 |
INTRODUCTION |
Nitric oxide (NO)1 is a
pluripotent regulatory gas with diverse biological effects on numerous
enzymes, receptors, structural proteins, and transcriptional factors
(1). Studies aimed at understanding the mechanisms by which NO has
these myriad actions have represented one of the most rapidly growing
areas of research during the last decade. Many reports have shown that
NO alters proteins by regulating gene transcription through alterations in promoter specific activity; i.e. NO has been reported to
suppress both Egr-1 (2) and PKG-I
(3) through control of their
respective promoters. A partial list of similarly inhibited gene
expression through regulation of promoter activity includes cytokines
(4), cytochrome P450 enzymes (5), growth factors (6) as well as
secondary inhibition of gene expression stimulatory by factors such as
1
,25(OH)2D3 (7) and estradiol (8). On the
other hand, the ability of nitric oxide to up-regulate promoter
activity has been less well reported, although many proteins are
increased after exposure to nitric oxide (9-11). Thus, the ability of
nitric oxide to inhibit promoters seems to be widespread.
We also found that nitric oxide inhibited the expression of a protein
we were interested in; when we turned to transcriptional assays, we
also measured effects of the permeable molecule on promoter-driven
firefly luciferase. However, while studying a series of promoter
deletions, we found that the effect of nitric oxide seemed to be
nonspecific. This led us to consider whether our findings might be
explained by a more general effect of NO on the luciferase reporter
system. Since the first descriptions of firefly luciferase as a highly
sensitive, rapid, and easy-to-perform assay (12-14), luciferase assay
has become perhaps the most commonly used method for monitoring
promoter activity. We chose three structurally different NO donors to
test both viral and eukaryotic promoters linked to the luciferase
reporter gene. Our experience, reported here, shows that NO donors
significantly repressed luciferase activity in a promoter-independent
fashion. Our data also show a direct effect of nitric oxide to shorten
the half-life of the luciferase message, revealing at least one
mechanism by which NO might have nonspecific effects on this reporter system.
 |
MATERIALS AND METHODS |
Materials--
Sodium nitroprusside (SNP),
2',2'-(hydroxynitrosohydrazono)bis-ethanimine (Deta/NO), and
(±)-(E)-4-ethyl-2-[(Z)-hydroxyimino]-5-nitro-3-hexen-1-yl-nicotinamide (NOR4) were obtained from Sigma/Cell Signaling & Neuroscience. pGL3-Control "pGL3-SV40 (SV40Enhancer)-Luc" and the pGL3-Promoter "pGL3-SV40-Luc" were purchased from Promega (Madison, WI).
pcDNA/CAT and LipofectAMINE were from Invitrogen. The 4xVDRE was
inserted into pTK-Luc vector. The macrophage colony stimulating factor (MCSF) promoter and the receptor activator of NF-
B ligand (RANKL) promoter were inserted into both pGL3-Basic and pGL3-Enhancer. pCAT-MCSF was generously provided by Dr. M. Harrington (Indiana University School of Medicine, Indianapolis, IN) (15). DNase I was
obtained from Ambion (Austin, TX). All other reagents were purchased
from Sigma or are specifically noted otherwise.
Cell Cultures and Transfection--
The ST-2 murine bone stromal
cell line was purchased from Riken Cell Bank (Tsukuba Science City,
Japan) and maintained in
-minimal essential medium with
10% FBS. HepG2 cells were obtained from American Type Culture
Collection (Manassas, VA) and grown in Dulbecco's modified Eagle's
medium supplemented with 10% fetal bovine serum. ST-2 cells and
HepG2 cells were seeded at a density of 150,000/well in six-well
plates. After 18-20 h, cells were transfected with 1.6 µg/well of
DNA and 8 µl of LipofectAMINE in the absence of serum. Five hours
later, an equal volume of medium with serum was added to bring the
final concentration of serum to 10%. The next day, media was changed,
and NO donors were added to cultures as specified.
Luciferase Assay--
To assess luciferase activity, the Promega
luciferase assay system was used. Briefly, reporter lysis buffer was
added and incubated at room temperature for 15 min before
centrifugation to remove cell debris. For the assay, 20 µl of cell
lysate was mixed with 100 µl of luciferase substrate, and light
emission was measured with the LumiCount Luminometer (PerkinElmer Life Sciences). In some experiments, cells were co-transfected with pcDNA3/CAT to allow for normalization to CAT activity. CAT
normalized results were not different from those normalized by protein
content (Bio-Rad detergent-compatible assay) or expressed as
luciferase activity alone.
CAT Assay--
ST-2 cells were transfected with CAT reporter
vector and treated with Deta/NO for 24 h. Cell lysates were
collected 48 h after transfection and heated at 60 °C for 10 min to inactivate endogenous deacetylase activity. For the assay, 100 µl of lysate was mixed with [14C]chloramphenicol and
n-butyryl coenzyme A at 37 °C overnight. Samples were
extracted with xylene and counted in a 2500 TR liquid scintillation
counter (PerkinElmer) as described in the Promega protocol.
RNA Isolation--
After ST-2 cells were treated with Deta/NO,
total RNA was extracted by TRIzol as described in the Invitrogen
protocol. DNase I treatment alone could not sufficiently eliminate
plasmid DNA contamination in our samples, as shown by persistent
presence of significant amplicons as assessed by nonsignificant
differences in cycle thresholds (CT values) between RNA
samples for negative control (i.e. not reverse-transcribed)
and samples that had been reverse transcribed. To decontaminate
preparations of plasmid DNA, RNA samples (5-6 µg) were incubated
with 30 U RsaI, which generated multiple cuts in the
luciferase coding region (particularly inside the luciferase PCR
amplicon; see below). To study possible NO effects on the CAT reporter
message, the lysates containing plasmid were cut with SspI.
After incubation for 1 h, the samples were heated at 65 °C for
10 min, and RNA was re-extracted with the RNAeasy mini kit (QIAGEN,
Valencia, CA). Finally, 10 units of DNase I (DNA-free DNase treatment
and removal reagents; Ambion) was added to each sample at 37 °C for
30 min followed by application of the DNase I removal reagent. The
samples were stored at
70 °C.
Real-time PCR--
Analysis of Luciferase, CAT, and 18 S
mRNA was performed using the iCycler (Bio-Rad). Reverse
transcription of 1 µg of total RNA was performed with random decamers
(Ambion) and superscript II reverse transcriptase (Invitrogen) in total
volume of 20 µl. For real-time PCR, amplification reactions were
performed in 25 µl containing primers at 0.5 µM and
dNTPs (0.2 mM each) in PCR buffer and 0.03 units of
Taq polymerase (Invitrogen) and SYBR-green (Molecular
Probes, Eugene, OR) at 1:150,000. Aliquots of cDNA were diluted
10-10,000-fold for 18 S and 5-625-fold for luciferase as well as CAT
to generate relative standard curves to which sample cDNA was
compared (16, 17). For luciferase, forward and reverse primers were
5'-GCC TGA AGT CTC TGA TTA AGT-3' and 5'-ACA CCT GCG TCG AAG T-3',
respectively, creating an amplicon of 96 bp (18). Real-time PCR to
identify CAT mRNA, represented by a 133-bp amplicon, used the
forward primer sequence 5'-GCG TGT TAC GGT GAA AAC CT-3' and the
reverse primer sequence 5'-GGG CGA AGA AGT TGT CCA TA-3'. For 18 S, an
amplicon of 345 bp was generated with forward primer 5'-GAA CGT CTG CCC
TAT CAA CT-3'and reverse primer 5'-CCA AGA TCC AAC TAC GAG CT-3' (17).
Standards and samples were run in triplicate. Dilution curves showed
that PCR efficiency was more than 90% for luciferase, CAT and 18 S
amplicons. Negative controls, such as samples without RT and PCR
mixtures lacking both cDNA and RNA were also set up for each
real-time PCR to ensure elimination of plasmid DNA. PCR product arising
from RNA samples that were not reverse-transcribed had CT
values of 27.9 ± 0.56 for luciferase, 28.4 ± 0.3 for CAT,
and 32.1 ± 0.95 for 18 S; this was not significantly different
from data generated from sample buffer alone with CT values
of 29.03 ± 0.38 for luciferase and 32.25 ± 0.99 for 18 S. Luciferase values were normalized for the amount of 18 S in
the same RT sample, which was also standardized on a dilution curve
from RT sample as described in the literature (16, 19).
Statistical Analysis--
Results are expressed as the mean ± S.E. Statistical significance was evaluated by Dunnett one-way
analysis of variance or t test (Prism; GraphPad Software,
San Diego, CA).
 |
RESULTS |
Nitric Oxide Donors Inhibit the Luciferase Expression of Multiple
Promoter Constructs--
Nitric oxide has been shown to repress the
activities of multiple promoters studied with luciferase reporter
plasmids, yet no specific sequences have been associated with this
down-regulation. To assess whether this effect might be nonspecific, we
tested several constructs, including those available commercially as well as some of particular interest to our laboratory. These are shown
in Fig. 1a and include both
those derived from pGL3-Basic and those where a SV40 enhancer was
placed downstream of the poly(A) signal as in the "pGL3-Enhancer"
vector.

View larger version (36K):
[in this window]
[in a new window]
|
Fig. 1.
SNP effect on luciferase activity.
Luciferase reporter constructs used in experiments are shown in
a. ST-2 cells were transfected with luciferase reporter
constructs: pGL3-CMV-Luc (b); pTK-4xVDRE-Luc (c);
pGL3-SV40-Luc (d); pGL3-MCSF-Luc (e); pGL3-SV40
(SV40Enhancer)-Luc (f), and pGL3-MCSF (SV40Enhancer)-Luc
(g). After treatment of SNP for 20 h, cell lysate was
collected for luciferase assay. Concentrations of SNP above 100 µM inhibited luciferase activity
(b-d); p < 0.01 shown by
asterisk. Inhibitory effect was seen in pGL3-SV40 (SV40Enhancer)-Luc
and pGL3-MCSF (SV40Enhancer)-Luc with SNP above 300 µM;
p < 0.01 (f-g).
|
|
ST-2 bone stromal cells were transfected with the luciferase reporter
constructs and treated with the nitric oxide donor SNP for 20 h
(overnight) at variable concentrations. Fig. 1, b-e, shows
that the luciferase activity in multiple constructs significantly decreased with the addition of 100 µM SNP. These
constructs represent both viral (CMV promoter, Fig. 1b; SV40
promoter, Fig. 1d), as well as a concatameric consensus
sequence representing the vitamin D response element associated with a
minimal thymidine kinase promoter (Fig. 1c) and
774-nucleotide sequence from the murine MCSF promoter (Fig.
1e) (20). Further reduction in luciferase activity was seen
with the addition of 300 µM SNP to cells transfected with
the constructs. When the SV40 enhancer was included as part of the
promoter construct, added downstream of the poly(A) signal, the
inhibitory effect of SNP was blunted with significant inhibition by
40% achieved only at 300 µM SNP; this was seen with both
the viral and the eukaryotic promoters studies in Fig. 1, f
and g.
To ascertain whether these general effects were confined to the
specific NO donor SNP, we also studied two other NO donors: Deta/NO,
which is a long lasting NO donor (half-life ~20 h at 37 °C) (21,
22) and NOR4, a short lasting NO donor (half-life ~1 h). ST-2 cells
were cultured in same condition as the SNP group above but treated with
Deta/NO or NOR4. The NOR4 was added twice because of its short
half-life, at 0 and 6 h. For data shown in Fig.
2, ST-2 cells were transfected with
pGL3-CMV-Luc (without SV40 enhancer) or pGL3-SV40 (SV40Enhancer)-Luc
before treatment with 0-300 µM of Deta/NO for 20-24 h.
Deta/NO at 300 µM decreased luciferase activities in both
reporter constructs. However, luciferase activity was inhibited
at 100 µM of Deta/NO in pGL3-CMV-Luc, in which there was
no SV40 enhancer downstream of the luciferase gene; activity was
similar at 100 µM SNP (Fig. 2a) but required 300 µM to significantly repress a construct containing
the SV40 enhancer (Fig. 2b). Similar results were found when
the donor NOR4 was studied along with SNP in ST-2 cell cultures
transfected with the pGL3-RANKL-Luc construct, again showing that 100 µm of this donor was able to repress the SV40 enhancer-less construct (Fig. 2c). These results demonstrate that commonly used NO
donors cause a significant decrease in luciferase expression in all
promoter constructs studied.

View larger version (33K):
[in this window]
[in a new window]
|
Fig. 2.
Comparison of NO donor effect on ST-2 cells
transfected with constructs with or without SV40 enhancer. ST-2
cells were transfected with pGL3-CMV-Luc, pGL3-SV40 (SV40Enhancer)-Luc,
or pGL3-RANKL-Luc (constructs shown in Fig. 1a) and treated
with NO donor for 20 h. Cell lysates were collected for luciferase
assay. a, there was a decrease in luciferase activity when
SNP or Deta/NO ( 100 µM) was added in pGL-CMV-Luc;
b, the inhibitory effect was right-shifted in pGL3-SV40
(SV40Enhancer)-Luc; c, luciferase activity decreased in a
dose dependent manner for SNP and NOR4 in the cultures transfected with
pGL3-RANKL-Luc. *, p < 0.01, significantly different
from untreated group.
|
|
Nitric Oxide Donors Fail to Inhibit the Same Promoters Driving a
CAT Reporter--
The inhibitory effect of NO donors on luciferase
activity in multiple unrelated promoter constructs could be explained
either by a direct effect of nitric oxide to inhibit promoter activity or a direct effect on luciferase mRNA processing or luciferase activity. To examine the first possibility, the effect of NO donors was
examined using two promoters studied previously in the luciferase reporters but here with a CAT reporter system: pcDNA3/CAT, which contained the CMV viral promoter, and pCAT-MCSF, containing the same
nucleotide sequence of the MCSF promoter studied in Fig. 1 (Fig.
3a).

View larger version (17K):
[in this window]
[in a new window]
|
Fig. 3.
Deta/NO does not affect CAT reporter
constructs. a, CAT constructs used in this experiment.
b, ST-2 cells were transfected with pcDNA3/CAT (CMV as
promoter) and treated with Deta/NO (0-500 µM) for
24 h. CAT activity remained at the same levels as it did in
control even with NO at 500 µM. c, ST-2 cells
were transfected with either pGL3-MCSF-Luc or pCAT-MCSF and treated
with 300 µM Deta/NO for 24 h. Cell lysates were
assayed for luciferase or CAT expression. There was a decrease in
luciferase expression in presence of NO but CAT levels were not changed
from the control group. This experiment was repeated three times with
similar data. *, p < 0.01, significantly different
from untreated group.
|
|
ST-2 cells were transfected with these two constructs. When cells
transfected with pcDNA/CAT (CMV promoter) were treated with Deta/NO
overnight (~48 h after transfection), CAT activities did not change
even when 500 µM of Deta/NO was added (Fig.
3b) in contrast with the inhibitory effect seen in Fig.
2a. We also compared the promoter activities when treated
with Deta/NO in either pCAT-MCSF or pGL3-MCSF-Luc. As shown in Fig.
3c, 300 µM of Deta/NO had no significant
effect on CAT activity (unchanged from control levels), whereas the
expected 50% reduction in luciferase activity was seen in the same
promoter driving the luciferase reporter. These results suggested that
the inhibitory effects of NO donors on luciferase activity were not
caused by NO effects on promoter-initiated transcription.
NO Donors Decrease Steady-state Luciferase mRNA Levels by
Decreasing Message Half-life--
We next investigated whether the NO
donor or its metabolites might directly inhibit luciferase activity in
the sample lysates. Cell lysates were made from ST-2 cells transfected
with pTK-4xVDRE-Luc. Before luciferase assay, samples were incubated
with different doses of the short half-life NO donor NOR4 for 30 min.
As shown in Fig. 4a,
luciferase activities were not changed with increasing NOR 4 added from
0 to 500 µM. We next examined whether a metabolic product
of the NO donor might affect luciferase activity in the lysate. ST-2
cell lysates were collected from cells previously transfected with
pGL3-MCSF-Luc and mixed with lysates made from ST-2 cells
untreated or treated for 24 h with 300 µM Deta/NO, the long acting NO donor. Assays showed that addition of ST-2 cell
lysates containing NO or its metabolites did not change luciferase activity (Fig. 4b).

View larger version (32K):
[in this window]
[in a new window]
|
Fig. 4.
NO does not directly affect luciferase
activity. a, NO does not directly affect luciferase assay.
ST-2 cells were transfected with pTK-4xVDRE-Luc plasmid. Cell lysates
were treated with NOR4 (0-500 µM) for 30 min before
luciferase assay. Data showed that NOR4 did not directly inhibit
luciferase activity. b, neither the NO donor nor its
metabolite inhibited luciferase activity. ST-2 cells were transfected
with pGL3-MCSF-Luc. Transfected ST-2 cell lysates were mixed with
reporter lysis buffer or untreated ST-2 cell lysate or lysates from
ST-2 cells treated with 300 µM of Deta/NO. Data showed
that there were no differences in luciferase activity among
groups.
|
|
We further examined whether NO donor repression of luciferase activity
was caused by changes in luciferase mRNA levels. For these
experiments, we used 300 µM of Deta/NO to treat ST-2
cells transfected with pGL3-MCSF-Luc. Real-time PCR analysis was used to measure amounts of luciferase mRNA that were normalized to 18 S
mRNA levels from real-time PCR in each sample. Care was taken to
rule out plasmid DNA contamination (from the transfection of plasmid)
in the RNA samples. In our hands, neither the protocol outlined by Cok
and Morrison (18), in which RNA samples were treated twice with DNase
I, nor the Ambion protocol, in which samples were heated before DNase I
digestion, was able to ensure that DNA contamination was removed: both
protocols showed the same threshold cycles of either RNA samples only
(without RT as negative control, equivalent amount of RNA for RT
reaction) or from RT products for luciferase detection (data not
shown). To overcome this obstacle, we added a new step in which samples
were treated with the endonuclease RsaI, which generated
multiple cuts in the luciferase gene, including one within the
luciferase PCR amplicon specified by the primers used for
amplification. RsaI was deactivated before continuing with
DNase I treatment. With this additional step, the average
CT value for luciferase from RT product was 20.2 ± 0.6 cycles compared with the average CT value of 27.9 ± 0.6 cycles from RNA sample (without RT). The luciferase CT value from contaminating plasmid DNA were sufficiently
shifted out of the range relevant for RNA analysis (23).
As shown in Fig. 5a, 300 µM Deta/NO caused a 25% decrease in steady-state
luciferase mRNA expression as measured by comparison with real-time
PCR products in cells not exposed to the NO donor. Our next step was to
examine whether the decrease in luciferase mRNA was caused by
changes in message stability.

View larger version (30K):
[in this window]
[in a new window]
|
Fig. 5.
NO donors affect luciferase mRNA by
decreasing message stability. a, luciferase mRNA levels
were measured after transfection with pGL3-MCSF-Luc and treatment with
Deta/NO (300 µM) for 24 h. Luciferase mRNA level
was significantly reduced to 74 ± 2.4% by Deta/NO treatment
compared with the control group. *, p < 0.0075, significantly different from untreated group. b, ST-2 cells
were transfected with pGL3-MCSF-Luc. After treatment with SNP (300 µM) for 2 h, actinomycin D (8 µg/ml) was added to
cultures. Total RNA was extracted at 0, 0.5, 1, and 2 h, and
followed by RT and real-time PCR. All luciferase data were normalized
with 18 S from the same sample. Half-life in the NO-treated group was
~40 min compared with ~75 min in the control group. This experiment
was repeated three times with similar data. c, cells were
transfected with pGL3-SV40 (SV40Enhancer)-Luc and treated as in
a, and the half-life of luciferase mRNA was prolonged in
presence of the SV40 enhancer to ~220 min in the control group.
d, ST-2 cells were transfected with pCAT-MCSF. CAT mRNA
expression was measured after treatment with Deta/NO (300 µM) for 24 h. CAT mRNA levels in the Deta/NO
treatment group were not significantly different from those of
untreated control cells.
|
|
Cells transfected with pGL3-MCSF-Luc construct were treated for 2 h with the NO donor SNP (300 µM). At this point,
actinomycin D was added at levels shown to inhibit further mRNA
transcription. In ST-2 cells, the control luciferase mRNA had a
very short half-life of about 75 min. In those cells treated with SNP,
the half-life was decreased to 45 min, a significant decrease seen in
two repeats of this experiment (Fig. 5b). Thus, the
substantial decreases in luciferase activity caused by NO donors are
reflected in the nearly 50% decrease in the stability of luciferase
mRNA. In contrast, luciferase mRNA half-life was longer (~220
min) in ST-2 cells transfected with pGL3-Control, in which the SV40
enhancer was placed downstream of the luciferase coding region and
poly(A) (Fig. 5c). Comparing luciferase mRNA half-life
between control and NO treatments, there was a slight decrease to
~200 min in the presence of 300 µM SNP.
As another control, CAT mRNA levels were also measured to evaluate
whether NO donors affected this message. ST-2 cells were transfected
with pCAT-MCSF and total RNA collected after treatment with Deta/NO
(300 µM) for 24 h. As shown in Fig. 5d,
CAT mRNA expression, measured by real-time PCR, was not changed in
the presence of the NO donor.
The NO Effect on Luciferase Activity Is Seen in HepG2
Cells--
HepG2, a hepatocyte carcinoma cell line was
used to evaluate whether the NO donor repression of luciferase was
restricted to the ST-2 bone stromal cell line. The constructs pGL3-SV40
(SV40Enhancer)-Luc and pGL3-MCSF (SV40Enhancer)-Luc were transfected
into HepG2 cells. The cells were then treated with NOR4 (0-300
µM) as was done previously in the ST-2 cell line. As
shown in Fig. 6a, high dose of
NOR4 (300 µm) decreased luciferase activity of the pGL3-SV40
(SV40Enhancer)-Luc as shown previously. The effect of the MCSF or SV40
promoter without the enhancer showed that SNP repressed activity at
less than 300 µm (Fig. 6b).

View larger version (19K):
[in this window]
[in a new window]
|
Fig. 6.
NO donor inhibits luciferase activity in
HepG2 cells. a, HepG2 cells were transfected with pGL3-SV40
(SV40Enhancer)-Luc and pGL3-MCSF (SV40Enhancer)-Luc and treated with
NOR4 (added two times) for 24 h. Luciferase activity was decreased
when 300 µM of NOR4 was added to cultures. b,
HepG2 cells transfected with pGL3-SV40-Luc or pGL3-MCSF-Luc were
treated with SNP (0-300 µM) for 20 h. Luciferase
activity decreased in a dose-dependent manner. *,
p < 0.05, significantly different from untreated
group.
|
|
 |
DISCUSSION |
Nitric oxide has been shown to modulate the gene expression of
many proteins through cGMP and cGMP independent pathways as well as
altering DNA binding capabilities of transcription factors through
S-nitrosylation (1, 24). Besides genes that seem to be
up-regulated, such as ecSOD (9), there are many genes whose expression is inhibited after treatment with nitric oxide (e.g. IGFBP-1 (6), cGMP-dependent protein kinase
I
(3), and the cytochrome P450 enzyme CYP3A4 (25)). Identification of the mechanism of gene repression often involves assessing the specific promoter activity in the presence of an NO donor. The luciferase gene is widely used in constructs employed to study promoter
and enhancer control of gene expression because luciferase measurement
is sensitive and responds quickly to changes in promoter activity (13,
14). Numerous citations suggest that nitric oxide, and NO donors,
strongly and dose-dependently inhibit promoters assayed
with a luciferase reporter. For instance, when NO donors were shown to
decrease vitamin D receptor-retinoid X receptor-VDRE complex formation,
the next study was to assay a VDRE fused to minimal thymidine
kinase promoter with a luciferase reporter: Deta/NO was shown to
decrease luciferase activity by 50% at 300 µM (7). A
repressive effect of NO on vitamin D stimulation of the
CYP3A4 gene was also suggested to rely on a transcriptional effect of NO; the authors showed that the NO donor NOR4 dose
dependently decreased the CYP3A4 promoter linked to a luciferase
reporter with an ED50 of around 300 µM (5,
25). Similarly, the expression of endogenous cGMP-dependent
protein kinase I
shown to be sensitive to NO, was studied with a
PKG-I
promoter/luciferase construct, revealing a
dose-dependent inhibition of luciferase activity after treatment with three structurally unrelated NO donors (3). In this
case, the activity of IGFBP-1 promoter/luciferase deletion constructs
was shown to be down-regulated by Deta/NO; although NO stimulated cGMP,
inhibitors of cGMP failed to block NO associated gene repression,
leading the authors to conclude that NO had an inhibitory effect
separate from cGMP (3). Reports such as these led to an examination of
NO involvement in gene repression in our laboratory. Consideration of
multiple promoter constructs in our laboratory showed many to be
sensitive to NO donor inhibition, leading us to examine the generality
of NO promoter repression.
To assess NO effect on the promoter-luciferase assay, we studied both
weak and strong promoters and those with an SV40 enhancer used to
increase sensitivity. As shown in Fig. 1, all the luciferase reporter
constructs we studied had decreased luciferase activity in the presence
of NO donors (including SNP, Deta/NO, and NOR4). Those lacking the SV40
enhancer, perhaps the most common type of construct used to study
promoter activity, were all inhibited by NO donors with apparent 50%
inhibition at ~300 µM. Similar results are presented
for other promoters studied with luciferase in the literature as cited
above. One group did show increased repression of the CYP2D6 promoter
compared with a control luciferase, in that case pGL3-control; however,
it seems that the control reporter contained the SV40 enhancer, whereas
their specific promoter did not (25). Our results show that constructs
containing the SV40 enhancer are less sensitive to the repressive
effect of the NO donor. Inclusion of the SV40 enhancer conveyed
resistance to suppression of luciferase activity until 300 µM (Fig. 2). Many firefly luciferase vectors are
marketed; for instance Promega's pGL3-control and the pGL3-enhancer
contain the SV40 enhancer, whereas the pGL3-basic and the pGL3-promoter
do not. Consideration of the SV40 enhancer region as a stabilizer that
is, perhaps, not sensitive to NO effects suggests that construct design
is important for analysis of NO effect: Sugawara inserted the
insulin-like growth factor binding protein 1 promoter into pGL3
promoter vector (6); Hara and Sellak used the pGL3-basic construct (3,
25). In those constructs derived from pGL3-Basic 100 µM
of SNP, Deta/NO, or NOR4 was sufficient to decrease luciferase activity.
One example from the literature was careful to show a control
luciferase construct that seems to be resistant to NO repression. In
this article by Sogawa et al., NO donors inhibited the
luciferase expression driven by a promoter containing four
hypoxia-response element (HRE3) sequences upstream of an SV40 promoter,
as well as one HRE sequence set before a VEGF promoter (26). The
inhibition of these HRE-containing sequences was highly significant at
low doses of SNP, with an ED50 under 10 µM.
The control luciferase reporter vector, also preceded by a SV40
promoter, was not inhibited by the NO donors
however, the luciferase
construct did differ from those containing HRE by the inclusion of an
SV40 enhancer downstream of luciferase coding frame and the poly(A)
signal (26). Despite this, it is likely that this extremely sensitive
repression is markedly different that the promoter responses we have
studied in this work.
We ruled out that NO donors caused a generalized decrease in gene
transcription by using two of the previously NO "inhibited" promoters linked to non-luciferase-based reporters. We had previously tried to use the
-galactosidase reporter, but this was also
significantly inhibited by Deta/NO in a dose-response manner in ST-2
cells in multiple promoter constructs (data not shown). As shown in
Fig. 3, NO failed to inhibit CAT expression driven by a viral or
eukaryotic promoter that was significantly "inhibited" by NO donors
in luciferase assay. The CAT assay results revealed that neither the
MCSF nor CMV promoter sequence was responsible for the NO-inhibition of luciferase activity. Thus, proper control constructs were able to
prevent misconceptions.
Because NO did not affect promoters in CAT reporter constructs and had
no direct effect on the luciferase assay, we considered whether NO's
effect on the luciferase activity might be through effects on altered
stability of the luciferase mRNA. After eliminating contaminating
reporter plasmid DNA with a two-step procedure involving a restriction
endonuclease cut followed by DNase I digestion, we found the luciferase
mRNA half-life to be in the range of 75 min. This was in agreement
with results from Xu et al. (27), who studied luciferase
after retroviral integration of the luciferase message finding a
half-life of 65 min. Our work confirms that luciferase mRNA has a
short half-life in the ST-2 cells, and that this half-life is further
decreased by exogenous nitric oxide. However, luciferase mRNA
half-life was increased to 220 min in constructs containing
the SV40 enhancer (Fig. 5c). In contrast, Cok and
Morrison's study (18) showed a message half-life of longer than
10 h from vector containing the SV40 enhancer region
indeed, essentially no mRNA was degraded during the 10 h of the study (18). It is not entirely clear whether luciferase mRNA stability is
completely embedded in the SV40 enhancer region. A Rous sarcoma virus-driven luciferase transfected into breast cancer cells
that does not have a SV40 enhancer seemed be resistant to degradation after 12 h of actinomycin D treatment (28). However, no mention was made of controlling for plasmid DNA contamination. The SV40 enhancer region was initially inserted into the luciferase reporter vectors to improve the sensitivity of the luciferase signal, giving an
increase of nearly 10-fold luciferase activity (13). This increased
sensitivity has been though to be caused by increased transport into
the nucleus (29) or perhaps by inhibition of gene silencing via
methylation (30).
In sum, nitric oxide is known to have widespread effects on gene
expression in almost every cell studied. The mechanisms involved in the
regulation of NO are complex and involve multiple effects through
variable signal cascades and alteration of protein nitrosylation and
oxidation. Our work reveals that NO also affects stability of mRNA,
particularly luciferase mRNA. Because luciferase seems to be a
fairly unstable transcript, as shown by a half-life of significantly
less than 80 min, it may be particularly susceptible to regulation
through altering message stability. Our results indicate that
luciferase reporter may not be a good choice for studying the activity
of promoters that seem to be regulated by nitric oxide. At the very
least, adequate controls, and appreciation of specifics of reporter
elements such as inclusion of activity enhancing sequences, are
necessary for any conclusion regarding significant effects of nitric oxide.