Nicotinic receptors mediate stress-nicotine detrimental interplay via dopamine cells' activity.

Epidemiological studies report strong association between mood disorders and tobacco addiction. This high comorbidity requires adequate treatment but the underlying mechanisms are unknown. We demonstrate that nicotine exposure, independent of drug withdrawal effects, increases stress sensitivity, a major risk factor in mood disorders. Nicotine and stress concur to induce long-lasting cellular adaptations within the dopamine (DA) system. This interplay is underpinned by marked remodeling of nicotinic systems, causing increased ventral tegmental area (VTA) DA neurons' activity and stress-related behaviors, such as social aversion. Blocking ß2 or α7 nicotinic acetylcholine receptors (nAChRs) prevents, respectively, the development and the expression of social stress-induced neuroadaptations; conversely, facilitating α7 nAChRs activation specifically in the VTA promotes stress-induced cellular and behavioral maladaptations. Our work unravels a complex nicotine-stress bidirectional interplay and identifies α7 nAChRs as a promising therapeutic target for stress-related psychiatric disorders.

BDNF-TrkB signaling through Erk1/2 MAPK phosphorylation mediates the enhancement of fear memory induced by glucocorticoids.

Activation of glucocorticoid receptors (GR) by glucocorticoid hormones (GC) enhances contextual fear memories through the activation of the Erk1/2(MAPK) signaling pathway. However, the molecular mechanism mediating this effect of GC remains unknown. Here we used complementary molecular and behavioral approaches in mice and rats and in genetically modified mice in which the GR was conditionally deleted (GR(NesCre)). We identified the tPA-BDNF-TrkB signaling pathway as the upstream molecular effectors of GR-mediated phosphorylation of Erk1/2(MAPK) responsible for the enhancement of contextual fear memory. These findings complete our knowledge of the molecular cascade through which GC enhance contextual fear memory and highlight the role of tPA-BDNF-TrkB-Erk1/2(MAPK) signaling pathways as one of the core effectors of stress-related effects of GC.

Disruption of the glucocorticoid receptor gene in the nervous system results in reduced anxiety.

The glucocorticoid receptor (Gr, encoded by the gene Grl1) controls transcription of target genes both directly by interaction with DNA regulatory elements and indirectly by cross-talk with other transcription factors. In response to various stimuli, including stress, glucocorticoids coordinate metabolic, endocrine, immune and nervous system responses and ensure an adequate profile of transcription. In the brain, Gr has been proposed to modulate emotional behaviour, cognitive functions and addictive states. Previously, these aspects were not studied in the absence of functional Gr because inactivation of Grl1 in mice causes lethality at birth (F.T., C.K. and G.S., unpublished data). Therefore, we generated tissue-specific mutations of this gene using the Cre/loxP -recombination system. This allowed us to generate viable adult mice with loss of Gr function in selected tissues. Loss of Gr function in the nervous system impairs hypothalamus-pituitary-adrenal (HPA)-axis regulation, resulting in increased glucocorticoid (GC) levels that lead to symptoms reminiscent of those observed in Cushing syndrome. Conditional mutagenesis of Gr in the nervous system provides genetic evidence for the importance of Gr signalling in emotional behaviour because mutant animals show an impaired behavioural response to stress and display reduced anxiety.

Genetic evidence of the programming of beta cell mass and function by glucocorticoids in mice.

AIMS/HYPOTHESIS: Prenatal exposure to excess glucocorticoids associates with low birthweight in rodents, primates and humans and its involvement in programming glucose homeostasis is suspected. Our aim was to further dissect the role of glucocorticoids on beta cell development and function in mice. METHODS: Using the model of maternal general food restriction during the last week of pregnancy, we thoroughly studied in the CD1 mouse-mothers and fetal and adult offspring--the pancreatic, metabolic and molecular consequences of maternal undernutrition associated with excess glucocorticoids. The specific involvement of the glucocorticoid receptor (GR) was studied in mutant fetuses lacking GR in pancreatic precursors or mature beta cells. RESULTS: Maternal general food restriction in the mouse is associated with decreased maternal glucose and increased corticosterone levels. Fetuses from underfed dams had increased corticosterone levels, decreased pancreatic endocrine gene expression but increased exocrine gene expression and a lower beta cell mass. The offspring of these dams had a low birthweight, permanent postnatal growth retardation and, as adults, impaired glucose tolerance, decreased beta cell mass (-50%) and massively reduced islet expression (-80%) of most of the genes involved in beta cell function (e.g. Pdx1, Sur1 [also known as Abcc8], insulin). Moreover, using mutant fetuses lacking GR in pancreatic precursors or beta cells we show that the deleterious effect of undernutrition on fetal beta cell development requires the presence of the GR in pancreatic precursor cells. CONCLUSIONS/INTERPRETATION: These results demonstrate the crucial role of excess fetal glucocorticoids and the importance of GR signalling in progenitor cells to programme beta cell mass and dysfunction.

The enhancement of stress-related memory by glucocorticoids depends on synapsin-Ia/Ib.

The activation of glucocorticoid receptors (GR) by glucocorticoids increases stress-related memory through the activation of the MAPK signaling pathway and the downstream transcription factor Egr-1. Here, using converging in vitro and in vivo approaches, respectively, GR-expressing cell lines, culture of hippocampal neurons, and GR genetically modified mice (GR(NesCre)), we identified synapsin-Ia/Ib as one of the effectors of the glucocorticoid signaling cascade. Stress and glucocorticoid-induced activation of the GR modulate synapsin-Ia/Ib through two complementary mechanisms. First, glucocorticoids driving Egr-1 expression increase the expression of synapsin-Ia/Ib, and second, glucocorticoids driving MAPK activation increase its phosphorylation. Finally, we showed that blocking fucosylation of synapsin-Ia/Ib in the hippocampus inhibits its expression and prevents the glucocorticoid-mediated increase in stress-related memory. In conclusion, our data provide a complete molecular pathway (GR/Egr-1/MAPK/Syn-Ia/Ib) through which stress and glucocorticoids enhance the memory of stress-related events and highlight the function of synapsin-Ia/Ib as molecular effector of the behavioral effects of stress.

Targeted transgenesis at the HPRT locus: an efficient strategy to achieve tightly controlled in vivo conditional expression with the tet system.

The tet-inducible system has been widely used to achieve conditional gene expression in genetically modified mice. To alleviate the frequent difficulties associated with recovery of relevant transgenic founders, we tested whether a controlled strategy of transgenesis would support reliable cell-specific, doxycycline (Dox)-controlled transgene expression in vivo. Taking advantage of the potent hypoxanthine-aminopterin-thymidine selection strategy and an embryonic stem (ES) cell line supporting efficient germ-line transmission, we used hypoxanthine phosphoribosyltransferase (HPRT) targeting to insert a single copy tet-inducible construct designed to allow both glucocorticoid receptor (GR) and beta-galactosidase (beta-Gal) expression. Conditional, Dox-dependent GR and beta-Gal expression was evidenced in targeted ES cells. Breeding ES-derived single copy transgenic mice with mice bearing appropriate tet transactivators resulted in beta-Gal expression both qualitatively and quantitatively similar to that observed in mice with random integration of the same construct. Interestingly, GR expression in mice was dependent on transgene orientation in the HPRT locus while embryonic stem cell expression was not. Thus, a conditional construct inserted in single copy and in predetermined orientation at the HPRT locus demonstrated a Dox-dependent gene expression phenotype in adult mice suggesting that controlled insertion of tet-inducible constructs at the HPRT locus can provide an efficient alternative strategy to reproducibly generate animal models with tetracycline-induced transgene expression.

Resetting of circadian time in peripheral tissues by glucocorticoid signaling.

In mammals, circadian oscillators reside not only in the suprachiasmatic nucleus of the brain, which harbors the central pacemaker, but also in most peripheral tissues. Here, we show that the glucocorticoid hormone analog dexamethasone induces circadian gene expression in cultured rat-1 fibroblasts and transiently changes the phase of circadian gene expression in liver, kidney, and heart. However, dexamethasone does not affect cyclic gene expression in neurons of the suprachiasmatic nucleus. This enabled us to establish an apparent phase-shift response curve specifically for peripheral clocks in intact animals. In contrast to the central clock, circadian oscillators in peripheral tissues appear to remain responsive to phase resetting throughout the day.

Genetic dissection of glucocorticoid receptor function in mice.

Upon hormone binding, the activated glucocorticoid receptor (GR) functions as a transcription factor via different modes of action to control gene expression. Recent gene-targeting studies in mice provide new insight into the role of GR in vivo and are helping decipher the molecular mechanisms underlying its actions.

The rat albumin promoter: cooperation with upstream elements is required when binding of APF/HNF1 to the proximal element is partially impaired by mutation or bacterial methylation.

We have characterized in the accompanying paper (P. Herbomel, A. Rollier, F. Tronche, M.-O. Ott, M. Yaniv, and M. C. Weiss, Mol. Cell. Biol. 9:4750-4758, 1989) six different elements in the albumin promoter. One of them, the proximal element (PE), is the binding site for a strictly liver specific factor, APF/HNF1. This binding site contains a bacterial DAM DNA methylase methylation target sequence which, when methylated, decreases the affinity of the protein for this element. When the different albumin promoter constructions were prepared in an Escherichia coli deoxyadenosine methylase-negative strain, the respective contributions of the elements to the overall promoter activity were strikingly different. An intact proximal element plus the TATA box gave almost full transcriptional activity in transient transfection experiments and only in differentiated hepatoma cells of line H4II, whereas the distal elements (distal element III [DEIII], the NF1-binding site DEII, and the E/CBP-binding site DEI) had become essentially dispensable. Mutations affecting the CCAAT box showed only a two- to threefold decrease. When PE was methylated, mutated, or replaced by the homologous element from the alpha-fetoprotein gene, activity in the context of the short promoter (PE plus the TATA box) was abolished. However, activity was restored in the presence of the upstream elements, showing that cooperation with factors binding to the CCAAT box and distal elements favors the functional interaction of the liver-specific APF/HNF1 factor with lower-affinity binding sites.

The rat albumin promoter is composed of six distinct positive elements within 130 nucleotides.

No fewer than six different positive regulatory elements concentrated within 130 base pairs constitute the rat albumin promoter, which drives highly tissue specific transcription in rat hepatoma cells in culture. Inactivation of each element led to a decrease in transcriptional efficiency: from upstream to downstream, 3- to 4-fold for distal elements III and II, 15-fold for distal element I, and 50-fold for the CCAAT box and the proximal element (PE). Three of these elements, distal elements III and II and, more crucially, the PE, were found to be involved in the tissue-specific character of transcription, with an additional negative regulation possibly superimposed at the level of the PE. Finally, our mapping of these regulatory elements in vivo entirely coincided with footprint data obtained in vitro, thereby allowing the tentative assignment of specific factors to the effects observed in vivo.

Regulation of albumin gene expression in hepatoma cells of fetal phenotype: dominant inhibition of HNF1 function and role of ubiquitous transcription factors.

Two widely used hepatoma cell lines, mouse BW1J and human HepG2, express gene products characteristic of fetal hepatocytes, including serum albumin, whereas reporter genes driven by the albumin promoter are expressed at very low levels compared with highly differentiated hepatoma cells. We have investigated the low albumin promoter activity in BW1J cells to understand differences in liver gene regulation between fetal and adult cells. Addition of the albumin upstream enhancer, or any other fragment of the albumin gene, failed to modify expression of the transfected promoter in BW1J cells. Analysis of cis elements of the albumin promoter showed that, in contrast to highly differentiated H4II cells, in BW1J cells the activity largely depends on ubiquitous transcription factors. Both BW1J and HepG2 cells produce the liver-enriched transcription factor HNF1; dimerization and DNA binding properties are identical to those of liver HNF1, yet the protein fails to show the anticipated transcriptional stimulatory activity. A transfected HNF1 expression vector strongly trans-activates the albumin promoter in HepG2 but only weakly in BW1J cells, and in hybrids (BW1J x Fao), inefficient HNF1 function is dominant. We conclude that hepatoma cells of the fetal phenotype are deficient in the use of HNF1 to drive transcription of the albumin gene and that they harbor a dominant modulator of HNF1 function.

Gene expression regulation following behavioral sensitization to cocaine in transgenic mice lacking the glucocorticoid receptor in the brain.

Several findings suggest that glucocorticoid hormones influence the propensity of an individual to develop cocaine abuse. These hormones activate two related transcription factors, the glucocorticoid receptor and the mineralocorticoid receptor. We have shown previously that mice carrying a mutation of the glucocorticoid receptor gene specifically in neural cells, glucocorticoid receptor knock-out in the brain, show a dramatic decrease in cocaine-induced self-administration and no behavioral sensitization to this drug, two experimental procedures considered relevant models of addiction. Here, we investigated in glucocorticoid receptor knock-out in the brain mice the consequences of this mutation at the level of the expression of neuropeptide, dopamine receptor and glutamate receptor subunit mRNAs. We quantified mRNA levels in the cortex, striatum and accumbens under basal conditions and following acute or repeated cocaine treatments. Our results show that, under basal conditions, neuropeptide (substance P, dynorphin) and dopamine receptor (D1, D2) mRNAs were decreased in glucocorticoid receptor knock-out in the brain mice in the dorsal striatum but not in the accumbens. However, cocaine-induced changes in the levels of these mRNAs were not modified in glucocorticoid receptor knock-out in the brain mice. In contrast, mutant mice showed altered response in mRNA levels of N-methyl-D-aspartate, GLUR5 and GLUR6 glutamate receptor subunits as well as of enkephalin following cocaine administration. These modifications may be associated to decrease of behavioral effects of cocaine observed in glucocorticoid receptor knock-out in the brain mice.

Impairment of mossy fiber long-term potentiation and associative learning in pituitary adenylate cyclase activating polypeptide type I receptor-deficient mice.

The pituitary adenylate cyclase activating polypeptide (PACAP) type I receptor (PAC1) is a G-protein-coupled receptor binding the strongly conserved neuropeptide PACAP with 1000-fold higher affinity than the related peptide vasoactive intestinal peptide. PAC1-mediated signaling has been implicated in neuronal differentiation and synaptic plasticity. To gain further insight into the biological significance of PAC1-mediated signaling in vivo, we generated two different mutant mouse strains, harboring either a complete or a forebrain-specific inactivation of PAC1. Mutants from both strains show a deficit in contextual fear conditioning, a hippocampus-dependent associative learning paradigm. In sharp contrast, amygdala-dependent cued fear conditioning remains intact. Interestingly, no deficits in other hippocampus-dependent tasks modeling declarative learning such as the Morris water maze or the social transmission of food preference are observed. At the cellular level, the deficit in hippocampus-dependent associative learning is accompanied by an impairment of mossy fiber long-term potentiation (LTP). Because the hippocampal expression of PAC1 is restricted to mossy fiber terminals, we conclude that presynaptic PAC1-mediated signaling at the mossy fiber synapse is involved in both LTP and hippocampus-dependent associative learning.

NFY or a related CCAAT binding factor can be replaced by other transcriptional activators for co-operation with HNF1 in driving the rat albumin promoter in vivo.

Like many eukaryotic genes, the rat albumin promoter contains a CCAAT consensus motif at position -80. In transfected H4II hepatoma cells the strength of this promoter depends to a large extent on the integrity of a hepatic nuclear factor 1 (HNF1) binding site located at position -60 and to a lesser extent on the CCAAT element. However, if the affinity for HNF1 is reduced, the CCAAT-box becomes essential for high, and tissue specific, promoter activity. We wished to determine which, among the different CCAAT binding factors co-existing in eukaryotic cells, was responsible for this co-operativity with HNF1. To this end we prepared a series of mutants of the CCAAT sequence and compared their effects on albumin promoter activity in vivo and on the binding of different CCAAT binding factors in vitro. Our results strongly suggest that a ubiquitous factor NFY (also designated CBF, ACF, CP1) interacts with this CCAAT element in vivo. We propose that during development NFY could facilitate transcription of the albumin gene in hepatocytes when the concentration of HNF1 is limiting. This co-operativity in transcriptional activation is not due to strict co-operativity in DNA binding between the two proteins and is not limited to NFY or a closely related factor, as the CCAAT-box can be replaced by AP1, SP1 or E2 target sites without significantly affecting the final activity.

Analysis of the distribution of binding sites for a tissue-specific transcription factor in the vertebrate genome.

Hepatocyte nuclear factor 1 (HNF1) is a dimeric homeoprotein expressed in hepatocytes and in a few other epithelial cells where it helps regulate the expression of a specific subset of genes. In an attempt to identify novel target genes for HNF1 and to assess the distribution of its target sites within the vertebrate genome, we performed a computer-assisted search within the available databases using a weighted matrix. Several hundred potential target sequences were identified within the GenBank and EMBL data banks. DNA binding assays demonstrated that more than 95%, of the new sites tested (52 sites among 54) bound HNF1. Surprisingly many HNF1 target sites were found in genes that are transcribed in cell types that do not contain the protein. On the other hand these sites are 2.5 to five times more frequent in hepatic genes than expected. It seems that the presence of HNF1 sites in liver-specific genes was favoured, but that no counter-selection occurred within the rest of the genome. HNF1 binding sites in liver genes are more often associated in clusters with sites for other transcription factors and the enrichment is more pronounced in promoter regions. We identified more than 100 liver specific genes that are potentially regulated by HNF1.

Inducible site-specific recombination in the brain.

The Cre/loxP recombination system allows the generation of tissue-specific somatic mutations in mice. Additional temporal control of somatic mutagenesis is highly desirable, as this would permit a more precise analysis of gene function in complex systems such as the central nervous system. Extending our previous studies, we compared several ligand-regulated recombinases, in which the ligand-binding domain (LBD) of the progesterone receptor or the estrogen receptor was fused to the Cre recombinase. A fusion protein between the Cre recombinase and a truncated LBD of the progesterone receptor was chosen to obtain inducible recombination in the brain. This fusion protein can be activated by the synthetic steroid RU486, but not by the physiological hormone progesterone. Its expression was targeted to the brain using regulatory sequences of the calcium-calmodulin-dependent kinase IIalpha or the Thy-1 gene. Application of RU486 to the mice induced Cre-mediated recombination of a lacZ reporter transgene in the cortex and hippocampus, showing that spatially and temporally controlled gene targeting can be mediated in the brain.

Differential promoter usage in prolactin receptor gene expression: hepatocyte nuclear factor 4 binds to and activates the promoter preferentially active in the liver.

Characterization of the rat PRL receptor (PRLR) gene has revealed three separate untranslated exon 1 sequences, each associated with a different transcription start site and 5'-flanking sequence. We show by RT-PCR that exon 1A is expressed primarily in liver but is also detectable in ovary and mammary gland. Exon 1B expression is observed exclusively in the ovary, whereas exon 1C is expressed in all three tissues. Transient transfection of luciferase reporter constructs containing parts of the 5'-flanking regions (0.3-1.1 kb) of exon 1A, 1B, and 1C, respectively, showed activity of the 1A promoter in Chinese hamster ovary (CHO) cells, the human hepatoma cell line, HepG2, and the rat hepatoma cell line, H4II, which was 10- to 14-fold increased compared with the activity of the promoter-less luciferase vector. No activity of the 1A promoter was detected in the human mammary cell line, T-47D. Relative to a vector containing the Simian virus 40 (SV40) promoter, the 1A promoter had 20% activity in H4II cells and 1-3% activity in CHO and HepG2 cells. The 1B promoter produced a 6.1-fold increase of luciferase activity in CHO cells (approximately 2% of the SV40 promoter), whereas no significant activity was detected in HepG2, H4II, and T-47D cells. The 1C promoter was strongly active in T-47D cells (approximately 64-fold over control) and moderately active in the other cell lines tested (9- to 13-fold over control). 5'-Deletion analysis of the 1A promoter revealed that a fragment containing -83/ +81 bp, relative to the transcription start site, was sufficient to drive transcription in hepatoma cells, whereas this construct was inactive in CHO cells. Cotransfection of CHO cells with the -83/+81 construct and an expression vector encoding the liver-enriched transcription factor, hepatocyte nuclear factor 4 (HNF4), revealed a dose-dependent transactivation of the proximal 1A promoter with a maximal stimulation of approximately 10-fold. Electrophoretic mobility shift assays showed binding of HNF4 to the sequence -14/+24 of the 1A promoter, and mutational analysis revealed that the sequence GGGCAAAGTCA at position +11/+21 is required for this binding. We conclude that the 1A, 1B, and 1C promoters of the PRLR gene are used in a cell type- dependent way that may play a role in differential hormonal regulation of the gene. In particular, we have shown that HNF4 operates on the proximal 1A promoter and may be responsible, in combination with other factors, for the increased activity of this promoter in adult female liver.

Characterization of the promoter of the human KAL gene, responsible for the X-chromosome-linked Kallmann syndrome.

We report on the first characterization of the human KAL promoter (pKAL), based on the analysis of a 2-kb fragment of the 5' flanking region. As determined by primer extension, transcription of the human KAL gene is initiated at two different sites in the quail embryonic neuroretina QNR/D cell line. The promoter region is G+C rich and contains a CCAAT box, two binding sites for the SP1 transcription factor and two AP2-binding sites, but no TATA box. It also shares a motif with several neural-specific genes. The ability of four deletion mutants to drive transcription of the heterologous chloramphenicol acetyltransferase (CAT)-encoding gene was determined in transfection experiments. The mutant containing the KAL sequence from nt +2 to -435 demonstrated a tissue-specific, although weak, transcriptional activity only in the quail embryonic neuroretina K2 and QNR/D cell lines. Longer constructs did not confer any activity. Therefore, we suggest that this 437-bp segment of pKAL constitutes a neural-specific promoter which could be negatively controlled by upstream sequences.

Mutagenesis of the glucocorticoid receptor in mice.

The glucocorticoid receptor is an ubiquitously expressed transcription factor involved in the regulation of many different physiological processes. Activated by glucocorticoids the receptor regulates transcription positively or negatively either by direct binding to DNA or by protein protein interactions. In order to define the role of the receptor during development and in physiology several mutations have been generated in the mouse. Mice with a disrupted glucocorticoid receptor gene die shortly after birth due to respiratory failure indicating an important role of the receptor in lung function. Transcription of genes encoding gluconeogenic enzymes in the liver is decreased, proliferation of erythroid progenitors is impaired and the HPA axis is strongly upregulated. To analyze molecular mechanisms of glucocorticoid receptor action in vivo a point mutation has been introduced into the mouse genome which allows to separate DNA-binding-dependent from DNA-binding-independent actions of the receptor. Mice homozygous for the point mutation survive indicating that DNA-binding of the receptor is not required for survival. Induction of glucoconegenic enzymes and proliferation of erythroid progenitors however is impaired. Interestingly, repression of corticotropin releasing factor (CRF) synthesis is maintained, whereas proopiomelanocortin (POMC) expression is upregulated. Since mice with a disrupted glucocorticoid receptor gene die shortly after birth attempts using the Cre/loxP-recombination system are made to bypass early lethality and to study the function of the receptor in defined cell types of adult animals.

Mice with targeted mutations of glucocorticoid and mineralocorticoid receptors: models for depression and anxiety?

Impaired corticosteroid receptor signaling is a key mechanism in the pathogenesis of stress-related psychiatric disorders such as depression and anxiety. Since in vivo expression and functional studies of corticosteroid receptors are not feasible in the human central nervous system, such analyses have to be done in animal models. Transgenic mice with mutations of corticosteroid receptors are promising tools, which allow us to investigate the role of these proteins in the pathogenesis of symptoms characteristic for depression and anxiety. This review summarizes the neuroendocrinological and behavioral findings that have been obtained in six different mouse strains with specific mutations that influence the expression or the function of the glucocorticoid or the mineralocorticoid receptor (MR). The analyses of these mice helped to define molecular concepts of how corticosteroid receptors regulate the activity of the hypothalamic-pituitary-adrenal (HPA) system. Furthermore, some of these mutant mice exhibited characteristic alterations in behavioral tests for anxiety and despair. However, so far, none of the mouse strains described here can be viewed as an animal model of a specific psychiatric disease defined by common diagnostic criteria. Using high throughput technologies for the identification of genes regulated by glucocorticoid receptor (GR) and MR in brain areas responsible for specific symptoms of stress-related disorders will yield potential new drug targets for the treatment of depression and anxiety.