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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 94 No. 4 (August 15), 1999:
pp. 1291-1299
Endothelin-1-Induced Interleukin-8 Production in Human Brain-Derived
Endothelial Cells Is Mediated by the Protein Kinase C and Protein
Tyrosine Kinase Pathways
By
R. Zidovetzki,
P. Chen,
M. Chen, and
F.M. Hofman
From the Departments of Biology and Neuroscience, University of
California, Riverside, CA; and the Department of Pathology, University
of Southern California School of Medicine, Los Angeles, CA.
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ABSTRACT |
We have previously demonstrated that endothelin-1 (Et-1) induces
human central nervous system-derived endothelial cells (CNS-EC) to
produce and secrete the chemokine interleukin 8 (IL-8). In the present
study, we use specific inhibitors and activators to elucidate the
signal transduction pathways involved in this process. Et-1-induced
IL-8 production was blocked by ETA receptor antagonist BQ610, but not by ETB receptor antagonist BQ788,
demonstrating that CNS-EC activation is initiated by Et-1 binding to
the ETA receptor. IL-8 mRNA expression is blocked by the
protein kinase C inhibitor bisindolylmaleimide or protein tyrosine
kinase inhibitors, genestein and geldanamycin, establishing the
involvement of the protein kinase C and protein tyrosine kinase
pathways in the activation process. The transcription factor, NF- B,
is involved in Et-1 activation as determined by specific inhibitors of
translocation and direct analysis of DNA-binding proteins. Neither
inhibition nor activation of cAMP-dependent protein kinase affected
IL-8 production in the absence or presence of Et-1. Similarly, no
effect was observed upon inhibition of protein phosphatases by okadaic acid. Thus, the signal transduction process induced by Et-1 in CNS-EC,
leading to increased mRNA IL-8 expression, is initiated by Et-1 binding
to ETA receptor followed by subsequent activation of
protein kinase C, protein tyrosine kinase, and NF-B. Because increased expression of Et-1 is associated with hypertension and stroke
and IL-8 is likely to be involved in the accumulation of neutrophils
causing tissue damage in ischemic/reperfusion injury, identification of
the mechanism involved in the Et-1-induced increase in IL-8 production
may have significant therapeutic value.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
ENDOTHELIN-1 (Et-1), a 21 amino acid
peptide produced by various cell types, including cerebral
microvessels, mediates vasoconstriction.1-3 This peptide
was reported to be increased in the plasma and cerebrospinal fluid of
patients with hypertension, ischemic stroke, and subarachnoidal
hemorrhage.4-7 Recent evidence demonstrated the pathologic
significance of Et-1, as shown by the correlation between reduced Et-1
activity and decreased lesion size in reperfusion injury after middle
cerebral artery occlusion in rats.8 In the investigation of
the mechanisms of Et-1-mediated pathology, we found that this peptide
induced the production of the chemokine interleukin-8 (IL-8) in human
central nervous system-derived endothelial cells (CNS-EC).9
IL-8, an  chemokine, is produced predominantly by
macrophages10-12 and endothelial cells13 and is
characterized by its potent and selective stimulatory effects on
neutrophils, which include chemotaxis,14
degranulation,15 respiratory burst,16 cell shape change, exocytosis of secretory vesicles and azurophil granules, expression of surface adhesion molecules, production of superoxide and
hydrogen peroxide reactive oxygen metabolites,17,18
activation of 5-lipogenase,19 and the release of cell
matrix resorbing gelatinase and elastase.18 Studies have
shown that ischemic brain injury involves the initial influx of
neutrophils and that depletion of these cells or blocking their entry
into the brain leads to a significant reduction in ischemic
damage.20,21 Et-1-induced IL-8 production may, therefore,
be involved in tissue damage during ischemia/reperfusion injury.
In the present work, we addressed the question of the intracellular
signal transduction pathway(s) used by Et-1 in inducing increased IL-8
production in CNS-EC. Biological effects of Et-1 are mediated by 2 types of Et receptors, ETA and ETB, and involve multiple intracellular signal transduction pathways, including activation of GTP-binding proteins, phospholipases, protein kinases and
phosphatases, and various transcription factors (see
Sokolovsky22 and Levin23 for reviews). It has
been suggested that a single ET receptor might evoke diverse signaling
pathways by activating multiple G proteins; these may, in turn,
activate single or multiple effectors, forming a network in which
various combinations of receptors and G proteins interact to activate
signal transduction pathways optimized for physiological regulation in
different cells.24 Moreover, the signal transduction upon
Et-1 binding to its receptor may differ among species and
tissues. The data presented here demonstrate that, in human
CNS-EC, Et-1 induces increased IL-8 mRNA and protein production via
ETA receptor, protein kinase C (PK-C)-dependent and
protein tyrosine kinase (PTK)-dependent pathways without involvement of
the cAMP-dependent pathway.
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MATERIALS AND METHODS |
Reagents.
The following reagents were purchased: Et-1 (Peninsula Lab, Belmont,
CA); ETA receptor antagonist, BQ610, and ETB
receptor antagonist, BQ788, from Alexis Corp (San Diego, CA);
cAMP-dependent protein kinase (PK-A) inhibitor, H-89, PK-C inhibitors,
bisindolylmaleimide-GF109203-X (GF) and Calphostin C; tyrosine kinase
inhibitors, genistein and geldanamycin, and inactive genistein analog
daidzen; and phosphoserine/phosphothreonine phosphatase inhibitor
okadaic acid from Calbiochem (La Jolla, CA); dibutyryl-cAMP;
phorbol-12-myristate-13-acetate (PMA); 4--phorbol (inactive analog
of PMA) and the NF-kB inhibitor, pyrrolidinedithiocarbamate (PDTC),
from Sigma (St Louis, MO). PK-C assay kits were purchased from GIBCO
Life Technologies (Gaithersburg, MD).
Cell culture.
CNS-EC were derived from human brain as previously described in
detail.25 Cells were cultured in RPMI-1640 medium (GIBCO Life Technologies, Grand Island, NY) supplemented with 100 ng/mL endothelial cell growth factor Endogro (VECTEC, Albany, NY), 2 mmol/L
L-glutamine, 10 mmol/L HEPES, 24 mmol/L sodium bicarbonate, 300 USP
units of heparin, 1% penicillin/streptomycin, and 10% fetal calf
serum (FCS). Endogro-free, 0.1% FCS medium was used 24 hours before
the experiment. The purity of CNS-EC (95%) was confirmed by
immunocytochemical staining for von Willibrand factor (vWF), glial
fibrillary acidic protein (GFAP) for astrocytes, and CD11b for the
macrophages, as previously described.25 Cells were used
until passage 4 or 5 only, because it was found that with increasing
passage number above 7, the intensity of vWF and  glutamyl
transpeptidase decreased.
RNase protection assay (RPA).
The radioactively labeled RNA antisense probes were prepared following
the manufacturer's protocol. Using the In Vitro Transcription Kit
(Pharmingen, San Diego, CA), 10 µL of [32P]UTP (3,000 Ci/mmol, 10 mCi/mL; NEN Research,Wilmington, DE) and 1 µL GACU pool
were added to the RPA Template Set (HCK-5), a human chemokine
multiprobe set including IL-8 and the housekeeping gene,
glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Pharmingen); also
included were T7 polymerase, dithiothreitol (DTT), RNAsin, and transcription buffer, as suggested by the manufacturer. The reaction was terminated by adding 2 µL of DNase for 30 minutes at
37°C. The probe was then extracted using Tris-saturated
phenol:chloroform:isoamylalcohol (25:24:1; GIBCO Life
Technologies) and chloroform:isoamylalcohol sequentially
and then was ethanol precipitated. The radiolabeled RNA pellet was
air-dried and solubilized with hybridization buffer. RNA from 2 to 3 × 106 cultured cells per experimental group was
isolated and prepared according to a modification of the acid phenol
method using the Trizol reagent (Life Technologies) as specified by the
manufacturer. Total RNA (10 µg) together with 6 × 105 cpm of probe was heat-denatured at 90°C and then
hybridized overnight at 56°C. Subsequently, the samples were
treated with the RNase cocktail, followed by proteinase K cocktails,
and then precipitated using ammonium acetate and ethanol. Air-dried
samples were solubilized in loading buffer, denatured at 90°C for 3 minutes, and then placed on ice. The protected fragments were resolved
in 5% acrylomide 8 mol/L urea gel (24 cm length); the gel was dried
and exposed using Hyper film (Amersham Life Science, Arlington Heights,
IL) at  70°C . The protected bands were observed for IL-8
(181 bp) and GAPDH (96 bp). The data are presented as the ratios of the spectrophotometric densities of IL-8 to GAPDH bands. The
manufacturer's recommended yeast tRNA negative and positive controls
were included in every RPA experiment.
Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis.
Endothelial cells were treated and total RNA was isolated as described
above. To synthesize cDNA, 1 µg of denatured total RNA was incubated
in 20 µL of reaction buffer (5 mmol/L MgCl2, 50 mmol/L
KCl, 10 mmol/L Tris-HCl, pH 8.3) with 2.5 µmol/L oligo(dT) primer and
murine leukemia virus reverse transcriptase (RT; 2.5 U/mL), which was
purchased as an RT-PCR kit (Perkin-Elmer, Foster City, CA). The reverse
transcription reaction was performed in 1 cycle: 42°C for 20 minutes, 99°C for 5 minutes, and 5°C for 5 minutes. PCR
reactions were performed using 5 µL of cDNA, which was diluted 1:4
for ETA and ETB receptors and 1:10 for
 -actin in the presence of the reaction buffer, a 0.3 µmol/L
concentration of each primer (Clontech, Palo Alto, CA), and 2.5 U of
taq polymerase (Perkin-Elmer) in a total volume of 50 µL.
These dilution factors were based on the log-linear relationship
between the mRNA and the signal intensity of the PCR product. The PCR
reaction was performed as follows: 94°C for 1 minute and 45 seconds
(1 cycle); 94°C for 15 seconds, 60°C for 30 seconds, and
72°C for 30 seconds (32 cycles); and 72°C for 6 minutes and
4°C for 5 minutes (1 cycle). PCR analysis was performed using the
human ETA receptor primers (sense primer [5'],
5'AGC TTC CTG GTT ACC ACT CAT CAA 3'; and antisense primer
[3'], 5' TCA ACA TCT CAC AAG TCA TGA G 3') and ETB receptor primers (sense [5'], 5' CGA GCT
GTT GCT TCT TGG AGT AG 3'; and antisense [3'], 5'
ACG GAA GTT GTC ATA TCC GTG ATC 3'). The sizes of
the ETA and ETB receptor PCR products are 714 and 701 bp, respectively. The human -actin primer, used as the control, was the following: sense primer (5'), 5' GTG GGG
CGC CCC AGG CAC CA 3'; and antisense primer (3'), 5'
CTC CTT AAT GTC ACG CAC GAT TTC 3'. The size of the PCR product
is 548 bp. The reverse transcriptase and PCR reactions were performed
in DNA thermal cycles (Perkin-Elmer Gene Amp PCR system 2400).
Ten-microliter PCR products were electrophoresed through a 1.5%
agarose gel and visualized by ethidium bromide staining. The gel
pattern was documented using the signal analytic system and the band
intensities were measured using the IP Lab gel program (BT Scientific
Technologies, Carlsbad, CA).
PK-C assay.
Endothelial cells were treated as described in the previous sections.
The PK-C assays were performed using PK-C assay kits obtained from
GIBCO Life Technologies. Briefly, the experimental treatments were terminated by replacing the medium with the cell extraction buffer (20 mmol/L Tris, 0.5 mmol/L EDTA, 0.5 mmol/L EGTA,
and 25 µg/mL each of aprotinin and leupeptin, pH 7.5) at room
temperature. The cells were then homogenized with a precooled dounce
homogenizer and the cytosol and membrane fractions were separated by
centrifugation (10,000g for 30 minutes) at 4°C. The supernatants were collected as the cytosol fraction. The pellets were
resuspended in 0.5 mL of extraction buffer with the detergent (1%
NP-40). Membrane fractions were obtained by centrifugation (10,000g for 10 minutes at 4°C) and collecting the
supernatants. PK-C in both membrane and cytosol fraction was partially
purified by ion exchange chromatography (DE-52 cellulose column). The
determinations of PK-C activity in the cytosol and membrane fractions
were performed according to the manufacturer's instructions. The
specific PK-C substrate was a synthetic peptide from myelin basic
protein (amino acids 4-14) with an acetylated N-terminal glutamine, and
the specific inhibitor was a peptide (amino acids 19-36) derived from
the same protein that binds to the pseudo substrate region of the
regulatory domain. The specific PK-C activity was determined as the
difference between phosphorylation of the PK-C specific substrate in
the absence or presence of the specific PK-C inhibitor. The data are presented as ratios of the membrane and the total (cytosol + membrane) PK-C activities.
Electrophoretic mobility shift assay (EMSA).
CNS-EC were grown to confluence in 100-mm Petri dishes (107
cells/dish), treated with Et-1 (10 8 mol/L), with
tumor necrosis factor (TNF; 10 pg/mL), or left untreated for 1 hour.
Cells were then washed twice with ice-cold phosphate-buffered saline
(PBS), scraped in 15 mL of PBS/plate, and centrifuged at 1,200 rpm for
10 minutes at 4°C. Cell pellets were then resuspended in 400 µL
of buffer A (10 mmol/L HEPES [pH 7.9], 1.5 mmol/L MgCl2, 10 mmol/L KCl, 0.5 mmol/L DTT, 0.5 mmol/L phenylmethyl sulfonyl fluoride [PMSF], 2 µg/mL leupeptin, and 2 µg/mL antipain) and placed on ice for 10 minutes; NP-40 (4%) was added to each tube to
reach a final concentration of 0.1%. The tubes were vortexed and
centrifuged at 14,000 rpm for 30 seconds at 4°C, and the
supernatants were removed. The nuclear pellet was resuspended in 50 µL of buffer C (20 mmol/L HEPES, 25% glycerol, 1.5 mmol/L
MgCl2, 300 mmol/L NaCl, 0.25 mmol/L EDTA, 0.5 mmol/L PMSF,
2 µg/mL leupeptin, and 2 µg/mL antipain), vortexed, and placed on
ice for 20 minutes. Samples were centrifuged at 14,000 rpm for 10 minutes at 4°C. Protein concentrations were determined using the
Bio-Rad protein assay kit (Bio-Rad Laboratories, Hercules, CA) with
bovine serum albumin as the standard. Nuclear extracts were diluted to
20 µg/µL with buffer C, aliquoted, and stored at 120°C.
EMSA was performed using Gel Shift Systems (Promega, Madison, WI). The
double-stranded oligonucleotides containing transcription factor
binding sites for NF-B and AP-1 used in this assay were commercially
available (Promega). The NF-B binding site oligonucleotide was
end-labeled with [-32P]ATP (ICN Pharmaceuticals Inc,
Costa Mesa, CA) by incubation with T4 Polynucleotide Kinase (Promega)
at 37°C for 10 minutes. The labeled probe was separated from
unincorporated nucleotide using G-25 spin columns (Amersham Pharmacia
Biotech, Piscataway, NJ). Nuclear extracts (20 µg) were incubated
with 10,000 cpm of labeled probe in a binding buffer containing 10 mmol/L Tris-HCl (pH 7.5), 4% glycerol, 1 mmol/L MgCl2, 0.5 mmol/L EDTA, 0.5 mmol/L DTT, 50 mmol/L NaCl, 0.05 mg/mL
poly(dI-dC).(dI-dC) for 30 minutes at 4°C. Unlabeled specific
(NF-B) or nonspecific (AP-1) binding site competitors, at 100-fold
excess, were added to the reaction at this time to serve as controls.
Reaction products were analyzed by electrophoresis using 5%
nondenaturing polyacrylamide gels (0.5× Tris-borate-EDTA buffer)
at 4°C for 2 hours. Gels were vacuum-dried and exposed to x-ray
film for 1 or 2 days.
Statistics.
Values were presented as the mean ± standard error of the mean
(SEM), unless otherwise stated. Statistical significance was evaluated
using the unpaired Student's t-test or ANOVA followed by
Bonferroni's test using ProStat software (Poly Software
International, Salt Lake City, UT); P < .05 was considered significant.
| |
RESULTS |
Both ETA and ETB receptors are expressed by
CNS-EC.
Et-1 action can be mediated via 2 main types of endothelin receptor,
ETA or ETB. It was originally postulated that
EC predominantly express ETB receptors26;
however, the presence of ETA receptors on the surface of
human CNS-EC has been reported.27 The RT-PCR results using
specific ETA and ETB receptor probes on 3 different primary CNS-EC cell cultures are shown in
Fig 1; these data demonstrate the
expression of mRNA for both receptor types by human CNS-EC. In
contrast, human umbilical vein endothelial cells (HUVECs) expressed only the ETB receptor mRNA (Fig 1).
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| Fig 1.
The expression of ETA and ETB
receptors on CNS-EC and HUVECs. RNA from cells from 3 different CNS-EC
cultures and 1 sample of HUVECs was extracted and examined using PCR
primers for ETA (lanes 1 through 4), ETB (lanes
5 through 8) receptors, and -actin (lanes 9 through 12). The CNS-EC
are grouped in lanes 1, 5, and 9; 2, 6, and 10; and 3, 7, and 11. HUVEC
RNA is shown in lanes 4, 8, and 12. M designates the marker lane.
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Et-1 activation in CNS-EC is mediated by the ETA
receptor.
The effect of Et-1 on IL-8 secretion by human CNS-EC has been
previously reported.9 We demonstrated that cells incubated with an optimal concentration of Et-1 (100 nmol/L) for 1 hour exhibited
an upregulation of IL-8 mRNA expression; Et-1-induced IL-8 protein was
detected within 24 hours. To ascertain the identity of the functional
Et-1 receptor type(s) in CNS-EC for IL-8 mRNA expression, we used a
specific ETA receptor antagonist, BQ610, or ETB
receptor antagonist, BQ788, concomitantly with Et-1. Cells were
pretreated with the Et-1 receptor antagonists for 20 minutes, followed
by incubation with Et-1 for 1 hour; subsequently, IL-8 mRNA was
analyzed using the RPA. The data for this and all other figures were
calculated as the ratios of IL-8 mRNA to GAPDH mRNA and are presented
in graphic form as relative mRNA values. Concentrations of receptor
antagonists, ranging from 200 nmol/L to 1 µmol/L, were previously
tested. Figure 2 demonstrates that
treatment with BQ610 at the lowest concentration (200 nmol/L)
completely abolished the IL-8 mRNA increase by Et-1, whereas BQ788 (200 nmol/L) had no effect. BQ610 and BQ788, at the highest concentration (1 µmol/L), gave essentially identical results. These data demonstrate
that Et-1 activation in CNS-EC is mediated by ETA receptor.
The data are representative of 3 experiments.
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| Fig 2.
The effect of Et receptor antagonists. The CNS-EC were
pretreated with 200 nmol/L ETA receptor antagonist, BQ610,
or 200 nmol/L ETB receptor antagonist, BQ788, for 20 minutes, followed by 1 hour of incubation with 100 nmol/L Et-1. In all
of the experiments presented, cells were treated with Et-1 for 1 hour.
Subsequently, RNA was isolated from the cells, and the RPA was
performed. The results are visualized by autoradiography; protected
fragments corresponding to IL-8 (181 bp) and GAPDH (96 bp) are shown
(A). The data in this and subsequent figures were calculated as the
ratios of IL-8 mRNA to GAPDH mRNA and are presented relative to the
control values (B). The data presented are representative of 1 of 3 replicate experiments performed. The error bars represent the SEM.
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PK-A-dependent pathway is not involved in Et-1-induced IL-8
production.
We have previously observed that Et-1 activates PK-A in human
CNS-EC,27a and others have shown that agents which increase cAMP increase IL-8 protein production.28 We therefore
tested the effects of PK-A activation or inhibition on Et-1-induced
IL-8 production in CNS-EC. Confluent cultures of CNS-EC were treated with Et-1 (100 nmol/L) alone or pretreated with the specific PK-A inhibitor, H-89 (0.5 µmol/L), or the PK-A-activating cAMP analog dibutyryl-cAMP (dBu-cAMP; 300 µmol/L) for 30 minutes, followed by the
addition of Et-1 for 1 hour (Fig 3). The
results show that direct activation of PK-A by dBu-cAMP did not affect
IL-8 mRNA expression; moreover, H-89 and dBu-AMP treatments did not
modify the Et-1-induced IL-8 mRNA synthesis, demonstrating that the
PK-A-dependent pathway is not involved in this effect of Et-1.
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| Fig 3.
The role of cAMP-dependent pathway. The CNS-EC were
pretreated for 30 minutes with 0.5 µmol/L of the specific PK-A
inhibitor, H-89, or with 300 µmol/L of PK-A-activating cAMP analog
dBu-cAMP, followed by 1 hour of incubation with 100 nmol/L Et-1. The
autoradiographic (A) and graphic data (B) are presented as described in
Fig 2.
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Et-1 increases PK-C activity.
The time course of the effect of 100 nmol/L Et-1 on membrane PK-C
activity in CNS-EC is shown on Fig 4. The
increase in PK-C activity was significant at 2 minutes, reached a
maximum at 10 minutes, sharply decreased by 20 minutes, and thereafter
slowly decreased to the longest observed time of 60 minutes, when the activity was still above the control levels (Fig 4). The effect was
completely inhibited by the presence of GF (1 µmol/L).
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| Fig 4.
PK-C activity of CNS-EC as a function of the duration of
Et-1 treatment. Et-1 concentration was 100 nmol/L, and the GF
concentration was 1 µmol/L. PK-C activity is expressed as a ratio of
the membrane to total activities. Solid line, in the absence of GF;
broken line, in the presence of GF.
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Involvement of PK-C in increased IL-8 mRNA expression.
Previous studies have reported that the PK-C-associated signal
transduction pathway may regulate IL-8 production in either a positive
or negative manner, depending on the cell type and the stimulus
involved.29-31 To test the involvement of the
PK-C-dependent signal transduction pathway in Et-1-induced IL-8 mRNA
expression, human CNS-EC were treated with Et-1 as described above or
incubated with the specific PK-C inhibitor GF (1 µmol/L) 30 minutes
before exposure to Et-1. The results in Fig
5 show that preincubation of the cells with GF completely eliminated
the Et-1-induced increase in IL-8 mRNA. Similar results were obtained
with another PK-C inhibitor, calphostin C (data not shown). To
determine the relevance of the early PK-C activation on the delayed
IL-8 response, GF was added to the cells 30 minutes after the initial
exposure of the cells to Et-1; the experiment was terminated after 1 hour of incubation, as described previously. The data showed that the addition of GF 30 minutes after exposure to Et-1 had no effect on
Et-1-induced IL-8 mRNA expression (Fig 5), demonstrating that the
early increase in PK-C activity is necessary and sufficient for IL-8
mRNA induction.
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| Fig 5.
The role of PK-C-dependent pathway. In 1 group
(GF+Et), the cells were pretreated for 30 minutes with 1 µmol/L of
the specific PK-C inhibitor GF, followed by 1 hour of incubation with
100 nmol/L Et-1. In another group (Et+GF), GF was added 30 minutes
after the initial treatment with Et-1. Cells were treated with Et-1 for
a total of 1 hour. The autoradiographic (A) and graphic data (B) are
presented as described in Fig 2.
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PTK pathway involved in Et-1 activation.
We then examined the role of PTKs in Et-1-induced IL-8 mRNA expression
in CNS-EC. Cells were treated with the PTK inhibitor genistein (37 µmol/L) for 15 minutes before the addition of Et-1; after 1 hour of
incubation, the experiment was terminated as described above. The data
in Fig 6A and B demonstrated that genistein
completely abolished the effect of Et-1, whereas the inactive genistein
analog daidzen (39 µmol/L) had no such effect. Another PTK inhibitor, geldanamycin (178 nmol/L), which specifically inhibits p60 c-src tyrosine kinase, also inhibited Et-1-induced IL-8 mRNA expression (Fig
6C). From these experiments, it was apparent that both the PK-C and PTK
pathways were involved in this Et-1 signaling pathway. Further studies
were performed to determine whether the effects of PK-C-dependent or
PTK-dependent pathways in IL-8 mRNA induction were sequential or
parallel. As shown in Fig 7, treatment of
CNS-EC for 1 hour with PK-C activator PMA (1 nmol/L) upregulated IL-8 mRNA expression; the inactive PMA analog 4--phorbol had no effect (data not shown). Pretreatment (15 minutes) with the PTK inhibitor genistein completely abolished the effect of PMA, demonstrating that
PTK activation is downstream from PK-C activation in this process.
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| Fig 6.
The role of PTKs. The cells were pretreated for 15 minutes with the specific PTK inhibitor genistein (37 µmol/L) or
the inactive genistein analog daidzen (39 µmol/L), followed by 1 hour
of incubation with 100 nmol/L Et-1; data are shown in an autoradiograph
(A) and graphically (B). CNS-EC were pretreated with geldanamycin (178 nmol/L) for 16 hours, followed by 1 hour of incubation with 100 nmol/L
Et-1. The autoradiograph of the RPA for IL-8 mRNA is shown (C).
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| Fig 7.
Sequential activation of PK-C and PTK. The cells were
treated for 1 hour with 1 nmol/L of the PK-C activator PMA or
pretreated for 15 minutes with the specific PTK inhibitor genestein (37 µmol/L) or the inactive genestein analog daidzen (39 µmol/L),
followed by the PMA treatment. The autoradiographic (A) and graphic
data (B) are presented as previously described.
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Protein phosphorylation in the signaling process is regulated by both
protein kinases and phosphatases. We tested the role of
serine/threonine phosphorylation on IL-8 mRNA expression in CNS-EC by
treating the cells with okadaic acid, a phosphoserine/phosphothreonine phosphatase inhibitor. CNS-EC were treated with okadaic acid (10 nmol/L) alone or 30 minutes before Et-1 treatment; the cells were then
incubated for 1 hour and analyzed for IL-8 mRNA expression. Figure 8 shows that okadaic acid at 10 nmol/L did not modify the IL-8-inducing effect of Et-1.
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| Fig 8.
The role of protein phosphatases. The CNS-EC were
pretreated for 30 minutes with 10 nmol/L of the phosphatase inhibitor
okadaic acid, followed by 1 hour of incubation with 100 nmol/L Et-1.
The autoradiographic (A) and graphic data (B) are presented as
previously described.
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NF-kB is involved in Et-1-induced IL-8 synthesis.
Finally, we examined the involvement of transcription factor NF-B in
the mechanism of Et-1 action. The cells were pretreated for 30 minutes
with NF-B inhibitor PDTC and then with Et-1 as described above. The
results in Fig 9 show that PDTC inhibited the Et-1-induced IL-8 mRNA increase in a concentration-dependent manner; complete inhibition was achieved at 30 µmol/L of PDTC. To
directly determine whether NF-B is involved in Et-1 activation, the
gel shift assay was performed. Figure 10
demonstrates that Et-1activation of CNS-EC induced NF-B
translocation and binding to DNA (lane 4). In the presence of the
unlabeled specific competitor, the shift was not observed (lane 5);
however, in the presence of the unlabeled nonspecific binding site to
transcription factor AP-1, the shift was apparent. TNF activation of
CNS-EC served as the positive control. These studies indicate that
NF-B is responsible, at least in part, for the Et-1-induced IL-8
production.
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| Fig 9.
Inhibitors of NF-B block Et-1 activation. Cells were
pretreated for 30 minutes with increasing concentrations of PDTC,
followed by 1 hour of incubation with 100 nmol/L Et-1. The
autoradiographic (A) and graphic data (B) are presented.
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| Fig 10.
Identification of NF-B as a transcription factor in
Et-1 activation. Nuclear extracts were prepared from CNS-EC incubated
in the absence (lanes 1, 2, and 3) or the presence of Et-1
(108 mol/L; lanes 4, 5, and 6) or TNF (10 pg/mL; lanes
7, 8, and 9) for 1 hour. A radiolabeled probe was incubated with
nuclear extract (10 µg of protein) for 30 minutes and protein-DNA
complexed was resolved by electrophoresis. Specific protein-DNA
complexes are indicated by arrows. Nuclear extract from lanes 1, 4, and
7 were analyzed for binding activity. Lanes 2, 5, and 8 contained a
100-fold molar excess of unlabeled double-stranded oligonucleotide
probe as a competitor. Lanes 3, 6, and 9 contained a 100-fold molar
excess of unlabeled nonspecific probe as competitor. The data presented
are from 1 of 3 representative experiments.
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| |
DISCUSSION |
This work extended our findings that Et-1 induced an upregulation of
IL-8 mRNA expression and protein production in human CNS-EC9 by focusing on the elucidation of the intracellular signal transduction pathway(s) involved in this process. We first identified the specific receptor class responsible for the
Et-1-induced IL-8 production in CNS-EC. Two distinct ET receptor
subtypes, ETA and ETB, have been cloned and
sequenced.32 EC in a variety of tissues, ie, liver, kidney,
and brain, possess predominantly ETB receptors, which
mediate vasodilation by the generation of endothelium-derived relaxing
factors and prostacyclins.26,32,33 ETA
receptors are predominantly found in peripheral tissues, especially in
vascular smooth muscle cells mediating vasoconstriction. Both types of
receptors were found in rat and human brain EC,34-36 where they mediate distinct functions. In agreement with the previous reports, we have found the expression of both receptor types by human
CNS-EC. Furthermore, our present results showed that ETA antagonist BQ610, but not ETB antagonist BQ788, inhibits
the observed effect of Et-1 effect, demonstrating that here the
ETA receptor is responsible for the initiation of the
signal transduction pathway to IL-8 production.
We next examined the role of specific signal transduction pathways
involved in Et-1-induced IL-8 production. It had been previously reported that increased intracellular cAMP levels caused increased IL-8
protein production in human monocytes,28 whereas in the present work inhibition of PK-A by H-89 did not affect Et-1-induced IL-8 increase. Our results agree with those of others30 who showed that, in human colon epithelial cells, H-89 did not affect the
IL-1-induced or TNF-induced IL-8 production; and this chemokine was
not stimulated by forskolin, an agent that upregulates cAMP. In
addition, White and Lee37 reported that inhibition of PK-A did not affect IL-1-induced IL-8 production by HUVEC. It is
interesting to note that we have previously found that Et-1 induces an
increase in intracellular cAMP levels and PK-A activity in human
CNS-EC.27a However, the present study demonstrates that, in
CNS-EC, PK-A activation is not relevant for the Et-1 induction of IL-8 production.
Our results demonstrated rapid activation of PK-C by Et-1. However,
activation of PK-C may increase or attenuate IL-8 production, depending
on both the cell type and stimulus involved. Thus, PMA induced IL-8
production in HUVEC,37,38 human
keratinocytes,29 human colon epithelial
cells,30 and human CNS-EC (this study). In HUVEC and human
colon epithelial cells, the PK-C inhibitors staurosporine or calphostin
inhibited PMA-induced37 or thrombin-induced38 IL-8 expression, but these inhibitors did not alter IL-1-induced or
TNF-induced IL-8 production,30,37,38 showing that, even in
identical cell populations, the same functional response can be
achieved by different stimuli via distinct pathways. PK-C inhibitors also block IL-8 mRNA expression in HL-6039 and in
fibroblasts.31 In the present study, we showed that human
CNS-EC require PK-C activation for Et-1-induced IL-8 mRNA expression,
and this activation takes place within the first 30 minutes of
stimulation. The addition of the PK-C inhibitor GF 30 minutes after
Et-1 stimulation clearly demonstrated that the short burst of PK-C
activity induced by Et-1 was necessary and sufficient for the
upregulation of IL-8 mRNA expression.
Similarly to PK-C, the role of PTK in inducing the IL-8 production is
cell type-dependent. Inhibition of PTK by various agents did not affect
IL-1-induced IL-8 production in HUVEC,37 whereas PTK inhibitors did inhibit IL-8 gene induction in a variety of other
model systems: by platelet activation factor in
fibroblasts,31 by IL-1 or TNF in human colon epithelial
cells,30 and by IL-1 in human bone marrow stromal
cells.40 The data here show that PTK is involved in the
upregulation of Et-1-induced IL-8 mRNA expression in human CNS-EC.
Furthermore, we demonstrated that, in these cells, PTK is directly
activated by PK-C and PMA-induced IL-8 increase is blocked by PTK
inhibitor genistein, which places PK-C activation upstream to PTK in
the activation process.
The inhibition of phosphoserine/phosphothreonine phosphatase in IL-8
mRNA expression is also cell type-dependent. Sonoda et al41
found that inhibition of phosphatases by okadaic acid or vanadate
dramatically increased IL-8 production by HL-60, but not by primary
monocytes. In the experiments presented here, okadaic acid alone did
not induce IL-8 mRNA increase in CNS-EC; furthermore, the presence of
okadaic acid did not affect the Et-1-induced mRNA increase,
demonstrating that, in the present system at the concentration of
okadaic acid used here (10 nmol/L), at least
phosphoserine/phosphothreonine phosphatase 2A is not involved in the
observed effects.
We next examined the role of the transcription factor, NF-B, in the
Et-1-induced IL-8 response. The IL-8 gene has several potential
binding sites for c-jun, NF-B, AP2, glucocorticoid receptor,
and NF-IL-6.42 It had been suggested that, depending on the
cell line, either the combination of NF-B and AP-1 or NF-B and
NF-IL6 is required.43 It has indeed been shown that, in
gastric cell lines, NF-B and AP-1 are sufficient for the IL-8 gene
activation,44 whereas in the fibrosarcoma line the
important factors are C/EBP and NF-B,42 and in
epithelial Caco2 cells NF-B, AP-1, and C/EBP.45 In the
present work, we demonstrated that NF-B activation is also involved
in the induction of IL-8 production by Et-1 in CNS-EC; however, other
transcriptional factors are also likely to be involved.
In conclusion, this study described, in part, the complex network of
intracellular signal transduction pathways used by Et-1 to effect IL-8
production. Both the elicited cellular responses and signal
transduction mechanisms used in performing a particular response are
cell type-dependent, necessitating the detailed investigation of the
pathway(s) involved for each specific response (eg, IL-8 production)
and each specific cell type (eg, human CNS-EC) and stimulus. In the
present work, we demonstrated that the Et-1- induced IL-8 mRNA
increase in human CNS-EC is initiated by binding of Et-1 to the
ETA receptor, followed by the subsequent activation of
PK-C, PTK, and NF-B. There are likely to be other enzymes and
factors involved in the signal transduction pathway leading to IL-8
production. However, it is only by the systematic characterization of
the components of this pathway that new therapeutic targets for the
regulation of this chemokine can be identified.
| |
ACKNOWLEDGMENT |
The authors thank Dr Mark Fisher for helpful comments and Dr Yueha Zhou
for her technical expertise.
| |
FOOTNOTES |
Submitted September 2, 1998; accepted April 12, 1999.
Supported by National Institutes of Health Grant No. PO1-NS31946.
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 F.M. Hofman, PhD, Department of Pathology,
University of Southern California, School of Medicine, 2011 Zonal
Ave, HMR 312, Los Angeles, CA 90033; e-mail: hofman{at}hsc.usc.edu.
| |
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