Together with the chromatin immunoprecipitation and biotinylated oligonucleotide pulldown data, the co-immunoprecipitation results also suggest the possibility that FoxM1 and FoxO3a cooperate to regulate ER␣ gene transcription.

Menstruation, or cyclic shedding of nonpregnant endometrial tissue with associated bleeding, occurs only in humans and a few other species. This breakdown of the endometrium in response to falling ovarian progesterone levels is a complex process, characterized by local leukocyte infiltration, expression and activation of matrix metalloproteinases, and apoptosis. Spontaneous decidualization (differentiation) of the stromal compartment precedes the cyclic shedding of the endometrium in various menstruating species but the mechanisms that link these processes are not understood. In this study, we identified FOXO1 as a key transcription factor responsible for mediating apoptosis of decidualized human endometrial stromal cells (HESCs) in response to progesterone withdrawal. We demonstrate that medroxyprogesterone acetate (MPA, a synthetic progestin) enhances the expression of FOXO1 in differentiating HESCs while simultaneously inducing cytoplasmic retention and inactivation of FOXO1. Withdrawal of MPA from decidualized HESCs results in rapid nuclear accumulation of FOXO1, increased BIM expression, a proapoptotic FOXO1 target gene, and cell death. Conversely, silencing of FOXO1 expression completely abolishes cell death induced by MPA withdrawal. In summary, the observation that differentiating HESCs become dependent on progesterone signaling for survival through induction and reversible inactivation of FOXO1 suggests a novel mechanism that links decidualization of the endometrium to menstruation.

Conclusions: Tamoxifen has been shown to inhibit ER␣-mediated cyclin D1 transcription, and acquired resistance to tamoxifen is associated with a shift to ER␣-independent cyclin D1 up-regulation. Taken together, our data show that TSA effectively induces cyclin D1 down-regulation through both ER␣-dependent and ER␣-independent mechanisms, providing an important new strategy for combating resistance to antiestrogens.

We used the estrogen-responsive MCF-7 breast cancer cell line as a relevant model to study the anti-proliferative effects of ICI182,780 and identified the negative cell cycle regulator p21Waf1 as a specific target of ICI182,780. Furthermore, silencing of the p21 Waf1 expression by small interfering RNA overcame the G 0 /G 1 cell cycle arrest induced by ICI182,780, suggesting that the induction of p21Waf1 expression has a direct role in mediating the ICI182,780-induced G 0 /G 1 arrest. We further demonstrated that the induction of p21Waf1 by ICI182,780 is mediated at transcriptional and gene promoter levels through the proximal Sp1 sites located near the transcription start site. Co-immunoprecipitation, DNA "pulldown," and chromatin immunoprecipitation experiments together showed that in cycling cells, estrogen receptor ␣ and histone deacetylase 1 (HDAC1) are recruited to the proximal Sp1 sites of the promoter to repress p21 Estradiol (E 2 ) 1 is the principle steroid mitogen for normal breast epithelial cells and has been shown to be important for the development and progression of de novo breast cancers. E 2 exerts its effect on target cells predominantly through binding to and activating the estrogen receptor ␣ (ER␣), which is a member of the steroid/thyroid hormone superfamily of liganddependent transcription factors. The ligand-bound receptor binds to promoter regions of estrogen-regulated genes, where it recruits co-activators or co-repressors as well as the transcriptional machinery necessary to modulate gene expression. The recruitment of ligand-bound ER to the promoter regions of target genes can occur directly through binding to specific DNA sequences in target genes known as estrogen response elements (EREs) or indirectly through interaction with other DNA-binding transcription factors, such as AP1 and Sp1 (1). In addition to estradiol, the transcription activity of ER can also be modulated by a group of ligands called selective estrogen receptor modulators (SERMs), which have agonist and/or antagonist functions depending on the target promoters and tissues. Some of these SERMs, including tamoxifen, raloxifene, and ICI182,780 (also clinically termed faslodex or fulvestrant), have been employed to antagonize estrogen actions in order to treat breast cancer and protect high risk individuals against breast cancer (2). However, SERMs, such as tamoxifen and raloxifene, also behave as agonists in certain tissues. In fact, the modes of action of SERMs are often promoter context-and cell type-specific. For example, whereas tamoxifen is an effective adjuvant treatment for breast cancer, it can also promote endometrial cancer. Recent gene array data showed that antiestrogens, such as tamoxifen, raloxifene, and ICI182,780, function by either partially or completely antagonizing the actions of estrogen on most genes in breast cancer cells (3). Although it was initially believed that anti-estrogens function merely by competing with endogenous estrogens for receptor binding, previous studies using mutated ERs and re...

All cardinal events during the reproductive cycle, including ovulation, implantation, and menstruation, are characterized by a profound tissue remodeling and an associated local inflammatory response. The ovarian hormone progesterone is a key modulator of inflammatory signals in reproductive tissues, but the underlying mechanisms are not well understood. In this study, we report that differentiating human endometrial stromal cells (ESCs) acquire resistance to interferon-gamma (IFNgamma)-dependent signal transducers and activators of transcription (STAT) 1 signaling, although phosphorylation, nuclear translocation, and binding of STAT1 to DNA, are unaffected. These observations prompted an investigation into the role of nuclear repressors of STAT1 signaling. We demonstrate that protein inhibitor of activated STAT-y is complexed to the progesterone receptor (PR) in human ESCs and that its ability to repress STAT1 signaling is dependent upon activation of PR in response to hormone binding. Conversely, IFNgamma and protein inhibitor of activated STAT-y synergistically inhibited PR-dependent transcription, demonstrating that the progesterone and IFNgamma signaling pathways engage in reciprocal transcriptional antagonism in human endometrium.

This chapter describes methods for studying downstream events of the PI3K/Akt signaling cascade, focusing on the FoxO transcription factors. These approaches also represent alternative means for gauging the phosphoinositide-3 kinase/Akt activity. We describe protocols for the fractionation of cytoplasmic and nuclear protein extracts and for studying transcription factor DNA-binding activity in vitro and in vivo.

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