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(Received for publication, January 22, 1996, and in revised form, April 1, 1996)
From the ABSTRACT The proteinase-activated receptor 2 (PAR-2)
belongs to the family of seven transmembrane region receptors, and,
like the related thrombin receptor, it is activated by specific
proteolytic cleavage of its extracellular amino terminus. It is not
known which proteinase is the physiological activator of the PAR-2, but
candidates can be found among the enzymes involved in the inflammatory
cascade systems. Here, we have studied the effects of various mediators
on the expression of the PAR-2 and the thrombin receptor in cultured
human umbilical vein endothelial cells. Stimulation with the cytokines
tumor necrosis factor INTRODUCTION Endothelial cells line the inside of all blood vessels and form a
dynamic element of the barrier between blood and surrounding tissues.
The unperturbed endothelium opposes coagulation and does not interact
with circulating cells except in special sites, e.g. the
high endothelial venules of lymph nodes. In inflammation the
endothelium, especially in postcapillary venules, becomes leaky and
expresses molecules that promote clot formation and the adherence and
subsequent transmigration of white blood cells (see Refs. 1 and 2 for
reviews).
Endothelial cells can be activated by inflammatory mediators like tumor
necrosis factor The proteinase-activated receptor 2 (PAR-2; Refs. 14, 15, 16) is a G
protein-coupled receptor that, like the thrombin receptor (17, 18), is
activated by proteolytic cleavage of its extracellular amino terminus.
Activation of this type of receptor occurs through the interaction of
the new amino terminus, the ``tethered ligand,'' with some other
region of the receptor (17, 19, 20). By Northern blot analysis, the
PAR-2 is found in many different organs (14, 16). One particular cell
type that expresses the PAR-2 is the human umbilical vein endothelial
(HUVE) cell,2 which also expresses the
thrombin receptor. It is not known which proteinase is the
physiological activator of the PAR-2, but potential candidates can be
found among the many proteinases activated and secreted in blood
coagulation and inflammation.
Here, we have tried to address the question of PAR-2 involvement in
inflammatory reactions by studying the regulation of this gene. We
analyzed in some detail the effects of TNF EXPERIMENTAL PROCEDURES Cell culture medium, glutamine, and antibiotics
were from Life Technologies, Inc. Calf serum was from HyClone.
Endothelial cell growth supplement and heparin, as well as LPS
(Escherichia coli serotype 0127:B8), phorbol 12-myristate
13-acetate (PMA), forskolin, and isobutylmethylxanthine (IBMX) were
from Sigma. Recombinant human TNF Umbilical cords were obtained from the
maternity ward at Lund University Hospital, and HUVE cells were
prepared essentially as described by Jaffe et al. (21).
Cells were grown in M199 medium supplemented with 10% fetal calf serum
and 10% newborn calf serum, heparin (20 IU/ml), streptomycin (50 µg/ml), penicillin (50 units/ml), amphotericin B (2 µg/ml), and
endothelial cell growth supplement (30 µg/ml). Endothelial cells were
identified by their characteristic cobblestone-like appearance and by
uptake of fluorescently labeled LDL particles (Biomedical
Technologies). By these criteria, cultures were judged essentially
pure. After isolation, cell cultures were expanded and then frozen and
stored at Three different DNA fragments were
used in Northern blot experiments: a 1.3-kilobase human PAR-2 cDNA
fragment (16), a 2.2-kilobase human thrombin receptor cDNA fragment
(17). and a 0.9-kilobase human glyceraldehyde phosphodehydrogenase
(GAPDH) cDNA fragment. Fragments were labeled with 32P
by random priming using the Rediprime kit and
[ For Northern blot studies, HUVE cells were
seeded in gelatin-coated 6-well tissue culture plates and grown to
confluence. Cells were then incubated for an additional 24 h in
medium without endothelial cell growth supplement. Stimulating agents
were diluted in fresh medium without endothelial cell growth supplement
and added to the cells. For the extended study on TNF Antibody 61-1 recognizes an epitope around the
activating cleavage site of the human thrombin receptor and has been
described previously (23). Antibody S14.8.2 was raised against a
peptide corresponding to residues threonine 29 through phenylalanine 59 of the human PAR-2 amino acid sequence with an extra cysteine added to
the peptide's carboxyl terminus. This antibody will be described in
detail elsewhere.3 Antibody specificity was
tested by fluorescence-activated cell sorting (FACS) analysis of COS-7
cells transfected with cDNAs encoding either the human PAR-2 or the
human thrombin receptor. COS-7 cells were transfected by the calcium
phosphate method and grown for 36 h. They were released from
culture dishes by treatment with 3 mM EDTA and incubated
with primary antibodies S14.8.2 or 61-1 (10 µg/ml) for 45 min,
washed, and incubated for 45 min with phycoerytrin-conjugated goat
anti-mouse IgG at a 1/200 dilution and then analyzed by FACS. For
measurements of receptor cell surface expression in HUVE cells,
antibodies were labeled with 125I by the chloramin T method
to a specific activity of about 1010 cpm/mg and purified on
Sephadex G-50 columns.
Endothelial cells were seeded in
gelatin-coated 12-well tissue culture plates and treated as for the
Northern blot experiments up to and including the stimulation. After a
20-h incubation period with stimulating agents, cells were washed twice
with ice-cold phosphate-buffered saline with 0.1% bovine serum albumin
and then left on ice for 30 min in this buffer. Labeled antibodies were
diluted to about 107 cpm/ml (
RESULTS In a series of experiments, changes in expression of the PAR-2 and
the thrombin receptor were studied at the mRNA level in cultured
HUVE cells. After stimulation for various time periods, total RNA was
extracted and analyzed by Northern blotting. Filters were probed with
PAR-2, thrombin receptor, and GAPDH cDNAs, where the latter was
used to normalize signals between samples.
First we tested the effects of
activating two major intracellular signaling pathways. Forskolin, an
activator of adenylyl cyclases, was used together with the
phosphodiesterase inhibitor IBMX (10 µM and 1 mM, respectively) to study the effect of elevating
intracellular cAMP concentrations. PMA, an activator of protein kinase
C, was tested at 20 ng/ml. The results, shown in Fig. 1,
indicate that the PAR-2 and the thrombin receptor are both
down-regulated in response to an elevation in cAMP concentration. After
20 h, the levels of mRNA were down by about 50% for both
receptors. Phorbol ester stimulation also caused a steady decrease over
time in thrombin receptor expression, whereas PAR-2 transcript levels
changed in a biphasic manner, with a near 3-fold increase at 4 h
that had returned almost to base line at the last time point. RNA
levels for both receptors appeared to increase over time in
unstimulated cells. Exactly what causes this change is unclear, but it
may be a response to the continuing growth factor deprivation.
Stimulation with IL-1
To support the above results on
PAR-2 gene induction, we examined the effects of increasing
concentrations of IL-1
It has been
shown that agents that elevate intracellular concentrations of cAMP
antagonize many of the proinflammatory actions of cytokines on
endothelial cells (24, 25, 26). It was therefore tested if the
phosphodiesterase inhibitor IBMX on its own could antagonize the
effects of IL-1
The expression of PAR-2 was
also followed over several days of treatment with 10 ng/ml TNF
The
different responses to cytokine stimulation displayed by the two
receptor genes do not necessarily reflect differences in the amounts of
receptor mobilized to the cell surface. The results are also compatible
with a model where the PAR-2 and the thrombin receptor genes are
regulated at different levels, the PAR-2 being primarily controlled by
its rate of transcription and mRNA degradation. It was therefore
also investigated whether stimulation resulted in increased amounts of
receptor protein at the cell surface. This was especially pertinent in
view of reports about the existence of an intracellular storage pool of
preformed thrombin receptor (27, 28, 29). To measure receptor protein on
the cell surface, we used antibodies raised against peptides made from
the receptor amino-terminal sequences. Antibody specificity was
analyzed by FACS of COS-7 cells that had been transfected with
cDNAs encoding either the PAR-2 or the thrombin receptor (Fig.
6). HUVE cells, stimulated as above for 20 h with
various substances, were incubated with radioiodinated antibodies to
either the PAR-2 or the thrombin receptor. Bound radioactivity was
quantitated after washings. The results are summarized in Fig.
7. Binding of antibodies against either receptor was
decreased after stimulation with forskolin + IBMX (10 µM
and 1 mM, respectively) or PMA (2 ng/ml). The effects of
cytokine treatment mirrored those seen at the mRNA level.
Stimulation with TNF
DISCUSSION The first proteinase-activated receptor to be described, the
thrombin receptor, is widely expressed with a preference for
mesenchymal tissues (30). The distribution of the PAR-2 is being
investigated, and it appears to have an almost complementary
distribution, being expressed mainly in various epithelial linings of
the body.4 In endothelial cells, the two
receptors are present together. Why do endothelial cells express both
of these receptors, likely to be important only in emergency
situations? Do they serve redundant functions and just respond to
different agents, or do they act at different stages of tissue
responses to injury?
Although endothelial cells differ between tissues and between segments
of the vascular tree, it appears that many types of endothelial cells
have a similar set of responses to inflammatory stimuli. In this study,
we used primary cultures of HUVE cells, partly because these cells are
easily available but also because they are an established model system
for the study of endothelial function, especially in inflammation,
which faithfully reflects many endothelial responses known from studies
on living animals.
Stimulation of HUVE cells with forskolin caused down-regulation of the
PAR-2 and the thrombin receptor alike, both at the mRNA and the
protein levels. Phorbol ester treatment, on the other hand, appeared to
detect a difference in the regulation of the two receptor genes.
Thrombin receptor mRNA levels decreased steadily with PMA
stimulation, whereas the amount of PAR-2 mRNA first rose to almost
3-fold the basal value before starting to decrease. After 20 h of
PMA stimulation, protein levels of both receptors were decreased. For
the thrombin receptor, the results agree with earlier observations from
experiments with human mesangial cells (31). Whether the differential
responses to phorbol ester treatment are physiologically relevant will
have to be investigated.
TNF When compared with other proteins that are induced by treatment with
TNF It has been shown that, in endothelial and other cells, the thrombin
receptor is stored in an endosomal compartment (27, 28, 29) from where it
can be mobilized after the receptors at the cell surface have been used
up. We are currently investigating whether the same is true for the
PAR-2. Because of this potential difference, it was important to
determine the levels of receptor protein at the cell surface in
parallel with measuring the amounts of mRNA. It appears that
neither cytokine nor LPS caused a net redistribution of thrombin
receptor from the endosome to the cell surface, ruling out the
possibility that induction of the thrombin receptor still occurred, but
on a different level from that governing PAR-2 expression.
From our results, it is not possible to draw any precise conclusions as
to the role or importance of the PAR-2 in inflammation. No attempts
were made to determine receptor numbers or the absolute levels of PAR-2
or thrombin receptor mRNA, but the relative induction by cytokines
of the PAR-2 compared with the thrombin receptor suggests that there is
an increasing need for a function served by PAR-2 during the mounting
of an inflammatory response. Exactly what this function is can only be
answered when we know which enzyme acts on the PAR-2. Further
investigations must also aim at studying the functional effects of
endothelial activation mediated by PAR-2.
FOOTNOTES REFERENCES
Volume 271, Number 25,
Issue of June 21, 1996
pp. 14910-14915
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
COMPARISON WITH THE THROMBIN RECEPTOR*
§,
Division of Molecular Neurobiology, The
Wallenberg Laboratory, Lund University, Sweden and ¶ COR
Therapeutics Inc., San Francisco, California 94080
or interleukin-1 as well as bacterial
lipopolysaccharide elevated the expression of PAR-2 in a
dose-dependent manner. The time course of induction after
cytokine stimulation was similar to those published for the adhesion
molecules intercellular adhesion molecule-1 and vascular cell adhesion
molecule-1. After 20 h of stimulation, PAR-2 mRNA and protein
levels were increased to 5-10-fold basal values, and, in the continued
presence of tumor necrosis factor , PAR-2 mRNA expression was
found to remain elevated for up to 4 days. In contrast, the thrombin
receptor gene was not induced by any of these inflammatory mediators.
The responses to phorbol ester treatment also differed between the two
genes. Thrombin receptor mRNA levels decreased steadily up to
20 h, whereas PAR-2 mRNA levels first rose to about 3-fold
basal values at 4 h before decreasing again. Cell surface protein
levels of both receptors were decreased after 20 h of phorbol
ester stimulation. Elevating intracellular cAMP levels by treatment
with forskolin resulted in decreased expression of both receptors, and
inhibition of cAMP degradation appeared to blunt the cytokine-induced
increase in PAR-2 expression. The induction of the PAR-2 by cytokine
treatment supports the concept of PAR-2 involvement in the acute
inflammatory response.
(TNF)1 and
interleukin-1 (IL-1) as well as by the bacterial substance
lipopolysaccharide (LPS). All three substances induce the production of
tissue factor and the down-regulation of thrombomodulin (3, 4, 5, 6), thus
initiating coagulation by the extrinsic pathway and removing an
important cofactor in the protein C anticoagulant system. Furthermore,
TNF, IL-1, and LPS signal endothelial cells to produce the leukocyte
chemoattractants macrophage chemotactic protein-1 (7) and interleukin-8
(8) and to expose at the surface the adhesion molecules E-selectin (9),
intercellular adhesion molecule-1 (ICAM-1, Ref. 10), and vascular cell
adhesion molecule-1 (VCAM-1, Ref. 11), surface proteins that bind
leukocyte receptors and act at different steps of white blood cell
extravasation (1, 2). TNF, IL-1, and LPS also cause an increased
permeability to macromolecules in endothelial cell monolayers (12,
13).
, IL-1, and LPS on the
expression in human endothelial cells of the PAR-2 and the thrombin
receptor, and we found that the PAR-2, but not the thrombin receptor,
is strongly induced by all three inflammatory mediators. The time
course of induction points to a possible role for the PAR-2 in the
later phases of the acute inflammatory response.
(1.54 × 107 units/mg)
and IL-1 (2.38 × 108 units/mg) were purchased from
Genzyme. Forskolin and PMA were dissolved in ethanol and kept as stock
solutions at 
20 °C. Cytokines were thawed and aliquoted upon
receipt and then stored at 70 °C. LPS was dissolved in
phosphate-buffered saline and kept as a stock solution at 20 °C,
and IBMX was dissolved in culture medium immediately before use.
130 °C until use (up to a few months). All experiments
were done at passage 3.
-32P]dCTP from Amersham Corp.
effects, fresh
medium with cytokine was added daily. After the indicated periods of
time, total RNA was prepared (22) and then separated on denaturing
formaldehyde agarose gels. The total amount of RNA retrieved from one
well was loaded in each lane. The gels were blotted to hybridization
membranes (Hybond C Extra, Amersham) by capillary transfer in 20 × SSC
(1 × SSC is 150 mM NaCl, 15 mM sodium citrate,
pH 7.0). Filters were sequentially hybridized to PAR-2, GAPDH, and
thrombin receptor probes. Before hybridization with the next probe,
filters were stripped in 0.1% SDS at 90 °C. Hybridizations were
performed at 42 °C in 50% formamide, 5 × SSPE (1 × SSPE is 150 mM NaCl, 10 mM sodium phosphate, 1 mM EDTA, pH 7.0), 10 × Denhardt's solution, 1% SDS, 100 µg/ml herring sperm DNA. Final washings were in 0.1 × SSC, 0.1% SDS
at 50 °C. Blots were then exposed to a Fuji phosphoimaging screen,
and signals were quantitated using equipment and software from the same
manufacturer. For each sample, signals obtained with the PAR-2 and
thrombin receptor probes were normalized to the GAPDH signals. The
results are expressed as -fold increase over control. Statistical
significance of changes in expression level was tested by analysis of
variance (ANOVA).
1 µg/ml) in
phosphate-buffered saline with bovine serum albumin and added to the
cells (250 µl/well). Calculating that there are roughly
105 cells in each well, this gives an approximately
100-fold molar excess of antibody over receptor even at expression
levels as high as 105 receptors per cell. Cells were
incubated with antibodies for 4 h at +4 °C, washed five times
in ice-cold phosphate-buffered saline with bovine serum albumin, and
then lysed in 0.2 M NaOH. Bound radioactivity was
quantitated in a 
-counter. Background binding in the presence of a
100-fold excess of cold antibody was around 1200 cpm/well for the PAR-2
antibody and for the thrombin receptor antibody around 4000 cpm/well.
Columns in Fig. 7 represent bound radioactivity after subtraction of
this background binding. Statistical significance of differences
between groups was analyzed by Student's t test.
Fig. 7.
Surface binding of PAR-2 and thrombin
receptor antibodies after stimulation. HUVE cells were stimulated
with the indicated substances for 20 h, and then binding of
PAR-2 (A) and TR antibodies (B) was
determined. The concentrations of the stimulating agents are as
described under ``Results.'' Each column represents the mean ± S.D. of three determinations from a single experiment. Unspecific
binding, determined in the presence of a 100-fold excess cold antibody,
has been subtracted. The experiment was repeated once with similar
results. In A, antibody binding after treatment with
forskolin/IBMX, TNF, or IL-1 was significantly different from
that to untreated control cells. Likewise, cells treated with cytokine + IBMX bound significantly less antibody than cells treated with
cytokine alone. In B, cells treated with forskolin/IBMX or
PMA bound significantly less antibody than untreated control
cells. (t test, = 0.01, p < 0.01).
Fig. 1.
Effects of forskolin and PMA on the
expression of the PAR-2 and the thrombin receptor mRNAs. HUVE
cells were stimulated with forskolin/IBMX (10 µM/1
mM) or PMA (20 ng/ml) or left untreated, and PAR-2
(A) and thrombin receptor (B) mRNA levels
were determined at the indicated times after addition of drugs.
Receptor mRNA signals have been normalized to GAPDH signals, and
the results are expressed as -fold increase compared with mean signal
at time 0. Each column represents the mean ± S.D. of three to six
determinations from two separate experiments. Below the plots,
representative examples are shown of the Northern blot signals. The
identities of the different bands are indicated at left.
Messenger RNA levels for both the PAR-2 and the thrombin receptor
changed significantly over time after treatment with either
forskolin/IBMX or PMA (ANOVA, = 0.01, p < 0.01).
or
TNF (both 10 ng/ml) or LPS (200 ng/ml) had dramatically different
effects on the expression of the two receptors. PAR-2 was strongly
induced by all three substances (Fig. 2). At 20 h
the mRNA levels had increased to 6-10 times basal values. In
contrast, thrombin receptor expression remained largely unaffected by
these treatments. In consequence of this, the remaining Northern blot
studies were limited to the examination of changes in PAR-2 mRNA
levels.
Fig. 2.
Effects of cytokines and LPS on PAR-2 and
thrombin receptor mRNA levels. HUVE cells were stimulated with
TNF (10 ng/ml), IL-1 (10 ng/ml), or LPS (200 ng/ml), and PAR-2
(A) and thrombin receptor (B) mRNA levels
were determined at the indicated time points. Results were calculated
as described in the legend to Fig. 1. Each column represents the
mean ± S.D. of four determinations from two separate experiments.
Below the plots representative examples are shown of the Northern blot
signals. The identities of the different bands are given at
left. Messenger RNA levels changed significantly over time for the
PAR2 after treatment with either cytokine or LPS and for the thrombin
receptor after treatment with IL-1 (ANOVA, = 0.01, p < 0.01).
, TNF, or LPS. Cells were stimulated for
4 h, and then mRNA was extracted and analyzed. Responses to
both cytokines reached a plateau level around 0.1 ng/ml, whereas LPS
concentrations had to be considerably higher, about 0.1 µg/ml (Fig.
3).
Fig. 3.
Dose dependence of the PAR-2 mRNA
induction by cytokines and LPS. HUVE cells were stimulated for
4 h with the indicated concentrations of TNF, IL-1, or LPS,
and then the PAR-2 mRNA levels were determined. The PAR-2 signals
were normalized to the GAPDH signals, and the results are expressed as
-fold increase compared with unstimulated cells. Each column represents
the mean ± S.D. of two determinations from a single experiment.
Representative Northern blot hybridizations are shown below the
diagram. The identities of the RNA bands are given at
left.
(1 ng/ml), TNF (10 ng/ml), or LPS (200 ng/ml) on
PAR-2 expression. The results are displayed in Fig. 4.
IBMX (1 mM) appeared to block completely the increase in
PAR-2 mRNA at earlier time points. The block was partially overcome
at 20 h. It was not investigated whether this is because IBMX only
slows down the rate of induction or if the increase at the last time
point reflects degradation of the phosphodiesterase inhibitor.
Fig. 4.
Effect of IBMX on the induction of the PAR-2
mRNA by cytokines and LPS. HUVE cells were stimulated with
TNF (10 ng/ml), IL-1 (1 ng/ml), or LPS (200 ng/ml) in the absence
(filled columns) or presence (open columns) of 1 mM IBMX, and PAR-2 mRNA levels were determined at the
indicated time points. Results were calculated as described in the
legend to Fig. 1. Each column represents the mean ± S.D. of two
determinations from a single experiment.
. The
medium with cytokine was changed daily to ensure a continuing
stimulation. As shown in Fig. 5, mRNA levels
increased steadily to a peak of about 14 times control values at
48 h and remained elevated over the following 2 days.
Fig. 5.
Time course of the induction of the PAR-2
mRNA by TNF. HUVE cells were stimulated with TNF (10 ng/ml) for the indicated times, and then PAR-2 mRNA levels were
determined. Results were calculated as described in the legend to Fig.
1. Each column represents the mean ± S.D. of four determinations
from a single experiment. Representative Northern blot hybridizations
are shown below the diagram. The identities of the RNA bands are given
at left.
(10 ng/ml) or IL-1 (0.5 ng/ml) increased
binding of PAR-2 antibodies about 5-fold, with no appreciable change in
thrombin receptor antibody binding. Addition of IBMX (1 mM)
with the cytokines caused a diminished increase in PAR-2 antibody
binding. In a single experiment, the appearance of PAR-2 protein was
followed at earlier time points after stimulation with TNF. The time
course was similar to that seen at the mRNA level (data not
shown).
Fig. 6.
Antibody specificity. COS-7 cells were
transiently transfected with cDNAs encoding either the PAR-2
(COS7-PAR2) or the thrombin receptor (COS7-TR). After 36 h, cells
were stained with antibodies against either the PAR2 (S14.8.2,
hatched areas) or the thrombin receptor (61-1, open
areas) and analyzed by FACS.
and IL-1 act together to orchestrate the acute inflammatory
response, and they do so mainly by inducing or reducing transcription
of a large number of genes in responding cells. LPS is a bacterial
substance but has been shown to have many proinflammatory effects in
common with TNF and IL-1. All these three substances caused a
marked increase in expression of the PAR-2, and the effect occurred at
concentrations similar to those needed to induce other
cytokine-responsive genes (3, 8, 10). The same treatments had no effect
on thrombin receptor expression. When the phosphodiesterase inhibitor
IBMX was added in combination with cytokines or LPS, it appeared to
inhibit the induction of the PAR-2. Others have shown that TNF
increases the activity of cAMP-specific phosphodiesterase in
endothelial cells, an effect that could be blocked by IBMX treatment,
which also elevated cAMP levels (25). Cyclic AMP was not measured in
the present study, but the results fit the pattern that cAMP and
cytokine signaling have opposite effects on many inflammatory
parameters. For example, elevation of cAMP inhibits TNF-induced
expression of E-selectin and VCAM-1 (24), as well as TNF-induced
endothelial leakage (25). On a molecular level, it has been shown that
cAMP and TNF competitively regulate the E-selectin promoter
(26).
, IL-1, or LPS, the PAR-2 groups with the slow responders. In
cultured HUVE cells, IL-1 and LPS induce tissue factor activity with
peak expression after 4 h of stimulation (3, 4), which returns to
basal values within 24 h. Induction of E-selectin by IL-1 follows
a similar time-course (9). Induction of the ICAM-1 and VCAM-1 by TNF
or IL-1 is slower, and expression remains elevated for at least 48 h in the continued presence of cytokine (10, 11). Here, PAR-2
expression reached a plateau level by 24 h and remained elevated
for 72 h with TNF stimulation, a time course similar to that of
the ICAM-1. Also like the ICAM-1, the PAR-2 was expressed at a low
level already in unstimulated endothelium, both by Northern blotting
and antibody binding.
§
To whom correspondence should be addressed: Division of Molecular
Neurobiology, The Wallenberg Neurocenter, Lund University,
S
lvegatan 17, S-22362 Lund, Sweden. Tel.: 46 46 2220586; Fax: 46 46 2220568; E-mail sverker.nystedt{at}mphy.lu.se.
1
The abbreviations used are: TNF, tumor
necrosis factor ; FACS, fluorescence-activated cell sorting; HUVE,
human umbilical vein endothelial; IBMX, isobutylmethylxanthine; ICAM-1,
intercellular adhesion molecule-1; IL-1, interleukin-1; PAR-2,
proteinase-activated receptor 2; PMA, phorbol 12-myristate 13-acetate;
VCAM-1, vascular cell adhesion molecule-1; ANOVA, analysis of variance;
GAPDH, glyceraldehyde phosphodehydrogenase; LPS,
lipopolysaccharide.
2
S. Nystedt, V. Ramakrishnan, and J. Sundelin,
unpublished observations.
3
Anna-Karin Larsson et al., manuscript
in preparation.
4
C. Nilsson, S. Nystedt, and J. Sundelin,
unpublished results.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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R. Ramachandran, L. R. Sadofsky, Y. Xiao, A. Botham, M. Cowen, A. H. Morice, and S. J Compton Inflammatory mediators modulate thrombin and cathepsin-G signaling in human bronchial fibroblasts by inducing expression of proteinase-activated receptor-4 Am J Physiol Lung Cell Mol Physiol, March 1, 2007; 292(3): L788 - L798. [Abstract] [Full Text] [PDF]
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 Y. Kida, H. Inoue, T. Shimizu, and K. Kuwano Serratia marcescens Serralysin Induces Inflammatory Responses through Protease-Activated Receptor 2 Infect. Immun., January 1, 2007; 75(1): 164 - 174. [Abstract] [Full Text] [PDF]
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K. Hirano The Roles of Proteinase-Activated Receptors in the Vascular Physiology and Pathophysiology Arterioscler. Thromb. Vasc. Biol., January 1, 2007; 27(1): 27 - 36. [Abstract] [Full Text] [PDF]
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W. Ruf Flow perturbation is linked to endothelial par signaling. Arterioscler. Thromb. Vasc. Biol., May 1, 2006; 26(5): 962 - 964. [Full Text] [PDF]
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F. Syeda, J. Grosjean, R. A. Houliston, R. J. Keogh, T. D. Carter, E. Paleolog, and C. P. D. Wheeler-Jones Cyclooxygenase-2 Induction and Prostacyclin Release by Protease-activated Receptors in Endothelial Cells Require Cooperation between Mitogen-activated Protein Kinase and NF-{kappa}B Pathways J. Biol. Chem., April 28, 2006; 281(17): 11792 - 11804. [Abstract] [Full Text] [PDF]
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M. Holzhausen, L. C. Spolidorio, R. P. Ellen, M.-C. Jobin, M. Steinhoff, P. Andrade-Gordon, and N. Vergnolle Protease-Activated Receptor-2 Activation: A Major Role in the Pathogenesis of Porphyromonas gingivalis Infection Am. J. Pathol., April 1, 2006; 168(4): 1189 - 1199. [Abstract] [Full Text] [PDF]
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 J. H. Cleator, W. Q. Zhu, D. E. Vaughan, and H. E. Hamm Differential regulation of endothelial exocytosis of P-selectin and von Willebrand factor by protease-activated receptors and cAMP Blood, April 1, 2006; 107(7): 2736 - 2744. [Abstract] [Full Text] [PDF]
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 E. A. Lidington, R. Steinberg, A. R. Kinderlerer, R. C. Landis, M. Ohba, A. Samarel, D. O. Haskard, and J. C. Mason A role for proteinase-activated receptor 2 and PKC-{epsilon} in thrombin-mediated induction of decay-accelerating factor on human endothelial cells Am J Physiol Cell Physiol, December 1, 2005; 289(6): C1437 - C1447. [Abstract] [Full Text] [PDF]
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F. Roviezzo, M. Bucci, V. Brancaleone, A. Di Lorenzo, P. Geppetti, S. Farneti, L. Parente, G. Lungarella, S. Fiorucci, and G. Cirino Proteinase-Activated Receptor-2 Mediates Arterial Vasodilation in Diabetes Arterioscler. Thromb. Vasc. Biol., November 1, 2005; 25(11): 2349 - 2354. [Abstract] [Full Text] [PDF]
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 U. Johansson, C. Lawson, M. Dabare, D. Syndercombe-Court, A. C. Newland, G. L. Howells, and M. G. Macey Human peripheral blood monocytes express protease receptor-2 and respond to receptor activation by production of IL-6, IL-8, and IL-1{beta} J. Leukoc. Biol., October 1, 2005; 78(4): 967 - 975. [Abstract] [Full Text] [PDF]
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F. Noorbakhsh, N. Vergnolle, J. C. McArthur, C. Silva, M. Vodjgani, P. Andrade-Gordon, M. D. Hollenberg, and C. Power Proteinase-Activated Receptor-2 Induction by Neuroinflammation Prevents Neuronal Death during HIV Infection J. Immunol., June 1, 2005; 174(11): 7320 - 7329. [Abstract] [Full Text] [PDF]
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M. Riewald and W. Ruf Protease-activated Receptor-1 Signaling by Activated Protein C in Cytokine-perturbed Endothelial Cells Is Distinct from Thrombin Signaling J. Biol. Chem., May 20, 2005; 280(20): 19808 - 19814. [Abstract] [Full Text] [PDF]
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J. Ahamed, M. Belting, and W. Ruf Regulation of tissue factor-induced signaling by endogenous and recombinant tissue factor pathway inhibitor 1 Blood, March 15, 2005; 105(6): 2384 - 2391. [Abstract] [Full Text] [PDF]
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 M. Steinhoff, J. Buddenkotte, V. Shpacovitch, A. Rattenholl, C. Moormann, N. Vergnolle, T. A. Luger, and M. D. Hollenberg Proteinase-Activated Receptors: Transducers of Proteinase-Mediated Signaling in Inflammation and Immune Response Endocr. Rev., February 1, 2005; 26(1): 1 - 43. [Abstract] [Full Text] [PDF]
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 A. Masamune, K. Kikuta, M. Satoh, N. Suzuki, and T. Shimosegawa Protease-Activated Receptor-2-Mediated Proliferation and Collagen Production of Rat Pancreatic Stellate Cells J. Pharmacol. Exp. Ther., February 1, 2005; 312(2): 651 - 658. [Abstract] [Full Text] [PDF]
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G. Busch, I. Seitz, B. Steppich, S. Hess, R. Eckl, A. Schomig, and I. Ott Coagulation Factor Xa Stimulates Interleukin-8 Release in Endothelial Cells and Mononuclear Leukocytes: Implications in Acute Myocardial Infarction Arterioscler. Thromb. Vasc. Biol., February 1, 2005; 25(2): 461 - 466. [Abstract] [Full Text] [PDF]
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W. O. Chung, S. R. Hansen, D. Rao, and B. A. Dale Protease-Activated Receptor Signaling Increases Epithelial Antimicrobial Peptide Expression J. Immunol., October 15, 2004; 173(8): 5165 - 5170. [Abstract] [Full Text] [PDF]
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ABSTRACT