Effect of ethanol on the regulation of corticotropin-releasing factor (CRF) gene expression

www.elsevier.com/locate/ymcne Mol. Cell. Neurosci. 29 (2005) 345 – 354

Effect of ethanol on the regulation of corticotropin-releasing factor (CRF) gene expression Zhongqi Li, Sang Soo Kang, Soon Lee, and Catherine Rivier* The Clayton Foundation Laboratories for Peptide Biology, The Salk Institute, 10010 N. Torrey Pines Road, La Jolla, CA 92037, USA Received 15 September 2004; revised 9 February 2005; accepted 6 April 2005

Ethanol stimulates hypothalamic – pituitary – adrenal axis activity in vivo. To determine the cellular and molecular mechanisms through which ethanol regulates corticotropin-releasing factor (CRF) gene expression, we compared the effect of ethanol and forskolin on CRF peptide secretion and messenger RNA levels in hypothalamic primary cell cultures, and on CRF promoter activity in the NG108-15 cell line. CRF secretion, mRNA levels, and gene transcription significantly increased in response to ethanol or forskolin. Mutation of the cAMPresponse element (CRE) reduced luciferase activity under basal conditions as well as in response to forskolin or ethanol. On the other hand, plasmid with five CRE repeats yielded dramatically elevated basal luciferase activity and significantly increased upregulation by ethanol. Inclusion of adenosine deaminase reduced the promoter response to ethanol. Finally a PKA inhibitor and a cAMP antagonist both decreased ethanol-induced CRF peptide secretion, gene expression, and transcription. These results suggest that ethanol upregulates CRF expression through cAMP/PKA-dependent pathways. D 2005 Elsevier Inc. All rights reserved.

Introduction Corticotropin-releasing factor (CRF), a 41 amino acid neuropeptide that has been isolated from the hypothalamus of numerous species including bovine, sheep, rat, mouse, and human (Keegan et al., 1994; Rivier et al., 1983; Shibahara et al., 1983; Vale et al., 1981), is widely distributed throughout the central nervous system (CNS) (Cummings et al., 1983) where it participates in the coordination of neuroendocrine, behavioral, and immune response to stress. More specifically, hypothalamic CRF plays a major role in the neuroendocrine responses that involve the hypothalamic – pituitary – adrenal (HPA) axis, which are characterized by the release of ACTH and corticosteroids (Herman and Cullinan, 1997; Herman et al., 1996; Sawchenko et al., 1996; Smagin et al., 2001). Appropriate secretion of CRF, ACTH, and corticosterone

* Corresponding author. Fax: +1 858 552 1546. E-mail address: [email protected] (C. Rivier). Available online on ScienceDirect (www.sciencedirect.com). 1044-7431/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.mcn.2005.04.002

is central to the maintenance of homeostasis and a healthy response to allostatic loads (Schulkin, 2003). On the other hand, pathological changes, such as when the HPA axis is either hypoor hyperresponsive to a stressor, lead to a large array of diseases (Schulkin, 2003). Consequently, understanding the mechanisms that mediate the influence of stressors on the HPA axis is very important. Ethanol is a known stimulator of the HPA axis (Rivier, 1996; Rivier et al., 1990) and as such, not only upregulates CRF, ACTH, and corticosterone levels, but also modifies the response of this axis to other stressors (Lee et al., 1999, 2000; Rivier and Lee, 2001). Ethanol therefore has the potential to influence the adaptive processes that normally allow mammalian organisms to remain healthy. While the influence of ethanol on the HPA axis is well known, the mechanisms responsible for these effects have remained surprisingly elusive, particularly at the cellular and molecular levels. Modulation of gene expression by extracellular signals can occur via activation of G protein-coupled membrane receptors, leading to increased levels of intracellular messengers such as cyclic adenosine monophosphate (cAMP) (Brindle and Montminy, 1992). Genes responsive to cAMP have a common DNA promoter sequence, the cAMP-responsive element (CRE) (Deutsch et al., 1988). Studies of the CRF gene have shown that the CRF promoter contains a CRE consensus sequence which has been identified as a functional regulatory element in human and rat CRF genes (Thompson et al., 1987; Vamvakopoulos et al., 1990), and we know that CRF transcription is regulated by modulation of intracellular cAMP and CRE binding (Burbach, 2002; Seasholtz et al., 1988; Spengler et al., 1992). For example, a study using human hepatocellular cells has shown that the activator of the protein kinase A (PKA), forskolin, increased the binding of CRE-binding protein (CREB) to the CRE in the CRF promoter (Wolfl et al., 1999). There is also evidence that ethanol influences the cAMP signal transduction pathway by increasing the cellular concentration of cAMP (Asher et al., 2002; Constantinescu et al., 2002; Diamond and Gordon, 1997; Kelly et al., 1995). Furthermore, acute exposure to ethanol increases adenosine receptor-activated cAMP production (Gordon et al., 1986), which in turn regulates the activity of PKA and influences a variety of cellular functions. However, very little is known regarding the participation of any of

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these mechanisms in the signaling pathways involved in the control of ethanol on CRF transcription. Investigators have used a variety of approaches in order to investigate the molecular mechanisms leading to altered CRF synthesis and release, and primary neuronal cultures, immortalized CRF lines (of hypothalamic or amygdala origin), or tumor lines, which are presently considered as the most useful in vitro models (Bayatti et al., 2003; Kasckow et al., 1997, 1999, 2003a,b; Mulchahey et al., 1999). Each has advantages and disadvantages, and in contrast to other peptides, both primary and immortalized cells have proved extremely difficult for the study of CRF. We therefore turned to a primary hypothalamic cell culture to investigate the effect of ethanol alone or in combination with either the PKA activator or inhibitor, on CRF mRNA expression and peptide release. Our original intent was to also use the primary cells for the transfection experiments. Gene delivery techniques can be largely divided into viral and non-viral methods, and each has strengths and weaknesses. We tested three commercially available chemical transfection agents but failed to obtain detectable and reliable reporter gene expression with any of them. Consequently, we chose the mouse neuroblastoma  rat glioma hybrid NG108-15 cell line to study CRF promoter activity assay because this clonal neuronal cell line displays many of the properties normally associated with differentiated neurons and had provided a useful model system of neuronal origin for studying molecular mechanisms of ethanol (Asher et al., 2002; Constantinescu et al., 2002; Diamond and Gordon, 1997; Gordon et al., 1986; Kelly et al., 1995). Collectively, the studies reported here provide the first evidence for the molecular mechanisms through which ethanol upregulates hypothalamic CRF.

Results Effects of ethanol and forskolin on CRF peptide release We first studied the time course of the CRF response to ethanol or forskolin in primary hypothalamic cell cultures. Twenty-fivemillimolar ethanol induced significant increases in CRF secretion 1 and 4 h post-treatment (Fig. 1). Twenty-five-micromolar forskolin also induced a significant and sustained increase in CRF peptide levels over the time course studied (Fig. 1). In contrast, no significant changes were found in intracellular CRF levels at any time (not shown). Finally, co-incubation of forskolin and ethanol resulted in a significant decreased CRF response, compared to forskolin alone (Fig. 1). Effect of ethanol and forskolin on CRF gene expression Figs. 2A and B illustrate the results of the RT-PCR analysis for CRF gene expression. CRF mRNA levels were normalized to GAPDH based on densitometric analysis. There were significant changes in CRF mRNA levels in response to the various treatments. Peak increase in the CRF mRNA response to 25 mM ethanol occurred at the 1-h time point (Fig. 2A). CRF mRNA levels significantly increased in response to forskolin or ethanol treatments, whereas GAPDH mRNA levels remained constant. Finally, and similar to what we had observed in Fig. 1, co-incubation of forskolin with 25 mM ethanol significantly inhibited the CRF mRNA response to forskolin alone (Fig. 2B).

Fig. 1. CRF peptide secretion by primary hypothalamic cell cultures. CRF peptide secretion was detected by RIA after treatment of cells with ethanol (EtOH, 25 mM), forskolin alone (Forsk, 25 AM), or forskolin and ethanol. Each point represents the mean T SEM from a single experiment that is representative of three separate experiments (N = 6 for each experiment). *P < 0.05, **P < 0.01, ethanol vs. control; ++P < 0.01, forskolin vs. control; ## P < 0.01, forskolin vs. forskolin + ethanol.

Effect of ethanol and forskolin on CRF promoter activity Previous studies had demonstrated that acute exposure to ethanol increased cAMP levels in NG108-15 cells (Gordon et al., 1986; Nagy et al., 1989), which is the reason why we chose this model for the promoter activity assay. NG 108-15 cells were transfected with the rat CRF promoter constructs pGL2-rcpi1500 or pGL2-rcpi498 and were subsequently treated with 25 AM forskolin and 25 mM ethanol. As shown in Figs. 3A and B, insertion of a 1.5-kb or 498-bp upstream region of the CRF promoter (pGL2-rcpi1500 and pGL2-rcpi498) increased promoter activity approximately 2.5-fold in response to forskolin stimulation, compared with untreated cells. A similar procedure using the same plasmids was performed with 25 mM ethanol. The maximum increase of the promoter activity was approximately 27% with pGL2-rcpi1500 and 37% with pGL2rcpi498 at 2 h after ethanol exposure (Fig. 3A, pGL2-rcpi1500, control: 4818 T 308, ethanol: 6127 T 489; Fig. 3B, pGL2rcpi498, control: 6175 T 431, ethanol: 8476 T 410). The induction of promoter activity by forskolin was significantly decreased when pGL2-rcpi1500 cells were exposed to ethanol for 2 h (Fig. 3A). The cAMP-dependent signal transduction pathway is thought to be critical in modulating CRF gene expression. To characterize the importance of the CRE site in mediating CRF transcriptional activation, the plasmid pGL2-rcpi498 was therefore modified to generate pGL2-rcpi498mCRE containing the mutated CRE. Furthermore, we created pGL2-rcpi498CRE5 with the addition of four repeats of CRE into pGL2-rcpi498 based on the previous report on that promoter with addition of more than four CRE repeats significantly increased the activity to forskolin (Suzuki et al., 1996). As shown in Table 1, this mutation and the addition of CRE significantly altered basal levels of the promoter activity. Neither ethanol nor forskolin had significant effects on promoter activity in the pGL2-rcpi498mCRE-transfected cells (Fig. 4). In contrast, both ethanol and forskolin induced a significant increase in luciferase activity in cells transfected with the pGL2-rcpi498CRE5 that has multiple CRE

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Fig. 2. Effect of forskolin (Forsk) and/or ethanol (EtOH) on CRF mRNA expression. Data are expressed as fold induction of control after normalized to GAPDH. (A) RT-PCR analysis of CRF gene expression after treatment with 25 mM ethanol. **P < 0.01, ethanol vs. control. (B) CRF gene expression after administration with 25 AM forskolin alone or in combination with 25 mM ethanol. Each point represents the mean T SEM from a single experiment that is representative of three separate experiments (N = 3 for each experiment). ++P < 0.01, forskolin vs. control; # P < 0.05, forskolin vs. forskolin + ethanol.

sequences (Fig. 5). When the effect of ethanol was compared to results obtained in the pGL2-rcpi498 transfection experiment (Fig. 3B), the increase produced by pGL2-rcpi498CRE5 was observed earlier and was sustained longer. These results support the hypothesis that CRE plays an important role in ethanol regulated CRF gene expression. Finally, to further elucidate the relationship between cAMP generation and ethanol, we examined CRF promoter activity when the extracellular adenosine was abolished by co-incubation of adenosine deaminase (ADA,

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Fig. 3. Regulation of CRF gene promoter activity by ethanol, forskolin, or combination of ethanol (EtOH) and forskolin (Forsk). Luciferase activity was measured after treatment with 25 mM ethanol, 25 AM forskolin, or 25 AM forskolin + 25 mM ethanol. The cells were transfected with either pGL2-rcpi1500 (A) or pGL2-rcpi498 (B). Each point represents mean T SEM from a single experiment that is representative of three separated experiments (N = 6 for each experiment). *P < 0.05, **P < 0.01, ethanol vs. control; ++P < 0.01, forskolin vs. control; ##P < 0.01, forskolin vs. forskolin + ethanol.

1 U/ml) with ethanol. As shown in Fig. 6A, inclusion of ADA significantly blocked ethanol-induced CRF promoter activity. However, the stimulatory effect of forskolin on CRF promoter activity was not significantly altered (Fig. 6B). Table 1 Basal levels of CRF promoter activity for NG108-15 cells after transfection Plasmid

Promoter activity

pGL2-rcpi498 pGL2-rcpi498mCRE pGL2-rcpi498CRE5

6843 T 290 3748 T 421TT 944,947 T 33,282TT

The data show basal levels of CRF promoter activity when transfected with different plasmids without any treatment. Each point represents the mean T SEM from a single experiment that is representative of 3 separate experiments. N = 6 for each experiment. TT P < 0.01, vs pGL2-rcpi498.

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Fig. 4. Effects of CRE mutation on the expression of luciferase activity of NG108-15 cells after treatment with ethanol (EtOH, 25 mM) or forskolin (Forsk, 25 AM). The cells were transfected with a pGL2-rcpi498mCRE that has mutations in the CRE sequence. Data are presented as mean T SEM from a single experiment that is representative of three independent experiments (N = 6 for each experiment). No significant difference was observed between treated groups.

Effect of the cAMP antagonist Rp-cAMP or the PKA inhibitor H89 on ethanol-induced CRF gene expression In order to determine the involvement of cAMP-PKA signaling pathways in mediating the influence of ethanol, we investigated the effect of the cAMP antagonist Rp-cAMP and the PKA inhibitor H89 on ethanol-stimulated CRF peptide secretion, CRF mRNA levels, and CRF promoter activity. Both Rp-cAMP (Figs. 7A and B) and H89 (Figs. 8A and B) abolished ethanol-induced increases in CRF peptide release and CRF mRNA levels in primary hypothalamic cells. The response of the CRF promoter to ethanol was also decreased by Rp-cAMP (Fig. 7C) and H89 (Fig. 8C) in NG108-15 cell. From these data we conclude that ethanol-induced

Fig. 6. Effect of adenosine deaminase (ADA, 1 U/ml) on 25 mM ethanol (EtOH) (A) or 25 AM forskolin (Forsk) (B) stimulated CRF promoter activity when NG108-15 cells were transfected with pGL2-rcpi498. Data are presented as mean T SEM from a single experiment that is representative of three independent experiments (N = 6 for each experiment). **P < 0.01, ethanol vs. ethanol + ADA.

CRF gene expression is mediated through cAMP-PKA signal transduction pathways.

Discussion

Fig. 5. Response of the CRF promoter containing the addition of 4 CRE repeats to ethanol (EtOH, 25 mM) and forskolin (Forsk, 25 AM) treatment. Data are presented as mean T SEM from a single experiment that is representative of three independent experiments (N = 6 for each experiment). *P < 0.05, **P < 0.01, ethanol vs. control; ++P < 0.01, forskolin vs. control.

The cellular processes underlying CRF gene expression in the hypothalamus are relatively difficult to study in the whole animal. Primary hypothalamic cell cultures have therefore been used as a model system to investigate the cellular and molecular mechanisms leading to changes in the activity of the hypothalamic neurons that produce this peptide (Hu et al., 1992; Widmaier et al., 1989). A culture system similar to the one we used has been previously shown to reflect physiological events also observed in the intact rodent (Costa et al., 2001; Hu et al., 1992; Widmaier et al., 1989), although we understand that such systems do not study the normal input from extrahypothalamic brain areas. Nevertheless, these studies suggested that cultured neonatal hypothalamic cells were suitable for the study of CRF synthesis and release. Because of the

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reported significant increase in basal CRF release measured in rat hypothalamus obtained during the first 10 postnatal days (Walker et al., 1986), we first used hypothalamic tissues taken from 6- to 7day-old rats that were then maintained in culture for 9 – 10 days. In order to establish the validity of this model in our laboratory, we first determined whether we could confirm previously published results (Hu et al., 1992; Redei et al., 1988; Widmaier et al., 1989) showing that activation of the PKA pathway by forskolin led to a significant increase in CRF release. This was further verified with RT-PCR analysis of CRF mRNA levels, and we report here that the expression of CRF mRNA significantly increased 1 h following forskolin treatment. These results suggest the involvement of cAMP-related second-messenger system events in this cell model. Consequently, they lend support to the concept that the primary hypothalamic cell culture system is a useful model for the investigation of the cellular and molecular mechanisms that play a role in regulating the activity of hypothalamic CRF neurons. Previous studies from our laboratory have shown that ethanol alters the activity of the HPA axis in laboratory animals (Rivier, 1996; Rivier and Lee, 1996; Rivier et al., 1990). Redei et al. (1988) reported that CRF release, measured by RIA, was significantly elevated after acute exposure to ethanol in an in vitro hypothalamic preparation system. Here, we provide further evidence for a positive regulation of CRF gene expression by ethanol. Indeed, 25 mM ethanol induced a significant increase in CRF secretion into the medium, measured 1 h or 4 h later. Consistent with the effects of ethanol on CRF peptide release, levels of CRF gene expression measured by RT-PCR analysis also increased 1 h after stimulation by ethanol. Collectively, these results suggest that ethanol upregulated both CRF biosynthesis and release. We then turned to some of the mechanisms that might be involved, and in this article we focus on the role of adenosine and cAMP-dependent pathways. Previous studies had indicated that ethanol increases cellular cAMP production, which subsequently activates the cAMP-PKA pathway (Asher et al., 2002; Constantinescu et al., 2002; Diamond and Gordon, 1997). We therefore hypothesized that this phenomenon might play a role in the ensuing CRF response. Ethanol is also known to rapidly increase extracellular adenosine concentrations in both the NG108-15 cell line (Nagy et al., 1989) and stimulates cAMP production in primary hypothalamic cells (Boyadjieva and Sarkar, 1997, 1999). Adenosine plays a role in many of the effects exerted by ethanol on the brain, but its receptor subgroups have different responses to adenosine in that activation of A1 or A2 receptor results in decreased or increased adenylate cyclase activity, respectively (Diamond and Gordon, 1997; Gubits et al., 1990). The membrane of the NG108-15 cells we used was shown to only carry A2 receptors, which can be activated by a high level of adenosine. This subsequently Fig. 7. Effect of the cAMP antagonist Rp-cAMP on ethanol-induced CRF peptide secretion, gene expression, and promoter activity. Primary hypothalamic cells and NG108-15 cells transfected with pGL2-rcpi498 were treated with 250 AM Rp-cAMP, followed 30 min later by 25 mM ethanol (EtOH). (A) CRF peptide secretion was detected by RIA after treatment with ethanol alone or in combination with Rp-cAMP. (B) On the basis of preliminary results, CRF mRNA levels were measured 1 h after treatment with ethanol alone or in combination with Rp-cAMP by RT-PCR. (C) CRF promoter activity was measured by luciferase assay after treatment with ethanol alone or in combination with Rp-cAMP. Each point or bar represents the mean T SEM of a single experiment that is representative of three separate experiments (N = 3 – 6 for each experiment). *P < 0.05, **P < 0.01, ethanol vs. ethanol + Rp-cAMP, +P < 0.05 control vs. ethanol.

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stimulates adenylyl cyclase and activates the cAMP pathway (Gubits et al., 1990; Yao et al., 2002, 2003). These observations suggest that this cell line is an appropriate model for our studies. Although there is evidence suggesting that cAMP signaling is an important target for ethanol action, there is little information linking ethanol-induced changes in expression of CRF gene to the cAMP pathway. A consensus CRE site has been found in the proximal promoter region of the CRF gene in rodents and humans (Thompson et al., 1987; Vamvakopoulos et al., 1990). Therefore, we decided to test the hypothesis that cAMP signaling plays a role in ethanol-induced CRF transcription. Using the NG108-15 cell line to study rat CRF promoter activity induced by forskolin and alcohol, we report here that both ethanol and forskolin, an activator of adenylate cyclase, significantly increased CRF promoter activity. The introduction of mutations into the CRE sequence resulted in a significant decrease in both basal (Table 1) and ethanol- or forskolin-induced promoter activities (Fig. 4). This indicates that the CRE acts as an activator for CRF gene expression and both forskolin and ethanol increased CRF gene transcription through this element. To further examine the role of the CRE on ethanol and forskolin-stimulated CRF gene transcription, we also investigated the role of multiple CRE in the response to ethanol and forskolin. First, we show here that the promoter with an addition of 4 repeats of CRE dramatically increased basal luciferase activity. This confirmed the role of CRE as the potential upregulating element for CRF gene expression. Second, we found that the CRF promoter activity responded maximally to ethanol stimulation when 4 CRE repeats had been added at 131 bp from the major transcription start site. Collectively, these results strongly implicate the CRE element in ethanol regulation of CRF gene transcription. They also provide a possible mechanism by which ethanol upregulates CRF gene transcription that is mediated by cAMP/CRE pathway. Indeed, subsequent experiments carried out with the cAMP antagonist Rp-cAMP or the PKA inhibitor H89 further demonstrated that ethanol-induced CRF peptide secretion, CRF mRNA levels, and CRF promoter activity were significantly inhibited by these antagonists. These results further support the concept that ethanol upregulates CRF gene expression through cAMP-PKA-dependent signal transduction pathways. Ethanol perturbs membrane components of the adenylyl cyclase signal transduction system and can alter neuronal cAMP generation (Kelly et al., 1995; Nagy et al., 1989). Acute ethanol exposure in vivo has been shown to activate the cAMP pathway in rodent brain regions, with subsequent increases in CREB phosphorylation (Yang et al., 1996). Ethanol also inhibits adenosine uptake through the nucleoside transporter, and the ensuing increase in extracellular adenosine levels leads to an increase in intracellular cAMP levels (Asher et al., 2002; Diamond and Gordon, 1997). As adenosine Fig. 8. Effect of the PKA inhibitor H89 on ethanol-induced CRF peptide secretion, gene expression, and promoter activity. Primary hypothalamic cells and NG108-15 cells transfected with pGL2-rcpi498 were treated with 10 AM H89, followed 30 min later by 25 mM ethanol (EtOH). (A) CRF peptide secretion was detected by RIA after treatment with ethanol alone or in combination with H89. (B) On the basis of preliminary results, CRF mRNA levels were measured 1 h after treatment with ethanol alone or in combination with H89 by RT-PCR. (C) CRF promoter activity was measured by luciferase assay after treatment with ethanol alone or in combination with H89. Each point or bar represents the mean T SEM of a single experiment that is representative of three separate experiments (N = 3 – 6 for each experiment). *P < 0.05, **P < 0.01, ethanol vs. ethanol + H89, +P < 0.05 control vs. ethanol.

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plays an important role in altered cAMP accumulation, we examined the involvement of extracellular adenosine in the response of the CRF promoter activity to ethanol by using adenosine deaminase (ADA), which breaks down adenosine. ADA treatment significantly reduced ethanol- but not forskolinstimulated CRF promoter activities. These data further supported the role of adenosine/cAMP in mediating the effects of ethanol on CRF and suggest that the adenosine-independent response of forskolin may reflect its effect on adenylyl cyclase itself. As A2 receptors are prominent in the hypothalamic region (Dohrman et al., 1997; Jarvis et al., 1989), our observation is consistent with the concept that A2 receptors are involved in the effects of ethanol observed in the intact animal (Naassila et al., 2002). It therefore seems reasonable to propose that ethanol may stimulate hypothalamic CRF synthesis and release through these receptors. Finally, we observed that ethanol inhibited the CRF response to forskolin. Interestingly, an interaction between ethanol and forskolin was first demonstrated when the former was used to solubilize the latter for in vitro experiments, and was found to inhibit the activity of this adenyl cyclase activator (Huang et al., 1982; Robberecht et al., 1983). At present, the mechanisms responsible for this interaction remain unknown. Forskolin was shown to directly act on the cyclic subunit of adenylyl cyclase and increase intracellular cAMP levels (Robberecht et al., 1983). High affinity binding sites for forskolin are associated with the activated complex of the catalytic subunit and the stimulatory G-protein (Gs) (Laurenza et al., 1989). As forskolin is a lipophilic molecule with limited solubility in water, the presence of other lipophilic agents like ethanol might affect its ability to bind to the targets. In summary we show here, using both a primary hypothalamic cell culture system and an assay based on CRF promoter activity, that ethanol upregulates CRF gene expression, and that a cAMP/ PKA-dependent signal transduction pathway is crucial for this response. Our results provide important insight into mechanisms of ethanol-induced CRF gene regulation and point to the usefulness of pharmacogenomic studies for understanding the mechanisms of action for ethanol. They also provide evidence that the two models we used, primary hypothalamic cells and transfection of the CRF promoter into NG108-15 cells, represent useful combined tools for cellular and molecular studies focused on CRF synthesis and release.

Experimental methods Reagents Ethanol (USP grade, 200 proof) was purchased from AAPER alcohol and Chemical Co. (Shelbyville, Kentucky). Forskolin and adenosine deaminase (ADA) were obtained from Sigma (St. Louis, MO). Rp-cAMP (adenosine-3V-5-cyclic phosphorothioate, Rpisomer) was purchased from LC laboratories (Woburn, MA) and H-89 was from Calbiochem (La Jolla, CA). Primary hypothalamic cell culture Pregnant adult Sprague – Dawley rats were allowed to give birth in our animal care facility at The Salk Institute. Brains were obtained at postnatal days 6 – 7, a period of rapid increase in the CRF content of the neonatal hypothalamus (Walker et al., 1986). The culture of hypothalamic cells was carried out using a

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previously described method (Papa et al., 1995; Widmaier et al., 1989). The pups were decapitated and hypothalamus were dissected at 4-C and placed into ice-cold Leibovitz medium (Invitrogen Inc., Carlsbad, CA) containing 0.6% glucose and 7.5 Ag/ml gentamicin (Invitrogen Inc., Carlsbad, CA). The hypothalamus was extended anteriorly about 1 mm rostral to the optic chiasm posteriorly by the region just rostral to the mammillary bodies, and laterally by the hypothalamic sulcus. The depth of the excised tissue was dorsally 3 mm. The tissues were washed twice with plating media (containing heat-inactivated 5% fetal bovine serum, 5% heat-inactivated horse serum prepared in Engle’s minimal essential medium with 0.6% glucose, 2 mM glutamine, 7.5 Ag/ml gentamicin. All these reagents are obtained from Invitrogen Inc. (Carlsbad, CA) and mechanically dissociated in a small volume of plating medium by gently pushing them through 20- and 22-gauge needles to disperse the tissue. The dispersed cells were washed through a sieve (with openings of 70 Am) and then centrifuged at 2000  g for 10 min. The cells were resuspended in plating medium and 2  106 cells per well were placed in 6-well plates (Costar, Cambridge, MA). The plates, previously coated with 20 Ag/ml poly-d-lysine (Sigma, St. Louis, MO), were incubated overnight at 37-C. After plating, the cells were incubated at 37-C in a 95% O2 5% CO2 incubator. Medium was replaced by fresh plating medium at day 3 and then changed every 2 days. The cells were kept in culture for 8 – 10 days before use. Stimulation experiments with primary hypothalamic cells All stimulation experiments were performed on cells that had been in culture for 9 – 10 days. On the day of the experiment, the cells were washed with Krebs bicarbonate buffer (KRB, pH 7.4) containing 2 mg/ml bovine serum albumin (BSA, Fraction V, Sigma, St. Louis, MO) and 2.8 mM glucose. The cells were then incubated with the following reagents, made up in KRB buffer: forskolin (25 AM), ethanol (25 mM), or a combination of forskolin (25 AM) with 25 mM ethanol, as well as a combination of PKA inhibitor H89 (10 AM) or Rp-cAMP (250 AM) with 25 mM ethanol. Control groups were incubated with KRB buffer alone. Once preliminary data were obtained, subsequent experiments were done with selected time points considered as most representative. For the CRF secretion experiment, the incubation buffer was collected at the end of incubation and stored at 20-C until the radioimmunoassay (RIA). For the measurement of intracellular CRF content, cells were lysed with 0.1% NP-40 in KRB buffer and sonicated using a Microson Ultrasonic Cell Disruptor XL 2000 (Misonix Inc., New York, NY) for 10 s at level 3. After centrifugation, the supernatant was stored at 20-C. All samples were lyophilized and reconstituted to 33% of the original volume with RIA buffer before use. The method of ethanol exposure to cells was similar to that described by Gordon et al. (1986). All culture plates were wrapped in Parafilm to prevent ethanol evaporation. The ethanol concentration was tested during the course of the experiment and was found to decrease by <10% over a 12-h period. CRF radioimmunoassay CRF was measured by RIA as previously described (Vale et al., 1983). The assay standard was synthetic human/rat CRF-41 that was dissolved in the RIA assay buffer (0.1 M NaCl, 0.05 M

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Na2HPO4 – NaH2PO4, 0.025 M EDTA, 0.1% Na Azide, 0.1% BSA, (crystalline), 0.05% Triton X-100, pH 7.5). Rabbit anti-rat CRF-41 (rc68), raised in our laboratory, was used at a final dilution of 1:700,000 in RIA buffer and incubated with 100 Al of samples or standards for 24 h at 4-C. 100 Al 125I iodinated TyrCRF-41 (labeled in our laboratory and exhibiting 20,000 – 25,000 cpm/min; Vale et al., 1983) was diluted in assay buffer with 0.5% normal rabbit serum and added to all samples for an additional 24 h. Sheep anti-rabbit gamma globulin was then added to the samples for an additional 30 min at room temperature. Samples were precipitated by centrifugation at 3000  g for 45 min, the supernatant was discarded and the radioactivity of the pellet was measured using a gamma counter. The ED80 of the assay is 5.5 T 0.10 pg/100 Al. CRF mRNA detection by reverse transcriptase-polymerase chain reaction (RT-PCR) Total RNA from primary cells was isolated using the Trireagent (Molecular Research Center, Inc., Cincinnati, OH) according to the manufacturer’s instructions. One milliliter per well of Tri-reagent was added to the 6-well plates. The cells were lysed directly in the culture plate by passing them several times through a pipette. Each homogenate was transferred to a 1.5-ml polypropylene centrifuge tube and 0.2 ml of chloroform was added at room temperature for 5 min. Samples were vigorously vortexed for 15 s, incubated at room temperature for 10 min, and centrifuged at 12,000  g for 15 min. The top aqueous layer, containing the RNA, was precipitated by addition of 0.5 volume (approximately 0.5 ml) of isopropanol, followed by centrifuging at 12,000  g for 8 min. The resulting white pellets were washed with 75% ethanol, and each pellet was then resuspended in 20 Al of RNase-free water. For the cDNA synthesis, 1.0 Ag of total RNA was reverse transcribed for 1 h at 37-C. The 20-Al reaction mixture contained 4 Al 5 first strand transcription buffer, 1 Al (0.5 Ag) of oligo(dT) primer, 1 Al 10 mM deoxynucleotide triphosphate, 8 U RNaseOUTi, and 100 U SuperScriptII (Invitrogen Inc., Carlsbad, CA). The PCR amplification was performed on 2 Al cDNA in a final volume of 50 Al including 5 Al 10 PCR buffer, 1.5 Al 50 mM MgCl2, 1 Al of 10 mM deoxynucleotide triphosphate, 2 Al of 10 pmol forward and reverse primers, and 1 U Taq polymerase (BIO-X-ACT DNA polymerase, Bioline UK Ltd.). The rat CRFspecific primers (forward: 5V-GAA GAG AAA GGG GAA AGG CAA AGA-3V, reverse: 5V-GCG GTG AGG GGC GTG GAG TT3V) were designed to amplify a 403-bp fragment across the first intron, ruling out the possibility of identical size bands resulting from genomic DNA contamination. PCR was performed in a PTC100i Programmable Thermal Controller (MJ Research. Inc., Watertown, MA). The initial denaturing step at 94-C for 5 min was followed by 35 cycles of amplification (denaturing for 45 s at 94-C, annealing for 45 s at 62-C for the first 5 cycles and 55-C for the following 30 cycles, primer extension at 72-C for 90 s). The final extension step took place for 7 min at 72-C. RT-PCR analysis of glyceraldehydes-3-phosphate dehydrogenase (GAPDH) mRNA level was performed as an internal control. GAPDH-specific primers (forward: 5V-AGA CAG CCG CAT CTT CTT GT-3V, reverse: 5V-CTT GCC GTG GGT AGA GTC AT-3V) were used to amplify a 207-bp fragment. All primers were purchased from Genbase Inc. (La Jolla, CA). PCR amplification products (20% of the reaction) were separated on a 2% agarose gel, and detected by ethidium bromide staining. The ratio of CRF to

GAPDH was quantified using Kodak 1D Image Analysis Software (Eastman Kodak Company, Rochester, NY). CRF promoter activity assay Plasmid constructs The promoter activity of the rat CRF upstream region was analyzed using a transient transfection assay. The luciferase reporter construct pGL2-rcpi1500 was generated by insertion of the 1.5-kb XbaI/KpnI DNA fragment containing rat CRF 5’ upstream region and part of exon 1, into the NheI/BglII site of pGL2-Basic (Promega, Madison, WI). This construct was cut with the restriction enzyme BglII and then self-ligated to generate the construct pGL2-rcpi498, which deleted the upstream region of 499 to 1403. The construct pGL2-rcpi498mCRE that contained 4-bp deletion in the CRE (TGACGTCA) located 223 to 230 upstream of CRF promoter was made by cutting with the restriction enzyme AatII, Klenow treatment, and followed by self-ligation. The construct pGL2-rcpi498CRE5 was created by introducing a 60-bp sequence that contained 4 CRE repeats into an ApaI site at 131 upstream promoter region of the rat CRF gene. To construct the CRF promoter with additional multiple CRE, the plasmid pGL2-rcpi498 was digested with ApaI. Synthesized oligonucleotides purchased from Genbase Inc. (La Jolla, CA) containing 4 repeat CRE (5V-CGT TGACGTCA CCACGT TGACGTCA CCACGT TGACGTCA CCACGT TGACGTCA CCA-3V, and 5VT G G T G A C G TC A A CG T G G T G A C G TC A A C G T G G TGACGTCA ACGTGG TGACGTCA ACG-3V) with ApaI site on either end were annealed and ligated to the unique ApaI site of the pGL2-rcpi498 to generate pGL2-rcpi498CRE5. The pGL2rcpi498CRE5 contained a total of 5 repeats of CRE elements. Restriction enzymes and Klenow enzyme were obtained from New England Biolabs (Beverly, MA). Transfection and luciferase assay The transient transfection assay was performed in NG108-15 cells. The cells were maintained in Dulbecco’s modified Eagle’s medium (Invitrogen Inc., Carlsbad, CA) supplemented with 10% heat-inactivated fetal bovine serum, and were transfected with PerFectini Transfection reagent (Gene Therapy System Inc., San Diego, CA). The cells were plated at a density of approximately 5  104/well in 24-well plates and transfected the next day with 0.75 Ag of reporter plasmid and 50 ng of RSV-h-gal (Promega, Madison, WI) as an internal control. To analyze the stimulatory effects of forskolin (25 AM), ethanol (25 mM), and the co-incubation of forskolin and ethanol on the CRF promoter activity, the NG 108-15 cells were transfected with reporter plasmids for 24 h, and fresh medium containing the reagents were added to the cells. To determine the role of extracellular adenosine and the effects of PKA inhibitor on ethanol-induced CRF promoter activity, the transfected cells were treated with 25 mM ethanol after preincubating 30 min with adenosine deaminase (ADA, 1 U/ml), H89 (10 AM), or Rp-cAMP (250 AM). Conditions for exposure to ethanol were based on procedures described in ‘‘Stimulation experiment on primary hypothalamic cells’’. The cells were harvested and assayed for luciferase and h-galactosidase activity after exposure to the above reagents for a different time period. The luciferase activity expressed from each reporter construct was normalized to the h-galactosidase activity.

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Statistical analysis Results are reported as mean T SEM. The significance between treatments and respective controls was assessed by one-way ANOVA followed by post hoc analysis using the Newman – Keul test. Differences were considered to be statistically significant at P < 0.05.

Acknowledgments The authors are indebted to Ms. Joan Vaughan (Salk Institute, La Jolla, CA) for preparing the CRF tracer and to Dr. Jean Rivier (Salk Institute, La Jolla, CA) for the gift of CRF. Research supported by NIH grants AA-08924 and AA-06420.

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