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INTRODUCTION |
The inducible isoform of nitric-oxide synthase
(NOS2)1 catalyzes the
production of nitric oxide (NO), which participates in the physiologic
and pathophysiologic regulation of multiple organ systems (1-5). One
disease process that is particularly affected by the overproduction of
NO is sepsis (6, 7). In sepsis, a severe underlying infection triggers
a cascade of events that may lead to intractable hypotension, multiple
organ system failure, and death (8, 9). Within this cascade,
inflammatory cytokines and vasoactive mediators promote the relaxation
of vascular smooth muscle cells and a decrease in vascular tone. NO is
a vasodilatory gas generated late in the sepsis cascade contributing to
the hypotension. The overproduction of NO during sepsis is generated
through the NOS2 pathway.
NOS2 is a highly inducible gene regulated at the level of
gene transcription (1, 10). Transactivation of the NOS2 gene is mediated by members of the nuclear factor (NF)-
B family of transcription factors, which bind to a site in the downstream portion
of the NOS2 5'-flanking sequence (
85 to
76) (11). Recently, we
demonstrated that high mobility group (HMG)-I(Y) protein, an
architectural transcription factor that binds to an AT-rich octamer
site (
61 to
54) close to the NF-
B binding site in the promoter
of the NOS2 gene, acts in concert with p50 and p65 to
facilitate NOS2 gene transactivation (12). Architectural transcription factors typically function by modifying the conformation of DNA, and thus provide a framework for the transcriptional machinery to operate. HMG-I(Y) does not drive transcription itself, but it
facilitates the assembly of a functional nucleoprotein complex. Our
studies revealed that binding of both HMG-I(Y) and NF-
B subunits in
the downstream 5'-flanking sequence of the NOS2 gene is
essential for the most potent activation of the promoter (12).
Moreover, we also demonstrated that HMG-I(Y) itself was up-regulated
(both at the mRNA and protein levels) by inflammatory cytokines
(13). Taken together, these studies suggest that HMG-I(Y) contributes to the transcriptional regulation of NOS2 by inflammatory mediators.
Previously, we and others have demonstrated that transforming growth
factor (TGF)-
1, a pleiotropic growth factor involved in a number of
physiologic processes (14-16), inhibited NOS2 expression in
vitro (17-19) and in vivo (20). The down-regulation of
NOS2, but not NOS3, in a rodent model of endotoxemia suggested that NOS
inhibition by TGF-
1 is more selective for the inducible isoform of
NOS (NOS2) in an experimental model of sepsis (20). In vascular smooth
muscle cells, inhibition of NOS2 by TGF-
1 occurs at the level of
gene transcription (19). However, the mechanism by which transcription
of the NOS2 gene is inhibited by TGF-
1 has not been fully elucidated.
We have shown previously that HMG-I(Y) is involved in the induction of
NOS2 by inflammatory mediators (12, 13). However, the ability of
HMG-I(Y) to be regulated by inhibitors of NOS2 expression is not known.
Furthermore, little is known about the down-regulation of HMG-I(Y) and
its subsequent effect on NOS2 gene transcription. The
present study was designed to determine (a) whether
TGF-
1, an inhibitor of NOS2 expression, also down-regulates the
expression of HMG-I(Y), and (b) the functional importance of
this TGF-
1-mediated down-regulation of HMG-I(Y) on promoter activity
of the NOS2 gene.
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EXPERIMENTAL PROCEDURES |
Reagents--
Bacterial lipopolysaccharide (LPS)
Escherichia coli (serotype 026:B6), actinomycin D, and
thioglycollate were all from Sigma Chemical Co. (St. Louis, MO).
Recombinant human interleukin (IL)-1
(Collaborative Biomedical,
Bedford, MA), human TGF-
1 (R & D Systems, Minneapolis, MN), and
mouse interferon (IFN)
(R & D Systems) were stored at
80 °C
until use.
Cell Culture--
Rat aortic smooth muscle cells (RASMC) were
harvested from male Harlan Sprague-Dawley rats (200-250 g) by
enzymatic dissociation according to the method of Gunther et
al. (21). The cells were cultured in Dulbecco's modified Eagle's
medium (JRH Biosciences, Lenexa, KS) supplemented with 10% fetal
bovine serum (FBS, HyClone, Logan, UT), penicillin (100 units/ml),
streptomycin (100 µg/ml), and 25 mM HEPES (pH 7.4)
(Sigma) in a humidified incubator at 37 °C. RASMC were passaged
every 4-5 days, and experiments were performed on cells 4-6 passages
from the primary culture. RAW 264.7 cells (mouse macrophage cell line
from American Type Culture Collection) were grown in Dulbecco's
modified Eagle's medium with supplements as described for RASMC,
except HEPES was omitted. Peritoneal macrophages were obtained from
C57/BL6 male mice (10 weeks of age) and cultured as described
previously by Aggarwal and Mehta (22).
Northern Blot Analysis--
To assess the effect of cytokine
treatment on HMG-I(Y) and NOS2 mRNA levels, cells were grown to
80% confluence in 100-mm Petri dishes (Becton Dickinson, Franklin
Lakes, NJ) and fed with fresh medium containing either 10 ng/ml
IL-1
, 500 ng/ml LPS, 100 units/ml IFN
, with or without 1 to 10 ng/ml TGF-
1 (IL-1
treatment required a decrease in FBS to 2% to
prevent cytokine inactivation). Dose-response experiments were
performed to select the optimal concentrations of inflammatory
mediators to induce or suppress NOS2 mRNA levels in RASMC or
macrophages (data not shown). Cells were harvested after 24 h. The
studies performed to assess the in vivo effects of LPS and
TGF-
1 treatments, along with experiments to select the appropriate
doses, were described in detail elsewhere (20). Briefly, rats received
LPS alone (4 mg/kg, intraperitoneally) or LPS followed by TGF-
1 (20 µg/kg, intraperitoneally). The rats were sacrificed 10 h later
for organ harvest.
Total RNA was extracted from rat spleen and cultured peritoneal
macrophages by guanidinium isothiocyanate extraction and centrifugation through cesium chloride gradient (23). Total RNA extraction from
cultured RASMC and RAW 264.7 cells was performed with the Qiagen RNeasy
midi kit (Qiagen, Valencia, CA). RNA was fractionated on a 1.3%
formaldehyde-agarose gel and transferred to nitrocellulose filters. The
filters were hybridized at 68 °C for 2 h with
32P-labeled mouse HMG-I(Y) (13) and rat NOS2 (19) probes.
The hybridized filters were then washed in 30 mM sodium
chloride, 3 mM sodium citrate, and 0.1% SDS solution at
55 °C, and autoradiographed with Kodak XAR film at
80 °C for
8-12 h or stored on phosphor screens for 2-4 h. To correct for
differences in RNA loading, the filters were washed in a 50% formamide
solution at 80 °C and rehybridized with a 32P-labeled
oligonucleotide probe complementary to 28 S ribosomal RNA. Images were
displayed, and radioactivity was measured on a PhosphorImager running
the ImageQuaNT software (Molecular Dynamics, Sunnyvale, CA).
Western Blot Analysis--
RASMC and RAW 264.7 cells were plated
in 150-mm tissue culture dishes (Becton Dickinson). When the cells were
80-90% confluent, 10% FBS medium was replaced with 0.4% bovine
serum medium. After 48 h, either vehicle, IL-1
(10 ng/ml,
RASMC), or LPS (0.5 µg/ml, RAW 264.7 cells) was added to the cells,
in the presence or absence of TGF-
1 (10 ng/ml). Cells were then
collected in phosphate-buffered saline solution 48 h later.
HMG-I(Y) protein was obtained from RASMC by acidic extraction followed
by trichloroacetic acid precipitation (24). The resulting protein
pellet was resuspended in 30 µl of high pressure liquid
chromatography-grade water and stored at
20 °C until it was
assayed. SDS-polyacrylamide gel electrophoresis (25) was performed by
mixing 10 µl of the resuspended acidic extract with 10 µl of
Laemmli sample buffer (2×). After 5 min of boiling, the samples were
resolved on a 15% polyacrylamide gel and transferred to polyvinylidene
difluoride (Immobilon-P, Millipore, Bedford, MA) overnight (26). The
membrane was blocked with 10% skim milk in 10 mM Tris-HCl,
150 mM NaCl, pH 8.0, 0.05% Tween-20 (TBS-T) for 1 h
at room temperature. It was then incubated with an anti-HMG-I(Y)
antibody (N19, Santa Cruz Biotechnology, Santa Cruz, CA) at 0.1 µg/ml
in 5% skim milk in TBS-T for 2 h at room temperature. After a
wash in TBS-T, the membrane was incubated with anti-goat, horseradish
peroxidase-conjugated IgG (Santa Cruz) under the same conditions used
for the primary antibody. After a final wash in TBS-T, the blot was
developed with an enhanced chemiluminescence kit (Amersham Pharmacia
Biotech, Arlington Heights, IL). Because the extraction also led to the
isolation of histone H1, under transfer conditions insufficient for
removal of H1 from the gel, we used the histone H1 band to correct for
differences in protein loading. After scanning, the intensities of the
HMG-I(Y) band and the Coomassie Blue-stained histone H1 band were
measured with IMAGE software (National Institutes of Health), and the
ratio of the two values was used to represent the normalized intensity of the HMG-I(Y) band.
Plasmids--
To evaluate the effect of IL-1
and TGF-
1 on
HMG-I(Y) transcription in vascular smooth muscle cells, plasmid
180
was used (27). This construct, cloned into pCAT-Basic, contained the second transcription start site of HMG-I(Y) (27, 28). Plasmid pGL2-Control contained the firefly luciferase gene driven by
an SV40 promoter and enhancer.
To evaluate NOS2 promoter activity in either vascular smooth muscle
cells or RAW 264.7 cells, we inserted 1485 bp of the 5'-flanking region
of the mouse NOS2 gene and the first 31 bp after the
transcription start site into pGL2-Basic to make iNOS(
1485/+31), as
described previously (29). To assess the effect of HMG-I(Y)
overexpression on NOS2 after treatment with TGF-
1 in both RASMC and
RAW 264.7, we constructed the HMGIYpcDNA3 expression vector by
cloning the coding sequence of mouse HMG-I(Y) cDNA into the
EcoRI site of pcDNA3 (Invitrogen). DNA mass was
normalized by adding the control plasmids as needed. Luciferase
activity was normalized by cotransfecting pCMV-
gal plasmid
(CLONTECH).
Transfections--
RASMC were transfected by a diethylaminoethyl
(DEAE)-dextran method (29). In brief, 500,000 cells were plated onto
100-mm tissue culture dishes and allowed to grow for 48-72 h (until
80-90% confluent). Then plasmids
180 and pGL2-Control (to correct
for differences in transfection efficiency) were added (5 µg each) to
the RASMC in a solution containing 500 µg/ml DEAE-dextran. RASMCs
were subsequently shocked with 10% dimethyl sulfoxide solution for 1 min and then allowed to recover in medium containing 10% heat-inactivated FBS. 12 h after transfection, RASMCs were placed in 2% FBS. RASMCs were then stimulated with vehicle, IL-1
(10 ng/ml) alone, or IL-1
plus TGF-
1 (10 ng/ml). After 24 h,
cell extracts were prepared by detergent lysis (Promega), and CAT
assays were performed as described (30, 31). Luciferase activity was measured with an EG&G AutoLumat LB953 luminometer (Gaithersburg, MD) and the Promega Luciferase Assay system to assess efficiency of
transfection. The ratio of CAT to luciferase activity in each sample
served as a measure of normalized CAT activity.
To investigate the effect of HMG-I(Y) overexpression on
TGF-
-mediated inhibition of NOS2 promoter activity by inflammatory stimuli, RASMC or RAW 264.7 were plated in 6-well cell culture plates
at 100,000/well and 70,000/well, respectively. The next day, cells were
transfected as described above using 1 µg/well iNOS(
1485/+31), 0.8 µg/well pCMV-
gal, and increasing amounts of HMGIYpcDNA3 (1, 2, or 3 µg/well). The corresponding empty vector was added when needed
to keep the DNA amount constant throughout the experiment. 18 h
after transfection, RAMC were treated with IL-1
(10 ng/ml) alone or
IL-1
plus TGF-
1 (0.5 ng/ml). RAW 264.7 were treated with LPS
alone (0.5 µg/ml) or LPS plus TGF-
1 (1 ng/ml). After an additional
24 h, cells were harvested by detergent lysis (Promega). In the
resulting cell extract, luciferase and
-galactosidase activity was
assayed as described previously (32).
Electrophoretic Mobility Shift Assay (EMSA)--
For the
preparation of the nuclear protein extract (29), RAW 264.7 cells were
grown to 80% confluence then stimulated with either vehicle, LPS (1 µg/ml), or LPS (1 µg/ml) plus TGF-
1 (10 ng/ml). LPS-stimulated
nuclear extract was subjected to electrophoretic mobility assay
analysis using a double-stranded oligonucleotide probe encoding region
87 to
52 of the NOS2 5'-flanking sequence (TGGGGACTCTCCCTTTGGGAACAGTTATGCAAAATA). Binding reactions were performed in a 25-µl volume containing 20,000 cpm of labeled probe, 10 µg of nuclear extract, 100 ng of poly(dG-dC)·poly(dG-dC)
(Sigma), 25 mM Hepes (pH 7.9), 50 mM KCl, 0.1 mM EDTA, 1 mM dithiothreitol, and 10%
glycerol, with or without oligonucleotide competitors. Reactions were
incubated for 20 min at room temperature, and DNA-protein complexes
were analyzed by electrophoresis on a 5% native polyacrylamide gel in
0.25 × Tris borate-EDTA (TBE) buffer at 4 °C. To characterize specific DNA-binding proteins, we incubated nuclear extracts with anti-HMG-I(Y) affinity-purified antibody (4 µg) (33) for 2 h at
4 °C before the addition of probe.
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RESULTS |
TGF-
1 Prevents the Induction of HMG-I(Y) and NOS2 mRNA by
IL-1
in RASMC--
Vascular smooth muscle cells are an important
target of proinflammatory cytokines during sepsis, and their relaxation
is an important determinant of the hypotension accompanying septic
shock. Thus, we initially assessed the effect of TGF-
1 on NOS2 and
HMG-I(Y) mRNA levels after IL-1
stimulation in RASMC. Fig.
1 shows that TGF-
1 not only prevented
the induction of NOS2 mRNA by IL-1
(white bars), but
it also prevented HMG-I(Y) induction (black bars). IL-1
induction of HMG-I(Y) mRNA was reduced by 96% after 24 h of
TGF-
1 treatment.

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Fig. 1.
Effect of TGF- 1 on
IL-1 -induced HMG-I(Y) and NOS2 mRNA levels
in RASMC. RASMC were treated with vehicle (V), IL-1
alone (IL, 10 ng/ml), or IL-1 plus TGF- 1
(IL+T, 1 ng/ml). Total RNA was extracted 24 h after
treatment, and Northern blot analysis was performed using 10 µg of
total RNA per lane. After electrophoresis, the RNA was transferred to
nitrocellulose filters, which were hybridized with
32P-labeled HMG-I(Y) and NOS2 probes. Filters were also
hybridized with a 32P-labeled oligonucleotide probe
complementary to 28 S ribosomal RNA to control for differences in
loading. The signal intensities were then plotted as -fold increases
compared with vehicle signal (mean ± S.E.) for HMG-I(Y)
(black bars) and NOS2 (white bars). Each
experiment was performed three times.
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To investigate the mechanism by which TGF-
1 decreased HMG-I(Y)
mRNA levels after their induction by IL-1
, we assessed HMG-I(Y) mRNA half-life and promoter activity (Fig.
2). To evaluate HMG-I(Y) mRNA
half-life, RASMC were cultured to 70% confluence. Cells were then
treated with either IL-1
alone or IL-1
plus TGF-
1 for 24 h. Actinomycin D was then added to the cells, and total RNA was
extracted as described at 0, 4, 8, 12, and 24 h. As reported previously (13), the half-life of HMG-I(Y) mRNA after IL-1
stimulation was ~12 h. The addition of TGF-
1 did not reduce
HMG-I(Y) mRNA half-life (Fig. 2A). The effect of
TGF-
1 on promoter activity of the HMG-I(Y) gene was
studied by transiently transfecting RASMC with plasmid
180. Cells
were then stimulated with vehicle, IL-1
, or IL-1
plus TGF-
1
for 24 h. TGF-
1 completely prevented the induction of HMG-I(Y)
promoter activity by IL-1
(Fig. 2B). Taken together,
these results suggest that TGF-
1 decreased the levels of HMG-I(Y)
mRNA by acting at the level of gene transcription.

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Fig. 2.
Effect of TGF- 1 on
HMG-I(Y) mRNA half-life and promoter activity. A,
half-life of HMG-I(Y) mRNA. Cells were treated with IL-1 alone
(IL, 10 ng/ml, filled circles) or IL-1 plus
TGF- 1 (IL+T, 1 ng/ml, open circles) for
24 h. After this incubation period, actinomycin D (10 µg/ml) was
added to the cells, and total RNA was extracted at the indicated times
(0, 4, 8, 12, and 24 h). Northern blot analysis was performed as
described in Fig. 1. The normalized signal intensity was then plotted
as a percentage of the 0-h value. The experiment was performed three
times. B, effect of TGF- 1 on IL-1 -induced promoter
activity of the HMG-I(Y) gene. A plasmid that contains the
HMG-I(Y) promoter and drives a CAT reporter, 180, was transfected
transiently into RASMC. Cells were then stimulated with vehicle
(V), IL-1 alone (IL, 10 ng/ml), or IL-1
plus TGF- 1 (IL+T, 1 ng/ml) for 24 h, after which the
cell extracts were harvested. Normalized CAT activity is shown as the
-fold induction from the activity of vehicle-treated cells (mean ± S.E., n = 4 in each group).
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TGF-
1 Prevents the Induction of HMG-I(Y) mRNA by LPS and
IFN
in Macrophages--
During sepsis, macrophages are activated by
endotoxin and other bacterial products to produce a battery of
mediators and proinflammatory cytokines (34). One of the main cytokines
produced by macrophages is IFN
(34), which may act in an autocrine
or a paracrine fashion. Both LPS and IFN
are potent inducers of NOS2
in macrophages (Fig. 3, white
bars). For this reason we wanted to investigate the effects of LPS
and IFN
on the expression of HMG-I(Y) in a mouse macrophage cell
line (RAW 264.7 cells) and in resident peritoneal macrophages. LPS
stimulation of RAW 264.7 cells (Fig. 3A) and peritoneal
macrophages (Fig. 3B) increased HMG-I(Y) mRNA 4- and
11-fold, respectively (black bars). This increase in
HMG-I(Y) message by LPS was decreased by TGF-
1 in both cell types.
NOS2 mRNA was also increased by LPS (14-fold in both RAW 264.7 and
peritoneal macrophages) and suppressed by TGF-
1 treatment
(white bars). IFN
stimulation of RAW 264.7 cells induced
HMG-I(Y) mRNA by 11-fold (Fig. 3C, black
bars) and NOS2 mRNA by 24-fold (white bars). In
peritoneal macrophages, the same cytokine induced a 5.5-fold increase
in HMG-I(Y) mRNA (Fig. 3D, black bars) and a
19-fold increase in NOS2 mRNA (white bars). TGF-
1
treatment was able to abolish the induction of HMG-I(Y) and NOS2
mRNA by IFN
in both cell types.

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Fig. 3.
Effect of TGF- 1 on
HMG-I(Y) and NOS2 mRNA levels in RAW 264.7 and peritoneal
macrophages after treatment with LPS or
IFN . RAW 264.7 cells (A) and
peritoneal macrophages (B) were treated with vehicle
(V), LPS alone (L, 0.5 µg/ml), or LPS plus
TGF- 1 (L+T, 10 ng/ml) for 24 h. RAW 264.7 cells
(C) and peritoneal macrophages (D) were also
treated with vehicle (V), IFN alone (IF, 100 units/ml), or IFN plus TGF- 1 (IF+T, 10 ng/ml) for
24 h. Total RNA was subsequently harvested in all experiments, and
Northern analyses were performed as described in Fig. 1. The signal
intensities were then plotted as -fold increases compared with vehicle
signal (mean ± S.E.) for HMG-I(Y) (black bars) and
NOS2 (white bars). Each experiment was performed three
times.
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Spleen is a macrophage-rich organ that responds to LPS administration
in vivo with a dramatic up-regulation of NOS2 expression (Refs. 29 and 35, and Fig.
4A). We decided to use the
spleen as a model to investigate the regulation of HMG-I(Y) in
macrophages in vivo, in response to LPS or LPS plus
TGF-
1. LPS administration in rats caused a 4-fold induction of
HMG-I(Y) transcript in the spleen (Fig. 4B). HMG-(Y)
mRNA levels were markedly reduced in rats receiving LPS plus
TGF-
1 compared with levels in rats receiving LPS alone (Fig.
4B). This effect of TGF-
1 on HMG-I(Y) mRNA levels was
similar to the suppression of NOS2 mRNA levels in rats receiving LPS and TGF-
1 (Fig. 4A).

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Fig. 4.
Effect of TGF- 1 on
LPS-induced NOS2 and HMG-I(Y) mRNA in vivo.
Conscious male Sprague-Dawley rats were injected with vehicle
(V, n = 2), LPS alone (L, 4 mg/kg
ip, n = 2), or LPS plus TGF- 1 (L+T, 20 µg/kg, n = 2). The rats were killed 10 h after
LPS administration, and total RNA was extracted from spleen tissue.
Northern blot analysis was performed as described in Fig. 1. The signal
intensities were then plotted as -fold increases compared with vehicle
signal (mean ± S.D.) for NOS2 (A) and HMG-I(Y)
(B).
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TGF-
1 Prevents the Induction of HMG-I(Y) Protein by IL-1
in
RASMC and LPS in Macrophages--
We next focused our attention on the
regulation of HMG-I(Y) protein by IL-1
in RASMC and by LPS in RAW
264.7 cells. We have shown previously that HMG-I(Y) protein is
maximally induced after 48 h of cytokine stimulation in RASMC
(13), thus this time point was used to assess the effect of TGF-
1 on
HMG-I(Y) protein induction. HMG-I(Y) protein levels were increased
7.5-fold by IL-1
in RASMC (Fig.
5A) and 5.5-fold by LPS in RAW
264.7 cells (Fig. 5B). When TGF-
1 was added, the
induction of HMG-I(Y) protein levels was decreased by 83%
(versus IL-1
, Fig. 5A) in RASMC and by 70%
(versus LPS, Fig. 5B) in RAW 264.7 cells.

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Fig. 5.
Effect of TGF- 1 on
HMG-I(Y) protein expression after treatment with
IL-1 or LPS in RASMC and RAW264.7 cells
respectively. A, RASMC were treated with vehicle
(V), IL-1 alone (IL, 10 ng/ml), or IL-1
plus TGF- 1 (IL+T, 1 ng/ml) for 48 h. B,
RAW 264.7 cells were treated with vehicle (V), LPS alone
(L, 0.5 µg/ml), or LPS plus TGF- 1 (L+T, 10 ng/ml) for 48 h. In both A and B, protein
was extracted from the cells and analyzed by Western blotting. A
representative blot is shown. The intensity of the band for HMG-I(Y)
was divided by the intensity of the band for histone H1. Normalized
signal intensities were plotted as the -fold induction from the
intensity of vehicle (mean ± S.E.). Each experiment was performed
twice.
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HMG-I(Y) Overexpression Rescues the TGF-
1-mediated
Down-regulation of NOS2 Gene Transactivation in RASMC and RAW 264.7 Cells--
We hypothesized that down-regulation of HMG-I(Y) may play a
role in mediating the inhibition of NOS2 expression by TGF-
1 in
response to inflammatory stimuli. To test this hypothesis, we
transiently cotransfected RASMC and RAW 264.7 cells with promoter construct iNOS(
1485/+31) and increasing concentrations of expression plasmid HMGIYpcDNA3 (0, 1, 2, 3 µg/well as described under
"Experimental Procedures"). Cells were then treated with either
vehicle, IL-1
(RASMC, Fig.
6A), or LPS (RAW 264.7 cells,
Fig. 6B), alone or in combination with TGF-
1. Compared
with cells receiving vehicle, the addition of IL-1
or LPS promoted a
significant increase in promoter activity of the NOS2 gene
in both RASMC (Fig. 6A) and RAW 264.7 cells (Fig.
6B), respectively. As expected, when vector-transfected cells (pcDNA3) were treated with TGF-
1, NOS2 promoter activity in response to IL-1
(RASMC) and LPS (RAW 264.7 cells) was
significantly reduced in RASMC (Fig. 6A) and RAW 264.7 cells
(Fig. 6B). HMG-I(Y)-transfected cells showed a
dose-dependent recovery of NOS2 promoter activity by both
IL-1
(Fig. 6A) and LPS (Fig. 6B). At a dose of
2 µg/well HMG-I(Y) expression plasmid, the responsiveness of the
NOS2 gene promoter activity was almost completely restored
in both RASMC (89% of IL-1
alone value, Fig. 6A) and RAW
264.7 cells (90% of the LPS alone value, Fig. 6B) despite
the presence of TGF-
1. To determine whether HMG-I(Y) could restore
other TGF-
1 down-regulated genes, we performed the same experiment
using the promoter for the matrix metalloproteinase-12 gene
(36). Overexpression of HMG-I(Y) did not rescue the TGF-
1-mediated
down-regulation of matrix metalloproteinase-12 promoter activity (data
not shown). These data suggest that recovery of NOS2 promoter activity
by HMG-I(Y) was a specific effect.

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Fig. 6.
Effect of HMG-I(Y) overexpression on
TGF- 1-mediated inhibition of NOS2 promoter
activity in RASMC and macrophages. RASMC (A) and RAW
264.7 cells (B) were transfected transiently with promoter
construct iNOS( 1485/+31) (1 µg/well) and an expression plasmid
encoding increasing amounts of HMG-I(Y) (0, 1, 2, or 3 µg/well, as
shown). The corresponding empty vector (pcDNA3) was added to keep
the total DNA content constant. After the transfection, RASMC were
treated with IL-1 (10 ng/ml) in the presence (+) or absence ( ) of
TGF- 1 (0.5 ng/ml). RAW 264.7 cells were treated with LPS (0.5 µg/ml) in the presence (+) or absence ( ) of TGF- 1 (1 ng/ml).
Normalized luciferase activity was plotted as the -fold induction from
the activity in cells receiving no IL-1 /LPS, no TGF- 1, and no
HMG-I(Y). Each experiment was performed at least twice, with a total
n = 6.
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Effect of TGF-
1 on HMG-I(Y) Binding to the Promoter of the NOS2
Gene--
We investigated the binding of HMG-I(Y) protein to the
downstream promoter sequence (region
87 to
52 of the NOS2
5'-flanking region, TGGGGACTCTCCCTTTGGGAACAGTTATGCAAAATA) of the
NOS2 gene by using nuclear extracts obtained from RAW 264.7 cells stimulated with LPS in the presence or absence of TGF-
1 for 4, 24, and 48 h. Following LPS stimulation, we observed the
appearance of a high molecular weight band that exhibited a clear
induction at 24 and 48 h after LPS (band 1, Fig.
7A). A second band was also evident (band 2). Band 2 decreased in intensity
in a time-dependent manner. The specificity of the two
bands was evaluated by adding a 100-fold molar excess of either the
unlabeled probe (identical competitor, IdC) or an unrelated
oligonucleotide (nonidentical competitor, NIdC). As shown in Fig.
7B, band 1 was abolished by the identical but not
the unrelated competitor. Band 2 was abolish by both
competitors. These data indicated that band 1 was the only
specific band. To investigate whether HMG-I(Y) was present in
band 1, the nuclear extracts from cells stimulated with LPS for 48 h were submitted for EMSA in the presence of either a
specific anti-HMG-I(Y) antibody or a control antibody (Fig.
7C). Band 1 supershifted in the presence of
anti-HMG-I(Y) antibody, but not control antibody, indicating that
HMG-I(Y) was present in the nucleoprotein complex binding to the
NOS2 promoter.

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Fig. 7.
Effect of TGF- 1 on
LPS-induced nuclear protein complex containing HMG-I(Y). EMSA were
performed with a 32P-labeled oligonucleotide probe from
region 87 to 52 of the NOS2 5'-flanking sequence, and nuclear
extracts from RAW 264.7 cells. A, cells were treated with
vehicle ( ) or LPS (+, 0.5 µg/ml) for increasing periods of time (as
indicated) prior to extraction of nuclear extract.
Arrowheads denote two bands (1 and 2)
consistent with DNA-protein complexes. B, EMSA was performed
on nuclear extract from LPS-treated (0.5 µg/ml) cells for 48 h.
Unlabeled competitors were added at a 100-fold molar excess.
IdC, identical competitor; and NidC, unrelated
competitor. Band 1 denotes a specific DNA-protein complex.
C, nuclear extracts from cells stimulated with LPS (0.5 µg/ml) for 48 h. EMSA was performed in the presence (+) of an
HMG-I(Y) antibody [ -HMG-I(Y)] or a control antibody. The
arrowhead denotes specific band 1. The
asterisk indicates supershifted complex. D,
nuclear extracts were harvested from cells stimulated with LPS (0.5 µg/ml) for increasing periods of time as indicated in the presence
(+) or absence ( ) of TGF- 1 (10 ng/ml).
|
|
Finally, we studied the binding of HMG-I(Y) to the promoter of the
NOS2 gene in RAW 264.7 cells that were treated with LPS alone or LPS plus TGF-
1 (Fig. 7D). TGF-
1 did not
influence the binding at 4 and 24 h, as shown by the comparable
intensities of the bands. In addition, the anti-HMG-I(Y) antibody was
still able to supershift band 1 in the TGF-
1-treated
nuclear extracts (data not shown), indicating that TGF-
1 was not
affecting HMG-I(Y) DNA binding properties at these time points. In
contrast, the intensity of band 1 was dramatically decreased
after 48 h of treatment with LPS plus TGF-
1 compared with LPS
alone. Interestingly, the Western blot analysis showed that, at the
same time point (48 h), HMG-I(Y) protein content was dramatically
decreased by TGF-
1 (Fig. 5B). These data suggest that the
decreased intensity of band 1 after 48 h of TGF-
1
was related to a decrease in HMG-I(Y) protein not an alteration in
binding affinity of the HMG-I(Y) protein.
 |
DISCUSSION |
HMG-I(Y) is a nonhistone chromosomal protein that, in conjunction
with transcription factors, including NF-
B, ATF-2/c-Jun, and IRF-1,
assembles nucleoprotein complexes important for the transcriptional
regulation of genes (37, 38). In particular, HMG-I(Y) has been shown to
be involved in the regulation of cytokine-responsive genes that are
critical for the mediation of inflammation (39-45). Previously, we
showed that HMG-I(Y) cooperates with NF-
B in the transcriptional
regulation of NOS2 by inflammatory cytokines and endotoxin in vascular
smooth muscle cells and macrophages (12). Moreover, HMG-I(Y) is
up-regulated by IL-1
and endotoxin in vitro and in
vivo, respectively (13). These data suggest that inflammatory mediators increase the expression of HMG-I(Y) protein, while promoting nuclear translocation of NF-
B at the same time, resulting in maximal
activation of NOS2 gene transcription. Thus, up-regulation of HMG-I(Y) appears to play an important role in the expression of NOS2
during inflammation.
The inflammatory response is a key component of host defense, but
excessive activation of the immune system and subsequent release of
vasoactive mediators (such as occurs in sepsis) may be fatal (8, 46).
The inflammatory process must therefore be regulated tightly in
vivo. The effects of proinflammatory cytokines are counterbalanced
by the production of anti-inflammatory mediators. Among these
anti-inflammatory factors, TGF-
1 has been shown to decrease
hypotension and LPS-induced mortality in rats when administered exogenously (20). TGF-
1 is also known to decrease macrophage responsiveness to LPS (47), and TGF-
1 can prevent NOS2 expression in
response to proinflammatory cytokines (5, 17, 19, 48). The effects of
TGF-
1 are mediated, in part, through Smad proteins that are
essential components in the signaling pathways of TGF-
1 receptors
(49). However, the effector(s) downstream of the TGF-
1 receptors
responsible for transcriptional down-regulation of the NOS2
gene is(are) not known.
In this study, TGF-
1 prevented the IL-1
induction of HMG-I(Y) and
NOS2 mRNA in RASMC (Fig. 1). The effect on HMG-I(Y) mRNA levels
did not involve a modification of transcript stability (Fig.
2A) but was related to a decrease in HMG-I(Y) promoter
activity in the presence of TGF-
1 (Fig. 2B). This effect
of TGF-
1 on HMG-I(Y) mRNA levels was not restricted to vascular
smooth muscle cells, because TGF-
1 down-regulated the HMG-I(Y)
message (similar to the NOS2 message) in macrophages stimulated with
inflammatory cytokines and endotoxin in vitro (Fig. 3) and
in vivo (Fig. 4). In agreement with mRNA levels,
TGF-
1 also down-regulated HMG-I(Y) protein in vascular smooth muscle
cells (Fig. 5A) and macrophages (Fig. 5B) induced
by IL-1
and endotoxin, respectively. These results provide the first
evidence that TGF-
1 can decrease HMG-I(Y) expression driven by
inflammatory stimuli.
We have suggested previously that induction of HMG-I(Y), and subsequent
transactivation of the NOS2 gene, may contribute to a
reduction in vascular tone during sepsis and other inflammatory disease
processes (12, 13). In addition, we have shown in the present study
that HMG-I(Y) and NOS2 expression are down-regulated by TGF-
1 in a
comparable manner after stimulation with inflammatory mediators. Thus,
we hypothesized that HMG-I(Y) content may be a limiting factor for NOS2
induction by endotoxin and proinflammatory cytokines. To test this
hypothesis, we overexpressed HMG-I(Y) in vascular smooth muscle cells
and macrophages in the presence of TGF-
1 to determine whether
HMG-I(Y) could restore inflammatory cytokine and endotoxin inducibility
of the NOS2 gene promoter. In both RASMC (Fig.
6A) and RAW 264.7 cells (Fig. 6B), overexpression of HMG-I(Y) was able to restore cytokine inducibility of the NOS2 promoter that was suppressed by TGF-
1. Taken together, these data
suggest that factors (such as TGF-
1) that decrease the amount of
endogenous HMG-I(Y) in cells limit the inducibility of NOS2 by
inflammatory mediators.
HMG-I(Y) has the potential to be post-translationally modified.
In vitro experiments have shown that phosphorylation of
HMG-I(Y) can decrease its affinity for DNA (50, 51). Thus, to determine whether HMG-I(Y) binding to the promoter of the NOS2 gene
was altered by TGF-
1, we performed EMSA. LPS induced the formation of a specific, high molecular weight complex in macrophages (Fig. 7,
A and B). Supershift experiments using an
anti-HMG-I(Y) antibody revealed the presence of HMG-I(Y) in this
complex (Fig. 7C). We have shown previously that NF-
B
binding to this portion of the NOS2 5'-flanking sequence (12) is
important for NOS2 gene transactivation; however, a direct
effect of TGF-
1 on NF-
B binding has not been demonstrated (29).
Thus, we wanted to determine whether an alteration in HMG-I(Y) binding
by TGF-
1 might be responsible for the suppression in NOS2
gene transactivation. TGF-
1 treatment did not modify the intensity
of the specific HMG-I(Y) binding complex after 4 and 24 h (Fig.
7D, lanes 2 and 4), suggesting that
DNA binding of HMG-I(Y) was not modified by TGF-
1. In contrast, the
dramatic decrease in intensity of this complex after 48 h of
TGF-
1 treatment (Fig. 7D, lane 6) corresponded
with a decreased in HMG-I(Y) protein content as shown by Western blot
analysis (Fig. 5). Taken together, these data suggest that a
down-regulation in HMG-I(Y) protein, not a disruption in HMG-I(Y)
binding, is accountable for the decreased nucleoprotein complex
responsible for transactivation of the NOS2 gene by
inflammatory mediators.
The present study provides further insight into the mechanism by which
TGF-
1 is able to down-regulate NOS2 gene transactivation by inflammatory mediators. Our data underscore the importance of
HMG-I(Y) in contributing to nucleoprotein complex formation for driving
transcription of the NOS2 gene. Moreover, we show that
inhibition of HMG-I(Y) transcription and subsequent decrease in
HMG-I(Y) protein by TGF-
1 significantly decreases NOS2 promoter activity, which can be restored by exogenous administration of HMG-I(Y). These data confirm the importance of HMG-I(Y) in regulating NOS2 gene transactivation by inflammatory mediators in
vascular smooth muscle cells and macrophages, cell types involved in
the pathophysiology of sepsis and other systemic inflammatory diseases.