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B kinases 
and 
in serum- and
IL-1-induced human VSMC proliferation
Division of Nephrology, Department of Medicine and Tupper Research Institute, New England Medical Center Hospitals, Tufts University School of Medicine, Boston, Massachusetts 02111
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Interleukin-1 (IL-1) is a potent vascular smooth muscle
cell (VSMC) mitogen, which can stimulate cells via activation of
nuclear factor-
B (NF-B) following phosphorylation of its
inhibitory subunit (IB). Because the proliferative effect of IL-1 is
additive with that of serum, the present studies assessed the role of
IB kinases (IKKs) and NF-B in both IL-1- and serum-induced VSMC proliferation. IL-1
(1 ng/ml) induced marked and persistent NF-B activation in VSMC that was maximal at 1 h and persisted for 3 days.
There was a 3-fold increase in DNA synthesis after acute IL-1 exposure
(24-96 h) and a 12-fold increase after chronic IL-1 exposure (>7
days). Electrophoretic mobility shift assay and supershift analysis
indicated that IL-1-induced NF-B complexes consisted of p65/p50
heterodimers and p50 homodimers. Human saphenous vein smooth muscle
cells (HSVSMC) were transiently cotransfected with expression plasmids
encoding a dominant negative mutant form of either IKK
or IKK, in
which K44 was mutated to A (K44A), and a green fluorescent
protein expression plasmid that allows identification of transfected
cells. IL-1 induced nuclear localization of p65 in 95% of cells
transfected with vector alone but in only 69% and 26% of cells
expressing IKK (K44A) or IKK (K44A), respectively. Likewise,
proliferation increased 3.2-fold in IL-1-treated HSVSMC which had been
transfected with vector alone, but only 2.2- and 1.5-fold proliferation
in HSVSMC expressing IKK (K44A) or IKK (K44A), respectively.
Although serum activated NF-B transiently, serum-induced
proliferation was markedly attenuated in HSVSMC expressing IKK
(K44A) and IKK (K44A) compared with HSVSMC transfected with vector
alone. The results support an essential role of IKKs in the
proliferative response of HSVSMC to IL-1 and to serum.
nuclear factor-B; dominant negative mutant; cell transfection; interleukin-1; vascular smooth muscle cells
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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INAPPROPRIATE PROLIFERATION of vascular smooth muscle cells (VSMC) is a common pathogenetic feature of vascular diseases, including primary atherosclerosis, postangioplasty restenosis, and vein graft disease. Numerous studies (15, 25, 27, 28) have documented the importance of 42- and 44-kDa mitogen-activated protein kinases (p42/p44 MAPK) in linking receptor activation by classic growth factors, such as platelet-derived growth factor (PDGF), basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), and thrombin, to VSMC proliferation. The proinflammatory cytokine interleukin-1 (IL-1) also induces VSMC proliferation. However, in contrast to classic growth factors, IL-1 does not activate p42/p44 MAPK, and the mechanisms of its proliferative actions are unknown.
The IL-1 signaling pathway that has been the most extensively
characterized to date culminates in the activation of the nuclear factor-B (NF-B) family of transcription factors (3). NF-B consists of dimers of the Rel family of proteins (including p50, p52,
p65, c-Rel, and RelB) and is found in the cytosol of cells bound to
specific inhibitors or IBs. The binding of IL-1 to the type I IL-1
receptor results in the activation of NF-B-inducing kinase (NIK)
(24). NIK in turn associates with the IB kinase (IKK) complex, which
contains two kinase subunits, IKK and IKK. IKK-induced
phosphorylation of IB triggers its subsequent polyubiquitination and degradation, and the loss of IBs is thought to be responsible for the initial rapid nuclear localization of NF-B and
transcriptional activation (17).
Activation of NF-B has been previously linked to the process of VSMC
proliferation in vitro and in vivo. NF-B is present in VSMC nuclei
in the fibrotic-thickened areas of human atherosclerotic lesions but is
not found in VSMC nuclei in normal blood vessels (8). Balloon injury of
rat carotid artery also results in rapid and transient activation of
NF-B in medial smooth muscle cells (SMC) (11). Direct experimental
assessment of the role of NF-B in SMC proliferation has been
difficult because of the lack of a specific cell-permeable NF-B
inhibitor. Antioxidants, including N-acetyl-L-cysteine and pentoxifylline, inhibit
serum- and thrombin-induced NF-B activation and proliferation in
bovine SMC (6, 9) but may also inhibit other signaling pathways.
Single-cell microinjection of either IB or double-stranded decoy
oligonucleotides, which bind NF-B and thereby retain it in an
inactive state in the cytosol (6), inhibited serum-induced
proliferation of bovine VSMC. Also, antisense oligonucleotides, which
inhibit p65 synthesis, attenuate thrombin-induced proliferation of
human VSMC (29) and neointima formation following balloon angioplasty
in rats (2). Together these studies provide evidence that NF-B is an
important component of mitogenic signaling by thrombin and serum in
bovine SMC. The present studies used the method of transient expression
of dominant negative kinases to specifically assess the role of
upstream IKKs in the mitogenic responses of human VSMC to IL-1 and to serum.
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METHODS |
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Culture of smooth muscle cells derived from human saphenous vein.
Primary cultures of smooth muscle cells derived from human saphenous
vein (HSVSMC) were obtained by explant technique from saphenous veins
harvested for coronary artery bypass surgery at New England Medical
Center. Cells were cultured in DMEM supplemented with 10% FCS,
glutamine, penicillin, streptomycin, and Fungizone (growth medium); and
the medium was changed twice a week. Chronic IL-1-stimulated cells were
cultured as above, except the growth medium was also supplemented with
IL-1 (1 ng/ml) for at least 7 days before the experiment. After
reaching confluence, HSVSMC were passaged with trypsin (0.05%) and
EDTA (0.53 mM) and then plated for experiments at a density of 15,000 cells/cm2.
Nuclear extract preparation and electrophoretic mobility shift
assay analysis.
Nuclear extracts were prepared from HSVSMC by standard methods (12).
Briefly, cells were lysed for 15 min on ice in buffer containing 10 mM
HEPES (pH 7.4), 1.5 mM MgCl2, 10 mM KCl, and 0.5 mM
dithiothreitol (DTT), supplemented with protease inhibitors (Complete,
Boehringer Mannheim), 1 mM NaF, and 1 mM sodium orthovanadate. NP-40
was added to a final concentration of 0.5%, and nuclei were pelleted
by centrifugation (16,000 g for 30 s). The crude nuclear pellets were lysed on ice for 30 min in buffer containing 20 mM HEPES
(pH 7.4), 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 20% glycerol, and 0.5 mM
DTT, supplemented with protease and phosphatase inhibitors, and were
then cleared by centrifugation and stored in aliquots at
70°C.
-light chain enhancer B site (Ig-B;
5'-cgcTTA
GAGAG) or the
proximal human intercellular adhesion molecule 1 (hICAM-1)
promoter B-like site (5'-gcgTTAGCTTGGAAATTCCGGAGC) were
obtained from Genosys. Complementary single-stranded oligonucleotides were annealed, and double-stranded oligonucleotides containing CGC and
GCG 5' protruding ends were labeled with
[-32P]dCTP and
[-32P]dGTP using the Klenow fragment of DNA
polymerase I. Binding reactions were performed using standard protocols
(35). Nuclear proteins (5 µg) were preincubated (15 min, 4°C)
with or without competitor oligonucleotides in buffer containing 25 mM
HEPES (pH 7.6), 40 mM KCl, 5 mM MgCl2, 1 mM DTT, 3 µg of
double-stranded poly(dI-dC), and 8% Ficoll. For supershift assays,
reactions were preincubated for 45 min with antisera specific for
members of the Rel family. Radiolabeled double-stranded
oligonucleotides (105 counts/min) were then added, and
reactions were incubated for 30 min at 4°C. Oligonucleotide-protein
complexes were resolved on a 4% polyacrylamide gel in buffer
containing 6.7 mM Tris, 3.3 mM sodium acetate, and 1 mM EDTA (pH 7.9).
The gel was dried and exposed to X-ray film at 70 °C.
Bromodeoxyuridine incorporation.
HSVSMC were plated (5,000 cells/well) into 96-well plates in growth
medium with or without IL-1. After 72 h, the medium was replaced
with DMEM supplemented with 5-bromo-2'-deoxyuridine (BrdU), 0.5%
FCS, insulin (1 µM), and transferrin (5 µg/ml), with or without IL-1. After 24 h, the cells were fixed and incubated sequentially with a monoclonal BrdU antibody conjugated with peroxidase, and with
tetramethylbenzidine as substrate. BrdU incorporation was measured as
the absorbance of the converted substrate at 405 nm.
Assay of NF-B activation and proliferation in
transfected HSVSMC.
HSVSMC (~2 × 106 cells) were cotransfected with 20 µg of pGreenLantern-1 (GIBCO), encoding enhanced green fluorescent
protein (GFP) as a marker of transfected cells, and 40 µg of pRK5,
containing either no insert or cDNA encoding a kinase-inactive form of
IKK or IKK in which K44 was mutated to A
(K44A). In other studies, HSVSMC were transfected concurrently with IKK (K44A) and IKK (K44A) expression plasmids. Cells were transfected by electroporation at 230 V and 960 µF, plated
on glass coverslips, incubated for 6 h in growth medium containing 5 mM
sodium butyrate (37), washed, and then grown in growth medium
overnight. Medium was changed to DMEM supplemented with 1% FCS with or
without IL-1 (2 ng/ml ) 24 h after electroporation.
B activation in GFP-positive cells was analyzed 30 min after
stimulation with IL-1 by assessing the localization of p65 by
immunostaining. Cells were washed in PBS, fixed in 3.7% formaldehyde, and permeabilized in 0.2% Triton X-100/PBS. Nonspecific binding sites
were blocked with 10% normal horse serum, and the cells were incubated
sequentially with polyclonal rabbit anti-p65 and Texas Red-coupled
donkey anti-rabbit IgG. Cells were then observed through a Nikon
Diaphot microscope equipped with epifluorescence. Localization of p65
was evaluated in 100 transfected cells per treatment group.
Proliferation in GFP-positive cells was determined by immunostaining
for the proliferation-associated Ki-67 antigen. Fresh medium with or
without IL-1 was added 48 h after stimulation with IL-1, and cells
were fixed 96 h after initial IL-1 exposure. Immunostaining was
performed as described above, except the cells were permeabilized with
0.2% Tween 20-PBS, the primary antibody was mouse monoclonal MIB-1
antibody (Immunotech), and the secondary antibody was Texas Red-coupled
donkey anti-mouse IgG. The presence of nuclear or nucleolar Ki-67
staining was assessed in 100 transfected cells per treatment group.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Time course of IL-1-induced HSVSMC proliferation.
The mitogenic effect of IL-1 is distinct in that initial exposure to
IL-1 can induce growth-inhibitory pathways under some experimental
conditions (18, 22). In contrast, exposure to IL-1 for >3 days is a
consistently effective mitogenic stimulus for human VSMC (5,
22). In the present study, proliferation, assessed as the
rate of incorporation of BrdU, was unchanged during the first 24 h of
IL-1 exposure but increased subsequently between 24 and 96 h (Fig.
1). There was an approximately threefold
increase in maximal stimulation of DNA synthesis that occurred
48-72 h after initial exposure to IL-1. BrdU incorporation
increased almost 12-fold in HSVSMC cultured chronically in medium
supplemented with IL-1. The high rate of DNA synthesis in HSVSMC
that were cultured continuously in IL-1-supplemented medium was only
minimally attenuated when IL-1 was excluded from the growth medium
for 72 h before the BrdU incorporation assay. Thus IL-1 induces
proliferation of HSVSMC by a mechanism that requires a delay of >24
h. Chronic exposure to IL-1 enhances proliferation more than acute
exposure, and the high proliferative rate of chronically treated cells
persists for >72 h following removal of IL-1.
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HSVSMC express p65/p50 heterodimers and p50 homodimers.
Electrophoretic mobility shift assay (EMSA) analysis was performed with
a classic B probe (Ig-B) that contained the B motif found
in the murine Ig light chain enhancer. Nuclear proteins prepared
from human VSMC that had been treated chronically with IL-1 bound
specifically to the classic B probe, forming complexes I and
II, which migrate as two distinct bands (Fig.
2A). Specificity of the DNA binding
complexes was demonstrated by the ability of a 100-fold excess of
nonlabeled B oligonucleotides to completely abolish both complexes
(Fig. 2A). The composition of NF-B complexes formed in
different cell types, or in response to different stimuli, can be
distinct due to the existence of multiple Rel proteins. The subunit
compositions of complex I and II were determined by testing the ability of a panel of antisera directed against different Rel proteins to bind to the complexes and thereby slow their migration through a polyacrylamide gel (Fig. 2C). Incubation with
antisera directed against p50 before addition of the radiolabeled probe slowed the migration of both complexes I and II, which
appeared as two lower mobility bands. Preincubation with antisera
directed against p65 abolished complex I but did not affect the
migration of complex II. These results indicate that
complex I consists of p65/p50 heterodimers, whereas complex
II consists of p50 homodimers. In additional experiments,
preincubation with antisera directed against p52, RelB, or c-Rel did
not affect the migration of either complex I or complex
II (data not shown). Also, Western blot analysis confirmed the
presence of increased levels of p65 and p50 in nuclear extracts of
HSVSMC that had been treated chronically with IL-1. NF-B
complexes of similar mobility were obtained when HSVSMC derived from
three different patients were exposed to IL-1 for 1 h (Fig.
2D). Together the data indicate that HSVSMC derived from
different patients express p65/p50 heterodimers, which are transcriptionally active, and p50 homodimers, which are thought to be
transcriptionally inactive (4), after either acute or chronic exposure
to IL-1.
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-B probe used binds p65/p50 and p50/p50 readily but may not
detect all NF-B complexes. For example, c-Rel/p65 heterodimers and
p65 homodimers bind preferentially to the B-like site found in the
proximal region of the human ICAM-1 promoter, but they bind poorly to
the Ig-B sequence (1, 33). To determine whether HSVSMC that have
been chronically stimulated with IL-1 produce distinct NF-B
complexes, we also performed EMSA analysis with a radiolabeled probe
containing the proximal hICAM-1 B-like sequence (1). Two complexes
that migrated with the same mobility as complex I and
II were observed with the hICAM-1 B probe; however, their
intensities were markedly reduced compared with the intensities obtained with the Ig-B probe (data not shown). The complex that migrated identically to complex I was displaced with
p65-specific antisera. These observations indicate that the hICAM-1
B probe also detects p65/p50 and p50/p50 dimers, but it binds these
dimers with lower affinity than does the classic B probe. This
conclusion is supported by the fact that a 100- to 500-fold excess of
unlabeled hICAM-1 B oligonucleotide attenuated but did not abolish
binding of NF-B proteins to the radiolabeled Ig-B probe (Fig.
2B). Thus we obtained no evidence that HSVSMC that have been
stimulated chronically with IL-1 express other Rel dimers that bind
preferentially to hICAM-1 B-like sites.
IL-1 induces persistent activation of NF-B, whereas
FCS induces transient activation.
Many stimuli activate NF-B transiently, whereas other inducers can
cause persistent NF-B activation. Levels of complexes I and
II were low but detectable in nuclear extracts prepared from
HSVSMC before stimulation with IL-1 (Fig.
3). The levels of both NF-B complexes
were markedly increased after 1 h of stimulation with IL-1 and
remained elevated after 72 h. In contrast to the persistent effect of
IL-1, exposure to 10% FCS induced transient NF-B activation, which
was marked at 1 h but returned to baseline by 2 h (Fig.
4).
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induced sustained activation of NF-B,
whereas acute exposure to FCS induced transient activation, we compared
the persistence of nuclear NF-B levels in HSVSMC that were cultured
chronically in medium supplemented with FCS alone versus medium
supplemented with FCS and IL-1. When HSVSMC were cultured in medium
supplemented with 10% FCS alone, nuclear levels of complexes I
and II were low but detectable 72 h after the last medium
change (Fig. 3). This basal NF-B activation may represent low
residual stimulation by serum-derived mitogens or constitutive
expression in cultured human VSMC. In contrast, nuclear extracts
prepared from HSVSMC that had been cultured chronically in medium
supplemented with 10% FCS and IL-1 (1 ng/ml) contained markedly
increased levels of complexes I and II 72 h following the last medium change. Thus culture of HSVSMC in the presence of
IL-1 and 10% FCS produces constant NF-B activation, whereas NF-B activation is not sustained when HSVSMC are cultured in standard growth medium. Nuclear B binding complexes returned to
baseline 72 h after removal of IL-1 from the medium (Fig. 3), in
contrast to proliferation, which remained elevated (Fig. 1).
Expression of kinase-inactive IKKs inhibits IL-1-induced
NF-B activation and proliferation of HSVSMC.
In transient cotransfection experiments, expression of dominant
negative mutant forms of IKK or IKK inhibited IL-1-induced NF-B activation in HSVSMC. HSVSMC were cotransfected with a
GFP-encoding expression vector (Fig.
5A) and either expression vector
alone (pRK5) or expression vector encoding IKK (K44A) or IKK
(K44A). NF-B activation in the transfected (GFP-positive) cell
population was assessed by immunostaining for p65. p65 was distributed
throughout the cytosol of nonstimulated cells and appeared
predominately in the nucleus after a 30-min exposure to IL-1 (Fig.
5A). In three experiments, nuclear p65 was detected in only
1.2% of GFP-positive cells that had been transfected with vector alone
and incubated in 1% FCS (Fig. 6A),
and this percentage was not significantly affected by transfection with
either IKK (K44A) or IKK (K44A) expression plasmids (nuclear p65
was detected in 0.5% and 1.0%, respectively, of GFP-positive cells).
After a 30-min exposure to IL-1, p65 localized to the nucleus in nearly
all HSVSMC that had been transfected with vector alone (nuclear p65 was
detected in 93% of GFP-positive cells). IL-1-induced nuclear
localization of p65 was moderately inhibited in HSVSMC expressing
IKK (K44A) and markedly inhibited in HSVSMC expressing IKK (K44A)
(nuclear p65 was detected in 67% and 25%, respectively, of
GFP-positive cells).
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(K44A) or IKK (K44A) (13 ± 4% and 12 ± 3%, respectively, of GFP-positive cells). IL-1 induced a 3.2-fold
increase in nuclear Ki-67 antigen expression; Ki-67 antigen was
detected in the nuclei of 38 ± 8% of vector-transfected GFP-positive
cells that had been incubated with IL-1 for 96 h. IL-1-induced nuclear
Ki-67 antigen expression was inhibited by 53% in HSVSMC transfected
with IKK (K44A) expression plasmid (25 ± 6% of GFP-positive
cells) and inhibited by 75% in HSVSMC transfected with IKK (K44A)
expression plasmid (18 ± 6% of GFP-positive cells). Normalized
values for Ki-67 staining in GFP-positive cells are shown in Fig.
6B.
As with expression of IKK (K44A) or IKK (K44A) alone, concurrent
expression of IKK (K44A) and IKK (K44A) likewise did not
attenuate the level of nuclear Ki-67 antigen in HSVSMC incubated in 1%
FCS (Fig. 7B). Concurrent
expression of IKK (K44A) and IKK (K44A) also did not attenuate
IL-1-induced Ki-67 more than expression of IKK (K44A) alone (Fig.
7B vs. Fig. 6B).
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Expression of kinase-inactive IKKs inhibits serum-induced HSVSMC
proliferation.
To test whether the inhibitory effect of catalytically inactive IKKs
was specific to IL-1-induced proliferation, we performed cotransfection
experiments as described above except that HSVSMC were stimulated with
10% FCS following electroporation. In a total of five experiments,
serum stimulation induced a 3.2-fold increase in the expression of
Ki-67 antigen (Fig. 7A). Among GFP-positive cells transfected
with vector alone, nuclear Ki-67 antigen was expressed in 11 ± 2% of
cells incubated with 1% FCS, and incubation in 10% FCS increased this
percentage to 35 ± 6% of cells. Transfection with IKK (K44A)
expression plasmid attenuated FCS-induced Ki-67 antigen expression by
74% (P < 0.03), an effect similar in magnitude to the
attenuation of IL-1-induced Ki-67 expression (75%; Fig. 6B).
Furthermore, concurrent expression of kinase-inactive IKK and IKK
attenuated FCS-induced proliferation to a greater extent than
expression of kinase-inactive IKK alone. FCS-induced Ki-67 antigen
expression was attenuated by 88% in HSVSMC that were cotransfected with both IKK (K44A) and IKK (K44A) expression plasmids (Fig. 7B; P < 0.01, n = 3 experiments).
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DISCUSSION |
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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The present studies provide evidence that IB kinase activity is
crucial to the proliferative responses of human VSMC to IL-1 and to
serum. The type I IL-1 receptor is part of a multimeric membrane
complex, which recruits TRAF6 (10), a member of the tumor necrosis
factor (TNF) receptor-associated factor family, following IL-1 binding.
TRAF6 then activates TAK-1, which in turn associates with and activates
NIK (24). NIK phosphorylates and activates two kinases, IKK and
IKK, both of which appear essential for the phosphorylation of
IB on serines 32 and 36 (13, 26, 34, 38, 39). This specific
phosphorylation of IB targets it for rapid ubiquitination and
degradation, resulting in the prompt liberation and nuclear
translocation of NF-B (32). Previous studies (34, 38) have shown
that mutation of a single conserved lysine within the kinase domain of
either IKK or IKK (K44 mutated to A) yields a
catalytically inactive enzyme that acts as a dominant negative
inhibitor of IL-1- and TNF-induced NF-B activation when expressed in
cells. In the present study, expression of catalytically inactive
IKK or IKK in human VSMC inhibited both IL-1-induced NF-B
activation and IL-1-induced proliferation. These results establish a
crucial role for IB kinase activity in the mitogenic response of
HSVSMC to IL-1.
The present results are consistent with the notion that both IKK and
IKK are essential for IL-1-induced phosphorylation and degradation
of IB and subsequent nuclear localization of p65. However, IKK
(K44A) was more effective than IKK (K44A) as an inhibitor of both
IL-1-induced NF-B activation and proliferation when expressed in
HSVSMC. In addition, concurrent expression of IKK (K44A) and IKK
(K44A) was not more effective than IKK (K44A) alone. In previous
studies, expression of IKK (K44A) has been consistently shown to
inhibit NF-B activation, whereas the effectiveness of IKK mutants
has been variable. IKK (K44A) was an effective inhibitor of
NF-B-dependent transcription in 293 cells, although it was somewhat
less effective than IKK (K44A) when equivalent amounts of plasmid
DNA were used in the transfection assay (34, 38). In other studies (26,
39), IKK (K44M) was also catalytically inactive, but its expression
produced little or no inhibition of TNF-induced nuclear translocation
of NF-B in HeLa cells. Similar results have been obtained using
targeted gene disruption. IL-1-induced NF-B activation was not
attenuated in fibroblasts prepared from mouse embryos that were
deficient in IKK (36) but was significantly attenuated in
fibroblasts prepared from IKK-deficient mouse embryos (21). However,
it is possible that the absence of IKK activity during embryonic
development results in a compensatory upregulation of IKK, thereby
masking a significant role of IKK that occurs in normal animals. Our
results, together with previous reports, suggest that catalytically
inactive IKK is a more reliable inhibitor of IL-1-induced NF-B activation.
NF-B binds to DNA as homo- or heterodimers of the Rel-related
proteins, including p50, p52, p65, c-Rel, and RelB (4, 23). p65, RelB,
and c-Rel are strong transcriptional activators, whereas p50 and p52
are weak transactivators that can form active dimers with p65, RelB,
and c-Rel. The existence of multiple Rel family members provides the
basis for potential diversity in the specific composition of NF-B
complexes produced in response to different inducers, in different cell
types, or in the same cell type of different individuals or different
species. In the present study, classic NF-B complexes consisting of
p50/p65 heterodimers and p50/p50 homodimers were induced in SMC derived
from saphenous veins of several different patients, following either
acute or chronic stimulation with IL-1. Also, nonclassic NF-B
complexes that bind preferentially to an hICAM-1 B-like
motif were not induced by either acute or chronic IL-1 stimulation.
These results are in agreement with previous studies (7) in which
HSVSMC expressed classic NF-B complexes after acute exposure to
IL-1. The present studies did not address the possibility that SMC
derived from different human vascular beds or from different species
express distinct NF-B complexes. It has been suggested (19) that SMC derived from bovine pulmonary artery express a heterodimer of p50 and a
novel SMC-specific Rel protein distinct from p65. Diversity of NF-B
complexes formed in different VSMC may contribute to differential
responses of these cells to inflammatory signals.
Although IL-1 elicits a rapid activation of NF-B in HSVSMC, as
occurs in other cell types, the mitogenic effect was delayed. An early
effect of IL-1-induced NF-B activation is induction of
cyclooxygenase-2 gene expression (5) and the subsequent generation of
prostanoids that can inhibit the early mitogenic response to IL-1 (22).
Thus production of growth inhibitory prostanoids may inhibit DNA
synthesis during the first 24-h exposure to IL-1, but prostanoid
synthesis may subside after 24 h (5), allowing the mitogenic effect of
IL-1 to be expressed. DNA synthesis then persisted for up to 96 h after
exposure to IL-1, as did NF-B activation. Nuclear levels of NF-B
were not enhanced during the chronic phase of IL-1 stimulation relative
to the acute phase and, thus, could not account for the enhanced
proliferative effect of chronic versus acute exposure to IL-1. It is
possible that the HSVSMC cultures used in the present study consisted
of a mixed population of IL-1-responsive and -nonresponsive cells. In
this case, chronic culture in IL-1-supplemented medium may select for IL-1-responsive cells and thereby elicit a greater mitogenic effect than acute exposure to IL-1.
Remarkably, expression of a dominant negative mutant form of IKK
markedly attenuated serum-induced proliferation, and concurrent expression of dominant negative mutant IKK attenuated serum-induced proliferation further. These results indicate that the activity of
IKK and IKK are also crucial for the mitogenic effect of serum-derived growth factors. These results agree with a previous report (6) that single-cell microinjection of IB or
double-stranded decoy oligonucleotides that bind and inactivate NF-B
largely attenuate serum-induced proliferation in bovine VSMC. Also,
antisense oligonucleotides that inhibit p65 synthesis attenuate
thrombin-induced proliferation of human VSMC (29). However, our studies
further identify upstream IB kinase activity as a crucial component
of serum-stimulated VSMC proliferation. It remains to be established whether serum-induced proliferation is dependent on constitutive or
serum-inducible IKK activity. The low levels of NF-B expressed in
the nucleus of nonstimulated cells may result from constitutive IKK
activity. Also, serum-induced NF-B activation in HSVSMC may be
mediated by a transient induction of IKK activity. The upstream pathways that are involved in either constitutive or serum-induced IB kinase activity in human VSMC are also not known. It is possible that serum activates NF-B via a pathway that involves sequential activation of NIK and IKK. NIK is structurally related to MEKK1 (MAPK kinase kinase-1), a kinase of the JNK (c-Jun
NH2-terminal kinase) cascade that is activated by the small
G proteins Rac and Cdc42 (14). MEKK1 can activate both IKK and
IKK (20, 30), presumably through phosphorylation of specific
conserved serine residues within the activation loops of the kinases.
Thus an alternative possibility is that serum-induced or constitutive NF-B activation involves MEKK1-induced activation of IKK and IKK.
In a previous study, we have shown that the mitogenic effect of IL-1 is
synergistic with that of serum. The addition of IL-1 to the culture
medium markedly enhances the proliferative rate of HSVSMC incubated in
the presence of high levels of FCS (20%) (5). This finding suggests
that IL-1 activates promitogenic signaling pathways that are not
optimally stimulated by serum-derived mitogens. The present study
documents that IL-1 induces persistent activation of NF-B, whereas
serum induces only transient activation. Also, HSVSMC grown in
IL-1-supplemented medium have continuously active NF-B, whereas
HSVSMC cultured in standard VSMC growth medium undergo only transient
activation of NF-B with each medium change. Persistent activation of
NF-B may account for the ability of both acute and chronic exposure
to IL-1 to enhance serum-induced proliferation of human VSMC. These
results are consistent with the previous observation that growth
factors, including serum, PDGF, bFGF, EGF, and thrombin, activate
NF-B in rat aortic SMC and that the more potent growth factors
elicit a stronger NF-B response (31). Together these studies suggest
that the magnitude and persistence of NF-B activation is rate
limiting for VSMC proliferation. However, given the important role of
p42/p44 and other MAPK pathways in VSMC proliferation, it is likely
that NF-B is a necessary but insufficient stimulus to VSMC
proliferation, as previously suggested (9).
In conclusion, the present studies provide evidence that the
proliferative response of human VSMC to IL-1 requires activation of
both IKK and IKK and persistent activation of NF-B. Although stimulation with serum induces only transient NF-B activation, IKK and IKK activity are also crucial to serum-induced HSVSMC proliferation. The upstream pathways that contribute to IKK activation in serum-stimulated HSVSMC remain to be established. Inhibition of IKKs
may represent a potential therapeutic alternative for inhibition of the
excessive VSMC proliferation that can contribute to the pathogenesis of
atherosclerosis and to the failure of vascular surgeries such as
balloon angioplasty and insertion of vascular access grafts.
| |
ACKNOWLEDGEMENTS |
|---|
We gratefully acknowledge Tawnya Gannon for technical assistance
and Dr. Tatiana Lebedeva for helpful advice concerning EMSA analysis.
Human recombinant IL-1 was kindly provided by Dr. Richard Dondero,
Cistron Biotechnology (Pine Brook, NJ), and IKK expression plasmids
were generously provided by Dr. David V. Goeddel (Tularik, South San
Francisco, CA). This work was supported by National Heart, Lung, and
Blood Institute Grant HL-47569.
| |
FOOTNOTES |
|---|
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: D. Beasley, New England Medical Center, Box 172, 750 Washington St., Boston, MA 02111 (E-mail: dbeasley{at}lifespan.org).
Received 4 August 1999; accepted in final form 24 November 1999.
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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