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INTRODUCTION |
Nonhistone chromosomal proteins of the high mobility group
(HMG)1 play a role in the transcriptional regulation of
mammalian genes, the promoter/enhancer
regions of which are close to AT-rich sequences (1-3). HMG proteins
contribute to the regulation of gene expression by serving as
architectural factors that alter chromatin structure (4). An important
member of this family is HMG-I(Y). HMG-I(Y) refers to two proteins,
HMG-I and HMG-Y, that are derived by alternative splicing of the same
gene (5). The biological functions of the two proteins are
indistinguishable. HMG-I(Y) binds to AT-rich regions in the minor
groove of DNA via motifs known as AT-hooks (6), thereby facilitating
the assembly of functional nucleoprotein complexes (enhanceosomes).
This assembly is promoted by modifying DNA conformation and by
recruiting nuclear proteins to an enhancer (7). By binding to DNA and
altering chromatin structure, HMG-I(Y) can have positive or negative
effects on transcription factor binding to promoter regions
(3, 8).
The role of HMG-I(Y) in enhanceosome assembly during viral induction of
the interferon (IFN)-
gene has been studied extensively (7, 9-11).
HMG-I(Y) binds to positive regulatory domains in the IFN-
enhancer,
reverses an intrinsic bend in the DNA, and recruits transcription
factors to their binding sites to have a positive effect on
transcription during viral stimulation. Factors recruited by HMG-I(Y)
include nuclear factor (NF)-
B, ATF-2/c-Jun, and IRF-1 (11, 12).
Other genes that require NF-
B and HMG-I(Y) for full transcriptional
activation include E-selectin (13, 14) and the chemokine melanoma
growth stimulatory activity/growth related protein
(MGSA/GRO
)
(15). E-selectin and MGSA/GRO
are cytokine-responsive genes critical
to mediation of inflammation. E-selectin affects the interaction of
leukocytes with the vascular wall during an inflammatory response (16),
and MGSA/GRO
is a potent chemoattractant for neutrophils (15). These
observations suggest that HMG-I(Y) may be important for full induction
of cytokine-driven promoters that require NF-
B for activation. Thus,
the ability of HMG-I(Y) to assemble higher order transcription factor
complexes may be important during systemic inflammatory responses.
Sepsis is a disease process that results from an overwhelming
inflammatory response due to severe infection (17-20). During Gram-negative bacterial infection, lipopolysaccharide (LPS) or endotoxin is released from the bacterial cell wall to activate the
immune cells of the host. These immune cells, which include macrophages, then release proinflammatory cytokines. When
proinflammatory cytokines, such as interleukin (IL)-1
and tumor
necrosis factor-
, are released in exaggerated amounts, the result is
hypotension and shock (17, 19). An important mediator of this
cytokine-induced hypotensive response is the potent vasodilator nitric
oxide (NO) (21, 22). Generation of NO under these circumstances is
regulated by the inducible isoform of NO synthase (iNOS or NOS2). As it does in the induction of IFN-
by viral stimulation and E-selectin and MGSA/GRO
by cytokine stimulation, NF-
B plays a critical role
in the activation of iNOS (23). Moreover, recent experiments have
emphasized the importance of NF-
B binding activity in the pathophysiology of sepsis (24).
In addition to a role for HMG-I(Y) in the transcriptional regulation of
genes, previous studies have shown an association between high levels
of HMG-I(Y) and the neoplastic transformation of cells. HMG-I(Y) is
up-regulated in malignant tumors (25-27), and its level of expression
correlates with the malignant phenotype of neoplasms in humans
(28-30). Little is known, however, about the regulation of HMG-I(Y)
expression in nontransformed or nonmalignant cells. For example, we do
not know how endotoxin and proinflammatory cytokines influence HMG-I(Y)
in vascular smooth muscle cells (which are important for the regulation
of vascular tone) or what the significance is of an up-regulation in
HMG-I(Y)
and its potential influence on genes, such as iNOS, that
regulate vascular tone
during systemic inflammatory processes.
In investigating the regulation of HMG-I(Y) in vascular smooth muscle
cells in vitro and in vivo, our goals in the
present study were to 1) determine whether the proinflammatory cytokine IL-1
regulated HMG-I(Y) mRNA and protein in primary cell
cultures, 2) determine whether such a response also occurred in smooth
muscle cells of the blood vessel wall after endotoxin stimulation
in vivo, 3) determine whether regulation of HMG-I(Y)
correlated with induction of iNOS, and 4) determine whether HMG-I(Y)
plays a role in the activation of iNOS gene transcription.
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EXPERIMENTAL PROCEDURES |
Cell Culture--
Rat aortic smooth muscle cells (RASMCs) were
harvested from male Sprague-Dawley rats (200-250 g) by enzymatic
dissociation according to the method of Gunther et al. (31).
The cells were cultured in Dulbecco's modified Eagle's medium (DMEM)
(J. R. H. Biosciences, Lenexa, KS) and supplemented with 10%
heat-inactivated 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. RASMCs were passaged every 4-5 days, and experiments were
performed on cells 4-6 passages from primary culture. Alveolar
macrophages were harvested from the lungs of male Sprague-Dawley rats
as described (32). Wright-Giemsa staining revealed that >95% of the
cells were normal lung macrophages. The cells were suspended in RPMI
1640 medium (J. R. H. Biosciences) supplemented with 2%
heat-inactivated fetal horse serum, penicillin (100 units/ml), and
streptomycin (100 µg/ml) in a humidified incubator at 37 °C.
Drosophila SL2 cells (ATCC, Manassas, VA) (33) were maintained at 23 °C in Schneider's insect medium (Sigma)
supplemented with 12% heat-inactivated FBS and gentamycin (50 µg/ml). SL2 cells were passaged every 4 days.
Northern Blot Analysis--
Total RNA was obtained from rat
aortas, after removal of adventitial tissue, and from cultured RASMCs
and rat alveolar macrophages by guanidinium isothiocyanate extraction
and centrifugation through cesium chloride (34). 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) and rat iNOS (35) 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 18 S ribosomal RNA. Images were
displayed and radioactivity was measured on a PhosphorImager running
the ImageQuant software (Molecular Dynamics, Sunnyvale, CA).
Plasmids--
To evaluate the effect of IL-1
on HMG-I(Y)
transcription in vascular smooth muscle cells, plasmid
180 was used
(36). This construct, cloned into pCAT-Basic, contained the second
transcription start site of HMG-I(Y) (36, 37). Plasmid pGL2-Control
contained the firefly luciferase gene driven by an SV40 promoter and
enhancer. To evaluate iNOS promoter activity in either vascular smooth
muscle cells or SL2 cells, we inserted 1485 base pairs of the
5'-flanking region of the mouse iNOS gene and the first 31 base pairs
after the transcription start site into pGL2-Basic to make
iNOS(-1485/+31), as described (38). Plasmid iNOS(-1485/+31 NF-
Bm),
which contained a mutated downstream NF-
B site (-85 to -83, GGG to
CTC) (38), was used to assess the specificity of NF-
B subunit
binding. Plasmid pPAC has been described elsewhere (10, 39). p50 and
p65 expression constructs were made by inserting the cDNAs coding
for NF-
B subunits p50 and p65 into the BamHI site of pPAC
(40). Expression vectors phsp82LacZ and pPACHMGI have been described
elsewhere (10). Plasmid pOPRSVI-CAT contained the chloramphenicol
acetyltransferase (CAT) gene driven by a Rous sarcoma virus-long
terminal repeats promoter, and plasmid -438SmLIM has been described
elsewhere (41).
Transfections--
RASMCs were transfected by a
diethylaminoethyl (DEAE)-dextran method (38). 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 RASMCs in a solution containing 500 µg/ml of 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. Twelve hours after
transfection, RASMCs were placed in 2% FBS. RASMCs were then
stimulated with vehicle or human recombinant IL-1
(10 ng/ml)
(Beckton Dickinson, Los Angeles, CA) for 24 h. Cell extracts were
prepared by detergent lysis (Promega), and CAT assays were performed by
a modified two-phase fluor diffusion method as described (42, 43).
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 assess the effect of distamycin A (Sigma) on iNOS promoter induction
by IL-1
, we transfected RASMCs by the DEAE-dextran method described
above. Plasmid iNOS(-1485/+31) or -438SmLIM (5 µg) was transfected
with pOPRSVI-CAT (to correct for differences in transfection
efficiency) into the RASMCs. Twelve hours after transfection, the cells
were placed in 2% FBS and incubated with the maximal stimulatory dose
of IL-1
(10 ng/ml) for 24 h in the presence or absence of
distamycin A (5 µM). The cells were subsequently harvested and analyzed for luciferase and CAT activity. In these experiments, the ratio of luciferase to CAT activity in each sample served as a measure of normalized luciferase activity.
SL2 cells were transfected by the calcium-phosphate method according to
Di Nocera and Dawid (44). In brief, SL2 cells were plated in 6-well
tissue culture dishes (Costar Corp.) 24 h before transfection.
Plasmids iNOS(-1485/+31) and iNOS(-1485/+31 NF-
Bm) were added at 1 µg/well. Plasmids p50-pPAC, p65-pPAC, and phsp82LacZ were added at
100 ng/well. Plasmid pPACHMGI was added at 0.1, 0.5, or 1 µg/well,
alone or in combination with p50-pPAC and p65-pPAC. Forty-eight hours
after the initial transfection, extracts from the SL2 cells were
prepared and luciferase activity was measured as described for RASMCs.
-Galactosidase assays, to assess transfection efficiency, were
performed as described elsewhere (45). The ratio of luciferase activity
to
-galactosidase activity in each sample served as a measure of
normalized luciferase activity.
Western Blot Analysis of HMG-I(Y) in RASMCs--
RASMCs were
plated in 150-mm tissue culture dishes (Beckton Dickinson, Franklin
Lakes, NJ). When the cells were 80-90% confluent, the 10% FBS medium
was replaced with 0.4% bovine serum medium. After 48 h, vehicle
or IL-1
(10 ng/ml) was added to the cells. RASMCs were then
collected in phosphate-buffered saline solution with a cell scraper
48 h later. HMG-I(Y) protein was obtained from RASMCs by acidic
extraction followed by trichloroacetic acid precipitation (46). 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 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. 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 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 Biotechnology) 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). 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 the
NIH Image software, and the ratio of the two values was used to
represent the normalized intensity of the HMG-I(Y) band.
Immunohistochemical Analysis of HMG-I(Y) Expression in Aortas
from LPS-treated Rats--
Aortic samples were collected and frozen.
Sections were cut to a thickness of 5 µm, fixed in 4%
paraformaldehyde, and washed in phosphate-buffered saline. Sections
were then incubated in 10% goat serum for 20 min at room temperature.
Sections were next incubated with anti-HMG-I(Y) antibody (N19, Santa
Cruz Biotechnology) at 0.25 µg/ml in phosphate-buffered saline-0.4%
Triton X-100 for 1 h at room temperature and then overnight at
4 °C. Control sections were incubated under the same conditions with
normal goat IgG (Research & Diagnostic Systems, Minneapolis, MN) at
0.25 µg/ml. After a wash, sections were incubated with biotinylated
rabbit anti-goat antibody (Vectastain ABC, Vector Laboratories,
Burlingame, CA) at 1.5 µg/ml in phosphate-buffered saline-0.4%
Triton X-100 for 1 h at room temperature. After they had been
incubated with avidin, the sections were developed with a peroxidase
3,3'-diaminobenzidine (DAB) kit (Vector Laboratories). To ensure
specificity of staining, we treated a series of sections from the same
animals with anti-HMG-I(Y) antibody N19 that had been preincubated for
1 h at room temperature with the synthetic peptide (40 µg/ml)
used to raise it. The peptide represents amino acids 2-20 of the
HMG-I(Y) sequence (ESSSKSSQPLASKQEKDGT, Santa Cruz Biotechnology).
Rat Model of Endotoxemia--
Male Sprague-Dawley rats (200-250
g) were treated with vehicle or Salmonella typhosa LPS (10 mg/kg intraperitoneally, Sigma). Nine hours later, we harvested the
aortas and processed them as described for use in Northern blot
analyses or immunohistochemical staining.
Statistics--
Data from the SL2 cell transfection experiments
were subjected to analysis of variance followed by Scheffe's test.
Significance was assumed at p < 0.05.
 |
RESULTS |
IL-1
Increased HMG-I(Y) mRNA in RASMCs in Primary
Culture--
Because the proinflammatory cytokine IL-1
is an
important downstream mediator of the vascular response to LPS, we
studied the effect of IL-1
on HMG-I(Y) gene expression in
vitro. Cultured RASMCs were exposed to various concentrations of
the cytokine for 24 h, and then total cellular RNA was extracted
for Northern blot analysis. IL-1
increased HMG-I(Y) mRNA
expression in a dose-dependent fashion (Fig.
1A), with a maximal effect at
10 ng/ml. We then studied the time course of the effect of IL-1
at
10 ng/ml. In comparison with vehicle-treated RASMCs, IL-1
-treated
RASMCs showed an initial increase in HMG-I(Y) mRNA after 6 h
(Fig. 1B). Peak induction of HMG-I(Y) message (6.2-fold)
occurred after 48 h of IL-1
stimulation, even though HMG-I(Y)
mRNA levels remained significantly elevated after 72 h of
IL-1
treatment. Pretreating the cells with the protein synthesis
inhibitor cycloheximide completely abolished induction of HMG-I(Y)
mRNA after IL-1
stimulation (Fig. 1C), indicating
that this induction depended on protein synthesis de novo.
To determine whether LPS could also have an effect on HMG-I(Y) in
cultured smooth muscle cells, we applied LPS directly to RASMCs. LPS
produced an increase in HMG-I(Y) message (data not shown), although
less dramatically than IL-1
. Previous studies have also shown that
the induction of iNOS in vascular smooth muscle cells is more
pronounced after stimulation with proinflammatory ctyokines than with
LPS (47, 48).

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Fig. 1.
Effect of IL-1 on HMG-I(Y) mRNA levels
in RASMCs in primary culture. A, induction of HMG-I(Y)
mRNA by IL-1 : dose response. RASMCs were treated with increasing
doses of IL-1 as indicated, and total RNA was extracted after
24 h of stimulation. B, induction of HMG-I(Y) mRNA
by IL-1 : time course. Cells were treated with vehicle (open
bars) or IL-1 (10 ng/ml) (filled bars), and total
RNA was extracted at the indicated times. C, effect of
protein synthesis inhibition on induction of HMG-I(Y) mRNA by
IL-1 . RASMCs were treated with cycloheximide (CHX) (10 µg/ml) for 30 min before the addition of IL-1 (10 ng/ml). Total
RNA was extracted after 12 h. In all three experiments, Northern
blot analysis was performed with 10 µg of total RNA per lane.
Representative blots are shown. In A and B, the
signal intensity of each RNA sample hybridized to the HMG-I(Y) probe
was divided by that of each sample hybridized to the 18 S probe.
Normalized signal intensities were plotted as the fold induction from
the 0 ng/ml intensity (A, mean ± S.D.) or the vehicle
intensity (B, mean ± S.D.). Each experiment was
performed at least twice, and points of peak HMG-I(Y) induction in
A and B were assessed three times.
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To determine the mechanism responsible for this increase in HMG-I(Y)
mRNA levels in RASMCs after IL-1
stimulation, we measured the
half-life of HMG-I(Y) mRNA in the absence or presence of IL-1
. Total cellular RNA was extracted at 0, 4, 8, 10, 12, and 24 h after stimulation with vehicle or IL-1
, and the HMG-I(Y) mRNA half-life was calculated. The half-life of the message was
approximately 12 h (Fig.
2A), and IL-1
did not
increase HMG-I(Y) mRNA stability. Thus, the increase in HMG-I(Y)
mRNA in response to IL-1
treatment could not be explained by a
change in mRNA stability after cytokine stimulation. We then
performed transient transfection experiments with HMG-I(Y) promoter
construct
180 (containing the second transcription start site) (36).
IL-1
increased HMG-I(Y) promoter activity in RASMCs after 24 h
of stimulation (Fig. 2B), suggesting that the induction of
HMG-I(Y) mRNA by IL-1
was due to an increase in gene
transcription. Analysis of a larger promoter construct (base pairs
-172 to +2322), containing the first three transcription start sites
of HMG-I(Y), produced no further increase in reporter activity compared
with plasmid
180 (data not shown). A promoter construct containing
more of the downstream sequence (base pairs +3771 to +4928), including
the fourth transcription start site of HMG-I(Y), was not responsive to
IL-1
stimulation (data not shown).

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Fig. 2.
Effect of IL-1 on HMG-I(Y) mRNA
half-life and promoter activity in RASMCs in primary culture.
A, half-life of HMG-I(Y) mRNA. Cells were treated with
vehicle (open circles) or IL-1 (10 ng/ml) (filled
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. Northern blot analysis was performed with 10 µg
of total RNA as described for Fig. 1. The signal intensity of each RNA
sample hybridized to the HMG-I(Y) probe was divided by that hybridized
to the 18 S probe. The normalized intensity was then plotted as a
percentage of the 0 h value against time (in log scale). This
experiment was performed three times. B, effect of IL-1
on HMG-I(Y) promoter activity. A plasmid that contains the HMG-I(Y)
promoter and drives a CAT reporter, 180, was transfected transiently
into RASMCs. Cells were then stimulated with vehicle (open
bar) or IL-1 (filled bar) 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.D., n = 4 in each group).
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IL-1
Induced HMG-I(Y) Protein Expression in RASMCs in Primary
Culture--
To see whether the increase in HMG-I(Y) mRNA levels,
which peaked 48 h after IL-1
treatment (Fig. 1A),
corresponded to an increase in HMG-I(Y) protein levels, we treated
cultured RASMCs with vehicle or IL-1
for 48 h. Proteins soluble
in 5% HClO4 were resolved by SDS-polyacrylamide gel
electrophoresis and analyzed by Western blotting with an antibody to
HMG-I(Y). IL-1
induced an increase in HMG-I(Y) protein levels
of 11.1-fold, as assessed by densitometry after normalization against
the levels of histone H1 (Fig. 3).

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Fig. 3.
Effect of IL-1 on HMG-I(Y) protein levels
in RASMCs in primary culture. Cells were stimulated with vehicle
(open bar) or IL-1 (10 ng/ml) (filled bar) for
48 h, and HMG-I(Y) protein extracted from the cells was 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 are plotted as the fold
induction from the intensity of the vehicle (mean ± S.D.). This
experiment was performed three times.
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Endotoxin Increased the Expression of HMG-I(Y) in Vascular Smooth
Muscle Cells in Vivo--
We intraperitoneally injected male
Sprague-Dawley rats (220-250 g) with vehicle or bacterial LPS. Nine
hours after treatment, the rats were killed, and abdominal aortas were
collected for immunohistochemical analysis with an anti-HMG-I(Y)
antibody. As shown in the top right panel of Fig.
4, LPS treatment induced a dramatic
up-regulation of HMG-I(Y) immunoreactivity in the vascular smooth
muscle and endothelial cells of the blood vessel wall. This observation
extends our findings in vitro by demonstrating that HMG-I(Y)
protein does indeed accumulate in response to LPS (and hence cytokine
stimulation) in vivo. Also, the pattern of staining
conformed to the expected nuclear localization of the HMG-I(Y) protein.
The bottom panels of Fig. 4 show aortic sections from the same animals
treated with HMG-I(Y) antibody that had been preincubated with the
peptide used as its immunogen. In the presence of the immunogen, one
would expect HMG-I(Y) immunoreactivity to be lost in areas of specific
staining. The absence of staining in the smooth muscle cells of the
vessel (Fig. 4, bottom panels) shows that the staining in
the top panels was specific. Staining in the endothelium was
not entirely lost after preincubation with the immunogen.

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Fig. 4.
Effect of LPS on HMG-I(Y) protein levels in
vascular smooth muscle cells in vivo. Rats were
injected intraperitoneally with vehicle or S. typhosa LPS at
a dose of 10 mg/kg. Nine hours later, the rats were killed and the
aortic tissue was removed. Aortas from rats receiving vehicle
(top left) (magnification, × 400) or LPS (top
right) (magnification, × 400) were stained with an anti-HMG-I(Y)
antibody. Tissue was also treated with anti-HMG-I(Y) antibody that had
been absorbed to its immunogen before the staining, both in
vehicle-treated (bottom left) (magnification, × 400) and
LPS-treated (bottom right) (magnification, × 400)
tissue.
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Coinduction of HMG-I(Y) and iNOS by Cytokines in Vascular Smooth
Muscle Cells and Macrophages--
The iNOS gene is overexpressed
dramatically when vascular smooth muscle cells are stimulated with
proinflammatory cytokines (35, 49), and this overexpression plays a
major role in the pathophysiology of endotoxemia (21, 22). Thus, we
investigated the relationship between HMG-I(Y) and iNOS gene expression
in vascular smooth muscle cells in response to stimulation by IL-1
(in vitro) and endotoxin (in vivo). RASMCs in
primary culture were treated with vehicle or IL-1
, and total
cellular RNA was extracted 6, 12, 24, 48, and 72 h thereafter. The
RNA was subjected to Northern analysis, and the blots were hybridized
sequentially with HMG-I(Y) and iNOS probes. The time courses of the
induction of HMG-I(Y) and iNOS mRNA by IL-1
(Fig.
5A) were remarkably similar. Both messages increased as early as 6 h after exposure to IL-1
, and peak induction occurred 48 h after exposure. To determine whether this coinduction of HMG-I(Y) and iNOS was also present in other
cell types, we stimulated alveolar macrophages with LPS in
vitro. HMG-I(Y) and iNOS mRNAs were both induced in alveolar macrophages after 6 h and 24 h of LPS administration (Fig.
5B). These in vitro results were confirmed fully
in vivo (Fig. 6) by Northern
blot analysis of RNA extracted from rat aortas that had been harvested
after 9 h of LPS treatment. HMG-I(Y) and iNOS mRNA levels
increased dramatically in aortic tissue after LPS stimulation. Basal
expression was low for both HMG-I(Y) and iNOS in vehicle-treated rats.

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Fig. 5.
Coinduction of HMG-I(Y) and iNOS mRNA in
RASMCs and macrophages in vitro. A, RASMCs
were treated with vehicle (-) or IL-1 (+, 10 ng/ml), and total RNA
was extracted at the indicated times. The experiment was performed
twice. B, rat alveolar macrophages were treated with vehicle
(-) or LPS (+, 5 µg/ml) and total RNA was extracted at the indicated
times. In both experiments (A and B), Northern
blot analyses were performed with 10 µg of total RNA per lane as
described for Fig. 1. The blots were hybridized with HMG-I(Y) and iNOS
probes and a probe for 18 S to assess equality of RNA loading.
Representative experiments are shown.
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Fig. 6.
Coinduction of HMG-I(Y) and iNOS mRNA by
LPS in rat aortas in vivo. Rats were treated
intraperitoneally with vehicle (-, 2 rats) or 10 mg/kg LPS (+, 2 rats). After 9 h, the rats were killed and the aortic tissue was
removed. Total RNA was extracted from the tissue, and Northern blot
analysis was performed as described for Fig. 1 with HMG-I(Y) and iNOS
probes. The blot was also hybridized with an 18 S probe to assess
equality of RNA loading.
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HMG-I(Y) Promoted Transactivation of the iNOS Gene--
Because
the patterns of HMG-I(Y) and iNOS mRNA induction after inflammatory
cytokine stimulation were similar, we determined whether HMG-I(Y) had a
causal role in transactivation of the iNOS promoter. NF-
B plays an
important role in cytokine induction of iNOS (23), as it does in the
induction of IFN-
, E-selectin, and MGSA/GRO
(10, 13, 15). We
transfected the iNOS promoter construct iNOS(-1485/+31) into
Drosophila SL2 cells, which were chosen because they contain
far less endogenous HMG-I(Y) than do mammalian cells (10). We
transfected into the SL2 cells, in conjunction with the iNOS promoter,
increasing concentrations of the HMG-I(Y) expression plasmid pPACHMGI,
with or without expression plasmids for the NF-
B subunits p50 and
p65. HMG-I(Y) alone had no significant effect on iNOS promoter activity
(Fig. 7A), whereas p50 and p65
alone produced a significant increase in promoter activity. In the
presence of p50 and p65, HMG-I(Y) produced a dramatic and
dose-dependent increase in iNOS promoter activity. These
data suggest that increasing concentrations of HMG-I(Y) potentiate
transactivation of the iNOS promoter by NF-
B. The NF-
B binding
site in the downstream portion of the iNOS 5'-flanking sequence (-85
to -76) has been shown to be critical for cytokine induction of the
iNOS promoter (38). Thus, we transfected iNOS promoter constructs
containing either an intact (iNOS(-1485/+31)) or a mutated downstream
NF-
B site (iNOS(-1485/+31 NF-
Bm)) into SL2 cells. We also
transfected into the cells expression plasmids for p50 and p65, in the
presence or absence of the HMG-I(Y) expression plasmid pPACHMGI, to
determine whether a mutated NF-
B site alters the ability of HMG-I(Y)
to transactivate iNOS. As shown in Fig. 7B, a mutated
downstream NF-kB site abolishes the ability of HMG-I(Y) to potentiate
iNOS transactivation by p50 and p65.

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Fig. 7.
Effect of HMG-I(Y) on transactivation of the
iNOS gene by NF- B. A, Drosophila SL2
cells were transfected transiently with the iNOS promoter construct
iNOS(-1485/+31) (1 µg/well), which drives a luciferase reporter
gene, along with increasing amounts (0.1, 0.5, or 1 µg/well) of
expression vectors containing HMG-I(Y) in the presence (+)
(filled bars) or absence (-) (open bars) of
expression vectors containing the NF- B subunits p50 and p65 (100 ng/well each). Normalized luciferase activities were plotted as the
fold induction from the activity of cells containing 0 µg of HMG-I(Y)
and no (-) p50-p65. Values represent the mean ± S.D.,
(n = 6). B, SL2 cells were transfected
transiently with iNOS promoter constructs (1 µg/well) containing
either an intact (iNOS(-1485/+31)) or a mutated downstream NF- B
site (iNOS(-1485/+31 NF- Bm)), along with expression vectors
containing p50 and p65 (100 ng/well each) in the presence (filled
bars) or absence (striped bars) of an expression vector
containing HMG-I(Y) (1 µg/well). Normalized luciferase activities
were plotted as fold induction. Values represent the mean ± S.D.
(n = 6). In both experiments (A and
B), the amount of DNA added to each sample was normalized
with vector DNA (pPAC). Transactivation of iNOS was expressed as a
measurement of luciferase activity normalized against -galactosidase
activity (which was used as an internal standard). ,
p < 0.05, or  , p < 0.001 versus 0 µg of HMG-I(Y) and no (-) p50-p65. ,
p < 0.05, or  , p < 0.001 versus the same amount of HMG-I(Y) and no (-) p50-p65. **,
p < 0.001 versus 0 µg of HMG-I(Y) and (+)
p50-p65.
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Distamycin A Inhibited iNOS Promoter Activity and NO Accumulation
Induced by IL-1
--
To determine whether HMG-I(Y) plays a role in
iNOS promoter transactivation by IL-1
in vascular smooth muscle
cells, we performed transfection experiments in the presence or absence
of distamycin A. Distamycin A is known to bind to AT-rich sequences
(clusters of at least four AT base pairs) in the minor groove of DNA
(3), and others have demonstrated that distamycin A interferes with the
binding of HMG-I(Y) to DNA (50). After transfection into RASMCs, the
promoter construct iNOS(-1485/+31) was induced markedly by IL-1
stimulation in the absence of distamycin A. However, coincubation with
distamycin A (5 µM) decreased IL-1
-induced iNOS
promoter activity by 43% (Fig.
8A). To ensure that this
effect of distamycin A was not related to a nonspecific inhibition of transcription initiation, we transfected a promoter construct from the
CRP2/SmLIM gene (
438SmLIM) into RASMCs and exposed the cells to
distamycin A or its vehicle. The CRP2/SmLIM gene is expressed in
vascular smooth muscle cells (41). Distamycin A had no effect on
CRP2/SmLIM promoter activity (data not shown). In addition, distamycin
A did not decrease the activity of pOPRSVI-CAT or pGL2-Control, plasmids driven by the Rous sarcoma virus and the SV40 promoters respectively (data not shown). Taken together, these data suggest that
inhibition of HMG-I(Y) binding to DNA suppresses full induction of the
iNOS promoter by IL-1
.

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Fig. 8.
Effect of distamycin A on iNOS promoter
activity and NO accumulation induced by IL-1 . A, RASMCs
were transfected transiently with the iNOS promoter construct
iNOS(-1485/+31) (5 µg). The cells were also cotransfected with
pOPRSVI-CAT (5 µg) to correct for differences in transfection
efficiency. The transfected cells were stimulated subsequently with
vehicle (open bar), IL-1 (10 ng/ml) (filled
bar), or a combination of IL-1 (10 ng/ml) and distamycin A (5 µM) (striped bar). Normalized luciferase
activity is shown as the fold induction from the activity of
vehicle-treated cells (mean ± S.E., n = 8 in each
group). B, RASMCs were stimulated with vehicle (open
bar), IL-1 (10 ng/ml) (filled bar), or a combination
of IL-1 (10 ng/ml) and distamycin A (5 µM)
(striped bar). NO2- accumulation is
shown as the fold induction from vehicle-treated cells (mean ± S.E., n = 6 in each group).
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To determine whether this suppression of iNOS promoter activity by
distamycin A translates into a decrease in NO accumulation, we
incubated RASMCs with distamycin A or vehicle for 24 h and then
stimulated the cells with IL-1
. The amount of NO produced by RASMCs
after 72 h of IL-1
stimulation was analyzed by measuring a
stable product of NO oxidation, NO2-
(nitrite), as described (35). Distamycin A (5 µM)
decreased IL-1
-induced NO2- accumulation
by 34% (Fig. 8B), an effect similar to the distamycin A-induced reduction in iNOS promoter activity.
 |
DISCUSSION |
HMG-I(Y) mRNA and protein levels are high in rapidly dividing,
undifferentiated mammalian cells (51). The level of HMG-I(Y) expression
correlates with the rate of cell proliferation, and elevated
concentrations of HMG-I(Y) are characteristic of transformed cells (25,
26). In fact, HMG-I(Y) may be a marker of metastatic aggressiveness in
tumors such as neoplasms of the thyroid (28) and prostate (29). Much
less is known about the regulation of HMG-I(Y) under non-growth-related
conditions, in vitro or in vivo. For example,
although HMG-I(Y) plays a role in the regulation of cytokine-induced
genes, to our knowledge there is nothing in the literature about the
ability of proinflammatory cytokines to regulate HMG-I(Y) expression in
cells in primary culture. We demonstrate here in cultured vascular
smooth muscle cells that the proinflammatory cytokine IL-1
increases
HMG-I(Y) mRNA levels in a dose- and time-dependent
manner (Fig. 1) and that the induction of HMG-I(Y) message by IL-1
is not related to a prolongation in mRNA stability (Fig. 2).
IL-1
does increase HMG-I(Y) promoter activity, suggesting that an
increase in gene transcription contributes to the induction of HMG-I(Y)
message. This increase in mRNA levels by IL-1
translates into an
increase in HMG-I(Y) protein levels (Fig. 3), as demonstrated by an
analysis of HMG-I(Y) protein expression in vascular smooth muscle cells.
To determine whether HMG-I(Y) is regulated in vivo by an
inflammatory stimulus, we administered LPS to rats. LPS promotes a
systemic inflammatory response in these animals, mimicking the pathophysiologic process of endotoxemia that occurs during sepsis in
humans (17, 20). In this process, immune cells are activated and
release a number of mediators and defense molecules, including proinflammatory cytokines (52). Although proinflammatory cytokines, such as IL-1
, help protect the host against infection, release of
exaggerated amounts of these cytokines can also have detrimental effects. One such effect, hypotension, is the result of proinflammatory cytokines stimulating production of vasodilatory mediators in the blood
vessel wall. Our interest in the work presented here was to determine
the effect of an inflammatory stimulus on HMG-I(Y) expression in the
vasculature. Immunohistochemical analysis of aortic tissue from rats
stimulated with LPS demonstrated a dramatic increase in HMG-I(Y)
staining within the blood vessel wall (Fig. 4). Also, preabsorption
experiments with the immunogen that was used to generate the HMG-I(Y)
antibody showed that this specific staining for HMG-I(Y) occurred
within the vascular smooth muscle cells of the blood vessel wall. To
our knowledge, this is the first demonstration that HMG-I(Y) can be
up-regulated by an inflammatory stimulus in vivo.
NO is a vasodilatory mediator critical to the hypotension of sepsis.
The enzyme responsible for NO synthesis during an inflammatory response
is iNOS. Previous studies have shown that stimulation with IL-1
(in vitro) and LPS (in vivo) increases iNOS in
vascular smooth muscle cells (35, 38, 49). We now demonstrate that induction of HMG-I(Y) correlates with induction of iNOS, both in
vitro (Fig. 5) and in vivo (Fig. 6). Because of their
similar expression patterns and the fact that HMG-I(Y) is known to
facilitate transcription of cytokine-driven genes that require NF-
B
for activation, we investigated the role of HMG-I(Y) in transactivation of the iNOS gene. Although HMG-I(Y) alone had no significant effect on
iNOS promoter activity, increasing concentrations of an HMG-I(Y) expression plasmid in the presence of p50 and p65 (subunits of NF-
B)
led to a dose-dependent increase in iNOS promoter activity (Fig. 7A). At higher concentrations, the HMG-I(Y) expression
plasmid potentiated the iNOS promoter response to p50 and p65, and this potentiation required an intact NF-
B site (
85 to
76) in the downstream portion of the iNOS 5'-flanking sequence (Fig.
7B). These data demonstrate for the first time that
HMG-I(Y), an architectural transcription factor, facilitates
transactivation of the iNOS gene by NF-
B. Furthermore, the
dose-dependent increase in iNOS promoter activity by
HMG-I(Y) suggests that the induction of HMG-I(Y) by an inflammatory
stimulus may have an important impact on iNOS gene regulation. This
concept was confirmed by our demonstration that distamycin A (an agent
that interferes with HMG-I(Y) binding to AT-rich sequences in the minor
groove of DNA) suppressed IL-1
-induced iNOS promoter activity and NO
accumulation in vascular smooth muscle cells (Fig. 8).
Our observation that proinflammatory cytokines are able to induce
HMG-I(Y) in vascular smooth muscle cells differs from the finding of
Thanos and Maniatis (9) in their study of NF-
B-dependent virus
induction of the IFN-
gene in human osteosarcoma cells. In their
study, viral infection did not induce transcription of the HMG-I(Y)
gene. Our observation that cytokines induce HMG-I(Y) gene transcription
in vascular smooth muscle cells may be related to our use of cells in
primary culture and our avoidance of a tumor-derived cell line (in
which HMG-I(Y) expression may have been higher at base line).
It has been suggested that the assembly of higher order nucleoprotein
complexes, consisting of different families of transcription factors,
may be a means of bringing together divergent signaling pathways to
activate a specific gene (10). The assembly of such complexes may
depend on architectural transcription factors like HMG-I(Y), which may
orchestrate this process. HMG-I(Y) is important in the regulation of
cytokine-driven genes, and we show in this report that HMG-I(Y) itself
is up-regulated by a proinflammatory stimulus in vascular smooth muscle
cells in primary culture and in aortic tissue in vivo.
Moreover, our experiments show that HMG-I(Y) may play an important role
in transactivation of the iNOS gene. This observation has relevance to
the pathophysiology of sepsis, an intense inflammatory response that
often results in hypotension and collapse of the circulatory system.