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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 93 No. 3 (February 1), 1999:
pp. 857-865
The MKK6/p38 Stress Kinase Cascade Is Critical for Tumor Necrosis
Factor- -Induced Expression of Monocyte-Chemoattractant
Protein-1 in Endothelial Cells
By
Matthias Goebeler,
Karin Kilian,
Reinhard Gillitzer,
Manfred Kunz,
Teizo Yoshimura,
Eva-B. Bröcker,
Ulf R. Rapp, and
Stephan Ludwig
From the Klinik und Poliklinik für Haut- und
Geschlechtskrankheiten and Institut für Medizinische
Strahlenkunde und Zellforschung (MSZ), Universität
Würzburg, Würzburg, Germany; and the Immunopathology
Section, Laboratory of Immunobiology, National Cancer
Institute-Frederick, Frederick, MD.
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ABSTRACT |
Monocyte chemoattractant protein-1 (MCP-1), a member of the C-C
subfamily of chemokines, is important for the local recruitment of
leukocytes to sites of inflammatory challenge. Here, we investigated endothelial signaling pathways involving members of the
mitogen-activated protein (MAP) kinase superfamily and studied their
role for MCP-1 expression in endothelium. We show that tumor necrosis
factor- (TNF-), a potent inflammatory activator of endothelium,
leads to activation of MAP kinases ERK, p38, and JNK in human umbilical vein endothelial cells (HUVEC). Contribution of MAP kinase pathways to
TNF--induced synthesis of endothelial MCP-1 was then studied by
pharmacologic inhibition and transient expression of dominant negative
or constitutively active kinase mutants using flow cytometry, Northern
blot, and luciferase reporter gene assays. Inhibition of Raf/MEK/ERK or
SEK/JNK pathways had no significant effect on MCP-1 levels, whereas
blocking the MKK6/p38 pathway by p38 inhibitors SB203580 or SB202190 or
by a dominant negative mutant of MKK6, the upstream activator of p38,
strongly inhibited TNF--induced expression of MCP-1. Consistent
with that finding, expression of wild-type or constitutively active
MKK6 significantly enhanced the effect of limiting TNF-
concentrations on MCP-1 synthesis. These data suggest a crucial role
for the MKK6/p38 stress kinase cascade in TNF--mediated endothelial
MCP-1 expression.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
MONOCYTE CHEMOATTRACTANT protein-1
(MCP-1), a member of the C-C chemokine family, attracts blood monocytes
both in vitro and in vivo.1,2 It is produced by a variety
of cells including vessel wall elements such as endothelial cells and
is thought to play a major role in the recruitment of leukocytes to
sites of prospective inflammation. Abundant amounts of MCP-1 have been
detected in diseases such as atherosclerosis, thus strongly suggesting
a role for the recruitment of monocytes to the evolving atheromatous
plaque.3,4 Besides subendothelial macrophages and smooth
muscle cells, endothelial cells have been considered to be the major
source of MCP-1 in atheromatous lesions.5 MCP-1 appears to
be important for activation of leukocyte subsets and triggers their
adhesiveness and transmigration through the endothelial layer.
Inhibition of induced MCP-1 expression is associated with reduced
transmigration of monocytes,6 as well as with diminished recruitment of T lymphocytes.7 Accordingly, neutralizing
antibodies against MCP-1 have been shown to inhibit T-cell recruitment
and cutaneous delayed-type hypersensitivity,8 as well as
pulmonary granuloma formation9 in rat models of
experimental inflammation, thus illustrating the importance of MCP-1 at
the onset of inflammatory processes.
Endothelial cells have been found to produce MCP-1 in response to
cytokines such as tumor necrosis factor- (TNF-), interleukins (IL)-1, 4, and 13, interferon- (IFN-), as well as by agents such
as thrombin (see Mantovani et al2,10 for review). However, the intracellular signaling pathways that couple endothelial activation from receptor levels to the level of MCP-1 expression are so far only
poorly understood.
A major mechanism through which signals from environmental stimuli are
transmitted to the nucleus involves activation of kinases related to
the mitogen-activated protein (MAP) kinase superfamily. To date, at
least three subgroups of MAP kinase family members have been identified
to be involved in a wide range of cellular responses to extracellular
signals.11 The first subgroup includes two isoforms of the
extracellular signal-regulated kinases, ERK1 and ERK2. ERKs are
strongly activated on stimulation of cells with mitogens such as
epidermal growth factor, platelet-derived growth factor, serum, and
phorbol-myristate acetate (PMA) through sequential activation of the
upstream kinases Raf and MEK. This classical mitogenic kinase cascade
plays an important role in mediating signals that control cell
proliferation, differentiation, or survival.12-14 In
contrast to the ERKs, the two other subgroups of the MAP kinase family,
namely the p38 and the c-Jun NH2-terminal kinase/stress-activated protein kinase (JNK/SAPK) subgroups, are only
weakly activated by mitogens, but are highly stimulated on exposure to
inflammatory cytokines such as TNF- and IL-1 and a wide variety of
environmental stress inducers such as lipopolysaccharides (LPS),
arsenite, anisomycin, heat shock, or ultraviolet (UV)
light.15 The upstream activator of p38 is the dual
specificity kinase MKK6, whereas JNK/SAPK is activated by MKK4/SEK.
Among the downstream targets of JNK and p38 are the transcription
factor c-jun and the MAPKAP-kinases 2 and 3, respectively. While the
physiologic role of the Raf/MEK/ERK cascade is well established, the
function of the stress-activated kinase cascades is far less
understood. p38 has originally been identified through the action of a
specific p38 inhibitor, SB203580, which partially blocks LPS-induced
TNF- and IL-1 biosynthesis in monocytes.16,17 Using the
same inhibitor, p38 has also been shown to be involved in regulation of
IL-6, granulocyte-macrophage colony-stimulating factor (GM-CSF), and human immunodeficiency virus (HIV)-long terminal repeat
(LTR)-dependent gene expression.18-20
So far, little is known about the role of the different MAP kinase
cascades in endothelial activation. Especially, the regulation of
endothelial chemokines such as MCP-1 by different MAP kinase pathways
is still an open issue.
Thus, we have analyzed the functional role of the different MAP kinase
cascades in MCP-1 transcription and synthesis in endothelium. Besides
the use of specific kinase inhibitors, we used a transient transfection
approach for human umbilical vein endothelial cells (HUVEC) to study
expression of endothelial MCP-1 in the presence of dominant negative or
constitutively active components of different signaling cascades. Here,
we present data to show that activation of the MKK6/p38 pathway is
important for expression of MCP-1, while SEK/JNK and Raf/MEK/ERK
cascades do not appear to be of major relevance.
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MATERIALS AND METHODS |
Reagents.
Human recombinant (r)TNF- was obtained from R & D Systems (Wiesbaden, Germany); phorbol 12-myristate 13-acetate
(PMA) from Serva (Heidelberg, Germany). Inhibitor SB203580 was kindly
provided by Dr J.C. Lee (Smith Kline Beecham
Pharmaceuticals, King of Prussia, PA) or purchased from Calbiochem (Bad
Soden, Germany). Inhibitors SB202190 and PD98059 were obtained from
Calbiochem. All other reagents were purchased from Sigma (Deisenhofen,
Germany) unless otherwise specified.
Cells and cell culture.
HUVEC were obtained from Clonetics (via Cell Systems, Remagen, Germany)
and cultured with endothelial basal medium (EBM;
Clonetics) supplemented with 2% fetal bovine serum, 1.0 µg/mL
hydrocortisone, 10 ng/mL human epidermal growth factor, 12 µg/mL
bovine brain extract, 50 µg/mL gentamicin, and 50 ng/mL amphotericin
B (ie, EGM). HUVEC were used between passages 3 and 5.
Immunoprecipitation and kinase assays.
Cells were lysed with 20 mmol/L Tris, pH 7.4, 137 mmol/L NaCl, 10%
(vol/vol) glycerol, 1% (vol/vol) Triton X-100, 2 mmol/L EDTA, 50 mmol/L sodium- -glycerophosphate, 20 mmol/L sodium pyrophosphate, 1 mmol/L Pefabloc (Merck, Darmstadt, Germany), 5 µg/mL aprotinin, 5 µg/mL leupeptin, and 5 mmol/L benzamidine (TLB buffer) at 4°C for
30 minutes. Cell debris was removed by centrifugation at
13,000g for 20 minutes. After quantification of the protein
content in the supernatants (Biorad Protein Assay; Biorad, Munich,
Germany), equal amounts were incubated with 25 µL protein A agarose
(Boehringer Mannheim, Mannheim, Germany) and 1 µg/mL
rabbit antisera against ERK1/ERK2, JNK (Santa Cruz, Heidelberg,
Germany), p38 (kindly provided by J. Han, The Scripps
Research Institute, La Jolla, CA) or an antiserum specific for both,
MAPKAP kinases 2 and 321 for 2 hours at 4°C. After
washing in TLB buffer supplemented with 500 mmol/L NaCl and,
thereafter, kinase buffer (25 mmol/L HEPES pH 7.5, 10 mmol/L
MgCl2, 25 mmol/L sodium--glycerophosphate supplemented with 5 mmol/L benzamidine, 1 mmol/L sodium orthovanadate, and 0.5 mmol/L dithiothreitol), each sample was incubated with 3pK K>M,
myelin basic protein (MBP), glutathione-S-transferase
(GST)-c-Jun(1-135) or Hsp27 as substrates for p38, ERK,
JNK, or MAPKAP kinases 2 and 3 in the presence of 100 µmol/L
unlabeled adenosine triphosphate (ATP), 5 µCi
[32P]-ATP, and kinase buffer in a volume of 20 µL
for 15 minutes at 30°C. Thereafter, reactions were terminated by
boiling in 5× Lämmli sodium dodecyl sulfate (SDS) sample
buffer for 4 minutes. Samples were subsequently subjected to
SDS-polyacrylamide gel electrophoresis (PAGE), blotted onto
nitrocellulose, and visualized by autoradiography. Western blot
analysis was performed to confirm equal loading of ERK1/ERK2, JNK, p38,
or MAPKAP kinases 2/3 proteins.
Plasmids and transient transfection procedures.
Plasmids used for transient transfections included KRSPA eukaryotic
expression vector,22 pGreen Lantern for expression of green
fluorescent protein (GFPS65T; Life Technologies, Eggenstein, Germany),
and KRSPA expression plasmids for wild-type MKK6, dominant negative
MKK6(Ala) or constitutively active MKK6(Glu) (kindly provided by Dr
R.J. Davis, Howard Hughes Medical School, University of
Massachusetts, Worchester, MA23). Dominant negative
versions of Raf (KRSPA Raf-C4B), ERK2 (KRSPA ERK2C3), SEK (KRSPA SEK
K>R), and SAPK (KRSPA SAPK KK>RR) were obtained from various
sources and have been described in detail in Ludwig et
al.21 The dominant negative p38 mutants [pCDNA3
p38(TY>AF), pCDNA3 p38(K>M)]24 were kindly provided by
J. Han (La Jolla, CA).
HUVEC were cultured to 50% to 60% confluence using Falcon 10-cm
tissue culture plates or 6-well plates (Becton Dickinson, Heidelberg,
Germany) before transient transfection, which basically followed a
diethyl aminoethyl (DEAE)-dextran protocol described by
Karmann et al.25 After washing with 1 mmol/L
HEPES/phosphate-buffered saline (PBS), cells were incubated with 20 µg (10-cm plates) or 5 µg DNA (6-well plates), 250 µg/mL
DEAE-dextran (Pharmacia, Freiburg, Germany) in 4 or 1 mL 1 mmol/L
HEPES/PBS for 30 minutes at 37°C. Thereafter, 6 or 1.5 mL EGM
containing 0.15 mmol/L chloroquine was added to each plate and cells
incubated for another 2.5 hours. Medium was then removed and cells
treated with 10% (vol/vol) dimethyl sulfoxide (DMSO) in EGM for 2.5 minutes. HUVEC were subsequently cultured for 36 hours in EGM and
finally stimulated with cytokines or other reagents as indicated.
Expression and function of the transfected kinase mutants was confirmed
in Western blots and immune complex kinase assays.
Flow cytometry.
Cell surface expression of intracellular adhesion molecule
(ICAM)-1 was performed as described
earlier.26,27 Briefly, HUVEC were incubated with 1%
(wt/vol) bovine serum albumin (BSA)/PBS for 30 minutes and then exposed
to mouse IgG1 monoclonal antibody (MoAb) against ICAM-1
(clone 84H10; Immunotech, via Dianova, Hamburg, Germany) or mouse
IgG1 of nonrelevant specificity (R & D Systems). After
several washes, cells were stained with F(ab')2
fragment of fluorescein isothiocyanate (FITC)-labeled rabbit antimouse IgG (Dako, Hamburg, Germany). Thereafter, propidium iodide was added to
allow exclusion of nonviable cells. Fluorescence was measured on 5,000 or 10,000 cells per sample using a FACScan (Becton Dickinson).
To determine expression of the chemokine MCP-1, an intracellular
staining procedure was applied. HUVEC were stimulated with cytokines
and other reagents as indicated in the presence of 2 µmol/L monensin.
Cells were washed, fixed with 4% (wt/vol) paraformaldehyde in PBS at
4°C for 20 minutes, and then incubated with phycoerythrin-labeled mouse MoAb against MCP-1 (mouse IgG1; clone 5D3-F7) or
corresponding phycoerythrin-labeled isotype control MoAb (PharMingen,
Hamburg, Germany), which had been diluted in permeabilization buffer
containing 1% fetal calf serum (FCS), 0.1% (wt/vol) saponin, PBS.
Fluorescence was then measured with a FACScan.
MCP-1 expression by HUVEC cotransfected with vectors expressing
dominant negative or constitutively active kinases and GFPS65T was
detected as follows. Thirty-six hours after transfection, cells were
exposed to cytokine and subsequently harvested. Cells were then
permeabilized as described above and successively stained with
unlabeled MoAb 5D3-F7 against MCP-1, biotin-SP-conjugated goat
antimouse IgG F(ab')2 and streptavidin-Cy-Chrome (PharMingen), respectively. Only those cells, which expressed GFPS65T (as detected in
the FL-1 channel), were considered for the detection of MCP-1 (as
measured in the FL-3 channel). Nonviable cells were excluded using
forward scatter (FSC) and side scatter (SSC) parameters.
Northern blot.
HUVEC were stimulated with TNF- in the absence or presence of
pharmacological inhibitors as indicated. For the assessment of an
inhibitory effect of SB202190 on mRNA stability, some samples were
treated with the transcriptional inhibitor, actinomycin D (Act D), for
distinct time intervals as indicated. Total cellular RNA was isolated
using a Quiagen RNeasy kit (Hilden, Germany) according to the
manufacturer's instructions. Denaturated total RNA (10 µg) was
separated on 1% agarose/formaldehyde gels and transferred to Hybond N+
membranes (Amersham Buchler, Braunschweig, Germany). Filters were UV
cross-linked and subsequently hybridized with a human MCP-1 cDNA
probe28 labeled with [32P]-deoxycytidine
triphosphate (dCTP) using the random primed DNA labeling
kit (Boehringer Mannheim, Mannheim, Germany). For control of RNA
loading of lanes, blots were either rehybridized with an actin probe or
densitometrically analyzed for 28S rRNA amounts using NIH Image 1.6 Software (NIH, Bethesda, MD). Autoradiography was
performed at  80°C using Amersham Hyperfilm.
Luciferase assays.
HUVEC were seeded in 6-well plates and transfected using the
DEAE-dextran protocol described above after having reached
subconfluence. The luciferase constructs used contained the proximal
promoter region and distal enhancer region of the human MCP-1 gene and have recently been described in detail.29 Transfections for luciferase assays were performed with 1 µg of reporter construct with
or without 4 µg of expression vectors containing MKK6(Ala), MKK6(Glu), p38(TY>AF), or p38(K>M) cDNAs. In a set of experiments, HUVEC were cotransfected with increasing amounts (1 µg, 4 µg, and 8 µg/per well) of MKK6(Ala) cDNA or corresponding empty expression vector. Thirty-six hours after transfection, cells were incubated with
TNF- in the absence or presence of pharmacological inhibitors for
another 12 hours as indicated. HUVEC of each well were then harvested
in 100 µL lysis buffer (50 mmol/L sodium
2-(N-morpholino)ethanesulfonic acid (MES) (Sigma), 50 mmol/L Tris-HCl,
pH 7.8, 10 mmol/L dithiothreitol, and 2% Triton X-100) for 30 minutes
at 4°C. The crude cell lysates were cleared by centrifugation, and
50 µL of precleared cell extracts were added to 50 µL luciferase
assay buffer containing 125 mmol/L sodium MES, 125 mmol/L Tris-HCl, pH
7.8, 25 mmol/L magnesium acetate, and 2 mg/mL ATP. Immediately after
the addition of 50 µL of 1 mmol/L D-luciferin (AppliChem, Darmstadt,
Germany) for each sample, the luminescence was detected using a LB96P
luminometer (Berthold, Bad Wildbach, Germany). Cell lysates were
normalized on the basis of protein contents; relative luciferase
activities of cells transfected with each cDNA expression vector were
based on activities of cells transfected with the same amount of empty
expression vector (KRSPA or pCDNA3). Data were obtained from four
independent experiments and are expressed as fold stimulation of
relative luciferase activity.
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RESULTS |
TNF--induced activation of endogenous MAP kinase cascades in
endothelial cells.
To study activation of endogenous MAP kinases by TNF-, HUVEC were
exposed for 0, 10, 30, 120, and 240 minutes to the cytokine. Kinase
activation was determined in immunecomplex kinase assays. Exposure of
HUVEC to TNF- induced a significant activation of stress kinases p38
and JNK, as well as of ERK within 10 minutes. p38 and ERK activities
decreased after 30 minutes, whereas JNK activity remained elevated up
to 240 minutes (Fig 1). Essentially similar
results, albeit weaker, were obtained when HUVEC were exposed
to IL-1, another potent inducer of endothelial activation (data not
shown). The effects of stress inducer sodium arsenite on p38 and JNK
kinase activity and of phorbol ester PMA on ERK activation were
analyzed for control purposes (Fig 1, see legend for detail), which
resulted in activation kinetics similar to those found in other cell
types.
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| Fig 1.
Activation kinetics of endogenous MAP kinases in HUVEC
after stimulation with TNF-, PMA, and arsenite. HUVEC were
stimulated with 5 ng/mL TNF-, 20 ng/mL PMA, or 0.25 mmol/L arsenite
for the time intervals indicated. Immunecomplex kinase assays were
performed as described in Materials and Methods using 3pK K>M,
GST-c-Jun(1-135), and myelin basic protein (MBP) as substrates for p38,
JNK, and ERK, respectively. Protein loads were controlled in Western
blot assays using appropriate antisera. On stimulation with PMA, a
strong activation of ERK was detected, which persisted over 240 minutes
(lower panel). Arsenite strongly induced p38 and JNK kinases with peak
levels after 240 minutes (upper and middle panels).
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The observation that all three MAP kinases, p38, JNK and ERK, are
activated by TNF-, one of the most potent physiological activators
of endothelium, does not prove their functional involvement in
endothelial activation. We therefore evaluated the potential role of
all three MAP kinase cascades for endothelial expression of MCP-1.
The p38 inhibitors, SB203580 and SB202190, inhibit MCP-1 synthesis.
To elucidate a potential contribution of different MAP kinase cascades
in TNF--induced expression of MCP-1, we studied the effects of
pharmacologic kinase inhibitors, namely the selective MAP kinase kinase
(MEK) inhibitor PD9805930 and the pyridinyl imidazoles,
SB203580 and SB202190,20,31 both inhibitors of p38. PD98059
acts by preventing activation of MEK by Raf, whereas the p38 inhibitors
function via blocking the active p38 kinase.20 The
inhibitors were used in a concentration that specifically blocked ERK
or p38 activity in other cells.21,32 Specific activity of
the inhibitors in HUVEC was verified by immunecomplex kinase assays
using downstream targets of MEK and p38, ie, ERK or MAPKAP kinases 2 and 3, respectively. A total of 20 µmol/L PD98059 efficiently blocked
activation of ERK in endothelial cells stimulated with PMA, but had no
effect on arsenite-induced activation of p38
(Fig 2A). A total of 20 µmol/L SB203580
strongly inhibited the activation of the p38 effectors MAPKAP kinases 2 and 3 in HUVEC either incubated with arsenite or PMA, whereas ERK
activation was not affected (Fig 2B). Essentially similar
results were obtained with SB202190 (Fig 2C).
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| Fig 2.
PD98059, SB203580, and SB202190 function as specific
kinase inhibitors in HUVEC. (A) Cells were left unstimulated or were
exposed to PMA (for 30 minutes) or arsenite (for 60 minutes) in the
presence or absence of 20 µmol/L PD98059 after 30 minutes of
pretreatment as indicated. After cell lysis, ERK or p38 were
immunoprecipitated and assayed for activity as described. (B) HUVEC
were left unstimulated or were stimulated with arsenite or PMA as
described above either without or with 30 minutes of preincubation with
20 µmol/L SB203580. After cell lysis, kinases MAPKAP kinases 2 and 3 or ERK were immunoprecipitated and assayed for activity as described in
Materials and Methods. (C) Experiments were performed as described in
(B), but with the use of 20 µmol/L SB202190 instead of SB203580.
Western blots show equal protein loading of the respective kinases.
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We analyzed the effect of all three inhibitors on the expression of the
endothelial chemokine MCP-1, which is upregulated during endothelial
activation. HUVEC were exposed to 20 µmol/L SB203580, SB202190, or
PD98059 before stimulation with TNF-. Cells were then processed for
flow cytometric analysis of MCP-1 using an intracellular staining
procedure. PD98059 did not significantly affect the TNF--induced
production of MCP-1 or adhesion molecule ICAM-1, which was included for
control purposes (Fig 3C). Both SB203580
and SB202190 also did not exert significant inhibitory effects on
ICAM-1 expression (Fig 3A and B). However, both p38 inhibitors had a
strong effect on TNF--induced MCP-1 production (up to 60%
inhibition), suggesting an essential role of the MKK6/p38 pathway for
synthesis of that chemokine.
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| Fig 3.
Effect of inhibitors SB203580, SB202190, and PD98059 on
MCP-1 and ICAM-1 expression. Cells were left untreated or pretreated
with 20 µmol/L SB203580 (A), 20 µmol/L SB202190 (B), or 20 µmol/L
PD98059 (C) for 30 minutes. Cells were stimulated with 1 ng/mL TNF-
for 12 hours in the presence or absence of inhibitors and subsequently
processed for flow cytometry analysis as described in Materials and
Methods. The mean ± SEM of relative fluorescence intensities of three
to six independent experiments is shown. Asterisks denote statistically
significant differences as compared with control (P < .05, Wilcoxon test).
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TNF--induced MCP-1 synthesis in HUVEC is partially blocked by
expression of a dominant negative mutant of MKK6.
To confirm the data obtained with pharmacologic inhibitors, we
developed a transient transfection approach to study the effect of
dominant negative mutants on synthesis of MCP-1. Efficiency of mutants
had been verified in various cell systems as
described.21,32,33 Cells transfected with the kinase
mutants were identified by coexpression of GFPS65T. Thirty-six hours
after transfection, cells were exposed to TNF- for another 12 hours
before detection of MCP-1. Expression was determined by flow cytometry
using Cy-Chrome-conjugated tertiary stage reagents as described in
Materials and Methods. Only those cells expressing GFPS65T detected in
the FL-1 channel were considered for analysis of the cotransfected
kinase inactive mutants. Additional experiments were performed in the
presence of 50 µg/mL polymyxin B sulfate to avoid LPS-induced
activation of endothelium. This helped to exclude that LPS potentially
contaminating the DNA preparations confounded the results.
Consistent with our findings obtained with the p38 inhibitors,
TNF--induced expression of MCP-1 was partially inhibited in cells
transfected with kinase inactive MKK6(Ala) (approximately 40%
inhibition), suggesting that MKK6 and its downstream kinase p38 are
involved in TNF--mediated upregulation of MCP-1
(Fig 4; Fig 5A
shows a set of representative fluorescence-activated cell sorting
[FACS] profiles). Dominant negative JNK (SAPK KK>RR) and a kinase
inactive mutant of its upstream activator MKK4/SEK (SEK K>R), as well
as dominant negative versions of Raf (Raf-C4B) and ERK (ERK2C3), did
not inhibit TNF--induced expression of MCP-1 (Fig 4), excluding a
critical role of both the SEK/JNK and the Raf/MEK/ERK cascade in MCP-1
synthesis.
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| Fig 4.
Effect of dominant negative kinase mutants on the
synthesis of MCP-1 by HUVEC. Cells were transfected in a 1:3 ratio with
pGreenLantern expressing GFPS65T and either empty expression vector
KRSPA or plasmids expressing dominant negative kinase mutants as
indicated and stimulated with 2 ng/mL TNF- for 12 hours.
Successfully transfected cells were analyzed for MCP-1 synthesis by
flow cytometry as described in Materials and Methods. The data shown
represent mean values ± SEM from four independent experiments. The
asterisk indicates a statistically significant difference as compared
with control (P < .05, Wilcoxon test).
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| Fig 5.
MCP-1 synthesis in cells expressing dominant negative
MKK6(Ala), MKK6 wild-type, or constitutively active MKK6(Glu) in the
presence or absence of TNF-. Cells were transfected in a 1:3 ratio
with pGreenLantern expressing GFPS65T and plasmids expressing either
empty expression vector KRSPA, KRSPA MKK6(Ala), KRSPA MKK6 wild-type,
or KRSPA MKK6(Glu). (A) Cells transfected with empty expression vector
or dominant negative MKK6, ie, MKK6(Ala), were either left unstimulated
or stimulated with 2 ng/mL TNF- for 12 hours. Successfully
transfected cells were analyzed for MCP-1 synthesis by flow cytometry
as described in Materials and Methods. Flow cytometry profiles of one
representative experiment are shown. Open profiles represent isotype
controls, shaded profiles represent cells labeled for MCP-1 expression.
Bold letters indicate the mean fluorescence intensities; additionally,
percentages of MCP-1-positive cells are given. (B) HUVEC transfected
with empty expression vector, wild-type MKK6, or a constitutively
active mutant of MKK6, ie, MKK6(Glu), were exposed to a limited
concentration of TNF- (0.2 ng/mL) and evaluated for MCP-1 expression
as indicated.
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Expression of wild-type or constitutively active MKK6 enhances
TNF--induced MCP-1 synthesis.
To further support these data, we analyzed whether overexpression of
MKK6 exerts positive effects on MCP-1 synthesis. When HUVEC are
stimulated with limiting concentrations of TNF-, expression of MKK6
wild-type enhanced synthesis of MCP-1, as shown by a significant upshift in the mean fluorescence intensity (bold letters), whereas transfection of a control vector had no effect (Fig 5B, lower panel,
middle). In addition, we tested a constitutive active mutant of MKK6,
which activates p38 in several cell types including HUVEC (data not
shown). Although expression of this mutant was not sufficient to induce
MCP-1 synthesis in unstimulated cells (Fig 5B, upper panel, left),
MCP-1 levels significantly increased in the presence of MKK6(Glu) when
cells were stimulated with limiting concentrations of TNF-.
The observation that specific p38 inhibitors and inactive or active
mutants of MKK6 negatively or positively interfere with the expression
of MCP-1 shows that the MKK6/p38 cascade is one of the crucial pathways
for TNF--induced synthesis of MCP-1.
The MKK6/p38 pathway enhances TNF--induced MCP-1 expression at
the mRNA level.
To elucidate whether regulation of MCP-1 synthesis by MKK6/p38 occurs
at the transcriptional level, Northern blot analysis was performed. As
shown in Fig 6A, stimulation of HUVEC with
TNF- strongly induced MCP-1 mRNA levels, whereas exposure of cells to SB203580 or SB202190 30 minutes before and during an 8-hour stimulation with TNF- markedly reduced MCP-1 mRNA levels. To analyze
whether the reduction of steady-state mRNA levels in the presence of
p38 inhibitor is due to an alteration of mRNA stability, the
experiments were performed in the presence of a transcriptional inhibitor, Act D. As shown in Fig 6B and C, the degree of decline of
MCP-1 mRNA amounts in HUVEC exposed to SB202190 is similar to that of
untreated cells. In both treated and untreated cells, approximately
20% of MCP-1 message is still present after 8 hours of transcriptional
inhibition, indicating that the p38 pathway is not significantly
involved in regulation of MCP-1 mRNA stability.
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| Fig 6.
Inhibition of TNF--induced MCP-1 mRNA synthesis by
p38 inhibitors, SB203580 and SB202190. (A) Total mRNA was extracted
from HUVEC exposed to 20 µmol/L SB203580 or SB202190 30 minutes
before and during an 8-hour period of stimulation with 2 ng/mL TNF-.
Control samples were incubated without p38 inhibitors in the absence or
presence of TNF- as indicated. MCP-1 mRNA levels were determined by
Northern blot analysis as described in Materials and Methods. Blots
were reprobed with an actin cDNA probe to control RNA loading. (B) To
assess the effect of p38 inhibition on stability of TNF--induced
MCP-1 mRNA, HUVEC were treated with TNF- in the absence or presence
of SB202190 as in (A). After 8 hours (time zero), Act D was added at 2 µg/mL and total RNA isolated at the time intervals indicated (in
minutes). Northern blot analysis was performed as described above. (C)
Autoradiography bands shown in (B) were analyzed by densitometry and
normalized to the RNA content as judged by the levels of 28S rRNA.
TNF--induced MCP-1 mRNA levels at time zero were set as 100%. The
decrease of MCP-1 mRNA amounts is shown after exposure to Act D for
different time intervals as indicated. Treatment with Act D alone had
no effect on the levels of MCP-1 mRNA (data not shown).
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The MKK6/p38 pathway mediates TNF--induced induction of the MCP-1
promoter.
Transcriptional regulation of the MCP-1 promoter by the MKK6/p38
pathway was studied using promoter-luciferase constructs. HUVEC were
transfected with a MCP-1 promoter/enhancer luciferase construct
containing the enhancer (between 2742 and 2513) and promoter regions (between 107 and +60) of the human MCP-1
promoter (Fig 7A, see Ueda et
al29 for details). Thirty-six hours after transfection,
HUVEC were stimulated with TNF- in the absence or presence of p38
inhibitors SB203580 or SB202190 (Fig 7B and 8A). Both inhibitors were found to
partially block TNF--induced transcription of the MCP-1
promoter/enhancer luciferase construct in a concentration-dependent
manner (Fig 8A and data not shown).
View larger version (32K):
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[in a new window]
| Fig 7.
Effect of p38 inhibitors and kinase mutants of the
MKK6/p38 signaling module on TNF--induced MCP-1 promoter
activity in HUVEC. (A) Schematic representation of the human MCP-1
promoter/enhancer construct with the SP1 and NF- B (A1 and A2) sites
marked. The proximal promoter and the distal enhancer region are
indicated by closed and open boxes. (B) An MCP-1 promoter/enhancer
luciferase construct was transfected into HUVEC according to a
DEAE-dextran protocol as described in Materials and Methods. After 36 hours, cells were stimulated with 1 ng/mL TNF- for 12 hours with or without a 30-minute pretreatment with 20 µmol/L
SB203580 as indicated. (C and D) HUVEC were cotransfected with MCP-1
promoter/enhancer luciferase constructs and kinase mutants
MKK6(Ala), MKK6(Glu), p38(TY>AF), or p38(K>M) as indicated.
Thirty-six hours later, cells were stimulated with 2 ng/mL TNF-.
Relative luciferase activity is expressed as fold stimulation as
described in Materials and Methods.
|
|
View larger version (21K):
[in this window]
[in a new window]
| Fig 8.
Concentration/response dependence of increasing amounts
of p38 inhibitor SB202190 or dominant negative MKK6 kinase mutant
MKK6(Ala) on TNF--induced MCP-1 promoter activity. HUVEC were
transfected with a MCP-1 promoter/enhancer luciferase construct as
described in Materials and Methods. (A) Thirty-six hours later, cells
were exposed to increasing concentrations of p38 inhibitor SB202190 as
indicated and stimulated with 1 ng/mL TNF- 30 minutes later for
another 12 hours. Luciferase activity was determined as described in
the legend to Fig 7. (B) HUVEC were cotransfected with the MCP-1
promoter/enhancer construct and increasing amounts of MKK6(Ala) as
indicated and stimulated with 2 ng/mL TNF- 36 hours later for
another 12 hours. Relative luciferase activity is expressed as fold
stimulation.
|
|
We then evaluated whether cotransfection of dominant negative or
constitutively active mutants of MKK6, ie, MKK6(Ala) or MKK6(Glu), influenced TNF--induced MCP-1 promoter-dependent transcription. As
shown in Figs 7C and 8B, the dominant negative mutant MKK6(Ala) inhibited TNF--induced expression of the reporter gene in a
concentration-dependent way, whereas MKK6(Glu) significantly enhanced
it. Similarly, dominant negative mutants of p38, ie, p38(TY>AF) and
p38(K>M), were able to significantly block MCP-1 promoter-dependent
transcription (Fig 7D). Although the levels of stimulation by TNF-
differ somewhat between distinct sets of experiments dependent on
vector background or potential variability of primary cells, the degree
of transcriptional inhibition or enhancement by the different mutants
or inhibitors greatly match the data obtained at the level of protein
synthesis. In conclusion, our data provide strong evidence that the
MKK6/p38 pathway accounts for at least 50% of the TNF--induced
signals controlling MCP-1 expression.
| |
DISCUSSION |
Mitogen- and stress-activated protein kinases of the MAP kinase family
respond to a plethora of extracellular signals integrating different
inputs, which may result in the functional reprograming of cellular
activities.20 These signals include factors such as
TNF-, which are delivered under conditions of inflammation. Our
studies were based on the observation that TNF- caused p38, JNK, and
ERK activation in endothelial cells. However, because cytokine-induced
activation of MAP kinases may exert pleiotropic effects on various
other cellular activities, a functional relevance for expression of
certain endothelial adhesion molecule genes or chemokines cannot
necessarily be deduced from these data.
We have now shown that the p38 inhibitors, SB203580 and SB202190,
inhibit the TNF--induced expression of MCP-1 at a transcriptional level. Besides the use of inhibitors, which may have potential side
effects, we followed an independent approach to evaluate the
contribution of each MAP kinase pathway to MCP-1 transcription and
synthesis. We used transient transfection assays with a panel of
dominant negative or constitutively active kinase mutants. Besides
promoter-reporter gene-based transfection experiments, our approach
allowed us to analyze the expression of endogenous proteins. Although
TNF- was found to be an efficient agonist of three different MAP
kinase cascades in HUVEC, only the MKK6/p38 pathway has now been
identified to be of major relevance for TNF--induced expression of
the MCP-1 gene. MCP-1 synthesis and transcription were affected to the
same degree by the different inhibitors and mutants of the MKK6/p38
pathway. Further, block of the p38 pathway does not significantly
affect MCP-1 mRNA stability, indicating that regulation of MCP-1
expression by the MKK6/p38 module mainly occurs at the transcriptional
level. This adds MCP-1 to the list of MKK6/p38-regulated genes such as
IL-6 or GM-CSF.18 A still open question is the
determination of the MKK6/p38-responsive regions of the MCP-1 promoter.
Structural features of the human MCP-1 promoter have recently been
described.29,34 While basal promoter activity is dependent
on a proximal promoter region containing an SP-1 site,
cytokine-inducible promoter activity is mainly mediated by a distal
enhancer region (Fig 7A). Two NF-B binding sites (A1 and A2) have
been identified in the distal enhancer region, which are crucial for
enhancer activity.29 Because another promoter-luciferase construct containing solely the proximal promoter region was neither responsive to TNF- or influenced by mutants or inhibitors of the
MKK6/p38 pathway (M.G. and S.L., unpublished observation, May
1998), it is most likely that the MKK6/p38-responsive
target sites are localized in the MCP-1 distal enhancer. Mutation of the A1 or A2 NF-B sites in this region resulted in loss of the enhancer activity,29 suggesting that MCP-1 regulation by
the cytokine-inducible MKK6/p38 pathway is also mediated by NF-B sites. Consistent with our observations, the MKK6/p38 cascade has been
reported to be involved in NF-B-dependent gene expression in
several other systems.18-20,35
MCP-1 transcription and synthesis were inhibited by roughly 50% to
60% of induced levels, indicating that at least half of the TNF-
signal leading to induction of MCP-1 expression is transmitted by the
MKK6/p38 signaling module. Using the p38 inhibitor, SB203580, similar
degrees of inhibition were observed for LPS-induced TNF- and IL-1
expression in monocytes,17 as well as for TNF--induced vascular cell adhesion molecule-1 (VCAM-1) expression in
HUVEC.36 Involvement of this pathway in TNF--induced
MCP-1 synthesis may be even higher taking into account that the
dominant negative mutants act in competition and do not completely shut
off the upstream signal32 and that the p38 inhibitors may
not be fully stable during incubation times of 12 hours.21
TNF- activates a variety of intracellular signaling pathways, which
may act in concert with the MKK6/p38 pathway to support MCP-1
expression in HUVEC. These include several other MAP kinase cascades
besides the MKK6/p38 pathway (Fig 1). However, according to our data,
the additional pathways, which account for full TNF--induced expression of the MCP-1 gene, are different from other MAP kinase signaling pathways, namely the Raf/MEK/ERK and the JNK/SAPK pathways.
The characterization of MCP-1 as a novel target gene of the MKK6/p38
cascade identifies the latter as an important mediator of physiologic
responses to TNF- signals in endothelial cells. This might be
important with respect to inflammatory dysfunctions. It has been shown
previously that inhibition of induced MCP-1 expression is associated
with reduced transmigration of monocytes through the vessel
wall,6 as well as with diminished recruitment of T
lymphocytes,7 indicating that the level of MCP-1 synthesis is critical for the course of inflammatory reactions.
The MKK6/p38 pathway might be further important in the control of
inflammatory responses in general, as this cascade appears to act on
several levels of leukocyte recruitment by endothelium. Besides the
effects on MCP-1 reported here, this pathway was discussed to be of
relevance for TNF--induced VCAM-1-36 and E-selectin expression in HUVEC,37 as well as for expression of the
proinflammatory cytokines TNF- and IL-1 in
monocytes.17 Thus, in analogy to the mitogenic Raf/MEK/ERK
cascade, the MKK6/p38 module might be considered to act as an
inflammatory pathway.
Finally, the identification of MCP-1 as a novel physiologic target for
the MKK6/p38 pathway might be important with respect to our
understanding of the mechanisms leading to the recruitment of monocytes
to the site of prospective plaque formation during atherosclerosis.
| |
ACKNOWLEDGMENT |
The authors thank Angelika Hoffmeyer and Christoph K. Weber for
critically reading the manuscript and Martina Gropengieer, Heide
Häfner, Sybille Schmid, and Atiye Toksoy for excellent technical assistance.
| |
FOOTNOTES |
Submitted June 30, 1998; accepted September 21, 1998.
Supported by Grants No. GO 811/1-1 and LU 477/2-3 from the Deutsche
Forschungsgemeinschaft (DFG) (to M.G. and S.L.).
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Stephan Ludwig, PhD, Institut
f. Med. Strahlenkunde u. Zellforschung (MSZ), University of
Würzburg, Versbacher Str. 5, D-97078 Würzburg, Germany; or
Matthias Goebeler, MD, Klinik für Haut und
Geschlechtskrankheiten, University of Würzburg,
Josef-Schneider-Str. 2, 97080 Würzburg, Germany.
| |
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Top
Abstract
Introduction