Studies indicate that estrogen receptor (ER)␣Cip1 and p27 Kip1 , which leads to a G 2 cell cycle arrest. These results demonstrate that ER␣ and ER␤ produce opposite effects in MCF-7 cells on cell proliferation and tumor formation. Natural or synthetic ER␤-selective estrogens may lack breast cancer promoting properties exhibited by estrogens in hormone replacement regimens and may be useful for chemoprevention of breast cancer.

The decline in estrogen levels during menopause is associated with increased cytokine production and inflammatory diseases. Estrogens exert anti-inflammatory effects by repressing cytokine genes, such as tumor necrosis factor-alpha (TNFalpha). The mechanisms involved in transcriptional repression by estrogens are virtually unknown. Here, we used chromatin immunoprecipitation to investigate how estrogens repress the autoinduction of the TNFalpha gene. TNFalpha assembled a transcriptional activation complex at the TNFalpha promoter that includes c-jun, p50-NFkappaB, p65-NFkappaB, CBP, Hsp90, and unliganded estrogen receptor (ER). Estradiol repressed TNFalpha gene expression by reversing the ligand-independent activation by ERalpha and the stimulatory actions of c-jun, NFkappaB, and CBP on transcription. Silencing of GRIP1 reversed the repression of TNFalpha and other cytokine genes by estradiol, demonstrating that GRIP1 is required for transcriptional repression and can act as a corepressor. Our study demonstrates that ERalpha is a TNFalpha-induced coactivator that becomes a repressor in the presence of estradiol by recruiting GRIP1.

Novel estrogenic therapies are needed that ameliorate menopausal symptoms and have the bone-sparing effects of endogenous estrogens but do not promote breast or uterine cancer. Recent evidence suggests that selective activation of the estrogen receptor (ER)-beta subtype inhibits breast cancer cell proliferation. To establish whether ERbeta-selective ligands represent a viable approach to improve hormone therapy, we investigated whether the estrogenic activities present in an herbal extract, MF101, used to treat hot flashes, are ERbeta selective. MF101 promoted ERbeta, but not ERalpha, activation of an estrogen response element upstream of the luciferase reporter gene. MF101 also selectively regulates transcription of endogenous genes through ERbeta. The ERbeta selectivity was not due to differential binding because MF101 binds equally to ERalpha and ERbeta. Fluorescence resonance energy transfer and protease digestion studies showed that MF101 produces a different conformation in ERalpha from ERbeta when compared with the conformations produced by estradiol. The specific conformational change induced by MF101 allows ERbeta to bind to an estrogen response element and recruit coregulatory proteins that are required for gene activation. MF101 did not activate the ERalpha-regulated proliferative genes, c-myc and cyclin D1, or stimulate MCF-7 breast cancer cell proliferation or tumor formation in a mouse xenograft model. Our results demonstrate that herbal ERbeta-selective estrogens may be a safer alternative for hormone therapy than estrogens that nonselectively activate both ER subtypes.

Increasing evidence suggests that fibroblast growth factors (FGFs) are neurotrophic in GnRH neurons. However, the extent to which FGFs are involved in establishing a functional GnRH system in the whole organism has not been investigated. In this study, transgenic mice with the expression of a dominant-negative FGF receptor mutant (FGFRm) targeted to GnRH neurons were generated to examine the consequence of disrupted FGF signaling on the formation of the GnRH system. To first test the effectiveness of this strategy, GT1 cells, a GnRH neuronal cell line, were stably transfected with FGFRm. The transfected cells showed attenuated neurite outgrowth, diminished FGF-2 responsiveness in a cell survival assay, and blunted activation of the signaling pathway in response to FGF-2. Transgenic mice expressing FGFRm in a GnRH neuron-specific manner exhibited a 30% reduction in GnRH neuron number, but the anatomical distribution of GnRH neurons was unaltered. Although these mice were initially fertile, they displayed several reproductive defects, including delayed puberty, reduced litter size, and early reproductive senescence. Overall, our results are the first to show, at the level of the organism, that FGFs are one of the important components involved in the formation and maintenance of the GnRH system.

Estrogenic effects are mediated through two estrogen receptor (ER) subtypes, ERα and ERβ. Estrogens are the most commonly prescribed drugs to treat menopausal conditions, but by nonselectively triggering both ERα and ERβ pathways in different tissues they can cause serious adverse effects. The different sizes of the binding pockets and sequences of their activation function domains indicate that ERα and ERβ should have different specificities for ligands and biological responses that can be exploited for designing safer and more selective estrogens. ERα and ERβ regulate different genes by binding to different regulatory elements and recruiting different transcription and chromatin remodeling factors that are expressed in a cell-specific manner. ERα-and ERβ-selective agonists have been identified that demonstrate that the two ERs produce distinct biological effects. ERα and ERβ agonists are promising new approach for treating specific conditions associated with menopause.

The role of estrogen receptor beta (ERβ) in breast cancer is unclear. ERβ is considered to have a protective role in breast cancer development based on findings demonstrating that ERβ expression inhibits ERα-mediated proliferation of breast cancer cells. We previously demonstrated that ERβ causes a ligand independent G2 cell cycle arrest in MCF-7 cells. To study the mechanisms of the ERβ-mediated G2 cell cycle arrest, we investigated its effects on the regulatory pathways responsible for the G2/M phase transition. We found that ERβ inhibits CDK1 activity, which is the critical determinant of the G2/M progression. CDK1 activity is modulated by both stimulatory and inhibitory factors. Cyclin B1 is the major activator of CDK1. ERβ inhibited the cell cycle-dependent stimulation of cyclin B1 mRNA and protein. GADD45A and BTG2 are two major inhibitors of CDK1, which have been implicated in breast tumor formation. Based on these findings, we explored if the expression pattern of GADD45A and BTG2 is affected by ERβ. We found that ERβ stimulates GADD45A and BTG2 mRNA levels. The induction of these two genes is caused by ERβ binding directly to these genes and recruiting c-jun and NCOA2. Our findings demonstrated that unliganded ERβ causes a G2 cell cycle arrest by inactivating CDK1 through the repression of cyclin B1 and stimulation of GADD45A and BTG2 expression. These results provide evidence that drugs that stimulate the production of unliganded ERβ may be effective new therapies to prevent breast cancer.

Hasdemir B, Mhaske P, Paruthiyil S, Garnett EA, Heyman MB, Matloubian M, Bhargava A. Sex-and corticotropin-releasing factor receptor 2-dependent actions of urocortin 1 during inflammation.

We studied the signaling pathways coupling gonadotropin-releasing hormone (GnRH) secretion to elevations in cAMP levels in the GT1 GnRH-secreting neuronal cell line. We hypothesized that increased cAMP could be acting directly by means of cyclic nucleotide-gated (CNG) cation channels or indirectly by means of activation of cAMP-dependent protein kinase (PKA). We showed that GT1 cells express the three CNG subunits present in olfactory neurons (CNG2, -4.3, and -5) and exhibit functional cAMP-gated cation channels. Activation of PKA does not appear to be necessary for the stimulation of GnRH release by increased levels of cAMP. In fact, pharmacological inhibition of PKA activity caused an increase in the basal secretion of GnRH. Consistent with this observation activation PKA inhibited adenylyl cyclase activity, presumably by inhibiting adenylyl cyclase V expressed in the cells. Therefore, the stimulation of GnRH release by elevations in cAMP appears to be the result of depolarization of the neurons initiated by increased cation conductance by cAMP-gated cation channels. Activation of PKA may constitute a negative-feedback mechanisms for lowering cAMP levels. We hypothesize that these mechanisms could result in oscillations in cAMP levels, providing a biochemical basis for timing the pulsatile release of GnRH.G onadotropin-releasing hormone (GnRH) secretion is controlled by a variety of regulatory mechanisms intrinsic to individual neurons or networks of GnRH-secreting neurons and by extrinsic regulatory mechanisms regulated by neurotransmitters released by efferent inputs to GnRH neurons. The development of the highly differentiated GT1 GnRH-secreting neuronal cell lines has provided a model to study the signaling mechanisms involved in the complex regulation of GnRH secretion (1). The GT1 cell lines were established from a hypothalamic tumor induced by genetic targeting of the expression of the oncogene encoding simian virus 40 T antigen to GnRH neurons in a transgenic mouse. The cells are highly differentiated and express and process GnRH at high levels (1, 2). The pulsatile release of GnRH appears to be an intrinsic property of individual or networks of GnRH neurons, since cultures of the GT1 cells release GnRH with a pulse frequency identical to that seen in castrate rodents (3-5). In vivo numerous efferent inputs to GnRH neurons release neurotransmitters that stimulate GnRH secretion (6). Neurotransmitters stimulating GnRH release from GT1 cells include bradykinin, dopamine (DA), endothelin, glutamate, neuropeptide Y, and norepinephrine (NE) (7-12).The current study is focused on the role of the cAMP signaling pathway in the regulation of GnRH secretion from GT1 cells. GT1 cells express both D1-DA receptors (8) and ␤1-adrenergic receptors (12) which are positively coupled to adenylyl cyclase (AC). Treatment of GT1 cells with DA or NE increased intracellular cAMP levels and stimulated GnRH secretion in a dose-dependent fashion (8, 12). The GnRH-releasing effects of DA and NE were mimicked by pharmacologically...