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Ethanol Induces Apoptotic Death of Developing -Endorphin Neurons via Suppression of Cyclic Adenosine Monophosphate Production and Activation of Transforming Growth Factor-1-Linked Apoptotic Signaling

Endocrine Program and Department of Animal Sciences, Rutgers, The State University of New Jersey, New Brunswick, New Jersey

Received July 18, 2005; accepted December 2, 2005

	Abstract

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The mechanism by which ethanol induces -endorphin (-EP) neuronal death during the developmental period was determined using fetal rat hypothalamic cells in primary cultures. The addition of ethanol to hypothalamic cell cultures stimulated apoptotic cell death of -EP neurons by increasing caspase-3 activity. Ethanol lowered the levels of adenylyl cyclase (AC)7 mRNA, AC8 mRNA, and/or cAMP in hypothalamic cells, whereas a cAMP analog blocked the apoptotic action of ethanol on -EP neurons. The AC inhibitor dideoxyadenosine (DDA) increased cell apoptosis and reduced the number of -EP neurons, and it potentiated the apoptotic action of ethanol on these neurons. -EP neurons in hypothalamic cultures showed immunoreactivity to transforming growth factor-1 (TGF-1) protein. Ethanol and DDA increased TGF-1 production and/or release from hypothalamic cells. A cAMP analog blocked the activation by ethanol of TGF-1 in these cells. TGF-1 increased apoptosis of -EP neurons, but it did not potentiate the action of ethanol or DDA actions on these neurons. TGF-1 neutralizing antibody blocked the apoptotic action of ethanol on -EP neurons. Determination of TGF-1-controlled cell apoptosis regulatory gene levels in hypothalamic cell cultures and in isolated -EP neurons indicated that ethanol, TGF-1, and DDA similarly alter the expression of these genes in these cells. These data suggest that ethanol increases -EP neuronal death during the developmental period by cellular mechanisms involving, at least partly, the suppression of cAMP production and activation of TGF-1-linked apoptotic signaling.

One possibility is that reduced cellular levels of cAMP activate transforming growth factor-1 (TGF-1) to induce neuronal apoptosis, because cAMP reduces TGF-1 gene transcription in pituitary cells (Pastorcic and Sarkar, 1997) and inhibits TGF-1-induced Smad3/4-dependent transcription in keratinocytes (Schiller et al., 2003). In addition, TGF-1 induces apoptosis of cerebellar granule neurons (De Luca et al., 1996) and the developing chick retina (Schuster et al., 2002). However, TGF-1 also can be neuroprotective in hippocampal and cortical neurons (Henrich-Noack et al., 1996; Scorziello et al., 1997). Furthermore, TGF-1 knockout mice showed increased neuronal cell death and microgliosis in mouse brain (Brionne et al., 2003). Hence, the mediatory role of TGF-1 in the apoptotic action of ethanol on hypothalamic neurons needed to be demonstrated.

Three different isoforms of TGF-s, TGF-1 to -3, have been described in mammalian cells and have been shown to have similar biological activities in many cells. In the developing nervous system, TGF-s are identified in many populations of postmitotic, differentiating neurons (Unnsicker et al., 1991; Krieglstein et al., 2000). TGF-1 is secreted in an inactive, latent form and is activated by acidification, alkalization, proteases, or heat (Roberts and Sporn, 1990). TGF- receptors are present in the hypothalamus and on -EP cells (Bouret et al., 2001). TGF-1-induced apoptosis in non-neuronal cells is associated with increased mitochondrial apoptotic proteins bcl-xs, bak, and bax, and decreased mitochondrial antiapoptotic proteins bcl-xL and bcl-2. It is believed that increased activity of proapoptotic peptides causes cytochrome c release to activate caspases and to cause cell death (Nass et al., 1996; Francis et al., 2000; Lee et al., 2002). It is not known whether TGF-1-induced signaling is required for apoptosis of hypothalamic cells, particularly -EP neurons.

In this study, we demonstrated that ethanol decreased cellular levels of cAMP and TGF-1-regulated apoptotic signaling to induce death of developing hypothalamic cells in cultures. Furthermore, we identified that -EP neurons are one of the hypothalamic cell types that were a target of ethanol.

	Materials and Methods

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Animal Use. Pregnant Sprague-Dawley female rats were obtained from Simonsen Laboratories (Gilroy, CA) and were used as the source of fetal rat brains for hypothalamic cell cultures. Animal surgery and care were performed in accordance with institutional guidelines and complied with the National Institutes of Health policy. The animal protocol (99-005) was approved by the Rutgers Animal Care and Facilities Committee.

Primary Cultures of Hypothalamic Cells and Treatments. Primary cultures of fetal hypothalamic cells were prepared from the mediobasal part of the hypothalamus (containing neuroendocrine neurons, including -EP, dopamine, thyrotropin-releasing hormone, and growth hormone-releasing hormone as well as containing glial cells; Brown, 1998). In brief, pregnant rats of the Sprague-Dawley strain (Simonsen Laboratories) at 18 to 20 days of gestation were sacrificed, and the fetuses were removed by aseptic surgical procedure. Brains from the fetuses were immediately removed; hypothalami were separated and placed in ice-cold Hanks' balanced salt solution containing antibiotic solution (100 U/ml penicillin, 100 µg/ml streptomycin, and 250 ng/ml amphotericin B), 0.1% bovine serum albumin, and 200 µM ascorbic acid (all from Sigma-Aldrich, St. Louis, MO). The block of hypothalamic tissue consisted of the mediobasal portion of the hypothalamus and extended approximately 1 mm rostral to the optic chiasma and just caudal to the mammillary bodies, laterally to the hypothalamic sulci, and dorsally to 2 mm deep. The hypothalamic cells were washed and then incubated at 37°C for 5 min using the same medium. After dispersion, the cells were plated at a density of 3.0 x 10⁶ cells per 25-mm² flask and at a density of 1.0 x 10⁶ cells per well in a 24-well plate. Both the flask and plate were coated with polyornithine at a concentration of 100 µg/ml and then incubated for 3 h. The cells were maintained in Dulbecco's modified Eagle's medium with 10% fetal calf serum at 37°C and 7.5% CO₂ in a humidified water-jacketed incubator for 2 days. After 2 days of plating, the medium was changed at 1-day intervals with serum-free, chemically defined medium (consisting of 30 nM selenium, 20 nM progesterone, 1 µM iron-free human transferrin, 100 µM putrescine, and 5 µg/ml insulin). On the 3rd day, the medium was removed, and the cultures were treated with vehicle or various doses of ethanol; TGF-1; dideoxyadenosine (DDA); dibutyryl-cAMP (dbcAMP); a caspase-3 blocker, Ac-DEVD-CHO; or staurosporin (STS) for 1, 2, or 4 days. All of the culture reagents were purchased from Sigma-Aldrich with the exception of fetal calf serum, TGF-1, and Ac-DEVD-CHO, which were purchased from Hyclone (Logan, UT), R&D Systems (Minneapolis, MN), and Calbiochem (San Diego, CA), respectively.

Immunocytochemistry. To identify apoptotic -EP neurons, hypothalamic cell cultures were double stained with terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL)-biotin labeling (Apoptosis kit; Roche Diagnostics, Indianapolis, IN) and immunoreactive -EP using an ABC kit (Vector Laboratories, Burlingame, CA) as described by us previously (De et al., 1994). The antibody for -EP was Y-10 (a gift from Dr. S. S. C. Yen, University of California, San Diego, CA) and used at a dilution of 1:1000. The immunoreactivities of Y-10 have been well characterized and found to be specific for -EP. We have shown previously that preincubation of the antiserum with an excess (100 µg/ml) of -EP antigens eliminated immunoreactive staining in hypothalamic cultures (De et al., 1994). Routine counts of cells exhibiting -EP immunoreactivities or combined TUNEL and -EP-like immunoreactivities were completed by two independent investigators. Approximately 200 to 500 total cells in each culture were counted, and the percentages of -EP or TUNEL and -EP-positive cells in each culture were determined. Colocalization of TGF-1 in -EP staining was carried out using a double-label method as described by us previously (Burns and Sarkar, 1993) using -EP antibody Y-10, TGF-1 goat antibody (1 µg/ml; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), biotinylated ABC reagents (Vector Laboratories), 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium as a coloring reagent for -EP staining, and diaminobenzidine as a coloring agent for TGF-1 staining. Preincubation of the antisera with an excess (100 µg/ml) of TGF-1 antigens reduced immunoreactive staining.

Protein and Apoptotic Enzyme Assays. Hypothalamic cell extracts were used to measure levels of nucleosome, caspase-3, and cAMP using a nucleosome ELISA kit (Oncogene Research Products, Boston, MA), a caspase-3 ELISA kit (Calbiochem), and a cAMP kit (Amersham Biosciences Inc., Piscataway, NJ) following instructions from the manufacturers. The supernatant samples of hypothalamic cell cultures treated with vehicle, ethanol, or DDA were used to assess TGF-1 release. The media samples were acidified using 4 mM HCl to activate a latent form of TGF- before measuring the peptide. The TGF-1 levels were determined by a Quantikine ELISA kit (R&D Systems) according to the kit's instructions. Protein contents of the cell extracts were determined using Bio-Rad DC Protein Assay reagents (Bio-Rad, Hercules, CA). Protein values were used to calculate the amount of cellular cAMP as picomoles per microgram of protein and caspase-3 activity as picomoles per minute per milligram of protein.

Laser Capture Microdissection of -EP Neurons. A rapid immunohistochemical staining protocol for -EP was developed to prevent the significant degradation of RNA because of prolonged incubation in aqueous media during the standard staining process. In brief, hypothalamic cell cultures maintained on glass slides were fixed with 4% paraformaldehyde for 5 min. The endogenous peroxidase activity in these cells was inhibited by incubating with 1% H₂O₂ in methanol for 10 min. In the presence of an RNase inhibitor (1 unit/µl), the culture slides were incubated with 10% blocking serum in 0.1 M phosphate buffer saline for 15 min, primary antibody (1:400 anti--EP; Y-10) for 90 min followed by secondary antibody (1:200; biotinylated anti-rabbit) for 30 min. The slides were then incubated with ABC solution (Vector Laboratories) made in 0.1 M phosphate buffer saline for 30 min and stained using diaminobenzidine. After -EP immunoreactivity was developed, the slides were completely dehydrated by incubating in graded ethanol solutions (75%, 30 s; 95%, 30 s; and 100% twice, 30 s) and xylene (twice; 1 min). From each culture slide, approximately 1000 individual positive cells were captured using the PixCell LCM system (Arcturus, Mountain View, CA). Laser spot size was set to 7.5 µm. The power amplitude and pulse duration of the PixCell laser were adjusted for each slide (65-75 mW, 750-850 ms). The thermoplastic film-coated caps containing the captured cells were incubated in proteinase K (2 mg/ml) solution made in lysis buffer (20 mM Tris-HCl, pH 8.0, 20 mM EDTA, and 2% sodium dodecyl sulfate) and examined under the microscope to ensure complete cell lysis. Total RNA from the LCM-captured cells was extracted using a Micro RNA isolation kit (Stratagene, La Jolla, CA) following the manufacturer's instructions. The genomic DNA from the RNA solution was removed by DNase I treatment, and the remaining RNA was amplified using a single round of linear amplification technique using the standard protocol with the RiboAmp RNA amplification kit (Arcturus). To assess the quality of RNA, culture slides from which the -EP cells had been captured were scraped and extracted for RNA. Obtained RNA was run on 1.2% agarose gel, which displayed 18S and 28S bands with slight smear, indicating the prominent RNA remained intact (data not shown). An additional sample with 2000 randomly captured cells, isolated using a procedure identical to that of the tested samples, was used to assess the quality of amplified antisense RNA (aRNA). After amplification, the aRNA from this sample was run on 1.2% agarose gel. The bulk of aRNA ranged from around 200 to 1700 bases, which was within the expected base length for amplified aRNA with the Ribo-Amp kit. The mRNA levels of ACs, TGF-1, and apoptosis regulatory genes in enriched -EP neurons were measured using quantitative real-time reverse transcription (RT)-PCR. Four independent samples were used for each group.

Real-Time Reverse Transcription-Polymerase Chain Reaction. Expression levels of various apoptotic and antiapoptotic genes in cultured cells were measured by quantitative real-time RT-PCR (TaqMan assay) using an ABI Prism 7700 sequence detector (Applied Biosystems, Foster City, CA). This assay is based on the 5' nuclease activity of TaqDNA polymerase for fragmentation of a dual-labeled fluorogenic hybridization probe. Total RNA was isolated from hypothalamic cultures treated with vehicle, ethanol, TGF-1, or DDA using the RNeasy mini kit (QIAGEN, Valencia, CA) and following the manufacturer's instructions. Total RNA (1 µg) was subjected to first-strand cDNA synthesis using the SuperScript First-Strand Synthesis system for RT-PCR (Invitrogen, Carlsbad, CA). cDNA was subjected to real-time RT-PCR using either the fluorogenic 5' nuclease assay (all the genes except AC7) or SYBR Green I double-stranded DNA binding dye chemistry (AC7), which were both provided by Applied Biosystems sequence detection systems. Gene-specific primers and fluorescent-labeled probes were designed using the application-based primer design software, Primer Express version 1.5 (Applied Biosystems) and were based on published GenBank sequences. These primers and probes are gene-specific as confirmed by the BLAST search and are listed in Table 1. We could not reliably detect the level of p27/kip mRNA in the cell extracts; therefore, this gene was not used in this study. Amplification was performed for one cycle of a sequential incubation at 50°C for 2 min and 95°C for 10 min, and subsequent 40 cycles of a consecutive incubation at 95°C for 15 s and 60°C for 1 min, except for AC7, which was detected by running 40 cycles at 95°C for 15 s and 52°C for 1 min, followed by 72°C for 1 min. The PCR products were run on a 1.5% agarose gel to verify the appropriate size of the amplicons. Relative quantity of mRNA was calculated by relating the PCR threshold cycle obtained from the tested samples to relative standard curves generated from a serial dilution of cDNA prepared from the total RNA. The mRNA level in each sample was normalized with the level of glyceraldehyde-3-phosphate dehydrogenase mRNA, which was measured by a control reagent (Applied Biosystems). Data shown in the tables are mean ± S.E.M. percentage of control values. Six to eight independent samples were used for each group.

Statistical Analysis. The data shown in the figures and text are mean ± S.E.M. Data comparisons between two groups were made using t tests, whereas comparisons among multiple groups were made using one-way analysis of variance. Post hoc tests involved the Student-Newman-Keuls test. A value of p < 0.05 was considered significant.

	Results

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Using fetal hypothalamic cells in primary cultures and DNA fragmentation assay, we have previously shown that long-term exposure to ethanol induces apoptotic death of hypothalamic cells during the developmental period (De et al., 1994). In this study, using cells from the mediobasal hypothalamus of fetal rats, we also found that exposure to ethanol doses of 50 to 150 mM for 2 or 4 days dose dependently increased the amount of DNA damage in the cells as determined by the nucleosome activity (a marker for DNA damage in the cells; Allen et al., 1997; Fig. 1A). Studies were conducted to determine whether ethanol-induced cell apoptosis in hypothalamic cultures involved activation of caspase-3. This enzyme is known to increase endonuclease activity to cause cell apoptosis (Cohen, 1997). As shown in Fig. 1B, ethanol dose dependently increased the activity of caspase-3 in hypothalamic cells after 2 and 4 days of treatment (Fig. 1B). We have shown previously that long-term treatment with 200 mM ethanol increases the number of apoptotic -EP neurons (as determined by Nissl and TUNEL stains) in developing hypothalamic cell cultures (De et al., 1994). In this study, we show that treatment with a moderate dose of ethanol (100 mM) for 2 or 4 days reduced the number of -EP-immunoreactive neurons in the hypothalamic cell cultures (Fig. 1, C-E). We also counted the number of -EP-immunoreactive cells stained with TUNEL (which identifies apoptotic cells; Allen et al., 1997) after 100 mM ethanol for different times. As shown in Fig. 1, F and G, many TUNEL-positive cells in cultures were -EP positive (green looked yellow when colocalized in red -EP cells). Furthermore, the number of TUNEL-positive -EP-immunoreactive cells was low in control cultures and in ethanol-treated cultures after 1 day of treatment but increased significantly after 2 and 4 days of ethanol treatment compared with controls (Fig. 1H). These results suggest that ethanol induces apoptotic death of hypothalamic cells, many of which are -EP neurons.

View larger version (41K): [in this window] [in a new window]   Fig. 1. Ethanol-induced apoptosis of -EP neurons via a caspase-3-dependent mechanism. A and B, mean ± S.E.M. of the nucleosome (A) and caspase-3 activity (B) in cultures that were treated with various doses of ethanol for 2 and 4 days. *, p < 0.05, compared with control; **, p < 0.05, compared with all other groups on the same day. C and D, representative microphotographs showing -EP-immunoreactive cells in hypothalamic cultures treated 2 days with vehicle (control; C) or ethanol (100 mM; D). Arrows indicate -EP-positive cells. -, 10 µm. E, change in the mean ± S.E.M. numbers of -EP neurons in cultures treated with ethanol (100 mM) or vehicle for 2 and 4 days. *, p < 0.05, compared with control, n = 3. F and G, representative microphotograph showing colocalization of -EP (red) and TUNEL (green) immunostaining in cells of hypothalamic cultures treated 2 days with vehicle (control; F) or ethanol (100 mM; G). Arrows indicate TUNEL-positive -EP cells. -,20 µm. H, mean ± S.E.M. of the percentage of -EP cells that were TUNEL-positive in cultures treated with 100 mM ethanol or vehicle for various times, n = 3. *, p < 0.05, compared with control on the same day. J and I, effects of various doses of caspase-3 blocker (Ac-DEVD-CHO; DEVD) on ethanol-induced alteration in the number of -EP neurons (I) and TUNEL-positive -EP cells (J) in hypothalamic cell cultures. *, p < 0.05, compared with control; **, p < 0.05, compared with all other doses of DEVD.

Additional experiments using the potent caspase inhibitor Ac-DEVD-CHO revealed that this blocker reduced the effect of ethanol on hypothalamic cell caspase-3 activity 2 days after treatment (n = 5-6; control, 25 ± 2 pmol/min/mg; 150 mM ethanol, 125 ± 6 pmol/min/mg; ethanol + 1.0 µM Ac-DEVD-CHO, 90 ± 5 pmol/min/mg; ethanol + 10.0 µM Ac-DEVD-CHO, 38 ± 2 pmol/min/mg; and ethanol + 100.0 µM Ac-DEVD-CHO, 5 ± 1 pmol/min/mg; p < 0.01, ethanol versus the rest of the groups), indicating that the blocker is effective in reducing the function of the enzyme in these cells. The blocker also reduced ethanol-induced nucleosome activity at 2 days in hypothalamic cells (n = 6; control, 0.4 ± 0.2 unit/ml; 150 mM ethanol, 3.3 ± 0.4 unit/ml; ethanol + 1.0 µM Ac-DEVD-CHO, 2.8 ± 0.3 unit/ml; ethanol + 10.0 µM Ac-DEVD-CHO, 0.8 ± 0.3 unit/ml; and ethanol + 100.0 µM Ac-DEVD-CHO, 0.6 ± 0.1 unit/ml; p < 0.001, ethanol versus the rest of the groups, except ethanol + 1.0 µM Ac-DEVD-CHO). Furthermore, the caspase blocker reduced the inhibitory action of ethanol on the number of -EP neurons (Fig. 1I) and on the stimulatory action of ethanol on the number of TUNEL-positive -EP neurons (Fig. 1J). These data suggest that ethanol induces death of -EP neurons via a caspase-3-dependent mechanism.

We tested the hypothesis that ethanol reduces cAMP activity to cause apoptosis in -EP neurons, by determining the effect of ethanol and the AC inhibitor DDA (Shoshani et al., 1999) on intracellular levels of cAMP; mRNA levels of AC6, -7, and -8; -EP cell numbers; and TUNEL-positive -EP neurons in hypothalamic cell cultures. We have chosen these isoforms of AC because our preliminary study identified AC6, -7, and -8 as the major isoforms of AC in the hypothalamic cells that are ethanol-sensitive (Yoshimura and Tabakoff, 1999; Chandler et al., 2004; Maas et al., 2005). We have also determined the effect of the cAMP analog dbcAMP on ethanol-induced alterations in the number of -EP neurons and TUNEL-positive -EP neurons in hypothalamic cell cultures. Measurement of intracellular levels of cAMP revealed that the dose (100 mM) of ethanol that induced apoptosis of -EP neurons significantly reduced cellular levels of cAMP in hypothalamic cell cultures after 2 or 4 days of treatment (Fig. 2A). Like ethanol, DDA also reduced the cellular levels of cAMP in these cell cultures. Both ethanol and DDA reduced the mRNA levels of AC6 after 4 days of treatment, but after 2 or 4 days of treatment, they reduced adenylyl cyclases 7 and 8 in hypothalamic cells (Fig. 2, B-D).

View larger version (35K): [in this window] [in a new window]   Fig. 2. The apoptotic action of ethanol is mediated by adenylyl cyclase-cAMP suppression in hypothalamic cells. A, effects of ethanol and the AC inhibitor DDA on cellular levels of cAMP in hypothalamic cells. Fetal hypothalamic cells were treated with ethanol (100 mM), DDA (100 µM), or vehicle (control) for 2 and 4 days. Cells were lyses and used for determination of cAMP. Data are mean ± S.E.M. obtained from six to eight observations. *, p < 0.001, significantly different from the vehicle-treated control group on the same day. B to D, mean ± S.E.M. of the mRNA levels of AC6 (B), AC7 (C), and AC8 (D) in cultures treated with ethanol (100 mM), DDA (100 µM), or vehicle (control) for 2 and 4 days. *, p < 0.05, compared with control, n = 6. E and F, dose-response effects of DDA in the absence (E) or in the presence (F) of ethanol on TUNEL-positive -EP cells. Hypothalamic cells were treated with various concentrations of DDA (10 and 100 µM) or vehicle (control) in the presence or absence of 100 mM ethanol for 2 days. Cells were processed to determine the number of TUNEL-positive -EP cells by immunocytochemical methods. *, p < 0.05, compared with control; **, p < 0.01, compared with the group treated with the other dose of DDA, n = 6. G and H, effects of dbcAMP on ethanol-induced increase in the number of TUNEL-positive -EP cells (G) and the change in the numbers of -EP neurons in hypothalamic cultures (H). Hypothalamic cells were treated with vehicle (control) or ethanol (100 mM) with or without dbcAMP (1.0 µM) for 2 days. Cells were used for immunocytochemical localization of the number of -EP neurons or the number of TUNEL-positive -EP cells. Data are mean ± S.E.M. obtained from four to six observations. *, p < 0.001, significantly different from the rest of the groups.

The cAMP analog dbcAMP, when simultaneously treated with ethanol, decreased the ability of ethanol to increase the number of TUNEL-positive -EP neurons (Fig. 2G) and to reduce the number of -EP neurons (Fig. 2H). dbcAMP has also been shown to reduce ethanol-induced apoptosis of -EP neurons (De et al., 1994). These results suggest that ethanol reduces intracellular levels of cAMP to induce apoptosis of -EP neurons.

The mediatory role of TGF-1 in the apoptotic actions of ethanol and DDA on hypothalamic cells and -EP neurons was studied by determining the TGF-1 immunoreactivity in hypothalamic cells and -EP neurons, by measuring changes in the levels of TGF-1 mRNA and the release of the TGF-1 peptide after treatments with ethanol and DDA in hypothalamic cells, by studying the effects of dbcAMP on ethanol-induced TGF-1 release, by evaluating the effect of TGF-1 with or without ethanol/DDA on -EP neurons' apoptosis, and by determining the effect of a TGF-1 neutralizing antibody on ethanol- and STS-induced apoptosis of hypothalamic cells and -EP neurons. In fetal hypothalamic cultures, many cells showed TGF-1 immunoreactivity, some of which were colocalized in -EP immunoreactive cells and some other uncharacterized cells (Fig. 3, A and B). Measurement of TGF-1 mRNA levels in cell extracts and TGF-1 protein levels in media samples from these cultures revealed that hypothalamic cells produced and secreted TGF-1 (Fig. 3, C and D). Both mRNA expression and the TGF-1 release were elevated 2 days after ethanol or DDA treatment, and the release of the peptide remained elevated 4 days after ethanol or DDA treatment. Because many -EP cells were TGF-1-immunoreactive, the observed increase in TGF-1 release from hypothalamic cells after ethanol or DDA treatment may represent changes in TGF-1 release from -EP neurons.

View larger version (40K): [in this window] [in a new window]   Fig. 3. The apoptotic action of ethanol is mediated by adenylyl cyclase-cAMP suppression, leading to TGF-1 increase in -EP cells in hypothalamic cultures. A, representative microphotograph showing colocalization of -EP (blue) and TGF-1 (brown) immunostaining in hypothalamic cells in cultures. Arrows indicate TGF-1-positive -EP cells. B, representative microphotograph showing decreased staining in a control section incubated with excess antigen TGF-1 and its antiserum after staining for -EP (blue). -, 20 µm. C, stimulatory effect of ethanol and DDA on TGF-1 mRNA levels in hypothalamic cells in primary cultures. Cultures were treated with or without ethanol (100 mM) or DDA (100 µM) for 2 or 4 days; cells were extracted and used for mRNA measurements using RT-PCR. *, p < 0.05, significantly different from the control group, n = 5 to 6 per group. D, stimulatory effect of ethanol and DDA on TGF-1 release from fetal hypothalamic cells in primary cultures in serum-free defined medium. Cultureswere treated with or without ethanol (100 mM) or DDA (100 µM) for 2 or 4 days. Media samples were obtained for a period of 24 h before the end of the treatment and used in the TGF-1 ELISA assay. *, p < 0.05, significantly different from the control group, n = 5 to 7 per group. E, effect of dbcAMP on ethanol-induced increase in TGF-1 release in hypothalamic cultures. Hypothalamic cells were treated with vehicle (control) or ethanol (100 mM) with or without dbcAMP (1.0 µM) for 2 days. Media samples were collected at 24-h intervals and assayed for TGF-1 levels. Data are mean ± S.E.M. obtained from four to six observations. *, p < 0.01, significantly different from the rest of the groups on the same treatment day. F, concentration-dependent effect of TGF-1 on -EP neuronal apoptosis as determined by calculating the percentage of the -EP cells that were TUNEL-positive after treatment with TGF-1 for 2 days. *, p < 0.05, significantly different from the control group; **, p < 0.05, significantly different from the 0.05 and 0.5 ng/ml TGF--treated groups, n = 4 per group. G, inability of TGF-1 to increase apoptosis of -EP neurons in the presence of ethanol (100 mM). Hypothalamic cultures were treated with various concentrations of TGF-1 in the presence of 100 mM ethanol for a period of 2 days. These cultures were processed for immunocytochemical localization of -EP neurons that are TUNEL-positive. The mean ± S.E.M. percentage of the -EP cells that were TUNEL-positive are shown, n = 4 per group. H, inability of TGF-1 to increase apoptosis of -EP neurons in the presence of DDA (10 µM). Hypothalamic cultures were treated with various concentrations of TGF-1 in the presence of 10 µM DDA for a period of 2 days. These cultures were processed for immunocytochemical localization of -EP neurons that are TUNEL-positive. The mean ± S.E.M. percentage of the -EP cells that were TUNEL-positive are shown, n = 4 per group.

Determination of -EP neuronal viability indicated that these cells were very sensitive to TGF-1, because treatment of TGF-1 in a dose range of 0.05 to 2.0 ng/ml for 2 days concentration dependently increased the number of apoptotic -EP neurons as determined by counting the number of TUNEL-positive -EP-stained cells (Fig. 3F). This concentration range of TGF-1 that caused -EP neuronal apoptosis is within the range that was released (0.3-1.0 ng/ml released during a period of 2 days) after the treatment with a neurotoxic dose of ethanol (Fig. 3, D and E). When the dose-response effects of TGF-1 on -EP neurons were determined in the presence of high dose of ethanol (100 mM), TGF-1 failed to maximize the ability of ethanol to induce apoptosis of -EP neurons (Fig. 3G), supporting the view that ethanol and TGF-1 may share common pathway to induce apoptosis of these neurons. Likewise, determination of the TGF-1 action on -EP neurons in the presence of DDA indicated that the peptide failed to potentiate the ability of DDA to induce apoptosis of -EP neurons (Fig. 3H), supporting the view that TGF-1 and DDA may use common pathway to induce apoptosis of these neurons. These data suggest that TGF-1 produced by hypothalamic cells during ethanol or DDA challenge has the ability to increase the apoptotic death of -EP neurons.

Further studies were conducted to determine the mediatory role of TGF-1 in the apoptotic action of ethanol by determining the effect of its neutralizing antibody on basal and ethanol-induced changes in nucleosome levels in hypothalamic cells, number of -EP neurons, and in TUNEL-positive -EP neurons. In hypothalamic cell cultures, the TGF-1 neutralizing antibody completely blocked the apoptotic action of ethanol (Fig. 4A), suggesting that TGF-1 might be important in mediating the apoptotic action of ethanol on these cells. Figure 4, B and C, show that TGF-1 neutralizing antibody blocked ethanol-stimulated loss of -EP neurons and the number of TUNEL-positive -EP neurons. TGF-1 neutralizing antibody alone did not affect the number of -EP neurons or the TUNEL-positive -EP neurons. TGF-1 neutralizing antibody also failed to alter staurosporine-induced increase in nucleosome activity in hypothalamic cells (Fig. 4D), suggesting that the antibody has a specific action on blocking ethanol-induced apoptotic death of -EP neurons. Together, these data suggest that the increased TGF-1 release caused by ethanol may be important in the mediation of the apoptotic action of ethanol on -EP neurons.

View larger version (39K): [in this window] [in a new window]   Fig. 4. The blocking effect of TGF-1 neutralizing antibody on ethanol-induced apoptosis of -EP neurons. Effect of TGF-1 neutralizing antibody (anti-TGF-1) on ethanol-induced -EP neuronal apoptosis was determined by measuring the nucleosome levels in hypothalamic cells (A), the changes in the -EP cell number (B), and the percentage of the -EP cells that were TUNEL-positive (C). Cells were treated for 2 days with vehicle (control), ethanol (100 mM), TGF-1 neutralizing antibody (2 µg/ml), or with ethanol (100 mM) and a TGF-1 neutralizing antibody (anti-TGF-1; 2 µg/ml). At the end of the experiment, cultures were used for the determination of nucleosome levels, -EP neuron numbers, and TUNEL-positive -EP neurons. *, p < 0.05, compared with the rest of the groups, n = 4 to 6 per group. D, inability of TGF-1 neutralizing antibody to inhibit STS-induced apoptosis in hypothalamic cells. Hypothalamic cells were treated with vehicle (control), anti-TGF-1 (2 µg/ml), STS (0.1 or 0.5 µM), STS (0.1 or 0.5 µM), and anti-TGF-1 (2 µg/ml) for 1 day. At the end of the experiment, cultures were used for the determination of nucleosome levels by ELISA, n = 4 to 5 per group. *, p < 0.05, compared with control. **, p < 0.05, compared with similar treatment but with a low dose of STS-treated groups.

View larger version (50K): [in this window] [in a new window]   Fig. 5. Expression profiles of apoptotic regulatory genes after treatment with ethanol, TGF-1, and the adenylyl cyclase antagonist DDA in LCM-captured -EP neurons. Two-day-old hypothalamic cultures were incubated with medium containing serum supplement and 100 mM ethanol, TGF-1 (2 ng/ml), DDA (100 µM), or vehicle (control) for 2 days. A and B, representative photos showing a hypothalamic culture immunostained for -EP antigen. Photo A shows arrow points on some -EP cells before LCM dissection in a representative culture. Photo B shows the same hypothalamic culture after microdissection. -, 10 µm. C, representative photograph of the thermoplastic film-coated caps that contain the captured cells. D to L, histograms showing changes in the levels of AC7 (D), AC8 (E), TGF-1 (F), bcl-2 (G), bcl-xL (H), bcl-xs (I), bax (J), bak (K), and capsase-3 (L) after treatment with ethanol, TGF-1, DDA, or control. *, p < 0.05, compared with the control group, n = 4 per group.

	Discussion

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In this study, both AC7 and AC8 mRNA levels were reduced in -EP neurons and in hypothalamic cells after ethanol treatment for 2 and 4 days. Similar treatments with ethanol reduced cAMP levels in hypothalamic cells. Ethanol has been shown in the short term to increase the effect of cAMP analogs on -EP production and release in hypothalamic cells, but in the long term (>24 h), it prevents the effects of cAMP analogs and hormones that affect various AC-bound receptors and -EP neuronal functions (De et al., 1994; Boyadjieva et al., 1997). Hence, long-term ethanol treatment might affect the activity of adenylate cyclases to reduce the levels of cAMP production in -EP neurons and other hypothalamic cells. It should be noted that ethanol also alters adenosine release to affect intracellular levels of cAMP in -EP neurons in hypothalamic cultures (Boyadjieva and Sarkar, 1999). Long-term exposure to ethanol has been shown to desensitize the adenosine-regulated cAMP production and -EP release from hypothalamic neurons. Hence, ethanol might reduce AC-bound receptors and desensitize the adenosine-regulated cAMP production to reduce intracellular levels of cAMP. The present data are consistent with the concept that one of the cellular adaptations to long-term ethanol use is the down-regulation of signaling pathways driven by AC-cAMP in several brain regions (Yoshimura and Tabakoff, 1999; Chandler et al., 2004; Mass et al., 2005). Ethanol is known to interact directly with the adenosine system by blocking nucleoside transporters in the cell membrane. The effect of this inhibition is an increase in extracellular adenosine levels and adenosine receptor activation (Diamond and Gordon, 1997). Long-term ethanol treatment has been shown to reduce cAMP levels because of the desensitization of stimulatory G protein-coupled receptors (such as adenosine A2 receptors) seen after prolonged receptor activation (Hack and Christie, 2003). Hence, the inhibitory action of ethanol on cAMP might involve G protein-coupled adenosine receptors in -EP neurons and other cells in the hypothalamic.

In this study, we found that ethanol and DDA reduced the level of AC-cAMP, whereas it increased gene expression and/or release of TGF-1 in hypothalamic cells. Furthermore, the cAMP analog prevented ethanol-induced TGF-1 release in hypothalamic cells. We found that -EP neurons in hypothalamic cultures express TGF-1 mRNA and proteins, and both ethanol and DDA increase the gene levels of this peptide in these neurons. These data provide the first evidence that ethanol increases TGF-1 production and/or release by reducing AC-cAMP levels in -EP neurons and other cells in the hypothalamic. How cAMP represses the TGF-1 system in hypothalamic cells is not clearly understood. Most cAMP gene targets identified to date contain one or more cAMP-responsive elements (CREs; Borrelli et al., 1992). We do not know whether the effects of cAMP on TGF-1 represent CREs binding to the negative elements of TGF-1 gene promoters. One should note that the TGF-1 promoter contains AP-2-like sequence elements (Geiser et al., 1991), which could potentially mediate cAMP responses (Imagawa et al., 1987). On the other hand, the TGF-1 promoter includes at least three AP-1 binding sites that seem to mediate the induction of the genes activated by phorbol esters (Kim et al., 1990). The distinction between 12-O-tetradecanoyl phorbol acetate responsive elements and CREs has been blurred by many instances of "cross-talk" between protein kinase A and protein kinase C pathways, and it is possible that cAMP signals affect expression of the TGF-1 gene through AP-1 binding sites present on its promoter.

We have shown here that TGF-1 induces apoptosis of immature -EP neurons. The apoptotic action of TGF-1 on these neurons was similar in magnitude to those found after ethanol and DDA. TGF-1 was ineffective in inducing apoptosis in the presence of a maximal dose of ethanol or DDA, suggesting the possibility of using a common cellular mechanism by these agents in causing apoptosis of -EP neurons. We also showed that a TGF-1 neutralizing antibody blocks the apoptotic action of ethanol in -EP neurons. Hence, the apoptotic action of ethanol on these cells seems to have resulted from increased TGF-1 production. The role of TGF-1 in the regulation of apoptosis has been reported in only a few nervous system structures: cultured rat immature cerebellar neurons maintained in low potassium (De Luca et al., 1996); the chick ciliary, dorsal root, retina, and spinal motor neurons (Krieglstein et al., 2000; Dunker et al., 2001); and now rat hypothalamic cells.

The data presented here also showed activation of apoptotic molecules and inactivation of antiapoptotic molecules that are regulated by TGF-1 in -EP neurons after ethanol and DDA treatments. TGF-1 has been shown to stimulate apoptosis by changing the expression of the bcl-2 class of mitochondrial proteins in non-neuronal cells (Francis et al., 2000). Of the proteins we examined, the proapoptotic proteins bcl-xs, bax, and bak act to stimulate apoptosis, whereas the antiapoptotic peptides bcl-2 and bcl-xL inhibit apoptotic mechanisms. When the activity of the proapoptotic peptides predominates, cytochrome c is released to activate caspases. In our study, ethanol, TGF-1, and DDA all reduced the mRNA levels of bcl-2 and bcl-xL, whereas increasing the levels of bax, bak, and/or bcl-xs in -EP neurons. Furthermore, an increase in the activity of caspase-3 in ethanol-treated cultures and caspase-3 mRNA in -EP neurons is consistent with the amplification of apoptosis because of a loss of bcl-2 and an increase in bak. These data provide evidence that long-term ethanol exposure acting directly or via reducing cellular levels of cAMP stimulates TGF-1 signaling that up-regulates proapoptotic proteins but suppresses antiapoptotic proteins to mediate the apoptotic action of ethanol on -EP neurons.

The ethanol neurotoxic action on developing hypothalamic cells may have long-term consequences, because fetal alcohol exposure is believed to cause behavioral abnormalities in alcohol-exposed offspring (Meyer and Riley, 1986). It has been shown that the neurotransmitter system that regulates the neuroendocrine response to stress is especially vulnerable to ethanol during the developmental period in rats. Behavioral and neurochemical studies indicate that defects in the ability of these rats to respond appropriately to stress seem to be because of alterations in the function of hypothalamic peptides (Weinberg et al., 1996). -EP is one of these peptides that participates in bringing about the body's homeostasis after a stress response (Plotsky, 1986). Hence, loss of -EP neurons can have serious consequences on the stress axis functions for the alcohol-exposed developing fetus.

	Acknowledgements

 
We acknowledge assistance of Dr. Sudhir Jain in various protein ELISA assays and of Alok De for immunocytochemistry.

	Footnotes

 
This work was supported by National Institutes of Health grant AA08757 (to D.K.S.).

This work was presented in part at the 2004 Annual Meeting of the Research Society on Alcoholism.

Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.

doi:10.1124/mol.105.017004.

ABBREVIATIONS: -EP, -endorphin; AC, adenylyl cyclase; TGF-1, transforming growth factor-1; DDA, dideoxyadenosine; dbcAMP, dibutyryl cAMP; Ac-DEVD-CHO, Ac-Asp-Glu-Val-Asp-aldehyde; STS, staurosporine; TUNEL, terminal deoxynucleotidyl transferase dUTP nick-end labeling; ABC, ATP-binding cassette; ELISA, enzyme-linked immunosorbent assay; LCM, laser capture microdissection; aRNA, antisense RNA; RT-PCR, reverse transcription-polymerase chain reaction; CRE, cAMP-responsive element; AP, adaptor protein.

Address correspondence to: Dr. Dipak K. Sarkar, Endocrinology Program and Department of Animal Sciences, 84 Lipman Dr., Rutgers, The State University of New Jersey, New Brunswick, NJ 08901. E-mail: sarkar{at}aesop.rutgers.edu

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