Glucocorticoids plus opioids up-regulate genes that ... - Springer Link

Cell Mol Neurobiol (2007) 27:651–660 DOI 10.1007/s10571-007-9151-3

ORIGINAL PAPER

Glucocorticoids plus opioids up-regulate genes that influence neuronal function Gregg R. Ward Æ Steven O. Franklin Æ Tonya M. Gerald Æ Krystal T. Dempsey Æ Darrel E. Clodfelter Jr. Æ Dan J. Krissinger Æ Kruti M. Patel Æ Kent E. Vrana Æ Allyn C. Howlett

Received: 21 September 2006 / Accepted: 13 April 2007 / Published online: 7 June 2007 Springer Science+Business Media, LLC 2007

Abstract (1) This study investigated the functional genomics of glucocorticoid and opioid receptor stimulation in cellular adaptations using a cultured neuronal cell model.(2) Human SH-SY5Y neuroblastoma cells grown in hormone-depleted serum were treated for 2-days with the glucocorticoid receptor-II agonist dexamethasone (30 nM); the l-opioid receptor

G. R. Ward S. O. Franklin T. M. Gerald K. T. Dempsey D. E. Clodfelter Jr. A. C. Howlett Neuroscience of Drug Abuse Research Program, Julius L. Chambers Biomedical/Biotechnology Research Institute, North Carolina Central University, Durham, NC 27707, USA D. J. Krissinger K. M. Patel K. E. Vrana Department of Pharmacology, Pennsylvania State University College of Medicine/Milton S. Hershey Medical Center, Hershey, PA 17033, USA A. C. Howlett (&) Department of Physiology & Pharmacology, Wake Forest University Health Sciences, Medical Center Blvd., Winston-Salem, NC 27157, USA e-mail: [email protected] Present Address: G. R. Ward Department of Biology, Division of Natural Sciences & Mathematics, Saint Augustine’s College, Raleigh, NC 27610, USA Present Address: K. T. Dempsey Biogen Idec, Research Triangle Park, NC 27709, USA

agonist [D-Ala2, N-Me-Phe4, Gly5-ol]-Enkephalin acetate (DAMGO; 1 nM); or dexamethasone (30 nM) plus DAMGO (1 nM). RNA was extracted; purified, reverse transcribed, and labeled cDNA was hybridized to a 10,000-oliogonucleotide-array human gene chip. Gene expression changes that were significantly different between treatment groups and were of interest due to biological function were verified by real-time reverse transcription polymerase chain reaction (RT-PCR). Five relevant genes were identified for which the combination of dexamethasone plus DAMGO, but neither one alone, significantly up-regulated gene expression (ANOVA, P < 0.05). (3) Proteins coded by the identified genes: FRS2 (fibroblast growth factor receptor substrate-2; CTNNB1 (b1-catenin); PRCP (prolyl-carboxypeptidase); MPHOSPH9 (M-phase phosphoprotein 9); and ZFP95 (zinc finger protein 95) serve important neuronal functions in signal transduction, synapse formation, neuronal growth and development, or transcription regulation. Neither opioid, glucocorticoid nor combined treatments significantly altered the cell growth rate determined by cell counts and protein. (4) We conclude that sustained l-opioid receptor stimulation accompanied by glucocorticoids can synergistically regulate genes that influence neuronal function. Future studies are warranted to determine if combined influences of glucocorticoid fluctuations and opioid receptor stimulation in vivo can orchestrate exagerated neuroadaptation to reinforcing drugs under chronic mild stress conditions.

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Keywords Dexamethasone DAMGO Glucocorticoid receptors Mu-opioid receptors SHSY5Y human neuroblastoma Real-time reverse transcription-polymerase chain reaction Synaptic plasticity

Cell Mol Neurobiol (2007) 27:651–660

provide some insights into gene expression that is uniquely vulnerable to stimulation of the opioid peptide system under conditions of stress.

Methods Introduction

Cell culture and drug treatments

The abuse of psychoactive agents leads to long-lived changes in synaptic communication among neurons in the brain that are critical to the reward mechanisms, craving, and other emotional responses associated with drug use (Nestler 2004). Changes in synaptic structure and function orchestrate the alteration in the sensitivity of neural circuits as a result of changes in gene expression in response to continued drug exposure (Nestler 2004). Gene expression and the gene products that have physiological, behavioral, cognitive and affective influences are of great importance in drug abuse (Gatley and Volkow 1998). Endogenous opioids have been linked to drug seeking behavior (de Wit and Stewart 1983; Stewart 1984; Stewart and Wise 1992; Shalev et al. 2002). The environmental influence of stress, mediated in part by adrenal corticosteroids, promotes drug use and contributes to relapse (Koob and Le Moal 2001; Piazza and Le Moal 1998; Shalev et al. 2002; Kreek 2002). In the present investigation, the interaction between mild stress levels of glucocorticoids and sustained stimulation of l-opioid receptors was examined in cultured SH-SY5Y neuronal cells. Gene expression changes were initially screened using cDNA microarray analysis, which provided an estimate of genes to be examined by reverse transcription and real-time polymerase chain reaction (PCR) analysis. Treatment with the glucocorticoid receptor (GR-II) agonist dexamethasone in combination with the l-opioid agonist DAMGO increased expression of five genes (FRS2, CTNNB1, PRCP, MPHOSPH9 and ZFP95) that were not induced by those concentrations of the two drugs alone. These target genes code for proteins involved in neuronal survival, differentiated neuronal properties, and synaptic development or plasticity. The identification of genes that are expressed in a synergistic manner by combined glucocorticoids and opioid peptides may

SH-SY5Y human neuroblastoma cells (American Type Culture Collection or ATCC, Manassas, VA) were cultured in 100 mm cell culture dishes in DMEM/Ham’s F12 media (Invitrogen, Carlsbad, CA) supplemented with 10% charcoal-treated fetal bovine serum (0.2 mm filtered; Cocalico Biologicals, Inc., Reamstown, PA), 1,000 units/ml penicillin G sodium and 1,000 ml/ml streptomycin (Invitrogen, Carlsbad, CA) for 6 days before drug treatment. The cells were treated for 48 h as follows: (1) Control (2-hydroxypropyl-b-cyclodextrin, Sigma, St. Louis, MO); (2) dexamethasone formulated as a complex with 2hydroxypropyl-b-cyclodextrin, thereby making it miscible with water (30 nM; Sigma, St. Louis, MO); (3) [D-Ala2, N-Me-Phe4, Gly5-ol]-Enkephalin acetate (DAMGO) (1 nM; Sigma, St. Louis, MO); and DAMGO (1 nM) plus dexamethasone (30 nM). The cells were harvested after 48 h of treatment, and the RNA was isolated using the Trizol method (Chomczynski and Sacchi 1987) (Invitrogen, Carlsbad, CA).

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cDNA microarray hybridization analysis and real-time reverse transcription polymerase chain reaction (RT-PCR) For the cDNA gene expression microarrays (n = 3 culture dishes, each was tested in duplicate) isolated RNA samples were purified using the RNeasy MinElute Cleanup Kit (Qiagen, Valencia, CA) and total RNA purity was confirmed on 1.0% formaldehyde/denaturing agarose gels (Agilent Technologies, Santa Clara, CA). cDNA was produced by reverse transcription using the SuperScript reverse transcriptase Indirect cDNA Labeling System (Invitrogen, Carlsbad, CA). A reference standard was prepared, comprised of equal amounts of cDNA from each of the four groups. The cDNA was conjugated to


Alexa Fluor 555/Green (Molecular Probes Inc., Eugene, OR) for reference standard or Alexa Fluor 647/Red (Molecular Probes Inc., Eugene, OR) for treatment groups and was hybridized to a spotted 10,000-oligonucleotide human gene (MWG Biotech AG, Germany) array chip (Pennsylvania State University College of Medicine/Milton S. Hershey Medical Center/JDRF Functional Genomics Core Facility). Fluorescence intensities were quantitated at the following excitation/emission wavelengths: Alexa Fluor 555 (555 nm/565 nm); Alexa Fluor 647 (650 nm/670 nm). The sample data were normalized to the reference standard data to control for variability within samples. The normalized gene probe intensities underwent Lowess normalization and gene expression filtration using GeneSpring software (Agilent Technologies, Santa Clara, CA). The drug treatment groups were analyzed using one-way analysis of variance (ANOVA) and genes were identified that exhibited 1.2-fold or greater differences between treatment groups. These genes were further categorized according to treatment group differences from control, or the dual-treatment group difference from control or either of the singletreatment groups (Dunnett’s two-sample comparisons). To validate genes of interest, SH-SY5Y cultures (n = 6–9 biological replicates) were treated, and RNA purified as described above. The MinElute purified RNA from the four treatment groups was reversed transcribed into cDNA using MultiScribe reverse transcriptase (Applied Biosystems, Foster City, CA) and random hexamers as the primer. PCR reactions were implemented in a 25-ml-reaction volume using TaqMan Universal PCR Master Mix (Applied Biosystems, Foster City, CA) according to the Applied Biosystems instructions. Real-time PCR was performed using a 7500 Real-time PCR System with primers and probes from 20X Assays-on-Demand Gene Expression Assay Mix (Applied Biosystems, Foster City, CA). 18S ribosomal RNA served as the reference standard gene, and no differences between treatment groups were observed for the reference standard 18S RNA. Relative gene expression differences were analyzed using the Relative Expression Software Tool-Multiple Condition Solver (RESTMCS) method ((Pfaffl et al. 2002)). Gene expression was determined by REST-MCS according to the following equation:

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ratio ¼

Etarget

DCPtargetðMeancontrolMeansampleÞ

ðEref ÞDCPref ðMeancontrolMeansampleÞ

The crossing point (or CP; similar to the threshold cycle or Ct) was used as an index of gene expression. The Real-time PCR efficiency (E) was assumed to be the optimal value E = 2.0 (based on Applied Biosystems optimization procedures). Finally, REST-MCS employed a Pair Wise Fixed Reallocation Randomisation Test to determine differences between control and treated samples. Unlike t-tests and ANOVA which assume normality, this test makes no assumptions about the distribution of the data yet retains the statistical power of these more standard parametric tests. Statistically significant differences between control and treated groups were determined at P < 0.05. Cell growth rate and total protein assay SH-SY5Y cells were treated in 6-well culture plates, and at the indicated times, harvested in PBS-EDTA. Cell number was quantitated using a hemocytometer, and protein was determined (Bradford 1976). Doubling times were calculated by exponential growth curve regression analysis (n = 6 experiments) and statistically significant differences were determined by one-way ANOVA using GraphPad Prism 4 for Windows (GraphPad Software, Inc., San Diego, CA). Western immunoblotting analysis After the 48 h drug treatment, SH-SY5Y cells were harvested, washed and resuspended in ice cold PBSEDTA containing protease inhibitor cocktail set III (10 ml/ml; EMD Biosciences, San Diego, CA). Cells were homogenized, and sedimented at 600 g for 3 min to remove unbroken cells. Protein was determined (Bradford 1976), and each sample was resuspended in sample buffer, heated at 1008C for 5 min and loaded (30 mg in 40 ml) onto a sodium dodecylsulfate-10% polyacrylamide (SDS-PAGE) gel for electrophoresis. The proteins were transferred onto a polyvinylidene fluoride membrane using Towbin’s transfer buffer, and blocked in TBS (10 mM Tris–HCl, pH 7.4, 150 mM NaCl) containing 0.05% Tween-20, 5% Carnation fatty acid free dried milk, and incubated in primary antibody (murine monoclonal against b1-catinen, 1:500 in blocking

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solution (Invitrogen, Inc., Carlsbad, CA); or rabbit polyclonal against FRS2, 1:500 in blocking solution (Abcam, Inc., Cambridge, MA). After incubation in bovine anti-mouse IgG-HRP conjugate, 1:1000 (Santa Cruz Biotechnology, Santa Cruz, CA) or goat anti-rabbit IgG-HRP conjugate, 1:1000 (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA), blots were visualized using Enhanced ChemiLuminescenceTM (ECL) (Amersham Biosciences, Piscataway, NJ). For loading controls, the PVDF blots were stripped using RestoreTM Western Blot Stripping Buffer (Pierce Biotechnology, Inc., Rockford, IL) and reprobed using mouse monoclonal antibodies against GAPDH 1:500 (Chemicon International, Inc., Temecula, CA). Densitometry was performed using an Alpha Innotech Imager (Alpha Innotech Corp., San Leandro, CA), and the data were analyzed (one-way ANOVA) using GraphPad Prism 4 (GraphPad Software, Inc., San Diego, CA).

molecular functional categories including signal transduction, binding, cell adhesion, transcription regulation, transport, cell communication, development, death, cell growth and maintenance, behavior, and apoptosis regulation. There was no enrichment of genes within a particular biological function category compared with the distribution of these genes in the total array. This suggests that the gene expression profiles after treatment with dexamethasone, DAMGO or the combination of both, may not involve the targeted regulation of a particular cellular function in the SH-SY5Y cells. Selection of target genes for further examination primarily focused on genes coding for identifiable proteins, particularly those involved in signal transduction and cellular regulation. Genes chosen for further investigation were those that were up-regulated by the combination of dexamethasone plus DAMGO, but not by a single drug treatment alone.

Results

Genes influenced by the combination of dexamethasone plus DAMGO

The SH-SY5Y cell line, chosen as the model for neurons, is a line cloned from the SK-N-SH human neuroblastoma (Biedler et al. 1978), and it has been widely investigated for signal transduction pathways that are regulated by opioid drugs (Costa et al. 1992; Rubovitch et al. 2003). SH-SY5Y cells possess both l- and d-opioid receptors (Kazmi and Mishra 1987), and so in order to limit the response to the l-opioid receptor, the l-selective agonist DAMGO was used to stimulate the cells. The concentration of DAMGO (1 nM) was that which increased Ca2+ and cyclic AMP in the parent SK-N-SH neuroblastoma line (Fields and Sarne 1997; Sarne et al. 1998). The response to corticosteroids was according to a protocol by which the GR-II agonist dexamethasone (30 nM) was reported to maximally stimulate a glucocorticoid response element reporter gene in the related SK-N-ME neuroblastoma cells that had been grown for 2 days in serum devoid of glucocorticoids prior to addition of drug (Herr et al. 2000). cDNA microarray experiments indicated that expression of selected genes was changed subsequent to treatment with dexamethasone (30 nM) alone, DAMGO (1 nM) alone, or dexamethasone (30 nM) plus DAMGO (1 nM) in combination. These genes were classified according to biological processes and

Real-time RT-PCR analysis of genes, selected as coding for identified proteins involved in biological functions, indicated the up-regulation of the expression of five genes subsequent to 48 h treatment of SHSY5Y human neuroblastoma cells with dexamethasone (30 nM) plus DAMGO (1 nM): FRS2 (fibroblast growth factor receptor (FGFR) substrate-2; Sucassociated neurotrophic factor 1); CTNNB1 (b1catenin; cadherin-associated protein b1); PRCP (prolyl-carboxypeptidase; prekallikrein activator; angiotensinase C); MPHOSPH9 (M-phase phosphoprotein 9, MPP9); and ZFP95 (Kruppel zinc finger protein 95 mouse homolog) (Fig. 1). The treatment with dexamethasone alone, or DAMGO alone, did not increase the expression levels. The basal expression of these genes was within a 10-fold range (Table 1), and the increase in response to the combined glucocorticoid plus l-opioid agonist was approximately 50% above basal for each of these genes (Fig. 1). To determine whether the combined treatment of dexamethasone plus DAMGO could influence the cell growth rate, the cell number and protein analysis was determined over several doubling times in culture. Neither the cell growth rate as detected by cell count (Fig. 2) nor protein level (Fig. 2, insert)

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Fig. 2 SH-SY5Y cell growth rate during drug treatment. Cells were grown as described in the text, and cell counts and protein levels were determined. Cell numbers were converted to a log scale for linear regression analysis, and the slopes were determined as follows (mean ± SEM, ·100): Control, 8.2 ± 0.48; Dex, 8.7 ± 1.0; DAMGO, 8.5 ± 1.1; 8.2 ± 0.89. The groups were not significantly different at P < 0.05 as determined by ANOVA (n = 6 individual experiments). Insert: Protein samples were quantified at days 3, 5, and 7, and the mean ± SEM are shown for Control, Dex, DAMGO, and Dex plus DAMGO. These groups were not significantly different at P < 0.05 as determined by ANOVA Fig. 1 Gene expression profiles for target cDNAs determined by real-time RT-PCR analysis. The data are presented as means ± SEM. *P < 0.05 Treatment versus control gene expression by REST-MCS. Fold-increases in response to combined dexamethasone plus DAMGO for each gene are shown compared with control expression as 1.0 (mean ± SEM, n = 7–9)

was influenced by the combined drug treatment or by either drug treatment alone. The doubling times calculated for treatments were (days) (3.7, control; 3.9, dexamethasone; 4.4, DAMGO; and 4.7, dexa-

methasone plus DAMGO. These values were not significantly different between treated groups and the control. This suggests that gene expression was not influenced by differences in cell cycle or growth rate between the treatment groups. Antibodies were available for b1-catenin and FRS2 to determine whether protein levels increased in SH-SY5Y cells using Western immunoblotting technique. Treatment with 30 nM dexamethasone plus 1 nM DAMGO did not significantly increase the

Table 1 Relative gene expression of targets Ct average

Ct average

Target gene 18Sa

DCt DCt

Target cDNA CTNNB1

Target cDNA 23.17 ± 0.09d

18S 7.82 ± 0.11

DCt 15.35 ± 0.11

DDCt 0 ± 0.11

2DDCt 1.00 ± 0.10

PRCP

25.06 ± 0.08

7.61 ± 0.20

17.46 ± 0.14

2.10 ± 0.14

0.23 ± 0.03

ZFP95

24.91 ± 0.10

7.61 ± 0.20

17.30 ± 0.15

1.95 ± 0.15

0.26 ± 0.03

MPHOSPH9

26.41 ± 0.17

7.63 ± 0.10

18.78 ± 0.18

3.42 ± 0.18

0.09 ± 0.01

FRS2

25.39 ± 0.17

7.58 ± 0.06

17.81 ± 0.17

2.45 ± 0.17

0.18 ± 0.03

b CTNNB1

Relative to CTNNB1c

a

All target cDNAs were normalized to the 18S reference via the determination of the difference between each target cDNA average Ct value from the respective 18S average Ct value b The normalized target cDNAs were normalized to the most abundant cDNA (CTNNB1) via the determination of the difference between each target cDNA (DCt) and the normalized CTNNB1 (DCtCTNNB1) c The relative expression level of each target cDNA was compared to that of the most abundant CTNNB1 cDNA using the expression 2DDCt d

All data were determined using the Comparative Ct method, and are presented as means ± SEM (n = 7–9)

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levels of b1-catenin or FRS2 protein in crude homogenates at 48 h in a manner commensurate with b1-catenin gene expression (data not shown). A small increase in b1-catenin but not FRS2 could be observed at an earlier (36 h) time point (Fig. 3). Studies using increased concentrations of dexamethasone or DAMGO, or the combination, on b1-catenin and FRS-2 protein levels suggested that the interaction is complex. High drug concentrations, 100 nM DAMGO, or 100 nM DAMGO plus 300 nM dexamethasone produced small increases in both high and low mobility bands, with the lower mobility (phosphoryated) band predominating (data not shown). Based upon these preliminary observations, we propose the testable hypothesis that increased transcription or stabilization of mRNA could provide a mechanism to compensate for the degradation of proteins that accompanies the signal transduction pathways involving these proteins. The sub-cellular translocation of b1-catenin and FRS-2, phosphorylation, as well as the regulation of b1-catenin levels by proteasome degradation pathways (Murase et al. 2002; Castelo-Branco et al. 2004; Liu et al. 2002; Li et al. 2004) are regulatory processes that would reduce the levels of protein in the total cellular pool, and would require an increase in mRNA to replenish spent proteins.

Fig. 3 Western blot analysis of b1-catenin and FRS2 protein levels. Crude homogenates were subjected to SDS-PAGE as described in the text, and probed with antibodies to b1-catenin, FRS2, with GAPDH as the loading control. (A) Homogenates following 36 h treatment with 30 nM dexamethasone plus 1 nM DAMGO. (B) Homogenates following 36 h treatment with 300 nM dexamethasone, 10 nM DAMGO, 100 nM DAMGO, or combined 300 nM dexamethasone plus 100 nM DAMGO. These are representative examples of several experiments

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Discussion Neuronal post-mitotic survival and synaptic plasticity functions of target proteins The combination of dexamethasone plus DAMGO, but neither drug alone, up-regulated the expression of the genes FRS2, CTNNB1, PRCP, MPHOSPH9 and ZFP95. The present findings and previous research (Murase et al. 2002; Hinsby et al. 2004; Yu and Malenka 2004) suggest that the increased expression of genes for proteins that are involved in signal transduction, cell communication, cell growth and development, cell adhesion and transcription regulation may influence neuronal cell survival and synaptic plasticity functions within the neuron. M-phase phosphoprotein MPP9 is a protein that is phosphorylated by cdc2/cyclin B1 kinase in M-phase of proliferating cells including human neuroblastoma (Vincent et al. 1997), where it is localized to the cytosol (Matsumoto-Taniura et al. 1996). MPP9 is restricted to the golgi in non-mitotic phases of the cell cycle (Matsumoto-Taniura et al. 1996), and is not detected in its phophorylated form in terminally differentiated neurons of the adult brain (Vincent et al. 1997). However, M-phase kinases and their phosphoprotein products appear in neurons within neurofibrillary tangles of Alzheimer’s disease, suggesting an association with neuronal cell death in these diseased non-mitotic neurons (Vincent et al. 1997). Since the glucocorticoid- and/or opioid-treatments failed to alter growth rate in the SH-SY5Y cells, the stimulated induction of MPP9 as a substrate for the M-phase kinases would be more consistent with a role in the regulation of neuronal survival rather than cell cycle progression. ZFP95 is a member of the Kruppel zinc finger protein family, and contains a SCAN motif and a transcription repressor KRAB A domain (Dreyer et al. 1999). As this transcription regulator has been shown to be expressed in human brain (Dreyer et al. 1999), it may serve a function in regulating neuronal differentiation, as shown for a related zinc finger transcription repressor in a zebra fish neurodevelopment model (Levkowitz et al. 2003). FRS2 serves as a myristylated, tyrosine-phosphorylated adaptor signaling molecule that mediates the converging FGFR and neural cell adhesion molecule (NCAM) pathways stimulating neurite outgrowth in


PC12 cells (Hinsby et al. 2004). Neurite extension resulted from either NCAM-mediated or fibroblast growth factor 2 (FGF2)-mediated FGF receptor-1 (FGFR-1) activation, FRS2 phosphorylation and scaffolding to Grb2 (Hinsby et al. 2004). Saffell and colleagues (1997) demonstrated that the overexpression of a dominant negative (dn) FGFR-1 totally ablated NCAM-mediated neurite extension, which suggests the involvement of FGFR-1 in NCAMmediated synaptic plasticity via the FRS2-Grb-RasRaf-ERK1/2 pathway in PC12 cells (Saffell et al. 1997). An alternative pathway to actin cytoskeleton assembly/disassembly and neurite outgrowth mediated by FRS2 might occur via the SRC non-receptor tyrosine kinase (Li et al. 2004). Riva and colleagues (1998) reported an increased synthesis of FGF2 protein, but a down-regulation of FGFR-1 mRNA subsequent to 24 h dexamethasone treatment of rodent cortical astrocytes, which would not be expected if the FGFR-1 and FRS2 were both on the same transcriptome regulated by glucocorticoids. Sandi and Loscertales (1999) demonstrated that a single corticosterone injection to rats could augment NCAM levels and signaling in the frontal cortex. Glucocorticoid induction of NCAM and FRS2 coordinately would be expected if both genes were on same transcriptome. b1-Catenin expression was increased in the nucleus accumbens subsequent to chronic (1 year) cocaine treatment in non-human primates, implicating this protein in the process of neuroadaptation to reinforcing drugs (Freeman et al. 2001). The b1catenin complex with N-cadherin is extremely vital to the process of early neuronal development, involving a sequence of events including neurite extension, axonal differentiation, dendritic arborization and synapse formation (Yap et al. 1997; Benson and Tanaka 1998; Tepass et al. 2000; Takai et al. 2003; Murase et al. 2002; Yu and Malenka 2004). b1Catenin complexes with the intracellular domain of N-cadherin cellular adhesion receptor proteins at the plasma membrane through tyrosine phosphorylation/ dephosphorylation reactions, and associates with the actin cytoskeleton through a-catenin (Yap et al. 1997; Lilien and Balsamo 2005). N-cadherin/b1-catenin complexes are found at axo-dendritic contact sites in the region of transmitter release, directly bordering postsynaptic densities in mature neurons (Uchida et al. 1996; Togashi et al. 2002; Abe et al. 2004).

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b1-catenin migrates from dendritic shafts into dendritic spines in rodent hippocampal slices in response to depolarization, thereby increasing the size and strength of synaptic contacts (Murase et al. 2002). The intracellular b1-catenin levels are under the control of phosphorylation by glycogen synthase kinase (GSK3) and casein kinase 1, leading to ubiquitination and degradation by the proteasome (Castelo-Branco et al. 2004; Liu et al. 2002). Wnt signalling can protect b1-catenin from this progression to degradation, thereby allowing b1-catenin to translocate to the nucleus and interact with transcription factors for Wnt target genes (Castelo-Branco et al. 2004). Thus, cellular protein levels are acutely regulated by the degradation pathway, explaining why we saw very little change in b1-catenin protein in the cellular lysates except at the 36 h time point. Prolyl-carboxypeptidase has been found in cell matrix, and functions as the prekallikrein activator (Moreira et al. 2002). This important peptidase can cleave precursors to produce the important neuropeptides angiotensin II and bradykinin (Moreira et al. 2002). Prolyl-carboxypeptidase serves a differentiated function of providing the mechanism for neuropeptide transmitter synthesis. Glucocorticoids and opioid peptides in the regulation of cell proliferation and survival The ability of the steroid and opioid treatments to alter cell cycle and cell survival was of interest because studies of glucocorticoid administration to rodents suggest that dosage and duration may determine whether neuronal survival may be compromised. In a study by Haynes and colleagues (2001), acute administration of 0.7–20 mg/kg (ip) dexamethasone to rats led to loss of neuronal microtubule integrity and apoptosis within certain brain regions of high sensitivity such as hippocampus and striatum. Repeated daily injections of 0.1 mg/kg (sc) dexamethasone to newborn rats produced dendritic vacuolation, but no changes in apoptosis markers (Tan et al. 2002). Treatment of proliferating SH-SY5Y human neuroblastoma cells with dexamethasone (100 nM to 10 mM for 48 h) resulted in a dosedependent increase in the production of reactive oxygen species and the mitochondrial membrane potential (Oshima et al. 2004). Neither proliferating nor differentiated SH-SY5Y cells underwent

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apoptosis at any time point (including 1 week exposure to high dose dexamethasone) (Oshima et al. 2004). Thus, sustained increased glucocorticoid levels may alter synaptic function, but does not commit neurons to apoptotic death, a point confirmed in our studies. The endogenous opioid peptide [Met5]-enkephalin inhibits cell cycle progression in a number of proliferating cells and cell lines by a mechanism that utilizes a nuclear opioid growth factor receptor, quite unrelated to the G-protein coupled l-opioid receptor (Zagon et al. 2002). Furthermore, concentrations of DAMGO 10-fold to 1000-fold greater than utilized in the present studies failed to compete for [Met5]enkephalin binding to the nuclear opioid growth factor receptor in tumor cell homogenates or purified fusion protein receptors, respectively (Zagon et al. 2002). Since the present studies found no significant change in the growth rate and total protein levels in the SH-SY5Y cell line upon exposure to DAMGO, the nuclear opioid growth factor receptor should not be considered as playing a role in the gene expression changes reported here. Synergism of dexamethasone plus DAMGO on gene expression In the present study, the observed synergy between l-opioid and GR-II stimulation could have resulted from the l-opioid receptor stimulation of the Fos-Jun (activator protein AP-1) promoter enhancing the dexamethasone-mediated activation of GR-II. Morphine increased the transcription of c-Fos in rat caudate-putamen, and Fos nucleoprotein levels remained for at least 3 h (Chang et al. 1988). Morphine also induced c-Jun protein in rodent caudate-putamen and deep neocortex (Garcia et al. 1995). Treatment of SH-SY5Y cells with acute (5–10 min) morphine or DAMGO resulted in ERK1/2 phosphorylation (Bilecki et al. 2005), which can lead to transcription factor regulation. ERK1/2 is also linked to stress-induced synaptic plasticity (Yang et al. 2004). The GR-II antagonist, RU-38486, attenuated long-term depression and reduced the hyperphosphorylation of ERK1/2, MEK1/2 and Raf in the hippocampi of rats that were exposed to short-term (*2 h) stress (tail shock and physical restraint) (Yang et al. 2004). In addition, the MEK1/2 inhibitor, U0126, attenuated long-term

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depression in the hippocampi of stressed rats (Yang et al. 2004). Using murine F9 testicular teratoma cells transfected with a DNA construct consisting of adjacent glucocorticoid response element (GRE) and AP-1 binding sites, Pearce and colleagues (1998) demonstrated a possible synergism between dexamethasone and AP-1 (either the c-Jun/c-Jun homodimer or the c-Jun/c-Fos heterodimer). Other studies have provided evidence for synergism between GR-II and AP-1 (c-Jun/c-Jun homodimer) activation (Diamond et al. 1990; Mittal et al. 1994). Clinical implications for chronic mild stress and opioid peptide activation The present investigation examined the interface between glucocorticoids and a sustained stimulation of l-opioid receptors in a model neuronal system in which drugs could be applied that might mimic the glucocorticoid components of a stress response simultaneously with opioid peptide activity. Chronic stress is known to modulate the rewarding effects of drugs of abuse as a result of neuroadaptation processes (Akiskal and McKinney 1973; Anisman and Zacharko 1982). Chronic stress that is both multifarious and capricious may prevent the onset of compensatory synaptic plasticity (Rodriguez Echandia et al. 1988). Chronic mild stress in a rat model increased the sensitivity of CNS reward mechanisms to amphetamine (Lin et al. 2002). Corticosterone mediated the experimental stress of intermittent footshock leading to reinstatement of cocaine seeking (Deroche et al. 1997; Mantsch and Goeders 1999). Therefore, knowledge of gene candidates influenced by the combined effects of stress and drugs of abuse is vital to establishing possible therapeutic interventions that may abate neuroadaptation changes associated with the disease of substance abuse. Further studies of the influence of chronic mild stress on reinforcement behaviors and concomitant gene expression changes in pertinent brain regions of behaving animals are warranted. Acknowledgments The authors thank Jenelle Jones, M.S. for assisting in the culturing of the SH-SY5Y human neuroblastoma cells and Western immunoblotting analysis. This work was supported by National Institute on Drug Abuse grants U24-DA12385 and R01-DA13770 (K.E.V.), K05DA00182 (A.C.H.) and the National Center on Minority Health and Health Disparities P21-MD00175.


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