(Received for publication, August 20, 1996)
From the Wilkinson Laboratory of the Kansas Cancer
Institute, the § Department of Pathology and Laboratory
Medicine, and the ¶ Department of Microbiology, Molecular Genetics
and Immunology, University of Kansas Medical Center,
Kansas City, Kansas 66160-7184
Mouse macrophages can be stimulated by interferon
(IFN)- and bacterial lipopolysaccharide (LPS) to produce nitric
oxide (NO) as the result of expression of the inducible NO synthase
(iNOS; EC 1.14.13.39) gene. The iNOS gene
promoter contains a candidate
-interferon- activated site (GAS). In
transfection studies reported here, it was demonstrated that a
luciferase reporter-gene construct, containing four synthetic copies of
the iNOS GAS, was inducible when transfected macrophages
were stimulated with either IFN-
, LPS, or a combination of the two.
Consistent with this finding were other transfection analyses, which
showed that responsiveness of the intact iNOS promoter to
these same agents was significantly reduced when two conserved
nucleotide positions within the GAS were mutated. Oligonucleotide
probes, which mimicked the iNOS GAS, formed a complex with
proteins that appeared in the nuclei of IFN-
or IFN-
+ LPS-treated macrophages within 30 min of stimulation, as shown by
electrophoretic mobility shift assay. LPS alone also caused the the
appearance of a nuclear protein capable of binding the iNOS
GAS-containing oligonucleotide; however, in contrast to binding induced
by IFN-
, approximately 2 h of stimulation with LPS were
required. The protein bound to the iNOS GAS-containing oligonucleotide reacted specifically with an antibody raised against Stat1
, regardless of the stimulus used. These data collectively support the conclusion that binding of Stat1
to the iNOS
promoter's GAS is required for optimal induction of the
iNOS gene by IFN-
and LPS.
Nitric oxide (NO),1 a short-lived,
free radical gas, is a multifunctional effector in many physiological
processes. Its biological effects include vasorelaxation (1),
inhibition of platelet aggregation (2), neurotransmission (3), as well
as microbial and tumor cell killing (4). In mammalian cells, production of NO from the substrate, L-arginine, is catalyzed by NO
synthase (NOS) (5). Three related genes encode isoforms of NOS in
different tissues. Neuronal and endothelial NOS are both
constitutively expressed and each is dependent upon
Ca2+/calmodulin for activity (6, 7). Mouse macrophages, and many other cell types, express an inducible isoform of NOS (iNOS) that
accumulates after cell activation by stimuli such as interferon- (IFN-
) plus bacterial lipopolysaccharide (LPS). This latter isoform functions independently of intracellular Ca2+ (8), because
it has calmodulin bound as an integral subunit. In addition to the
beneficial functions noted above, NO production has been associated
with tissue damage (9) and septic shock (10). Given the wide spectrum
of positive and negative effects that NO has, study of iNOS
gene expression is of particular importance.
The mouse iNOS promoter has been extensively characterized
(11, 12) and is known to contain two transcriptional regulatory regions, an enhancer and a basal promoter, within which a number of
response elements have been localized. Those known to be active include
B sites located both in the enhancer (13, 14) and basal promoter
(13, 14, 15); two juxtaposed enhancer-linked IFN-stimulated response
elements (ISREs), the distal one of which is a strong activator (16,
17), while the proximal one is a weak activator of transcription (16)
and an octamer element in the basal promoter
(14).2 The candidate
-interferon-activated site (GAS), located in the enhancer
(nucleotide positions
942 to
934), has been reported in one study
to be nonfunctional in regulating iNOS gene expression (17).
An extensive evaluation of this finding has not been established, however.
GAS elements are known to bind homodimers of a phosphorylated form of
the 91-kDa transcription factor, Stat1 (STAT stands for
signal transducer and activator of
transcription; reviewed in Darnell et al. (19))
which is a latent cytoplasmic protein. Treatment of cells with IFN-
causes tyrosine phosphorylation of Stat1
by the IFN-
receptor-associated Janus kinases 1 and 2. Subsequently, phosphorylated
Stat1
forms homodimers and translocates into the nucleus where it
induces transcription of GAS-containing genes. Other STAT proteins,
e.g. Stat3-6, which are induced by growth factors and
cytokines other than IFN-
, bind to DNA elements that are closely
related to the GAS (20, 21).
The present study demonstrates that the candidate iNOS GAS
is, indeed, necessary for optimal IFN--, LPS-, and IFN-
+ LPS-mediated induction of the iNOS promoter. Furthermore,
Stat1
, unlike Stats 3-6, is able to bind the iNOS GAS in
IFN-
-, LPS-, or IFN-
+ LPS-stimulated RAW 264.7 mouse
macrophages. These data provide evidence for the direct involvement of
Stat1
in the transcriptional induction of the mouse iNOS
gene.
The macrophage-like cell line RAW
264.7 (American Type Culture Collection no. TIB-71, Rockville, MD) was
used in all studies. Cells of this line were cultured in spinner flasks
containing 25 mM Hepes-buffered RPMI 1640 medium
(Sigma, which contained 10% (v/v) Fetal Clone I
bovine serum product (HyClone Laboratories, Logan, UT), 2 mM glutamine (ICN Biomedicals, Inc., Irvine, CA), 100 µg/ml streptomycin (Sigma), and 100 units/ml
penicillin (APOTHECON, Princeton, NJ). The lipid A-rich fraction II of
LPS was phenol-extracted from Escherichia coli O111:B4 and
obtained from List Biological Laboratories, Inc. (Campbell, CA).
Recombinant mouse IFN- (1.27 × 106 antiviral
units/mg) was provided by Schering-Plough through the American Cancer
Society (Atlanta, GA). Endotoxin was undetectable in all culture media
and reagents, at a sensitivity of 50 pg/ml, as determined by the
Limulus amebocyte assay (Associates of Cape Cod, Woods Hole,
MA). Restriction enzymes and T4 polynucleotide kinase were obtained
from New England BioLabs (Beverly, MA). [
-32P]ATP
(specific activity, 4,500 mCi/mmol) was from ICN Biomedicals, Inc.
The plasmid pGLH/H2 (12), containing the full-length iNOS promoter cloned into the pGL2 Basic luciferase reporter gene vector (Promega, Madison, WI), was used for oligonucleotide-directed mutagenesis of the GAS and ISRE using the Unique Site Elimination (U.S.E.) mutagenesis protocol from Pharmacia Biotech Inc., according to the manufacturer's instructions. After mutagenesis, both the enhancer and basal promoter of each construct were sequenced to confirm that the appropriate mutations were isolated and that no ectopic mutations within other iNOS functional elements had been introduced.
Four copies of the iNOS gene's GAS and two copies of the interferon regulatory factor-1 (IRF-1) gene's GAS (both synthesized by Integrated DNA Technologies, Coralville, IA) were each cloned upstream of a thymidine kinase basal promoter linked to the luciferase reporter gene of pGL2-Basic. The identity of each construct was confirmed by DNA sequence analysis. All plasmid DNA was purified by two sequential CsCl/ethidium-bromide equilibrium centrifugations.
Transfection and Transient Expression of Luciferase Reporter Gene ConstructsEach construct (3 pmol) was transfected into RAW 264.7 cells by electroporation (12, 22). A human growth hormone expression vector, pXGH5, was cotransfected (0.6 pmol/transfection) as an internal
control of transfection efficiency. Cells from three electroporations
were pooled to eliminate differences between individual transfections
and the mixture then equally divided among 12 wells of a 24-well
cluster dish (Corning CoStar Corp., Cambridge, MA). They were next
allowed to adhere to the substratum for 2 h before the medium was
changed. After 72 h (which were needed to reduce background levels
of luminescence), the medium was again changed and triplicate cultures
were incubated for 8 h with medium alone or medium that contained
either 100 units/ml IFN-, 100 ng/ml LPS, or a combination of the two
stimuli. The cells were then assayed for luciferase activity and human
growth hormone production, as described previously (12). The results of
human growth hormone assays showed that there was no significant difference in the relative efficiency of transfection between constructs (data not shown), consistent with earlier reports (12, 13,
22). All data are reported as fold induction, which was calculated by
dividing the relative light units of each stimulated culture with that
of the corresponding unstimulated control culture and averaging three
independent experiments. The average is reported along with the
standard error of the mean.
Each pair of complementary single-stranded
oligonucleotides (Life Technologies, Inc., Custom Primers) was
end-labeled by T4 polynucleotide kinase and annealed, as described by
Muroi et al. (23). Unlabeled, annealed oligonucleotides were
used as cold competitors. The top strand sequence of each
oligonucleotide used was: iNOS GAS, 5-CTTTTCCCCTAACAC-3
;
mutated GAS (GASmt), 5
-CTTT
CCCCTA
CAC-3
(the
mutated nucleotides are underlined); and NF-IL6,
5
-TCACATTGTGCAATCTTAATAAGG-3
. The oligonucleotide that contained the
NF-IL6 binding site was used as a nonspecific competitor. Cell cultures
were incubated in medium alone or treated with either 100 units/ml
IFN-
, 100 ng/ml LPS, or 100 units/ml IFN-
plus 100 ng/ml LPS for
the times indicated in the legends to Figs. 2 and 3. Nuclear extracts
were prepared from these cultures as described by Sadowski and Gilman (24) and modified by adding sucrose to a final concentration of 7.5%
immediately after cell homogenization (25). Binding reactions were
performed in a total volume of 30 µl by preincubating 10-20 µg of
nuclear extract (see legends to Figs. 3 and 4 for specific quantities)
for 5 min with reaction buffer (20 mM Hepes, pH 7.9, 40 mM KCl, 1 mM MgCl2, 0.1 mM EGTA, 0.1 mM dithiothreitol) that contained
4% Ficoll, 4 µg/ml poly(dI-dC), 1 mg/ml bovine serum albumin, and
0.05% Nonidet P-40. 32P-Labeled oligonucleotide (0.05 pmol) was then added, and the incubation was continued for 25 min. For
competition or supershift assays either a 40-fold excess of unlabeled
oligonucleotide or 1 µg of antibody, respectively, was added to the
reaction mixture following the preincubation period. Incubation was
then continued for 10 min before the labeled oligonucleotide was added,
after which the incubation was extended for another 15 min. All
reactions were allowed to proceed at room temperature. A 5% native
polyacrylamide gel, containing 0.2 × TBE (18 mM Tris
base, 18 mM boric acid, 0.4 mM EDTA), was
pre-electrophoresed at 350 V at 4 °C until the current fell to about
7 mA. Twenty-five µl of each reaction mixture were then loaded, and
the gel was electrophoresed for 2 h and 15 min.
Given that the
iNOS GAS has been described as nonfunctional (17), a simple,
direct test of its activity was sought to assess the validity of this
earlier observation. The approach chosen was to test expression of a
linked reporter gene using synthetic promoters. Two luciferase
reporter-gene constructs were made. One of these contained four tandem
copies of the iNOS GAS and the second, two tandem copies of
the IRF-1 GAS as a positive control. Each was linked to a
synthetic thymidine kinase (TK) basal promoter. RAW 264.7 macrophages were transfected with each of these reporter constructs,
stimulated with either IFN-, LPS, or the combination of IFN-
+ LPS, and assayed for luciferase activity (Table I). The
four iNOS GAS copies mediated an approximate 3-fold increase in luciferase reporter gene activity when transfected cells were stimulated with IFN-
, a 1.6-fold increase when stimulated with LPS,
and more than a 3-fold increase when stimulated with IFN-
+ LPS.
These results strongly suggested that the iNOS GAS is
functional in response to each of these three stimuli. The inductive
capacity of the IRF-1 GAS was greater than that of the
iNOS GAS. This was evidenced by the fact that the
IRF-1 GAS construct, which had only two elements driving the
TK promoter, compared with four in the iNOS GAS
construct, yielded more than twice the fold induction when either
IFN-
alone or IFN-
+ LPS were used as stimuli. By contrast, there
was no significant difference (p = 0.27 by Student's t test) between the fold induction observed with the two
constructs using LPS alone as stimulus.
|
To
determine whether the iNOS GAS is functional in the
environment of the iNOS promoter, mutational analyses were
undertaken. Three constructs were made (one containing the wild-type
iNOS promoter, a second containing the iNOS
promoter bearing a two-nucleotide mutation in the GAS, and a third
containing the iNOS promoter bearing a two-nucleotide
mutation in the strong, distal ISRE) and transfected into RAW 264.7 macrophages. This was followed by stimulation of cell cultures with
IFN-, LPS, or IFN-
+ LPS. As shown in Fig. 1, RAW
264.7 macrophages transfected with a construct containing the GAS
mutation showed a significant 2-3-fold decrease in luciferase
production, depending upon the stimulus, when compared with cells
transfected with the wild-type construct. The reporter construct
containing the mutated strong, distal ISRE also showed decreased
responsiveness to these mediators. The extent of reduction was found to
be equivalent to that seen for the mutated GAS. This not only indicated
that the GAS is functional in regulating mouse iNOS gene
expression, but also that it is as important as the strong, distal
ISRE.
IFN-
In view of the results reported above, it was
predicted that the iNOS GAS should bind one or more nuclear
proteins after cellular stimulation with IFN-, LPS, or a combination
of the two. To test this prediction, electrophoretic mobility shift
assays were performed using nuclear extracts from RAW 264.7 macrophages. By using a 15-base pair probe that contained the
iNOS GAS, a specific binding complex (denoted by the
arrowhead in Fig. 2, A-C) was
detected in stimulated, but not unstimulated, cells (Fig. 2,
A-C, compare lanes 2 with lanes 1,
respectively). The specificity of this binding complex was demonstrated
by the fact that unlabeled iNOS GAS oligonucleotide could
compete effectively for binding (Fig. 2, A-C, lanes
3), while an unrelated NF-IL6 oligonucleotide could not (Fig. 2,
A-C, lanes 5). Additional confirmation of
specificity was obtained by using an oligonucleotide in this
competition study that contained a mutated iNOS GAS sequence
(the same two mutations introduced into the luciferase reporter-gene
construct used for mutational analysis, above); this oligonucleotide
also did not compete for binding (Fig. 2, A-C, lanes
4). In addition to the specific complex, we also detected
nonspecific binding complexes both in unstimulated and stimulated cells
(Fig. 2, A-C, indicated by the bracket).
In RAW 264.7 macrophages treated with either IFN- or IFN-
+ LPS,
the appearance of the specific binding complex was rapid, occurring
within 30 min of stimulation (Fig. 2, A and C).
However, in LPS-stimulated macrophages this binding complex was not
observed at 30 min (data not shown) but, rather, required approximately 2 h of stimulation before it became detectable (Fig.
2B). Furthermore, the relative amount of binding activity
induced by LPS was greatly reduced, compared with that stimulated by
IFN-
or IFN-
+ LPS, as indicated by the fact that more nuclear
extract, and longer exposure times were required to achieve the
autoradiographic result shown in Fig. 2B (refer to the
legend in Fig. 2).
To identify the nuclear protein(s) that specifically
bound to the iNOS GAS, we next performed antibody supershift
assays. Antibodies specific for each of five members of the STAT family of transcription factors (Stat1, Stat3-6), which are known to be
activated either by IFN or by other cytokines or hormones (26, 27, 28, 29, 30) and
known to bind sequences related to the GAS, were used in these
supershift assays. Antibody against Stat1 was the only one able to
supershift the specific binding complex, and it could do so in assays
of nuclear extracts from cells stimulated with either of the two
stimuli, or by the combination of both (Fig. 3,
A-C, indicated by the open
arrowhead). It was concluded from these results that the specific
binding complex identified by EMSA contains Stat1
.
The results presented are important because they show that the
enhancer-linked GAS is, in fact, active in the induction of transcription of the mouse macrophage iNOS gene in response
to LPS ± IFN-. In addition, it has been shown here that the
transcription factor Stat1
binds to this GAS and that such binding
occurs regardless of whether RAW 264.7 cells are stimulated with
IFN-
or LPS. Finally, the results shown here indicate that while
both IFN-
and LPS treatment of mouse macrophages cause the binding
of Stat1
to the iNOS gene, there is a temporal difference
between the two.
Mouse macrophages can be induced by the combination IFN- + LPS to
produce NO even at concentrations of these two mediators that, by
themselves, are ineffective. In other words, the two mediators function
synergistically (31). This would seem to suggest that IFN-
and LPS
induce separate, complementary signaling pathways that are both
required for optimal expression of the iNOS system. However, at
high concentrations of LPS alone (i.e. greater than 10 ng/ml), iNOS gene expression and NO production occur in a
dose-dependent manner. Upon first consideration, this would
seem inconsistent with the hypothesis that IFN-
and LPS generate
independent signals. It has recently been demonstrated, however, that
high levels of LPS also induce mouse macrophages to produce IFN-
,
and that autocrine/paracrine stimulation by this mediator is critical
to the LPS-mediated production of NO (32, 33).
The delayed induction of Stat1 binding by LPS alone, compared with
IFN-
alone or IFN-
+ LPS, is consistent with the fact that
autocrine/paracrine IFN-
plays a role in LPS-mediated activation of
mouse macrophages for NO production. The likely reason that lower GAS
binding activity is generated by IFN-
is that most of the
tyrosine-phosphorylated Stat1
produced by this stimulus is
sequestered into the transcription factor ISGF-3, although small
amounts of Stat1
homodimers are detectable in type-I IFN-stimulated cells (34). ISGF-3 binds to a subset of ISREs that differ in sequence
from the iNOS ISRE. IFN-
, by contrast, induces
predominantly GAS-binding homodimers of Stat1
(19, 20). Thus, we
propose that the lower level of iNOS promoter induction
caused by LPS (and autocrine/paracrine IFN-
) versus
IFN-
+ LPS is likely the result of lower availability of Stat1
homodimers generated by LPS which, in turn, are needed to bind the GAS
maximally. It should be noted, however, that other autocrine/paracrine
factors produced by LPS-stimulated mouse macrophages, such as IL-6 and
IL-10, also cause the tyrosine-phosphorylation of Stat 1
(reviewed
in Finbloom and Larner (20)). It is possible, therefore, that these
mediators could be the source of a portion of the GAS binding activity
seen in LPS stimulated RAW 264.7 macrophages.
In addition to a direct role for Stat1 in iNOS gene
activation, this transcription factor can also act indirectly. For
example, it is required for induction of the IRF-1 gene
(35), which encodes the IRF-1 transcription factor that binds the two
juxtaposed ISREs in the iNOS promoter's enhancer. In
IRF-1 knockout mice, IFN-
+ LPS induces virtually no iNOS
mRNA accumulation or NO production. This is in spite of the fact
that all other transcription factors necessary for iNOS gene
induction (i.e. NF-
B, Stat1
, and octamer binding
factors) are ostensibly available to interact with
cis-regulatory elements of the iNOS gene. In work
reported elsewhere (13), we have similarly shown that mutation of the
enhancer-linked
B element of the iNOS promoter abolishes
responsiveness of the enhancer not just to LPS, but to added IFN-
as
well. This is in spite of the fact that NF-
B itself is not activated
by IFN-
. The targeted disruption of the Stat1 gene also
abolishes macrophage NO production in response to IFN-
+ LPS (36).
However, it cannot be concluded from this result whether the effect is
attributable directly to the absence of Stat1
or, indirectly to a
relative lack of IRF-1.
Taken together, the above observations suggest that transcription
factors that interact with the iNOS enhancer must do so cooperatively. Stated another way, iNOS gene expression
appears to require the simultaneous presence of all transcription
factors that bind its enhancer; when all are present transcription is enhanced, and when any one of them is absent transcription either remains off or is greatly reduced. This is consistent with the observation that the expression of iNOS in mouse macrophages is population-based (37, 38). Increases in iNOS expression are achieved by
increasing the number of cells within the population that express the
iNOS gene and, therefore, iNOS protein, indicating that
expression of the iNOS system in individual cells is either fully on or
off. This suggests that in the absence of maximal stimulation, such as
by the combination of IFN- + LPS, at least one transcription factor
controlling iNOS gene expression may be limiting. By this
reasoning, full expression of iNOS protein would only be achieved by
those cells in the population that exceed threshold levels of the
limiting factor(s). Stat1
may be that factor because increasing
numbers of cells, in a given population, can be induced to express the
iNOS gene when increasing concentrations of either IFN-
(37) or -
(38) is added to LPS-stimulated cell cultures.
In conclusion, a minimum of six, cis-acting regulatory
elements are now known to control regulation of the mouse
iNOS gene, two ISREs (16, 17), two B elements (13, 14, 15),
an octamer element (14),2 and as shown here, a GAS. These
are bound by transcription factors either activated or induced by LPS
plus one of the interferons. The picture that emerges is one of complex
regulation of the mouse iNOS gene, the product of which can
be both beneficial (4, 18) and detrimental (9, 10) to the host.
Although we do not yet understand the manner in which the transcription
factors necessary for gene induction interact on the iNOS
promoter, there are several indications that they must do so
cooperatively. Identification of the various transcription factors
involved and discovery of how they interact with one another on and,
perhaps off, the iNOS promoter is essential to understanding
transcriptional control of the mouse iNOS gene and,
therefore, regulation of NO production.
We thank Drs. Charlotte Zhang and Fang Fan for their helpful advice and Mari Lynn Lovelace for administrative assistance.