From the Division of Neurobiology, Barrow Neurological Institute, Phoenix, Arizona 85013
Received for publication, October 10, 2002, and in revised form, February 12, 2003
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ABSTRACT |
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Nicotine exposure can have long lasting effects
on nervous system function, some of which must contribute to nicotine
dependence. Up-regulation, an increase in numbers of
radioligand-binding nicotinic acetylcholine receptors (nAChR), occurs
on exposure to nicotine at high concentrations. To determine whether
altered gene expression might account for long term changes and
up-regulation following nicotine exposure, we assessed effects of
1 h of 1 mM nicotine exposure on alteration of
gene expression in the neuron-like SH-SY5Y neuroblastoma clonal line.
Repeat and cross-controlled microarray analyses yielded a list of 17 genes from the initially screened ~5,000 whose expression was
consistently altered following nicotine treatment. Subsequent
quantitative, real time reverse transcriptase PCR analyses
confirmed altered expression in 14 of 16 genes tested. Further, the
general nAChR antagonist, d-tubocurarine, blocked all but two of the
observed changes in gene expression, indicating that these changes are
dependent on nAChR activation. Use of other antagonists revealed that
nAChR subtypes can differentially affect gene expression. The genes
affected code for proteins that may be broadly categorized into four
groups: transcription factors, protein processing factors, RNA-binding
proteins, and plasma membrane-associated proteins. Our results suggest
that nicotinic activation of nAChR may have a broad role in affecting
cellular physiology through modulating gene expression.
Nicotinic acetylcholine receptors
(nAChR)1 are ligand-gated
cation channels implicated in a variety of neuronal functions, including memory processing (1), neurotransmitter release (2), cell
survival (3), and synaptic plasticity (4). Nicotine is a tobacco
alkaloid that acts acutely to stimulate nAChR channel opening, as does
the endogenous nAChR ligand, the neurotransmitter acetylcholine. This
functional response is a transient event thought only to alter
electrical activity in excitable cells. However, some nAChR are
expressed in nonexcitable tissues. Moreover, longer term psychological
and physiological effects of nicotine on the brain and body also must
reflect interactions with nAChR, but only if there are lasting
consequences of ligand binding and/or ion channel opening. An improved
understanding of both the molecular signaling cascades initiated by
short or longer term interactions of nicotine with nAChR and the
specific targets of those signaling cascades is essential to enhanced
perspectives on normal physiological roles of nAChR.
Nicotine is known to affect the expression of several genes. Among
these is the gene coding for tyrosine hydroxylase, which is involved in
a rate-limiting step in catecholamine synthesis (5, 6), as well as
genes involved in the regulation of food intake and energy expenditure,
such as neuropeptide Y, orexins, and their receptors (7, 8). In
addition, nicotine, like other substances of abuse, such as cocaine and
alcohol, induces the expression of immediate early genes such as
c-fos and junB in various brain regions
(9-11). Nicotine also up-regulates the mRNA levels of
c-fos and c-jun in the neuronal SH-SY5Y cell
line.2 Because these
immediate early genes function as transcription factors, their
nicotine-mediated up-regulation suggests that nicotine may regulate the
expression of additional genes in SH-SY5Y cells.
Nicotine activates the mitogen-activated protein kinase (MAPK)
signaling pathway in a variety of tissues and cell types (12-15). Recent work indicates that nicotine also activates this signaling pathway in SH-SY5Y cells (16). Further, nicotine and MAPK signaling pathways affect many of the same cellular processes, such as cell survival and memory processing (1, 3, 17, 18).
Beyond the role of nicotine in activating the MAPK cascade and early
immediate gene expression, little is known about the specific genes
that nicotine may regulate. We therefore investigated the effects of
nicotine exposure on gene expression in the SH-SY5Y cell line using a
microarray-based approach to identify candidate nicotine-regulated
genes. We show that nicotine, at a concentration that induces
up-regulation of nAChR, a process that has been implicated in nicotine
dependence and tolerance, affects the expression of a wide range of
genes that code for proteins with seemingly diverse functions.
Collectively, these results demonstrate that nicotine can modulate the
gene expression profile of a neuron-like cell line and suggest that
some of the cellular and physiological effects of nicotine may result
from these nAChR-mediated effects.
Drug Treatment and RNA Isolation--
The SH-SY5Y human
neuroblastoma-derived cell line was grown to 90% confluence and
treated with 1 mM nicotine for 1 h at 37 °C under
standard incubation conditions (95% humidity, 5% CO2) (19). Messenger RNA was isolated using the Oligotex®
Direct mRNA isolation kit (Qiagen) following the manufacturer's protocol. In a repeat experiment for RT-PCR analyses (see below), SH-SY5Y cells at 90% confluence were treated with 1 mM
nicotine alone, 100 µM d-tubocurarine (d-TC) alone, or 1 mM nicotine plus 100 µM d-TC for 1 h at
37 °C. Total RNA was isolated using TRIzol® reagent
(Invitrogen). To assess nAChR subtype-specific effects on gene
expression, SH-SY5Y cells were grown to 90% confluence and treated
with 1 mM nicotine, 1 µM Synthesis of Fluorescent cDNA and Hybridization to Microarray
Slides--
Microarray analyses were performed by the University of
Arizona Cancer Center Microarray Core Facility on a fee-for-service basis. Briefly, for both the control sample and the nicotine-treated sample, 4 µg of mRNA was reverse transcribed using avian
myeloblastosis virus reverse transcriptase, incorporating
Cy3-dUTP into the control sample cDNA and Cy5-dUTP into the
nicotine-treated sample cDNA. In separate reverse transcription
reactions for an independent hybridization, the control sample cDNA
was labeled with Cy5-dUTP, and the nicotine-treated sample cDNA was
labeled with Cy3-dUTP. These reciprocal labeling reactions were
performed twice and hybridized to individual arrays, yielding four
replicate sets of "dye switch" data designed to control for any
bias caused by selectivity of dye staining intensity of specific cDNAs.
The labeled cDNA probes were purified using the
QIAquick® PCR purification kit (Qiagen). The purified
cDNA was then mixed with a hybridization buffer containing 15 µg
of poly(dA), 6 µg of yeast tRNA, 15 µg of CoT 10 DNA, 2×
Denhardt's, 2.7× SSC, and 0.2% SDS, denatured, and hybridized for
16 h at 62 °C on a 5K cDNA microarray. The cDNAs
imprinted on these arrays are available from Research Genetics. For
detailed information about the microarray used in these experiments and
the genes represented on the array refer to
azcc-microarray.arl.arizona.edu/index.php3. Following hybridization, the slides were washed for 15 min in 0.5× SSC, 0.01% SDS and for 15 min in 0.06× SSC, 0.01% SDS, followed by a final 5-min wash with
0.06× SSC. The slides were then scanned for fluorescence emission from
each spot on the array at 570 and 670 nm for Cy3 and Cy5, respectively.
Normalization of Fluorescence Intensity and Analysis of
Expression Levels--
In microarray studies performed at the Arizona
Cancer Center microarray facility, median intensity of a given spot of
1.4-fold above background intensity in both the Cy5 and Cy3 channels
was required for calculation of expression ratios. Background intensity was assessed by measuring fluorescence from a spot containing no
cDNA. The raw levels of fluorescence for each spot were corrected for this background fluorescence and analyzed. The fluorescence intensity values in the two fluorescence channels were normalized relative to a set of housekeeping genes. Lastly, the standard deviation
of the mean of the log10 of the ratios for the housekeeping genes was used to calculate 40, 60, 80, 90, 95, and 99% confidence intervals, against which the ratios of signal intensities for all other
spots on the arrays with fluorescence above background were compared.
The statistical significance of the changes identified were calculated
using a paired, two-tailed t test comparing the observed
expression ratios for the four replicates to a reference value of 1.
Quantitative, Real Time Polymerase Chain Reaction--
For this
analysis, 5 µg of DNase I-treated total RNA from the untreated
SH-SY5Y cells and from each of the drug-treated cells was reverse
transcribed using SuperScript® II reverse transcriptase
(Invitrogen). Following reverse transcription, each sample was diluted
such that cDNA corresponding to that produced from 10 ng of total RNA
was used in subsequent PCRs. PCRs were performed using the
LightCycler® (Roche Molecular Biochemicals), which allows
real time monitoring of the increase in PCR product concentration after
every cycle based on the fluorescence of the double-stranded DNA
specific dye SYBR green (20, 21). The number of cycles required to produce a detectable product above background was measured for each
sample. These cycle numbers were then used to calculate fold differences in the starting mRNA level for each sample using the following method. First, the cycle number difference for vimentin, a
housekeeping gene, was determined in the control sample and the
appropriate drug-treated sample. This difference was referred to as
Nicotine Consistently Alters the Expression of a Diverse Set of
Genes--
To assess the extent to which nicotine modulates gene
expression in neuronal cells, we have performed analyses using a
microarray containing cDNAs corresponding to ~5,000 different
genes. These results provide the foundation for more exhaustive whole
genome screening. However, here we have focused on a smaller number to identify novel nicotine-regulated genes and firmly establish the extent
to which nicotine alters the expression of these genes. For these
analyses, the neuroblastoma-derived SH-SY5Y cell-line was treated with
1 mM nicotine for 1 h. Treatment with 1 mM
nicotine maximally induces a long lasting up-regulation in numbers of
nAChR radioligand-binding sites in SH-SY5Y cells, and this
up-regulation has been implicated in long lasting effects of nicotine
exposure, such as nicotine dependence and tolerance (22). Messenger RNA isolated from these cells or from control cells was used in the microarray analyses. Based on previous results investigating the effects of nicotine on gene expression (23), we anticipated that
nicotine may have subtle effects on gene expression in the SH-SY5Y cell
line. For this reason, we performed reciprocal fluorescent labeling of
the control and nicotine-treated mRNA populations in duplicate,
yielding a data set consisting of four independently hybridized,
cross-controlled microarrays (see "Materials and Methods"). Multiple replicate hybridizations using reverse labeled samples are
necessary to elucidate significant changes in gene expression in the
1.2-1.6-fold range (see Ref. 24 and references therein). These
multiple hybridizations and reciprocal labeling experiments were then
assessed to identify the most consistent and significant (reproducible
changes in effects between experiments) changes in gene expression.
We compared all of the data at the 40% confidence interval (see
"Materials and Methods") for genes whose expression was
consistently up-regulated or down-regulated in all four replicate
hybridizations. We selected the 40% confidence interval to avoid
excluding subtle gene expression changes. Using this comparison we
identified a list of 17 consistently altered genes (Table
I). Statistical analyses using Student's
t test showed these changes to be significant at the
p < 0.05 level for all but one of these genes (RAB6A). The number of affected genes increased to 51 when comparing only three
arrays and to 392 genes if only one reciprocal labeling experiment was
used. Thus, multiple replicate arrays using reverse labeled samples
were necessary to minimize the number of false positives from the large
data set and to confirm identification of candidate genes whose
expression was changed subtly but reproducibly. Importantly, because
there was some variability in expression ratios between hybridizations,
it is possible that significant but subtle changes in gene expression
were lost as more replicates were added to the analysis. The genes
identified and the significance of their sensitivity to nicotine
exposure will be discussed in greater detail below. However, it is
notable that there were increases in expression of only 3 of the 17 genes, whereas expression of the other 14 genes was repressed by
nicotine treatment. Also notable was the low magnitude of these changes
resulting from nicotine treatment, including 26-38% increases in gene
expression for the three up-regulated genes and a greater than 2-fold
decrease in expression for only one of the 14 down-regulated genes.
Nicotine Alters Gene Expression through nAChR Activation--
We
used a pharmacological approach both as another way to test the
significance and specificity of changes in gene expression and, because
nicotine readily crosses the cell membrane, to assess whether nicotine
altered gene expression through nAChR-dependent or
nAChR-independent pathways. In the course of these studies, we also
obtained sample replicates and analyzed them by real time RT-PCR to
confirm the results of the microarray surveys. The SH-SY5Y cells were
treated with 1 mM nicotine alone, 100 µM d-TC
(a general nAChR antagonist) alone, or the combination of 100 µM d-TC plus 1 mM nicotine. We then utilized
quantitative, real time RT-PCR using total RNA as the template to
verify and replicate the gene expression changes observed in the
microarray experiments (see "Materials and Methods"). Vimentin
mRNA was chosen as the control for normalization because its
expression level was unchanged in the microarray analyses (
The RT-PCR analyses confirmed statistically significant changes in
expression of 14 of the 16 consistently altered genes that were tested
(Table II; RAB6A and regulatory factor X-associated protein expression
was not significantly changed; RT-PCR analysis of effects on expression
of an expressed sequence tag was not performed). Each primer pair was
checked for specificity by melting curve analysis and agarose gel
electrophoresis to ensure that only specific product was quantitated
(Fig. 2 and data not shown). In most
cases, the magnitude of the change observed in the RT-PCR experiments
was comparable with or greater than that seen on the microarrays.
Importantly, pretreatment with d-TC blocked or reversed the
nicotine-induced changes in mRNA levels for all of the genes identified (including a trend toward blockade of the effects of nicotine on ZFR expression) except for the FEZ1 variants 1 and 2 (Table
II). These results implicate nAChR function in the observed nicotinic
modulation of mRNA levels. Further, these results argue that the
observed modulation of gene expression in the presence of nicotine is a
specific effect of this drug acting at nAChR.
In some cases, the modulation of gene expression observed using
quantitative, real time RT-PCR was in the opposite direction to that
observed in the microarray analysis (compare matrin3, FEZ1, TFPI2, and
EGR1 in Tables I and II). Importantly, this apparent discrepancy does
not reflect variation between the mRNA samples used for the
microarrays and the replicate total RNA samples used for RT-PCR
studies, because matrin3, which showed slight induction on nicotine
exposure in the microarray experiments, was found in RT-PCR analyses of
both mRNA and total RNA samples to be comparably repressed by
nicotine treatment (Table II). Expression ratios were low for these
four genes but still in the range for the 10 other genes for which
RT-PCR and microarray studies showed concordance. Absolute expression
levels were low (data not shown) for FEZ1, TFPI2, and EGR1 but not for
matrin3. However, the 1.9-fold increase in EGR1 expression that is seen
here in the quantitative, real time RT-PCR experiments with SH-SY5Y
cells following 1 h of 1 mM nicotine treatment (Table
II) is in concordance with results of another study showing ~2.8-fold
induction of EGR1 in PC12 cells following 1 h of 200 µM nicotine treatment (25). Although we cannot fully
explain the differences between the microarray and RT-PCR results, we
consider that findings from quantitative, real time RT-PCR analyses,
especially when corroborated by antagonist sensitivity of the effects
of nicotine, are more reliable than more raw microarray results.
Therefore, we used RT-PCR findings for our interpretations if there was
ambiguity in results from the two types of analyses. Nevertheless, our
observations underline the importance of secondary verification of
microarray results and indicate potential complications in deriving
gene expression profiling conclusions based solely on microarray analyses.
Nicotine Modulates the Levels of a Variety of mRNAs Coding for
Plasma Membrane-associated Proteins--
The RT-PCR analyses confirmed
that nicotine treatment subtly but significantly altered the levels of
a variety of classes of mRNAs. Nicotine repressed the expression of
mRNAs that code for plasma membrane associated proteins, including
contactin 1 and protein-tyrosine phosphatase receptor
Interestingly, nicotine also repressed the expression of another
membrane-associated protein TFPI2. TFPI2 codes for a serine protease
inhibitor that functions as a tumor suppressor and is repressed in
invasive cells of many tumor types (29-32). Thus, 1 h of
continuous nicotine treatment represses the expression of a known tumor
suppressor gene in the SH-SY5Y cell line.
Nicotinic Receptor Stimulation Alters Transcription Factor
Expression--
Nicotine affected the expression of multiple mRNAs
coding for proteins that either are known to be involved in
transcription or that are implicated in transcription based on
homology. The early growth response 1 (EGR1) mRNA is up-regulated
(Table II). Results from the microarray analysis suggested that
nicotinic stimulation down-regulated EGR1 expression (Table I).
However, the expression levels of EGR1 are only slightly above
background in the microarray analysis, decreasing the reliability of
the observed expression ratios using this technique (see
"Discussion"). The induction of EGR1 seen in the RT-PCR experiments
(Table II) is consistent with prior observations showing induction of
immediate early genes, such as c-fos, c-jun, and
junB in SH-SY5Y cells and in various brain regions in
response to nicotine (9-11, 33). Further, the 1.9-fold increase in
EGR1 mRNA level observed here is consistent with the 2.8-fold
induction seen in PC12 cells following 1 h of 200 µM
nicotine treatment (25). Both cocaine and amphetamine have been shown
to induce EGR1 expression in multiple brain regions. The identification
of nicotine as an additional EGR1-activating drug in a neuronal cell
line provides further evidence for a common regulatory mechanism for
these drugs of abuse and suggests that the SY-SY5Y cell line may be
useful for elucidating the common signaling pathways that these drugs activate.
The expression of several additional genes implicated in transcription
was changed in response to nicotine. In contrast to EGR1, nicotine
repressed the MLL3, p53-induced gene 7 (PIG7/LITAF1), and
retinoblastoma binding protein 6 (RBBP6) genes (Table II). Alteration
of these genes that are implicated directly or indirectly in
transcription raises the possibility that either these genes could be
involved in regulating the expression of the other genes identified
here or that there may be additional genes whose expression may be
affected in response to nicotine.
Nicotine Affects the Expression of Genes Coding for Protein
Processing Factors and RNA-binding Proteins--
Nicotine also altered
the expression of two genes involved in protein processing, ubiquitin
ligase E3A (UBE3A) and chromosome 2 open reading frame 2 (C2orf2). UBE3A exhibits brain specific maternal imprinting.
Loss of function mutations in this gene are associated with Angelman
syndrome (for review see Ref. 34), a disease in which seizures are
common. Repression of this gene suggests an additional possible
underlying mechanism for the observation that high doses of nicotine
and various nAChR antagonists can induce seizures in animal models
(35-40). C2orf2 encodes a protein of unknown function. However,
this protein contains numerous conserved domains including four WD
repeats and a highly conserved serine protease domain suggestive of a
role in protein processing (41).
Nicotine also alters the expression of two RNA-binding proteins,
matrin3 and ZFR, potentially implicating nAChR in RNA processing events. The ZFR protein is essential for murine embryonic development (42). However, the human ZFR mRNA is highly expressed in adult brain (43), suggesting that this gene has important functions beyond
development. Matrin3 is a member of a large family of RNA-binding proteins and is a nuclear matrix protein known to be involved in the
nuclear retention of A-to-I edited mRNA (44). The potential role of
these genes in nAChR function and in the physiological effects of
nicotine is currently unclear. Regardless, repression of these
mRNAs in response to nicotine suggests that nicotine may affect RNA metabolism.
Different nAChR Subtypes Have Varying Effects on Gene
Expression--
The SH-SY5Y cell line expresses
Results showed that
Antagonist effects on nicotine repression or induction of other genes
were more complex. Repression of PTP
There were instances where Microarray Analyses Can Elucidate Nicotine-dependent
Changes in Gene Expression--
The current findings indicate that
exposure of neuronal SH-SY5Y cells to nicotine at a concentration that
produces maximal nAChR up-regulation has relatively subtle effects on
the expression of a range of genes coding for proteins with diverse
functions. These effects can be identified using a microarray-based
approach provided that sufficient replicate, reciprocal labeling
experiments are performed to separate the true responses from the
background noise. The microarray approach is useful for generating
hypothesis about what genes are affected by a given treatment. However,
subsequent RT-PCR experiments are required to independently validate
the microarray results, especially when there is a low test/control expression ratio or when mRNA levels are only slightly above
background. Further validation of results using pharmacological studies
also is suggested when possible. In this study, quantitative, real time
RT-PCR experiments confirmed altered expression following nicotine
exposure for 14 of the 16 genes tested base on microarray findings
(Table II). Ten of these genes are altered in a manner consistent with
the observed expression ratios from the microarray analyses. In
contrast, RT-PCR experiments to detect the mRNAs for matrin3, FEZ1,
TFPI2, and EGR1 showed that the expression levels of these genes were
altered in a direction opposite to that observed in the microarray
analyses. This finding could reflect low expression ratios (Table I)
and, except for matrin3, low absolute levels of mRNA expression for
these genes in SH-SY5Y cells (data not shown). Nevertheless,
pharmacological studies indicated nicotinic receptor antagonist
sensitivity of effects of nicotine on expression of 13 of the 14 genes
confirmed by RT-PCR analyses. Collectively, the results of this study
illustrate the utility of microarrays as screening devices. Moreover,
the results also underscore the need for more comprehensive RT-PCR
studies to validate and extend the results of microarray analyses. In addition, pharmacological approaches can be valuable to provide further
verification and illumination of observations.
Effects of Nicotine on Gene Expression Involve nAChR
Activation--
Nicotine rapidly crosses the plasma membrane and
therefore could affect gene expression either through a
nAChR-dependent signaling pathway or through a
nAChR-independent pathway. To distinguish between these possibilities,
we determined whether nicotine could modulate gene expression when
nAChR activity was blocked by the general nAChR antagonist d-TC. The
critical observation suggesting that nAChR activation is involved in
the nicotine-dependent modulation of gene expression is
that d-TC blocks most of the observed nicotine-induced changes in gene
expression. This finding verifies that the observed subtle changes in
gene expression resulting from nicotine exposure are a specific effect
of this drug acting at nAChR. Further, this finding implies that there
may be signaling pathways leading from the nAChR to the nucleus to
affect gene expression (see below). However, it is possible that some
of the observed effects on mRNA levels could result from
post-transcriptional mechanisms.
Individual nAChR Subtypes Differentially Affect Gene
Expression--
The SH-SY5Y cells express homomeric
The assessment in this study of effects of nAChR antagonists alone
should be, but is not, routine practice. Many studies showing the
effects of antagonist plus agonist treatments, such as blockade of or
failure to block nicotinic agonist effects and synergy with nicotinic
agonist effects, need to be replicated with concomitant assessments of
antagonist effects alone to help elucidate the bases for ligand
actions. Had our studies not examined the effects of antagonists alone,
interpretation of the results would have been misleadingly simplified.
What Are the Signaling Pathways Leading from nAChR Activation to
Altered Gene Expression?--
Our data suggest that at least two
initial nAChR-mediated signals can modulate gene expression. Although
d-TC blocked the majority of the effects of nicotine on gene
expression, the levels of several mRNAs were affected by d-TC
alone, and this effect was not reversed when nicotine and d-TC were
used in conjunction. For DHFR, d-TC repressed expression comparably to
nicotine. For contactin 1, matrin3, and UBE3A, d-TC elicited an
increase in mRNA levels, whereas nicotine reduced mRNA levels.
Additionally, Could Changes in Contactin 1 mRNA Levels Be Involved in
Nicotine-induced nAChR Up-regulation?--
One of the interesting
aspects of nAChR function is the phenomenon of up-regulation. When
SH-SY5Y cells are continuously exposed to nicotine, there is an early
transient decrease in the total number of assembled, cell surface nAChR
that display radioligand binding. However, numbers of total
radioligand-binding sites increase immediately, reflecting an increase
in intracellular pools (22). Over time, the decline in surface receptor
numbers reverses, perhaps reflecting renewal and then later
up-regulation of cell surface pools replenished from the increased
intracellular pool of precursors. The mechanisms underlying this
response are poorly understood.
Nicotine treatment significantly reduced the expression of the
contactin 1 mRNA in as little as 1 h (Tables I and II and Fig.
2). Contactin physically interacts with many different proteins on the
cell surface, including voltage-gated Na+ channels (26,
27), protein-tyrosine phosphatase receptors (25, 47-49), contactin
associated protein (50), and Fyn receptor tyrosine kinase (51).
Interaction of contactin with voltage-gated Na+ channels
has been shown to increase the cell surface expression of fully
assembled and functional channels (26, 27). This observation suggests
an intriguing possible explanation for nAChR cell surface regulation.
If contactin regulates the surface expression of nAChR in a manner
similar to that of the voltage-gated Na+ channels, then one
would predict that repression of contactin 1 mRNA would result in
reduced surface expression of nAChR. Consistent with this model,
contactin mRNA is down-regulated following 1 h of nicotine
exposure, a time in which surface expression of nAChR is also
significantly reduced. Interestingly, after 24 h of nicotine
exposure surface nAChR have returned nearly to pretreatment levels
(22). This correlates with a 1.4-fold up-regulation of contactin 1 mRNA following 24 h of continuous exposure to 1 mM nicotine (data not shown). Future work will be aimed at elucidating the
potential role of contactin 1 in the regulation of nAChR function.
Summary--
Nicotine exposure has reproducible, but sometimes
relatively subtle, effects on gene expression in a neuron-like cell
line. These gene expression changes can be classified into three
general groups based on the effects of nAChR antagonists. Further, many of these effects are pharmacologically specific and appear to be
mediated by traditional nAChR channel function. Other effects of
nicotine on gene expression may result from alternative, yet nAChR-dependent, mechanisms. Our results demonstrate the
utility of microarrays in this type of analysis to identify candidate genes where subtle changes in gene expression, as would be predicted to
result from drug exposure, occur. Our results also highlight some of
the caveats in interpreting the results from such an approach, emphasizing the importance of secondarily verifying consistent changes
in gene expression. From these studies come tangible suggestions and
targets for future investigation as to how nicotine affects gene
expression in the nervous system, potentially adding to ways in which
this drug exerts its physiologically relevant effects.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-cobratoxin, 1 µM
-cobratoxin plus 1 mM nicotine, 3 µM mecamylamine, or 3 µM mecamylamine plus 1 mM nicotine for 1 h at 37 °C. Total RNA was
isolated using TRIzol® reagent (Invitrogen). RNA
concentrations were measured by spectrophotometry and adjusted
accordingly. Only RNA samples with an
A260/A280
ratio > 1.4 were used for microarray hybridizations and RT-PCR experiments.
H. Next, the cycle number difference for the gene of
interest was determined in the control sample and the appropriate
drug-treated sample, yielding another value,
I. The cycle
number difference for the gene of interest was then corrected for
slight differences in the amount of total RNA in control and
drug-treated samples by subtracting
H from
I, yielding a new value,
K. The expression ratio for the gene of interest was then calculated as
2
(
K) for genes that were induced
and as
(2
K) for genes that were repressed. The
expression ratios reported are the averages of three to eight replicate
PCRs. The statistical significance was calculated using a paired,
two-tailed t test comparing the cycle number difference for
the gene of interest (
I) to that of vimentin
(
H) across all replicates. Specificity of each primer
pair was confirmed by melting curve analysis and agarose gel
electrophoresis. The primers were designed using Primer3 software
(bioinformatics.weizmann.ac.il/cgi-bin/primer/primer3.cgi) and
subsequently checked for specificity using BLAST
(www.ncbi.nlm.nih.gov/genome/seq/HsBlast.html).
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
Microarray analyses reveal numerous, consistent, nicotine-induced
alterations of gene expression
1.03 ± 0.31). In addition, subsequent RT-PCR experiments wherein the
levels of vimentin mRNA were compared with the
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA showed
that both mRNAs were present at comparable levels in all drug-treated samples relative to the control sample (Fig.
1 and Table
II (GAPDH row)). Further, when normalized
to the amount of total RNA in each sample, none of the drug treatments
altered the expression of either GAPDH or vimentin. This result is
consistent with previous studies using human coronary artery
endothelial cells where GAPDH expression was unchanged in response to
nicotine (23). These combined results indicated that vimentin mRNA
expression was unaltered in response to nicotine.
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Fig. 1.
Housekeeping gene expression is unaltered in
response to both nicotine and d-tubocurarine. Shown is an inverted
image of an ethidium-stained agarose gel of RT-PCR samples
corresponding to the product present after 22 cycles of PCR for both
vimentin (left) and GAPDH (right).
nic, untreated control; +nic, samples treated
with 1 mM nicotine for 1 h; d-TC, samples
treated with 100 µM d-tubocurarine for 1 h;
d-TC + nic, samples treated with 1 mM nicotine
and 100 µM d-tubocurarine for 1 h. The fold change
values were calculated as described under "Materials and Methods"
and correspond to the fold change relative to the untreated
(
nic) sample.
Real time RT-PCR studies verify that nicotine modulates the expression
of numerous genes
K values (see "Materials and
Methods") was used to calculate a range of fold induction or
repression for each sample to determine the reliability of the results.
Each range column represents the range of values for the drug-treated
sample to the left of that column.
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Fig. 2.
The RT-PCR products are specific for each
gene. Each PCR generates only one product of the predicted size,
indicating the specificity of the primers used. The specific genes
targeted in the PCRs are indicated above the lanes. nic,
untreated control; +nic, samples treated with 1 mM nicotine for 1 h; d-TC, samples treated
with 100 µM d-tubocurarine for 1 h; d-TC + nic, samples treated with 1 mM nic and 100 µM d-tubocurarine for 1 h. Quantitation of the
amount of product was performed in real time as described under
"Materials and Methods." Calculated expression ratios for each
drug-treated sample relative to the control sample are indicated
beneath each lane. These values represent the average values over at
least three PCRs.
(PTP
).
Interestingly, the contactin 1 protein physically interacts with PTP
(26) as well as with voltage-gated Na+ channels (27, 28).
These observations suggest interesting possibilities for roles of
contactin and PTP
in nAChR up-regulation (see "Discussion").
Another interesting observation is that whereas nicotine exposure
repressed expression, d-TC significantly induced UBE3A expression and
showed a distinct trend to induction of contactin 1 expression (Table
II). This induction was not reversed when nicotine and d-TC were used
in conjunction, suggesting at least two possible explanations. First,
there may be a low basal rate of spontaneous nAChR channel opening that
helps to sustain the expression levels for these genes in the untreated
case. Application of the nAChR antagonist may prevent this spontaneous
channel opening, thereby resulting in increased expression.
Nicotine-mediated channel opening would have the opposite effect,
resulting in reduced expression. A second possibility is that binding
of either agonist or antagonist to the nAChR may induce alternative
conformational changes in the receptor that either activate or inhibit
a regulatory pathway. Distinguishing between these possibilities will
be a goal of future work.
3,
5,
7,
2, and
4 nAChR subunits that assemble to form various
3*-nAChR subtypes or homomeric
7-nAChR (19, 45). To
determine which receptor subtypes mediate the observed gene expression
changes, we treated SH-SY5Y cells with nicotine in the presence of
either 1 µM
-cobratoxin or 3 µM
mecamylamine.
-Cobratoxin is thought to be a specific ligand
for nAChR containing
1 or
7 subunits. A 1 µM dose
of
-cobratoxin will specifically inhibit responses to nicotine of
7-nAChR expressed by SH-SY5Y cells. Although interactions of mecamylamine (or of d-tubocurarine) at non-nAChR targets cannot be
entirely discounted, mecamylamine at low micromolar doses
selectively inhibits nAChR containing
3 or
4 subunits. The
mecamylamine inhibition profile for SH-SY5Y cells suggests that
3
4*-nAChR represent the primary
3*-nAChR subtype present (19).
Mecamylamine at a concentration of 3 µM would be expected
to inhibit about 80% of the nicotinic responses of
3
4*-nAChR and
only 20% of
7-nAChR responses to nicotine (46).
-cobratoxin blocked nicotine-mediated repression
of C2orf2 and RBBP6 (Table III).
This result suggests that specific activation of
7-nAChR is both
necessary and sufficient to down-regulate both of these
mRNAs. In contrast, none of the observed changes in
C2orf2 or RBBP6 gene expression were specifically blocked by
mecamylamine at a 3 µM concentration. However, either
-cobratoxin or mecamylamine affected the nicotine-mediated
repression of multiple genes. For contactin1, MLL3, UBE3A, ZFR, and
DHFR, both
-cobratoxin and mecamylamine prevented
nicotine-dependent repression of gene expression. These
results implied that simultaneous signaling through both
7-nAChR and
3*-nAChR subtypes was required for nicotine-dependent
repression of these mRNAs. In addition, MLL3 showed a
trend toward induction in response to the combination of
mecamylamine plus nicotine.
nAChR subtypes differentially affect gene expression
-Cobratoxin, samples treated
with 1 µM
-cobratoxin for 1 h;
-Cobratoxin + nicotine, samples treated with 1 µM
-cobratoxin and 1 mM nicotine for 1 h; Mecamylamine, samples treated
with 3 µM mecamylamine for 1 h, Mecamylamine + nicotine, samples treated with 3 µM mecamylamine and 1 mM nicotine for 1 hr. The numbers in bold represent the
average fold changes across multiple independent PCR reactions (from
three to five reactions). The Range columns were calculated as for
Table II. Several samples wherein the observed fold changes were over
3-fold have accompanying relatively large standard deviations. Serial
dilution experiments in which the GAPDH mRNA was amplified
indicated that the standard deviation increases as the difference in
starting mRNA level between the control and test samples increases
(data not shown), thereby possibly explaining this observation.
, PIG7, and cDNA DKFZp564F112 (represented by GenBankTM accession number
N28268) were not blocked by either
-cobratoxin or mecamylamine. The
combined set of observations that PTP
, PIG7, and N28268 repression
are blocked when all nAChR subtypes are inhibited by d-TC (Table II)
but not when
7- or
3
4*-nAChR are inhibited alone suggests that
nicotine-mediated signaling through either receptor subtype is
sufficient to modulate expression of these genes. However, it should be
noted that we cannot rule out the possibility that the remaining 20%
of functional
3
4*-nAChR in the presence of 3 µM
mecamylamine may be sufficient to repress these genes in response to
nicotine. Alternatively, repression of these genes could occur through
additional
3* subtypes that are not inhibited by mecamylamine at
a 3 µM concentration.
-cobratoxin exposure alone affected
mRNA expression comparably to the effects of nicotine (Table III).
This effect of
-cobratoxin alone also complicates the interpretation of findings for contactin 1, MLL3, DHFR, and UBE3A, where a block of
the effects of nicotine by
-cobratoxin was seen. Similarly, there were instances where mecamylamine exposure alone affected mRNA
expression comparably to nicotine, thereby possibly explaining why
mecamylamine did not block the effects of nicotine on C2orf2. Additionally, the effect of mecamylamine alone complicates the interpretation of findings for DHFR and UBE3A, where a block of the
effects of nicotine by mecamylamine was seen. These results imply that
the combined effects of agonist and antagonist acting at nAChR can have
differing effects on gene expression than either agonist or antagonist
acting alone. Further, assuming that there are no effects of these
antagonists on other targets, these results suggest that changes in the
conformation of the nAChR that result from antagonist or agonist
binding may play a role in activating signaling pathways that
ultimately result in alterations of gene expression.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
7-nAChR and
heteromeric
3*-nAChR subtypes. Experiments wherein
7-nAChR and
3*-nAChR subtypes were differentially inhibited using either 1 µM
-cobratoxin or 3 µM mecamylamine
yielded four important observations (see Table
IV for a summary of antagonist studies in
RT-PCR analyses). First,
-cobratoxin prevented the
nicotine-dependent down-regulation of both C2orf2
and RBBP6 mRNAs. This result indicates that down-regulation of
these mRNAs results exclusively from activation of the
7-nAChR. Second, both
-cobratoxin and mecamylamine blocked, reduced, or reversed the repression of matrin3, contactin 1, TFPI2, MLL3, UBE3A,
DHFR, and ZFR mRNAs (Table IV). This finding suggests that simultaneous nicotinic activation of both
7-nAChR and
3*-nAChR is
required to repress these mRNAs. Third, neither antagonist prevented nicotine-mediated repression of the N28268 (cDNA DKFZ in
Table IV) and PIG7 genes. However, d-TC, a general nAChR antagonist,
prevented nicotinic effects on expression of these genes, suggesting
that signaling through either
7- or
3*-nAChR is sufficient to
affect expression of these messages. Nevertheless, we cannot currently
rule out either of the possibilities that the 20% of active
3
4-nAChR that remain functional in the presence of 3 µM mecamylamine are sufficient to regulate these genes or that additional
3* subtypes may regulate these genes. Fourth, the
three nAChR antagonists alone were able to alter the expression of some
of the genes identified (see Tables II-IV for a summary of results).
Although there is a formal possibility that antagonists could be
affecting ongoing non-nAChR signaling that modulates gene expression,
this result implies that there exists a nAChR-dependent pathway to repress gene expression that is independent of nAChR channel
opening.
Summary of nicotine treatment-induced gene expression changes and their
sensitivity to blockade by nicotinic antagonists
-cobratoxin (cbtx), or mecamylamine (meca) alone
(columns 2-5, respectively); or effect of nicotine in the presence of
d-TC,
-cobratoxin, or mecamylamine (columns 6-8 under the
horizontal bar labeled Nicotine above). Group 1: Gene expression
changes in response to nicotine that are either blocked or reversed by
d-TC,
-cobratoxin, or mecamylamine. Group 2: Gene expression changes
that are blocked by either d-TC or cbtx. Group 3: Gene expression
changes that are blocked or reversed by d-TC only. The
indicates
significant induction alone. The
indicates significant repression
alone.
indicates nicotinic effects that are significantly blocked
in the presence of the indicated antagonist.
indicates nicotinic
effects that are reversed in the presence of the indicated antagonist.
Any symbol in parentheses indicates a trend toward the indicated effect
that did not reach statistical significance.
-cobratoxin and mecamylamine alone affected the
expression of multiple genes. These observations suggest several
possibilities. First, there may be at least two
nAChR-dependent signaling pathways. One pathway may be
dependent on channel opening and subsequent ion flow, and a second
pathway may be activated by changes in nAChR conformation that result
from either agonist or antagonist binding. In support of the first
possibility,
7-nAChR are highly permeable to calcium, and in the
SH-SY5Y cell, activation of
7-nAChR has been shown to activate the
extracellular signal-regulated kinase 1/2 through a
calcium-dependent mechanism (16). Nicotine has been shown
to activate this pathway, which ultimately affects gene expression. In
contrast, the observation that antagonists alone can significantly
repress expression of some genes supports the view that alternative
conformational states of the nAChR can affect the expression of some
genes. Previous studies have not determined the effects of nAChR
antagonists on activation of the MAPK pathway. It is therefore unclear
whether mecamylamine, d-TC, or
-cobratoxin could
activate the MAPK signaling pathway, thereby possibly explaining their
effects on gene expression through a common signaling pathway.
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FOOTNOTES |
---|
* This work was supported in part by endowment and/or capitalization funds from the Men's and Women's Boards of the Barrow Neurological Foundation, by the Robert and Gloria Wallace Foundation, by National Institutes of Health Grant R01-NS40417, by Arizona Disease Control Research Commission Grant 10011, and by Philip Morris Inc. through an External Research Program postdoctoral fellowship (to T. D.), and was conducted in part in the Charlotte and Harold Simensky Neurochemistry of Alzheimer's Disease Laboratory.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Div. of Neurobiology,
Barrow Neurological Inst., 350 West Thomas Rd., Phoenix, AZ 85013. Tel.: 602-406-3398; Fax: 602-406-4172; E-mail:
rlukas@chw.edu.
Published, JBC Papers in Press, February 14, 2003, DOI 10.1074/jbc.M210389200
2 L. Lucero and R. J. Lukas, unpublished observation.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
nAChR, nicotinic
acetylcholine receptor(s);
MAPK, mitogen-activated protein kinase;
RT, reverse transcriptase;
d-TC, d-tubocurarine;
GAPDH, glyceraldehyde-3-phosphate dehydrogenase;
PTP, protein-tyrosine
phosphatase receptor
;
EGR1, early growth response 1;
PIG7, p53-induced gene 7;
RBBP6, retinoblastoma binding protein 6;
UBE3A, ubiquitin ligase E3A;
C2orf2, chromosome 2 open reading frame 2;
TFPI2, tissue factor pathway inhibitor 2;
ZFR, zinc finger RNA-binding
protein;
MLL3, myeloid/lymphoid or mixed lineage leukemia 3;
FEZ, fasciculation and elongation protein
, Zygin;
DHFR, dihydrofolate reductase.
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