Selective deletion of Pten in theca-interstitial cells leads to androgen excess and ovarian dysfunction in mice.

Theca cell-selective Pten mutation (tPtenMT) in mice resulted in increases in PDK1 and Akt phosphorylation, indicating an over-activation of PI3K signaling in the ovaries. These mice displayed elevated androgen levels, ovary enlargement, antral follicle accumulation, early fertility loss and increased expression of Lhcgr and genes that are crucial to androgenesis. These abnormalities were partially reversed by treatments of PI3K or Akt inhibitor. LH actions in Pten deficient theca cells were potentiated. The phosphorylation of Foxo1 was increased, while the binding of Foxo1 to forkhead response elements in the Lhcgr promoter was reduced in tPtenMT theca cells, implying a mechanism by which PI3K/Akt-induced upregulation of Lhcgr in theca cells might be mediated by reducing the inhibitory effect of Foxo1 on the Lhcgr promoter. The phenotype of tPtenMT females is reminiscent of human PCOS and suggests that dysregulated PI3K cascade in theca cells may be involved in certain types of PCOS pathogenesis.

Mouse germline restriction of Oct4 expression by germ cell nuclear factor.

The POU-domain transcription factor Oct4 is essential for the maintenance of the mammalian germline. In this study, we show that the germ cell nuclear factor (GCNF), an orphan nuclear receptor, represses Oct4 gene activity by specifically binding within the proximal promoter. GCNF expression inversely correlates with Oct4 expression in differentiating embryonal cells. GCNF overexpression in embryonal cells represses Oct4 gene and transgene activities, and we establish a link to transcriptional corepressors mediating repression by GCNF. In GCNF-deficient mouse embryos, Oct4 expression is no longer restricted to the germ cell lineage after gastrulation. Our studies suggest that GCNF is critical in repressing Oct4 gene activity as pluripotent stem cells differentiate and in confining Oct4 expression to the germline.

A-ring reduced metabolites of 19-nor synthetic progestins as subtype selective agonists for ER alpha.

It has previously been demonstrated that 19-nor contraceptive progestins undergo in vivo and in vitro enzyme-mediated A-ring double bond hydrogenation. Bioconversion of 19-nor progestins to their corresponding tetrahydro derivatives results in the loss of progestational activity and acquisition of estrogenic activities and binding to the ER. Herein, we report subtype-selective differences in ligand binding and transcriptional potency of nonphenolic synthetic 19-nor derivatives between ER alpha and ER beta. In this study, we have examined both ER- and PR-mediated transcriptional activity of a number of A-ring chemically reduced derivatives of norethisterone and Gestodene. Double bond hydrogenation decreased the transcriptional potency of norethisterone and Gestodene through both PR isoforms with a 100- to 1,000-fold difference, respectively. In terms of the effects of norethisterone and Gestodene and their corresponding 5 alpha-dihydro (5 alpha-norethisterone and 5 alpha-Gestodene), or 3 alpha,5 alpha-tetrahydro or 3 beta,5 alpha-tetrahydro derivatives (3 alpha,5 alpha-norethisterone/3 alpha,5 alpha-Gestodene and 3 beta,5 alpha-norethisterone/3beta,5 alpha-Gestodene, respectively) on estrogen-mediated transcriptional regulation, the 3 beta,5 alpha-tetrahydro derivatives of both norethisterone and Gestodene showed the highest induction when HeLa cells were transiently transfected with an expression vector for ER alpha. This activity could be inhibited with tamoxifen. These compounds did not activate gene transcription via ER beta, and none of them showed antagonistic activities through either ER subtype. The 3 beta,5 alpha-tetrahydro derivatives of both norethisterone and Gestodene were active in other cells in addition to HeLa cells and activated reporter expression through the oxytocin promoter. In summary, two ER alpha selective agonists have been identified. These compounds, with ER alpha vs. ER beta selective agonist activity, may be useful in evaluating the distinct role of these receptors as well as in providing useful insights into ER action.

Loss of orphan receptor germ cell nuclear factor function results in ectopic development of the tail bud and a novel posterior truncation.

The dynamic embryonic expression of germ cell nuclear factor (GCNF), an orphan nuclear receptor, suggests that it may play an important role during early development. To determine the physiological role of GCNF, we have generated a targeted mutation of the GCNF gene in mice. Germ line mutation of the GCNF gene proves that the orphan nuclear receptor is essential for embryonic survival and normal development. GCNF(-/-) embryos cannot survive beyond 10.5 days postcoitum (dpc), probably due to cardiovascular failure. Prior to death, GCNF(-/-) embryos suffer significant defects in posterior development. Unlike GCNF(+/+) embryos, GCNF(-/-) embryos do not turn and remain in a lordotic position, the majority of the neural tube remains open, and the hindgut fails to close. GCNF(-/-) embryos also suffer serious defects in trunk development, specifically in somitogenesis, which terminates by 8.75 dpc. The maximum number of somites in GCNF(-/-) embryos is 13 instead of 25 as in the GCNF(+/+) embryos. Interestingly, the tailbud of GCNF(-/-) embryos develops ectopically outside the yolk sac. Indeed, alterations in expression of multiple marker genes were identified in the posterior of GCNF(-/-) embryos, including the primitive streak, the node, and the presomitic mesoderm. These results suggest that GCNF is required for maintenance of somitogenesis and posterior development and is essential for embryonic survival. These results suggest that GCNF regulates a novel and critical developmental pathway involved in normal anteroposterior development.

The oestrogenic effects of gestodene, a potent contraceptive progestin, are mediated by its A-ring reduced metabolites.

Gestodene (17 alpha-ethynyl-13 beta-ethyl-17 beta-hydroxy-4, 15-gonadien-3-one) is the most potent synthetic progestin currently available and it is widely used as a fertility regulating agent in a number of contraceptive formulations because of its high effectiveness, safety and acceptability. The observation that contraceptive synthetic progestins exert hormone-like effects other than their progestational activities, prompted us to investigate whether gestodene (GSD) administration may induce oestrogenic effects, even though the GSD molecule does not interact with intracellular oestrogen receptors (ER). To assess whether GSD may exert oestrogenic effects through some of its neutral metabolites, a series of experimental studies were undertaken using GSD and three of its A-ring reduced metabolites. Receptor binding studies by displacement analysis confirmed that indeed GSD does not bind to the ER, whereas its 3 beta,5 alpha-tetrahydro reduced derivative (3 beta GSD) interacts with a relative high affinity with the ER. The 3 alpha,5 alpha GSD isomer (3 alpha GSD) also binds to the ER, though to a lesser extent. The ability of the A-ring reduced GSD derivatives to induce oestrogenic actions was evaluated by the use of two different molecular bioassays: (a) transactivation of a yeast system co-transfected with the human ER alpha (hER alpha) gene and oestrogen responsive elements fused to the beta-galactosidase reporter vector and (b) transactivation of the hER alpha-mediated transcription of the chloramphenicol acetyl transferase (CAT) reporter gene in a HeLa cells expression system. The oestrogenic potency of 3 beta GSD was also assessed by its capability to induce oestrogen-dependent progestin receptors (PR) in the anterior pituitary of castrated female rats. The results demonstrated that 3 beta GSD and 3 alpha GSD were able to activate, in a dose-dependent manner, the hER alpha-mediated transcription of both the beta-galactosidase and the CAT reporter genes in the yeast and HeLa cells expression systems respectively. In both assays the 3 beta derivative of GSD exhibited a significantly greater oestrogenic effect than its 3 alpha isomer, while unchanged GSD and 5 alpha GSD were completely ineffective. Neither 3 beta GSD nor 3 alpha GSD exhibited oestrogen synergistic actions. Interestingly, the pure steroidal anti-oestrogen ICI-182,780 diminished the transactivation induced by 3 beta GSD and 3 alpha GSD in the yeast expression system. Furthermore, administration of 3 beta GSD resulted in a significant increase of oestrogen-dependent PR in the anterior pituitaries of castrated rats in comparison with vehicle-treated animals. The characteristics of the 3 beta GSD-induced PR were identical to those induced by oestradio benzoate. The overall results demonstrate that 3 beta GSD and its 3 alpha isomeric alcohol specifically bind to the ER and possess a weak intrinsic oestrogenic activity, whereas unmodified GSD does not. The data contribute to a better understanding of the GSD mechanism of action and allow the hypothesis to be advanced that the slight oestrogenlike effects attributable to GSD are mediated by its non-phenolic, tetrahydro reduced metabolites.

Germ cell nuclear factor is a response element-specific repressor of transcription.

We have shown that the orphan receptor Germ Cell Nuclear Factor (GCNF) binds to a direct repeat of the sequence AGGTCA with zero base pair spacing (DR0). Here, we further characterize the binding characteristics of GCNF. We demonstrate that GCNF binds specifically to DR0s as a homodimer, and does not bind with high affinity to DR1-DR6 sequences. GCNF is the first nuclear receptor shown to bind specifically to DR0s. The wild type GCNF is unable to transactivate the reporter plasmid DR0(2)tkCAT. Lacking a ligand to activate GCNF, we fused the activation domain from the viral protein VP16 to GCNF, and observed activation of DR0(2)tkCAT. This activation is specifc to DR0s, and is not observed when that sequence is replaced by DR1-DR6 sequences. In addition GCNF does not transactivate through an SF-1 response element. At increasing concentrations, wild type GCNF is able to repress basal transcription. Repression is again specific to DR0s. The preference of GCNF for the DR0 sequence both in vitro and in transfections suggests that GCNF defines a novel nuclear receptor signaling pathway.

The mouse bone morphogenetic protein-4 gene. Analysis of promoter utilization in fetal rat calvarial osteoblasts and regulation by COUP-TFI orphan receptor.

Bone morphogenetic protein-4 (BMP-4) is one of a member of related polypeptides that are important in bone formation and other developmental processes. We isolated the BMP-4 gene from a mouse genomic library and characterized the exon-intron structure and one of the candidate promoters. Two alternative 5'-noncoding exons, 1A and 1B, were identified by reverse transcription polymerase chain reaction assays. Quantitative competitive polymerase chain reaction using Exon 1A, Exon 1B, and Exon 3 primers indicate the 1A-containing transcript is the primary BMP-4 mRNA expressed in bone cell cultures. Primer extension analysis supports that 1A is the major promoter utilized in bone cell cultures as well as in 9.5-day mouse embryos. 1A promoter activity indicate selective DNA regions functional in bone cells. We found potential regulatory response regions in the 1A 5'-flanking region of the BMP-4 gene for the chicken ovalbumin upstream-transcription factor I (COUP-TFI). Specific binding to the COUP-TFI response regions in the BMP-4 1A promoter was demonstrated. By co-transfection of a COUP-TFI expression plasmid with the BMP-4 1A promoter in fetal rat calvarial osteoblasts, we demonstrated that COUP-TFI inhibits the BMP-4 promoter activity. This suggests that COUP-TFI could act as a silencer for BMP-4 transcription in vivo.

A rapid one-step method to purify baculovirus-expressed human estrogen receptor to be used in the analysis of the oxytocin promoter.

We have produced a truncated form of the human estrogen receptor (hER) as a fusion protein with glutathione S-transferase (GST) in Spodoptera frugiperda (Sf) cells using the baculovirus expression vector (BEV) system. The protein is correctly produced and can be purified from crude whole-cell extracts by a single-step, batch-wise affinity-purification procedure. We show that this GST-hER fusion protein binds at its DNA-binding site specifically and in a hormone-inducible manner. Furthermore, we used the purified hER to analyze the complex estrogen response element (ERE) in the promoter of the oxytocin-encoding gene.

Negative regulation by the R2 element of the MHC class I enhancer in adenovirus-12 transformed cells correlates with high levels of COUP-TF binding.

The transcriptional down-regulation of the major histocompatibility complex (MHC) class I antigens in adenovirus type 12 (Ad12) transformed cells gives them the potential to escape immunosurveillance and to form tumors. The enhancer of the class I promoter is the target of transcriptional repression which is mediated by the E1A gene of Ad12. The R2 region within the class I enhancer acts as a negative element in Ad12-transformed cells and exhibits a stronger binding activity than is observed in nontumorigenic Ad5-transformed cells, which are not reduced in class I expression. The R2 element contains a nuclear hormone receptor half-site consensus sequence, AGGTCA, which is required for both the binding activity and the ability of R2 to act as a negative element in Ad12-transformed cells. In this study, we show that an orphan hormone receptor protein, COUP-TF, contributes to the differential R2 binding activity observed between Ad12- and Ad5-transformed cells. Additionally, COUP-TF was shown to bind as a dimer to the R2 element and to use the consensus AGGTCA as one half-site and its 3' flanking sequence as a probable second degenerate half-site. Since COUP-TF can act as a transcriptional repressor, we suggest that the higher COUP-TF binding activity to the R2 element in Ad12-transformed cells contributes to down-regulation of class I transcription and, consequently, tumorigenesis.

Repression of estrogen-dependent stimulation of the oxytocin gene by chicken ovalbumin upstream promoter transcription factor I.

The orphan receptor chicken ovalbumin upstream promoter transcription factor I (COUP-TF I) fully prevented not only the activation of the oxytocin gene by retinoic acid and thyroid hormone but also completely repressed the estrogen-dependent stimulation in transfected P19 EC cells. DNase I footprinting showed that the COUP-TF I protein bound to the 5'-flanking region of the oxytocin gene at the site of the distal composite hormone response element, which mediates the responses to estrogen, retinoic acid, and thyroid hormone. Electrophoretic mobility shift assay using this composite hormone response element as probe showed that COUP-TF I and the estrogen receptor competed for binding but did not form a heterodimer. The binding by COUP-TF I was stronger than the binding of the estrogen receptor. Thus, the mechanism of repression involves occupancy of integrated binding sites. By mutagenesis of the composite hormone response element, the COUP-TF I binding site and the estrogen response element could be separated, resulting in functional dissociation of the repressive action of COUP-TF I and the induction by estrogen. The results show that repression of gene expression by COUP-TF I is not limited to receptors that act through heterodimerization but also extends to the homodimer-forming estrogen receptor in a context-dependent manner. This interaction between COUP-TF I and the estrogen receptor may provide a physiological mechanism of selective antagonism of gene regulation by estrogens.