Genome-Wide Expression Analysis Reveals Diverse Effects of Acute Nicotine Exposure on Neuronal Function-Related Genes and Pathways

Previous human and animal studies demonstrate that acute nicotine exposure has complicated influences on the function of the nervous system, which may lead to long-lasting effects on the behavior and physiology of the subject. To determine the genes and pathways that might account for long-term changes after acute nicotine exposure, a pathway-focused oligoarray specifically designed for drug addiction research was used to assess acute nicotine effect on gene expression in the neuron-like SH-SY5Y cells. Our results showed that 295 genes involved in various biological functions were differentially regulated by 1 h of nicotine treatment. Among these genes, the expression changes of 221 were blocked by mecamylamine, indicating that the majority of nicotine-modulated genes were altered through the nicotinic acetylcholine receptors (nAChRs)-mediated signaling process. We further identified 14 biochemical pathways enriched among the nicotine-modulated genes, among which were those involved in neural development/synaptic plasticity, neuronal survival/death, immune response, or cellular metabolism. In the genes significantly regulated by nicotine but blocked by mecamylamine, 13 enriched pathways were detected. Nine of these pathways were shared with those enriched in the genes regulated by nicotine, including neuronal function-related pathways such as glucocorticoid receptor signaling, p38 MAPK signaling, PI3K/AKT signaling, and PTEN signaling, implying that nAChRs play important roles in the regulation of these biological processes. Together, our results not only provide insights into the mechanism underlying the acute response of neuronal cells to nicotine but also provide clues to how acute nicotine exposure exerts long-term effects on the nervous system.

Although nicotine dependence is attributed mainly to repeated and chronic nicotine exposure, acute administration also can evoke noticeable physiological effects. For example, in humans, acute nicotine exposure, while significantly increasing the plasma nicotine concentration (Argacha et al., 2008), modulates a series of physiological processes such as aortic wave reflection, arterial stiffness, and plasma asymmetrical dimethylarginine (ADMA) concentration (Adamopoulos et al., 2009). Experimentally, acute nicotine administration can significantly increase activation in multiple regions of mouse brain (Suarez et al., 2009) and induce spinal motor circuit output in embryonic zebrafish (Thomas et al., 2008). Previous studies have demonstrated the beneficial effect of acute nicotine exposure on cognitive performance (Foulds et al., 1996;Phillips and Fox, 1998;Ernst et al., 2001;Kumari et al., 2003), as well as its antidepressant effect (Tizabi et al., 2009).
Various genes and biochemical processes are involved in the biological response to acute nicotine exposure. These include immediate-early genes such as c-FOS, c-JUN, NURR77, and EGR1 (Ichino et al., 1999(Ichino et al., , 2002Salminen et al., 1999) and the dendritically targeted early response genes activity-regulated

IntroductIon
Nicotine is the dependence-inducing constituent underlying tobacco smoking. Through interactions with nicotinic acetylcholine receptors (nAChRs) in the central nervous system (CNS), nicotine exposure not only stimulates the mesocorticolimbic dopamine system in the outer shell of the nucleus accumbens (NAc) and other brain regions Balfour, 1992, 1997;Gaddnas et al., 2001;Tammimaki et al., 2006) but also modulates the release of other neurotransmitters such as norepinephrine, serotonin, and GABA (Kenny et al., 2001;Wonnacott, 2006, 2009). Prolonged nicotine treatment can regulate the expression of genes/ proteins involved in various functions such as ERK1/2 and CREB (Brunzell et al., 2003), as well as their downstream targets such as c-FOS and FOSB (Pagliusi et al., 1996;Nisell et al., 1997;Soderstrom et al., 2007). Further, biochemical pathways underlying various physiological processes; e.g., MAPK signaling, phosphatidylinositol phosphatase signaling, growth factor signaling, and ubiquitinproteasome pathways, are modulated by nicotine (Tang et al., 1998;Konu et al., 2001;Li et al., 2004). Through its direct or indirect interaction with these genes and biological pathways, nicotine is involved in the regulation of various physiological processes, such as learning and memory, angiogenesis, energy metabolism, synaptic function, response to oxidative stress, and addiction (Harkness and rnA IsolAtIon Total RNA was isolated separately from each of the drug-treated and control cultures using Trizol (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. To eliminate potential residual DNA contamination, the RNA samples were treated with RNase-free DNase I at 37°C for 30 min followed by inactivation at 65°C for 10 min. The RNA concentration of each sample was measured at an optical density of 260 nm, and the integrity of the RNA was checked by examination of 28S and 18S ribosomal bands using agarose-formaldehyde gel electrophoresis.

MIcroArrAy productIon
A pathway-focused oligoarray designed specifically for the study of drug addiction and related disorders was used. Briefly, 3,565 genes essential to CNS activities for the maintenance of neuronal homeostasis, as well as those associated with the neuron response to addictive substances such as nicotine, alcohol, and cocaine, were selected on the basis of an earlier version of a pathway-focused cDNA microarray (Konu et al., 2004a) and an extensive literature survey. These genes covered most of the major pathways related to cell metabolism, genetic information processing, cellular signaling transduction, neuron-related disease, and cell communication. OligoWiz 1 was used to design the oligonucleotide for each gene. The average length of these oligonucleotides was 59.2 ± 3.8 bases (mean ± SD), with a GC content of 0.53 ± 0.05 and a T m of 76.4 ± 1.7°C. The oligonucleotides and 10 control clones were synthesized by SIGMA Genosys (Sigma-Aldrich, St. Louis, MO, USA) and spotted at a concentration of 40 μM in 3× SSC and 1.5 M betaine buffer onto CMT-GAPS II slides (Corning Life Sciences, Lowell, MA, USA) using an OmniGrid MicroArrayer OGR-03 (GeneMachines, San Carlos, CA, USA).

cdnA probe lAbelIng, hybrIdIzAtIon, And scAnnIng
To reduce the potential influence of any gene-specific dye effect, a universal reference design was used in the microarray analysis. For each experimental group, six independent cultures were prepared. Another 12 independent control cultures were used as "universal controls." For each sample, 10 μg of total RNA was extracted for cDNA synthesis. Following purification with phenol:chloroform extraction and isopropanol precipitation, total RNA was dissolved in 28 μl of water, mixed with 4 μl of 10× buffer, 4 μl of 10 mM dTTPfree dNTP, 1 μl of 10 mM dTTP, 2 μl of 1 mM cyanine 3-dUTP (for either nicotine-treated or control sample) or cyanine 5-dUTP (for universal control sample; Enzo, Farmingdale, NY, USA), and 1 μl of Klenow fragment (50 units/μl), then incubated at 37°C for 3 h prior to purification with a QIAquick PCR kit (Qiagen, Valencia, CA, USA). The cyanine 3-labeled sample (either nicotine-treated or control) cDNA probes were mixed with the cyanine 5-labeled universal control cDNA probe and added to 7.5 μl of 20× SSC, 3 μg of CotI DNA, 3 μg of polyA, and 0.5 μl of 10% SDS adjusted to a final volume of 50 μl. The mixture was applied to the pathwayfocused oligonucleotide microarray and hybridized overnight at 60°C. Slides were washed in 1× SSC and 0.2% SDS at 60°C for 5 min followed by washing in 0.1× SSC and 0.2% SDS and in 0.1× SSC at room temperature for 10 min each. Hybridized slides were scanned cytoskeleton-associated protein (ARC) and dendrin in specific brain regions of rats (Schochet et al., 2005(Schochet et al., , 2008Schmitt et al., 2008). Acute nicotine exposure not only increases the activity of dopaminergic neurons and promotes dopamine release (Levin and Rose, 1995), but also enhances dopamine turnover and metabolism (Grenhoff and Svensson, 1988). The administration of a single dose of nicotine enhances the synthesis and release of striatal dynorphin, a component of the circuit promoting negative motivational and affective states, possibly under the regulation of dopamine and glutamate (Isola et al., 2009). Acute nicotine exposure may impair nNOS-dependent dilation of cerebral arterioles via a mechanism related to the increase in oxidative stress (Arrick and Mayhan, 2007). Even a few minutes of nicotine exposure can significantly suppress arachidonic acid signaling in various brain regions in rats (Chang et al., 2009). In mice, acute administration of a low dose of nicotine increases phosphor-ERK1/2 immunoreactivity in the NAc, prefrontal cortex, and some other regions innervated by the mesolimbic dopamine pathway (Barik and Wonnacott, 2009).
Clearly, acute nicotine exposure can evoke multiple effects in the neuronal system, but its action mechanism is not completely understood. The neuron-like human neuroblastoma cell line SH-SY5Y endogenously expresses various nAChR subunits (e.g., α3, α5, α7, β2, and β4; Gould et al., 1992;Peng et al., 1994), which are assembled to form different types of nAChRs, such as α3 * -nAChR or homomeric α7-nAChR receptors. The cells therefore provide a suitable in vitro model to investigate alterations in nAChRs in the presence of cholinergic ligands and have been widely used in nicotine-related study. In an earlier study, 17 genes with diverse biological functions were found to be significantly regulated in SH-SY5Y cells after exposed to 1 mM nicotine for 1 h (Dunckley and Lukas, 2003). The authors further showed that most of these genes were regulated via nAChRs-mediated mechanisms. However, because of the limitation of array technologies and bioinformatics tools at that time, a detailed analysis, especially at the biological pathway level, was impossible. With the rapid progress in the fields of genomics and bioinformatics, a detailed and comprehensive profiling of the genes and pathways responsive to acute nicotine treatment becomes feasible and necessary. In this study, by using an established and well-tested in vitro model in the field, we investigated the relation between acute nicotine exposure and neuronal function by examining the genes and biochemical pathways differentially regulated by nicotine in SH-SY5Y cells using a customized pathway-focused microarray Wei et al., 2011).

MAterIAls And Methods cell culture And drug treAtMent
The human neuroblastoma SH-SY5Y cells were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA) and cultured in a 1:1 mixture of ATCC-formulated Eagle's Minimal Essential Medium and F12 medium supplemented with 10% fetal bovine serum (GIBCO Invitrogen, Grand Island, NY, USA) at 37°C in a humidified atmosphere of 5% CO 2 . At about 80% confluence, cells were treated with 1 mM nicotine (calculated as free base), 3 μM mecamylamine (nicotinic receptor antagonist), 1 mM nicotine in combination with 3 μM mecamylamine, or no drug (controls) for 1 h. For each experimental group, six independent cultures were prepared (N = 6). was done as described previously (Gutala et al., 2004;Konu et al., 2004a). Briefly, PCR was carried out in a volume of 25 μl using the TaqMan assay on the ABI 7000 Sequence Detection System. The 18S ribosomal RNA was used as an internal control to normalize the expression patterns of target genes. Data analysis was performed using a comparative C t method (Winer et al., 1999). Five independent cell cultures were prepared under the same conditions used for the microarray experiments for both the nicotine treatment and control groups (N = 5).

Western blotting analysis
The protein concentration of MAPK14 (p38) was measured in both nicotine-treated (1 mM for 1 h) and control samples. After appropriate treatment, cells were harvested using RIPA buffer followed by incubation on ice for 30 min. The cells were centrifuged at 12,000×g for 15 min to remove debris, and the total protein content of the lysates was determined using the Bio-Rad protein assay (Bio-Rad, Chicago, IL, USA). The samples were boiled at 90°C for 5 min and then cooled on ice. Twenty micrograms of protein was fractionated on 10% SDS-polyacrylamide gel (containing 30% acrylamide and 0.8% bisacrylamide) in Tris-glycine buffer containing SDS at 80 V for approximately 1.5 h. The separated proteins were then transferred to a PVDF membrane (Millipore, Bedford, MA, USA) overnight at 30 V at 4°C. The membrane was blocked for 1 h at room temperature with 5% non-fat dry milk diluted in TBST buffer and incubated overnight with primary antibody at 4°C. After being washed three times in TBST, the membranes were subjected to secondary antibody conjugated to horseradish peroxidase for 1 h at room temperature. Immunoreactivity was detected using an enhanced chemiluminescence kit (Bio-Rad), and the preparations were exposed to X-ray film. The MAPK14 antibody was from Calbiochem (San Diego, CA, USA), and antiα-tubulin monoclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) was used to detect the housekeeping protein α-tubulin. The MAPK14 immunoreactivity bands were quantified using densitometry. After the films were developed, they were scanned on a Microtek ScanMaker i800 (Microtek Lab Inc., Cerritos, CA, USA) with ScanWizard 5.5 at a resolution of 600 dpi for quantitative analysis with ImageQuant 5.1 (Molecular Dynamics, Sunnyvale, CA, USA). The relative MAPK14 values were normalized to α-tubulin, and the significance of the difference between the nicotine-treated and control samples was determined by t-test via MATLAB (The Mathworks, Natick, MA, USA). Four independent cell cultures were prepared under the same conditions used for the microarray experiments for both nicotine treatment and control groups (N = 4).

results gene expressIon chAnges In response to Acute nIcotIne exposure
In the nicotine-treated group, 295 genes were significantly modulated (p < 0.05; Table A1 in Appendix), with FDR values ≤0.258. Among these genes, 98 had p-values <0.01, which corresponded to an FDR value of 0.155. Of the 295 differentially expressed genes, 221 showed no significant changes (p ≥ 0.05) in the cells treated with nicotine and mecamylamine, suggesting these genes were regulated through the mecamylamine-modulated nicotinic receptors.
using the ScanArray Gx microarray scanner, and the intensities of each probe were quantified with the ScanArray Express microarray analysis system (PerkinElmer, Waltham, CA, USA).

dAtA norMAlIzAtIon And stAtIstIcAl AnAlysIs
After scanning the arrays, we obtained the raw hybridization intensity for each element printed on the array and then used the background-subtracted median intensity of each spot for further statistical analysis. The two replicates on each chip were initially processed separately. The weakest 5% of spots and the saturated spots in each replicate were discarded. An intensity-dependent method, locally weighed linear regress (Lowess), was used to normalize the data of each replicate (Yang et al., 2002). To minimize experimental error, genes with six or fewer valid measurements were removed from further analysis, and the two technical replicates per chip were averaged to obtain the measurement of each gene for a given sample. The normalized data from each drug treatment group were compared with the normalized data of controls, and significantly regulated genes were identified by unpaired twotailed Student's t-test. To control the multiple comparison error, the false discovery rate (FDR) was calculated by the method of Benjamini and Hochberg (1995) via MATLAB (The Mathworks Inc., Natick, MA, USA). The genes significantly regulated by each drug treatment were analyzed by ingenuity pathway analysis (IPA) 2 with the goal of revealing the enriched biochemical pathways. The core of IPA is the ingenuity pathways knowledge base (IPKB), which contains the biological function, interaction, and other related information of a curated gene set and more than 330 biochemical pathways. This pathway-based software is designed to identify global canonical pathways, dynamically generated biological networks, and global functions from a given list of genes. Basically, the genes with their symbols, corresponding GenBank Accession Numbers, or both were uploaded into the IPA and compared with the genes included in each canonical pathway using the whole gene set of IPKB as the background. All the pathways with one or more genes overlapping the candidate genes were extracted. In IPA, each of these pathways was assigned a p-value, which denoted the probability of overlap between the pathway and input genes, via Fisher's exact test. Because a relatively large number of pathways were examined, multiple comparison correction for the individually calculated p-values was necessary to make reliable statistical inferences. The FDR was again calculated with the method of Benjamini and Hochberg (1995).

Quantitative real-time RT-PCR
The quantitative real-time RT-PCR (qRT-PCR) was carried out for eight representative genes selected from the aforementioned differentially expressed genes: cyclin d3 (CCND3), cell division cycle 42 (CDC42), death domain-associated protein 6 (DAXX), mitogen-activated protein kinase 14 (MAPK14), mads box transcription enhancer factor 2, polypeptide D (MEF2D), nerve growth factor receptor (NGFR), phospholipase A2, group IVA (PLA2G4A), and transformation-related protein 53 (TRP53). All primers and TaqMan probes for the eight genes were purchased from Applied Biosystems (ABI, Foster City, CA, USA), and qRT-PCR analysis treatment group, suggesting that mecamylamine may have either no significant effect or a synergistic effect with nicotine on the expression changes in these genes.

pAthwAys enrIched In the genes regulAted by nIcotIne
As shown above, genes and biological processes involved in different functions were regulated by nicotine exposure. To further identify the biological themes underlying these genes, the biochemical pathways enriched in the nicotine-regulated genes were investigated. Two sets of genes were analyzed by IPA; i.e., those regulated significantly by 1 h of nicotine exposure (N = 295), and those with expression significantly changed by 1 h of nicotine with the effect being blocked by mecamylamine (N = 221). The second gene set included only those with p-values ≥0.05 during co-treatment with nicotine and mecamylamine. Although in this set, some genes may have considerable fold changes, the selection was based on p-value only.
Results from IPA analysis showed that 14 pathways were significantly enriched among the genes regulated by nicotine (p < 0.05; FDR < 0.05; Figure 1; Table 1), and 13 pathways were enriched in the second gene set. Among these pathways, nine were identified for both gene sets; i.e., glucocorticoid receptor signaling, p38 MAPK signaling, PI3K/AKT signaling, PTEN signaling, acute-phase response signaling, ERK/MAPK signaling, Toll-like receptor signaling, VEGF signaling, and T-cell receptor signaling. For the gene set regulated by nicotine treatment, five pathways were uniquely identified; i.e., mitochondrial dysfunction, PPAR signaling, IL-6 signaling, hepatic fibrosis/hepatic stellate cell activation, and death receptor signaling. The pathways uniquely identified in the second gene set were CD28 signaling in T-helper cells, CD40 signaling, role of NFAT in regulation of the immune response, and IL-8 signaling.
Of the 295 genes significantly regulated by nicotine treatment, 74 were differentially expressed (p < 0.05) in the cells treated with nicotine + mecamylamine ( Table A1 in Appendix). Most of these genes showed the same trend of regulation as those in the nicotine pathways were assigned p-values <0.05, they are not reported here because of the relatively high FDR value after multiple comparison correction (with the minimum FDR being 0.262).
As shown in Table 1, multiple genes in each pathway were significantly regulated by nicotine exposure. Although the expression changes of some genes within each pathway were not statistically significant, they showed consistent patterns with those significantly modulated. One of the pathways, p38 MAPK signaling, is shown in Figure 2. Genes involved in this pathway were extensively regulated in response to nicotine exposure. The expression In the pathways identified for the two gene sets, the genes affected were not identical ( Table 1). For the nicotine + mecamylaminetreated samples, the pathways included only genes whose expression was modulated by nicotine exposure but blocked by simultaneous treatment with the two chemicals. For the nicotine-treated samples, the pathways also included genes whose expression was modulated in both nicotine-and nicotine + mecamylamine-treated samples.
We also performed pathway analysis on the 74 genes significantly regulated in both nicotine-treated and the nicotine + mecamylamine-treated samples via IPA. Although a few  Figure 3 shows the fold change in activity along with the standard deviation of the eight genes in response to nicotine treatment relative to the controls, as detected by microarray and quantitative RT-PCR. These analyses revealed significant up-regulation of seven genes and down-regulation of CDC42, which is consistent with the result from the microarray analysis. Furthermore, a comparison of the fold changes of each gene detected by the two molecular techniques revealed a correlation coefficient of 0.87 (p = 0.0068), indicating overall measurement of gene expression from the two approaches was consistent. Although only eight representative genes were selected for verification by qRT-PCR, a comparison of the two techniques demonstrates that the results from our microarray analyses were highly reproducible and reliable.
Furthermore, we used Western blotting to determine the total amount of MAPK14 (p38 MAPK) protein in lysates of cells treated with 1 mM nicotine and control samples. The results revealed significant induction (35.5%; p = 0.03) of MAPK14 expression in nicotine-treated samples (Figure 4).

dIscussIon
In this study, we profiled gene expression in the SH-SY5Y cell line in response to 1 h of 1 mM nicotine exposure using a pathwayfocused oligonucleotide microarray. Treatment with 1 mM nicotine maximally induces the up-regulation of numbers of nAChR radioligand-binding sites in SH-SY5Y cells, and this up-regulation of a few genes, such as DAXX, MKP1/5, MAPK14, and MEF2, was significantly modulated by acute nicotine exposure (p < 0.05). Although the expression of other genes; e.g., ASK1, MKK4, and HPK1, was not significantly regulated under the experimental conditions, they showed patterns consistent with those of significantly regulated genes.

Expression verification of representative genes
In an independent experiment involving the same treatments as in the microarray analysis, qPCR was performed on eight representative genes; i.e., CCND3, CDC42, DAXX, MAPK14, MEF2D, NGFR, PLA2G4A, and TRP53. All these genes were significantly regulated by nicotine exposure ( Table A1 in Appendix), and each gene was part of one or more enriched pathways associated with nicotine treatment ( Table 1). For example, CCND3 is part of the IL-8 signaling pathway; CDC42 of CD28 signaling and PTEN signaling pathways; DAXX of the p38 MAPK signaling pathway; MAPK14 of multiple pathways such as glucocorticoid receptor signaling, p38 MAPK signaling, acute-phase response signaling, and Toll-like receptor signaling; MEF2D of p38 MAPK signaling that plays a role of NFAT in regulation of the immune response; NGFR of PTEN signaling, acute-phase response signaling, PPAR signaling, and IL-6 signaling; PLA2G4A of p38 MAPK and ERK/MAPK signaling; and TRP53 of the PI3K/AKT signaling pathway. Genes shown in green are down-regulated, whereas those shown in red are induced by acute nicotine treatment. The genes significantly regulated by nicotine are those with closed blue edges. To demonstrate the expression pattern of the pathway, all the genes with expression ratios >1.1 or <0.90 relative to the controls are also shown. The genes in white with light gray edges are either not regulated by nicotine (expression ratios between 0.9 and 1.1) or were not measured in our analysis. The pathway was drawn by the IPA program. has been implicated in the long-lasting effects of nicotine exposure, such as dependence and tolerance (Ke et al., 1998;Lukas, 2003, 2006). Given that the purpose of this study was to determine the pharmacologic effects of nicotine using an in vitro system, we adopted a 1-mM concentration for nicotine, as used in a previous study (Dunckley and Lukas, 2003). However, the current study is neither a simple modeling of the physiological conditions of smokers, nor repeat earlier studies in the same model. Instead, it focuses on exploring the molecular response at the pathway level to acute nicotine exposure that may maximally activate the function of molecules such as nAChRs and their downstream genes in SH-SY5Y cells.
Nicotine evokes its physiological effects mainly by binding with nAChRs. As a broad-spectrum antagonist of nAChRs, mecamylamine at a concentration of 3 μM can block most of the responses of α3*-nAChR to nicotine, as well as a fraction of the α7-nAChR responses (Chavez-Noriega et al., 1997;Papke et al., 2001;Lukas, 2003, 2006). Of the 295 genes significantly regulated by nicotine, 221 were not differentially expressed during co-treatment of nicotine + mecamylamine, providing evidence of direct involvement of nAChR in the process of modulation of SH-SY5Y cells by nicotine. Thus, our results indicate that the changes in 75% (221/295) of the differentially expressed genes in response to nicotine are attributable to direct interaction with nAChRs, especially α3*-nAChR receptors, which is consistent with the conclusions drawn from previous studies Lukas, 2003, 2006). However, it is difficult to confirm a clear relation between nicotinic receptor subtype and gene expression from our findings because of the existence of different subtypes of nAChRs on the membrane of SH-SY5Y cells. These receptors have different properties, such as sensitivity to nicotine, permeability to calcium, and propensity to desensitize. It is possible that the regulation of some genes is related to multiple subtypes of nAChRs, including those α3or α7-containing receptors. Partially blocking the function of some types of receptors (e.g., α3*and α7-containing) may reduce the overall activation of nAChRs, which may lead to more subtle regulation of some genes compared with the response when mecamylamine is not applied. On the other hand, our results do not imply that nAChRs play little or no role in the regulation of genes significantly regulated in both nicotine and nicotine + mecamylamine treatment. The expression changes of these genes may have multiple explanations. A small fraction of α3*-nAChR receptors remain functionally active under the influence of mecamylamine, which may be sufficient to regulate the expression of some genes. At the same time, α7 and other types of nAChR receptors may be responsible for the expression change of some other genes as well. It is also possible that the expression changes of some genes in response to nicotine are caused by mechanisms not directly involving nAChRs, as nicotine can readily cross the plasma membrane and affect intracellular processes through an nAChR-independent mechanism.
Our analysis showed that acute nicotine exposure has effects on the expression of a large number of genes with diverse functions ( Table A1 in Appendix). Among them are genes involved in the ubiquitin-proteasome pathway, such as proteasome subunit beta 4 (PSMB4), proteasome 26S subunit non-ATPase 2 (PSMD2), ubiquitin-activating enzyme E1 Chr X (UBE1X), ubiquitin-conjugating enzyme E2A  The SH-SY5Y cells were treated for 1 h with or without (Controls) 1 mM nicotine. After treatment, protein was purified from the lysates and analyzed by Western blotting. In this figure, the MAPK14 concentration was determined using a specific monoclonal antibody. The histogram shows the relative expression of MAPK14 normalized to expression of α-tubulin, which demonstrated that the protein concentrations in nicotine-treated cells are significant higher than those in controls (p < 0.03). Data are presented as mean ± SEM (N = 4).
(NRXN1) was down-regulated in both treatment groups. This and another member of the same family, NRXN3, were associated with smoking behaviors in several recent genetic studies (Bierut et al., 2007;Nussbaum et al., 2008;Novak et al., 2009). Although our data provide no further information on the mechanisms by which these genes are regulated, it is clear that acute nicotine exposure is responsible for their expression changes.
Consistently, the pathways enriched in nicotine-regulated genes were involved in many biological processes, such as neural development/synaptic plasticity, cell survival/death, immune response, and cellular metabolism. Of the identified pathways, nine were enriched in genes significantly regulated by nicotine, as well as those regulated by nicotine but blocked by mecamylamine, indicating these pathways were regulated mainly through nAChR-dependent mechanisms. Among these pathways, acute-phase response signaling, Toll-like receptor signaling, and T-cell receptor signaling play important roles in the immune response. Although the other pathways; i.e., glucocorticoid receptor signaling, p38 MAPK signaling, PI3K/AKT signaling, PTEN signaling, ERK/MAPK, and VEGF signaling, are involved in a large range of physiological processes, all of them represent key pathways for maintaining proper neuronal functions, such as cellular signaling and proliferation and differentiation of neurons. The modulation of the neuronal functionrelated pathways may have relatively long-lasting consequences. One example is that adolescent rats display different long-term neuroadaptive responses to acute nicotine than do adult animals, which is suggested to be related to the immature or still-developing plasticity mechanisms in the prefrontal cortex of young animals (Schochet et al., 2004). Among these pathways, particularly interesting are the glucocorticoid receptor and p38 MAPK signaling.
For the glucocorticoid receptor signaling pathway, 21 genes were significantly regulated by acute nicotine treatment ( Table 1). The glucocorticoid receptor, GCCRα (also known as nuclear receptor subfamily 3, group c, member 1; NR3C1) was slightly suppressed by nicotine (fold change 0.83; p-value = 0.09). The regulation of these genes clearly indicates that the glucocorticoid receptor signaling pathway is modulated by acute nicotine exposure. Glucocorticoids, cortisol in humans, and corticosterone in the rodent, are secreted by the adrenal cortex and belong to the family of steroid hormones. Glucocorticoids and their receptors are essential for the development and survival of vertebrates (Cole et al., 1995). All the cellular responses to glucocorticoids are attributed to their binding to the intracellular glucocorticoid receptor (GCCR) and the translocation of the complexes to the nucleus, which then modulates gene expression through diverse mechanisms (Dittmar et al., 1997(Dittmar et al., , 1998Robinson-Rechavi et al., 2001). Although the activity of the GCCR is directly related to gene transactivation, considerable cross-talk occurs between the GCCR and a cohort of molecules to mediate their functions as transcriptional regulators. Studies on animals and humans have outlined the importance of glucocorticoids in mediating drug-seeking behavior and the rewarding properties of psychostimulant drugs (Piazza and Le Moal, 1997;Marinelli and Piazza, 2002). For example, in mice, activation of glucocorticoid receptors increases the propensity to self-administer cocaine by acting on the postsynaptic dopaminoceptive neurons of the dopaminergic system (Ambroggi et al., 2009). It was also found that learning and memory deficits in cocaine-dependent humans are associated with higher cortisol concentrations (Fox et al., 2009). The (UBE2A), ubiquilin 1 (UBQLN1), and ubiquitin-specific protease 28 (USP28). Earlier studies showed that genes involved in the ubiquitin-proteasome pathway were regulated in SH-SY5Y cells by 1 mM nicotine after both 1-and 24-h treatment Lukas, 2003, 2006). In the current study, more genes related to this pathway were found to be modulated by nicotine exposure. The ubiquitinproteasome pathway is regulated by nicotine in cell lines (Konu et al., 2004b;Ficklin et al., 2005;Wang et al., 2007), animal models Li et al., 2004;Rezvani et al., 2007;Vadasz et al., 2007;Wang et al., 2009), and human smokers (Mexal et al., 2005). Thus, the changes in ubiquitin-proteasome-related genes observed in this study may not be specific for the relatively high nicotine concentration; instead, they may be caused by mechanisms similar to those discovered in previous studies.
Multiple genes involved in mitochondrial functions also were found to be regulated by nicotine; these include acyl-coenzyme A thioesterase 3 (ACATE3), ATP synthase subunit g (ATP5L), mitochondrial ribosomal protein S7 (MRPS7), NADH dehydrogenase (ubiquinone) 1 alpha subcomplex 4 (NDUFA4), and RISP. Previous studies have demonstrated that nicotine exposure can modulate the morphology (Onal et al., 2004) and function of mitochondria, such as protein turnover (Katyare and Shallom, 1988), enzyme activity (Xie et al., 2005), and generation of reactive oxygen species (Cormier et al., 2001;Soto-Otero et al., 2002). Recently, we showed that nicotine exposure regulates the electron transport system in multiple brain regions of rats (Wang et al., 2009). The results from the current study indicate that acute nicotine exposure also can regulate the expression of mitochondrial genes, potentially leading to the modulation of mitochondrial functions. In addition, several genes involved in synaptic transmission were identified, such as neuron-specific gene family member 1 (NSG1), synuclein alpha (SNCA), synapsin 2 (SYN2), synaptotagmin 2 (SYT2), synaptotagmin 7 (SYT7), and tachykinin 2 (TAC2). The expression of NSG1 in mouse cortical neurons is regulated by treatment with nicotine, alcohol, or both . SNCA is suggested as a candidate associated with alcohol, cocaine, and methamphetamine dependence (Kobayashi et al., 2004;Bonsch et al., 2005;Mash et al., 2008;Spence et al., 2009). The phosphorylation of synapsin is regulated by cocaine in a region-specific manner in rat brain (Edwards et al., 2007). The regulation of these synaptic transmission-related genes indicates that even a short nicotine exposure may have important effects on neuronal functions.
Among the 74 genes significantly regulated by both nicotine and nicotine + mecamylamine treatment, some have already been reported to be involved in the cellular response to nicotine exposure. Activating transcription factor 2 (ATF2), a DNA-binding protein that binds to cAMP response elements, was suppressed in both treatment conditions and is involved in mediating the activation of tyrosine hydroxylase transcription under nicotine treatment in PC12 cells (Gueorguiev et al., 2006). Nerve growth factor (NGF), a member of the neurotrophin family, is an essential mediator of neuronal activity and synaptic plasticity (Levi-Montalcini, 1987; Thoenen et al., 1987). Both NGF and its receptor, NGFR, were upregulated in the two treatment groups. Nicotine has been reported to regulate the expression of NGF and NGFR in cell lines or tissues (Terry and Clarke, 1994;Jonnala et al., 2002;Garrido et al., 2003;Hernandez and Terry, 2005;French et al., 2006). Neurexin 1 of various cell types, being stimulatory at low concentration and inhibitory at high concentration (Pollman et al., 1999;Ghosh et al., 2005). The observed mRNA decrease of TGFB2 and its receptor, TGFBR2, may indicate an increase in cell proliferation, although this remains to be established. Our analysis further showed a moderate enhancement of MAPK14 as judged by both mRNA and protein expression. MEF2 is one of the many downstream targets regulated by MAPK14. In our analysis, the transcription of two members of the MEF2 family was induced in the nicotine-treated samples; i.e., MEF2D (fold change 1.35; p = 0.008) and MEF2C (fold change 1.27; p = 0.177). MEF2 genes are highly expressed in neurons of the CNS (Heidenreich and Linseman, 2004;Shalizi and Bonni, 2005) and play important roles in neuronal differentiation and maintenance. Blocking the function of MEF2 genes may result in the death of neurons, whereas their expression can promote neuronal survival (Mao et al., 1999;Li et al., 2001;Gaudilliere et al., 2002). Although the activity of MEF2 is regulated mainly via its phosphorylation by MAPK14 or other kinases, considering the expression induction of MAPK14 and MEF2 genes, it may be reasonable to assume the activity of MEF2 is enhanced by acute nicotine exposure in SH-SY5Y cells.
In an earlier study, Dunckley and Lukas (2003) analyzed the gene expression patterns in SH-SY5Y cells treated with nicotine under similar experimental conditions (i.e., 1 mM nicotine for 1 h) with different microarray platforms. Among the 17 genes identified, eight had valid expression measurements in our analysis, and their expression patterns were largely consistent in the two studies. While Dunckley and Lukas (2003) performed a more detailed investigation of the relation between the nicotine-regulated genes and nAChRs, we here provide a much more broad and comprehensive analysis and description of the biological processes/pathways underlying responses to acute nicotine exposure. Results from the two studies also indicate that more detailed and comprehensive information about the interaction between acute nicotine and SH-SY5Y cell line could be obtained.

conclusIon
By profiling the gene expression pattern in SH-SY5Y cell lines treated acutely with nicotine, we have identified a set of significantly modulated genes. Further, we showed that the majority of these differently expressed genes were regulated through the nAChRmediated mechanism. From these significantly regulated genes, we have detected the enriched biochemical pathways, which included those potentially involved in the neuronal response to addictive drugs such as nicotine, including glucocorticoid receptor and p38 MAPK signaling. These findings can help us to understand the molecular mechanisms underlying the development of nicotine addiction. On the other hand, the identification of these genes and pathways further indicates that the physiological consequences of acute nicotine exposure are complicated. Thus, further investigation is necessary in order to obtain a more detailed and comprehensive understanding of the interaction between acute nicotine treatment and the nervous system.

AcknowledgMents
This project was in part supported by NIH grants DA-13783 and DA-12844 to Ming D. Li. The authors thank Dr. David L. Bronson for his excellent editing of this manuscript. neuronal circuitry adaptations induced by heroin self-administration were correlated with elevated glucocorticoid production in certain brain regions of rats (Weber et al., 2009). Corticosteroid receptor modulation of the mesoaccumbens dopamine neurotransmission is believed to be a key neurobiological mechanism mediating the effects of stress in addiction (Tye et al., 2009). Nicotine can also interact with glucocorticoid receptor signaling cascades directly or indirectly. Furthermore, the relations between nicotine and glucocorticoids are bidirectional; i.e., not only can nicotine affect the hypothalamic-pituitary-adrenal (HPA) axis and circulating glucocorticoids, but adrenal steroids can modulate nicotine's behavioral and physiological actions (Caggiula et al., 1998). Nicotine administration elevates plasma corticosterone in animals (Caggiula et al., 1998). Similarly, both cigaret smoking and nicotine infusion rapidly increase circulating cortisol concentrations in humans (Caggiula et al., 1998). In human smokers, the degree of nicotine addiction is reflected by changes in the sensitivity of central glucocorticoid receptors (Reuter et al., 2004). Nicotine administration to pregnant animals can lead to fetal overexposure to maternal glucocorticoid and result in fetal adrenocortical dysfunction and other diseases after birth (Chen et al., 2007). The significant regulation of the corticosteroid receptor signaling pathway by acute nicotine exposure provides additional evidence of the importance of this pathway in nicotine addiction, and the significantly regulated genes in this pathway are excellent candidates for further investigation. At the same time, the cross-talk between glucocorticoid receptor signaling and other pathways, such as MAPK signaling and PI3K/AKT signaling (Clark and Lasa, 2003;Gupta et al., 2007), implies that the regulation of this pathway indirectly induces modulations in other biological processes.
The MAPK signaling pathway is a major modulator of cell growth, division, and differentiation (Adams and Sweatt, 2002) and plays a key role in mediating neuronal activation induced by dopamine, glutamate, and drugs of abuse (Jiao et al., 2007). Nicotine activates the MAPK signaling pathway in a variety of tissues and cell types, including SH-SY5Y cells (Heusch and Maneckjee, 1998;Tang et al., 1998;Dajas-Nakayama et al., 2001;Wang et al., 2001;Bailador et al., 2002). Furthermore, many cellular processes are affected by both nicotine and MAPK signaling, such as cell survival and memory processing (Levin and Simon, 1998). Despite these known interactions between MAPK signaling and nicotine exposure, our work has shown a more detailed pattern of the effect of acute nicotine exposure on this pathway. A number of genes were significantly regulated by nicotine exposure (Table 1). There were also genes in this pathway that were regulated to a lesser extent (Figure 2). Whereas nicotine exposure decreases the amount of mRNA for TGF-beta 2 (TGFB2), TGFBR2, interleukin 1 receptor (IL1R), and interleukin 1 receptor-associated kinase 1 (IRAK), as well as a couple downstream genes, it also induces the expression of a number of genes, such as death-associated protein 6 (DAXX), MAPK14 (p38 MAPK), and mads box transcription enhancer factor 2 polypeptide D (MEF2D). The regulation pattern of this pathway clearly indicates the cross-talk and interaction between different cascades. Earlier studies showed that nicotine treatment decreases the amount of TGF-beta mRNA in different tissues. For example, nicotine treatment significantly suppresses the release of TGF-beta 1 in bovine aortic endothelial cells (Cucina et al., 1999) and several other cell lines (Lane et al., 2005). This protein has a bimodal dose-dependent effect on the proliferation