DNA demethylation induces SALL4 gene re-expression in subgroups of hepatocellular carcinoma associated with Hepatitis B or C virus infection.

Sal-like protein 4 (SALL4), an embryonic stem cell transcriptional regulator, is re-expressed by an unknown mechanism in poor prognosis hepatocellular carcinoma (HCC), often associated with chronic hepatitis B virus (HBV) infection. Herein, we investigated the mechanism of SALL4 re-expression in HBV-related HCCs. We performed bisulfite sequencing PCR of genomic DNA isolated from HBV-related HCCs and HBV replicating cells, and examined DNA methylation of a CpG island located downstream from SALL4 transcriptional start site (TSS). HBV-related HCCs expressing increased SALL4 exhibited demethylation of specific CpG sites downstream of SALL4 TSS. Similarly, SALL4 re-expression and demethylation of these CpGs was observed in HBV replicating cells. SALL4 is also re-expressed in poor prognosis HCCs of other etiologies. Indeed, increased SALL4 expression in hepatitis C virus-related HCCs correlated with demethylation of these CpG sites. To understand how CpG demethylation downstream of SALL4 TSS regulates SALL4 transcription, we quantified by chromatin immunoprecipitation (ChIP) assays RNA polymerase II occupancy of SALL4 gene, as a function of HBV replication. In absence of HBV replication, RNA polymerase II associated with SALL4 exon1. By contrast, in HBV replicating cells RNA polymerase II occupancy of all SALL4 exons increased, suggesting CpG demethylation downstream from SALL4 TSS influences SALL4 transcriptional elongation. Intriguingly, demethylated CpGs downstream from SALL4 TSS are within binding sites of octamer-binding transcription factor 4 (OCT4) and signal transducer and activator of transcription3 (STAT3). ChIP assays confirmed occupancy of these sites by OCT4 and STAT3 in HBV replicating cells, and sequential ChIP assays demonstrated co-occupancy with chromatin remodeling BRG1/Brahma-associated factors. BRG1 knockdown reduced SALL4 expression, whereas BRG1 overexpression increased SALL4 transcription in HBV replicating cells. We conclude demethylation of CpGs located within OCT4 and STAT3 cis-acting elements, downstream of SALL4 TSS, enables OCT4 and STAT3 binding, recruitment of BRG1, and enhanced RNA polymerase II elongation and SALL4 transcription.

Convergence of multiple mechanisms of steroid hormone action.

Steroid hormones modulate a wide array of physiological processes including development, metabolism, and reproduction in various species. It is generally believed that these biological effects are predominantly mediated by their binding to specific intracellular receptors resulting in conformational change, dimerization, and recruitment of coregulators for transcription-dependent genomic actions (classical mechanism). In addition, to their cognate ligands, intracellular steroid receptors can also be activated in a "ligand-independent" manner by other factors including neurotransmitters. Recent studies indicate that rapid, nonclassical steroid effects involve extranuclear steroid receptors located at the membrane, which interact with cytoplasmic kinase signaling molecules and G-proteins. The current review deals with various mechanisms that function together in an integrated manner to promote hormone-dependent actions on the central and sympathetic nervous systems.

Dopamine agonist signalling in the hypothalamus of female rats is independent of calcium-dependent kinases.

We have previously demonstrated that dopamine agonist, SKF38396 (SKF), can substitute for progesterone in the facilitation of female reproductive behaviour in oestradiol benzoate-primed female rats and mice. We also reported that both progesterone- and SKF-initiated signalling were mediated by the cAMP-dependent protein kinase A signal transduction cascade. As the rapid effects of progesterone are also mediated by calcium-dependent kinases, calcium- and calmodulin-dependent kinase (CaMKII) and protein kinase (PKC), we sought to determine whether SKF-initiated signalling also recruited calcium as a second messenger. We measured the changes in the activation of CaMKII and PKC in the ventromedial nucleus (VMN) of the hypothalamus and preoptic area (POA) of the rat brain, which are the two regions implicated in the regulation of female reproductive behaviour in rodents. We measured the basal activities representing the activation of the kinases by in vivo treatments, as well as the total kinase activities assayed in the presence of exogenous cofactors in vitro. We report that, in contrast to progesterone-initiated signalling, there was no recruitment of calcium by SKF in the hypothalamus, as shown by the absence of changes in CaMKII activities in the VMN and POA. Furthermore, SKF-treatment resulted in a rapid increase in calcium-independent basal PKC activity in the VMN but not the POA. These rapid changes were not the result of changes in PKC protein levels or phosphorylation status. These data indicate that progesterone- and SKF-recruit distinct signalling molecules within the same regions of the brain to activate region-specific signal transduction pathways.

A biological role for the gonadotrophin-releasing hormone (GnRH) metabolite, GnRH-(1-5).

Gonadotrophin-releasing hormone (GnRH) was first isolated in the mammal and shown to be the primary regulator of the reproductive system through its initiation of pituitary gonadotrophin release. Subsequent to its discovery, this form of GnRH has been shown to be one of many structural variants found in the brain and peripheral tissues. Accordingly, the original form first discovered and cloned in the mammal is commonly referred to as GnRH-I. In addition to the complex regulation of GnRH-I synthesis, release and function, further evidence suggests that the processing of GnRH-I produces yet another layer of complexity in its activity. GnRH-I is processed by a zinc metalloendopeptidase EC 3.4.24.15 (EP24.15), which cleaves the hormone at the covalent bond between the fifth and sixth residue of the decapeptide (Tyr(5)-Gly(6)) to form GnRH-(1-5). It was previously thought that the cleavage of GnRH-I by EP24.15 represents the initiation of its degradation. Here, we review the evidence for the involvement of GnRH-(1-5), the metabolite of GnRH-I, in the regulation of GnRH-I synthesis, secretion and facilitation of reproductive behaviour.

Steroid hormone action in the brain: cross-talk between signalling pathways.

Ovarian steroid hormones, oestradiol and progesterone, modulate neuroendocrine functions in the central nervous system, resulting in alterations in physiology and behaviour. The classical model of steroid hormone action assumes that these neural effects are predominantly mediated via their intracellular receptors functioning as 'ligand-dependent' transcription factors in the steroid-sensitive neurones regulating genes and genomic networks with profound behavioural consequences. Studies from our laboratory demonstrate that, in addition to their cognate ligands, intracellular steroid receptors can be activated in a 'ligand-independent' manner by the neurotransmitter dopamine, which alters the dynamic equilibrium between neuronal phosphatases and kinases. A high degree of cross-talk between membrane-initiated signalling pathways and the classical intracellular signalling pathways mediates hormone-dependent behaviour in mammals. The molecular mechanisms, by which a multitude of signals converge with steroid receptors to delineate a genomic level of cross-talk in brain and behaviour are discussed.

Signaling mechanisms in progesterone-neurotransmitter interactions.

Ovarian steroid hormones, estradiol and progesterone, modulate neuroendocrine functions in the CNS resulting in alterations in physiology in female mammals. Classical model of steroid hormone action assumes that these neural effects are predominantly mediated via their intracellular receptors functioning as "ligand-dependent" transcription factors in the steroid-sensitive neurons regulating genes and genomic networks with profound behavioral consequences. Steroid receptors are phosphoproteins and steroid hormone-dependent, receptor-mediated transcription is dependent on the state of phosphorylation of the cognate receptors and/or their co-regulator proteins. Studies from our laboratory have demonstrated that in addition to the steroid hormones, intracellular steroid receptors can be activated in a "ligand-independent" manner by neurotransmitters that can alter the dynamic equilibrium between neuronal phosphatases and kinases. Using biochemical and molecular approaches we have elucidated that the signaling cascade initiated by neurotransmitter, dopamine, converges with steroid hormone-initiated pathway to regulate neuroendocrine pathways associated with reproductive behavior. Signal transduction via protein phosphorylation is common to the molecular pathways through which steroid hormones and neurotransmitters mediate their physiological effects in the CNS involving a high degree of cross-talk and reinforcement among rapid, membrane-initiated pathways at the G-protein level and the classical intracellular signaling pathways at the transcriptional level in mammals. The molecular mechanisms, by which a multitude of signals converge with steroid receptors to delineate a genomic level of cross-talk, provide new avenues for understanding the role of steroid hormones in brain and behavior.

Induction of Fos in the accessory olfactory system by male odors persists in female mice with a null mutation of the aromatase (cyp19) gene.

The ability of odors from soiled male bedding to induce neuronal Fos-immunoreactivity (IR) in sensory neurons located in both the apical and basal zones of the vomeronasal organ (VNO) and in two segments of the VNO-projection pathway, the anterior nucleus of the medial amygdala and the bed nucleus of the stria terminalis (BNST), was significantly reduced in adult, ovariectomized, estrogen-treated female mice with a homozygous null mutation of the cyp19 gene (ArKO) which encodes the estrogen biosynthetic P450 enzyme, aromatase. However, a significant odor-induced activation of Fos-IR was seen in other segments of the VNO-projection pathway of ArKO females, including the accessory olfactory bulb (AOB) granule cell layer, the posterior-dorsal medial amygdala (MePD), and the medial preoptic area (MPA). These results suggest that the VNO/accessory olfactory pathway to the hypothalamus was functional in ArKO females even though they had presumably been exposed to less estrogenic stimulation than wild-type (WT) control females throughout development and until the time that estrogen treatment was begun in adulthood. Thus, the hypothesis of Toran-Allerand [Prog. Brain Res. 61 (1984) 63] that female-typical features of neuroendocrine and behavioral function require perinatal exposure to estrogen was not supported, at least for the VNO/accessory olfactory system.

Progesterone receptor and dopamine receptors are required in Delta 9-tetrahydrocannabinol modulation of sexual receptivity in female rats.

Ovarian steroids, estrogen and progesterone, influence the sensitivity of certain neural processes to cannabinoid treatment by modulation of brain dopaminergic activity. We examined the effects of the active ingredient of cannabis, Delta(9)-tetrahydrocannabinol (THC), on sexual behavior in female rats and its influence on steroid hormone receptors and neurotransmitters in the facilitation of sexual receptivity. Our results revealed that the facilitatory effect of THC was inhibited by antagonists to both progesterone and dopamine D(1) receptors. To test further the idea that progesterone receptors (PR) and/or dopamine receptors (D(1)R) in the hypothalamus are required for THC-facilitated sexual behavior in rodents, antisense and sense oligonucleotides to PR and D(1)R were administered intracerebroventricularly (ICV) into the third cerebral ventricle of ovariectomized, estradiol benzoate-primed rats. Progesterone- and THC-facilitated sexual behavior was inhibited in animals treated with antisense oligonucleotides to PR or to D(1)R. Antagonists to cannabinoid receptor-1 subtype (CB(1)), but not to cannabinoid receptor-2 subtype (CB(2)) inhibited progesterone- and dopamine-facilitated sexual receptivity in female rats. Our studies indicate that THC acts on the CB(1) cannabinoid receptor to initiate a signal transduction response that requires both membrane dopamine and intracellular progesterone receptors for effective induction of sexual behavior.

Solar photo-oxidative disinfection of drinking water: preliminary field observations.

The feasibility of using solar photo-oxidation to inactivate faecal bacterial contaminants in drinking water has been evaluated under field conditions in India and South Africa. Freshly drawn samples from all six test water sources were low in dissolved oxygen, at 13-40% of the air saturation value. However, vigorous mixing followed by exposure to full-strength sunlight in transparent plastic containers (1-25 l capacity) caused a rapid decrease in the counts of faecal indicator bacteria, giving complete inactivation within 3-6 h, with no evidence of reactivation. These results demonstrate that solar photo-oxidation may provide a practical, low-cost approach to the improvement of drinking water quality in developing countries with consistently sunny climates.

Requirement for DARPP-32 in progesterone-facilitated sexual receptivity in female rats and mice.

DARPP-32, a dopamine- and adenosine 3',5'-monophosphate (cAMP)-regulated phosphoprotein (32 kilodaltons in size), is an obligate intermediate in progesterone (P)-facilitated sexual receptivity in female rats and mice. The facilitative effect of P on sexual receptivity in female rats was blocked by antisense oligonucleotides to DARPP-32. Homozygous mice carrying a null mutation for the DARPP-32 gene exhibited minimal levels of P-facilitated sexual receptivity when compared to their wild-type littermates. P significantly increased hypothalamic cAMP levels and cAMP-dependent protein kinase activity. These increases were not inhibited by a D1 subclass dopamine receptor antagonist. P also enhanced phosphorylation of DARPP-32 on threonine 34 in the hypothalamus of mice. DARPP-32 activation is thus an obligatory step in progestin receptor regulation of sexual receptivity in rats and mice.

Reduction of mucin-1 expression during the receptive phase in the rat uterus.

Mucin-1 (Muc1), an integral membrane mucin, is expressed on the apical surface of uterine epithelial cells (UE) of various species. Loss of Muc1 is believed to be necessary for embryo attachment. Muc1 expression is markedly reduced in luminal epithelia during the receptive phase in mice, baboons, and pigs. In the present study, we examined Muc1 expression during the rat estrous cycle and at Day 5 of pregnancy, the time of embryo attachment. In contrast to findings in the mouse, indirect immunofluorescence revealed that uterine Muc1 protein expression was unaltered during the estrous cycle. However, similar to what is observed in the mouse and other species, Muc1 protein decreased at Day 5 of pregnancy in luminal UE. The decrease in Muc1 expression was specific to luminal UE and did not occur in glandular UE. A partial cDNA corresponding to the cytoplasmic tail region of rat Muc1 was generated by a reverse transcription-polymerase chain reaction (RT-PCR) strategy. This cDNA sequence is 89% and 91% identical to the corresponding region of mouse Muc1 at the nucleotide and amino acid levels, respectively. The predicted sequence of rat Muc1 protein has 70-90% identity to the Muc1 protein sequence obtained in other species. Semiquantitative RT-PCR experiments indicated that the mRNA encoding rat Muc1 decreased 57% at Day 5 as compared with the levels found at estrus. This value included mRNA from both luminal and glandular UE and so may underestimate the relative decrease in mRNA in the luminal compartment. In conclusion, we have determined that the levels of rat Muc1 protein and mRNA decrease in the luminal UE at the time of implantation, a pattern similar to that seen in the mouse, baboon, and pig. This supports the general theory that reduction of Muc1 expression is necessary for embryo implantation.

Progesterone receptor function from a behavioral perspective.

Hormonal induction of sexual receptivity in ovariectomized female mice can be effectively reinstated by sequential administration of estradiol and progesterone. In this regard, mice appear to be similar to other rodents. While it is generally accepted that hypothalamic progesterone receptors function as estradiol-induced transcription factors in the induction of sexual receptivity in rats, hamsters, and guinea pigs, relatively little is known about their role in the mouse, a species which exhibits genotypic and strain differences in the responsiveness to steroid hormones. Using a transgenic mouse carrying a null mutation for the progesterone receptor by gene targeting, we examined the role of the progesterone receptor as a coordinator of key regulatory events in the induction of sexual receptivity. A concordance between hypothalamic progesterone receptor levels and behavioral responsiveness was established by comparing the homozygous mutant, heterozygous mutant, and wild-type littermates. The behavioral and biochemical findings reveal the importance of estradiol-induced progesterone receptors for the expression of sexual behavior in female mice. The behavioral response of the two parental mouse strains from which the recombinant genotype was generated was also examined. As an extension of our earlier studies on the ligand-independent activation of progesterone receptors by neurotransmitters, the behavioral effect of dopamine in the facilitation of sexual receptivity in mice was also examined. The studies provide further evidence that steroid hormone receptors function as general transcription factors to achieve the integration of neural information in the central nervous system, and they assign a more important role for progesterone receptors than hitherto envisioned.

Dopamine requires the unoccupied progesterone receptor to induce sexual behavior in mice.

Using the recently generated mutant mice strain (PRKO) carrying a null mutation for the progesterone receptor (PR) gene by gene targeting, we examined the critical role of PR as a coordinator of key regulatory events involved in the steroid hormone and dopamine-facilitated sexual behavior in female mice. In vitro one-point binding analyses of estradiol benzoate (EB)-induced cellular PRs and immunohistochemistry of PR in the mediobasal hypothalamus demonstrated a reduction in binding in the homozygous females, equivalent to background levels seen in EB-unresponsive tissue. The biochemical findings correlated well with the behavioral observations, with the wild type females exhibiting high levels of lordosis, while the homozygous females showed minimal lordosis in response to mating by male mice. As a critical validation of our earlier studies on ligand-independent activation of PRs by dopamine, we examined the facilitation of sexual behavior by a dopamine agonist in the null mutants. Wild type females having the full complement of PRs exhibited high levels of lordosis, while the homozygous females showed minimal lordosis in response to dopamine. To determine whether this reduced response was due to a general lack of ability to express lordosis, mice were treated with another neurotransmitter, serotonin. No significant difference in the serotonin-facilitated lordosis response was observed between the wild type and the homozygous females. We conclude that multiple signal transduction pathways coexist in the neuroendocrine system for reproductive behavior, with PR acting as a transcriptional mediator for dopamine, as well as progesterone, to achieve integration of neural communication in the central nervous system.

Mice lacking progesterone receptor exhibit pleiotropic reproductive abnormalities.

Although progesterone has been recognized as essential for the establishment and maintenance of pregnancy, this steroid hormone has been recently implicated to have a functional role in a number of other reproductive events. The physiological effects of progesterone are mediated by the progesterone receptor (PR), a member of the nuclear receptor superfamily of transcription factors. In most cases the PR is induced by estrogen, implying that many of the in vivo effects attributed to progesterone could also be the result of concomitantly administered estrogen. Therefore, to clearly define those physiological events that are specifically attributable to progesterone in vivo, we have generated a mouse model carrying a null mutation of the PR gene using embryonic stem cell/gene targeting techniques. Male and female embryos homozygous for the PR mutation developed normally to adulthood. However, the adult female PR mutant displayed significant defects in all reproductive tissues. These included an inability to ovulate, uterine hyperplasia and inflammation, severely limited mammary gland development, and an inability to exhibit sexual behavior. Collectively, these results provide direct support for progesterone's role as a pleiotropic coordinator of diverse reproductive events that together ensure species survival.

Inhibition of rat sexual behavior by antisense oligonucleotides to the progesterone receptor.

To test further the idea that sexual behavior in rodents is mediated via the progesterone receptor (PR) in the ventromedial nucleus of the hypothalamus, antisense and sense oligonucleotides to progesterone receptor were administered intracerebroventricularly into the third cerebral ventricle of ovariectomized estrogen-primed animals. Progesterone-facilitated sexual behavior was inhibited in animals treated with antisense oligonucleotides, with proceptive and receptive responses being minimal or completely suppressed. Sexual behavior was not altered by control sense oligonucleotides. In vitro binding assays of the cytosol progesterone receptors demonstrated a 52.2% reduction of PRs in the hypothalamus of animals that received antisense oligonucleotides, suggesting a reduction in PR synthesis. These data suggest that a threshold level of estrogen-induced hypothalamic PR is critical in the regulation of progesterone-facilitated sexual behavior in female rats.

Convergent pathways for steroid hormone- and neurotransmitter-induced rat sexual behavior.

Estrogen and progesterone modulate gene expression in rodents by activation of intracellular receptors in the hypothalamus, which regulate neuronal networks that control female sexual behavior. However, the neurotransmitter dopamine has been shown to activate certain steroid receptors in a ligand-independent manner. A dopamine receptor stimulant and a D1 receptor agonist, but not a D2 receptor agonist, mimicked the effects of progesterone in facilitating sexual behavior in female rats. The facilitory effect of the neurotransmitter was blocked by progesterone receptor antagonists, a D1 receptor antagonist, or antisense oligonucleotides to the progesterone receptor. The results suggest that in rodents neurotransmitters may regulate in vivo gene expression and behavior by means of cross-talk with steroid receptors in the brain.

Nitric oxide mediates sexual behavior in female rats.

Nitric oxide (NO), an active free radical formed during the conversion of arginine to citrulline by the enzyme NO synthase (NOS), mediates vasorelaxation, cytotoxicity, and neurotransmission. Neurons containing NOS (NOergic) are located in the hypothalamus. These NOergic neurons control the release of several hypothalamic peptides. Release of NO from these NOergic neurons stimulates pulsatile release of luteinizing hormone-releasing hormone (LHRH) in vivo and LHRH release in vitro. LHRH not only induces LH release, which induces ovulation, but also facilitates female sexual behavior. Sexual behavior can be induced reliably in estrogen-primed ovariectomized female rats by progesterone (P). This behavior consists of proceptive behavior to attract the male and the assumption of a clear characteristic posture, lordosis, when mounted by the male. To ascertain the role of NO in the control of sexual behavior in female rats, an inhibitor of NOS, NG-monomethyl-L-arginine was microinjected into the third cerebral ventricle (3V) of conscious, ovariectomized, estrogen-primed rats with indwelling cannulae. NG-Monomethyl-L-arginine (10-1000 micrograms) prevented P-facilitated lordosis when administered intracerebroventricularly into the 3V, 20 min prior to the 3V injection of P. NG-Monomethyl-D-arginine, which does not inhibit NOS, did not inhibit lordosis under the same experimental conditions. Microinjection into the 3V of sodium nitroprusside (SNP), which spontaneously releases NO, facilitated lordosis in estrogen-primed rats in the absence of P. The facilitation of lordosis induced by either P or SNP was prevented by intracerebroventricular injection of hemoglobin, which binds NO. Lordosis facilitated by P or SNP was blocked by injection of LHRH antiserum into the 3V. The results are interpreted to mean that the P-facilitated lordosis response is mediated by LHRH release. Furthermore, since NO release from SNP also facilitates lordosis in the absence of P and this response could be blocked by LHRH antiserum, we conclude that P brings about the release of NO, which stimulates LHRH release that facilitates lordosis. Thus, the results indicate that NO induces LHRH release and that LHRH then plays a crucial role in mediation of sexual behavior in the female rats.

Initiation and maintenance of in vitro decidualization are independent of hormonal sensitization in vivo.

The effects of in vivo hormonal sensitization on the competence of uterine stromal (US) cells to decidualize in vitro were assessed. In vitro differentiation of uterine stroma isolated from Day 4 pregnant rats, sensitized to respond to a decidual stimulus, was compared to that in nonsensitized immature, castrated or cycling rats. The initiation of in vitro decidualization--as monitored by the expression of the decidual markers desmin and laminin in rat US cells--was independent of the hormonal status of the animal from which the cells were isolated and occurred in the absence of serum in the medium. Differentiation was accelerated in high-density cultures where contact inhibition suppressed proliferation and decreased the extent of cell growth. The extent to which in vitro decidualization imitates in vivo stromal cell differentiation was assessed by comparing decidualization in the rabbit, a species with only a limited decidual cell response, and in the rat. US cells isolated from nonpregnant rabbits differentiated in vitro by expressing laminin, but not desmin. Indirect immunofluorescence of frozen uterine sections from pregnant and nonpregnant rabbits validated in vitro differentiation as a faithful reflection of the in vivo program of decidualization. Although the program of US cell differentiation may vary between the species, initiation of differentiation in vitro appeared to be independent of hormonal preparation in vivo for both the species examined.