From the Critical Care Medicine Department, Warren Grant Magnuson Clinical Center, National Institutes of Health, Bethesda, Maryland 20892
Received for publication, December 20, 2002 , and in revised form, May 6, 2003.
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Recently, we described a GC box element that responds to
NO independent of cGMP or NO-mediated
transcription factor damage
(18). Previous investigations
had shown that NO donors, but not cGMP, up-regulated tumor
necrosis factor (TNF
) in human peripheral blood mononuclear
cells (5) and neutrophil
preparations (6). Furthermore,
differentiated U937 cells, a human monoblastoid line, transfected with
inducible NO synthase produced increased amounts of
TNF
(19). Because U937
cells lack soluble guanylate cyclase, this cell line was subsequently used as
a model system to explore cGMP-independent gene regulation by
NO in phagocytes
(19,
20). Initial studies linked
NO up-regulation of TNF
mRNA and protein with
decreases in intracellular concentrations of cAMP
(20). Notably,
NO effects were mimicked by H89, an inhibitor of
cAMP-dependent protein kinase (PKA), and blocked by dibutryl cAMP
(Bt2cAMP), a PKA activator
(18,
20). PKA modulates gene
transcription by phosphorylating nuclear proteins such as cAMP-response
element (CRE) binding protein or Sp1
(21). The TNF
promoter
has corresponding binding sites for both of these transcription factors
(22). However, in reporter
gene experiments, NO and cAMP responses were lost only when
the Sp site (proximal GC box) was either deleted or mutated
(18). NO and
H89 were found to decrease, whereas Bt2cAMP increased Sp1 binding
to the TNF
GC box (18).
Thus Sp1 and its proximal GC box-binding site functioned as a genomic
NO sensor and repressed TNF
transcription.
Proximal Sp factor binding sites are found in numerous genes that, unlike
TNF, lack TATA and CCAAT transcriptional start sites. In TATA- and
CCAAT-less promoters, proximal Sp factor binding sites are necessary elements
in the recruitment, positioning, and stabilization of the transcriptional
complex (23,
24). For these genes, in
contrast to TNF
, Sp factors often act as transactivators and
NO-induced reductions in binding would be expected to
turn-off rather than turn-on transcription.
The current investigation sought to generalize the NO
responsive, GC box paradigm to other types of human promoters and to
characterize how these NO sensors might function
differentially in the context of larger composite regulatory elements. We
tested whether NO-Sp factor-GC box signaling and promoter
regulation might also apply to a Sp factor-dependent gene that lacks TATA and
CCAAT motifs. Furthermore, we investigated the mechanisms by which
NO-induced decreases in Sp factor binding might lead to
either gene activation or repression. For the first question,
NO regulation of endothelial NO synthase
(eNOS) was studied in a human-hybrid endothelial cell line. The eNOS promoter
lacks a TATA box, but has two overlapping Sp factor binding sites that are
important for basal transcriptional activity
(25,
26). Unlike TNF, the
proximal GC box of eNOS is not flanked by sequences that bind strong
transactivating factors (26).
For the second question, elements flanking the Sp binding site of TNF
,
AP1, and AP2, were evaluated for their role in determining the directionality
of NO-induced promoter responses.
![]() |
EXPERIMENTAL PROCEDURES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Western BlottingEAhy 926 cells, a human-hybrid endothelial line generously contributed by Dr. John B. Graham, North Carolina University (27), were lysed after incubation for 24 h with one of the following reagents or combinations: NAP (control, 500 µM), SNAP (500 µM), Bt2cAMP (100 µM), and H89 (15 µM). Western blotting for eNOS was performed as described previously (19, 28). Briefly, soluble proteins (20 µg) from these different conditions were separated on 420% Tris glycine SDS gels (Novex, San Diego, CA) and transferred to polyvinylidene difluoride membranes. After overnight blocking with 5% nonfat dry milk containing 0.05% Tween 20 at 4 °C, membranes were treated with anti-eNOS 1:1,000 and developed with anti-mouse IgG conjugated to horseradish peroxidase.
Plasmid ConstructionThe two-plasmid reporter gene system
used in this investigation has been described previously
(18,
29). This reporter gene system
is free of cryptic cAMP response elements and amplifies signals from low
activity mutant promoters
(29). Briefly, promoters of
interest were inserted into the first plasmid, promoterless ptTA, to induce
production of a transactivator (tTA) containing a tetracycline response
element binding domain. This fusion protein binds to the second plasmid,
pUHG10.3CAT (kindly provided by Dr. Rob Hooft van Huijsduijnen, Glaxo
Institute for Molecular Biology, Geneva, Switzerland), thereby transactivating
expression of a reporter gene, chloramphenicol acetyltransferase (CAT). The
following promoter constructs were studied: human eNOS using wild
type peNOS (from 1322 to +22 of the eNOS gene) and a Sp mutant
peNOS(mSp), both graciously provided by Dr. Kenneth Wu, University of Texas,
Houston Medical School (25);
human TNF using wild type pTNF (from 393 to +93 of the
TNF
gene), a gift from Dr. James S. Economou, UCLA School of Medicine
(18,
22), an AP1 mutant pTNF(mAP1),
a Sp mutant pTNF(mSp), an AP2 mutant pTNF(mAP2), and finally an AP1 and Sp
double mutant pTNF(mAP1)/(mSp); an artificial
AP1/(Sp)3 promoter
(18) using wild type
p(AP1)/(Sp)3, an AP1 mutant p(mAP1)/(Sp)3, and a Sp
triple mutant p(AP1)/(mSp)3; and two short artificial promoters
spanning the AP1/Sp consensus sequence of the TNF
promoter (71
to 45) in both the forward and reverse orientation, p(AP1)/(Sp) and
p(Sp)/(AP1). Mutants of pTNF were generated using site-directed mutagenesis
kits (Clontech, Palo Alto, CA). The wild type artificial promoter and its
mutants were created by inserting synthetic double-stranded oligonucleotides,
containing the corresponding sequences, into plasmid ptTA. The structures and
site mutations for these promoters are outlined in
Fig. 1. Mutated sequences were
searched against the TRANSFACTM data base and did not match known binding
sites for human transcription factors
(30).
|
Cell Transfection and CAT AssayWild type and mutant
constructs of the human eNOS promoter were transfected into the human-hybrid
endothelial cell line EAhy 926
(27) using DOTAP transfection
kits (Roche Diagnostics) according to the manufacturer's protocol. Wild type
and mutant TNF and artificial promoter constructs were transfected into
the human monoblastoid U937 cell line using previously described methods
(18). After transfection, U937
cells were incubated for 16 h with PMA (100 nM) to differentiate
them into a TNF
-producing phenotype. CAT activity for all reporter gene
experiments was measured after incubation for 24 h in the presence of various
reagents by enzyme-linked immunosorbent assay kit (Roche Diagnostics). In all
transfections,
-galactosidase expressed by a cotransfected internal
pSV
-Gal control (Promega, Madison, WI) was used to normalize CAT.
Promoter activity was calculated for each promoter construct as fold induction
of CAT expression compared with the CAT expression obtained using the
promoterless ptTA plasmid.
Electrophoretic Mobility Shift Assays (EMSA)U937 cells were
incubated with PMA (100 nM) for 16 h. Differentiated U937 or
EAhy 926 cells were cultured for 24 h with one of the
following reagents or combinations: NAP (control; 500 µM), SNAP
(500 µM), Bt2cAMP (100 µM), and H89 (15
µM). EMSA were performed with 15 µg of nuclear extract and
double-stranded DNA probes were labeled with 32P using previously
described methods (31). The
probes used included: Sp probe representing 108 to 83 section of
the eNOS promoter (5'-GGGATAGGGGCGGGGCGAGGGCCAGC-3'); AP1-Sp probe
representing the 71 to 44 section of the TNF promoter
(5'-GCTGGTTGAATGATTCTTTCCCCGCCCT-3'); and the Sp-AP2 probe
representing the 52 to 25 section of the TNF
promoter
(5'-CCCCGCCCTCCTCTCGCCCCAGGGACAT-3'). For supershift assays, 2
µg of the corresponding antibody was added to the binding reaction mixture
and incubated for 20 min on ice prior to addition of the labeled probe. For
competition assays, 100-fold molar excess of cold probe was added 10 min prior
to the corresponding hot probe.
Chromatin Immunoprecipitation (ChIP) AssayU937 cells were
incubated with PMA (100 nM) for 16 h. Differentiated U937 or
EAhy 926 cells were cultured for 24 h with one of the
following reagents: NAP (control; 500 µM), SNAP (500
µM), or Bt2cAMP (100 µM). The ChIP
assay was performed according to the manufacturer's instructions (Upstate Cell
Signaling Solutions, Charlottesville, VA). Briefly, cells were cross-linked
with 1% formaldehyde for 10 min at room temperature. Glycine was added to a
final concentration of 0.125 M to stop cross-linking. After washing
with ice-cold phosphate-buffered saline, cells were lysed for 10 min (1
x 106 cells/200 µl of SDS lysis buffer). The chromatin was
sheared by sonication 4 times for 40 s at one-third of the maximum power with
1 min cooling on ice between each pulse. Cross-link released total chromatin
was quantitated to determine the starting amount of DNA present in different
samples (input chromatin). The remaining chromatin fractions were pre-cleared
with salmon sperm DNA/protein A-agarose for 1 h followed by
immunoprecipitation with either anti-Sp1 or anti-Sp3 (Upstate Biotechnology
Inc.) overnight at 4 °C. Immune complexes were collected with salmon sperm
DNA/protein A-agarose for 1 h, washed 5 times, and finally eluted in 1% SDS,
0.1 M NaHCO3. Sp1- or Sp3-DNA cross-linking was reversed
at 65 °C overnight and digested with 100 µg of proteinase K at 45
°C for 1 h. DNA was then recovered in diethylpyrocarbonate-H2O
for PCR using phenol/chloroform extraction and ethanol precipitation. PCRs
were performed using the following promoter-specific primers: TNF-
(127 to +29), 5'-CCACTACCGCTTCCTCCAGAT-3' and
5'-CTGTCCTTGCTGAGGGAGCGT-3'; eNOS (170 to 7),
5'-CGGGCGTGGAGCTGAGGCTT-3' and
5'-CCAGCAGAGCCCTGGCCTT-3'. The PCR products were analyzed by
electrophoresis on 2% agarose gels, stained with SYBR Green I (Molecular
Probes), and quantified with Kodak Image Station 440 (Eastman Kodak Co.).
Statistical AnalysisData are presented as mean ± S.E. of at least three independent experiments. All p values are two-sided unless noted otherwise, and considered significant if less than 0.05. Densitometry results from eNOS Western blots were analyzed by two-way analysis of variance (ANOVA), followed by Dunnett's post-hoc test to find which conditions significantly differed from the common control. Reporter gene experiments within each promoter were analyzed using two-way ANOVA (the two factors being group and experiment). If the overall ANOVA was significant, Fisher's least significant difference method was used to determine which groups differed. Stringently accounting for multiple comparisons was not considered critical because it was expected a priori (18) that the SNAP, H89, and SNAP/H89-treated groups would behave similarly as would the control, Bt2cAMP, and SNAP/Bt2cAMP-treated groups. Comparisons of control promoter activity across multiple wild type and mutant constructs were performed using two-way ANOVA, followed by the stringent post-hoc method of Games-Howell. ChIP assay results were analyzed using two-way ANOVA, followed by Bonferroni-adjusted pairwise comparisons. For pattern-response comparisons performed on the reporter gene element order and ChIP experiments, data was first rescaled to a common mean and then subjected to a 3-way ANOVA, computing the interaction term.
To evaluate reciprocal binding effects at adjacent AP1 and Sp or Sp and AP2 binding sites, Bonferroni-adjusted p values were computed for each condition separately by testing whether the change from control for Sp was in the opposite direction to the change from control for AP1 (or AP2). Evidence for a reciprocal effect was then examined over all conditions by summing these 5 p values and comparing this result to that expected under the null hypothesis of no reciprocal effect.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
NO Responsiveness of the Human eNOS Promoter;
Dependence on the Proximal GC BoxNext we used a reporter gene
system to determine whether NO and H89 decrease eNOS
expression by suppressing transcription and, if so, whether these effects are
transmitted through the proximal GC box element. To this end, we investigated
the responses of both wild type and mutant constructs
(Fig. 1). As shown in
Fig. 3, SNAP, H89, or both
decreased, whereas Bt2cAMP increased eNOS promoter activity
(p < 0.0002 for all pairwise comparisons to control). Furthermore,
Bt2cAMP blocked the down-regulatory effect of NO
(p = 0.3 compared with Bt2cAMP alone). Mutation of the GC
box, previously shown to bind Sp1
(25,
26), reduced overall promoter
activity, and similar to TNF
(18), significantly reduced
its ability to respond to either NO or cAMP pathway signals
(p = 0.0012 compared with wild type promoter). These observations
further confirm that the proximal GC box is an important transactivator of the
human eNOS promoter. In addition, the results demonstrate that
NO can negatively regulate eNOS transcription through this
pivotal element.
|
Effect of NO on Sp Proteins; Binding to the
Proximal GC Box of the eNOS PromoterOur previous studies
demonstrated that NO decreased intracellular cAMP levels
leading to reduced Sp1 binding to the human TNF promoter
(18,
20). Notably, this effect of
NO on Sp1 binding was simulated by PKA inhibition. To
investigate whether the effect of NO on eNOS promoter
activity occurred through a similar mechanism, EMSA were performed using
32P-labeled probe containing the Sp consensus sequence of the
proximal eNOS promoter (Fig.
4). Nuclear extracts from endothelial EAhy 926
cells formed two main complexes (a major Sp complex and a minor band) with the
probe (Fig. 4B). The
presence of Sp1 in the major complex was demonstrated by anti-Sp1 polyclonal
antibody, which partially supershifted the band
(Fig. 4B).
Furthermore, unlabeled Sp oligonucleotide was shown to compete off protein
from the major complex. Incubation with SNAP, H89, or SNAP plus H89
(Fig. 4A) similarly
decreased the formation of the Sp major complex compared with nuclear extract
from control cells (p < 0.01 for all). Conversely,
Bt2cAMP increased Sp factor binding
(Fig. 4A; p
< 0.01) and interfered with the ability of SNAP to reduce Sp complex
formation (Fig. 4A;
p = 0.12 compared with Bt2cAMP alone by paired t
test).
|
To obtain direct in vivo evidence of a NO effect
on Sp protein binding to the GC box in the proximal eNOS promoter, a ChIP
assay was performed using EAhy 926 endothelial cells. PCR
primers for the ChIP assay were designed to amplify a 163-bp sequence
encompassing the proximal GC box in the eNOS promoter. As seen in
Fig. 4, C and
D, the GC box-containing region of eNOS binds Sp1 and to
a lesser degree Sp3 protein in vivo. Importantly, effects of
NO and Bt2cAMP on protein binding were
significantly different from each other for both Sp1 (p = 0.026) and
Sp3 (p = 0.03). Furthermore, Sp1 and Sp3 had highly similar patterns
of response across the three conditions (p = 0.89 for a difference).
These results by in vitro EMSA and in vivo ChIP assay
demonstrate, as previously shown for TNF
(18), that NO
decreases Sp1 binding to the proximal GC box element of the human eNOS
promoter.
NO Responsiveness of the TNF
Promoter; Role of AP1 and AP2 Sites Flanking the GC BoxNext,
we re-examined the TNF
promoter to determine whether GC box flanking
sequences somehow cause Sp1 or other Sp proteins to behave functionally as
transcriptional repressors. As shown in
Fig. 5, the NO
donor, SNAP, and the PKA inhibitor, H89, both increased while the PKA
activator, Bt2cAMP, decreased the wild type TNF
promoter
activity (p < 0.0001 for all). Note that all responses are
opposite to those seen for eNOS. Consistent with its function as a repressor
element, mutation of the Sp binding site enhanced TNF
promoter activity
(p < 0.01 comparing pTNF(mSp) to control pTNF). Furthermore, the
up-regulatory effect of NO on the TNF
promoter was
blocked by either the addition of Bt2cAMP (p > 0.20
compared with Bt2cAMP alone) or Sp mutation (p = ns for
overall test of group differences by ANOVA). In contrast, AP1 mutation, as
seen in Fig. 5, left, the
TNF
promoter NO-responsive, but completely reversed
the direction of responses to both NO and cAMP pathway
signals (eNOS-like pattern) compared with the wild type promoter; SNAP and H89
alone and in combination decreased, but Bt2cAMP increased the
activity of pTNF(mAP1) (p < 0.0001 for each compared with
control). Although the AP2 mutation (pTNF(mAP2)) decreased overall promoter
activity, the pattern of responses to NO, H89, and
Bt2cAMP were unchanged compared with that of the wild type promoter
(Fig. 5). Double mutations of
AP1 and Sp (pTNF(mAP1/mSp), like the Sp mutation alone, rendered the
TNF
promoter unresponsive to either NO or
Bt2cAMP (Fig. 5).
These data support the conclusion that the GC box (Sp factor binding site) in
the proximal TNF
promoter functions as a NO sensor,
whereas the upstream AP1 site controls response polarity.
|
Functional Analysis of Sp and AP1 Binding Sites in Artificial
PromotersNext, we used an artificial promoter
p(AP1)/(Sp)3-tTA (Fig.
1) to further explore the behavior of NO- and
cAMP-responsive GC box motifs. Three potential Sp protein binding sites were
used in the artificial construct to create a promoter with sufficient length
and activity (18). As shown in
Fig. 6A, SNAP, H89, or
SNAP plus H89 increased (p < 0.0001 for each), whereas
Bt2cAMP or SNAP plus Bt2cAMP decreased (p <
0.001 for both) the activity of the wild type artificial promoter construct
(TNF promoter-like responses). Likewise, mutation of the AP1 site
reversed the direction of all observed responses; SNAP, H89, or SNAP plus H89
decreased (p < 0.0001 for all), while Bt2cAMP or SNAP
plus Bt2cAMP increased (p < 0.0001 for both) the
activity of the AP1 mutant promoter (Fig.
6A). Thus, the AP1 mutant demonstrated eNOS-like
responses to NO and cAMP pathway signals. Again, simultaneous
mutation of all Sp sites abolished the effects of SNAP, H89, and
Bt2cAMP (Fig.
6A).
|
Unlike the TNF promoter, disruption of the AP1 site in the
artificial construct did not decrease, but rather slightly increased base-line
artificial promoter activity. This suggests that the AP1 mutation may have
increased cooperation among the tandem Sp sites. Consistent with this concept,
simultaneous mutation of all Sp sites did not increase base-line
transcription, indicating that the transcriptional activity of this construct
reflects the net effect of Sp repression and transactivation across the three
sites. Nonetheless, the responses of this artificial construct to
NO and cAMP analogs were similar to those of the TNF
promoter and the same functional relationship between AP1 and the GC box
elements was demonstrated.
To investigate whether the AP1 site and GC box order affects
NO responsiveness, two additional artificial promoter
constructs were studied. The first, p(AP1)/(Sp)-tTA, contains the 26-bp
sequence from the TNF promoter (71 to 45) that spans the
AP1 site and GC box. For p(Sp)/(AP1)-tTA, an identical sequence was used but
inverted, placing the GC box upstream to the AP1 site
(Fig. 1). As shown in
Fig. 6B, SNAP
increased (p < 0.001), while Bt2cAMP or SNAP plus
Bt2cAMP decreased the activity of both promoters (p <
0.001), a pattern of responses identical to those of the TNF
promoter.
Interestingly, element order was not important for module function. The
overall pattern of response to NO and cAMP analogs was
preserved independent of the element order (p = 0.71 for a
difference).
A Binding Interaction Analysis of AP1, Sp, and AP2 Sites in the
Proximal TNF PromoterTo further investigate how
AP1, Sp, and AP2 sites may interact to convert NO signals
(through decreased Sp protein binding) from off to on, we studied
transcription factor binding behavior in the presence and absence of
NO and cAMP pathway signals. EMSA were performed using
combined AP1-Sp or Sp-AP2 32P-labeled probes spanning corresponding
sequences in the TNF
promoter. Nuclear extracts for these experiments
were prepared from PMA-differentiated U937 cells after incubation under
various conditions (Fig.
7).
|
Two major DNA-protein complexes were detected with both probes. The AP1-Sp
probe formed Sp and AP1 binding complexes
(Fig. 7B) and the
Sp-AP2 probe formed Sp and AP2 binding complexes
(Fig. 7D). The
presence of Sp1, AP1, and AP2 in their corresponding complexes was confirmed
by super-shift assays with specific antibodies. Sequence specificity was
demonstrated using appropriate competition assays with cold probes
(Fig. 7, B and
D). Interestingly, blocking Sp protein binding to the
32P-labeled probe with excess cold Sp oligonucleotide increased AP1
binding in vitro (Fig.
7B). Conversely, blocking AP1 binding with excess cold
AP1 oligonucleotide increased Sp protein binding
(Fig. 7B). Thus, even
simple competition testing supported the notion that strong binding
interactions exist between these two sites. Likewise, incubation with SNAP,
H89, or SNAP with H89 similarly decreased Sp binding, but reciprocally
increased AP1 binding (Fig. 7, A
and B; one-sided p < 0.0001 for an overall
reciprocal Sp/AP1 effect). Although NO has been reported to
enhance DNA binding of AP1 through a cGMP-dependent signaling pathway
(8), this effect cannot be
invoked here because U937 cells have been shown to lack
NO-sensitive guanylate cyclase
(19,
20). However, in cells with an
intact NO-cGMP signaling pathway, combined
NO effects through cGMP-dependent increases in AP1 binding
and cGMP-independent decreases in Sp protein binding might be expected to
synergistically activate TNF-like promoters.
As expected for the combined Sp-AP2 probe, SNAP or H89, or both
consistently decreased whereas Bt2cAMP increased Sp protein binding
(Fig. 7, C and
D). However, none of these reagents significantly altered
AP2 binding, and therefore changes in Sp protein binding were not reciprocally
mirrored by AP2 (one-sided p = 0.5). Collectively, these results
suggest that AP1 and Sp factor sites in the proximal TNF promoter
antagonize each other by competing for binding space and demonstrate that
NO inhibition of Sp protein binding reciprocally enhances AP1
binding, thus increasing promoter activity.
Effect of NO on the in Vivo Binding of Sp1
and Sp3 to the Proximal TNF PromoterTo further
investigate NO effects on Sp protein binding to the proximal
GC box of the TNF
promoter in vivo, ChIP assays were performed
on PMA-differentiated U937 cells using PCR primers designed to amplify a
156-bp sequence encompassing the proximal GC box in the TNF
promoter.
Consistent with the in vitro EMSA results, NO and
Bt2cAMP had directionally opposite effects that differed
significantly from each other (Fig.
8) for both Sp1 (p = 0.006) and Sp3 (p =
0.0004). Again, the pattern of effects on Sp1 and Sp3 across the three
conditions were very similar (p = 0.85 for a difference), suggesting
that both Sp1 and Sp3 may be involved in GC box-mediated TNF
gene
regulation by NO.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Here, we describe a modular NO-responsive logic gate-like
mechanism containing a GC box-actuator variably coupled to an adjacent AP1
inverter element that determines response polarity. This putative regulatory
module was shown to decrease eNOS but increase TNF expression through
similar effects on Sp factor binding. For both promoters, GC box disruption
abolished NO and cAMP responsiveness. In contrast, mutating
the AP1 site in the TNF
promoter maintained promoter sensitivity to
NO and cAMP signaling, but completely reversed the direction
of their effects. These structure-function relationships were further
confirmed using artificial promoter constructs and supported by both in
vitro EMSA and in vivo ChIP experiments. Sp factor binding sites
were found to be necessary for NO sensitivity while the
presence or absence of an AP1 site determined whether NO
produced on (TNF
-like) or off (eNOS-like) responses, respectively.
NO- and cAMP-induced changes in promoter activity correlated
with strong reciprocal binding interactions between AP1 and Sp proteins. Thus,
nearby regulatory sequence and corresponding nuclear protein binding events
can switch the behavior of GC box motifs from that of activator to repressor
without fundamentally altering its binding relationship with Sp factor family
members. Together, these results suggest a general logic whereby secondary
inputs not only modulate output, but also can convert off signals to on.
The finding that NO and cAMP can regulate eNOS through its proximal, canonical Sp1 binding site has several important implications for vascular physiology. First, it suggests a negative feedback loop mechanism whereby eNOS can turn off its own transcription. Furthermore, unfettered NO production by inducible NOS might down-regulate eNOS through this pathway in conditions associated with vascular inflammation such as sepsis and atherosclerosis (37, 38). Finally, vasoactive mediators that affect intracellular cAMP such as angiotensin II and catecholamines (39) may thereby alter eNOS expression in the vasculature.
The relationship between cAMP-induced PKA activation and increases in Sp factor binding that were repeatedly demonstrated in this investigation are not invariably supported by the existing literature. Sp1 dephosphorylation in some reports has been associated with increased Sp1 binding to GC box motifs (40, 41). Importantly, eNOS, itself, has been shown to be induced by protein phosphatase 2A-mediated dephosphorylation of Sp1 (25). In contrast, C-terminal Sp1 phosphorylation has been associated with its activation during the cell cycle (42). This suggests that Sp1 phosphorylation or dephosphorylation might have different effects depending on the site specificity of the enzyme involved. Consistent with our observations, Rohlff et al. (18, 21, 43) and others have demonstrated that Sp1 is a cAMP- and PKA-responsive transcription factor, which can be activated by phosphorylation (21, 42, 44).
Notably, the regulation of Sp factor-dependent genes through PKA could
potentially explain some effects of cAMP signaling on cell growth and
inflammation. For example, previous reports have shown that cAMP signaling
suppresses TNF transcription
(29,
45,
46). The current investigation
identifies the proximal GC box element as a final target for this
anti-inflammatory effect. However, it is important to note that GC box effects
on TNF
promoter activity, as reported here, were generally modest
relative to control values. The TNF
promoter has numerous cis-elements
including NF-
B, Ets, CRE, NF-AT, and C/EBP
that contribute to and
control its transcriptional activation
(47,
48).
An important aspect of the current experiments is that the consequences of
NO or cAMP signaling-induced changes in Sp protein binding to
GC box elements was placed in a larger context. A nearby binding site and the
presence or absence of sequence-specific protein binding to this adjacent
element was shown to ultimately determine whether Sp factor-dependent events
would activate or repress transcription. Although the Sp protein family has
been reported to interact with many transcription factors including
NF-B (49), AP1
(50), AP2
(51), NF-Y
(43), and Egr-1
(52), our data provide details
of a flexible mechanism that can be configured to produce either up- or
down-regulation in response to a single environmental signal. Furthermore, we
identify two forms of this molecular circuit in naturally occurring, but
dissimilar promoters. The version of this regulatory module with Sp-dependent
repressor function may provide a useful model for understanding similar
observations in other genes with putative GC box repressor elements
(5357).
This paradigm may also have relevance for cAMP signaling events that have been
shown to exert opposite effects depending on the activation state of secondary
transcription factors (46).
Collectively, our results imply a promoter logic that can generate off, on, or
indifferent responses depending on the physical and functional presence of
secondary inputs.
![]() |
FOOTNOTES |
---|
To whom correspondence should be addressed: Critical Care Medicine Dept.,
Bldg. 10, Rm. 7D43, Warren Grant Magnuson Clinical Center, National Institutes
of Health, Bethesda, MD 20892, Tel.: 301-496-9320; Fax: 301-402-1213; E-mail:
rdanner{at}nih.gov.
1 The abbreviations used are: NO, nitric oxide; TNF,
tumor necrosis factor
; PKA, cAMP-dependent protein kinase;
Bt2cAMP, dibutyryl cAMP; CRE, cAMP-response element; eNOS,
endothelial nitric-oxide synthase; PMA, phorbol 12-myristate 13-acetate; SNAP,
S-nitroso-N-acetylpenicillamine; NAP,
N-acetyl-D-penicillamine; tTA, tetracycline-controlled
transactivator; CAT, chloramphenicol acetyltransferase; EMSA, electrophoretic
mobility shift assay; ChIP, chromatin immunoprecipitation; ANOVA, analysis of
variance.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|