From the Department of Molecular Medicine, Beth
Israel Deaconess Medical Center, Harvard Medical School, Boston,
Massachusetts 02215, the § Laboratory for Systems Biology
and Medicine at Research Center for Advanced Science and
Technology, the University of Tokyo, Tokyo 153-8904, Japan, and
the ¶ Joslin Diabetes Center, Harvard Medical School, Boston,
Massachusetts 02215
Received for publication, September 3, 2002, and in revised form, December 2, 2002
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ABSTRACT |
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We recently demonstrated that thrombin induces
the expression of vascular adhesion molecule-1 (VCAM-1) in endothelial
cells by an NF- Vascular adhesion molecule-1
(VCAM-1)1 is a 110-kDa cell
surface glycoprotein that is expressed in cytokine-activated
endothelial cells (1). The VCAM-1 promoter, originally cloned and
characterized in cultured endothelial cells (2), represents a
potentially valuable tool for dissecting the molecular mechanisms of
endothelial cell activation. Several studies have demonstrated the
importance of two tandem NF- In a recent report, we showed that the incubation of endothelial cells
with thrombin or the PAR-1 agonist, thrombin receptor activation
peptide (TRAP), resulted in increased VCAM-1 mRNA levels and VCAM-1
promoter activity (8). Not surprisingly, a mutation of the tandem
NF- In this study, we provide evidence for the importance of PKCs in
coupling PAR-1 signaling to NF- Several isoforms of PKC have been identified in cultured endothelial
cells, including PKC- We show here that thrombin stimulates binding of NF- Materials--
Human thrombin and TRAP (SFLLRNPNDKYEPF) were
obtained from Sigma (St. Louis, MO). Rottlerin, PD98059, SB203580,
LY294002, bisindolylmaleimide I (BIM), Gö6976 were obtained from
Calbiochem (San Diego, CA). Myristoylated PKC- Cell Culture--
Human umbilical vein endothelial cells (HUVEC)
and human coronary artery smooth muscle cells (HCASMC) (Clonetics, La
Jolla, CA) were cultured in EGM-2 MV and SmGM-2 complete media,
respectively. HUVEC and HCASMC were used within the first eight passages.
Plasmids--
The construction of the VCAM-1-luc plasmid and
GATA4-TK-luc was previously described (8). To generate
NF- RNA Isolation, Northern Blot Analysis, and RNase Protection
Assays--
HUVEC were serum-starved in EBM-2 MV medium containing
0.5% FBS. 18 h later, HUVEC were pretreated for 30 min with
PD98059, SB203580, LY294002, BIM, Gö6976, or myristoylated
PKC- Transfections and Analysis of Luciferase Activity--
HUVEC
were transfected using FuGENE 6 reagent (Roche Molecular Biochemicals,
Indianapolis, IN) as previously described (8). Briefly, HUVEC (1 × 105 cells/well) were seeded in 12-well plates 18-24 h
before transfection. 0.5 µg of the reporter gene construct and 50 ng
of a control plasmid containing the Renilla luciferase
reporter gene under the control of a cytomegalovirus (CMV)
enhancer/promoter (pRL-CMV) (Promega, Madison, WI) were incubated with
2 µl of FuGENE 6. 24 h later, the cells were washed with
phosphate-buffered saline two times and cultured for 18 h in
serum-starved medium (EBM-2 plus 0.5% FBS). The serum-starved cells
were preincubated for 30 min with rottlerin, PD98059, SB203580,
LY294002, BIM, Gö6976, or myristoylated PKC- Nuclear Extracts and Electrophoretic Mobility Shift
Assays--
Nuclear extracts were prepared as previously described (8,
21). Briefly, double-stranded oligonucleotides were labeled with
[ Thrombin Induces VCAM-1 mRNA Expression in Endothelial Cells
through PI3K, PKC, and p38 MAPK-dependent Signaling
Pathways--
In a previous study, we demonstrated that thrombin
induces VCAM-1 mRNA levels and VCAM-1 promoter activity in primary
human endothelial cells and that this effect is mediated by tandem
NF- Thrombin-mediated PI3K-dependent Induction of VCAM-1
mRNA in Endothelial Cells Is Mediated by PKC-
In addition to Akt, a number of other signaling molecules lie
downstream of PI3K. Some of these mediators, such as p90RSK, are
activated by MAPK, and therefore should be inhibited by PD98059. In
contrast, activation of PKC isoforms may be regulated in a PI3K/PDK-1-dependent, MAPK-independent manner. Indeed,
overexpression of a dominant negative form of PKC- Thrombin-mediated PI3K-dependent Induction of VCAM-1
Promoter Activity in Endothelial Cells Is Mediated by PKC- Thrombin-mediated Induction of NF- Thrombin-mediated Induction of GATA Binding to the VCAM-1 Promoter
Is Regulated by a PI3K- and PKC- Thrombin-mediated Transactivation of a Minimal Promoter Containing
Tandem VCAM-1 NF- Thrombin Fails to Induce VCAM-1 mRNA Expression in Vascular
Smooth Muscle Cells--
It is well established that thrombin
signaling occurs in VSMC. Moreover, IL-4 has been shown to induce
VCAM-1 mRNA in VSMC (23, 24). In the next set of experiments, we
wished to determine whether thrombin induces VCAM-1 expression in VSMC.
As shown in Fig. 7A, the
incubation of HCASMC with 10 ng/ml IL-4, but not 1.5 units/ml thrombin,
resulted in increased levels of VCAM-1 mRNA. In contrast to these
results, thrombin and TRAP, but not IL-4, resulted in a marked increase
in tissue factor expression in HCASMC, with maximal levels
occurring at 4 h (Fig. 7, B-D). Taken together, these
results suggest that thrombin-mediated induction of VCAM-1 mRNA is
specific to endothelial cells and that the absence of response in VSMC
is attributable to a defect at the post-receptor level.
Thrombin Fails to Induce NF- We have shown that thrombin induces VCAM-1 expression specifically
in endothelial cells via PI3K-PKC-B- and GATA-dependent mechanism. In the
present study, we describe the signaling pathways that mediate this
response. Thrombin stimulation of the VCAM-1 gene and promoter in human umbilical vein endothelial cells was inhibited by preincubation with
the phosphatidylinositol 3-kinase inhibitor, LY294002, the protein
kinase C (PKC)-
inhibitor, rottlerin, a PKC-
peptide inhibitor,
or by overexpression of dominant negative (DN)-PKC-
. In
electrophoretic mobility shift assays, thrombin-mediated induction of
NF-
B p65 binding to two NF-
B motifs in the upstream promoter region of VCAM-1 was blocked by LY294002 and rottlerin, whereas the
inducible binding of GATA-2 to a tandem GATA motif was inhibited by
LY294002 and the PKC-
peptide inhibitor. In co-transfection assays,
thrombin stimulation of a minimal promoter containing multimerized
VCAM-1 NF-
B sites was inhibited by DN-PKC-
but not DN-PKC-
. In
contrast, thrombin-mediated transactivation of a minimal promoter
containing tandem VCAM-1 GATA motifs was inhibited by DN-PKC-
but
not DN-PKC-
. Finally, thrombin failed to induce VCAM-1 expression in
vascular smooth muscle cells. Taken together, these data suggest that
the endothelial cell-specific effect of thrombin on VCAM-1 expression
involves the coordinate activity of PKC-
-NF-
B and
PKC-
-GATA signaling pathways.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B elements located at position
77 and
63, relative to the transcriptional start site, in transducing the response to inflammatory mediators (2-4). Other studies have provided
evidence for the role of co-stimulators in mediating cytokine response,
including Sp1 (5), activating protein-1 (6), and interferon
regulatory factor-1 (7).
B motif blocked the response to thrombin. Moreover, in
electrophoretic mobility shift assays, thrombin induced the binding of
p65 homodimers to the two adjacent NF-
B sites in the VCAM-1
promoter. A more interesting finding was that a mutation of a tandem
GATA motif (located at
244 and
259) also attenuated the thrombin
response. Consistent with these results, thrombin treatment resulted in
increased binding of GATA-2 to the VCAM-1 promoter. These data
suggested that thrombin-mediated induction of VCAM-1 involves the
coordinate activity of NF-
B p65 and GATA-2 and raised new questions
as to how the PAR-1 receptor was linked to the downstream transcription
factors and whether or not the PAR-1-VCAM-1 pathway was specific to
endothelial cells.
B- and GATA-dependent
expression of VCAM-1 in endothelial cells. The PKCs comprise a family
of structurally related serine/threonine protein kinase isoforms that
play a key role in divergent signaling pathways and cellular functions
(9-11). The PKCs are classified into three subgroups, based on
structural differences in their regulatory domains and mode of
activation. The conventional or classic PKCs (cPKC-
, -
1, -
2, and -
) are activated by
Ca2+, diacylglycerol (DAG), and phorbol esters; the novel
PKCs (nPKC-
, -
, -
, and -
) are activated by DAG and phorbol
esters, but not Ca2+; and the atypical PKCs (aPKC-
and
-
) are activated by Ca-, DAG-, and phorbol ester-independent
mechanisms. PKC isoforms are differentially distributed between
tissues, cell types, and subcellular compartments. Moreover, the
various PKC isoforms vary in their response to extracellular signals,
substrate specificities, and cellular functions.
, -
, -
, -
, and -
(12, 13). In
contrast, PKC-
1 and PKC-
are undetectable in endothelial cells
(12, 13). The various PKC isoforms have been shown to differ in their
spatial distribution both in resting endothelial cells and in response
to extracellular mediators (12). PKCs have been implicated in multiple
endothelial cell functions, including expression of adhesion molecules
(14), and endothelin-1 (15), proliferative response to growth factors
(16), shear stress signaling (17), and angiogenesis (18).
B p65 homodimers
via a PI3K- and PKC-
-dependent pathway and
binding of GATA-2 through a PI3K- and PKC-
-dependent
signaling cascade. Moreover, we demonstrate that thrombin induces
VCAM-1 expression in endothelial cells but not vascular smooth muscle
cells (VSMC). Taken together, these findings provide novel information
about cell type-specific thrombin signaling.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
pseudosubstrate
peptide inhibitor was obtained from BIOMOL (Plymouth, PA), and human
IL-4 was from Peprotec (Rocky Hill, NJ).
B8-TK-luc, a double-stranded oligonucleotide
containing four copies of the tandem NF-
B motifs from the VCAM-1
promoter was cloned into XhoI-digested TK-luc plasmid
vector. Insert direction was confirmed by automated DNA sequencing. The
DN (kinase-dead)-PKC-
and DN-PKC-
expression plasmids were
obtained from Pradip Majumder (Dana Farber Cancer Institute, Boston,
MA) and Christopher Carpenter (Beth Israel Deaconess Medical Center,
Boston, MA), respectively.
peptide at the doses indicated and then incubated in the
absence or presence of 1.5 units/ml human thrombin for 4 h.
Alternatively, HUVEC were infected with adenoviruses encoding the
cDNAs of
-galactosidase, dominant negative T308A, S473A-Akt
(DN-Akt), constitutively active Gag-Akt (CA-Akt), dominant negative
PKC-
(DN-PKC-
), or wild-type PKC-
(WT-PKC-
). The
recombinant viruses DN-Akt and CA-Akt were a kind gift of Kohjiro Ueki
(Joslin Diabetes Center, Boston, MA). The recombinant viruses
DN-PKC-
and WT-PKC-
were generated as previously described (19).
Infections were carried out at a multiplicity of infection of 20 for
-galactosidase, CA-Akt, and DN-Akt and a multiplicity of infection
of 10 for green fluorescence protein, DN-PKC-
, and WT-PKC-
for
12 h. Infected cells were grown in complete medium for another
12 h, serum-starved in 0.5% FBS for 12 h, and then treated
in the absence or presence of 1.5 units/ml thrombin for 4 h.
HCASMC were serum-starved in SmGM-2 medium containing 0.5% FBS.
18 h later, cells were incubated in the absence or presence of 10 ng/ml human IL-4, 1.5 units/ml human thrombin, or 20 nmol/ml TRAP.
HUVEC and HCASMC were harvested for total RNA at the times indicated,
using the TRIzol reagent (Invitrogen, Gaithersburg, MD). 10 µg of
total RNA was electrophoresed on the 1.4% agarose formaldehyde gel,
transferred to a nylon membrane, and hybridized for 18 h at
42 °C with 106 cpm/ml [32P]dCTP-labeled
human VCAM-1 or tissue factor cDNA probes. The signals were
quantified with Image (National Institutes of Health), and statistical
analyses were carried out using the Student t test. Cell
migration assays were carried out by modified Boyden chamber, as
previously described (20).
peptide inhibitor
and then incubated with 1.5 units/ml thrombin for 6 h. The cells
were lysed and assayed for luciferase activity using the
Dual-luciferase reporter assay system (Promega) and Lumat LB 9507 luminometer (Berthold, Gaithersburg, MD). For co-transfections, 0.4 µg of the reporter gene construct, 0.4 µg of the PKC expression vector, and 50 ng of pRL-CMV were incubated with 2 µl of FuGENE 6. The cells were then incubated in the presence or absence of 1.5 units/ml thrombin for 6 h, at which time cells were lysed and
assayed for luciferase activity as described above.
-32P]dCTP and Klenow fragment and purified by spun
column (Amersham Biosciences, Piscataway, NJ). 10 µg of HUVEC nuclear
extracts was incubated with 10 fmol of 32P-labeled probe, 1 µg of poly(dI-dC), and 3 µl of 10× binding buffer (100 mM Tris HCl (pH 7.5), 50% glycerol, 10 mM
dithiothreitol, 10 mM EDTA) for 20 min at the room
temperature, followed by 30 min at 4 °C. The following
oligonucleotides sequences were used for probes: VCAM-1 NF-
B
motifs, 5'-TGCCCTGGGTTTCCCCTTGAAGGGATTTCCCTCCGCCTC-3'; VCAM-1 GATA
motifs, 5'-ATTGTCCTTTATCTTTCCAGTAAAGATAGCCTTTT-3'. To test the
effect of antibodies on DNA-protein binding, nuclear extracts were
preincubated with antibodies of p65 (Santa Cruz Biotechnology, Santa
Cruz, CA) for 30 min at room temperature or with antibodies to GATA-2,
GATA-3, or GATA-6 (Santa Cruz) for 1 h at 4 °C. DNA-protein
complexes were resolved on a 5% non-denaturing polyacrylamide gel
containing 5% glycerol in 0.5× TBE (50 mM Tris, 50 mM boric acid, and 1 mM EDTA). The loaded gel
was fixed with 10% methanol and 10% acetic acid then
autoradiographed. Electrophoretic mobility shift assays were carried
out in triplicate, using independent preparations of nuclear extracts.
The signals were quantified with NIH Image, and statistical analyses
were carried out using the Student t test.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B and GATA motifs (8). In this study, we wished to delineate the
signaling pathways that couple the thrombin receptor to the inducible
binding of NF-
B and GATA. To that end, we employed Northern blot
analyses to assay for thrombin-mediated induction of VCAM-1 mRNA in
the absence or presence of various protein kinase inhibitors.
Thrombin-mediated induction of VCAM-1 mRNA expression was inhibited
53% by 1 µM BIM (Fig. 1,
lane 4), 67% by 5 µM BIM (Fig. 1, lane
5), 89% by 10 µM LY294002 (Fig. 1, lane
7), 95% by 50 µM LY294002 (Fig. 1, lane
8), 22% by 2.5 µM SB203580 (Fig. 1, lane
10), and 53% by 20 µM SB203580 (Fig. 1, lane
11). In contrast, thrombin stimulation of VCAM-1 was unaltered by
10-50 µM PD98059 (Fig. 1, lanes 13 and
14). Consistent with these results, thrombin-mediated induction of the 1.8-kb human VCAM-1 promoter (spanning the region between
1716 and +119, relative to the start site of transcription) was inhibited 95% by LY294002, 65% by BIM and 67% by SB203580 but
not by PD98059 (data not shown). Taken together, these findings suggest
that thrombin stimulation of VCAM-1 is mediated by PI3K-, PKC-, and p38
MAPK-dependent, ERK1/2-independent signaling
pathway(s).
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Fig. 1.
Thrombin induces VCAM-1 mRNA expression
in primary endothelial cells by a PI3K-, PKC-, and p38
MAPK-dependent signaling pathway. Northern blot
analyses of VCAM-1 in serum-starved HUVECs preincubated for 30 min with
1 µM BIM (lane 4), 5 µM BIM
(lanes 3 and 5), 10 µM LY294002
(lane 7), 50 µM LY294002 (lanes 6 and 8), 2.5 µM SB203580 (lane 10),
20 µM SB203580 (lane 9 and 11), 10 µM PD98059 (lane 13), and 50 µM
PD98059 (lanes 12 and 14), and treated in the
absence ( ) or presence (+) of 1.5 units/ml thrombin for 4 h. The
results are representative of three independent experiments.
B, quantification of Northern blot analyses. Densitometry
was used to calculate the ratio of VCAM-1 and 28 S signals. The results
show the means and standard deviations of -fold induction relative to
untreated cells obtained from three independent experiments.
and PKC-
but
Not Akt--
PI3K lipid products have been shown to activate several
downstream substrates. Perhaps the best understood and most widely studied of these targets is Akt. To determine whether the thrombin signal was dependent on Akt, endothelial cells were infected with adenovirus expressing
-galactosidase, constitutively active Akt (CA-Akt), or dominant negative Akt (DN-Akt). Thrombin-mediated induction of VCAM-1 was not inhibited by the overexpression of DN-Akt
(Fig. 2A, lane 6). Moreover,
the overexpression of CA-Akt failed to induce VCAM-1 expression (Fig.
2A, lane 3). As a positive control, the
overexpression of the DN-Akt adenovirus was shown to inhibit
insulin-induced glycogen synthase kinase-3 in L6 myoblast cells (data
not shown). More importantly, DN-Akt inhibited vascular epidermal
growth factor-mediated induction of endothelial cell migration, whereas
CA-Akt was sufficient in inducing endothelial cell migration (Fig.
2B). These findings, which are similar to those of a
previous study (22), suggest that the DN-Akt is functioning as a
dominant negative in cultured endothelial cells. Therefore, we conclude
that Akt is neither necessary nor sufficient for mediating the thrombin
response.
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Fig. 2.
Thrombin-mediated, PI3K-dependent
up-regulation of VCAM-1 in endothelial cells is mediated by
PKC- and PKC-
, but
not AKT. A, Northern blot analyses of VCAM-1 in HUVECs
infected with adenovirus expressing
-galactosidase (lanes
1 and 2), constitutively active Akt (lanes 3 and 4), or dominant negative Akt (lanes 5 and
6), treated in the absence (
) or presence (+) of 1.5 units/ml thrombin for 4 h. B, migration assay, as
measured by modified Boyden apparatus, of endothelial cells expressing
either
-galactosidase (Adv), dominant negative Akt, or
constitutively active Akt, treated in the absence or presence of 50 ng/ml vascular epidermal growth factor for 4 h. Results represent
the mean ± S.D. of three independent experiments. C,
Northern blot analyses of VCAM-1 in HUVECs infected with adenovirus
green fluorescent protein (lanes 1 and 2), wild
type PKC-
(lanes 3 and 4), or dominant
negative PKC-
(lanes 5 and 6), treated in the
absence (
) or presence (+) of 1.5 units/ml thrombin for 4 h.
D, uninfected HUVECs were serum-starved, preincubated for 30 min with 1 µM Gö6976 (lanes 3 and
4), 10 µM rottlerin (lanes 5 and
6), or 10 µM myristoylated PKC-
peptide
inhibitor (lanes 7 and 8), and then treated in
the absence (
) or presence (+) of 1.5 units/ml thrombin for 4 h.
The results are representative of two independent experiments.
resulted in
significant attenuation (70%) of thrombin response (Fig.
2C, lane 6). Moreover, the overexpression of
wild-type PKC-
resulted in super-induction of VCAM-1 by thrombin (1.8-fold) (Fig. 2C, lane 4). Consistent with
these results, the preincubation of HUVEC with a myristoylated PKC-
peptide inhibitor resulted in a profound reduction (75%) of
thrombin-mediated VCAM-1 stimulation (Fig. 2D, lane
8). Interestingly, the addition of the PKC-
inhibitor,
rottlerin, resulted in complete loss (97%) of thrombin-mediated
induction of VCAM-1 (Fig. 2D, lane 6). Together, these data suggest that thrombin stimulation of VCAM-1 is mediated by
PI3K-dependent, PKC-
and PKC-
signaling pathways.
Finally, the addition of the classic PKC inhibitor, Gö6976,
resulted in partial inhibition (38%) of the thrombin response (Fig.
2D, lane 4), supporting a role for one of the
classic PKC isoforms, namely cPKC-
, -
1,
-
2, or -
.
and
PKC-
--
To establish a role for PKC-
and PKC-
in mediating
thrombin stimulation of the VCAM-1 promoter, HUVECs were transiently transfected with the VCAM-1-luc plasmid and treated with thrombin in
the presence or absence of PKC isoform-selective inhibitors. As shown
in Fig. 3, thrombin-mediated induction of
VCAM-1 promoter activity was almost completely inhibited by rottlerin
and the myristoylated PKC-
peptide inhibitor (93 and 80%
inhibition, respectively) and partially inhibited by Gö6976 (53%
inhibition). These findings are consistent with those of the Northern
blot assays. Together, the data suggest that PKC-
and PKC-
lie
downstream of PI3K in the PAR-1-VCAM-1 signaling pathway.
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Fig. 3.
Thrombin-mediated induction of VCAM-1
promoter activity in endothelial cells is mediated by
PKC- and PKC-
.
HUVECs were transiently transfected with VCAM-1-luc plasmid,
preincubated for 20 min with 1 µM Gö6976, 10 µM rottlerin, or 10 µM myristoylated
PKC-
peptide inhibitor, and then treated in the absence or presence
of 1.5 units/ml thrombin for 6 h. The results show the means ± S.D. of luciferase light units (relative to untreated cells)
obtained in triplicate from four independent experiments. Luciferase
light units were corrected for transfection efficiency as described
under "Experimental Procedures." *, p < 0.001 compared with thrombin treatment, no inhibitor.
B Binding to the VCAM-1
Promoter Is Regulated by a PI3K- and PKC-
-dependent
Signaling Pathway--
We have previously shown that thrombin-mediated
induction of VCAM-1 involves the inducible binding of p65 homodimers
and GATA-2 to tandem NF-
B and GATA motifs, respectively (8). To
determine the role of the PI3K, PKC-
, and PKC-
signaling pathways
in mediating DNA-protein binding, we carried out electrophoretic
mobility shift assays. In the first set of experiments, a radiolabeled
double-stranded oligonucleotide probe containing the two adjacent
NF-
B sites in the VCAM-1 promoter was incubated with nuclear
extracts derived from untreated and thrombin-treated HUVECs. Incubation
with nuclear extracts from untreated or thrombin-treated HUVECs
resulted in specific DNA-protein complexes (open arrows,
Fig. 4). These DNA-protein complexes were
inhibited by the addition of a 50-fold molar excess of unlabeled
self-competitor but not by the same concentration of unlabeled NF-
B
mutant competitor (data not shown). The faster migrating DNA-protein
complex (closed arrow) was nonspecific, because it was
inhibited by the addition of both wild type and mutant competitors
(data not shown). As previously reported (8), the addition of thrombin
resulted in a marked increase in DNA binding activity (Fig. 4; compare
lane 3 with lane 2). Also consistent with our
previous results, the addition of anti-p65 antibody resulted in a
supershift of the specific DNA-protein complexes (asterisk, Fig. 4; lane 13). Thrombin-mediated induction of NF-
B
binding was inhibited ~75% by preincubation of cells with rottlerin
or LY294002 (Fig. 4, lanes 4 and 7), and 22-24%
by preincubation with Gö6976 or BIM, respectively (Fig. 4,
lanes 8 and 9). In contrast, NF-
B DNA-protein
complexes were unaffected by preincubation with the PKC-
peptide
inhibitor, PD98059, or SB203580 (Fig. 4, lanes 5,
6, and 10). Together, these results suggest that
thrombin-mediated binding of NF-
B to the VCAM-1 promoter is
regulated by a PI3K, PKC-
-dependent pathway.
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Fig. 4.
Thrombin induces binding of
NF- B p65 homodimers to the VCAM-1 promoter via
a PI3K, PKC-
-dependent signaling
pathway. A, electrophoretic mobility shift assays were
performed with 32P-labeled NF-
B probe in the absence
(lane 1) or presence of 10 µg of nuclear extract from
HUVECs preincubated for 30 min with vehicle (lanes 2 and
3), 10 µM rottlerin (lane 4), 10 µM myristoylated PKC-
inhibitor (lane 5),
50 µM PD98059 (lane 6), 50 µM
LY294002 (lane 7), 5 µM BIM (lane
8), 1 µM Gö6976 (lane 9), and 20 µM SB203580 (lane 10) and then treated in the
absence (
) or presence (+) of 1.5 units/ml thrombin. The open
arrow indicates specific DNA-protein complexes. The closed
arrow indicates nonspecific DNA-protein complex. For supershift
analysis, thrombin-treated HUVECs were incubated with antibody against
p65 (lane 13). The asterisk indicates the
super-shifted complex. B, quantification of NF-
B binding.
The results show the means ± S.D. of specific signal (relative to
untreated cells) obtained from three independent experiments. *,
p < 0.05;
, p = 0.08, thrombin
treatment, no inhibitor.
-dependent Signaling
Pathway--
In the next set of experiments, a radiolabeled
double-stranded oligonucleotide probe encompassing the tandem GATA
motif in the VCAM-1 promoter was incubated with nuclear extracts
derived from untreated and thrombin-treated HUVEC. As expected, the
incubation of probe with nuclear extract from thrombin-treated cells
resulted in increased GATA binding (closed and open
arrows, Fig 5). The DNA-protein
complexes were specific as defined by inhibitor studies (data not
shown). Moreover, the complexes were inhibited by preincubation with
the anti-GATA-2 antibody from Santa Cruz Biotechnology (Fig. 5,
lane 13) but with anti-GATA-3 or anti-GATA-6 antibodies
(data not shown). Thrombin stimulation of GATA-2-DNA binding activity was inhibited ~82% by preincubation of cells with the PKC-
peptide inhibitor and LY294002 (Fig. 5, lanes 5 and
7) and 25% by preincubation with SB203580 (Fig. 5,
lane 8). In contrast, GATA binding activity was unaltered by
preincubation of cells with rottlerin, PD98059, Gö6976, or BIM
(Fig. 5 lanes 4, 6, 9, and
10, respectively). Together, these results suggest
that thrombin-mediated induction of GATA binding is regulated by a
PI3K- and PKC-
-dependent signaling pathway.
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Fig. 5.
Thrombin induces binding of GATA-2 to the
VCAM-1 promoter via a PI3K,
PKC- -dependent signaling
pathway. A, electrophoretic mobility shift assays were
performed with 32P-labeled GATA probe in the absence
(lane 1) or presence of 10 µg of nuclear extract from
HUVECs preincubated for 30 min with vehicle (lanes 2 and
3), 10 µM rottlerin (lane 4), 10 µM myristoylated PKC-
inhibitor (lane 5),
50 µM PD98059 (lane 6), 50 µM
LY294002 (lane 7), 20 µM SB203580 (lane
8), 1 µM Gö6976 (lane 9), and 5 µM BIM (lane 10) and then treated in the
absence (
) or presence (+) of 1.5 units/ml thrombin. The open
arrow indicates specific DNA-protein complexes. The closed
arrow indicates the nonspecific DNA-protein complex. For
supershift analysis, thrombin-treated HUVECs were incubated with
antibody against GATA-2 (lane 13). The asterisk
indicates the super-shifted complex. B, quantification of
GATA binding. The results show the means ± S.D. of specific
signal (relative to untreated cells) obtained from three independent
experiments. *, p < 0.05 compared with thrombin
treatment, no inhibitor.
B or GATA Motifs Is Inhibited by DN-PKC-
and
DN-PKC-
, Respectively--
To more definitively establish a
connection between the PKC isoforms and downstream transcription factor
activity, HUVEC were co-transfected with a luciferase reporter plasmid
containing either two copies of the tandem VCAM-1 GATA motif or four
copies of the tandem VCAM-1 NF-
B motif linked to the minimal herpes
simplex virus TK promoter (GATA4-TK-luc and
NF-
B8-TK-luc, respectively) and expression plasmids for
either DN-PKC-
or DN-PKC-
. As shown in Fig.
6A,
NF-
B8-TK-luc activity was stimulated 5.3-fold by the
thrombin treatment. Thrombin-mediated induction of
NF-
B8-TK-luc was significantly inhibited (64%) by
DN-PKC-
, but not by DN-PKC-
. In contrast,
GATA4-TK-luc was stimulated 2.1-fold by thrombin, an effect
that was completely inhibited by DN-PKC-
, but not the DN-PKC-
(Fig. 6B). Together with the previous results, these findings suggest that thrombin activates the PKC-
-NF-
B and
PKC-
-GATA signaling pathways in endothelial cells.
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Fig. 6.
Thrombin-mediated induction of
NF- B8-TK- and
GATA4-TK-promoter activities is inhibited by
DN-PKC-
and DN-PKC-
,
respectively. A, HUVECs were transiently co-transfected
with 0.4 µg of NF-
B8-TK-luc and 0.4 µg of expression
plasmid for either DN-PKC-
or DN-PKC-
, then treated in the
absence (
) or presence (+) of 1.5 units/ml thrombin for 6 h.
B, HUVECs were transiently co-transfected with 0.4 µg of
GATA4-TK-luc and 0.4 µg of expression plasmid for
DN-PKC-
or DN-PKC-
. The results show the means ± S.D. of
luciferase light units (relative to untreated cells) obtained in
triplicate from four independent experiments.
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Fig. 7.
Thrombin fails to induce VCAM-1 mRNA
expression in vascular smooth muscle cells. Northern blot analyses
of VCAM-1 (A) or tisue factor (B-D) in
serum-starved HCASMC treated with 10 ng/ml IL-4 (A and
D), 1.5 units/ml thrombin (A and B),
or 20 nmol/ml TRAP (C) for the indicated times. The results
are representative of two independent experiments.
B and GATA Binding to the VCAM-1
Promoter in Vascular Smooth Muscle Cells--
NF-
B is expressed
ubiquitously and has been shown to transduce extracellular signals in
endothelial cells and VSMC alike. In contrast, GATA-2 is a marker for
endothelial cells and is not expressed in VSMC. Therefore, we predicted
that the endothelial cell-type-specific nature of the thrombin-VCAM-1
response would reflect differences in GATA binding activity. To test
this hypothesis, we carried out electrophoretic mobility shift assays
with nuclear extracts from HCASMC and radiolabeled probes spanning
either the tandem NF-
B or GATA motifs. As predicted, the incubation
of the GATA probe with nuclear extracts from untreated,
thrombin-treated or IL-4-treated HCASMC did not result in specific
DNA-protein complexes (Fig.
8A). The complex indicated by
the closed arrow was nonspecific, in that it was inhibited
by the addition of both wild type and GATA mutant competitor
oligonucleotides and was unaltered by preincubation with anti-GATA2,
GATA3, and GATA6 antibodies (Fig. 8A, lanes 5-7,
and data not shown). More surprising was the finding that thrombin did
not induce binding of NF-
B to the tandem NF-
B sites. As shown in
Fig. 8B, incubation of the NF-
B probes with nuclear
extracts from untreated HCASMC resulted in specific DNA-protein
complexes (open arrow, lane 2), similar to the
pattern observed with HUVEC-derived nuclear extract (see Fig. 4).
However, in contrast to HUVEC, the incubation of HCASMC with thrombin
did not stimulate NF-
B binding (Fig. 8B). Nor did the addition of thrombin alter the composition of the NF-
B complex, as
evidenced by supershift experiments (Fig. 8B, lanes
10-12). Consistent with previous reports, the incubation of
HCASMC with IL-4 did not stimulate NF-
B binding. Taken together,
these findings suggest that in VSMC there is an uncoupling
between PAR-1 and the binding activity of NF-
B and GATA-2.
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Fig. 8.
Thrombin fails to induce
NF- B and GATA binding to the VCAM-1 promoter
in vascular smooth muscle cells. A, electrophoretic
mobility shift assays were performed with 32P-labeled GATA
probe in the absence (lane 1) or presence of 10 µg of
nuclear extract from untreated HCASMC (lane 2) or HCASMC
treated with 1.5 units/ml thrombin (lane 3) or 10 ng/ml IL-4
(lanes 4-7). To test the effect of antibodies on the
DNA-protein complexes, IL-4-treated HCASMC nuclear extracts were
incubated with antibodies against GATA-2 (lane 5), GATA-3
(lane 6), and GATA-6 (lane 7). The closed
arrow indicates nonspecific DNA-protein complex. B,
electrophoretic mobility shift assays were performed with
32P-labeled NF-
B probe in the absence (lane
1) or presence of 10 µg of nuclear extract from untreated HCASMC
(lane 2), HCASMC treated with 1.5 units/ml thrombin (lane 3), or 10 ng/ml IL-4 (lanes
4 and 7-12) HCASMC. In competition assays, a 50-fold
molar excess of unlabeled wild type (lane 8) or mutant
(lane 9) NF-
B probe was added to the reaction mixture.
The open arrow indicates the specific DNA-protein complex.
The closed arrow indicates the nonspecific DNA-protein
complex. In super-shift experiments, IL-4-treated HCASMC were incubated
with antibodies against p65 (lane 10), Rel B (lane
11), and c-Rel (lane 12). The asterisk
indicates the super-shifted complex. The results are representative of
two independent experiments.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-NF-
B and PI3K-PKC-
-GATA signaling pathways (summarized in Fig.
9). In addition, our findings implicate a
role for the classic PKC isoform, PKC-
. These data are novel in that
they demonstrate the involvement of classic, novel, and atypical PKC
isoforms at the level of a single gene promoter, establish a link
between PKC and GATA binding activity, and provide evidence for cell
type-specific thrombin signaling.
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Fig. 9.
Model. Schematic shows the endothelial
cell-specific effect of thrombin on VCAM-1 expression.
The observation that thrombin stimulates VCAM-1 through a
PKC--NF-
B p65 pathway is consistent with the results described for the intercellular adhesion molecule (ICAM)-1 gene (25). Together,
these data contrast with the majority of published reports, in which
PKC-
is implicated as the principle activator of NF-
B (26, 27).
For example, in NIH-3T3 cells the expression of wild type or activated
PKC-
was shown to stimulate IKK
and increase NF-
B activity
(28, 29), whereas the overexpression of DN-PKC-
had the opposite
effect (29). In addition to its role as an IKK
kinase, PKC-
may
directly phosphorylate and activate p65 and or c-Rel via an interaction
with the transactivation domain (27). Tumor necrosis factor (TNF)-
was shown to induce the expression of ICAM-1 in endothelial cells via a
PKC-
-NF
B-p65-homodimer-dependent mechanism (13).
Genetic evidence for the role of PKC-
in mediating NF-
B
activation was obtained from studies of mice that are null for the
PKC-
gene (30). The targeted disruption of PKC-
resulted in
impaired cytokine (TNF-
and IL-1)-induced phosphorylation of p65 and
NF-
B transcriptional activity in embryonic fibroblasts and reduced
IKK activation in lung tissue (30). Together with the results of the
ICAM-1 study, our findings suggest that the link between PKC-
and
NF-
B may be relatively specific to PAR-1 signaling in endothelial cells.
Rottlerin, which has been widely used as a PKC--selective inhibitor,
was recently reported to uncouple mitochondria and thereby reduce the
levels of intracellular ATP in a variety of non-endothelial cell types
(31). The latter finding emphasizes the need for caution in
interpreting studies with this compound. However, it should also be
pointed out that rottlerin was shown to indirectly inhibit
stimulus-induced phosphorylation of PKC-
(31). Moreover, the finding
that rottlerin inhibited thrombin-induced binding of p65 NF-
B, but
not GATA-2, suggests that this compound exerts some degree of
specificity in HUVEC at the level of DNA-protein interactions. Finally,
and most importantly, the results of the co-transfection experiments
with DN-PKC-
and DN-PKC-
add strong support to our conclusion
that thrombin induces VCAM-1 expression via a
PKC-
-NF-
B-dependent signaling pathway.
The mechanism by which PKC- activates NF-
B and VCAM-1 expression
was not specifically addressed in this study. In the case of the ICAM-1
gene, thrombin-mediated activation of PKC-
was shown to stimulate
p38 MAPK, thereby resulting in increased transcriptional activity of
NF-
B (25). The importance of p38 MAPK in mediating the
transactivation potential of p65 is supported by studies in fibrosarcoma cells, in which TNF
-mediated induction of IL-6 involved a p38 MAPK-sensitive NF-
B pathway (32). In the present study, thrombin stimulation of VCAM-1 was partly inhibited by SB203580, suggesting that p38 MAPK also plays a role in the PAR-1-VCAM-1 pathway.
Thrombin stimulation of NF-B8-TK-luc was not completely
abolished (64% inhibition) by co-transfection with the DN-PKC-
. These results raise the possibility that other signaling pathways are
involved in transducing the thrombin signal at the level of NF-
B
activity. Consistent with this hypothesis is the observation that
inhibition of classic PKC with Gö6976 partially blocked thrombin
induction of VCAM-1 mRNA and promoter activity as well as NF-
B
binding. An alternative explanation is that the dominant negative
(kinase-dead) PKC-
failed to completely inhibit endogenous PKC-
activity in the co-transfection assays.
In contrast to the established function of NF-B in transducing
extracellular signals, GATA binding proteins are widely viewed as
constitutively active transcription factors involved in mediating cell
type-specific gene expression and lineage determination. Only recently
has it become evident that the GATA family of proteins may also play a
role in mediating inducible gene expression. GATA DNA-binding activity
and/or GATA mRNA expression has been shown to increase in response
to a number of mediators, including insulin-like growth factor 1 (33),
follicle stimulating hormone (34), endothelin-1 (35), IL-3 (36), IL-4
(37), and thrombin (8). GATA activity has been reported to decrease in
response to other mediators such as estrogen (38) and transforming
growth factor-
(39). Together, these observations suggest that
GATA-2 may function as a signal transducer or immediate early gene,
coupling short term changes in the extracellular environment to long
term changes in gene expression.
The present study is the first to establish a link between PKC- and
the GATA family of transcription factors. The mechanism by which
PKC-
activates GATA binding remains to be determined. Although
PKC-
has been shown to activate ERK1/2 in response to a variety of
signals (40-43), the inability of PD98059 to prevent thrombin-mediated
induction of VCAM-1 argues against a role for ERK1/2 in the
thrombin-VCAM-1 pathway. The preincubation of HUVEC with SB203580
resulted in partial inhibition of inducible GATA binding, suggesting
that MAPK p38 may play a role in the PKC-
-GATA-2 pathway. It is
conceivable that PKC-
induces GATA binding activity by altering the
redox state of the cell. Alternatively, PKC-
may directly
phosphorylate GATA-2, resulting in increased DNA-binding and
transcriptional activity. It is interesting to note that PKC-
has
been located in both the cytosol and the nucleus in resting endothelial
cells, in contrast to PKC-
, which was exclusively localized to the
cytosol (12). The addition of thrombin resulted in a rapid nuclear
translocation of PKC-
, an effect that was not observed with basic
fibroblast growth factor (12). These results suggest that
thrombin-activated PKC-
may translocate to the nuclear compartment
where it activates GATA-2.
Several lines of evidence argue that PI3K-PDK1 lies upstream of
thrombin-mediated PKC--NF-
B and PKC-
-GATA-2 interactions. First, the response of VCAM-1 to thrombin was abrogated in the presence
of the PI3K inhibitor, LY294002. Second, the addition of this inhibitor
resulted in a significant reduction of NF-
B and GATA-2 binding.
Third, the overexpression of dominant negative or constitutively active
Akt failed to alter VCAM-1 expression in the absence or presence of
thrombin, pointing to the involvement of another downstream substrate.
Finally, although PDK1 has been shown to phosphorylate the activation
loop of all PKCs (44, 45), only PDK1-mediated activation of PKC-
and
PKC-
is dependent on PI3K (46-51).
In contrast to our results, Rahman et al. (52) reported a
role for Akt in PAR-1-mediated induction of PKC--NF-
B. It is difficult to reconcile the differences between these two studies. Our
investigation focused on the role of DN-Akt in inhibiting endogenous
VCAM-1 mRNA levels in endothelial cells, whereas the previous study
employed co-transfections with DN-Akt and a synthetic NF-
B or ICAM-1
promoter construct (52). It is formally possible that different
signaling pathways mediate PAR-1 stimulation of endogenous VCAM-1
mRNA and NF-
B promoter activity. Indeed, we found that the
overexpression of DN-Akt in HUVECs failed to abrogate thrombin
stimulation of ICAM-1 (data not shown). It is interesting to point out
that many of the co-transfection experiments employed in the previous
report involved a construct that contains 5 NF-
B elements from the
long terminal repeat of the human immunodeficiency virus (HIV) promoter
(52). Previous studies have demonstrated important differences between
the VCAM-1- and HIV-containing NF-
B DNA sequences (53). For example,
the NF-
B elements from the VCAM-1 promoter are transactivated by p65
homodimers, whereas those from the HIV promoter are transactivated by
p65-p50 heterodimers (53). It is conceivable that thrombin signals
through different pathways induce NF-
B binding to the HIV and VCAM-1
consensus sites. Another interpretation of the discrepancy in results
between these two studies is that our DN-Akt lacked inhibitory
activity. However, several lines of evidence argue against this
hypothesis. First, previous studies have demonstrated that the inactive
phosphorylation mutant of Akt functions effectively as a
dominant-negative (22, 54, 55). Moreover, overexpression of the DN-Akt
adenovirus inhibited insulin-induced glycogen synthase kinase-3 in L6
myoblast cells. Finally, we demonstrated that DN-Akt inhibited vascular epidermal growth factor-mediated induction of endothelial cell migration, whereas CA-Akt was sufficient in inducing endothelial cell
migration. In summary, our data argue strongly against a role for Akt
in mediating the thrombin response of VCAM-1.
Although receptor tyrosine kinases are known to activate PI3K- or
PI3K-
, G protein-coupled receptors (GPCR) have been shown to
stimulate a distinct subclass of PI3K, PI3K-
, through an interaction of heterotrimeric G proteins with the catalytic subunit p100
(56,
57). Importantly, GPCR-coupled PI3K-
is capable of activating PKC-
(40). Based on these observations, we propose that the PI3K-
isoform may be responsible for mediating thrombin's effect on the
PKC-
-NF-
B and PKC-
-GATA-2 pathways.
The failure of thrombin to induce VCAM-1 expression in VSMC suggests
that the PAR-1-VCAM-1 signaling pathway is specific to endothelial
cells. Previous studies have established the presence of functional
thrombin receptors on VSMC (58-64). Consistent with these reports, we
demonstrated that thrombin stimulated tissue factor mRNA levels in
human coronary artery VSMC, an effect that was mimicked by the PAR-1
agonist, TRAP. These data suggest that the discordance in VCAM-1
response to thrombin in endothelial cells and VSMC is attributed to
differences at the post-receptor level. Because GATA-2 expression is
restricted to endothelial cells, and because GATA-2 binding is both
necessary and sufficient for thrombin-mediated induction of VCAM-1 in
endothelial cells (8), we predicted that the lack of response in VSMC
would be attributable to the absence of GATA-2. However, in
electrophoretic mobility shift assays, thrombin failed to induce the
binding not only of GATA-2 but also NF-B p65 homodimers to the
VCAM-1 promoter. Taken together, these results suggest that thrombin
induces VCAM-1 mRNA specifically in endothelial cells and that the
failure to stimulate VCAM-1 in VSMC is attributable to a "defect"
in the pathway somewhere downstream of the PAR-1 receptor and upstream of the NF-
B and GATA transcription factors. It is interesting to
speculate that VSMCs express low levels of the GPCR-coupled PI3K-
isoform or lack the appropriate repertoire of G proteins to activate
PKC-
-NF-
B and PKC-
-GATA-2 pathways. In support of the former
hypothesis, thrombin has been shown to induce only a weak and transient
phosphorylation of Akt compared with PDGF in VSMC (65).
An analysis of the differences between the regulation of the VCAM-1 and
ICAM-1 genes and between thrombin and TNF- signaling offers
interesting contrasts in signaling mechanisms. In the case of ICAM-1,
both thrombin and TNF-
stimulate expression through the inducible
binding of NF-
B p65 homodimers. However, thrombin exerts its effect
via PKC-
(and to a lesser extent, PKC-
), whereas TNF-
signals
through PKC-
(25). In the case of VCAM-1, thrombin and TNF-
induce expression through the coordinate interaction of NF-
B p65
homodimers and GATA-2 (8). Here we show that thrombin stimulation of
NF-
B binding is mediated by PKC-
, whereas that of GATA-2 binding
is dependent on PKC-
. It will be interesting to determine whether
TNF-
, like thrombin, induces VCAM-1 expression through
PKC-
-NF-
B p65 and PKC-
-GATA-2 pathways or whether, like the
ICAM-1 gene, TNF-
mediates its effect predominantly through the
PKC-
isoform. Moreover, it would be valuable to study endothelial
cells that are null for PKC-
or PKC-
. Based on the existing data,
one might predict that a deficiency in PKC-
would lead to impaired
ICAM-1 and VCAM-1 response to thrombin, whereas an absence of PKC-
might prevent TNF-
-mediated induction of ICAM-1 (via NF-
B) and
thrombin stimulation of VCAM-1 (through GATA-2).
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Christopher Carpenter for helpful suggestions, Alex Toker for critical review of the manuscript, Kohjiro Ueki for invaluable input, and Katherine Spokes for technical assistance.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grants HL 60585-04, HL 63609-02, and HL 65216-03.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. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Beth Israel
Deaconess Medical Center, RW-663, Boston, MA 02215. Tel.: 617-667-1031; Fax: 617-667-2913; E-mail: waird@caregroup.harvard.edu.
Published, JBC Papers in Press, December 18, 2002, DOI 10.1074/jbc.M208974200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
VCAM-1, vascular
cell adhesion molecule-1;
DAG, diacylglycerol;
VSMC, vascular smooth
muscle cells;
TRAP, thrombin receptor activation peptide;
BIM, bisindolylmaleimide I;
HUVEC, human umbilical vein endothelial cells;
HCASMC, human coronary artery smooth muscle cells;
CMV, cytomegalovirus;
ICAM-1, intercellular adhesion molecule-1;
TNF-, tumor necrosis factor-
;
GPCR, G protein-coupled receptor;
PI3K, phosphatidylinositol 3-kinase;
PKC, protein kinase C;
cPKC, classic PKC;
nPKC, novel PKC;
aPKC, atypical PKC;
IL-4, interleukin-4;
DN, dominant negative;
FBS, fetal bovine serum;
WT, wild type;
MAPK, mitogen-activated protein kinase;
ERK, extracellular signal-regulated
kinase;
CA, constitutively active;
HIV, human immunodeficiency virus;
PAR-1, protease-activated receptor-1.
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