(Received for publication, August 29, 1996, and in revised form, October 10, 1996)
From the Vascular Research Division, Department of
Pathology, Brigham and Women's Hospital and Harvard Medical School,
Boston, Massachusetts 02115 and the ** Howard Hughes Medical
Institute, and Program in Molecular Medicine, Department of
Biochemistry and Molecular Biology, University of Massachusetts Medical
School, Worcester, Massachusetts 01605
E-selectin expression by endothelium is crucial
for leukocyte recruitment during inflammatory responses.
Transcriptional regulation of the E-selectin promoter by tumor necrosis
factor (TNF
) requires multiple nuclear factor-
B (NF-
B)
binding sites and a cAMP-responsive element/activating transcription
factor-like binding site designated positive domain II (PDII). Here we
characterize the role of the stress-activated family of
mitogen-activated protein (MAP) kinases in induced expression of this
adhesion molecule. By UV cross-linking and immunoprecipitation, we
demonstrated that a heterodimer of transcription factors ATF-2 and
c-JUN is constitutively bound to the PDII site. TNF
stimulation of
endothelial cells induces transient phosphorylation of both ATF-2 and
c-JUN and induces marked activation of the c-JUN N-terminal kinase
(JNK1) and p38 but not extracellular signal-regulated kinase (ERK1).
JNK and p38 are constitutively present in the nucleus, and DNA-bound
c-JUN and ATF-2 are stably contacted by JNK and p38, respectively.
MAP/ERK kinase kinase 1 (MEKK1), an upstream activator of MAP kinases, increases E-selectin promoter transcription and requires an intact PDII
site for maximal induction. MEKK1 can also activate NF-
B -dependent gene expression. The effects of dominant
interfering forms of the JNK/p38 signaling pathway demonstrate that
activation of these kinases is critical for cytokine-induced E-selectin
gene expression. Thus, TNF
activates two signaling pathways, NF-
B and JNK/p38, which are both required for maximal expression of E-selectin.
The recruitment of leukocytes from the circulation into the extravascular space is critical for inflammatory responses and repair of tissue injury. The process of leukocyte emigration involves several steps (reviewed in Refs. 1, 2). The initial interaction between leukocytes and endothelium appears to be transient, resulting in the rolling of leukocytes along the vessel wall. The rolling leukocytes then become activated by local factors generated by the endothelium, resulting in their arrest and firm adhesion to the vessel wall. Finally, the leukocyte transmigrates the endothelium. These complex processes are regulated in part by specific endothelial-leukocyte adhesion molecules. The initial rolling interactions are mediated by the selectins, while firm adhesion and diapedesis appear to be mediated by the interaction of integrins on the surface of leukocytes with immunoglobulin gene superfamily members expressed by endothelial cells.
Expression of some of the endothelial-leukocyte adhesion molecules is
dynamically regulated at sites of leukocyte recruitment. These changes
in surface proteins provide the endothelial cell with a mechanism of
regulating cell-cell interactions during recruitment of specific types
of leukocytes. For example, endothelial expression of E-selectin is
dramatically induced at sites of inflammation. The E-selectin gene is
transcriptionally silent in quiescent endothelial cells and is rapidly
and transiently transcribed when endothelial cells are activated with
tumor necrosis factor (TNF
)1 or
interleukin-1
(3). Transcription of E-selectin peaks in 1-2 h and
returns to base-line levels by 12 h post-induction. Previous
studies of the E-selectin promoter identified several promoter elements
or positive regulatory domains (PDs) necessary for TNF
responsiveness (4). DNA binding studies reveal a requirement for
nuclear factor-
B (NF-
B) and a small group of other
transcriptional activators (reviewed in Ref. 5). Three of the elements
(PDI, -III, and -IV) contain NF-
B recognition sequences. One of
these, PDI, is a consensus NF-
B site and has been shown to bind
p50/p65 heterodimers (6). The other two
B elements (PDIII and PDIV) are immediately adjacent to each other, and at least one, and perhaps
both, are also occupied by the p50/p65 heterodimer (4, 7, 8). The
fourth required region, PDII, contains an element similar to the cAMP
response element/activating transcription factor element (CRE/ATF). The
sequence, TGAC
TCA, varies at one site (underlined) from
the consensus CRE/ATF sequence (TGACGTCA) and is identical to a
functional site in the c-JUN promoter (9, 10). Previous
studies using recombinant proteins have shown that the PDII element can
be occupied by a variety of members of the ATF family of transcription
factors, including ATF-2, ATF-a, ATF-3, and c-JUN (11, 12). Although
the nature of the ATF family members bound to this element in
TNF
-activated endothelial cells is uncertain, ATF-2 appears to be
essential for E-selectin expression in that ATF-2 homozygous null mice
are defective in E-selectin induction (13). In addition to the
transcriptional activators, cytokine-induced E-selectin expression
depends upon the chromatin architectural protein, high mobility group
protein I(Y) (HMGI(Y)), which binds specifically to several sites in
the promoter and potentiates binding of NF-
B to either PDIII or PDIV and binding of ATF-2 to PDII (4, 8).
The best-studied signaling pathway involved in TNF activation of
E-selectin gene transcription is the NF-
B/I
B
system (reviewed in Refs. 14-17). Constitutively present in all cells, the p50/p65 subunits of NF-
B are held in the cytoplasm by one or more members of
a family of inhibitors, including p100, p105, I
B
, and I
B
. In endothelial cells, TNF
stimulation results in phosphorylation of
I
B
, which targets the inhibitor for ubiquitination and
degradation by the proteasome (18-20). Loss of I
B
results in
nuclear translocation of the p50/p65 heterodimer, where it binds its
recognition sequences and activates transcription.
In contrast to the NF-B/Rel family of proteins, other classes of
transcription factors reside constitutively in the nucleus but require
an activation signal. The family of mitogen-activated protein (MAP)
kinases are important mediators of signals transduced from the cell
surface to transcription factors in the nucleus (for review see Ref.
21). Multiple MAP kinases with different substrate specificities are
activated by distinct extracellular stimuli. Recent studies revealed
two novel subgroups, the c-JUN N-terminal kinase (JNK) protein kinases
(also called stress-activated protein kinases) and p38 kinases which
are activated in response to TNF
, interleukin-1
, bacterial
lipopolysaccharide, and UV light (22-30). The JNK protein kinases
and the related p38 kinases can phosphorylate the N-terminal activation
domain of c-JUN on serines 63 and 73 and ATF-2 on threonines 69 and 71 (27, 31, 32) as well as other substrates (33). Activation of the
JNK/p38 protein kinases involves a kinase cascade in which the upstream activator MAP kinase kinase kinase (MEKK) phosphorylates and activates MAP kinase kinase 3, 4, and 6 (MKK 3, 4 and 6), which in turn phosphorylate and activate JNK and p38 MAP kinases (34-37).
Ultimately, phosphorylation of c-JUN and ATF-2 by JNKs increases
the transactivating properties of both of these proteins (31, 32,
36, 38-41).
To further understand how transcription of the E-selectin gene is
induced in response to cytokines, we examined the role of the PDII
element and its associated transcriptional activators to act as a
signaling target for the JNK/p38 MAP kinase pathway. Herein we show
that c-JUN/ATF-2 heterodimers are the preferred complex bound at the
E-selectin PDII site. TNF stimulation of endothelial cells results
in transient enhanced phosphorylation of both c-JUN and ATF-2, and
concomitant activation of JNK and p38 kinases. Moreover, an upstream
activator of the JNK signaling pathway, MEKK1 (42, 43), can activate
E-selectin as efficiently as TNF
, and activation requires that the
PDII element be intact. Overexpression of phosphorylation-defective
ATF-2 or kinase-inactive JNK can block TNF
and MEKK1 activation of
the E-selectin promoter. Additionally, overexpression of MEKK1
activates
B-dependent reporter gene expression, an
effect which is decreased by a dominant negative form of a member of
the JNK signaling pathway and by MAP kinase phosphatase. Thus, TNF
and MEKK1 activate at least two signaling pathways, NF-
B/I
B
and JNK/p38 MAP kinases, which converge on the E-selectin promoter and
confer maximal cytokine responsiveness to the gene.
Human endothelial cells
obtained from collagenase-digested umbilical veins (HUVEC) (44) were
cultured in M199 with 20% fetal bovine serum, 100 µg/ml porcine
intestinal heparin, 50 µg/ml endothelial mitogen, 50 units/ml
penicillin, and 50 µg/ml streptomycin, and 25 mM HEPES in
gelatin-coated plates. Bovine aortic endothelial cells (BAEC) were
isolated and maintained in culture using previously described
procedures (45). For experimental cytokine induction, confluent
monolayers of endothelial cells were exposed to recombinant human
TNF (Genentech, San Francisco, CA) at a final concentration of 100 units/ml in complete media.
Following experimental treatment of HUVECs, nuclear and cytosolic extracts were prepared as described previously (20) The resulting crude nuclear extracts were diluted 1:1 with buffer D (20 mM HEPES (pH 7.9), 100 mM KCl, 0.2 mM EDTA, 20% glycerol, 0.5 mM DTT). All buffers additionally contained the following mixture of protease inhibitors and phosphatase inhibitors: 1.0 mM phenylmethanesulfonyl fluoride (PMSF), 10 µg/ml each leupeptin and aprotinin, 1.5 µg/ml pepstatin A, 1.0 mM sodium fluoride, and 1.0 mM sodium orthovanadate.
UV Cross-linkingPhotoreactive labeled DNA probes were
prepared by annealing coding or noncoding strand templates with
complementary primers and filling in with the Klenow fragment of DNA
polymerase 1 in the presence of [-32P]dATP,
[
-32P]dCTP, dGTP, and 1:1 dTTP and
5-bromo-2
-deoxyuridine 5
-triphosphate (Sigma) (6,
46). Probes were designed to incorporate 5-bromo-2
-deoxyuridine 5
-triphosphate into the CRE/ATF-like site within PDII.
Oligonucleotides utilized were template (lower strand)
5
GTACAATGATGTCAGAAACTCTGTC3
and primer (upper strand)
5
GACAGAGTTTC3
. Binding reactions (20 µl) contained 15 µg of
nuclear extract protein, binding buffer (10 mM Tris (pH
7.5), 50 mM NaCl, 1 mM DTT, 1 mM
EDTA, 5% glycerol), 0.5 µg of poly[dI·dC], 0.5 µg of salmon
sperm DNA, and 106cpm of labeled DNA. Reactions were
incubated at room temperature for 20 min. Samples were UV-irradiated
for 15 min using a transilluminator (Fotodyne Inc, New Berlin, WI). For
immunoprecipitation, radiolabeled adducts from four binding reactions
were pooled and diluted to 200 µl with RIPA buffer (10 mM
Tris-Cl (pH 8.0), 150 mM NaCl, 0.5% sodium dodecyl
sulfate, 1% Nonidet P-40, and 1% deoxycholate supplemented with 200 µg/ml bovine serum albumin, 0.1 mg/ml salmon sperm DNA, 1.0 mM PMSF, 1 µg/ml each leupeptin, aprotinin, and pepstatin
A, 1 mM sodium fluoride, and 1 mM sodium
orthovanadate) or ELB (50 mM HEPES (pH 7.9), 250 mM NaCl, 0.1% Nonidet P-40, 5 mM EDTA, 0.5 mM DTT, supplemented with 200 µg/ml bovine serum albumin,
0.1 mg/ml salmon sperm DNA, 1 mM PMSF, 1 µg/ml each
leupeptin, aprotinin, and pepstatin A, 1 mM sodium
fluoride, and 1 mM sodium orthovanadate) and incubated with
2 µl of antisera (1 h on ice) followed by 20 µl Protein A-Sepharose
(1 h at 4 °C with shaking). Immune complexes were washed three times
with RIPA or ELB buffer and analyzed on 10% SDS-polyacrylamide gels.
Duplicate sets of radiolabeled adducts were denatured prior to
immunoprecipitation by heating at 100 °C for 3 min in 0.5% SDS.
ATF-2 and c-JUN polyclonal rabbit antisera were purchased from Santa
Cruz Biotechnology Inc. (Santa Cruz, CA), p38 (HOG) rabbit antisera was
purchased from Upstate Biotechnology Inc. (Lake Placid, NY), and JNK
antisera was as described (31). JNK and p38 MAP kinases were expressed in the yeast Pichia pastoris using vectors purchased from
Invitrogen Corp. JNK1 and p38 MAP kinase expression were induced with
methanol using procedures recommended by the manufacturer
(Invitrogen).
A biotinylated double-stranded
oligonucleotide (Integrated DNA Technologies, Inc. Coralville, IA)
spanning the cytokine response region of the E-selectin promoter (156
to
78) was coupled to Dynabeads M-280 streptavidin (Dynal, Lake
Success, NY) according to the manufacturer's recommendations. The
E-selectin-coupled matrix (250 µg) was incubated with 25 µl of
endothelial nuclear extract in binding buffer (50 mM NaCl,
5 mM MgCl2, 10 mM Tris (pH 7.5), 1 mM DTT, 1 mM EDTA, 0.25 µg/ml
poly[dI·dC], and 5% glycerol) for 20 min at room temperature with
mixing every 5 min to keep the Dynabeads in suspension. The non-bound
or flow-through fractions were collected, and the Dynabeads with bound
extract proteins were washed four times in 1 × binding buffer
containing 0.5 µg/ml poly[dI·dC]. Proteins bound to the Dynabeads
were solubilized in 1 × Laemmli sample buffer, boiled, and
subjected to SDS-polyacrylamide gel electrophoresis, followed by
transfer to nitrocellulose membranes. Membranes were probed with
antisera to Sp1, p65, ATF-2, c-JUN (Santa Cruz Biotechnology, Santa
Cruz, CA), and p50 (provided by Nancy Rice, NCI, Frederick, MD) at
dilutions ranging from 1:2500-1:10,000, followed by enhanced
chemiluminescent detection as described below.
Extracts were prepared from
control and TNF-treated HUVECs in which the media were replaced with
media containing 1% fetal calf serum for 1 h prior to
stimulation. Cells were solubilized with Triton lysis buffer (TLB, 20 mM Tris (pH 7.4), 1% Triton X-100, 10% glycerol, 137 mM NaCl, 2 mM EDTA, 25 mM
-glycerophosphate, 1 mM sodium orthovanadate, 2 mM pyrophosphate, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin). Extracts were centrifuged at
14,000 × g for 15 min at 4 °C. The JNK, p38, or ERK
protein kinases were immunoprecipitated by incubation for 1 h at
4 °C with specific rabbit polyclonal antibodies bound to Protein
A-Sepharose (Pharmacia Biotech Inc.). The rabbit polyclonal JNK and p38
antibodies have been described (31) The immunoprecipitates were washed
twice with TLB and twice with kinase buffer (20 mM HEPES
(pH 7.4), 20 mM
-glycerophosphate, 20 mM
MgCl2, 2 mM dithiothreitol, 0.1 mM sodium orthovanadate). The kinase assays were initiated by the addition
of 1 µg of substrate protein and 50 µM
([
-32P]ATP) (10 Ci/mmol) in a final volume of 22 µl.
The reactions were terminated after 15 min at 30 °C by addition of
Laemmli sample buffer. Control experiments demonstrated that the
phosphorylation reaction was linear with time for at least 30 min under
these conditions. The phosphorylation of the substrate proteins was examined by SDS-polyacrylamide gel electrophoresis followed by autoradiography.
HUVECs
(approximately 1.5 × 106) were untreated or treated
with TNF for 15 min, rinsed three times in ice-cold
phosphate-buffered saline, and harvested in RIPA lysis buffer (20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 2 mM EDTA, 1% deoxycholate, 1% Triton X-100, 0.25% SDS, 1 mM PMSF, 0.1 mM DTT, 10 µg/ml each leupeptin
and aprotinin, 1.5 µg/ml pepstatin, 1 mM each sodium
vanadate and sodium fluoride). Both samples were divided equally to
three tubes, and 5 µl of ATF-2 rabbit antiserum (Santa Cruz
Biotechnology Inc.) was added to each. The samples were incubated
overnight at 4 °C with rocking and precipitated with 50 µl of
Protein A slurry for 4 h, and then washed three times in RIPA
buffer. The pellets were then washed an additional two times in
phosphatase buffer (50 mM HEPES (pH 7.5), 1 mM
DTT, 1 mM MgCl2, 1 mM PMSF, 10 µg/ml each leupeptin and aprotinin, 1.5 µg/ml pepstatin) to remove
the phosphatase inhibitors. The Protein A pellet was resuspended in 50 µl phosphatase buffer, and 10 mM NaF was added to one
tube to provide a negative control. Protein phosphatase 2A (PP2A, 0.5 units; UBI) or calf intestinal alkaline phosphatase (1 unit; Boehringer
Mannheim) were then added, and the samples were incubated at 37 °C
for 2 h. After incubation the samples were washed once in RIPA
buffer, resuspended in 20 µl of SDS sample buffer, boiled, and
fractionated by 8% SDS-polyacrylamide gel electrophoresis. The gel was
transferred to nitrocellulose and immunoblotted with ATF-2 mouse
monoclonal antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA) as
described below.
Nuclear extracts from TNF-treated human
umbilical vein endothelial cells were electrophoresed on 8%
SDS-polyacrylamide gels and transferred to nitrocellulose in 25 mM Tris, 192 mM glycine, 5% methanol at 100 V
for 1 h. ATF-2 and c-JUN rabbit antisera (Santa Cruz Biotechnology
Inc.) were used at 1:5,000 dilution. Antiserum specific for the
phosphorylated form of c-JUN (Ser63) was obtained from New
England BioLabs (Beverly, MA) and used at 1:1000 dilution. Polyclonal
antisera to MKK3 and MKK4 (Santa Cruz, Biotechnology Inc. Santa Cruz,
CA) were used at 1:1000 dilution. Immunoreactive proteins were detected
according to the enhanced chemiluminescent protocol (Amersham Corp.)
using 1:10,000 horseradish peroxidase-linked donkey anti-rabbit
secondary antiserum. Blots were exposed to film for 1-10 min.
Bovine aortic endothelial cells (BAECs) were
transfected, harvested, and assayed for reporter proteins as described
previously (4). Relative transfection efficiency was determined by
cotransfection with pTK-GH (Promega) (4 µg). For experimental
cytokine induction, confluent monolayers of endothelial cells were
exposed to recombinant human TNF (Genentech, San Francisco, CA) at a
final concentration of 200 units/ml in complete media. A fragment
containing the region
578 to +35 of the E-selectin promoter was
generated by polymerase chain reaction amplification as described
previously (4), gel-purified, and subcloned into the SmaI
site of the reporter plasmid pCAT3. The mPDII reporter includes a
4-base pair mutation in the PDII ATF-2 site (gtcaATCA) generated as
described previously (4). The expression vectors for ATF-2 and ATF-2
[Ala69/71] (32), MEKK1 (33, 42, 43), MKK4 (Ala) (36), and
JNK1 [Ala183Phe185] (27) have been described.
Transfection of Chinese hamster ovary cells was done as described
previously (36). Transfection efficiency was monitored using a control
plasmid (pCH110, Pharmacia LKB Biotech). Luciferase reporter and
-galactosidase activities were measured as described previously
(32). The mouse MKP-1 expression plasmid was generously provided by Dr.
N. K. Tonks (47).
The E-selectin promoter contains a
functional CRE/ATF-like element which is necessary for cytokine
responsiveness (4, 9, 13). DNA binding studies with recombinant
proteins demonstrated that ATF-2, ATF-a, ATF-3, and a variety of
heterodimers are all capable of binding the E-selectin CRE/ATF-like
element (9). To fully define the preferential occupants of PDII in
control and TNF-activated endothelial cells, we used UV
cross-linking followed by immunoprecipitation. Nuclear extracts from
human umbilical vein endothelial cells (HUVECs) were subjected to UV
irradiation to cross-link the bound proteins to a photoreactive PDII
probe and then immunoprecipitated with antibodies to c-JUN and ATF
proteins. Shown in Fig. 1A are the results
with antiserum to ATF-2 and c-JUN using extracts from HUVECs untreated
or treated with TNF
for 12 h. Under denaturing conditions,
antiserum to ATF-2 immunoprecipitated predominantly ATF-2. Antisera to
c-JUN immunoprecipitated predominantly c-JUN. Other antisera, including
that raised to ATF-a, ATF-3, or p65, failed to immunoprecipitate any
photoreactive species.2 This pattern of
adducts was observed in nuclear extracts from untreated endothelial
cells (Fig. 1A) and from cells treated with TNF
for 15 min and 1 h.2 Thus, ATF-2 and c-JUN are constitutively
associated with the promoter and are components of the cytokine-induced
E-selectin enhancer complex.
Immunoblotting of Proteins Bound to the Cytokine Response Region of the E-selectin Promoter
The transcriptional activators binding
the positive regulatory domains in the E-selectin promoter were defined
by their ability to bind isolated promoter elements. To determine if
the activators present in nuclear extracts would bind to the complete
E-selectin cytokine response region, a biotinylated affinity matrix
spanning the complete E-selectin enhancer was generated and coupled to streptavidin-coated magnetic beads. This biotinylated probe was incubated with nuclear extracts from control or TNF-treated (15 min)
HUVECs and washed extensively. Transcription factor complexes were
released from the magnetic beads by boiling in SDS sample buffer and
detected by immunoblotting. As shown in Fig. 1B, Sp1 binding
was minimal, demonstrating that the association of transcriptional activators with the complex was specific. Additionally, binding of both
the p65 and p50 subunits of NF-
B was TNF
-inducible. ATF-2 and
c-JUN from both control and TNF
-treated cells were bound, confirming
results obtained by UV cross-linking with the isolated PDII site (Fig.
1A). These results demonstrate that p50 and p65 are
inducibly recruited to an intact E-selectin cytokine response region
that contains constitutively bound ATF-2 and c-JUN. These findings are
consistent with previous structural and functional studies
characterizing the transcription factors bound to isolated positive
regulatory domains (4, 6-8).
Both JNK and p38 MAP kinases can phosphorylate ATF-2
while c-JUN is phosphorylated by JNK (27, 31, 32). To evaluate the
activity of p38, JNK, and ERK MAP kinases in response to TNF, kinase
activity in immunoprecipitates was measured in whole cell extracts from
control and TNF
-treated HUVECs using immune complex kinase assays.
As shown in Fig. 2A, both p38 and JNK kinases
were strongly and transiently activated within 15 min of TNF
stimulation. In contrast, ERK protein kinase was basally active and
minimally affected by TNF
(Fig. 2A). These findings
suggest that the TNF receptor preferentially initiates signaling events
in endothelial cells leading to phosphorylation and activation of p38
and JNK kinases, rather than initiating the ERK MAP kinase system.
To determine whether these changes in kinase activity were due to
alterations in levels or changes in subcellular location of p38 or JNK
protein, Western blot analysis was performed on subcellular fractions
from control and TNF-treated HUVECs. As shown in Fig. 2B,
p38 and JNK1 MAP kinases are constitutively present in both the
cytosolic and nuclear compartments of fractionated cultured endothelial
cells. Using the immunoprecipitation kinase assay, we have detected
TNF
-induced JNK-1 activity in both cytosolic and nuclear extracts.
Most of the JNK-1 activity was in the cytosol, and peak activity of the
kinase in both the nucleus and cytosol was observed at 15 min after
cytokine addition.3 Thus the rapid changes
in kinase activity seen in endothelial cells following TNF
treatment
(Fig. 2A) were not due to alterations in levels or changes
in subcellular location of p38 or JNK protein. The upstream activating
kinases, MKK3 and MKK4, are also present in both the cytosol and
nucleus of HUVECs (Fig. 2B). The distribution of these
kinases did not change with TNF
stimulation. In contrast to the
findings with the kinases, dramatic nuclear accumulation of p65 was
seen in response to TNF
stimulation (Fig. 2B), as reported previously (6). The constitutive presence of these kinases in
the nuclear compartment indicates that their activation may occur in
the nucleus of endothelial cells.
Selective activation of the JNK and p38 MAP kinases in
response to TNF suggests that the cytokine may increase
phosphorylation of transcription factor targets of these protein
kinases in endothelial cells. The phosphorylation of ATF-2 and c-JUN
increases the ability of these proteins to act as transcriptional
activators. To determine if ATF-2 and c-JUN were phosphorylated in
endothelial cells in response to TNF
, cytosolic and nuclear extracts
were prepared from control and TNF
-induced HUVECs and immunoblotted.
ATF-2 was undetectable in the cytosolic fraction (data not shown) while constitutively expressed in the nucleus (Fig.
3A). ATF-2 from cells exposed to TNF
for
15 min exhibited a modest retardation in mobility when compared with
ATF-2 from control cells (Fig. 3A, compare lanes
1 and 2). The time course of the ATF-2 mobility shift
closely correlated with the kinase activation (Fig. 2A). To
investigate whether this mobility shift was due to a change in
phosphorylation status, extracts from HUVECs stimulated for 0 or 15 min
with TNF
were immunoprecipitated with an ATF-2 antibody, treated
with phosphatases, and immunoblotted with a second ATF-2 antiserum. In
the presence of the phosphatase inhibitor, sodium fluoride (Fig.
3B, lanes 1 and 4), treatment with
protein phosphatase-2A (PP2A), a phosphoserine/threonine-specific
enzyme, did not alter the pattern of migration seen in the absence of
PP2A (Fig. 3A, lanes 1 and 2). In the absence of
sodium fluoride, PP2A treatment clearly resulted in a downward shift in
the mobility of ATF-2 from TNF
-treated cells (lane 5) but
did not affect ATF-2 mobility from control cells (lane 2).
Treatment with calf intestinal phosphatase resulted in a downward shift
of ATF-2 from both untreated and TNF
-stimulated cells (lanes
3 and 6), indicating that ATF-2 in cultured endothelial
cells is basally phosphorylated. The differences in migration and the
differences in sensitivity to the two phosphatases indicated that ATF-2
is hyperphosphorylated in response to TNF
stimulation of endothelial
cells, consistent with findings obtained with IL-1 treated and
UV-irradiated cells (32).
Similarly prepared nuclear extracts were analyzed for phosphorylation
of c-JUN. A polyclonal antibody to a synthetic phosphopeptide corresponding to residues 59-67 of human c-JUN was obtained. This antibody detects only phosphorylated c-JUN (Fig. 3C) and is
reported not to cross-react with the phosphorylated forms of JunD or
JunB (New England BioLabs, Beverly, MA). As shown in Fig.
3C, c-JUN is constitutively present in HUVEC nuclei and,
like ATF-2, is rapidly and transiently phosphorylated in response to
TNF. Shown for comparison, NF-
B p65 translocates into the nucleus
following TNF
treatment. Translocation of p65 into the nucleus
occurs simultaneously with phosphorylation of ATF-2 and c-JUN. Taken
together, these data indicate that TNF
-activated JNK and p38 MAP
kinases can phosphorylate the components of the transcription factor
heterodimer associated with the E-selectin PDII regulatory domain.
The ability of ATF-2
and c-JUN from unstimulated endothelial cells to bind to the PDII site,
the constitutive presence of JNK and p38 MAP kinases and their upstream
activators in the nucleus, and the transient activation of the kinases
in response to TNF suggested that phosphorylation of ATF-2 and c-JUN
may take place while the heterodimer is actively bound to the
E-selectin promoter. To explore this possibility, UV cross-linking and
immunoprecipitation were employed. The radiolabeled photoreactive PDII
probe was incubated with HUVEC nuclear extract and exposed to UV light
to covalently couple ATF-2 and c-JUN to the labeled DNA (see Fig. 1).
These extracts were subjected to immunoprecipitation with antibodies to
JNK, p38, ATF-2, or c-JUN under less stringent conditions (ELB buffer)
than those used in previous studies (RIPA buffer, Fig. 1).
Immunoprecipitation with anti-JNK (Fig. 4, lanes
1-4) resulted in a doublet of apparent molecular mass of 45-55
kDa. The lower band of the doublet comigrates with the band produced by
precipitation with anti-c-JUN (lane 12). The pattern of
bands seen with anti-JNK did not change with TNF
stimulation,
including up to 12 h (data not shown). Preimmune sera failed to
precipitate any DNA-protein adducts (lane 5). Thus, results
with anti-JNK show that JNK can interact with c-JUN, while bound to
DNA.
A similar approach was used to explore whether p38 and ATF-2 are
associated. When anti-p38 was used in the UV cross-linking and
immunoprecipitation protocol, a broad band was observed (Fig. 4,
lanes 6, 7) which comigrated with the band seen with
precipitation of ATF-2 (lane 11). Again, this pattern did
not change with various conditions of TNF stimulation. When the
cross-linked sample was boiled in SDS prior to addition of p38
antibody, the DNA-protein adduct was not precipitated (Fig. 4,
lane 8), indicating that the immunoprecipitated species
(lanes 6, 7) which co-migrates with ATF-2 (lane
11) is the result of protein-protein interactions. These results
with anti-p38 show that p38 associates with ATF-2 while ATF-2 is bound
to DNA. To further evaluate the specificity of the immunoprecipitation
reaction, the kinase antibodies were incubated with yeast lysates, from
either control yeast or yeast programmed to produce the respective
kinase, prior to being added to the cross-linked reaction. Strikingly,
incubation of anti-p38 with p38 containing lysate completely abolished
the ability of the antibody to precipitate the DNA-protein adduct (Fig.
4, compare lanes 9 and 10). Similarly, incubation
of anti-JNK with JNK containing yeast lysate diminished the intensity
of DNA-protein adduct compared with antibody incubated with control
lysate (data not shown). These results indicated that the JNK and p38
kinases are capable of stable interactions with DNA-bound substrates,
c-JUN and ATF-2, respectively. In addition, although both p38 and JNK
kinases can both phosphorylate recombinant ATF-2, p38 appeared to be
the preferred species associating with ATF-2 when the ATF-2/c-JUN
heterodimer was bound to DNA.
JNK kinases contain a
Thr-Pro-Tyr motif and must be phosphorylated on Thr and Tyr for
activation (27). Like the ERK group of MAP kinases, activation of the
JNK and p38 kinases involves a kinase cascade in which the upstream
kinase MAP kinase kinase kinase activates MAP kinase kinase 3, 4, and 6 (MKK3, 4 and 6) which in turn activate the JNK and p38 MAP kinases (31,
32, 34-37). To determine if activation of the JNK/p38 kinase pathway in response to TNF is required for induction of E-selectin
transcription, transient transfections were undertaken to overexpress
members of this pathway in bovine aortic endothelial cells (BAEC).
Increasing amounts of an expression plasmid coding for the upstream
activator, MEKK1 (33, 42, 43), were transfected into BAECs with an E-selectin-promoter reporter plasmid (p-578), and the effects were
compared with that of TNF
stimulation (Fig.
5A). As described previously (4), TNF
stimulation resulted in approximately 4-fold induction of E-selectin
promoter activity and a plasmid bearing a mutation in the CRE/ATF
sequence in PDII (p-578mPDII) was significantly less active (Fig.
5A). In addition, the lowest amount (100 ng) of MEKK1 was
capable of stimulating expression from the wild-type E-selectin
promoter, and induction occurred in a dose-dependent
manner. The mutated E-selectin promoter responded less well to MEKK1
and only to the higher doses. These data indicate that an upstream
activator of the JNK pathway can activate E-selectin transcription. In
addition, we investigated whether a catalytically inactive form of JNK1
would act as a dominant inhibitor of TNF
and MEKK1 activation of the
E-selectin promoter. Transient transfection assays were performed with
a JNK1 mutant in which the sites of activating Thr and Tyr were
replaced with Ala and Phe (27). As shown in Fig. 5B, the
mutant JNK1 is capable of blocking a portion of the TNF
response and
most of the response to the lowest dose of MEKK1. As increasing amounts
of MEKK1 are expressed, the inhibition by mutant JNK1 is diminished.
This may represent the activation of other signaling pathways (42, 43).
The ability of catalytically inactive JNK1 to block TNF
and MEKK1
induction suggests that this kinase is important for TNF
induction
of E-selectin transcription.
A Phosphorylation-defective Mutant of ATF-2 Blocks Cytokine-induced Expression of E-selectin
The JNK/p38 kinases phosphorylate the
N-terminal activation domains of c-JUN and ATF-2. To determine the
importance of ATF-2 phosphorylation in TNF-induced expression of
E-selectin, we tested whether a phosphorylation-defective mutant of
ATF-2 inhibits E-selectin promoter activity. Plasmids expressing either
wild-type ATF-2 or a phosphorylation-defective mutant of ATF-2 (ATF-2
[Ala69, 71]) were transfected into BAECs with the p-578
reporter plasmid and challenged with TNF
. The cytokine activated the
expression of the E-selectin promoter-reporter about 5-fold (Fig.
5C). The mutant ATF-2 [Ala69, 71] diminished
TNF
induction of the E-selectin promoter. The presence of wild-type
ATF-2 did not affect the TNF
response of the E-selectin promoter
(Fig. 5C). To determine the importance of ATF-2
phosphorylation in MEKK1-induced expression of E-selectin, cells were
stimulated with MEKK1 at levels that do not activate a construct with a
mutation in PDII (Fig. 5A). Under these conditions,
activation in response to co-transfected MEKK1 was potentiated by
wild-type ATF-2 but not by the mutant ATF-2 [Ala69, 71]
(Fig. 5C). Partial suppression by the dominant negative
mutant may be due to the high levels of ATF-2 in endothelial cells.
These data demonstrate that phosphorylation of ATF-2 is necessary for full activation of the E-selectin promoter by TNF
and MEKK1.
Maximal activation of the E-selectin promoter
by MEKK1 involves phosphorylation of ATF-2 and requires an intact PDII
site. However, like TNF, MEKK1 still activates an E-selectin
promoter reporter in which the PDII site is mutated but the NF-
B
sites (PDI, III, and IV) are intact (Fig. 5A). These
findings suggest that MEKK1 may also serve as an upstream activator of
NF-
B. To directly test this hypothesis,
NF-
B-dependent promoters which lack ATF-2 and c-JUN
binding sites were evaluated for the ability to respond to MEKK1
overexpression. VCAM-1 is a cell adhesion molecule which, like
E-selectin, is highly inducible by TNF
in endothelial cells (1). The
VCAM-1 promoter contains two tandem consensus NF-
B binding sites
(45, 48). Transfection of BAECs with the MEKK1 expression vector
resulted in increased activity of a promoter-reporter plasmid
containing the two isolated
B elements from the VCAM-1 promoter. The
level of activation of the reporter construct by MEKK1 was similar to
that seen with TNF
(Fig. 6A). These
findings are consistent with recent studies in which MEKK1-induced
transcription from other
B-dependent reporters and
co-expression of I
B
inhibited MEKK1-induced transcriptional activity (49, 50). Taken together, the results demonstrate that the
MEKK kinase system can activate NF-
B-dependent gene expression but do not define the signaling pathway.
The activation state of NF-B is determined by association with a
cytoplasmic inhibitor, such as I
B
. Phosphorylation of I
B
results in the ubiquitination, dissociation, and subsequent degradation
of the inhibitor by the proteasome proteolytic pathway (18-20,
51-53). The signaling pathways that couple MEKK1 to the activation of
the I
B
kinase remain elusive. To explore these events, we
utilized two approaches that build on the observation that MEKK1
activates
B-dependent transcription. Expression of MEKK1
increased activity of a
B-dependent reporter in a model system by about 14-fold (Fig. 6B), consistent with the
kinase-dependent induction of the authentic E-selectin
promoter (Fig. 5A) and other
B-dependent
reporters (Fig. 6A and Refs. 49, 50). In the first approach
to investigate the signaling pathways that couple MEKK1 to NF-
B
activation, a dominant negative form of a MAP kinase kinase was used to
block signaling from MEKK1. Cotransfection of a dominant negative
mutant in the MEKK1 signaling pathway should inhibit MEKK1-activated
B-dependent reporter gene expression, if these events
are coupled. MEKK1 activates MKK4 that phosphorylates both JNK and p38
(34, 35). A dominant negative mutant of MKK4, MKK4(Ala), inhibited the
ability of MEKK1 to activate the
B-luciferase reporter (Fig.
6B). The partial inhibition may be due to an inefficient inhibitory effect of the MKK4 mutant and/or high levels of MKK4 activity in the transfected cells. Nevertheless, these findings substantiate MEKK1-activated NF-
B transcription and demonstrate a
role of the components of the MEKK1 kinase cascade in NF-
B-mediated transcription. In the second approach to investigating the signaling pathways that couple MEKK1 to NF-
B activation, we examined whether the MAP kinase phosphatase, MKP-1, affected the ability of MEKK1 to
activate the
B-luciferase reporter. MKP-1 has been reported to
dephosphorylate ERK, JNK, and p38 kinases (30, 31, 47, 54).
Coexpression of MKP-1 with MEKK1 resulted in a decrease in activation
of the
B-reporter in this system (Fig. 6B). Taken together, these findings link MEKK1 activation and NF-
B gene expression.
The current study has identified an additional pathway of kinase
activation in endothelial cells that occurs simultaneously with the
previously described pathway of NF-B activation (Fig. 7). Both pathways are required for full activation of
E-selectin gene transcription in response to TNF
. In endothelial
cells, TNF
interaction with a TNF
receptor (TNFR1) (55) may
induce coupling with receptor-associated proteins and generation of
various TNF-induced signals (56, 57). These initial events lead to activation of both JNK/p38 kinases and NF-
B. One signaling pathway results in phosphorylation, ubiquitination, and degradation of I
B-
by the proteasome (18-20, 51-53, 58) with nuclear
accumulation of NF-
B. Concomitantly, the second set of
TNF
-induced events leads to activation of the JNK and p38 kinases,
resulting in phosphorylation of ATF-2 and c-JUN (22-32). These two
pathways are rapidly activated and converge on the E-selectin promoter
to result in full cytokine responsiveness of this gene (Fig. 7).
Although both pathways are activated simultaneously, there are notable
differences in the duration of activation. Nuclear translocation of
NF-B in endothelial cells treated with TNF
occurs by 15 min and
persists over many hours when TNF
is continuously present.
E-selectin transcription requires continuous presence of the activating
cytokine and the continuous presence of NF-
B in the nucleus (3, 59).
As shown in the present study, activation of JNK/p38 MAP kinases and
the subsequent phosphorylation of ATF-2 and c-JUN also occurs within 15 min. In contrast to NF-
B, both JNK and p38 kinase activity and
phosphorylation of ATF-2 and c-JUN are transient events.
Phosphorylation of c-JUN and ATF-2 by the kinases increases their
ability to activate transcription without affecting their DNA binding
or dimerization properties (10, 32, 38-40). The precise role of
transient phosphorylation in transcriptional activation is unclear but
may involve recruitment of co-activators such as p300 and the
CRE-binding protein (60-62). Phosphorylation may be a prerequisite for
recruitment of some components of the basal transcriptional apparatus.
Once transcription is initiated, sustained phosphorylation of ATF-2 and
c-JUN may no longer be required. Dephosphorylation may be accomplished
by phosphatases, whose levels or activity may also be regulated by TNF
.
A key regulatory event in the ERK MAP kinase activation pathway is
where the kinases are located within the cell. The ERKs are present in
the cytoplasm of quiescent cells and translocate into the nucleus
following activation (63-65). In contrast, the present study shows
that in cultured endothelial cells, JNK and p38 MAP kinases, as well as
two of the upstream activators, MKK3 and 4, are constitutively present
in the nucleus. The constitutive presence of the JNK/p38 MAP kinases
and the corresponding transcriptional activators in the nucleus of
endothelial cells suggest that TNF-induced phosphorylation events
may occur in a DNA-bound substrate-kinase complex. We have demonstrated
the presence of JNK and p38 kinases constitutively associated with the
ATF-2 and c-JUN heterodimers bound to the E-selectin PDII element.
These data are consistent with earlier reports that show that
epitope-tagged JNK can interact with recombinant c-JUN, regardless of
whether the JNK1 has been activated (22, 27, 30, 66). Our studies
extend these observations and show that the authentic kinases present
in endothelial nuclear extracts can associate with their intact
transcription factor substrates while bound to DNA. Such associations
have been postulated to occur and could serve to target the kinases to
phosphorylate additional protein substrates bound to neighboring
elements. These associations may be characteristic of certain types of
inducible genes. Several other genes induced by inflammatory mediators
have functional elements for ATF-2 or ATF-2-c-JUN heterodimers,
including c-JUN, urokinase, and
-interferon (67-70). The
similarities between the E-selectin promoter and the other promoters
suggest that activation of the JNK/p38 families of MAP kinases in the
nucleus may be a common theme in the regulation of genes involved in
various inflammatory and ischemic responses associated with
cardiovascular diseases (71).
The JNK/p38 MAP kinase and NF-B signaling systems may represent two
distinct but interactive signal transduction pathways. The JNK/p38 MAP
kinase and NF-
B signaling systems may emanate directly from distinct
TNF receptor-associated proteins (55-57). Our data supports the
concept that cross-talk occurs between the pathways of NF-
B and
JNK/p38 MAP kinase activation. MEKK1 was shown in other cell types to
be activated by TNF
(72). The findings in this paper are consistent
with recent studies that demonstrate MEKK1-induced transcription from
other
B-dependent reporters and inhibition of
MEKK1-induced transcriptional activity by co-expression of I
B
(49, 50). Taken together, these results demonstrate that the MEKK
kinase system can activate NF-
B-dependent gene
expression but do not define the signaling pathway. To examine the
signaling pathways that couple MEKK1 to the activation of the I
B
kinase, we utilized two approaches that build on the observation that
MEKK1 activates
B-dependent transcription. MEKK1 activation of NF-
B-dependent gene transcription was
inhibited by a dominant negative form of MKK4 and by a phosphatase
(MKP-1) that dephosphorylates ERK, JNK, and p38 kinases. These findings substantiate MEKK1-activated NF-
B transcription and demonstrate a
role of the components of the MEKK1 cascade in NF-
B-mediated transcription. Moreover, MEKK1-driven phosphorylation of JNK MAP kinases may be involved in activation of NF-
B. However, a direct effect of these kinases on I
B
is unlikely, since the inhibitor is
not a substrate for these MAP kinases.4
Whether the component(s) of the NF-
B/I
B
signaling pathway are
directly targeted by JNK or p38 is unknown. Nonetheless, activation of
MEKK1 and subsequent downstream signaling events involving both the
NF-
B system and the JNK/p38 MAP kinases may be important for
TNF
-induced gene expression.
Various stimuli and cellular stresses that activate the JNK/p38 MAP kinases are also physiological activators of E-selectin expression. Our findings demonstrate the importance of this signaling pathway in the activation of the E-selectin gene. The acute induction of E-selectin expression at sites of inflammation may be attributed to the combination of these two rapidly activated kinase cascades, which converge on the E-selectin gene promoter to ensure immediate responsiveness. The identification of a second signaling pathway provides another mechanistic link between the endothelial environment, E-selectin gene expression, and the early events in leukocyte adhesion and may be a means of generating specificity in the pattern of adhesion molecule expression at sites of inflammatory responses.
We thank Kay Case for her excellent technical assistance.