(Received for publication, August 31, 1995; and in revised form, October 5, 1995)
From the
The production of inducible nitric oxide synthase (iNOS) within
vascular smooth muscle (VSM) cells following exposure to
proinflammatory cytokines is a major cause of the vasorelaxation and
hypotension of septic shock. We have defined the cytokine-responsive
element of the murine iNOS promoter, transfected into a VSM cell line,
and the role of the NF-B/Rel family of proteins in iNOS gene
activation in these cells. The combination of interleukin-1,
interferon-
, and tumor necrosis factor-
stimulates promoter
activity by a factor of 8.1-fold; single cytokines show little
activity, while pairs of cytokines produce an intermediate effect.
Using a series of promoter deletion mutants, we have defined the
cytokine-responsive element from position -890 to -1002;
this region contains an NF-
B-binding site as well as a number of
interferon response elements. Nuclear proteins from cytokine-stimulated
VSM cells which bind to an oligonucleotide containing this
B site
are composed of p65 together with an unidentified protein of 50 kDa,
which is not a known Rel family member. A promoter mutant with a 2-base
pair change within this
B site, which abolishes NF-
B binding,
has an activity of only approximately 34% (S.E. ± 1.5) of the
wild-type promoter. In addition, protein binding to this site is
abolished by a specific inhibitor of NF-
B activation, which also
abrogates iNOS activity. Residual inducibility in such mutant promoters
is attributable to the presence of an independently functioning
downstream
B site (-85 to -75). The mechanism by which
NF-
B activates the iNOS promoter in VSM cells in response to
cytokines appears to be markedly different to that operative in
macrophages in response to lipopolysaccharide.
Nitric oxide (NO) ()is a potent
vasorelaxant(1) , but also plays a role in physiological
processes as diverse as neurotransmission (2) and host
defense(3) . There are three types of enzymes which synthesize
NO, the NO synthases (4) . Of these, two are constitutive, but
the third is normally produced only following transcriptional
activation of its gene(5, 6) . This inducible NO
synthase (iNOS) is produced in a variety of cell types following
stimulation with a number of different factors, the most important of
which are proinflammatory cytokines and lipopolysaccharide (4, 7) . Normal VSM produces no NO in the resting
state. However, following stimulation with cytokines, such as
interleukin-1 (IL-1), interferon-
(IFN-
), and tumor necrosis
factor-
(TNF-
), iNOS is
synthesized(8, 9, 10) , leading to NO
production, vasorelaxation, and the hypotension so characteristic of
septic shock(11, 12) .
The promoter of the murine
gene for iNOS contains numerous potential sites for the binding of a
number of different transcription factors (13) . This
1.7-kilobase pair DNA element confers inducibility by IFN- and
lipopolysaccharide (LPS) in macrophages. A key region of the promoter
in mediating the response to LPS is a downstream
B site, which
extends from position -85 to -76(14) . The synergic
effect of IFN-
, however, requires the presence of the 5` region of
the promoter which has not been functionally mapped in any
detail(13, 15) . All studies on the promoter of iNOS
have so far been performed in macrophages and have described the effect
of only LPS and IFN-
.
A number of different cell types can
produce iNOS after stimulation by LPS and IFN-, as well as
proinflammatory cytokines such as IL-1, TNF-
, and
IL-2(10) . Cytokines are rarely produced in isolation, and
their ability to interact with one another increases the range of
biological effects which they mediate. Given the complexity of the iNOS
promoter and the necessity to integrate the stimulatory effects of a
number of proinflammatory cytokines, different mechanisms might apply
in cell types other than macrophages and following different stimuli.
Of particular interest is the control of iNOS gene activity within
VSM cells where the production of NO plays an important part in the
pathophysiology of septic shock. We chose to analyze the effects of
three proinflammatory cytokines, TNF-, IFN-
, and IL-1 on iNOS
promoter function, since these cytokines are all produced during septic
shock from a variety of different causes(16) , and are known to
synergize in the production of iNOS from VSM cells(10) . In
contrast to the promoter function in macrophages, we find that an
upstream
B site is of key importance in mediating the synergic
effect of cytokines on iNOS gene activity within VSM cells.
Figure 2: A, NOS activity in A7r5 cells following various cytokine treatments. Values are the means of three determinations; error bars are ± 1 S.E. of the mean. The nitrite levels for untreated cells have been subtracted in all cases and did not differ significantly from medium alone. B, iNOS promoter reporter gene activity within vascular smooth muscle cells following various cytokine treatments. Cytokines were used at the concentrations as in Panel A. Values are expressed as the -fold increase in CAT activity seen following cytokine treatment relative to the CAT activity seen with no added cytokines. Results are the means of three to five estimations; error bars are ± 1 S.E. of the mean.
A schematic diagram of the murine iNOS promoter region is
shown in Fig. 1with the putative transcription factor-binding
sequences highlighted. This complete sequence was cloned upstream of
the bacterial CAT gene in the reporter plasmid pCAT-basic, to produce
an iNOS reporter vector, pNOS-CAT. This vector was transfected into
vascular smooth muscle cells. CAT activity from such transfected cells
after a variety of different cytokine stimuli is shown in Fig. 2B. Cytokine stimulation of CAT activity from this
construct was analogous to nitrite output under the same conditions.
When added singly, only IL-1 showed a significant stimulation of CAT
activity of 3-fold over basal levels (S.E. ± 0.47). However, the
combination of two or three cytokines together increased the levels of
CAT activity in the transfectants, to a maximum of 8.1 (S.E. ±
0.86)-fold over basal levels with the combination of all three
cytokines. Pairs of cytokines showed levels of stimulation broadly
intermediate between single cytokines and the combination of all three;
the addition of TNF- to IL-1, however, made no significant
difference to the result obtained with IL-1 alone. The interaction of
the cytokines in these experiments was borderline between an additive
and a synergistic effect.
Figure 1:
Schematic
representation of the 5` upstream elements of the murine iNOS gene.
Putative transcription factor binding elements are illustrated:
activator protein-1 (AP-1), TNF response element (TNF-RE), ISRE, GAS, IFN- response element (IFN-
RE), and basal transcription complex recognition
site (TATA).
Figure 3: A CAT activity following transfection of vascular smooth muscle cells with plasmids 1.1 (Panel A) and 2.1 (Panel B). Values are expressed as the -fold increase in CAT activity seen following cytokine treatment relative to the CAT activity seen with no added cytokines. Cytokine concentrations are as in Fig. 2. Values are the means of three determinations; error bars are ± 1 S.E. of the mean.
Figure 4: CAT activity of a series of deletion mutants of the iNOS promoter. Mutant plasmid designations are shown to the left with the boundaries of the mutation shown measured relative to the mRNA start site. A schematic diagram of this region of the promoter is shown above with the various putative transcription factor-binding sites, and their orientation (5` to 3`) is indicated by the arrow. CAT activity is measured as the -fold increase in activity seen following addition of all three cytokines in the concentrations shown in Fig. 2; it is expressed as a percentage of the value achieved with the full-length iNOS promoter reporter gene construct (pNOS-CAT) in the same experiment. Values shown are for one experiment; similar results were obtained on two further repeats.
Mutants extending into the 3` end of this region of the promoter
were also analyzed. Virtually full induction following cytokine
treatment was seen in mutant 7, which contains a deletion extending
from position -480 to -890, to the 3` side of the
interferon regulatory sites. However, when the area deleted extended to
position -979 (mutant 43), the level of induction following
cytokine treatment dropped to one-third of the level seen with the
full-length promoter (Fig. 4). This removes the GAS/ISRE/IRF-1
sites and the adjacent B site. Thus, these combined sets of
mutants show that a 112-base pair region of the promoter extending from
position -890 to -1002 is required for full iNOS
transcriptional activity following treatment of vascular smooth muscle
cells with IL-1, TNF-
, and IFN-
.
Figure 6:
EMSA using nuclear extract (2 µg) from
A7r5 cells and an oligonucleotide containing the upstream B site
(-957 to -979). A, nuclear extract from untreated
cells (lane a) or cytokine-treated cells (IL-1, TNF-
,
IFN-
; lanes b-d) was mixed with no added competitor
DNA (lanes a and b); or with a 5-fold molar excess of
unlabeled nonspecific competitor DNA (an oligonucleotide containing the
upstream GAS/ISRE sites; lane c); or a 5-fold molar excess of
unlabeled specific DNA (the same oligonucleotide as used for labeling; lane d). The specific shifted band is shown with an arrow; a nonspecific band is indicated by an arrowhead. B, the same radiolabeled
NF-
B-containing oligonucleotide was mixed with a nuclear extract
of A7r5 cells which had received no treatment (lane a), the
cytokine mixture, as described in Panel A (lane c),
or this cytokine mixture with 60 µM PDTC, following a
10-min preincubation with the same concentration of the drug (lane
b).
Figure 5:
The -fold stimulation of CAT activity in
VSM cells of reporter gene constructs with either wild-type or mutant
B sequences. The -fold stimulation in CAT activity following IL-1,
TNF-
, and IFN-
stimulation is shown relative to that seen
with no cytokine additions, expressed as a percentage of the value
achieved with the wild-type sequence (pNOS-
B). Bars represent the mean of two separate experiments; the error bars are 1 S.E. of the mean.
Figure 7:
EMSA using 2 µg (lanes a, c, e, g, and i) or 4 µg (lanes b, d, f, h, and j)
of nuclear extract from A7r5 cells and the same oligonucleotide as used
in Fig. 6. Cells were treated for 2 h with the following
reagents: IL-1 (lanes a and b); IFN- (lanes
c and d); TNF-
(lanes e and f);
LPS (2 µg ml
; lanes g and h);
or a mixture of IL-1, IFN-
, and TNF-
(lanes i and j). The specific shifted band is shown with an arrow;
a nonspecific band is indicated by an arrowhead.
Figure 8:
EMSA performed as in Fig. 7with
nuclear extracts (2 µg) from cytokine-treated A7r5 cells (lanes
a and b) or peritoneal macrophages treated with 2 µg
ml LPS for 2 h (lanes c-e). The
following additions were made to the incubations; none (lanes c and f), anti-p65 (lanes a and d),
anti-p50 (lanes b and e). The specific shifted band
in the A7r5 cells is shown by the long arrow. The short
arrows indicate the supershifted bands
The molecular composition of the
specific protein-DNA complex produced in VSM cells on the upstream
B site was further analyzed by cross-linking the proteins in this
complex to the radiolabeled DNA probe and analyzing the components on
an SDS-polyacrylamide gel (Fig. 9). As expected, the main
protein component of the complex had a molecular mass in reasonable
agreement with the molecular mass of p65 complexed to a short DNA
fragment (upper band). However, we also reproducibly found an
additional protein component of molecular mass of about 50 kDa as part
of this complex (lower band). The identity of this protein is
not known.
Figure 9:
Autoradiograph of a SDS-polyacrylamide
gel analysis of proteins cross-linked to the radiolabeled NF-B
probe. The specific band produced from vascular smooth muscle cell
nuclear cell extracts as seen in Fig. 8, long arrow,
was cut from the gel, and the proteins contained within it were
cross-linked to the radiolabeled probe by ultraviolet irradiation.
These proteins were analyzed by SDS-polyacrylamide gel electrophoresis
and autoradiographed. Molecular mass markers in kilodaltons are shown
to the left.
A particular feature of iNOS activity within VSM cells is
that full activity requires the presence of combinations of cytokines (Fig. 2B). This additive/synergic effect of IL-1,
TNF-, and IFN-
is also seen on the transcriptional rate
following cytokine treatment (Fig. 2A). Such
differences that do exist between changes in levels of NO output and
promoter activity may be attributable to
post-transcriptional/translational modifications. We were careful to
choose concentrations of cytokines which were saturating for their
stimulatory effect on iNOS production and that fall within the range
found in disease states. This suggests that each cytokine produces a
unique effect in enhancing iNOS gene transcription which cannot be
substituted by increasing the concentration of another cytokine. Since
NO is highly potent its production needs to be tightly regulated, such
that it is only produced under the appropriate conditions. Relying on
more than one signal for gene activation reduces the likelihood of
inappropriate transcription of the gene.
The 1.7-kilobase pair 5`
upstream sequence of the iNOS gene has only been shown to function as a
promoter within macrophages(13, 15) . We have shown
here that the same sequence functions as a cytokine-inducible promoter
within VSM cells. We have defined an enhancer type element responsible
for the maximal effect of these three cytokines as a 112-base pair
region extending from position -890 to -1002, which
contains consensus sequences for a B site, a GAS/ISRE element and
binding sites for IRF1.
We have provided evidence that the iNOS
promoter is regulated differently in VSM cells stimulated with IL-1,
TNF-, and IFN-
compared to macrophages stimulated with LPS
and IFN-
(summarized in Table 1). The combination of IL-1,
TNF-
, and IFN-
increased the transcriptional rate of the
promoter within VSM cells by a factor of 8.1. This compares to an
enhancement of 44-fold reported for the same promoter following LPS and
IFN-
treatment within the macrophage cell line RAW
264(13) . These results correlate with the output of NO from
each of these cells: RAW 264 cells produce approximately 7.5 nmol of
nitrite h
per 10
cells, while A7r5 cells
produce about 2 nmol of nitrite h
per 10
cells. (
)The effects of LPS on the transcription of iNOS
within macrophages are mediated through the binding of NF-
B/Rel
proteins to the downstream
B site(14) . In contrast to
those results most of the stimulatory effects of the three cytokines
IL-1, TNF-
, and IFN-
are mediated through an upstream
112-base pair element. The 112-base pair element described in this
study contains an NF-
B binding sequence, which when deleted or
mutated by two nucleotides, reduces the promoter's activity to
34% of the wild type ( Fig. 4and Fig. 5). This residual
activity is virtually completely removed when the second downstream
B site is mutated as well (Fig. 5). However, mutation of
the downstream
B site alone (mutant
pNOS-
B
) only reduces activity to 65% of the
wild type. Thus although both
B sites contribute to full iNOS gene
activity in VSM cells, the upstream site is more important. Moreover,
since promoter constructs mutated in the downstream site alone retain
considerable promoter activity (Fig. 5), this indicates that
there is no absolute requirement for this site for effective promoter
activity within VSM cells. Inhibition of NF-
B activation with PDTC
prevents NOS production within vascular smooth cells and inhibits the
binding of a nuclear factor to sequences containing the upstream
B
site (Fig. 6).
NF-B activation is crucial to iNOS
transcriptional activation in both macrophages, after IFN-
and LPS
stimulation, and in vascular smooth muscle cells after TNF-
, IL-1,
and IFN-
treatment. However, a different
B site is involved
in each case. The
B site binding protein in VSM cells consists of
p65 together with a protein of molecular mass of 50 kDa, which is not
recognized by antibodies to p50, p52, relB, p65 or c-rel ( Fig. 8and data not shown). Western blots have indicated
that p105 (the precursor of p50) is not processed in A7r5 cells
following TNF-
and IL-1 stimulation (data not shown). The antibody
to p52 is known to recognize rat p52 in Western blots and supershift
experiments,
but in fact, immunoprecipitation and Western
analysis have indicated that A7r5 cells are devoid of p52 (data not
shown). It is an intriguing possibility, therefore, that this
unidentified 50-kDa protein may represent another rel family
member, but clearly purification and cloning of the cDNA of this
protein will be required to establish its nature. It is unlikely that
this protein is a breakdown product of p65; while the presence of this
protein is consistently demonstrable by UV cross-linking, no
heterogeneity in the electrophoretic mobility of the protein-DNA
complex has ever been observed.
In the cells used in this study, LPS
gives little additional increase over the induction of iNOS activity
seen with the combination of three cytokines, and has little effect on
its own. ()In addition, LPS did not produce NF
B binding
activity in these cells (Fig. 7). In some primary VSM cell
preparations, LPS is active(8) , while in others it is
not(10) . This may reflect the presence or absence of the LPS
receptor CD14 within the cell population(21) .
The region we
have delineated as necessary for the cytokine induction of iNOS
transcription by TNF-, IL-1, and IFN-
contains two consensus
sequences for IRF-1. This is a transcription factor whose level is
increased following stimulation of cells with
IFNs(22, 23) . Mice with a targeted disruption of the
IRF-1 gene fail to induce iNOS within macrophages, suggesting that
IRF-1 is essential for iNOS activation in these cells(24) .
Studies using the iNOS promoter transfected into the macrophage cell
line RAW 264 have shown that sequences within the 5` portion of the
promoter mediate the synergic effect of IFN-
with LPS on iNOS gene
transcription(15, 13) . This effect was lost when
sequences from position -1029 to -913 were
deleted(15) ; however, the 3` limit of this element was not
defined. Recent studies have shown the importance of IRF-1 binding to a
site at position -913 to -923 in the induction of iNOS
within macrophages by IFN-
(25) . It remains to be
determined if IRF-1 is involved in the induction of iNOS transcription
in vascular smooth muscle cells. However, the close proximity of the
IRF-1 binding sites and the
B site in this enhancer suggests that
interactions may occur between transcription factors binding to these
sites, as has been shown for a similar region in the HLA class I heavy
chain gene promoter(26) . Closely adjacent to the IRF-1 sites
is an ISRE/GAS site (Fig. 1). IFN-
has been shown to
promote phosphorylation of the transcription factor
Stat91(27, 28) , which then undergoes translocation to
the nucleus where it binds to the GAS site and activates
transcription(27, 29) . We are currently examining the
role that Stat91 and IRF-1 might play in the induction of iNOS by
IFN-
.
The production of NO within vascular smooth muscle produces profound vasodilatation. This may be beneficial in a local area of inflammation, by facilitating the delivery of inflammatory cells and immune effectors such as antibody and complement. However, the need for a tight control of this process is demonstrated by the widespread inappropriate vasodilatation that occurs in septic shock, leading to hypotension, multiorgan failure, and death(16) . The need for the simultaneous presence of several proinflammatory cytokines to produce maximal iNOS production ensures that the iNOS protein within smooth muscle is only produced as part of a vigorous inflammatory response, hopefully near the site of the inflammatory stimulus. The current study has begun to unravel the molecular mechanisms underlying this cooperative effect.