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INTRODUCTION |
The transforming growth factor-
(TGF-
)1 superfamily of
growth factors and cytokines is involved in a wide variety of
physiological and pathophysiological processes in the cardiovascular
system, and the signaling mechanisms utilized by this class of
effectors are rapidly being elucidated. The discovery of Smad proteins
and the demonstration that they can mediate many of the transcriptional effects of these growth factors has been an important advance (1-3).
The central roles of these proteins in mediating TGF-
responses in
cells is highlighted by the demonstration that mutations in Smads 2, 3, and 4 have been causally linked to specific malignancies, and
disruption of the Smad2 and Smad4 genes in mice
results in early embryonic lethality (4, 5). As the detailed molecular mechanisms of Smad protein signaling have emerged a number of functional interactions between these proteins and other signaling pathways have been reported. For instance, recent work has demonstrated that the classic mitogen-activated protein kinase pathway (MAPK/ERK) can negatively regulate bone morphogenic
protein/Smad1-dependent transcriptional responses. This
appears to occur as a result of the phosphorylation of Smad1 by a
member of the Erk kinases and subsequent translocation of Smad1 out of
the nucleus (6). In contrast to this inhibitory effect, the
stress-activated protein kinase pathway (i.e. the SAPK/JNK
pathway) has been implicated as a positive regulator of certain
Smad-dependent effects. Previous data from several groups
have documented the importance of AP-1 and related transcriptional
effectors in some TGF-
-mediated transcriptional responses, and a
recent report demonstrated a direct functional interaction between
Smad3 and c-Fos/c-Jun in mediating TGF-
-dependent transcription (7, 8).
Recently, signals derived from growth factor receptors containing
tyrosine kinase activities were shown to be capable of modulating Smad-dependent effects. This was suggested to occur as a
result of activation of a kinase downstream of MEK-1, an upstream
activator of the classic MAPK/ERK kinase pathway, resulting in the
phosphorylation of Smad2 (9). In addition, a variety of other kinases
have been implicated in TGF-
signaling, such as TAK-1 and TAB,
although their precise roles have not been elucidated (10-12). Taken
together, these data indicate that multiple signal transduction
cascades may modulate Smad signaling in cells and begin to provide
possible mechanisms by which signals derived from non-TGF-
family
members may impact TGF-
superfamily responses. Furthermore, these
data suggest that Smad proteins may be involved in the transduction of
diverse signals in cells. In this report we demonstrate that MEKK-1, a
MAPK kinase kinase that is an upstream activator of the SAPK/JNK
pathway, is capable of selectively activating
Smad2-dependent transcriptional activity independently of
TGF-
in cultured endothelial cells. These data demonstrate a
functional interaction between the SAPK/JNK and Smad signaling pathways
and suggest that Smad protein signaling may be modulated by MEKK-1 or
related kinases in endothelial cells.
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EXPERIMENTAL PROCEDURES |
Cells, Reagents, and Constructs--
Primary bovine aortic
endothelial cells (BAEC) were isolated as described and cultured in
Dulbecco's modified Eagle's media supplemented with 10%
heat-inactivated bovine calf serum, 2 mM L-glutamine, 250 units/ml penicillin G, and 250 µg/ml
streptomycin (13). These were utilized at passages 3-12. COS-7 cells
were maintained in the same medium. The p3TP promoter, Smad protein expression constructs, Smad-GAL4/VP16 fusion constructs, and CBP fusion
constructs have been described previously (14). The plasminogen activator inhibitor-1 promoter (P800) was provided by David Loskutoff, dominant negative MEKK-1 by Roger Davis, and activated TAK-1 expression plasmid by Eisuke Nishida. The active MEKK-1, MEK-1, and protein kinase
A expression plasmids were obtained from Stratagene. The activated
MEKK-1 construct consists of amino acids 380-672 of MEKK-1. The
activated MEK-1 construct consists of the 2218/E222 derivative that has
had amino acids 32-51 deleted. The c-Jun and Elk1 fusion proteins
consist of amino acids 1-223 of c-Jun and 307-427 of Elk1 fused to
the Gal4 DNA binding domain, respectively. The CRE, AP-1, and
NF-
B-dependent promoters consist of four tandem CRE
sites (AGCCTGACGTCAGAG), seven tandem AP-1 sites (TGACTAA), or four
tandem NFKB sites (TGGGGACTTTCCGC) cloned upstream of a minimal TATA
box and luciferase reporter gene. The Smad2 mutants pMSmad2*P and
pMSmad2C*P have been described previously (14). For the labeling
experiments, a Flag epitope replaced the Gal4 binding domain, and these
were subcloned into the expression vector pCI (Promega).
Transfections, Reporter Assays, and
Immunoprecipitations--
For transient transfections, cells were
seeded at 50-70% confluence and transfected using LipofectAMINE (Life
Technologies, Inc.) for 5 h. The cells were allowed to recover
overnight in media containing 0.2% serum. Following approximately
18 h of incubation, luciferase and
-galactosidase activity were
measured (Tropix). All results are reported as luciferase activity
(relative light unit) normalized to cotransfected
-galactosidase
activity (expressed from cotransfected, constitutive expression
constructs, e.g. cytomegalovirus
-galactosidase,
phosphoglycerol kinase
-galactosidase) and are representative of at
least three independent experiments. A typical transfection utilized
0.25-0.5 µg of reporter gene plasmid and 5 ng of active kinase
(e.g. MEKK-1 and MEK-1) expression plasmid. Total DNA and
total amount of expression plasmid were equalized by the addition of
appropriate control plasmids.
Antibodies directed against the epitopes used in the
immunoprecipitations and Western blots were obtained from commercial suppliers (Boehringer Mannheim, Santa Cruz Biotechnology, Eastman Kodak
Co.). The anti-CBP/P300, MEKK-1, and MEK-1 antisera were obtained from
Santa Cruz Biotechnology. Cell lysates were made approximately 24 h after transfection in 1% Triton X-100, 150 mM NaCl, 50 mM Tris-HCl, pH 8.0, with protease inhibitors, and immunoprecipitations were performed overnight at 4 °C. Proteins were
resolved on 10-12% SDS-polyacrylamide denaturing gels, transferred to
nitrocellulose by electroblotting, and probed with appropriate antisera
at 1/1000-1/2000 as indicated in the figures. Individual proteins were
detected with a secondary antibody coupled to peroxidase and visualized
with chemiluminescence (ECL).
Metabolic Labeling--
COS-7 cells were transfected as above,
allowed to recover overnight, and subsequently incubated in low
phosphate media containing [32P]orthophosphate (500 µCi/ml) for 3 h at 37 °C. For the TGF-
stimulation, active
TGF-
at 5 ng/ml (R & D Systems) was added for the final 30 min. The
cells were washed, lysed, and immunoprecipitated with anti-epitope
antisera as above. Proteins were resolved on a 10% denaturing
acrylamide gel and electroblotted to nitrocellulose, and
autoradiography was performed. Western blots with anti-epitope antisera
were performed to control for protein levels.
Immunofluorescence--
BAEC were transfected as above. The
confluent monolayers were then washed and fixed in 2% paraformaldehyde
for 10 min and permeabilized with 1% Nonidet P-40 for 1 min. After
pre-blocking with nonspecific goat serum, the primary (anti-epitope)
antibodies were applied at 1/500 for 1 h. The monolayers were then
washed and incubated with appropriate secondary antibodies coupled to fluorescein isothiocyanate. The monolayers were visualized with a Nikon
Microphot-FXA fluorescence microscope and imaged with a Power
Macintosh-based image analysis system (Oncor).
Shear Stress Stimulation--
BAEC were exposed to a steady
laminar shear stress stimulus utilizing a modified cone-plate flow
system that has been described in detail previously and is a well
validated method for exposing cultured cells to defined fluid
mechanical stimuli (15, 16). For these experiments the BAEC were
transfected as described above and subsequently plated to 12 individual
"coverslips" (per experiment) that were simultaneously subjected to
the LSS (flow) stimulus for 18 h. LSS at approximately 10 dynes/cm2 was achieved by utilizing a 0.5° cone rotating
at a rate of 100 rpm. Identical coverslips maintained under standard
(static) culture conditions served as controls. All flow
experiments utilized the same culture medium as the static experiments.
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RESULTS |
MEKK-1 Selectively Activates Smad-dependent
Transcription--
To investigate the role of non-receptor kinases in
the regulation of Smad signaling in endothelial cells, we examined the ability of constitutively activated forms of MEK-1, MEKK-1, and TAK-1
to regulate two TGF-
responsive promoters, the plasminogen activator
inhibitor-1 promoter (P800) and p3TP. MEK-1 is an activator of the
classic MAPK/ERK pathway, and MEKK-1 is an activator of the stress or
SAPK/JNK pathway (17, 18). TAK-1 is a kinase that has been implicated
in TGF-
signaling and is capable of activating components of the
SAPK/JNK pathway, but its precise role is unknown (12). In preliminary
titration experiments in cultured bovine aortic endothelial cells
(BAEC), a ratio of active kinase to reporter gene plasmid of 1 to 100 was found to be sufficient to reproducibly activate transcription, and
under these conditions only MEKK-1 was reproducibly capable of
activating the TGF-
-responsive promoters P800 and p3TP. Comparable
levels of active MEK-1 or active or wild type TAK were unable to
reproducibly activate these promoters, although active TAK-1 could
stimulate the P800 promoter when expressed at very high levels (data
not shown). Fig. 1 demonstrates the
response of the p3TP and P800 promoters to coexpression of active
MEKK-1, active MEK-1, or a constitutively active TGF-
type-1
receptor (actT
R1). Both promoters are induced significantly by the
active TGF-
1 receptor and active MEKK-1 but not by active MEK-1.
When these kinases are expressed in combination with the activated
TGF-
receptor, MEKK-1 appears to stimulate the activity of both
promoters in a manner additive to the TGF-
receptor, whereas MEK-1
has little effect. To explore the mechanisms of these effects in more
detail, the p3TP promoter was stimulated with either active T
R1 or
active MEKK-1, and the effects of coexpression of a dominant negative
form of MEKK-1 or Smad7, an inhibitory Smad protein, was examined. As
shown in Fig. 1C, the induction of p3TP by the active
TGF-
receptor was partially inhibited by the dominant negative
MEKK-1 and was potently inhibited by coexpression of Smad7. Similarly,
the induction of p3TP by active MEKK-1 was inhibited by coexpression of
both dominant negative MEKK-1 and Smad7. Fig. 1E is a series
of Western blots demonstrating the expression levels of the activated
kinases utilized in these experiments. At the level of expression
plasmids used (5 ng) we reproducibly observed a level of activated
MEKK-1 or MEK-1 protein approximately 3-5 times that of the
corresponding endogenous protein, and the relative levels of the two
overexpressed active kinases (MEKK-1 and MEK-1) appeared comparable.
Taken together, these results suggest that maximal transcriptional
induction of the P800 and p3TP promoters by the active TGF-
1
receptor requires the action of MEKK-1 as well as Smad proteins. In
addition, the observation that Smad7 can block active MEKK-1-mediated
stimulation of these promoters suggests that Smad proteins are playing
a role in the MEKK-1-mediated transcriptional stimulation.

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Fig. 1.
MEKK-1-mediated stimulation of
TGF- -responsive promoters in cultured
endothelial cells. A and B, bovine aortic
endothelial cells (BAEC) were transfected with the plasminogen
activator inhibitor-1 promoter (P800) or p3TP-lux promoter, together
with the indicated combinations of activated receptor and or kinases,
and reporter gene activity was assayed 18 h later. All results are
expressed as luciferase activity normalized to cotransfected,
constitutively expressed -galactosidase measured in the same sample.
All transfection conditions were normalized to contain the same
quantity of expression plasmid to control for nonspecific effects.
C and D, the p3TP promoter was induced by active
TGF- receptor or active MEKK-1, and the ability of increasing
amounts (1:1, 1:2.5, 1:5) of dominant negative MEKK-1 (MEKKDN) or Smad7
expression to inhibit induction was assessed. Error bars
represent ± 1 S.D. of the mean for independent replicates, and
the experiments shown are representative of at least three independent
experiments. E, Western blots demonstrating the relative
levels of active MEKK-1, MEK-1, T bR1, and TAK-1 typically achieved in
these transient transfection experiments. Lanes 1, 3, 5, and
7 are untransfected controls, and lanes 2, 4, 6, and 8 have been transfected with the active MEKK-1, MEK-1,
T R1, or TAK-1 constructs, respectively. In lanes 1 and
2 the endogenous MEKK-1 is seen as is the truncated active
kinase. The lower molecular weight band seen in lane 1 is a
nonspecific band that does not compete off with MEKK-1 peptide (data
not shown). In lanes 3 and 4 both the endogenous
and active MEK-1 kinases are seen. In lanes 5-8, the active
T R1 or active TAK-1 proteins are visualized by probing with the
anti-hemagglutinin epitope antibody that does not detect the endogenous
proteins. These blots all contain equivalent amounts of protein and
were developed for comparable amounts of time.
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To investigate this hypothesis directly, we utilized a Gal4-based
transcriptional system. We have previously shown that this system
faithfully recapitulates ligand (TGF-
)-induced,
Smad-dependent activation of transcription in cultured
endothelial cells (14). Bovine aortic endothelial cells (BAEC) were
cotransfected with constructs encoding fusion proteins between the Gal4
DNA binding domain and either full-length or C-terminal MH2
domains of Smad2, -4, -6, and -7 together with a
Gal4-dependent luciferase reporter gene and constitutively
activated forms of MEKK-1 or MEK-1. As shown in Fig.
2, MEKK-1 markedly stimulates
Smad2-dependent transcription in this system. The low basal
level of luciferase activity observed in the absence of MEKK-1 probably
reflects a small amount of endogenous TGF-
activation in the
endothelial culture system. Smad4 demonstrates a reproducibly lower
level of basal transcriptional activity in this system, and this is not
enhanced in the presence of MEKK-1. Gal4 constructs containing only the
C-terminal MH2 domain (along with the linker region) of
these proteins (pM2C and pM4C) demonstrated significantly increased
basal levels of transcriptional activity and were also markedly
stimulated by MEKK-1. This observation is consistent with previous data
demonstrating that the transcriptional activation domain is contained
within the C-terminal half of the Smad protein (MH2 domain)
and indicates that MEKK-1 can stimulate Smad2- and
Smad4-dependent transcription in the absence of the inhibitory MH1 domain. In contrast to Smad2 and Smad4,
neither Smad6 nor Smad7 demonstrated any significant basal or
MEKK-1-induced transcriptional activity in endothelial cells. Moreover,
overexpression of a constitutively activated form of MEK-1 does not
stimulate the transcriptional activity of any Smad construct tested in
this system (Fig. 2A). Fig. 2B demonstrates that
the MEKK-1-mediated stimulation of GAL4-Smad2 (pM2)-mediated
transcription is inhibited by coexpression of either the dominant
negative MEKK-1 or Smad7. Taken together, these results indicate that
MEKK-1 appears capable of selectively stimulating
Smad2-dependent transcriptional activity in BAEC and that
Smad7 can interfere with this process.

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Fig. 2.
MEKK-1 can stimulate
Smad-dependent transcription in cultured endothelial
cells. A, BAEC were transfected with various human Smad
proteins fused to the Gal4 DNA binding domain (e.g. pM2,
pM4, etc.) together with a Gal4-dependent promoter and the
indicated active kinases. Gal4-Smad2-dependent
transcription is markedly stimulated by coexpression of active MEKK-1,
as is the activity of the C-terminal half of Smad2 fused to Gal4 BD
(pM2C). B, Gal4-Smad2 (pM2) was stimulated with active
MEKK-1, and the ability of increasing coexpression of a dominant
negative form of MEKK-1 (MEKKDN) or Smad7 to inhibit this activation is
assessed. All Gal4-Smad fusion proteins are expressed at comparable
levels in this system as assessed by anti-Gal4 Western blot (data not
shown).
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MEKK-1 has been reported to modulate the activity of a number of signal
transduction pathways in addition to the SAPK/JNK pathway such as
NF-
B/I
B and p53-mediated events (19-21). To examine the
selectivity of our endothelial cell system, we performed a number of
controls to assess the specificity of the observed MEKK-1-mediated stimulation. Conditioned media from endothelial cells expressing active
MEKK-1 consistently failed to stimulate p3TP or GAL4-Smad2-mediated transcription, and inclusion of a neutralizing antibody to soluble TGF-
in the media of the transfection studies (in quantities sufficient to inhibit the activity of exogenously added active TGF-
1
at 1.0 ng/ml) did not inhibit MEKK-1-mediated stimulation of
Smad-dependent transcription (data not shown). These
results indicate that MEKK-1 is not inducing the expression of TGF-
which can then act in a paracrine manner.
To ensure that MEKK-1 activation of Smad2 and Smad4 was not the result
of promiscuous signaling or indiscriminate stimulation of the general
transcriptional apparatus in this overexpression system, we examined
the response of a series of well characterized control promoters and
transcriptional effectors. As shown in Fig. 3, both constitutively activated kinases
(MEKK-1 and MEK-1) demonstrated a selective pattern of promoter
activation when expressed at these levels in BAEC (see Fig.
1E). Active MEKK-1 potently stimulated a coexpressed
AP-1-dependent promoter, whereas an
NF-
B-dependent promoter was also stimulated but to much
lesser degree. Active MEKK-1 was unable to significantly stimulate a
CRE-dependent promoter, as compared with a constitutively
activated form of protein kinase A, a known activator of
CRE-dependent transcription (Fig. 3). Thus, expression of
active MEKK-1 appears to be eliciting the expected pattern of promoter
specificity in this endothelial cell context. To examine further the
selectivity of transcription factor activation in our system, we
examined the ability of active MEKK-1 and MEK-1 to stimulate the
activity of fusion constructs between the Gal4 DNA binding domain and
the activation domains of the transcription factors c-Jun, Elk-1, and
VP-16 (pMJun, pMElk-1, and pMVP16, respectively). Active MEKK-1
markedly stimulated the transcriptional activity of pMJun but, at best,
was only a modest activator of pMElk-1 in our system (Fig. 3).
Conversely, active MEK-1 overexpression potently activated pMElk-1 but
not pMJun, consistent with the role of MEK-1 as an activator of the
classic MAPK (ERK) signal transduction pathway. Neither of these
kinases had a significant effect on the high basal transcriptional
activity of the GAL4-VP16 fusion protein. Taken together, these data
demonstrate that when carefully expressed at relatively low levels in
cultured endothelial cells, constitutively activated forms of both
MEKK-1 and MEK-1 consistently demonstrate a pattern of promoter and
transcription factor activation that is selective, and under these
conditions, only MEKK-1 appears capable of significantly stimulating
Smad2-dependent transcription.

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Fig. 3.
Overexpression of active MEKK-1 or MEK-1
results in a selective pattern of promoter and transcription factor
activation in cultured endothelial cells. A, the
indicated active kinases were cotransfected into BAEC with an AP-1,
NF- B, or CRE-dependent promoter, and the activation of
transcription was assessed by reporter gene assay. MEKK-1 potently
activates the AP-1-dependent promoter (approximately
10-fold) while activating the NF- B-containing promoter to a lesser
degree (2-3-fold). Active MEK-1 did not activate any of the promoters
to a significant degree. B, the ability of active MEKK-1 and
MEK-1 to activate c-Jun, ELK-1, or the VP16 activation domains was
assessed in a Gal4-based assay. These proteins were fused to the Gal4
DNA binding domain and coexpressed with the kinase and a
Gal4-dependent promoter. Active MEKK-1 potently activates
c-Jun, and to a lesser degree ELK-1 under these conditions. Active
MEK-1 can activate ELK1-mediated transcription. Neither kinase has a
significant effect on VP16-mediated transcription under these
conditions.
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MEKK-1 Transcriptional Stimulation Correlates with a Change in the
Phosphorylation State of Smad2--
Considerable experimental evidence
supports a model of Smad signaling that involves phosphorylation of
certain Smad proteins (e.g. Smad2) on a series of C-terminal
serines (termed the SSXS motif) by the kinase present in
TGF-
family type 1 receptors (22, 23). To investigate the role of
this C-terminal SSXS motif of Smad2 in the transcriptional
response to active MEKK-1 described above, we generated two mutant
Gal4-Smad2 fusion constructs (pM2*P and pM2C*P) that lack these serine
residues. We have previously shown that the full-length Smad2 mutant
(pM2*P) is unresponsive to TGF-
in BAEC, whereas the truncated Smad2
mutant (pM2C*P) retains considerable transcriptional activation ability
(14). When these constructs were cotransfected into BAEC together with a constitutively activated form of MEKK-1, the full-length mutant Smad2
was not stimulated (Fig. 4). In contrast,
pM2C*P possessed a significant basal level of transcriptional activity
that was markedly enhanced by MEKK-1 (Fig. 4). MEK-1 overexpression
resulted in no significant stimulation of either Smad2 mutant (Fig.
4).

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Fig. 4.
Transcriptional activation of Smad2 by MEKK-1
correlates with a change in the phosphorylation state of Smad2 and does
not require the C-terminal SSXS motif.
A, Gal4 fusion proteins of Smad2 with the C-terminal
SSXS motif deleted (pM2*P) or the MH2 domain of
Smad2 with the SSXS deleted (pM2C*P) were examined for their
transcriptional response to active MEK-1 or MEKK-1. The latter is
significantly stimulated by active MEKK-1 expression. B,
wild type Smad2 or mutant Smad2 lacking the SSXS motif
(Smad2*P) tagged with a Flag epitope were expressed by transient
transfection, and the cells were subsequently metabolically labeled
with [32P]orthophosphate. Stimuli consisted of soluble
TGF- (5 ng/ml) for 30 min or cotransfection of the indicated
activated receptor or kinases. Smad2 phosphorylation was assessed by
immunoprecipitation with anti-Flag antiserum and autoradiography. The
No tx lane is a no transfection control; the
control lane is no treatment. The bottom
panels confirm comparable protein expression of Flag-Smad2. The
bar graphs represent quantification of phosphorylation by
densitometry of the autoradiograms and corresponding Western blots.
These results are representative of two independent experiments.
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To determine if MEKK-1 stimulation of Smad2 transcriptional activity
also correlated with a change in its phosphorylation state, we
performed a series of in vivo labeling experiments. Cells
were transfected with epitope-tagged Smad2 expression constructs along
with constitutively activated forms of the TGF-
type I receptor
(T
RI), MEKK-1, or MEK-1 and metabolically labeled in the presence of
[32P]orthophosphate. As shown in Fig. 4, Smad2
demonstrated a basal level of phosphorylation when overexpressed. In
the presence of either soluble TGF-
or active T
RI, the
phosphorylation state of Smad2 increases although the soluble TGF-
ligand stimulus consistently results in a lower level of Smad2
phosphorylation in this system. This is in agreement with previous data
describing the role of the TGF-
type I receptor kinase in the
phosphorylation of Smad2 (22). In the presence of MEKK-1, the
phosphorylation state of Smad2 significantly increases as compared with
either T
RI or the untreated control, whereas active MEK-1 only
modestly increased Smad2 phosphorylation.
To determine if the MEKK-1-mediated increase in the phosphorylation
state of Smad2 occurs on the SSXS motif known to be the substrate for the TGF-
type I receptor, we examined the
phosphorylation of the Smad2 mutant in which the SSXS motif
has been deleted (Smad2*P). As with wild type Smad2, when Smad2*P was
overexpressed there is a low but detectable level of basal
phosphorylation. In contrast to wild type Smad2, however, the
phosphorylation state of Smad2*P was not significantly altered by
either soluble TGF-
or coexpression of the active T
RI but is
enhanced by coexpression of active MEKK-1. These results suggest that
MEKK-1 activity can result in the phosphorylation of Smad2 (either by
MEKK-1 itself or a yet to be defined kinase) and that this
phosphorylation event can occur at a site or sites distinct from the
C-terminal SSXS motif that is the site of type 1 receptor
kinase-mediated phosphorylation. Given the ability of active MEKK-1 to
stimulate the transcriptional activity of Gal4 Smad2C and Smad2C*P
(Figs. 2A and 4A), the sites of MEKK-1-induced phosphorylation likely reside in the linker region or the C-terminal MH2 domain of Smad2. Although the SSXS motif
appears to be dispensable for the MEKK-1-mediated stimulation of Smad2
demonstrated here, these data do not exclude some role for this site in
the wild type Smad2 protein stimulated with active MEKK-1.
MEKK-1 Stimulates Smad-Smad Interactions, Nuclear Localization of
Smad Proteins, and Interaction of Smad2 with a Required Transcriptional
Coactivator--
Homo- and heterotypic interactions between Smad2 and
Smad4 are thought to be critical for nuclear localization and
subsequent stimulation of transcriptional events in response to TGF-
(1-3). To determine if MEKK-1 can regulate Smad-Smad interactions, we employed mammalian two-hybrid and coimmunoprecipitation approaches. In
the mammalian two-hybrid system, protein-protein interactions are
detected by fusing one test protein to the Gal4 DNA binding domain,
fusing a second test protein to the strong transcriptional activation
domain of the VP16 protein, and coexpressing these with the
GAL4-dependent reporter. An interaction between these two
proteins results in activation of a Gal4-dependent
luciferase reporter by the VP16 activation domain. By using Gal4 and
VP16 fusion constructs of Smad2 and Smad4, we investigated the ability of MEKK-1 to regulate Smad-Smad interactions in endothelial cells. As
shown in Fig. 5A, this system
demonstrates some basal level of interaction between Smad2 and itself,
as well as with Smad4, in the absence of any stimulation (see
"control" condition in pM2 with VP-Smad2 or VP-Smad4 compared with
pVP alone). This result likely reflects both an increased association
between the Smad proteins due to overexpression and the presence of
small amounts of TGF-
in the culture system. However, the
constitutively activated form of MEKK-1 markedly enhances both
homotypic Smad2-Smad2 and heterotypic Smad2-Smad4 interactions.
Interestingly, active MEKK-1 did not significantly stimulate
Smad4-Smad4 interactions in this assay (data not shown). Expression of
active MEK-1, which did not stimulate the transcriptional activity of
Smad2 or Smad4, had no effect on Smad-Smad interactions in this
endothelial two-hybrid system (Fig. 5A).

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Fig. 5.
Active MEKK-1 can stimulate homotypic
Smad2-Smad2 and heterotypic Smad2-Smad4 interactions in BAEC.
A, two-hybrid analysis of the ability of Gal4-Smad2 to
interact with either Smad2 or Smad4 fused to the VP16 activation
domain, respectively (VP-Smad2, VP-Smad4), when coexpressed in
endothelial cells with the indicated active kinases. MEKK-1 can
stimulate Smad2-Smad2 interactions and Smad2-Smad4 interactions in this
system. (Note: these experiments are typically performed with 1/10th to
1/50th the amount of Gal4 fusion expression plasmid to minimize any
background transcriptional activity of the Gal4 fusion proteins
themselves.) B, both active TGF- receptor (actT bR1)
and active MEKK-1, but not active MEK-1, can stimulate Smad2 and Smad4
to interact as assessed by coimmunoprecipitation (IP) in
BAEC. Smad4 tagged with a hemagglutinin epitope and Smad 2 tagged with
a Flag epitope were coexpressed with the indicated kinases, and
precipitations were performed with antibodies against the Flag epitope.
Coprecipitating Smad4 was detected by Western blot with the
anti-hemagglutinin antibody (upper panel). The lower
two panels confirm comparable protein expression.
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To demonstrate Smad-Smad interactions in cell extracts, we performed a
series of coimmunoprecipitations. Epitope-tagged Smad2 and Smad4 were
coexpressed with active T
R1, MEK-1, TAK-1, or MEKK-1 in endothelial
cells, and the interactions between Smad2 and Smad4 were assessed by
immunoprecipitating Smad2 and probing the immunocomplexes for Smad4. As
can been seen in Fig. 5, there is no detectable interaction between
these two proteins in this assay in the absence of stimulation
(control), but in the presence of a constitutively activated form of
the TGF-
type I receptor (T
RI), significant amounts of Smad4
coimmunoprecipitate with Smad2. This is consistent with previous data
demonstrating TGF-
receptor-stimulated interaction between these two
Smad proteins (22). As shown in Fig. 5, active MEKK-1 also stimulates
this interaction between Smad2 and Smad4, whereas active MEK-1 or
TAK did not stimulate any detectable Smad2-Smad4 association under these conditions. These data are consistent with the mammalian two-hybrid data presented above and demonstrate that MEKK-1 can selectively and specifically stimulate homotypic Smad2-Smad2 and heterotypic Smad2-Smad4 interactions in BAEC.
To determine if MEKK-1 enhancement of Smad2 and Smad4 interactions
leads to changes in the subcellular localization of these proteins, we
utilized immunofluorescence microscopy. Epitope-tagged Smad2 or Smad4
was transfected into BAEC and visualized by immunofluorescence. As can
be seen in Fig. 6, both Smad2 and -4 demonstrate diffuse staining in unstimulated cells, a pattern
consistent with a predominantly cytoplasmic localization of these
proteins. When a constitutively activated form of MEKK-1 is
cotransfected with either Smad2 or Smad4, both proteins exhibit
predominant nuclear staining. Cotransfection of active MEK-1 did not
significantly alter the pattern of Smad staining in these cells. To
ensure that these effects were not simply the result of overexpression
of Smad proteins, we expressed Smad7, a distinct Smad that is
unresponsive to MEKK-1 transcriptional stimulation (Fig. 2). As shown
in Fig. 6, Smad7 exhibits diffuse, predominantly cytoplasmic staining,
which is unaltered in the presence of active MEKK-1.

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Fig. 6.
Coexpression of active MEKK-1 with Smad
proteins in BAEC stimulates the nuclear localization of Smad2 and Smad4
but not Smad7. Immunofluorescence was performed on monolayers of
BAEC cotransfected with combinations of Smad expression constructs
(indicated on the left) and activated kinases (indicated at
the top). The cells were fixed, permeabilized, stained with
antibodies against the epitopes on the Smad proteins, and then
visualized with secondary antibodies coupled to fluorescein
isothiocyanate by fluorescence microscopy. Regions of the confluent
monolayer containing representative stained cells are shown (adjacent
cells are untransfected and thus appear unstained). Smad2 and Smad4,
but not Smad7, demonstrated a predominant nuclear localization in
active MEKK-1-expressing cells.
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We and others(14, 24, 25) have recently shown that Smad2 and Smad4 are
among the growing number of regulated transcriptional activators that
interact with the mammalian transcriptional coactivator, CREB-binding
protein (CBP). To determine if MEKK-1 could stimulate Smad2 and
Smad4 interactions with CBP, we employed the mammalian two-hybrid
system. As shown in Fig. 7, MEKK-1
selectively stimulates an interaction between Smad2 and the C-terminal
549 amino acids of CBP. This interaction was specific for Smad2 as
MEKK-1 did not significantly stimulate interaction between Smad4 and
any region of CBP in this two-hybrid system (data not shown). To
confirm these results biochemically, we performed immunoprecipitations with an anti-CBP/P300 antisera on cell extracts derived from
endothelial cells expressing tagged Smad constructs and active T
R1,
MEKK-1, TAK, or MEK-1. As shown in Fig. 7, in the presence of either
active TBR1 or active MEKK-1, both Smad2 and Smad4 can be demonstrated to coimmunoprecipitate with CBP/P300.

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Fig. 7.
Active MEKK-1 stimulates the interaction
between Smad2 and the transcriptional coactivator CBP in BAEC.
A, two-hybrid analysis of Gal4-Smad2 (pM2) with the five
domains of the coactivator CBP fused to VP16 in endothelial cells.
Active MEKK-1 specifically stimulates the interaction of Smad2 with the
carboxyl domain of CBP in endothelial cells. B, this
interaction is confirmed by coimmunoprecipitation (IP) of
Smad2 and Smad4 with CBP in the presence of active MEKK-1. Lysates from
BAEC expressing Smads 2 and 4 and the indicated active kinases were
immunoprecipitated with a pooled anti-CBP/P300 antisera, and
coprecipitating Smads were detected by Western blotting. The
lower band present in all lanes is the heavy chain of the
anti-CBP/P300 antisera (IgH). The bottom panels
confirm comparable expression of the Smad2 and -4 proteins in this
experiment.
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Laminar Shear Stress Can Stimulate Smad2-mediated Transcriptional
Activity in Endothelial Cells--
To determine whether endogenous
MEKK-1 expressed in endothelial cells is involved in the regulation of
Smad2 activity, we examined the ability of steady laminar shear stress,
a physiologically relevant fluid mechanical stimulus and a known
modulator of endogenous MEKK-1 in endothelial cells (26, 27), to
stimulate Smad-2 transcriptional activity. As shown in Fig.
8, 18 h of steady laminar shear
stress (LSS) at a magnitude of 10 dynes/cm2 reproducibly
stimulated the transcriptional activity of the Gal4-Smad2 construct,
and this effect could be almost completely inhibited by coexpression of
the dominant negative MEKK-1. LSS also stimulated the transcriptional
activity of a Gal4-Jun construct consistent with previous work
demonstrating the regulation of this transcription factor by shear
stress in endothelial cells. These data indicate that the action of
endogenous MEKK-1 is playing an important role in the activation of
Smad2 by this biomechanical stimulus (LSS) in endothelial cells and
suggest that Smad proteins may be capable of transducing diverse
stimuli in these cells.

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Fig. 8.
LSS can stimulate Smad2 and c-Jun-mediated
transcriptional activity in endothelial cells. The Gal4-Smad2 or
Gal4-Jun constructs together with the Gal4 reporter were cotransfected
into BAEC in the presence or absence of varying amounts of the dominant
negative MEKK-1 as in Fig. 1. The cells were subsequently stimulated
with 18 h of steady laminar shear stress at 10 dynes/cm2 in a well characterized in vitro flow
system as described under "Experimental Procedures." The LSS
stimulus reproducibly stimulated the transcriptional activity of both
Gal4-Smad2 and Gal4-Jun, and this was significantly inhibited by
coexpression of increasing amounts of the dominant negative
MEKK-1.
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DISCUSSION |
The discovery of Smad proteins has been a major advance in our
understanding of TGF-
superfamily signaling. Recent data suggest that these proteins may interact with a variety of other signaling pathways in cells and thus may be involved in the transduction or
modulation of other stimuli as well (6, 8, 9, 28). We have demonstrated
that a constitutively active form of MEKK-1 can selectively activate
Smad-dependent transcription in cultured endothelial cells
in the absence of exogenous TGF-
stimulation. MEKK-1 activation of
Smad2-mediated transcription correlated with an increase in
phosphorylation of Smad2 even in the absence of the C-terminal
SSXS motif of Smad2 that is the site of TGF-
type-1 receptor-induced phosphorylation, further supporting the fact that
MEKK-1 induced activation of Smad2 can occur independently of TGF-
.
Activation of Smad2 by MEKK-1 resulted in an enhanced interaction with
Smad4, nuclear localization of both Smad2 and Smad4, and a stimulation
of Smad-transcriptional coactivator (CBP) interactions. These data thus
describe a novel mechanism for the initiation of Smad protein signaling
in endothelial cells.
The functional interactions between the classic mitogen (MAPK/ERK)- and
stress (SAPK/JNK)-activated kinase pathways and Smad proteins are
likely to be complex. Previous work has demonstrated that both pathways
can be implicated in TGF-
transcriptional responses (29-31). Atfi
and colleagues (29, 30) reported that a dominant negative form of
MEKK-1 could inhibit TGF-
-mediated induction of the p3TP promoter in
HepG2 cells, and this same group subsequently reported that dominant
negative MEKK-1 could inhibit Smad3/4-mediated transcription. Recently
Zhang and colleagues (8) demonstrated a direct interaction between
Smad3/4 and c-Jun and c-Fos, two transcription factors that are among
the targets of the MAPK/SAPK pathways. These data suggest that there
are important stimulatory interactions between Smad proteins and the
SAPK/JNK pathway in the context of TGF-
signaling. Our results
demonstrating that MEKK-1, a MAPKKK in this pathway, can specifically
stimulate Smad2-mediated transcription suggest an additional
stimulatory interaction between these pathways. Interestingly, both
hepatocyte growth factor and epidermal growth factor that signal
through receptor tyrosine kinases have also been reported to activate Smad2-mediated signaling independently of TGF-
. This effect was demonstrated to be due to a phosphorylation event on Smad2 but did not
appear to require the presence of Smad4 (9). Our findings are
consistent with a model whereby Smad2 is the target of MEKK-1 or a
downstream kinase, and this stimulates Smad2 to interact with Smad4,
translocate to the nucleus, interact with the transcriptional coactivators CBP/P300, and activate transcription. In this regard the
events downstream of Smad2 activation mediated by active MEKK-1 appear
similar to those induced by TGF-
stimulation. Thus, MEKK-1-mediated stimulation of Smad2 may be an additional or alternate mechanism of
activation of Smad-mediated transcriptional events in endothelial cells.
The MEKK-1 mediated stimulation of Smad2-dependent
transcription described here is likely due to the direct action of this or another downstream kinase on Smad2 rather that simply due to transcriptional synergy between AP-1 and Smad2 since we have
demonstrated a change in the phosphorylation state of Smad2 in active
MEKK-1 expressing cells. Furthermore, this correlates with the ability of MEKK-1 to stimulate the transcriptional activity of a mutant form of
Smad2 that is not a substrate for TGF-
type 1 receptor-mediated phosphorylation (Smad2C*P). In addition, expression of active TAK-1
under conditions where this kinase could activate transcription of
GAL4-c-Jun fusion proteins or an AP-1 dependent promoter, did not
reproducibly activate Smad2-mediated transcription (data not shown).
Thus, activation of Smad2-mediated transcription and
c-Jun/AP-1-mediated transcription can be dissociated in this
endothelial system. Finally, active MEKK-1 selectively stimulated Smad2
interactions with itself and with Smad4 and CBP, but did not stimulate
Smad4 homotypic interactions or Smad4-CBP interactions in the
endothelial 2-hybrid system (Figs. 5 and 7, and data not shown). Taken
together these data suggest that Smad2 is a primary target of this
novel intracellular kinase-mediated regulation.
A potential limitation of these studies is the use of constitutively
activated forms of signaling kinases that can be associated with
nonspecific or promiscuous effects. However, the MEKK-1-mediated activation we observe is selective since active MEK-1, a distinct upstream kinase in the MAPK pathway, or active TAK-1, a kinase implicated in TGF-
signaling, failed to demonstrate the ability to
activate Smad2 when overexpressed at comparable levels as activated forms. TAK-1 was identified as a TGF-
-activated kinase and has been
reported to activate the TGF-
responsive promoter plasminogen activator inhibitor-1 in mink lung epithelial cells (12). In endothelial cells in our hands this activation required very high levels of active TAK-1 expression, and under these conditions, we
observed activation of a variety of other promoters such as the
cytomegalovirus and SV-40 promoters. Interestingly, active TAK-1 was
able to activate an AP-1-dependent promoter when expressed in endothelial cells (data not shown), consistent with previous data
demonstrating the ability of this kinase to stimulate JNK activity
(11). However, under these conditions we did not observe reproducible
Smad protein activation. Similarly, active MEK-1 could activate
ELK-1-dependent transcription but not
Smad-dependent transcription in our system. Thus, although
these results do not exclude roles for TAK-1 or MEK-1 in Smad-mediated
signaling, we were unable to demonstrate selective stimulation of
Smad-mediated signaling by these kinases in endothelial cells.
To address the question of whether endogenous MEKK-1 is involved in
Smad protein-mediated signaling, we utilized a dominant negative form
of the protein to inhibit the action of the endogenous kinase. As shown
in Fig. 1, the dominant negative MEKK-1 can partially inhibit
TGF-
-mediated transcriptional induction suggesting that endogenous
MEKK-1 is involved in this process. These data are consistent with
previous data demonstrating the role of this kinase in TGF-
signaling (30). To examine the ability of other activators of
endogenous MEKK-1 to modulate Smad signaling in endothelial cells, we
stimulated the cells with a physiologic fluid mechanical stimulus,
steady laminar shear stress, at 10 dynes/cm2. This
biomechanical stimulus has been demonstrated to elicit sustained
activation of MEKK-1 in endothelial cells in vitro (26, 27).
Under these conditions, we observed a reproducible stimulation of
Smad2-mediated transcriptional activity that was inhibited by
coexpression of the dominant negative MEKK-1. In contrast, when we
examined the response to TNF-
, a distinct activator of MEKK-1, we
failed to see any significant stimulation of Smad2-mediated transcription (data not shown). Taken together, these data suggest that
endogenous MEKK-1 is playing a role in Smad2-mediated transcriptional activation in endothelial cells and that the context in which MEKK-1 is
activated is likely to be an important determinant of its ability to
modulate Smad protein signaling.
The ability of Smad7 to inhibit MEKK-1-mediated stimulation of
Smad2-dependent transcriptional activation points to a
novel role for this inhibitory Smad. We have previously demonstrated that Smad7 is induced by biomechanical forces such as steady laminar shear stress in endothelial cells and can bind to the activated type 1 TGF-
receptor and inhibit its ability to phosphorylate Smad2 (13,
32). In the context of active MEKK-1 expressing endothelial cells,
Smad7 may be utilizing an analogous mechanism to interact with
intracellular kinases and block their ability to phosphorylate Smad2.
Furthermore, flow-regulated Smad7 expression may be an important locus
of cross-talk between biomechanical stimuli such as fluid shear
stresses and cytokine/growth factor stimuli such as TGF-
and related
species in endothelial cells (33).
In summary, we have demonstrated that Smad2-mediated transcriptional
activation can be initiated by coexpression of an active form of
MEKK-1. These data together with previous data from other groups
suggest that multiple mechanisms may exist to modulate Smad protein
signaling in cells and that this important family of intracellular
signaling molecules may be involved in more than just TGF-
superfamily signaling. Given the many stimuli that have been
demonstrated to activate the SAPK/JNK pathway and MEKK-1 (17, 26, 34),
Smad proteins may be involved in modulating the cellular response to
diverse signals ranging from humoral effectors such as growth factors,
to biomechanical stimuli such as physical stresses. As such, this class
of endothelial-expressed signaling proteins may participate importantly
in the orchestration of complex biological processes such as
atherogenesis, vascular remodeling, angiogenesis, and vascular development.