Identification of Genetic and Genomic Influences on Progressive Ethanol Consumption in Diversity Outbred Mice.

Genetic factors play a significant role in the risk for development of alcohol use disorder (AUD). Using 3-bottle choice intermittent access ethanol (IEA), we have employed the Diversity Outbred (DO) mouse panel as a model of alcohol use disorder in a genetically diverse population. Through use of gene expression network analysis techniques, in combination with expression quantitative trait loci (eQTL) mapping, we have completed an extensive analysis of the influence of genetic background on gene expression changes in the prefrontal cortex (PFC). This approach revealed that, in DO mice, genes whose expression was significantly disrupted by intermittent ethanol in the PFC also tended to be those whose expression correlated to intake. This finding is in contrast to previous studies of both mice and nonhuman primates. Importantly, these analyses identified genes involved in myelination in the PFC as significantly disrupted by IEA, correlated to ethanol intake, and having significant eQTLs. Genes that code for canonical components of the myelin sheath, such as Mbp, also emerged as key drivers of the gene expression response to intermittent ethanol drinking. Several regulators of myelination were also key drivers of gene expression, and had significant QTLs, indicating that genetic background may play an important role in regulation of brain myelination. These findings underscore the importance of disruption of normal myelination in the PFC in response to prolonged ethanol exposure, that genetic variation plays an important role in this response, and that this interaction between genetics and myelin disruption in the presence of ethanol may underlie previously observed behavioral changes under intermittent access ethanol drinking such as escalation of consumption.

Identification of quantitative trait loci and candidate genes for an anxiolytic-like response to ethanol in BXD recombinant inbred strains.

Genetic differences in acute behavioral responses to ethanol contribute to the susceptibility to alcohol use disorder and the reduction of anxiety is a commonly reported motive underlying ethanol consumption among alcoholics. Therefore, we studied the genetic variance in anxiolytic-like responses to ethanol across the BXD recombinant inbred (RI) mouse panel using the light-dark transition model of anxiety. Strain-mean genetic mapping and a mixed-model quantitative trait loci (QTL) analysis replicated several previously published QTL for locomotor activity and identified several novel anxiety-related loci. Significant loci included a chromosome 11 saline anxiety-like QTL (Salanq1) and a chromosome 12 locus (Etanq1) influencing the anxiolytic-like response to ethanol. Etanq1 was successfully validated by studies with BXD advanced intercross strains and fine-mapped to a region comprising less than 3.5 Mb. Through integration of genome-wide mRNA expression profiles of the mesocorticolimbic reward circuit (prefrontal cortex, nucleus accumbens and ventral midbrain) across the BXD RI panel, we identified high priority candidate genes within Etanq1, the strongest of which was Ninein (Nin), a Gsk3ß-interacting protein that is highly expressed in the brain.

Inhibition of monoacylglycerol lipase reduces nicotine withdrawal.

BACKGROUND AND PURPOSE: Abrupt discontinuation of nicotine, the main psychoactive component in tobacco, induces a withdrawal syndrome in nicotine-dependent animals, consisting of somatic and affective signs, avoidance of which contributes to drug maintenance. While blockade of fatty acid amide hydrolase, the primary catabolic enzyme of the endocannabinoid arachidonoylethanolamine (anandamide), exacerbates withdrawal responses in nicotine-dependent mice, the role of monoacylglycerol lipase (MAGL), the main hydrolytic enzyme of a second endocannabinoid 2-arachidonylglycerol (2-AG), in nicotine withdrawal remains unexplored. EXPERIMENTAL APPROACH: To evaluate the role of MAGL enzyme inhibition in nicotine withdrawal, we initially performed a genetic correlation approach using the BXD recombinant inbred mouse panel. We then assessed nicotine withdrawal intensity in the mouse after treatment with the selective MAGL inhibitor, JZL184, and after genetic deletion of the enzyme. Lastly, we assessed the association between genotypes and smoking withdrawal phenotypes in two human data sets. KEY RESULTS: BXD mice displayed significant positive correlations between basal MAGL mRNA expression and nicotine withdrawal responses, consistent with the idea that increased 2-AG brain levels may attenuate withdrawal responses. Strikingly, the MAGL inhibitor, JZL184, dose-dependently reduced somatic and aversive withdrawal signs, which was blocked by rimonabant, indicating a CB1 receptor-dependent mechanism. MAGL-knockout mice also showed attenuated nicotine withdrawal. Lastly, genetic analyses in humans revealed associations of the MAGL gene with smoking withdrawal in humans. CONCLUSIONS AND IMPLICATIONS: Overall, our findings suggest that MAGL inhibition maybe a promising target for treatment of nicotine dependence.

Molecular and neurologic responses to chronic alcohol use.

This chapter provides an overview of current knowledge on the molecular and clinical aspects of chronic alcohol effects on the central nervous system. This drug is almost ubiquitous, widely enjoyed socially, but produces a diverse spectrum of neurologic disease when abused. Acutely, alcohol interacts predominantly with γ-aminobutyric acid-A (GABA-A) and N-methyl-d-aspartate (NMDA) receptors, but triggers diverse signaling events within well-defined neural pathways. These events result in adaptive changes in gene expression that ultimately produce two major states: addiction and toxicity. Epigenetic modifications of chromatin could lead to long-lived or even transgenerational changes in gene expression, thus producing aspects of the heritability of alcohol use disorders (AUD) and long-term behaviors such as recidivism. The diverse clinical syndromes produced by chronic alcohol actions in the central nervous system reflect the molecular pathology and predominantly involve aspects of tolerance/withdrawal, selective vulnerability (manifest as central pontine myelinolysis, Marchiafava-Bignami disease), and additional environmental factors (e.g., thiamine deficiency in Wernicke-Korsakoff's syndrome). Additionally, deleterious aspects of chronic alcohol on signaling, synaptic transmission, and cell toxicity lead to primary alcoholic dementia. Genetically determined aspects of myelin structure and alcohol actions on myelin gene expression may be a prominent molecular mechanism resulting in a predisposition to, or causation of, AUD and multiple other neurologic complications of chronic alcohol. The dramatic progress made in understanding molecular actions of alcohol holds great promise for our eventual treatment or prevention of AUD and neurologic complications resulting from chronic alcohol abuse.

The α3ß4* nicotinic ACh receptor subtype mediates physical dependence to morphine: mouse and human studies.

BACKGROUND AND PURPOSE: Recent data have indicated that α3ß4* neuronal nicotinic (n) ACh receptors may play a role in morphine dependence. Here we investigated if nACh receptors modulate morphine physical withdrawal. EXPERIMENTAL APPROACHES: To assess the role of α3ß4* nACh receptors in morphine withdrawal, we used a genetic correlation approach using publically available datasets within the GeneNetwork web resource, genetic knockout and pharmacological tools. Male and female European-American (n = 2772) and African-American (n = 1309) subjects from the Study of Addiction: Genetics and Environment dataset were assessed for possible associations of polymorphisms in the 15q25 gene cluster and opioid dependence. KEY RESULTS: BXD recombinant mouse lines demonstrated an increased expression of α3, ß4 and α5 nACh receptor mRNA in the forebrain and midbrain, which significantly correlated with increased defecation in mice undergoing morphine withdrawal. Mice overexpressing the gene cluster CHRNA5/A3/B4 exhibited increased somatic signs of withdrawal. Furthermore, α5 and ß4 nACh receptor knockout mice expressed decreased somatic withdrawal signs compared with their wild-type counterparts. Moreover, selective α3ß4* nACh receptor antagonists, α-conotoxin AuIB and AT-1001, attenuated somatic signs of morphine withdrawal in a dose-related manner. In addition, two human datasets revealed a protective role for variants in the CHRNA3 gene, which codes for the α3 nACh receptor subunit, in opioid dependence and withdrawal. In contrast, we found that the α4ß2* nACh receptor subtype is not involved in morphine somatic withdrawal signs. CONCLUSION AND IMPLICATIONS: Overall, our findings suggest an important role for the α3ß4* nACh receptor subtype in morphine physical dependence.

Genetic variation within the Chrna7 gene modulates nicotine reward-like phenotypes in mice.

Mortality from tobacco smoking remains the leading cause of preventable death in the world, yet current cessation therapies are only modestly successful, suggesting new molecular targets are needed. Genetic analysis of gene expression and behavior identified Chrna7 as potentially modulating nicotine place conditioning in the BXD panel of inbred mice. We used gene targeting and pharmacological tools to confirm the role of Chrna7 in nicotine conditioned place preference (CPP). To identify molecular events downstream of Chrna7 that may modulate nicotine preference, we performed microarray analysis of α7 knock-out (KO) and wild-type (WT) nucleus accumbens (NAc) tissue, followed by confirmation with quantitative polymerase chain reaction (PCR) and immunoblotting. In the BXD panel, we found a putative cis expression quantitative trait loci (eQTL) for Chrna7 in NAc that correlated inversely to nicotine CPP. We observed that gain-of-function α7 mice did not display nicotine preference at any dose tested, whereas conversely, α7 KO mice demonstrated nicotine place preference at a dose below that routinely required to produce preference. In B6 mice, the α7 nicotinic acetylcholine receptor (nAChR)-selective agonist, PHA-543613, dose-dependently blocked nicotine CPP, which was restored using the α7 nAChR-selective antagonist, methyllycaconitine citrate (MLA). Our genomic studies implicated a messenger RNA (mRNA) co-expression network regulated by Chrna7 in NAc. Mice lacking Chrna7 demonstrate increased insulin signaling in the NAc, which may modulate nicotine place preference. Our studies provide novel targets for future work on development of more effective therapeutic approaches to counteract the rewarding properties of nicotine for smoking cessation.

Using genome-wide expression profiling to define gene networks relevant to the study of complex traits: from RNA integrity to network topology.

Postgenomic studies of the function of genes and their role in disease have now become an area of intense study since efforts to define the raw sequence material of the genome have largely been completed. The use of whole-genome approaches such as microarray expression profiling and, more recently, RNA-sequence analysis of transcript abundance has allowed an unprecedented look at the workings of the genome. However, the accurate derivation of such high-throughput data and their analysis in terms of biological function has been critical to truly leveraging the postgenomic revolution. This chapter will describe an approach that focuses on the use of gene networks to both organize and interpret genomic expression data. Such networks, derived from statistical analysis of large genomic datasets and the application of multiple bioinformatics data resources, potentially allow the identification of key control elements for networks associated with human disease, and thus may lead to derivation of novel therapeutic approaches. However, as discussed in this chapter, the leveraging of such networks cannot occur without a thorough understanding of the technical and statistical factors influencing the derivation of genomic expression data. Thus, while the catch phrase may be "it's the network … stupid," the understanding of factors extending from RNA isolation to genomic profiling technique, multivariate statistics, and bioinformatics are all critical to defining fully useful gene networks for study of complex biology.

Chloride intracellular channels modulate acute ethanol behaviors in Drosophila, Caenorhabditis elegans and mice.

Identifying genes that influence behavioral responses to alcohol is critical for understanding the molecular basis of alcoholism and ultimately developing therapeutic interventions for the disease. Using an integrated approach that combined the power of the Drosophila, Caenorhabditis elegans and mouse model systems with bioinformatics analyses, we established a novel, conserved role for chloride intracellular channels (CLICs) in alcohol-related behavior. CLIC proteins might have several biochemical functions including intracellular chloride channel activity, modulation of transforming growth factor (TGF)-ß signaling, and regulation of ryanodine receptors and A-kinase anchoring proteins. We initially identified vertebrate Clic4 as a candidate ethanol-responsive gene via bioinformatic analysis of data from published microarray studies of mouse and human ethanol-related genes. We confirmed that Clic4 expression was increased by ethanol treatment in mouse prefrontal cortex and also uncovered a correlation between basal expression of Clic4 in prefrontal cortex and the locomotor activating and sedating properties of ethanol across the BXD mouse genetic reference panel. Furthermore, we found that disruption of the sole Clic Drosophila orthologue significantly blunted sensitivity to alcohol in flies, that mutations in two C. elegans Clic orthologues, exc-4 and exl-1, altered behavioral responses to acute ethanol in worms and that viral-mediated overexpression of Clic4 in mouse brain decreased the sedating properties of ethanol. Together, our studies demonstrate key roles for Clic genes in behavioral responses to acute alcohol in Drosophila, C. elegans and mice.

Alcohol trait and transcriptional genomic analysis of C57BL/6 substrains.

C57BL/6 inbred mice have been widely used as research models; however, widespread demand has led to the creation of several B6 substrains with markedly different phenotypes. In this study, we report that two substrains of C57BL/6 mice, C57BL/6J (B6J) and C57BL/6NCrl (B6C), separated over 50 years ago at two different breeding facilities differ significantly in alcohol consumption and alcohol preference. The genomes of these two substrains are estimated to differ by only 1-2% of all gene loci, providing a unique opportunity to extract particular expression signatures between these substrains that are associated with quantifiable behavioral differences. Expression profiling of the cortex and striatum, hippocampus, cerebellum and the ventral brain region from alcohol-naïve B6C and B6J mice showed intervals on three chromosomes that are enriched in clusters of coregulated transcripts significantly divergent between the substrains. Additional analysis identified two genomic regions containing putative copy number differences between the substrains. One such region on chromosome 14 contained an estimated 3n copy number in the B6J genome compared with B6C. Within this interval, a gene of unknown function, D14Ertd449e, was found to be both associated with alcohol preference and vary in copy number across several inbred strain lineages. H2afz, Psen1, Wdfy1 and Clu were also identified as candidate genes that may be involved in influencing alcohol consumption.

RhoA, encoding a Rho GTPase, is associated with smoking initiation.

We used microarray analysis of acute nicotine responses in mouse brain to choose rationale candidates for human association studies on tobacco smoking and nicotine dependence (ND). Microarray studies on the time-course of acute response to nicotine in mouse brain identified 95 genes regulated in ventral tegmental area. Among these, 30 genes were part of a gene network, with functions relevant to neural plasticity. On this basis and their known roles in drug abuse or synaptic plasticity, we chose the genes RhoA and Ywhag as candidates for human association studies. A synteny search identified human orthologs and we investigated their role in tobacco smoking and ND in a human case-control association study. We genotyped five and three single nucleotide polymorphisms from the RhoA and Ywhag genes, respectively. Both single marker and haplotype analyses were negative for the Ywhag gene. For the RhoA gene, rs2878298 showed highly significant genotypic association with both smoking initiation (SI) and ND (P = 0.00005 for SI and P = 0.0007 for ND). In the allelic analyses, rs2878298 was only significant for SI. In the multimarker haplotype analyses, significant association with SI was found for the RhoA gene (empirical global P values ranged from 9 x 10(-5) to 10(-5)). In all multimarker combinations analyzed, with or without inclusion of the single most significant marker rs2878298, identical risk and protective haplotypes were identified. Our results indicated that the RhoA gene is likely involved in initiation of tobacco smoking and ND. Replication and future model system studies will be needed to validate the role of RhoA gene in SI and ND.

Microarrays: lost in a storm of data?

Microarray expression profiling is instrumental to our understanding of the function of the genome. Resolution of functionally relevant expression patterns will require the analysis of large data sets compiled from multiple investigators. For this and other reasons, I argue that it is crucial for array data to be publicly shared in a format as close to the 'raw data' as possible. Issues such as protection of intellectual property, ensuring quality of the data, and the format and timing for sharing array data are also discussed.

Identification of novel ethanol-sensitive genes by expression profiling.

Chronic exposure to ethanol or other addicting drugs causes long-lasting, deleterious behavioral responses, such as tolerance, dependence, sensitization, and addiction. Changes in brain gene expression are thought to be a critical component of these behavioral adaptations. Our laboratory and others have utilized cultured neuronal cells as model systems for studying gene regulation by ethanol. Recently, the use of non-biased, high-throughput approaches to studying gene expression has allowed identification of gene regulation "patterns," rather than single genes responding to ethanol. This review will discuss how expression-profiling approaches can be used to identify functional changes occurring in neural cells with chronic exposure to ethanol.

Expression profiling of neural cells reveals specific patterns of ethanol-responsive gene expression.

Adaptive changes in gene expression are thought to contribute to dependence, addiction and other behavioral responses to chronic ethanol abuse. DNA array studies provide a nonbiased detection of networks of gene expression changes, allowing insight into functional consequences and mechanisms of such molecular responses. We used oligonucleotide arrays to study nearly 6000 genes in human SH-SY5Y neuroblastoma cells exposed to chronic ethanol. A set of 42 genes had consistently increased or decreased mRNA abundance after 3 days of ethanol treatment. Groups of genes related to norepinephrine production, glutathione metabolism, and protection against apoptosis were identified. Genes involved in catecholamine metabolism are of special interest because of the role of this pathway in mediating ethanol withdrawal symptoms (physical dependence). Ethanol treatment elevated dopamine beta-hydroxylase (DBH, EC 1.14.17.1) mRNA and protein levels and increased releasable norepinephrine in SH-SY5Y cultures. Acute ethanol also increased DBH mRNA levels in mouse adrenal gland, suggesting in vivo functional consequences for ethanol regulation of DBH. In SH-SY5Y cells, ethanol also decreased mRNA and secreted protein levels for monocyte chemotactic protein 1, an effect that could contribute to the protective role of moderate ethanol consumption in atherosclerotic vascular disease. Finally, we identified a subset of genes similarly regulated by both ethanol and dibutyryl-cAMP treatment in SH-SY5Y cells. This suggests that ethanol and cAMP signaling share mechanistic features in regulating a subset of ethanol-responsive genes. Our findings offer new insights regarding possible molecular mechanisms underlying behavioral responses or medical consequences of ethanol consumption and alcoholism.

Gene expression in human alcoholism: microarray analysis of frontal cortex.

BACKGROUND: Changes in brain gene expression are thought to be responsible for the tolerance, dependence, and neurotoxicity produced by chronic alcohol abuse, but there has been no large scale study of gene expression in human alcoholism. METHODS: RNA was extracted from postmortem samples of superior frontal cortex of alcoholics and nonalcoholics. Relative levels of RNA were determined by array techniques. We used both cDNA and oligonucleotide microarrays to provide coverage of a large number of genes and to allow cross-validation for those genes represented on both types of arrays. RESULTS: Expression levels were determined for over 4000 genes and 163 of these were found to differ by 40% or more between alcoholics and nonalcoholics. Analysis of these changes revealed a selective reprogramming of gene expression in this brain region, particularly for myelin-related genes which were down-regulated in the alcoholic samples. In addition, cell cycle genes and several neuronal genes were changed in expression. CONCLUSIONS: These gene expression changes suggest a mechanism for the loss of cerebral white matter in alcoholics as well as alterations that may lead to the neurotoxic actions of ethanol.

Characterization of promoter elements mediating ethanol regulation of hsc70 gene transcription.

Chronic exposure to ethanol increases transcription of the molecular chaperone Hsc70 in NG108-15 neuroblastoma X glioma cells. This and other ethanol-induced changes in gene expression may contribute to central nervous system tolerance and dependence in alcoholics. Here, we characterized sequences in the hsc70 promoter that are required for ethanol-induced transcriptional regulation. Deletion analysis of the hsc70 promoter showed that the 74-base pair region proximal to the transcription start site was sufficient for ethanol responsiveness. Point mutation or deletion of a consensus Spl-binding site at -67/-61 base pairs greatly reduced the induction by ethanol. Hsc70 promoter constructs with diminished ethanol responsiveness in NG108-15 cells similarly had decreased transcriptional activation by exogenous Sp1 in Drosophila SL2 cells. Some artificial promoter constructs containing multiple Sp1 sites were highly responsive to ethanol, but others were not, suggesting that the organization of the proximal promoter region was an additional factor that affected the ethanol response. Gel mobility shift analysis confirmed that an Sp1-like protein bound to the -67/-61 consensus Sp1 site. However ethanol exposure did not alter Sp1 DNA-binding activity. Together, our findings show that ethanol induction of Hsc70 requires a functional Sp1-binding site. Additional proximal promoter elements may also play a role in determining whether an Sp1-containing promoter will respond to ethanol.

Cloning and characterization of the rat and human phosducin-like protein genes: structure, expression and chromosomal localization.

We isolated and characterized the rat gene encoding phosducin-like protein (PhLP), a putative heterotrimeric G protein modulator. The transcription start site was mapped by primer extension. The putative promoter region lacked a TATA sequence but contained a potential initiator element. Two splice variants were identified by RT-PCR of rat brain RNA, potentially generating either the full length or an amino-truncated protein. Only the full-length protein was immunodetected in all mouse tissues surveyed. Comparison of the conceptual translation product of the rat PhLP gene with those from human and Drosophila clones shows a striking conservation in the amino-terminal region of PhLP from these species. This contrasts with the relatively low degree of homology between PhLP and phosducin in this region, suggesting a functional role for this portion of the PhLP protein. Finally, we mapped the human PhLP gene by PCR analysis of somatic cell hybrids and the Stanford G3 radiation hybrid panel. The human PhLP gene (PDCL) is located on chromosome 9, linked to the polymorphic markers D9S1876 and D9S1674 (66-71 cM).

Phosducin-like protein (PhLP), a regulator of G beta gamma function, interacts with the proteasomal protein SUG1.

Phosducin-like protein (PhLP) and phosducin are highly homologous proteins that interact with the beta gamma subunits of guanine nucleotide binding proteins. While phosducin has a well-characterized role in retinal signal transduction, PhLP function remains unclear. To further understand the function of PhLP, we have examined other potential protein:protein interactions with PhLP using the yeast two-hybrid system. PhLP was found to interact with a mouse homologue of the yeast SUG1, a subunit of the 26S proteasome which may also indirectly modulate transcription. This interaction was further confirmed by an in vitro binding assay and co-immunoprecipitation of the two proteins in overexpression studies. Inhibition of proteasome function by lactacystin led to accumulation of high molecular weight, ubiquitin-immunoreactive protein precipitated by PhLP antiserum. We suggest that PhLP/SUG1 interaction may target PhLP for proteasomal degradation.

Interaction of phosducin-like protein with G protein betagamma subunits.

Phosducin-like protein (PhLP), a widely expressed ethanol-responsive gene (Miles, M. F., Barhite, S., Sganga, M., and Elliott, M. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 10831-10835), is a homologue of phosducin, a known major regulator of Gbetagamma signaling in retina and pineal gland. However, although phosducin has a well characterized role in retinal phototransduction, function of the PhLP remains unclear. In this study we examine the ability of PhLP to bind Gbetagamma dimer in vitro and in vivo. Using PhLP glutathione S-transferase fusion proteins, we show that PhLP directly binds Gbetagamma in vitro. Studies with a series of truncated PhLP fusion proteins indicate independent binding of Gbetagamma to both the amino- and C-terminal halves of PhLP. Protein-protein interactions between Gbetagamma and PhLP are inhibited by the alpha subunit of Go and Gi3, suggesting that PhLP can bind only free Gbetagamma. Finally, we show that PhLP complexes, at least partially, with Gbetagamma in vivo. Following overexpression of epitope-tagged PhLP together with Gbeta1gamma2 proteins in COS-7 cells, a PhLP-Gbetagamma complex is co-immunoprecipitated by monoclonal antibody directed against the epitope tag. Similarly, polyclonal anti-PhLP antibody co-precipitates endogenous PhLP and Gbetagamma proteins from NG108-15 cell lysates. These data are consistent with the hypothesis that PhLP is a widely expressed modulator of Gbetagamma function. Furthermore, because alternate forms of the PhLP transcript are expressed, there may be functional implications for the existence of two Gbetagamma-binding domains on PhLP.

Interaction of ethanol with inducers of glucose-regulated stress proteins. Ethanol potentiates inducers of grp78 transcription.

GRP78, a molecular chaperone expressed in the endoplasmic reticulum, is a "glucose-regulated protein" induced by stress responses that deplete glucose or intracisternal calcium or otherwise disrupt glycoprotein trafficking. Previously we showed that chronic ethanol exposure increases the expression of GRP78. To further understand the mechanism underlying ethanol regulation of GRP78 expression, we studied the interaction between ethanol and classical modulators of GRP78 expression in NG108-15 neuroblastoma x glioma cells. We found that, in addition to increasing basal levels of GRP78 mRNA ("induction"), ethanol produced greater than additive increases in the induction of GRP78 mRNA by the "classical" GRP inducers A23187, brefeldin A, and thapsigargin ("potentiation"). Both the ethanol induction and potentiation responses modulated grp78 gene transcription as determined by stable transfection analyses with the rat grp78 promoter. Ethanol potentiated the action of all classical inducers of grp78 transcription that were studied. In contrast, co-treatment with the classical GRP inducers thapsigargin and tunicamycin produced only simple additive increases in grp78 promoter activity. Transient transfection studies with deletion mutants of the rat grp78 promoter showed that cis-acting promoter sequences required for ethanol induction differ from those mediating responses to classical GRP inducers. Furthermore, linker-scanning mutations of the grp78 promoter suggested that the ethanol potentiation response required a cis-acting promoter element different from those involved in induction by ethanol or classical inducing agents. While the ethanol induction response required 16-24 h to be detectable, ethanol potentiation of thapsigargin occurred within 6 h. The potentiation response also decayed rapidly after ethanol removal. In addition, the protein kinase A inhibitor Rp-cAMPS and protein phosphatase inhibitor okadaic acid both increased ethanol potentiation of thapsigargin while Sp-cAMPS, an activator of protein kinase A, decreased ethanol potentiation. Taken together, our findings suggest two mechanisms by which ethanol regulates grp78 transcription, both differing from the action of classical GRP inducers such as thapsigargin. One mechanism (potentiation) involves a protein phosphorylation cascade and potentiates the action of classical GRP inducers. In contrast, GRP78 induction by ethanol involves promoter sequences and a mechanistic pathway separate from that of the ethanol potentiation response or classical GRP78 inducers. These studies show that ethanol produces a novel and complex regulation of grp78 transcription which could be of particular importance during neuronal exposure to GRP-inducing stressors as might occur with central nervous system injury.