ovaries may not be appropriately differentiated and are unable to respond properly to gonadotropin stimulation.

GnRH-Regulated Expression of Jun and JUN Target Genes in Gonadotropes Requires a Functional Interaction between TCF/LEF Family Members and β-Catenin

GnRH regulates gonadotrope function through a complex transcriptional network that includes three members of the immediate early gene family: Egr1, Jun, and Atf3. These DNA-binding proteins act alone or in pairs to confer hormonal responsiveness to Cga, Lhb, Fshb, and Gnrhr. Herein we suggest that the transcriptional response of Jun requires a functional interaction between the T-cell factor (TCF)/lymphoid enhancer factor (LEF) family of DNA-binding proteins and beta-catenin (officially CTNNB1), a coactivator of TCF/LEF. Supporting data include demonstration that GnRH increases activity of TOPflash, a TCF/LEF-dependent luciferase reporter, in LbetaT2 cells, a gonadotrope-derived cell line. Additional cotransfection experiments indicate that a dominant-negative form of TCF7L2 (TCFDN) that binds DNA, but not beta-catenin, blocks GnRH induction of TOPflash. Overexpression of AXIN, an inhibitor of beta-catenin, also reduces GnRH stimulation of TOPflash. Transduction of LbetaT2 cells with TCFDN adenoviruses diminishes GnRH stimulation of Jun mRNA without altering expression of Egr1 and Atf3, two other immediate early genes that confer GnRH responsiveness. Reduction of beta-catenin in LbetaT2 cells, through stable expression of short hairpin RNA, also selectively compromises GnRH regulation of Jun expression and levels of JUN protein. Finally, overexpression of TCFDN attenuates GnRH regulation of Cga promoter activity, a known downstream target of JUN. Together, these results indicate that GnRH regulation of Jun transcription requires a functional interaction between TCF/LEF and beta-catenin and that alteration of either impacts expression of JUN downstream targets such as Cga.

Kinetic folding of Haloferax volcanii and Escherichia coli dihydrofolate reductases: haloadaptation by unfolded state destabilization at high ionic strength.

Salts affect protein stability by multiple mechanisms (e.g., the Hofmeister effect, preferential hydration, electrostatic effects and weak ion binding). These mechanisms can affect the stability of both the native state and the unfolded state. Previous equilibrium stability studies demonstrated that KCl stabilizes dihydrofolate reductases (DHFRs) from Escherichia coli (ecDHFR, E. coli DHFR) and Haloferax volcanii (hvDHFR1, H. volcanii DHFR encoded by the hdrA gene) with similar efficacies, despite adaptation to disparate physiological ionic strengths (0.2 M versus 2 M). Kinetic studies can provide insights on whether equilibrium effects reflect native state stabilization or unfolded state destabilization. Similar kinetic mechanisms describe the folding of urea-denatured ecDHFR and hvDHFR1: a 5-ms stopped-flow burst-phase species that folds to the native state through two sequential intermediates with relaxation times of 0.1-3 s and 25-100 s. The latter kinetic step is very similar to that observed for the refolding of hvDHFR1 from low ionic strength. The unfolding of hvDHFR1 at low ionic strength is relatively slow, suggesting kinetic stabilization as observed for some thermophilic enzymes. Increased KCl concentrations slow the urea-induced unfolding of ecDHFR and hvDHFR1, but much less than expected from equilibrium studies. Unfolding rates extrapolated to 0 M denaturant, k(unf)(H(2)O), are relatively independent of ionic strength, demonstrating that the KCl-induced stabilization of ecDHFR and hvDHFR1 results predominantly from destabilization of the unfolded state. This supports the hypothesis from previous equilibrium studies that haloadaptation harnesses the effects of elevated salt concentrations on the properties of the aqueous solvent to enhance protein stability.

Estrogen Responsiveness of the TFIID Subunit TAF4B in the Normal Mouse Ovary and in Ovarian Tumors

ABSTRACT Estrogen signaling in the ovary is a fundamental component of normal ovarian function, and evidence also indicates that excessive estrogen is a risk factor for ovarian cancer. We have previously demonstrated that the gonadally enriched TFIID subunit TAF4B, a paralog of the general transcription factor TAF4A, is required for fertility in mice and for the proliferation of ovarian granulosa cells following hormonal stimulation. However, the relationship between TAF4B and estrogen signaling in the normal ovary or during ovarian tumor initiation and progression has yet to be defined. Herein, we show that Taf4b mRNA and TAF4B protein, but not Taf4a mRNA or TAF4A protein, are increased in whole ovaries and granulosa cells of the ovary after exposure to 17beta-estradiol or the synthetic estrogen diethylstilbestrol and that this response occurs within hours after stimulation. Furthermore, this increase occurs via nuclear estrogen receptors both in vivo and in a mouse granulosa cancer cell line, NT-1. We observe a significant increase in Taf4b mRNA in estrogen-supplemented mouse ovarian tumors, which correlates with diminished survival of these mice. These data highlight the novel response of the general transcription factor TAF4B to estrogen in the normal ovary and during ovarian tumor progression in the mouse, suggesting its potential role in regulating actions downstream of estrogen stimulation.

Steroid Receptors in the Uterus and Ovary

The coordinated functions of the ovary and reproductive tract are critical to mammalian fertility. Receptors for ovarian steroids within the uterus and ovary are central mediators of ovulation, implantation, and gestation, thus understanding of cellular localization and mechanisms involved in their functions in these tissues is essential knowledge to appreciate and further study pregnancy. Here, we have reviewed details regarding the basic structures and mechanisms of the steroid receptors. We have described steroid receptors found within the ovary and uterus, and summarized numerous studies utilizing both natural and elegantly engineered genetic models and steroid ligands that reveal much regarding the mechanisms and roles of estrogen, progesterone, androgen, and glucocorticoid receptors in these tissues.

Expression of Human NSAID Activated Gene 1 in Mice Leads to Altered Mammary Gland Differentiation and Impaired Lactation

Transgenic mice expressing human non-steroidal anti-inflammatory drug activated gene 1 (NAG-1) have less adipose tissue, improved insulin sensitivity, lower insulin levels and are resistant to dietary induced obesity. The hNAG-1 expressing mice are more metabolically active with a higher energy expenditure. This study investigates female reproduction in the hNAG-1 transgenic mice and finds the female mice are fertile but have reduced pup survival after birth. Examination of the mammary glands in these mice suggests that hNAG-1 expressing mice have altered mammary epithelial development during pregnancy, including reduced occupancy of the fat pad and increased apoptosis via TUNEL positive cells on lactation day 2. Pups nursing from hNAG-1 expressing dams have reduced milk spots compared to pups nursing from WT dams. When CD-1 pups were cross-fostered with hNAG-1 or WT dams; reduced milk volume was observed in pups nursing from hNAG-1 dams compared to pups nursing from WT dams in a lactation challenge study. Milk was isolated from WT and hNAG-1 dams, and the milk was found to have secreted NAG-1 protein (approximately 25 ng/mL) from hNAG-1 dams. The WT dams had no detectable hNAG-1 in the milk. A decrease in non-esterified free fatty acids in the milk of hNAG-1 dams was observed. Altered milk composition suggests that the pups were receiving inadequate nutrients during perinatal development. To examine this hypothesis serum was isolated from pups and clinical chemistry points were measured. Male and female pups nursing from hNAG-1 dams had reduced serum triglyceride concentrations. Microarray analysis revealed that genes involved in lipid metabolism are differentially expressed in hNAG-1 mammary glands. Furthermore, the expression of Cidea/CIDEA that has been shown to regulate milk lipid secretion in the mammary gland was reduced in hNAG-1 mammary glands. This study suggests that expression of hNAG-1 in mice leads to impaired lactation and reduces pup survival due to altered milk quality and quantity.

Abstract A81: Estrogen-responsiveness of the TFIID subunit TAF4B and its potential function in ovarian cancer, epigenetic regulation and meiotic DNA repair

Female infertility affects approximately 10.9% of women ages 15-44 in the US, and the molecular mechanisms leading to this disorder are multifaceted and varied. We have previously demonstrated that the gonadal-enriched TFIID subunit TAF4B, a paralog of the general transcription factor TAF4A, is required for fertility in mice. Female mice deficient for TAF4B exhibit a phenotype resembling premature ovarian failure, including early oocyte loss, follicular atresia and severely reduced granulosa cell proliferation when treated with 17β-estradiol. The inability of estrogen to stimulate granulosa cell proliferation led us to hypothesize that TAF4B is involved in an estrogen signaling pathway within the ovary. A large percentage of Taf4b -knockout oocytes die by apoptosis immediately after birth, and this germ cell loss is attenuated by estrogen supplementation, further suggesting a connection between TAF4B and estrogen. Taf4b -knockout ovaries also display deregulated epigenetic marks, which can affect DNA repair during meiotic homologous recombination. Furthermore, estrogen is known to regulate epigenetic changes in the ovary, leading us to hypothesize that the estrogen rescue may occur via modulation of the epigenetic state and consequent repair of double-strand breaks during meiosis. Here, we show that Taf4b mRNA and TAF4B protein expression are upregulated by estrogens in whole ovaries and purified granulosa cells of the ovary and that this increase occurs via nuclear estrogen receptors. We observe significant increases of Taf4b mRNA in estrogen-exposed mouse ovarian tumors, and the mice exposed to estradiol had significantly diminished survival compared to those receiving a placebo pellet. Combined with the fact that epigenetic deregulation and DNA repair processes play a key role in tumorigenesis, these results suggest that in addition to fertility defects, TAF4B could also affect ovarian tumorigenesis later in life. Our preliminary data suggest that the loss of oocytes in Taf4b -knockout ovaries may occur due to deficiencies in epigenetic regulation and/or a deficiency in DNA repair during meiotic prophase, since DNA repair and meiosis related genes are significantly reduced in Taf4b -knockout ovaries. Future studies will determine if estrogen treatment of neonatal Taf4b -knockout ovaries ameliorates these epigenetic and DNA repair deficits, leading to the observed oocyte rescue, and will explore if ovarian tumorigenesis is altered in the absence of TAF4B. Citation Format: Jennifer R. Wardell, Kathryn J. Grive, Kendra M. Hodgkinson, April K. Binder, Kimberly A. Seymour, Lindsay A. Lovasco, Ken S. Korach, Barbara C. Vanderhyden and Richard N. Freiman. Estrogen-responsiveness of the TFIID subunit TAF4B and its potential function in ovarian cancer, epigenetic regulation and meiotic DNA repair. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr A81.