Methylation of the Corticotropin Releasing Hormone Gene Promoter in BeWo Cells: Relationship to Gene Activity.

Corticotropin releasing hormone (CRH) production by the human placenta increases exponentially as pregnancy advances, and the rate of increase predicts gestational length. CRH gene expression is regulated by cAMP in trophoblasts through a cyclic AMP-response element (CRE), which changes its transcription factor binding properties upon methylation. Here we determined whether methylation of the CRH proximal promoter controls basal and cAMP-stimulated CRH expression in BeWo cells, a well-characterized trophoblastic cell line. We treated the cells with 8-Br-cAMP and the DNA methyltransferase inhibitor 5-aza-2' deoxycytidine (5-AZA-dC) and determined the effects on CRH mRNA level and promoter methylation. Clonal bisulfite sequencing showed partial and allele independent methylation of CpGs in the CRH promoter. CRH mRNA expression and the methylation of a subset of CpGs (including CpG2 in the CRE) increased spontaneously during culture. 8-Br-cAMP stimulated CRH expression without affecting the increase in methylation. 5-AZA-dC decreased methylation and augmented 8-Br-cAMP-stimulated CRH expression, but it blocked the spontaneous increase of CRH mRNA level. We conclude that the CRH promoter is a dynamically and intermediately methylated genomic region in BeWo cells. Promoter methylation did not inhibit CRH gene expression under the conditions employed; rather it determined the contribution of alternative cAMP-independent pathways and cAMP-independent mechanisms to CRH expression control.

Effects of corticotrophin releasing hormone (CRH) on cell viability and differentiation in the human BeWo choriocarcinoma cell line: a potential syncytialisation inducer distinct from cyclic adenosine monophosphate (cAMP).

BACKGROUND: Placental production of corticotrophin releasing hormone (CRH) rises exponentially as pregnancy progresses, and has been linked with the onset of normal and preterm labour. CRH is produced in syncytiotrophoblast cells and production is increased by glucocorticoids and cAMP. It remains unclear whether cAMP acts by inducing differentiation of cytotrophoblasts and/or through induction of syncytialisation. As CRH can stimulate cAMP pathways we have tested whether a feed-forward system may exist in placental cells during syncytialisation. METHODS: The choriocarcinoma BeWo cell line was treated with cAMP, CRH or vehicle. Cell viability was determined by MTT assay, while apoptosis was analysed by DAPI staining and by FACS. Differentiation was measured by assaying message for hCG and ERVW-1 (syncytin1) by qRT-PCR, as well as the respective protein by ELISA. Fusion of BeWo cells was assessed by co-staining cell membrane and nuclei with CellMask and Hoechst 33342. CRHR1 and CRHR2 mRNA levels were measured by qRT-PCR. RESULTS: We show that cAMP has an inductive effect on syncytialisation, as evidenced by induction of hCG secretion, by ERVW-1 mRNA expression and by formation of multinuclear cells. CRH mRNA expression was found to increase prior to the changes in the other syncytialisation markers. cAMP had an inhibitory effect on BeWo cell viability, but exogenous CRH did not. However, CRH did mimic the differentiation inducing effect of cAMP, suggesting a link between CRH and cAMP signalling in syncytialisation. We also found that treatment of BeWo cells with exogenous CRH resulted in elevated cellular CRHR1 levels. CONCLUSIONS: This study suggests a positive feed-forward role exists for CRH in trophoblast cell differentiation, which may underlie the exponential rise in CRH observed as gestation advances.

Toxicity of a novel anti-tumor agent 20(S)-ginsenoside Rg3: a 26-week intramuscular repeated administration study in rats.

The purpose of this study is to investigate the potential subchronic toxicity of 20(S)-Ginsenoside Rg3(Rg3), by a 26-week repeated intramuscular administration in rats. Rg3 was administrated to rats at dose levels of 0, 4.2, 10.0 or 20.0 mg/kg/day. There was no treatment-related mortality and, at the scheduled autopsy, dose-dependent increases in the absolute and relative spleen weights, of both the 10.0 mg/kg and 20.0 mg/kg dose groups were observed. Absolute and relative kidney weights were significantly elevated in the female 10.0 mg/kg dose group and in the male 20.0 mg/kg dose group. Hematological investigations revealed a dose-dependent increase in the total white blood cell (WBC) count and in the percentage of neutrophils, but a decrease in the percentage of lymphocytes, in rats treated with doses of 10.0/20.0 mg/kg. These effects were completely reversible during the recovery period, and no other adverse effects were observed. It was concluded that the 26-week repeated intramuscular dose of Rg3 caused increases in the spleen and kidney weights, WBC counts and in the percentage of neutrophils, but a decrease in the percentage of lymphocytes, with doses of 10.0 or 20.0 mg/kg/day. The no-observed-adverse-effect level for rats was considered to be 4.2 mg/kg/day.

Tissue specific epigenetic differences in CRH gene expression.

Corticotropin Releasing Hormone (CRH), a 41-amino acid peptide, is a major regulator of hypothalamic-pituitary-adrenal axis function. CRH also has important roles in several processes pertaining to pregnancy and parturition, including being a possible regulator of gestational length and predictor of pre-term birth. Regulation of the CRH promoter exhibits some tissue-specificities, the most well characterized example being glucocorticoids, which can stimulate placental CRH production but suppress hypothalamic CRH. In the last decade there has been growing interest in the role of epigenetic regulation of gene expression. Modification of the structure of chromatin is an example of epigenetic change affecting gene expression. We have found that inhibition of histone deacetylases results in an increase in CRH expression in the AtT20 pituitary cell line, but a decrease in CRH expression in the placenta. In this paper we review tissue specific differences in CRH gene expression, and discuss how epigenetic chromatin modification mechanisms can relate to tissue specific differences in expression of CRH.

Toxicity of a novel anti-tumor agent 20(S)-ginsenoside Rg3: a 26-week intramuscular repeated administration study in Beagle dogs.

The potential subchronic toxicity of a dammarane-type triterpenoid saponin with antitumor effect, 20(S)-Ginsenoside Rg3, was studied repeated intramuscular administration in Beagle dogs over a 26-week period. 20(S)-Ginsenoside Rg3 was administrated intramuscularly at 0, 0.70, 2.86 or 7.20 mg/kg/day doses for 26 weeks in both male and female dogs (n = 4 for male and female dogs for each dose). During the test period as well as during the 8-week recovery period, clinical signs, mortality, body weights, food consumption, respiratory frequency, electrocardiogram, ophthalmoscopy, urinalysis, hematology, serum biochemistry, gross findings, organ weights and histopathology were examined. In dogs treated with doses of 2.86 or 7.20 mg/kg, hematological investigations revealed a dose-dependent increase in the total white blood cell (WBC) count and in the percentage of neutrophils, but a decrease in the percentage of lymphocytes. These effects were completely reversed during the recovery period, and no other adverse effects were observed. The no-observed-adverse-effect levels for both male and female dogs were considered to be 7.20 mg/kg/day.

MyD88 and TRIF mediate the cyclic adenosine monophosphate (cAMP) induced corticotropin releasing hormone (CRH) expression in JEG3 choriocarcinoma cell line.

BACKGROUND: Classically protein kinase A (PKA) and transcription factor activator protein 1 (AP-1) mediate the cyclic AMP (cAMP) induced-corticotrophin releasing hormone (CRH) expression in the placenta. However enteric Gram (-) bacterial cell wall component lipopolysaccharide (LPS) may also induce-CRH expression via Toll like receptor (TLR)4 and its adaptor molecule Myd88. Here we investigated the role of MyD88, TRIF and IRAK2 on cAMP-induced CRH promoter activation in JEG3 cells in the absence of LPS/TLR4 stimulation. METHODS: JEG3 cells were transfected with CRH-luciferase and Beta-galactosidase expression vectors and either empty or dominant-negative (DN)-MyD88, DN-TRIF or DN-IRAK2 vectors using Fugene6 (Roche). cAMP-induced CRH promoter activation was examined by using a luminometer and luciferase assay. Calorimetric Beta-galactosidase assays were performed to correct for transfection efficiency. Luciferase expression vectors of cAMP-downstream molecules, CRE and AP-1, were used to further examine the signaling cascades. RESULTS: cAMP stimulation induced AP-1 and CRE promoter expression and led to dose-dependent CRH promoter activation in JEG3 cells. Inhibition of MyD88 signaling blocked cAMP-induced CRE and CRH promoter activation. Inhibition of TRIF signaling blocked cAMP-induced CRH but not CRE expression, while inhibition of IRAK2 did not have an effect on cAMP-induced CRH expression. CONCLUSION: MyD88 and TRIF exert direct regulatory effect on cAMP-induced CRH promoter activation in JEG3 cells in the absence of infection. MyD88 most likely interacts with molecules upstream of IRAK2 to regulate cAMP-induced CRH expression.

Phytoestrogens induce differential estrogen receptor beta-mediated responses in transfected MG-63 cells.

Phytoestrogens may function as partial agonists or antagonists of estrogen in many tissues including bone. Five phytoestrogens, belonging to the isoflavones and the flavonoids groups, were assayed in the human MG-63 osteoblastic cell line for their ability to stimulate transcriptional activity of an estrogen-response element (ERE)-luciferase reporter gene via the estrogen receptor beta (ERbeta). Although MG-63 cells were shown to express endogenous estrogen receptors, estradiol (E2) did not affect transcriptional activity of an ERE reporter in these cells. However, E2 did activate the ERE-reporter significantly in MG-63 cells where ERbeta was overexpressed. The isoflavones, genistein and daidzein, caused a dose-dependent increase in the ERE-reporter activity in MG-63 cells overexpressing ERbeta. Among the flavonoids, kaempferol activated ERE-reporter activity, whereas puerarin inhibited ERE-reporter transcription in cells overexpressing ERbeta. Quercetin had no effect on ERE-reporter activity over a concentration range of 10(-10)-10(-6) mol/l. The ERE-reporter activity induced by daidzein, genistein, and kaempferol was blocked by both ICI 182780 and 4-hydroxytamoxifen and partly blocked by puerarin. Our results demonstrated that different phytoestrogens exhibited differential transcription activity of an ERE-reporter via ERbeta-mediated mechanisms in MG-63 cells.

Lipopolysaccharide stimulation of trophoblasts induces corticotropin-releasing hormone expression through MyD88.

OBJECTIVE: We hypothesized that intrauterine infection may lead to placental corticotrophin-releasing hormone (CRH) expression via Toll-like receptor signaling. STUDY DESIGN: To test this hypothesis JEG3 cells were stimulated with lipopolysaccharide (LPS), chlamydial heat shock protein 60, and interleukin (IL)-1. CRH expression was assessed by reverse transcription polymerase chain reaction (RT-PCR). The signaling mechanisms that were involved were examined in transient transfection experiments with beta-galactosidase, CRH-luciferase, cyclic adenosine monophosphate (AMP) response element-luciferase, dominant-negative (DN)-myeloid differentiation primary response gene (MyD88) and DN-toll-IL-1-receptor domain containing adapter inducing interferon (TRIF) vectors. Luciferase activity was determined by luciferase assay. Beta-galactosidase assay was performed to determine transfection efficiency. RESULTS: LPS, chlamydial heat shock protein 60, and IL-1 stimulation led to CRH expression in the JEG3 cells. LPS-induced CRH expression was not due to the autocrine effect of LPS-induced IL-1 because the supernatant from LPS-conditioned JEG3 cells did not induce CRH expression in the naïve cells. DN-MyD88, but not DN-TRIF, blocked the LPS-induced CRH expression. The cAMP response element did not play a role in LPS-induced CRH expression. CONCLUSION: Toll-like receptor signaling 4 may induce placental CRH expression through MyD88.

Phorbol ester stimulates corticotropin-releasing hormone gene promoter activity through a cAMP regulatory element in primary placental cells.

Placental corticotropin-releasing hormone (CRH) plays an important role in the mechanisms controlling human pregnancy and parturition, and several endogenous factors are known to regulate placental CRH gene expression. In this article, the authors investigate the regulation of the CRH gene's promoter activity by a protein kinase C (PKC) activator, phorbol-12-myristate-13-acetate (PMA), in primary cultures of placental cells. The PMA stimulation of the CRH gene promoter activity was dose dependent, and further studies, including progressive deletion and mutation analysis of the CRH promoter, localized the region essential for PMA responsiveness to a consensus cyclic adenosine monophosphate regulatory element (CRE). Furthermore, estradiol treatment resulted in decreases of both basal and PMA-stimulated promoter activity when the CRE element was present but had no effect when the CRE element was absent. Thus, PMA stimulates CRH gene transcriptional activity through the CRE, suggesting that cross-talk between PKC and protein kinase A signaling pathways targets this regulatory element in placental cells.

Corticotrophin releasing hormone and the timing of birth.

Corticotrophin-releasing hormone (CRH) is the hypothalamic peptide that controls the function of the pituitary-adrenal axis in response to stress. CRH is also expressed abundantly in the human placenta and is present in high concentrations in maternal and fetal plasma during late pregnancy. During pregnancy, CRH derived from the placenta is thought to play a crucial role in the regulation of fetal maturation and the timing of delivery, and CRH has also been implicated in the control of fetal-placental blood flow. Elevated CRH concentrations, as compared with gestational age matched controls, occur in patients in preterm labour. The exponential curve depicting the CRH increase is shifted to the left in women who will subsequently deliver preterm and to the right in women who will deliver post dates. This has led to the suggestion that CRH production is linked to a placental clock which determines the length of gestation. Clinically, maternal plasma CRH concentrations may be useful in identifying women at high risk of preterm delivery and CRH antagonists may be useful in preventing preterm labour. As significant CRH production by the placenta is restricted to primates, future research must take into account the species specificity of the mechanisms regulating parturition. A number of significant gaps remain in our knowledge of the function of this peptide in pregnancy. This review examines the current evidence regarding the role of CRH in human parturition.

Advances in understanding corticotrophin-releasing hormone gene expression.

Glucocorticoids inhibit corticotrophin-releasing hormone (CRH) gene expression in the hypothalamic paraventricular nucleus (PVN), but stimulate expression in the placenta. In AtT20 cells (a model of PVN CRH production) cAMP produces a high level of promoter activity. Cyclic AMP stimulation occurs through the cAMP response element (CRE) and the caudal type homeobox protein response element (CDXARE). The CRE acts as part of a cAMP response unit that includes the hybrid steroid response element (HRE), ecdysone response element (EcRE), metal-responsive transcription factor-1 response element (MTFRE), ying yang 1 response element (YY1RE) and negative glucocorticoid response element (nGRE). Cyclic AMP acts on the HRE, EcRE and MTFRE to block YY1RE mediated inhibition of the CRE. Glucocorticoids acting at the nGRE inhibit cAMP activation of the CRE. In placental cells the CRH promoter has low intrinsic basal activity and cAMP causes a modest increase in activity. Stimulation by glucocorticoids and cAMP and inhibition by estrogen and estrogen receptor alpha occurs through the CRE. In AtT20 cells multiple response elements coordinate a response to cAMP and glucocorticoids while in placental cells the CRE acts in isolation. These differences in promoter function lead to responses that meet specific physiological needs.

Steroid hormone mediated regulation of corticotropin-releasing hormone gene expression.

Corticotropin-releasing hormone (CRH), a 41 amino acid polypeptide, is expressed in many regions of central nervous system and peripheral tissues and mediates many physiological functions. Abnormal production of CRH is involved in some pathological processes. Among various endogenous factors that modulate CRH production, steroid hormones control CRH production by regulating its gene transcription. Although there is no classical steroid hormone response element in the CRH promoter, steroid hormones regulate CRH gene expression through protein-protein interaction or by binding directly to response elements.

Identification of a family of DNA-binding proteins with homology to RNA splicing factors.

We describe a unique family of human proteins that are capable of binding to the cAMP regulatory element (CRE) and that are homologous to RNA splicing proteins. A human cDNA was isolated that encodes a protein with a distinctive combination of modular domain structures: 2 leucine-zipper-like domains, a DNA-binding zinc-finger-like domain, an RNA-binding zinc-finger-like domain, and 2 coiled-coil protein-protein interaction domains. It also has a serine-arginine-rich domain, commonly found in proteins involved in RNA splicing. The protein was discovered using the CRE as bait in a yeast 1-hybrid assay. It was then shown to bind specifically to the CRE in vitro using gel shift assays. We have named the protein CRE-associated protein (CREAP). We show that it is widely expressed in human tissues but is highly expressed in several fetal tissues and in several regions of the adult brain. CREAP is closely related to 2 human proteins of unknown function. CREAP shows significant homology with a small nuclear ribonucleoprotein of yeast, Luc7p, involved in 5' splice site recognition. The 3 human CREAP proteins form a unique family with the potential to act as transcription factors that link to RNA processing.

Estrogen receptor-mediated down-regulation of corticotropin-releasing hormone gene expression is dependent on a cyclic adenosine 3',5'-monophosphate regulatory element in human placental syncytiotrophoblast cells.

Placental CRH plays a major role in the mechanisms controlling human pregnancy and parturition. Understanding how placental CRH production is regulated is therefore of importance. Previously we have shown that placental expression of CRH peptide and mRNA are inhibited by estrogens, in contrast to the stimulatory effects of estrogen on hypothalamic CRH production. Our current study found that in placental cells cotransfected with a CRH promoter construct and an estrogen receptor-alpha expression vector results in a differential regulation whereby 17beta-estradiol (E2) decreased and the putative pure estrogen antagonist, ICI 182780, increased CRH promoter activity. Sequential deletion of the CRH promoter indicated that the region between -248 and -213 bp was essential for the effect of both E2 and ICI 182780. This region contains a consensus cAMP regulatory element (CRE) that is a requirement for E2- and ICI 182780-mediated activity because the CRE motif can confer E2 inhibition on a heterologous promoter such as rabbit beta-globin. Mutation of the CRE resulted in a complete reversal of E2 and ICI 182780 regulatory effects. In summary, our results demonstrate that a consensus CRE is required for the action of estrogen receptor ligands in human placental syncytiotrophoblast cells.

Complex regulatory interactions control CRH gene expression.

Glucocorticoids inhibit corticotrophin releasing hormone (CRH) production in the hypothalamus but stimulate production from the placenta. To identify key elements regulating the CRH gene, mouse pituitary tumor-derived cells (AtT20 cells) were used as a hypothalamic model in an analysis of the CRH promoter. Two cAMP responsive elements were identified: (I) a consensus cAMP response element (CRE) and (II) a previously unrecognized caudal-type homeobox response element (CDXRE). Glucocorticoids inhibit only the component of cAMP-stimulation occurring via the CRE through an action involving a negative glucocorticoid response element (nGRE). We also identified two regions that, in the absence of the nGRE, can be stimulated by glucocorticoids: (I) the CRE and (II) a region between -213 to -99bps. Electrophoretic mobility shift assays identified binding of the transcription factors CREB and Fos at the CRE in AtT20 cells, whereas CREB and cJun were detected in placental cells. In addition, a novel CRE-binding transcription factor has been identified that is expressed in the brain and in placenta. A model is presented whereby CRH gene regulation is mediated via tissue specific expression of transcription factors.

Novel glucocorticoid and cAMP interactions on the CRH gene promoter.

Glucocorticoids inhibit corticotrophin releasing hormone (CRH) production in the hypothalamus but stimulate production from the placenta. We have sought to identify the key elements regulating the CRH gene. Mouse pituitary tumour-derived cells (AtT20 cells) were used in deletion and mutational analyses of the CRH promoter. Two cAMP responsive elements were identified: (I) a consensus cAMP response element (CRE) and (II) a previously unrecognised caudal-type homeobox response element (CDXRE). Glucocorticoids inhibit only the component of cAMP-stimulation occurring via the CRE through an action involving a negative glucocorticoid response element (nGRE). We also identified two regions that, in the absence of the nGRE, can be stimulated by glucocorticoids: (I) the CRE and (II) a region between -213 and -99 bps. Electrophoretic mobility shift assays (EMSAs) identified binding of the transcription factors CREB and Fos at the CRE in AtT20 cells while CREB and cJun were detected in placental cells. Tissue specific expression of transcription factors may mediate regulation of the CRH gene.

Estrogen represses whereas the estrogen-antagonist ICI 182780 stimulates placental CRH gene expression.

CRH and estrogens, produced by placental trophoblasts, have been suggested to play pivotal roles in the control of human parturition. Estrogen has been shown to affect hypothalamic CRH expression. Therefore, we evaluated 17 beta-estradiol (E2) in the regulation of CRH gene expression in placental cells. E2 inhibited CRH mRNA expression in a dose-dependent manner, which paralleled the decrease in CRH protein levels in culture media. A complete estrogen receptor (ER) antagonist, ICI 182780, not only blocked repression of CRH mRNA levels by E2, but up-regulated CRH mRNA and protein synthesis. An ER alpha-mixed agonist/antagonist and ER beta antagonist, 4-hydroxytamoxifen, also down-regulated CRH gene expression. Using quantitative RT-PCR, we found that placental trophoblasts express predominantly the ER alpha form of the receptor. Transient transfection assays conducted in the choriocarcinoma cell line JEG-3 demonstrated that E2 repressed CRH promoter activity, whereas the antagonist ICI 182780 up-regulated CRH promoter activity when ER alpha was cotransfected. These studies demonstrate that E2 represses placental CRH gene expression through an ER alpha-mediated mechanism. Estrogen may therefore modulate placental CRH production, influencing the rate of rise of maternal plasma CRH concentrations and potentially the length of gestation.