Studying the subcellular localization and DNA-binding activity of FoxO transcription factors, downstream effectors of PI3K/Akt.

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.

The Forkhead box M1 protein regulates the transcription of the estrogen receptor alpha in breast cancer cells.

In this study, we have identified the Forkhead transcription factor FoxM1 as a physiological regulator of estrogen receptor alpha (ERalpha) expression in breast carcinoma cells. Our survey of a panel of 16 different breast cell lines showed a good correlation (13/16) between FoxM1 expression and expression of ERalpha at both protein and mRNA levels. We have also demonstrated that ectopic expression of FoxM1 in two different estrogen receptor-positive breast cancer cell lines, MCF-7 and ZR-75-30, led to up-regulation of ERalpha expression at protein and transcript levels.

Progestins regulate the expression and activity of the forkhead transcription factor FOXO1 in differentiating human endometrium.

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.

Histone deacetylase inhibitor trichostatin A represses estrogen receptor alpha-dependent transcription and promotes proteasomal degradation of cyclin D1 in human breast carcinoma cell lines.

Purpose: Estrogen receptor alpha (ERalpha)-positive breast cancer cell lines are up to 10 times more sensitive than ERalpha-negative cell lines to the antiproliferative activity of the histone deacetylase inhibitor trichostatin A (TSA). The purpose of the study was to investigate the mechanisms underlying this differential response.

Experimental Design And Results: In the ERalpha-positive MCF-7 cell line, TSA repressed ERalpha and cyclin D1 transcription and induced ubiquitin dependent proteasomal degradation of cyclin D1, leading primarily to G(1)-S-phase cell cycle arrest.

ICI182,780 induces p21Waf1 gene transcription through releasing histone deacetylase 1 and estrogen receptor alpha from Sp1 sites to induce cell cycle arrest in MCF-7 breast cancer cell line.

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 p21Waf1 expression by small interfering RNA overcame the G0/G1 cell cycle arrest induced by ICI182,780, suggesting that the induction of p21Waf1 expression has a direct role in mediating the ICI182,780-induced G0/G1 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.

Steroid receptor action.

The ovarian hormones oestradiol and progesterone exert their actions on target cells predominantly through the binding and activation of the oestrogen receptor (ER) and progesterone receptor (PR), respectively. These receptors are members of the steroid/thyroid hormone superfamily of ligand-dependent transcription factors and bind to the control regions (promoters) of specific genes, where they recruit co-activators or co-repressors and the transcriptional machinery necessary to elicit gene expression. The ability of a nuclear receptor to modulate gene transcription is further dependent on its interaction with other transcription factors, which in turn can be regulated by either distinct or multiple cytoplasmic signalling pathways.

Convergence of interferon-gamma and progesterone signaling pathways in human endometrium: role of PIASy (protein inhibitor of activated signal transducer and activator of transcription-y).

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.