Glucocorticoid receptors in dopaminoceptive neurons, key for cocaine, are dispensable for molecular and behavioral morphine responses.

BACKGROUND: Psychostimulants and opiates trigger similar enduring neuroadaptations within the reward circuitry thought to underlie addiction. Transcription factors are key to mediating these enduring behavioral alterations. The facilitation of these maladaptive changes by glucocorticoid hormones suggests that the glucocorticoid receptor (GR), a transcription factor involved in the stress response, could be a common mediator of responses to pharmacologically distinct classes of abused drugs. METHODS: We employed mouse models carrying GR gene inactivation in either dopamine or dopaminoceptive neurons to determine the involvement of this transcription factor in behavioral responses to cocaine and morphine. We then combined microarray analysis, drug-elicited immediate early gene induction, and in vivo microdialysis to elucidate the molecular underpinnings of these responses. RESULTS: Inactivating GR within dopaminoceptive neurons markedly reduces cocaine-induced conditioned place preference and the expression of locomotor sensitization. In striking contrast, GR had no effect on behavioral morphine responses in either dopaminoceptive or dopamine neurons. The dopaminoceptive mutation engenders alterations in the expression of striatal genes that are implicated in glutamatergic transmission and plasticity. Within the nucleus accumbens, impaired cellular responses to cocaine are conspicuous; a pronounced deficit in cocaine-elicited extracellular dopamine release, expression of the key IEGs c-Fos and Zif268, and phosphorylation of extracellular signal-regulated kinases 1/2 in mutants were observed. In contrast, these molecular and neurochemical changes were not observed in response to morphine, mirroring the lack of effect on behavioral responses to morphine. CONCLUSION: Combined behavioral and molecular approaches have identified a subset of neurons in which GR differentially influences cocaine- and morphine-induced responses.

Characterization of glucocorticoid receptor and hepatocyte nuclear factor 4alpha (HNF4alpha) binding to the hnf4alpha gene in the liver.

Hepatocyte nuclear factor 4alpha (HNF4alpha) plays a crucial role in hepatocyte differentiation, liver organogenesis and regulation of liver functions. In mouse liver, HNF4alpha is expressed from two promoters, P1 and P2, the latter being very weakly active and only in the embryo. Previously, using transfection assays we identified an enhancer upstream of P1 that mediates both HNF4alpha transactivation and glucocorticoid induction and showed that HNF4alpha1, originated from P1, represses activity of the P2 promoter, possibly through its indirect recruitment to the promoter. However, glucocorticoid receptor (GR) binding to the enhancer was not shown and HNF4alpha binding to P2, first reported in isolated human hepatocytes, was not confirmed in mouse liver. Here, to analyse glucocorticoid inducibility and auto-regulation of the hnf4alpha gene in the liver, we accurately mapped and quantitatively assessed GR and HNF4alpha binding to enhancer and HNF4alpha recruitment to the P2 promoter using chromatin immunoprecipitation (ChIP) and real-time PCR. We proved that GR binds to enhancer from embryonic day (E) 17.5 onward and HNF4alpha even earlier. We showed that HNF4alpha binds to P2 independently of the activation function (AF) 1 domain in adult liver. We mapped the binding region between -400 and -200 bp upstream of the transcription start site. Although Sp1 binds within this region in vitro, we did not find evidence of a role of this factor in HNF4alpha recruitment. Our results suggest that, in the liver, HNF4alpha expression may be induced by glucocorticoids around birth and positive auto-regulation of the gene may take place early in development. They support a model of P2 repression involving HNF4alpha recruitment to promoter, possibly through interaction with several promoter-bound factors.

Expression of the alpha7 isoform of hepatocyte nuclear factor (HNF) 4 is activated by HNF6/OC-2 and HNF1 and repressed by HNF4alpha1 in the liver.

The hepatocyte nuclear factor (HNF) 4alpha gene possesses two promoters, proximal P1 and distal P2, whose use results in HNF4alpha1 and HNF4alpha7 transcripts, respectively. Both isoforms are expressed in the embryonic liver, whereas HNF4alpha1 is almost exclusively in the adult liver. A 516-bp fragment, encompassing a DNase I-hypersensitive site associated with P2 activity that is still retained in adult liver, contains functional HNF1 and HNF6 binding sites and confers full promoter activity in transient transfections. We demonstrate a critical role of the Onecut factors in P2 regulation using site-directed mutagenesis and embryos doubly deficient for HNF6 and OC-2 that show reduced hepatic HNF4alpha7 transcript levels. Transient transgenesis showed that a 4-kb promoter region is sufficient to drive expression of a reporter gene in the stomach, intestine, and pancreas, but not the liver, for which additional activating sequences may be required. Quantitative PCR analysis revealed that throughout liver development HNF4alpha7 transcripts are lower than those of HNF4alpha1. HNF4alpha1 represses P2 activity in transfection assays and as deduced from an increase in P2-derived transcript levels in recombinant mice in which HNF4alpha1 has been deleted and replaced by HNF4alpha7. We conclude that although HNF6/OC-2 and perhaps HNF1 activate the P2 promoter in the embryo, increasing HNF4alpha1 expression throughout development causes a switch to essentially exclusive P1 promoter activity in the adult liver.

Comparative genomic analysis of the HNF-4alpha transcription factor gene.

Hepatocyte nuclear factor-4alpha (HNF-4alpha), the gene for the maturity-onset diabetes of the young type 1 (MODY1) form of type 2 diabetes mellitus (T2DM), is within the T2DM-linked region on chromosome 20q12-q13.1 and consequently, is a positional candidate gene for T2DM. Mutations in the coding region of HNF-4alpha are rare in diabetes affected subjects. Altered regulation of HNF-4alpha gene expression, controlled by distant enhancer sequences, may contribute to the development of type 2 diabetes. Comparative sequence analysis was performed between 13 kb of genomic DNA 5' to the P1 promoter sequences of the human, mouse, and rat HNF-4alpha coding sequences. Three regions, located at -10.5 kb (295 bp in length), -6.25 kb (421 bp in length), and -5.36 kb (263 bp in length), have significant sequence identity between the species. These three regions were functionally characterized using the chloramphenicol acetyltransferase (CAT) reporter assay, in which the conserved 5' regions of mouse HNF-4alpha were cloned in front of the herpes simplex virus thymidine kinase promoter driving transcription of the CAT gene. A fragment containing the 421 bp conserved region significantly increased CAT activity in differentiated rat hepatoma cells (13.7-+/-1.9-fold control), while only a modest increase in CAT activity was observed in pancreatic cells (2.5-+/-0.9-fold control; 1.6-+/-0.1-fold control) and dedifferentiated hepatoma cells (1.7-+/-0.4-fold control). The remaining two conserved regions increased CAT activity minimally in pancreatic (1.1-+/-0.1-fold control to 1.9-+/-0.1-fold control) and hepatic (1.6-+/-0.5-fold control to 2.3-+/-0.4-fold control) cell lines. Denaturing high-performance liquid chromatography (DHPLC) was used to search for sequence variants in DNA from 259 T2DM individuals. Two single nucleotide polymorphisms (SNPs) were identified, both of which increased CAT activity in the insulinoma cell lines in the CAT reporter assay (1.4-fold increase over wild-type; 1.7-fold increase over wild-type). These results suggest that comparative sequence analysis can efficiently identify regulatory elements and that sequence variants in regulatory elements of HNF-4alpha can contribute to altered HNF-4alpha gene expression.