A reporter assay for target validation in primary neuronal cultures.

The deposition of beta-amyloid peptides (Abeta42 and Abeta40) in neuritic plaques is one of the hallmarks of Alzheimer's disease (AD), and genes modulating their brain levels and neuronal effects could result in future disease modifying therapies. The causal association of candidate targets with AD is of paramount importance in current drug discovery, as a lack of efficacy of many candidate drugs is often due to inadequate validation of their pharmacological target. In Alzheimer's as well as in other neurodegenerative diseases, in vitro target validation is hampered by the difficulty of transfecting primary neuronal cultures and assaying the effects of genes on neuronal viability. Here we describe a rapid, sensitive and simple reporter-based assay for the validation of genes putatively associated with Abeta-mediated neurotoxicity, which can in principle be extended to the validation of targets in the context of other neuronal insults. The assay is suitable for the generation of robust and reproducible data in primary neuronal cultures allowing the dissection at a molecular level of complex pathways activated by the toxic insult in a cellular context that more closely represents the real disease situation.

Delta-CHr improves the identification of anemic syndromes and the evaluation of hemoglobin synthesis.

Reticulocyte hemoglobin content (CHr) is considered an index of iron status, helpful in the differential diagnosis of microcytoses. Its potential can be enhanced by comparing CHr dynamic reference values (CHr-e: expected CHr), which are proportional to the MCVr variations occurring in micro- or macrocytosis, with measured CHr values. We demonstrate that the difference between measured CHr and CHr-e (DeltaCHr) is helpful to differentiate the anemic syndromes and, in particular, beta-thalassemia vs. presumable sideropenia. DeltaCHr can also indicate when to interrupt iron supplementation. DeltaCHr allows an insight into the erythropoiesis of thalassemic and sideropenic subjects, pointing out the reduced hemoglobin production and ineffective erythroid activity in these conditions.

Engineering of a mouse for the in vivo profiling of estrogen receptor activity.

In addition to their well known control of reproductive functions, estrogens modulate important physiological processes. The identification of compounds with tissue-selective activity will lead to new drugs mimicking the beneficial effects of estrogen on the prevention of osteoporosis and cardiovascular or neurodegenerative diseases, while avoiding its detrimental proliferative effects. As an innovative model for the in vivo identification of new selective estrogen receptor modulators (SERMs), we engineered a mouse genome to express a luciferase reporter gene ubiquitously. The constructs for transgenesis consist of the reporter gene driven by a dimerized estrogen-responsive element (ERE) and a minimal promoter. Insulator sequences, either matrix attachment region (MAR) or beta-globin hypersensitive site 4 (HS4), flank the construct to achieve a generalized, hormoneresponsive luciferase expression. In the mouse we generated, the reporter expression is detectable in all 26 tissues examined, but is induced by 17beta-estradiol (E2) only in 15 of them, all expressing estrogen receptors (ERs). Immunohistochemical studies show that in the mouse uterus, luciferase and ERs colocalize. In primary cultures of bone marrow cells explanted from the transgenic mice and in vivo, luciferase activity accumulates with increasing E(2) concentration. E2 activity is blocked by the ER full antagonist ICI 182,780. Tamoxifen shows partial agonist activity in liver and bone when administered to the animals. In the mouse system here illustrated, by biochemical, immunohistochemical, and pharmacological criteria, luciferase content reflects ER transcriptional activity and thus represents a novel system for the study of ER dynamics during physiological fluctuations of estrogen and for the identification of SERMs or endocrine disruptors.

Estrogen prevents the lipopolysaccharide-induced inflammatory response in microglia.

After neuronal injury and in several neurodegenerative diseases, activated microglia secrete proinflammatory molecules that can contribute to the progressive neural damage. The recent demonstration of a protective role of estrogen in neurodegenerative disorders in humans and experimental animal models led us to investigate whether this hormone regulates the inflammatory response in the CNS. We here show that estrogen exerts an anti-inflammatory activity on primary cultures of rat microglia, as suggested by the blockage of the phenotypic conversion associated with activation and by the prevention of lipopolysaccharide-induced production of inflammatory mediators: inducible form of NO synthase (iNOS), prostaglandin-E(2) (PGE(2)), and metalloproteinase-9 (MMP-9). These effects are dose-dependent, maximal at 1 nm 17beta-estradiol, and can be blocked by the estrogen receptor (ER) antagonist ICI 182,780. The demonstration of ERalpha and ERbeta expression in microglia and macrophages and the observation of estrogen blockade of MMP-9 mRNA accumulation and MMP-9 promoter induction further support the hypothesis of a genomic activity of estrogen via intracellular receptors. This is the first report showing an anti-inflammatory activity of estrogen in microglia. Our study proposes a novel explanation for the protective effects of estrogen in neurodegenerative and inflammatory diseases and provides new molecular and cellular targets for the screening of ER ligands acting in the CNS.

Estrogen blocks inducible nitric oxide synthase accumulation in LPS-activated microglia cells.

Estrogens are thought to play a protective role against neurodegeneration through a variety of mechanisms including the activation of growth factors and neurotransmitter synthesis, the control of synaptic plasticity and functions, and the blockade of oxidative reactions. We here propose a novel mechanism to explain the neuroprotective effects of estradiol by showing that estrogens may antagonize nitric oxide synthase activity and reduce the accumulation of nitrites and nitrates consequent to various inflammatory stimuli. The potential anti-inflammatory activity of estradiol is analyzed in vitro in cells in culture including primary cultures of microglia and in vivo in a well-known model of inflammation.

Oestrogen prevention of neural cell death correlates with decreased expression of mRNA for the pro-apoptotic protein nip-2.

We have recently identified nip-2 as a gene target for 17beta-oestradiol activity in the neuroblastoma SK-ER3 cells expressing the oestrogen receptor (ER) alpha. Here we show 17beta-oestradiol treatment of neuroblastoma and rat embryo neurones in culture blocks the increase in nip-2 mRNA induced by apoptotic stimuli and prevents cell death as indicated by cell counting, 3,(4,5-dimethylthiazol-2-yl)2,5-diphenil-tetrazoliumbromi de and DNA fragmentation assays. Neither of these effects are observed in the presence of the specific ER antagonist ICI 182,780, and are absent in neuroblastoma cells not expressing ER. We propose that nip-2 plays a relevant role in neural cell apoptosis and that a decrease in its expression is instrumental for the oestrogen anti-apoptotic effect described here. The experimental evidence presented supports the recent hypothesis of a protective role of oestrogens in neurodegenerative diseases such as Alzheimer's disease and highlights the importance of the development of new ER ligands for the prevention of neural cell damage.

Estradiol induces differential neuronal phenotypes by activating estrogen receptor alpha or beta.

Estrogens are female sex steroids that have a plethora of effects on a wide range of tissues. These effects are mediated through two well characterized intracellular receptors: estrogen receptor alpha and beta (ERalpha and ERbeta, respectively). Because of their high structural homology, it has been argued whether these two receptors may elicit differential biochemical events in estrogen target cells. Here we examine the effect of 17beta-estradiol-dependent activation of ERalpha and ERbeta on neurite sprouting, a well known consequence of this sex hormone action in neural cells. In SK-N-BE neuroblastoma cells transfected with ERalpha or ERbeta, 17beta-estradiol induces two distinct morphological phenotypes. ERalpha activation results in increased length and number of neurites, whereas ERbeta activation modulates only neurite elongation. By the use of chimeric receptors we demonstrate that the presence of both transcription activation functions located in the NH2-terminus and COOH-terminus of the two ER proteins are necessary for maintaining the differential biological activity reported. ERalpha-dependent, but not ERbeta-dependent, morphological changes are observed only in the presence of the active form of the small G protein Rac1B. Our data provide the first clear evidence that, in a given target cell, ERalpha and ERbeta may play distinct biological roles and support the hypothesis that 17beta-estradiol activates selected intracellular signaling pathways depending on the receptor subtype bound.

Expression of the estrogen-regulated gene Nip2 during rat brain maturation.

The present study stems from previous observations demonstrating that in the neuroblastoma cell line SK-ER3 the mRNA content of the pro-apoptotic gene Nip2 is decreased following treatment with estradiol. We investigate the content of Nip2 mRNA during the maturation of rat embryo brain and we show that Nip2 mRNA is very low at embryo day 15 and steadily increases up to day 20. At day 21 Nip2 mRNA is decreased almost to the low levels observed in the mature brain. Studies in neurons from rat embryo at day 18 show that Nip2 mRNA content is significantly decreased by exposure to estradiol at 1 nM concentration demonstrating that the observations previously done in the SK-ER3 neuroblastoma cell line can be reproduced in neurons in culture. The finding that estrogens may modulate the activity of Nip2 gene activity may be of relevance not only with regard to the effects of estradiol on brain maturation, but also for the understanding of the neuroprotective effects recently described for this hormone.

Identification of estrogen target genes in human neural cells.

In mammals, estrogens have a multiplicity of effects ranging from control of differentiation of selected brain nuclei, reproductive functions, sexual behavior. In addition, these hormones influence the manifestation of disorders like depression and Alzheimer's. Study of the cells target for the hormone has shown that estrogen receptors (ERs) are expressed in all known neural cells, including microglia. In view of the potential interest in the use of estrogens in the therapy of several pathologies of the nervous system, it would be of interest to fully understand the mechanism of estrogen activity in the various neural target cells and get an insight on the molecular means allowing the hormone to display such a variety of effects. We have proposed the use of a reductionist approach for the systematic understanding of the estrogen activities in each specific type of target cell. Thus, we have generated a model system in which to study the activation of one of the known (ERs), estrogen receptor alpha. This system allowed us to identify a number of novel genes which expression may be influenced following the activation of this receptor subtype by estradiol (E(2)). We here report on data recently obtained by the study of one of these target genes, nip2, which encodes a proapoptotic protein product. We hypothesize that nip2 might be an important molecular determinant for estrogen anti-apoptotic activity in cells of neural origin and represents a potential target for drugs aimed at mimicking the E(2) beneficial effects in neural cells.

Estrogen and progesterone induction of survival of monoblastoid cells undergoing TNF-alpha-induced apoptosis.

Induction of apoptosis of mononucleated cells is a physiological process for regulating the intensity of the immune response. The female steroid hormones estrogen (E2) and progesterone (Prog) are known to modulate the reactivity of the immune system; recently it has been demonstrated that they can regulate induction of apoptosis of endothelial cells and osteoblasts. TNF-alpha-mediated induction of apoptosis has been well characterized in myeloid cells. We investigated whether E2 and Prog could interfere with TNF-alpha-induced apoptosis of the monoblastoid U937 cell line. Treatment with E2 or Prog increased survival and prevented apoptosis induced by TNF-alpha in both undifferentiated and macrophage-like PMA-differentiated U937 cells, as assessed by trypan blue exclusion cell counting, thymidine incorporation, AnnexinV labeling, followed by flow cytometry and DNA fragmentation studies. This effect can be associated with the activation of specific hormone receptors, since we observed the expression of the estrogen receptor alpha (ER-alpha), ER-beta, and progesterone receptor (PR) mRNAs; the ER-alpha protein expression was confirmed by immunocytochemical analysis. In addition, hormone-mediated survival against apoptosis was concentration dependent, reaching the half-maximal effect at 10 nM and blocked by the ER antagonist ICI 182,780 in undifferentiated cells, further supporting a receptor-mediated mechanism of cell survival. Other steroid receptor drugs such as Raloxifene, RU486, or the ICI 182,780 in PMA-differentiated cells displayed agonist activity by preventing TNF-alpha-induced apoptosis as efficiently as the hormones alone, providing further evidence to the notion that steroid receptor drugs may manifest agonist or antagonist activities depending on the cellular context in which they are studied. Treatment with E2 was also associated with a time-dependent decrease in the mRNA level of the proapoptotic Nip-2 protein, supporting the hypothesis that hormone responsiveness of U937 cells is mediated by target gene transcription. Together, these results demonstrate that ER and PR can be activated by endogenous or exogenous ligands to induce a genetic response that impairs TNF-alpha-induced apoptosis in U937 cells. The data presented here suggest that the female steroid receptors play a role in regulation of the immune response by preventing apoptosis of monoblastoid cells; this effect might have important consequences in the clinical use of steroid receptor drugs. --Vegeto, E., Pollio, G., Pellicciari, C., Maggi, A. Estrogen and progesterone induction of survival of monoblastoid cells undergoing TNF-alpha-inuced apoptosis.

The novel progesterone receptor antagonists RTI 3021-012 and RTI 3021-022 exhibit complex glucocorticoid receptor antagonist activities: implications for the development of dissociated antiprogestins.

We have identified two novel compounds (RTI 3021-012 and RTI 3021-022) that demonstrate similar affinities for human progesterone receptor (PR) and display equivalent antiprogestenic activity. As with most antiprogestins, such as RU486, RTI 3021-012, and RTI 3021-022 also bind to the glucocorticoid receptor (GR) with high affinity. Unexpectedly, when compared with RU486, the RTI antagonists manifest significantly less GR antagonist activity. This finding indicates that, with respect to antiglucocorticoid function, receptor binding affinity is not a good predictor of biological activity. We have determined that the lack of a clear correlation between the GR binding affinity of the RTI compounds and their antagonist activity reflects the unique manner in which they modulate GR signaling. Previously, we proposed a two step "active inhibition" model to explain steroid receptor antagonism: 1) competitive inhibition of agonist binding; and 2) competition of the antagonist bound receptor with that activated by agonists for DNA response elements within target gene promoters. Accordingly, we observed that RU486, RTI 3021-012, and RTI 3021-022, when assayed for PR antagonist activity, accomplished both of these steps. Thus, all three compounds are "active antagonists" of PR function. When assayed on GR, however, RU486 alone functioned as an active antagonist. RTI 3021-012 and RTI 3021-022, on the other hand, functioned solely as "competitive antagonists" since they were capable of high affinity GR binding, but the resulting ligand receptor complex was unable to bind DNA. These results have important pharmaceutical implications supporting the use of mechanism based approaches to identify nuclear receptor modulators. Of equal importance, RTI 3021-012 and RTI 3021-022 are two new antiprogestins that may have clinical utility and are likely to be useful as research reagents with which to separate the effects of antiprogestins and antiglucocorticoids in physiological systems.

Oligonucleotide squelching reveals the mechanism of estrogen receptor autologous down-regulation.

Antisense oligos complementary to the 5'-end, but not to the 3'-end, of the estrogen receptor (ER) messenger RNA caused a paradox accumulation of ER protein in MCF-7 cells. The same effect was observed after treatment of the cells with the corresponding sense oligos. The oligos interfering with ER down-regulation were demonstrated to specifically bind the ER with affinities in the nanomolar range. It is, therefore, proposed that the ER up-regulation induced by the oligos might be due to squelching of the ER (or ER-inducible proteins) from their binding site located in the 5'-end of the ER gene. We also report that transcriptionally inactive ER mutants can undergo down-regulation, and that in denaturing gels, the migration profile of ER-oligo and ER-estrogen-responsive element complexes are dissimilar. We, therefore, propose that ER can interact with DNA in different ways and at different binding sites. These observations might have important pharmacological consequences, since specific drugs could be devised to induce the ER conformation necessary to perform only selected tasks of the ER transcriptional repertoire.

SK-ER3 neuroblastoma cells as a model for the study of estrogen influence on neural cells.

The neuroblastoma SK-ER3 cell line obtained by stable transfection of the human SK-N-BE cell line is proposed as a model for the study of estrogen receptor activity in cells of neural origin. In the SK-ER3 cell line the estrogen receptor, once activated, initiates a differentiation program leading to growth arrest, morphological changes, and acquisition of the dopaminergic phenotype. In the absence of estrogens, this program can be triggered by IGF-I, which can activate the unliganded estrogen receptor via the ras-pathway. It is proposed that this model system might recapitulate the events occurring in vivo during the differentiation of the nervous system and that IGF-I may play an important role in the activation of estrogen receptor at the very early stage of brain development affecting the differentiation of a number of hypothalamic and extrahypothalamic brain regions.

Mapping and characterization of the functional domains responsible for the differential activity of the A and B isoforms of the human progesterone receptor.

In humans, the biological response to progesterone is mediated by two distinct forms of the progesterone receptor (human (h) PR-A, 94 kDa and hPR-B, 114 kDa). These two isoforms are transcribed from distinct estrogen-inducible promoters within a single copy PR gene; the only difference between them is that the first 164 amino acids of hPR-B (B-upstream sequence) are absent in hPR-A. In most cell lines such as MCF-7 (human breast cancer cells), CV-1 (monkey kidney fibroblasts), and HeLa (human cervical carcinoma cells), hPR-A functions as a transcriptional repressor, whereas hPR-B functions as a transcriptional activator of progesterone-responsive genes. Interestingly, in these cell contexts, hPR-A also acts as a trans-dominant repressor of the transcriptional activity of other steroid hormone receptors. In contrast to hPR-A, which functions predominantly as a ligand-dependent transcriptional repressor, we show in this study that the A isoform of the chicken PR (cPR-A) lacks this trans-dominant repressor function and is a transcriptional activator in all contexts examined. By constructing chimeras between the N-terminal domains of the chicken and human PR, we mapped the trans-dominant repressor function of hPR-A to the first 140 amino acids of the protein. Notably, when this 140-amino acid "repressor" domain is placed onto chicken PR-A, the activity of the latter changes from a transcriptional activator to a repressor. Interestingly, however, this "repressor domain" is necessary, but not sufficient, for trans-repression as it is inactive when it is tethered to a heterologous protein. This suggests that the trans-repression function is comprised not only of the repressor domain of hPR-A but also requires the context of the receptor to function. The identification of a discrete inhibitory region within hPR-A which is transferable to another receptor implies that this region interacts with a set of transcription factors or adaptors that are distinct from those recognized by hPR-B, the identification of which will be required to define the mechanism by which hPR-A modulates steroid hormone receptor transcriptional activity. Thus, although chickens and humans both produce two very similar forms of the progesterone receptor, it is clear from these studies that the mechanism of action of progesterone in these two systems is quite different.

16 alpha-substituted analogs of the antiprogestin RU486 induce a unique conformation in the human progesterone receptor resulting in mixed agonist activity.

Previously, we have shown that agonists and antagonists interact with distinct, though overlapping regions within the human progesterone receptor (hPR) resulting in the formation of structurally different complexes. Thus, a link was established between the structure of a ligand-receptor complex and biological activity. In this study, we have utilized a series of in vitro assays with which to study hPR pharmacology and have identified a third class of hPR ligands that induce a receptor conformation which is distinct from that induced by agonists or antagonists. Importantly, when assayed on PR-responsive target genes these compounds were shown to exhibit partial agonist activity; an activity that was influenced by cell context. Thus, as has been shown previously for estrogen receptor, the overall structure of the ligand-receptor complex is influenced by the nature of the ligand. It appears, therefore, that the observed differences in the activity of some PR and estrogen receptor ligands reflect the ability of the cellular transcription machinery to discriminate between the structurally different complexes that result following ligand interaction. These data support the increasingly favored hypothesis that different ligands can interact with different regions within the hormone binding domains of steroid hormone receptors resulting in different biologies.

Cross-coupling between insulin and estrogen receptor in human neuroblastoma cells.

Insulin is a well known mitotic agent for neuroblastoma cells. Human SK-N-BE neuroblastoma cells stably transfected with the estrogen receptor, however, undergo growth arrest and differentiation when treated with insulin. These effects were shown to be due to an insulin-dependent activation of the unliganded estrogen receptor. Here, we demonstrate that this activation involves the AF-2 COOH-terminal domain of the estrogen receptor and that the communication between estrogen and insulin receptor systems occurs via selected and specific transduction signals. In fact, by the use of dominant negative and dominant positive mutants we demonstrate that p21ras is essential for insulin and estrogen receptor coupling. With pharmacological tools, we prove that PI 3'kinase does not contribute to this cross-talk and that protein kinase C triggers transduction signals that act in synergism with p21ras. These results prove the intricacy of all these intracellular paths of communication. The finding that, in neuroblastoma cells, selected signal transduction systems are involved in the insulin-dependent activation of estrogen receptor is of particular interest considering that estrogen receptor might restrict the role played by insulin during the differentiation of neural cells and interfere with its proliferative potential while allowing its regulation of other functions related to cell survival.

Expression of early genes in estrogen induced phenotypic conversion of neuroblastoma cells.

Estrogens are known to modulate the growth rate and differentiation state of a number of cells. In uterine, as well as in mammary tumor cells, estrogen-dependent proliferation and differentiation are correlated to a series of biochemical responses, including increased expression of proto-oncogenes such as: c-fos, c-jun and c-myc. Since estrogens were shown to regulate the proliferation and the differentiation state of cells of nervous origin, the aim of the present study was to investigate whether these effects were associated to changes in the expression of early genes. In the model system utilized, the human cell line SK-ER3, an increase in c-fos mRNA and Fos protein without change of c-jun and related genes mRNA concentration was observed after short term treatment with 17 beta-estradiol (E2). A significant decrease of c-fos, c-jun and jun-D proto-oncogene mRNA levels were found after prolonged hormonal treatment. The exposure to the hormone did not determine any change in N-myc expression. Since the three protooncogene mRNAs are rapidly induced following estrogen treatment in other cell systems and target tissues, it is concluded that the estrogen-induced differentiation of neuroblastoma cells is correlated to a pattern of expression of early genes that might be peculiar for the activity of this hormone in neural cells.

Specificity of action of a herpes virus VP16/tetracycline-dependent trans-activator in mammalian cell cultures.

In this work, we have studied the activity of a tetracycline modulatable trans-activator (tTA) generated by fusing the DNA binding domain of the tetracycline repressor to the trans-activation domain of the Herpes simplex virus protein 16 (HSV VP16) (plasmid pUHD15-1Neo). In the three different cell lines studied (HTC, rat hepatoma; T47D, human breast cancer; SK-N-BE, human neuroblastoma), the expression of the luciferase gene under the control of a tetracycline operator sequence (plasmid pUHC13-3) was used as a control of the incorporation and the functionality of the trans-activator. Clones selected from these cells responded in a time and dose-dependent manner to the withdrawal of tetracycline. In all these clones, the tTA trans-activator not only modulates the activity of the luciferase gene, but also modulates the activity of a number of endogenous proteins, including C/EBP beta, the glucocorticoid receptor (GR), and SP1. In the transfected cells, the level of these transcription factors was strongly inhibited in the presence of tetracycline and was highly increased after tetracycline removal. Electrophoresis mobility shift assay (EMSA) and footprint experiments proved that the induced proteins are perfectly efficient in binding the DNA. Their transcriptional activity was also determined. In HTC/A9 cells, the level of the chloramphenicol acetyltransferase (CAT) expression driven by the promoter of the alpha 1-glycoprotein (AGP) gene was strongly enhanced at 72-84 hr following removal of tetracycline from the growth media. The accumulation of the endogenous AGP mRNA also increased at 84 hr. In the T47D/TA11 and SK-N-BE/C2.6 cells, a general activation of protein synthesis was also evidenced.

Insulin-like growth factors activate estrogen receptor to control the growth and differentiation of the human neuroblastoma cell line SK-ER3.

The neuroblastoma cell line SK-ER3, which is stably transfected with the estrogen receptor (ER), was used to study the effect of insulin and insulin-like growth factors (IGF-I and IGF-II) on growth and morphological differentiation induced by estrogens. The data demonstrate that insulin and related growth factors control the growth and morphological differentiation of the cell line expressing the ER, but not of the parental cell line. Effects elicited by the growth factors in SK-ER3 cells can be blocked by ER antagonists. Transient transfection studies further confirm an effect of the IGFs in modulation of ER-activated promoters. The results presented support the hypothesis of the existence of cross-talk between membrane and intracellular receptors and provide evidence for physiological consequences of the activation of such a pathway of communication. The present study is of particular interest with regard to the theory of prenatal involvement of the ER in maturation of nerve cells. It could, in fact, be hypothesized that IGF-I and IGF-II, present in high concentrations in the developing brain, might activate the ER expressed in several embryonic brain nuclei.