Vascular Biology Research Centre, Biomedical Sciences Division, King's College London, Kensington, London W8 7AH, United Kingdom
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ABSTRACT |
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Work from this and other laboratories has identified a role for
protein tyrosine kinases in interleukin-1 (IL-1
)- and tumor necrosis factor-
(TNF-
)-induced responses in endothelial cells. In this study, we show that activation of human umbilical vein endothelial cells (HUVEC) by IL-1
leads to increased tyrosine phosphorylation of several proteins including one with a molecular mass
of ~42 kDa. This protein was identified as
p42mapk by Western blot analysis.
Tyrosine phosphorylation and catalytic activation of
p42mapk by IL-1
was transient,
reaching maximal levels after 30 min and returning to basal levels by
120-300 min. Activation of
p42mapk in HUVEC was also observed
in response to TNF-
or to the protein kinase C (PKC)-activating
phorbol ester phorbol 12-myristate 13-acetate (PMA). Pretreatment of
HUVEC with IL-1
or TNF-
prevented reactivation of
p42mapk by either cytokine but did
not affect subsequent activation in response to PMA. Activation of
p42mapk by PMA was significantly
reduced by the PKC inhibitor Ro-31-8220 and completely inhibited by the
protein tyrosine kinase inhibitor genistein. Genistein, but not
Ro-31-8220, attenuated IL-1
- and TNF-
-induced
p42mapk activation. Taken
together, the results of this study demonstrate 1) that
p42mapk is transiently activated
in HUVEC by IL-1
and TNF-
, 2)
that this activation is PKC independent, and
3) that a genistein-inhibitable tyrosine kinase may be an upstream regulator of cytokine-induced p42mapk activation in human
endothelium.
cytokines; signal transduction; protein kinases; phosphatases
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INTRODUCTION |
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THE PROINFLAMMATORY CYTOKINES interleukin-1 (IL-1) and
tumor necrosis factor (TNF)- induce a range of identical responses in vascular endothelial cells (EC) (44), including induction or
upregulation of leukocyte adhesion molecule expression (11); secretion
of inflammatory mediators such as nitric oxide (NO), prostacyclin, and
platelet-activating factor (9, 10, 19); and regulation of pro- and
antithrombotic functions (4, 42, 48). A major response to IL-1 and
TNF-
in EC is rapid activation of the transcription factor NF
B,
which is involved in the regulation of many of the genes responsible
for the above effects (12), but the nature of the postreceptor signals
evoked by these cytokines remains unclear.
In many different cell types, IL-1 and TNF- promote phosphorylation
on serine or threonine (Ser/Thr) of a number of intracellular substrates including the 27-kDa small heat shock protein (20), the
epidermal growth factor (EGF) receptor (6), talin (45), the cap-binding
protein (21), and unidentified 65- and 74-kDa proteins (53). Recently,
Ser/Thr kinase activity has been found associated with the IL-1
receptor in T cells (36) and the p60 TNF-
receptor in U-937 cells
(14, 58), although the downstream mode of action of these kinases
remains poorly defined. One non-receptor-associated Ser/Thr
kinase activated by IL-1 and TNF-
is protein kinase C (PKC) (41, 50,
51), and in EC, some responses to these cytokines can be mimicked by
treatment of cells with the PKC-activating phorbol ester phorbol
12-myristate 13-acetate (PMA) (31). The role of PKC as an effector
molecule induced by IL-1 or TNF-
in EC is questionable, however,
because downregulation of PKC activity by long-term treatment with PMA
does not affect responses to either cytokine (40, 48). In EC, IL-1 and
TNF-
have also been shown to activate the separate Ser/Thr
kinase protein kinase A (PKA), via elevation of intracellular adenosine
3',5'-cyclic monophosphate (cAMP) (17, 56). In addition,
IL-1 and TNF-
induce the hydrolysis of sphingomyelin to ceramide
(50), leading to activation of a 97-kDa Ser/Thr-specific
ceramide-dependent kinase (32).
In addition to activation of Ser/Thr kinases, IL-1 and TNF-
have been shown to increase tyrosine phosphorylation in several cell
types including melanoma cells (24), U-937 cells (23), and EC (8, 63).
Furthermore, responses to IL-1 such as activation of NF
B in melanoma
cells (24) and NO synthesis in smooth muscle cells (34) can be
attenuated by the selective inhibitors of protein tyrosine kinases
(PTK) genistein (2) and herbimycin A (57). In EC, use of these
inhibitors has suggested a role for PTK in TNF-
-induced plasminogen
activator inhibitor-1 production (59), IL-1-induced endothelin
production (26), cyclooxygenase activity (8), and leukocyte adhesion
molecule expression and function induced by both cytokines (1, 37, 63).
Recent studies have shown that members of the mitogen-activated protein
kinase (MAPK) family, including
p44mapk and
p42mapk (ERK1 and ERK2,
respectively), are activated by IL-1 and TNF- in a variety of
different cell types (60, 61, 66-68). MAPKs are
Ser/Thr-specific kinases that are activated by phosphorylation on residues Thr-183 and Tyr-185 by members of a family of
dual-specificity kinases named MAPK/ERK kinases (MEKs; Refs. 13, 52,
67, 69). Upstream activators of MEK include the kinase c-Raf-1 (29), which is activated via ligand engagement of receptors with intrinsic tyrosine kinase activity (33). In contrast, activation of MAPKs by IL-1
and TNF-
appears to require a MEK kinase (MEKK1) that is distinct
from c-Raf-1 (5, 66). In EC, activation of
p42mapk has been observed as an
intracellular response to shear stress (43) and a number of vasoactive
agonists including 1-oleoyl-lysophosphatidic acid (LPA), bradykinin,
histamine, and bacterial lipopolysaccharide (LPS) (3, 16, 38). Because
many of the cellular responses of EC to LPS, IL-1, and TNF-
are
similar (44), it is possible that elements of the intracellular signals
evoked by LPS and the cytokines are shared. Therefore, we have
investigated the effects of IL-1
and TNF-
on the activation of
MAPK in human umbilical vein endothelial cells (HUVEC) to understand
more fully the intracellular signaling responses to these cytokines in
EC.
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MATERIALS AND METHODS |
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Reagents.
The human recombinant cytokines IL-1 and TNF-
were purchased from
R & D Systems (Oxford, UK). The phorbol esters phorbol 12-myristate
13-acetate (PMA) and 4
-phorbol 12,13-didecanoate (4
-PDD) were
from Sigma (Poole, UK). The antiphosphotyrosine monoclonal antibody
PY20 and the anti-p42/p44mapk
monoclonal antibody MK-12 were purchased from Affiniti Research Products (Nottingham, UK). The phosphospecific
p42/p44mapk antibody was obtained
from Promega (Southampton, UK). The peroxidase-conjugated goat
anti-mouse and anti-rabbit immunoglobulin G were purchased from Pierce
(Rockford, IL). The PKC inhibitor Ro-31-8220 was generously provided by
Dr. Trevor Hallam (Roche Products), and
1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7), genistein, and
daidzein were from Calbiochem (La Jolla, CA). Reagents for sodium
dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) were
from Bio-Rad (Hemel Hempstead, Herts, UK) or National Diagnostics
(Hessle, Hull, UK). Enhanced chemiluminescence (ECL) Western blot
detection reagent, Hyperfilm-ECL film, and
[
-32P]ATP were
obtained from Amersham (Bucks, UK), and Rennaissance 4CN Plus was from
Du Pont (Dreieich, Germany). The specific
anti-p42mapk antibody (antiserum
122) was kindly provided by Prof. Chris Marshall (Institute of Cancer
Research, London, UK). Culture media were purchased from Sigma or Life
Technologies (Paisley, Scotland, UK). All other reagents were obtained
from Sigma or BDH (Poole, Dorset, UK) at the highest available grade.
Cell culture. Endothelial cells from human umbilical veins were isolated as previously described (18). Primary cultures of HUVEC were grown in medium 199 (Sigma) supplemented with 10% (vol/vol) fetal calf serum, 10% (vol/vol) newborn calf serum, 4 mM glutamine, 100 U/ml penicillin, 100 U/ml streptomycin, and 20 mM NaHCO3 at 37°C in 5% CO2-95% air atmosphere in 25-mm2 tissue-culture flasks (Becton Dickinson, Plymouth, UK) that had been precoated with 1% gelatin. When confluent (1 × 106 cells/flask), primary cultures were trypsinized using phosphate-buffered saline (PBS) containing 0.1% trypsin-0.025% EDTA, plated into 75-cm2 tissue-culture flasks (Becton Dickinson), and grown in the above medium containing 90 mg/ml heparin sodium and 20 mg/ml endothelial cell growth factor (ECGF). For experiments, confluent first passage HUVEC (3 × 106 cells/flask) were plated onto 60-mm tissue-culture dishes (Nunc, Roskilde, Denmark) at a cell density of 5 × 104/ml in growth factor-containing medium and grown to confluence (1 × 106 cells/dish). All experiments were performed using second passage HUVEC up to 72 h after reaching confluence. Cytokine-induced p42mapk activation (and adhesion molecule expression) were quantitatively similar over degrees of confluence ranging between 70 and 100% (unpublished observations).
Immunoblot analysis of tyrosine-phosphorylated proteins and
p42mapk.
Confluent HUVEC monolayers in 60-mm dishes were serum deprived and ECGF
deprived for 12-16 h before experimentation in medium 199 containing 5 mM glutamine. Experiments were terminated by aspiration of
cell supernatant followed by washing twice with ice-cold
PBS containing 200 mM sodium orthovanadate. Cells were then lysed in buffer containing 63.5 mM tris(hydroxymethyl)aminomethane (Tris) · HCl (pH 6.8), 10% glycerol, 2% SDS, 5%
-mercaptoethanol, 1 mM sodium orthovanadate, 1 mM
4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF), and 50 µg/ml
leupeptin, after which lysates were boiled for 10 min and then
centrifuged at 10,000 g for 10 min.
Proteins in cell lysates were separated by SDS-PAGE (10%) and
electrotransferred onto Immobilon-P membrane (Millipore, Bedford, MA).
The molecular weight markers used were either SeeBlue prestained standards (Novel Experimental Technology, San Diego, CA) or Rainbow colored protein markers (Amersham Life Sciences, Bucks, UK). Membranes were blocked for 4 h in TBST [50 mM Tris, 150 mM NaCl, 0.02%
(vol/vol) Tween-20, pH 7.4] containing 3% (wt/vol) bovine serum
albumin (BSA; Advanced Protein Products, Brierley Hill, UK) and
subsequently probed overnight with either antiphosphotyrosine (PY20; 1 µg/ml), anti-p42/p44mapk (MK-12;
1:25,000 dilution), or antiphosphospecific
p42/p44mapk (1:5,000 dilution) in
TBST/0.3% (wt/vol) BSA. Blots were then washed in TBST (8 × 15 min) and incubated in TBST/0.3% (wt/vol) BSA containing horseradish
peroxidase-conjugated goat anti-mouse or anti-rabbit immunoglobulin G
as appropriate (1:10,000 dilution; 1 h). After the blots were further
washed (8 × 15 min) in TBST, immunoreactive bands were visualized
using either ECL reagent or Renaissance 4CN Plus according to the
manufacturer's instructions. Blots probed with PY20 that were to be
further probed with MK-12 were stripped using a buffer containing 62.5 mM Tris · HCl (pH 6.7), 2% SDS, and 0.7%
-mercaptoethanol at 50°C for 30 min.
Assay of MAPK activity.
Confluent monolayers of HUVEC in 60-mm dishes were prepared for
experiments as described above. After washing twice with ice-cold PBS
containing 200 µM sodium orthovanadate, monolayers were incubated on
ice for 5 min in 200 ml of buffer containing 10 mM
Tris · HCl (pH 7.4), 150 mM NaCl, 2 mM ethylene
glycol-bis(-aminoethyl
ether)-N,N,N',N'-tetraacetic acid (EGTA), 10 µg/ml leupeptin, 10 µg/ml aprotinin, 1 mM AEBSF, 2 mM dithiothreitol, and 1 mM sodium orthovanadate. Cell layers were then
scraped using a rubber policeman and transferred to microcentrifuge
tubes (Beckman Instruments, Palo Alto, CA). Lysates were then sonicated
(4 × 10 s; 4°C) followed by centrifugation (35,000 g) for 20 min at 4°C. The
supernatant was stored at
70°C until assayed for MAPK
activity using the BIOTRAK
p42/p44mapk assay system (Amersham
Life Sciences) according to the manufacturer's instructions. MAPK
activity in 15 µl of cell lysates was measured as the amount of
32P incorporated per minute into a
peptide based on the Thr-669 phosphorylation site of the EGF receptor.
Immunoprecipitation and immune complex kinase assay.
Confluent, quiescent HUVEC in 60-mm dishes were washed and treated as
described for the immunoblotting studies. Cells were lysed in ice-cold
lysis buffer [20 mM Tris · HCl (pH 7.5), 2 mM EDTA, 0.5 mM EGTA, 0.5% Triton X-100, 50 mg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride, 500 µM sodium orthovanadate, 250 µM
NaF, and 0.1% -mercaptoethanol], scraped into 1.5-ml
microcentrifuge tubes, and sonicated (4 × 5 s; on ice). After
centrifugation (9,000 g, 1 min,
4°C), lysates were immunoprecipitated with 3 µl of rabbit anti-ERK2 antiserum (122) for 3 h at 4°C with constant rotation. Immune complexes were captured with protein A/G-agarose (3 h, 4°C)
and washed three times in lysis buffer followed by a further wash in
lysis buffer supplemented with 20 mM
MgCl2. Immune complex kinase
activity was assayed using 5 µCi
[
-32P]ATP (50 µM), 20 mM MgCl2, and 1 mg/ml
myelin basic protein (MBP) for 20 min at 30°C. Reactions were
terminated by the addition of 25 µl of 2× sample buffer, and
MBP phosphorylation was visualized by SDS-PAGE (12%) followed by
autoradiography.
Statistical analysis. Student's t-test was used to compare means of groups of data. P < 0.05 was considered statistically significant.
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RESULTS |
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IL-1 and TNF-
activate
p42mapk in HUVEC.
Stimulation of quiescent HUVEC with 100 U/ml IL-1
caused increased
tyrosine phosphorylation of a 42-kDa protein that was detectable after
15 min, maximal at 30 min, and returned to basal levels after 180 min
(Fig.
1A).
When antiphosphotyrosine immunoblots were reprobed using an antibody
that recognized both p42mapk and
p44mapk, only one band migrating
at 42 kDa was observed (data not shown). The 42-kDa
tyrosine-phosphorylated protein was confirmed as
p42mapk by gel mobility-shift
analysis (Fig. 1B), which revealed
an upper, electrophoretically slower band corresponding to
phosphorylated p42mapk. The
intensity of the upper band in control samples varied between experiments but never exceeded 15% of the intensity of the lower band
when measured by densitometry (data not shown). In response to IL-1
,
the intensity of the upper band increased time dependently, reaching
maximal levels at 30 min. Thereafter, the intensity decreased and
reached basal levels within times ranging from 120 to 300 min in
separate experiments.
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Activation of p42mapk by PMA.
The PKC-activating phorbol ester PMA (100 nM) also induced tyrosine
phosphorylation of a 42-kDa protein that was detectable after 5 min and
remained phosphorylated at 120 min (Fig.
4). PMA also time-dependently increased
tyrosine phosphorylation of at least six other proteins in HUVEC,
whereas the inactive PMA analog 4-PDD (100 nM) did not affect
tyrosine phosphorylation of any protein at either 15 or 120 min (data
not shown). As depicted in Fig. 4, mobility-shift analysis identified
the 42-kDa protein as p42mapk, and
the appearance of the upper band corresponded with the time course of
increased tyrosine phosphorylation as assessed by antiphosphotyrosine immunoblotting (data not shown). Activation of
p42mapk was dose dependent, with
maximal effects at 30 min observed in the presence of 100 nM PMA; no
activation of p42mapk was observed
in response to 100 nM 4
-PDD at 15 or 120 min (Fig. 4).
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Effects of cytokine pretreatment on subsequent
p42mapk activation.
To investigate the effects of cytokine pretreatment on subsequent
activation of p42mapk in HUVEC,
cells were incubated for 4 h with either medium alone, IL-1 (100 U/ml), or TNF-
(100 U/ml) and then for a further 30 min with either
IL-1
(100 U/ml) or PMA (100 nM) or for 15 min with TNF-
(100 U/ml) (Fig.
5A).
After pretreatment with medium alone, similar levels of activated
p42mapk were observed in response
to IL-1
, TNF-
, and PMA. In contrast, pretreatment for 4 h with
either IL-1
or TNF-
prevented reactivation of
p42mapk by either cytokine when
compared with medium (control)-treated cells. Pretreatment with either
cytokine did not affect the ability of PMA to reactivate
p42mapk. Similar results were
observed when HUVEC were treated for times ranging from 2 to 8 h with
cytokines (data not shown). We also investigated the recovery of
reactivation of p42mapk in
IL-1
-treated cells by incubating HUVEC for 2 h with IL-1
(100 U/ml) followed by removal of cytokine-containing medium and replacement
with IL-1
-free medium. HUVEC were then treated for 30 min with 100 U/ml IL-1
at 2, 4, and 6 h after removal of the original
IL-1
-containing medium. As shown in Fig.
5B, addition of fresh IL-1
immediately after removal of the original IL-1
did not lead to
activation of p42mapk. In
contrast, 2 h after removal of IL-1
, subsequent incubation with
IL-1
caused partial reactivation of
p42mapk. Maximal reactivation was
observed 4 h after removal, but this did not reach the level of
activation observed in medium-treated cells.
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Effects of protein kinase inhibitors on activation of
p42mapk by PMA, IL-1, and
TNF-
.
The effects of protein kinase inhibitors on PMA- and IL-1
-activated
p42mapk are shown in Fig.
6. Pretreatment of HUVEC for 15 min with
the selective PKC inhibitor Ro-31-8220 (1 µM) caused a marked
inhibition of p42mapk
phosphorylation in response to 100 nM PMA (Fig.
6A,
top) but did not affect
phosphorylation in response to 100 U/ml IL-1
(Fig. 6B,
top). PMA-induced catalytic activity
of p42mapk was also reduced by 1 µM Ro-31-8220 from 1.20 ± 0.01 to 0.38 ± 0.01 pmol
phosphate/min (87 ± 1% inhibition; Fig.
6A,
bottom), whereas activity induced by
IL-1
was enhanced by 157 ± 1% from 1.95 ± 0.02 to 3.06 ± 0.10 pmol phosphate/min by 1 µM Ro-31-8220 (Fig.
6B,
bottom). Phosphorylation of
p42mapk in response to PMA was
completely inhibited by pretreatment of HUVEC for 15 min with the PTK
inhibitor genistein (100 mM) and partially inhibited by the less active
analog daidzein (100 µM; Fig. 6A,
top). Daidzein reduced PMA-induced
p42mapk activity to 0.71 ± 0.05 pmol phosphate/min (52 ± 1% inhibition), but
activity was reduced by genistein to 0.16 ± 0.01 pmol phosphate/min (111 ± 1% inhibition), which was lower than activity in either control cell lysates (0.26 ± 0.01 pmol phosphate/min) or from cells
treated with 100 µM 4
-PDD (0.19 ± 0.01 pmol phosphate/min; Fig. 6A,
bottom). Phosphorylation of
p42mapk induced by IL-1
(100 U/ml) was unaffected by 100 µM daidzein but was significantly reduced
by 100 µM genistein (Fig. 6B,
top). Activity in response to
IL-1
was reduced to 1.15 ± 0.04 pmol phosphate/min by
genistein (48 ± 2% inhibition) but was not affected by 100 µM
daidzein (1.83 ± 0.04 pmol phosphate/min; Fig.
6B,
bottom). None of the inhibitors was
cytotoxic for HUVEC at the times and concentrations used when measured
using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide as previously described (37).
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DISCUSSION |
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In this report, we have shown that stimulation of HUVEC with the
proinflammatory cytokines IL-1 or TNF-
leads to rapid activation of the Ser/Thr kinase
p42mapk as evidenced by increased
tyrosine phosphorylation, reduced mobility on SDS-PAGE, enhanced EGF
receptor peptide phosphorylation in cell lysates, and increased MBP
phosphorylation in p42mapk
immunoprecipitates. We have also shown that this activation can be
inhibited by genistein, a selective inhibitor of PTKs (2). Previous
studies have shown that responses by EC to IL-1
and TNF-
can be
inhibited by genistein (8, 26, 59, 63), but to date, no specific
intracellular signaling mechanism involving genistein-sensitive
tyrosine phosphorylation in response to these cytokines has been
identified in EC. The results of this study represent the first
demonstration of such a mechanism, although the specific upstream,
postreceptor events leading to
p42mapk activation in HUVEC remain
to be determined.
Activation of p42mapk by TNF-
was first reported by Kohno et al. (27), who described rapidly
increased tyrosine phosphorylation of two proteins with molecular
masses of 41 and 43 kDa in human fibroblasts. These were later
identified as p42 and p44mapk (60,
61), and subsequent workers demonstrated their activation by TNF-
in
mouse macrophages (66, 68) and by IL-1
in various human and mouse
cell lines (7). Activation of
p42mapk after stimulation of
receptor tyrosine kinases and G protein-coupled receptors occurs via
phosphorylation of the dual-specific kinase MEK by the Ser/Thr
kinase c-Raf-1 (29, 33), whereas activation by TNF-
in mouse
macrophages is via phosphorylation of the MEK-1 isoform of MEK by the
c-Raf-1-independent MEK kinase MEKK1 (66, 67). In addition to p42 and
p44mapk, IL-1
and TNF-
activate jun NH2-terminal kinase
(JNK) and p38mapk, which are
distantly related to p42 and
p44mapk (5, 30, 46, 54).
Activation of JNK by IL-1
and TNF-
is also MEKK1 dependent but
involves activation of a JNK kinase (JNKK) named MAPK kinase (MKK)4 in
place of MEK (5, 15, 65). Several recent studies have demonstrated the
cytokine-induced activation of JNK and
p38mapk in endothelial cells (25,
39, 47), but further study will be required to identify which, if any,
of these upstream mechanisms operate in HUVEC, leading to activation of
p42mapk by IL-1
and TNF-
.
The results of our study demonstrate that cytokine-induced p42mapk activation in HUVEC requires an upstream tyrosine kinase, but the nature and location of this genistein-inhibitable kinase remains to be determined. Further evidence for this is the ability of peroxovanadate, a potent inhibitor of protein tyrosine phosphatases, to induce rapid activation of p42mapk in HUVEC (Wheeler-Jones, Houliston, and Pearson, unpublished data). It is possible that genistein directly inhibits tyrosine phosphorylation of p42mapk, although it has been suggested that the site of action of genistein is not MEK but before the interaction of c-Raf-1 with Ras-GTP (55). In the case of c-Raf-1-independent activation of p42mapk, genistein may inhibit an unidentified PTK upstream of MEKK1, and because MEKK1 has also been shown to bind to Ras-GTP (49), a genistein-sensitive PTK regulating Ras activation may be involved in cytokine-induced activation of p42mapk. However, inhibition of PMA-activated p42mapk in HUVEC by genistein suggests direct inhibition of MEK, as PKC is known to phosphorylate and activate c-Raf-1 (28). Because c-Raf-1 activates MEK directly, the only kinase capable of phosphorylating p42mapk on tyrosine in the pathway evoked by PMA is MEK itself. Identification of the MEK isoforms activated by cytokines and PMA in HUVEC and their relative succeptibilities to genistein will be required before any firm conclusions can be made about the nature of the genistein-inhibitable kinase.
Activation of p42mapk in response
to PMA indicates a potential role for PKC in cytokine-induced
activation; however, we do not believe that PKC is involved for the
following reasons. First, the time courses of activation of
p42mapk in response to cytokines
and PMA were significantly different, suggesting activation of distinct
signaling mechanisms by each stimulant. Second, treatment of HUVEC for
4 h with IL-1 and TNF-
did not affect subsequent activation of
p42mapk by PMA but did prevent
reactivation by each cytokine. If cytokines and PMA activated the same
signaling mechanism in HUVEC, downregulation of this pathway
leading to cross-inhibition of reactivation by separate cytokines would
be expected to prevent reactivation by PMA. The long time course of
p42mapk activation by PMA
prevented detection of any effects of PKC downregulation by long-term
PMA incubation (data not shown). Finally, PMA-induced activation of
p42mapk was inhibited by the
selective PKC inhibitor Ro-31-8220, whereas Ro-31-8220 failed to
inhibit activation in response to either IL-1
or TNF-
. Although
we observed increased catalytic activity of
p42mapk by IL-1
in
Ro-31-8220-pretreated cells, which may have been via inhibition of a
PKC-activated MAPK phosphatase (62), no increased phosphorylation of
p42mapk was detected by
mobility-shift analysis, suggesting that the increased activity was
because of a nonspecific effect of the inhibitor. Interestingly, the
nonspecific Ser/Thr kinase inhibitor H-7, which we (37) and
others (31, 48) have shown to inhibit adhesion molecule expression in
response to IL-1
and TNF-
in HUVEC, failed to inhibit activation
of p42mapk by either cytokine. It
therefore seems that the H-7-dependent kinase required for activation
of HUVEC lies on a distinct signaling pathway from that leading to
p42mapk activation, for example,
by being located upstream of NF
B activation that is required for
adhesion molecule expression (12, 39, 47).
As discussed above, the different time courses of
p42mapk activation in response to
cytokines and PMA suggest involvement of separate signaling pathways.
Furthermore, the marked differences between cytokine- and PMA-induced
p42mapk activation may underlie
differences in the outcome of endothelial activation by these separate
stimuli. Previous studies of PC12 neuronal cells have shown that
separate cellular responses to tyrosine kinase receptor activation
including differentiation and proliferation are determined by the
duration of MAPK activation (35). We also observed differences in the
time taken to reach maximal activation in response to the individual
cytokines. Maximal phosphorylation in response to TNF- occurred
after 15 min, but IL-1
-induced activation was slower, reaching a
maximum at 30 min. Although the initial events induced by the cytokines
leading to p42mapk activation are
unknown, serine-specific kinase activities associated with the IL-1
type 1 receptor (36) and the p60 TNF-
receptor (14, 58) have been
reported. It may be that the different time courses of
p42mapk activation in HUVEC are
because of differences in activation of these or other
receptor-associated kinases. It is interesting to note that, like
p42mapk activation by IL-1
and
TNF-
in HUVEC, the TNF-
-receptor- and IL-1
-associated kinases
are not inhibited by H-7 (36, 58). However, although the initial
signaling processes evoked by separate cytokines may be different,
cross-inhibition of p42mapk
activation by pretreatment with cytokines suggests that later events
induced by IL-1
and TNF-
leading to
p42mapk activation overlap.
The anti-p42/p44mapk antibody
employed for the mobility-shift analyses in this and an earlier study
in HUVEC (64) detected only the p42 isoform of MAPK, whereas we have
detected both MAPK isoforms in rat aortic smooth muscle cells
(Houliston, Pearson, and Wheeler-Jones, unpublished data). This finding
is in agreement with the recent reports of Pearce et al. (43) and
McLees et al. (38), who demonstrated the presence of only
p42mapk in HUVEC and the human
endothelial cell hybrid line EAhy 926, respectively, using different
antisera. Immunoblot analysis using an antiphosphotyrosine antibody
also demonstrated phosphorylation of only one protein of 42 kDa in
response to cytokines and no other proteins of a similar molecular mass
(Fig. 1A). However, an antiserum
raised against the active, dually phosphorylated forms of p42 and
p44mapk detected both forms of
MAPK in HUVEC (Fig. 3B) and,
moreover, showed that treatment with either IL-1 or TNF-
enhanced
the phosphorylation state of both p42 and
p44mapk. Interestingly, Arditi et
al. (3) recently demonstrated activation of both p42 and
p44mapk in bovine and human brain
microvascular ECs in response to LPS, which together with our results
shows a diversity in expression of MAPK isoforms in endothelial cells
derived from separate vascular beds and also indicates an
antibody-dependent variation in the ability to detect p42/p44 MAP
kinases and their activation.
In conclusion, we have demonstrated the activation of a tyrosine
phosphorylation-dependent signaling pathway in human ECs in reponse to
IL-1 and TNF-
. The precise nature of the upstream kinases leading
to activation of p42mapk, along
with the downstream effects of
p42mapk activation, remains
unknown, but further study of this pathway will be crucial for a full
understanding of the effects of these cytokines on endothelial cells.
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ACKNOWLEDGEMENTS |
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We thank the midwives and delivery staff at St. Mary's Hospital, London, for help in obtaining the umbilical cords. We also thank Dr. Trevor Hallam and Prof. Chris Marshall for the kind gifts of Ro-31-8220 and antiserum 122, respectively.
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FOOTNOTES |
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This work was supported by the Arthritis and Rheumatism Council, the British Heart Foundation, and Ono Pharmaceuticals. C. P. D. Wheeler-Jones is a British Heart Foundation Intermediate Research Fellow.
Present address and address for reprint requests: M. J. May, Section of Immunobiology, Yale University School of Medicine, Rm. FMB 409, 310 Cedar St., PO Box 208011, New Haven, CT 06520-8011.
Received 31 January 1997; accepted in final form 25 November 1997.
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