Protein kinase C
regulates heterologous
desensitization of thrombin receptor (PAR-1) in endothelial
cells
Weihong
Yan,
Chinnaswamy
Tiruppathi,
Hazel
Lum,
Renli
Qiao, and
Asrar B.
Malik
Department of Pharmacology, College of Medicine, University of
Illinois, Chicago, Illinois 60612
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ABSTRACT |
We studied the effects of protein kinase C (PKC) activation on
endothelial cell surface expression and function of the proteolytically activated thrombin receptor 1 (PAR-1). Cell surface PAR-1 expression was assessed by immunofluorescence (using anti-PAR-1 monoclonal antibody), and receptor activation was assessed by measuring increases in cytosolic Ca2+ concentration in
human dermal microvascular endothelial cells (HMEC) exposed to
-thrombin or phorbol ester,
12-O-tetradecanoylphorbol-13-acetate (TPA).
Immunofluorescence showed that thrombin and TPA reduced the cell
surface expression of PAR-1. Prior exposure of HMEC to thrombin for 5 min desensitized the cells to thrombin, indicating homologous PAR-1
desensitization. In contrast, prior activation of PKC with TPA produced
desensitization to thrombin and histamine, indicating
heterologous PAR-1 desensitization. Treatment of cells with
staurosporine, a PKC inhibitor, fully prevented heterologous desensitization, whereas thrombin-induced homologous desensitization persisted. Depletion of PKC
isozymes
(PKC
I and
PKC
II) by transducing cells
with antisense cDNA of PKC
I
prevented the TPA-induced decrease in cell surface PAR-1 expression and
restored ~60% of the cytosolic Ca2+ signal in response to
thrombin. In contrast, depletion of PKC
isozymes did not affect the
loss of cell surface PAR-1 and induction of homologous PAR-1
desensitization by thrombin. Therefore, homologous PAR-1
desensitization by thrombin occurs independently of PKC
isozymes,
whereas the PKC
-activated pathway is important in signaling heterologous PAR-1 desensitization in endothelial cells.
endothelium; protein kinase C isozymes; 12-O-tetradecanoylphorbol-13-acetate
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INTRODUCTION |
THROMBIN ACTS on its receptor, proteolytically
activated thrombin receptor 1 (PAR-1) (4, 14, 34), on vascular
endothelial cells to stimulate production of prostacyclin (35),
increase the cytosolic Ca2+ signal
(11, 17, 20, 21, 27), and induce expression of cell surface adhesion
proteins, P-selectin and intercellular adhesion molecule-1 (ICAM-1)
(31). Thrombin activates PAR-1 by binding to the receptor and inducing
cleavage of PAR-1 at the extracellular
NH2 terminus between
Arg-41 and Ser-42 (34). The NH2 terminus functions as a
"tethered ligand," since synthetic peptides [e.g.,
SFLLRNPNDKYEPF or thrombin receptor activation peptide (TRAP)]
corresponding to the new NH2
terminus substitute for the tethered ligand (34).
Desensitization of PAR-1 is regulated by phosphorylation of PAR-1 (5).
Protein kinase C (PKC) activators (e.g., phorbol esters) inhibited
thrombin-mediated responses in human leukemic cells and platelets,
suggesting that desensitization of PAR-1 was the result of
PKC-dependent phosphorylation (5, 33, 38). Coexpression of G
protein-coupled receptor kinase 3 (GRK-3) with PAR-1 in
Xenopus oocytes also prevented
thrombin-mediated signals, indicating that GRK-3-induced PAR-1
phosphorylation could also mediate receptor desensitization (16).
Because of the possibility that PKC-induced phosphorylation of PAR-1
may be an important signal regulating desensitization, we investigated
the role of PKC in the endothelial cell surface expression and function
of PAR-1. The results showed that the PKC
-activated pathway is
critical in mediating the loss of cell surface PAR-1 and inhibiting
PAR-1-activated Ca2+ signaling and
contributes to the induction of heterologous desensitization of PAR-1.
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MATERIALS AND METHODS |
Materials.
12-O-tetradecanoylphorbol-13-acetate (TPA), staurosporine,
histamine, hydrocortisone, and fibronectin were purchased from Sigma
Chemical (St. Louis, MO). Human
-thrombin was a gift from Dr. J. Fenton II (New York State Department of Health, Albany, NY). MCDB-131
medium, trypsin-EDTA, and
L-glutamine were purchased from
GIBCO (Grand Island, NY), fetal bovine serum (FBS) from HyClone (Logan,
UT), epidermal growth factor from Collaborative Biomedical Products
(Bedford, MA), and rhodamine-labeled affinity-purified antibodies to
mouse immunoglobulin G (IgG) from Kirkegaard and Perry Laboratories
(Gaithersburg, MD). TRAP was synthesized as a COOH-terminal amide (32).
Cell culture.
Human microvascular endothelial cells (HMEC), a human dermal
microvascular endothelial cell line, were obtained from Dr. Edwin W. Ades (National Center for Infectious Diseases, Center for Disease Control, Atlanta, GA) (1). The cells were grown in endothelial basal
medium MCDB-131 supplemented with 10% FBS, 10 ng/ml epidermal growth
factor, 2 mM L-glutamine, and 1 µg/ml hydrocortisone. HMEC were used as target recipient cells for
transduction. PA317 cells, the amphotropic cell line derived from
NIH/3T3 cells (no. CRL-9078, American Type Culture Collection,
Rockville, MD), were used as packaging cells (22). PA317 cells were
cultured in Dulbecco's modified Eagle's medium supplemented with 4.5 g/l glucose and 10% FBS. PA317 and HMEC were cultured on tissue
culture dishes, passaged every 2-3 days, and maintained in an
incubator at 37°C in 5% CO2
atmosphere.
PKC
I plasmid
construction and transduction of HMEC.
The 2.3-kilobase Xho I DNA fragment
containing the entire cDNA for rat
PKC
I was isolated from plasmid
MT-PKC
I (a gift from Dr. John
Knopf, Genetics Institute, Andover, MA). The antisense PKC
I cDNA fragment
(PKC
I-AS) was inserted into the
cloning site of the retroviral plasmid vector pLNCX between two viral
long terminal repeats. The vector pLNCX, which has been described by Miller and Rosman (23), was obtained from Dr. A. D. Miller (Fred Hutchinson Cancer Research Center, Seattle, WA). A neomycin
phosphotransferase (neo) gene
contained in pLNCX confers vector resistance to the antibiotic G418 and
provides the basis for screening transfected cells. Transcription of
inserted antisense PKC
I cDNA
was driven by immediate-early promoter of human cytomegalovirus. The
pLNCX vector and pLNC-PKC
I-AS
were introduced into the PA317 packaging cells through
liposome-mediated transfection described in the manufacturer's
protocol (Life Technologies, Gaithersburg, MD). PA317 cells were
screened with 500 µg/ml G418 and grown to confluence (25). The
conditioned medium containing replication incompetent retroviral
particles was used to transduce HMEC. Conditioned medium was filtered
and mixed with HMEC growth medium (1:1). The mixture was used to
incubate HMEC, which were plated at 5 × 105 cells/60-mm dish 1 day before
the incubation. On the next 2 days, HMEC were washed with Hanks'
balanced salt solution and incubated with a 1:1 mixture of fresh
producer PA317 cell conditioned medium and regular HMEC growth medium.
HMEC were screened with 500 µg/ml G418. The surviving cells were
expanded and used for experiments.
Western blot analysis.
Control and transduced cells were cultured in 100-mm dishes. When cells
were confluent, they were washed twice with phosphate-buffered saline
(PBS), pH 7.4, containing 0.1 mM phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, and 1 µg/ml aprotinin. After they were washed, cells were scraped and centrifuged at 3,000 g for 10 min. The cell pellet was
suspended in PBS containing 0.1 mM phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, and 1 µg/ml aprotinin. Total cell proteins (60 µg) were separated on sodium dodecyl sulfate-polyacrylamide gel
electrophoresis and transferred to a nitrocellulose membrane. The
membrane was blocked with 5% nonfat dry milk in
tris(hydroxymethyl)aminomethane (Tris)-buffered saline with 0.05%
Tween 20 (TBST) for 3 h. After three rinses with TBST, the membrane was
incubated with monoclonal antibody (MAb) to PKC
(Transduction Labs,
Lexington, KY) in TBST with 1% nonfat dry milk overnight at 4°C
(this MAb recognizes PKC
I and
PKC
II isoforms). The membrane
was then washed three times with TBST and incubated with secondary
antibody (anti-mouse IgG conjugated with peroxidase; Amersham,
Arlington Heights, IL). Antibody-reacting protein bands were identified
using an enhanced chemiluminescence (ECL) kit (Amersham) following the
manufacturer's protocol.
Immunofluorescence.
MAb ATAP2 prepared against the PAR-1
NH2-terminal sequence
(SFLLRNPNDKYEPF) was a gift from Dr. Lawrence F. Brass (University of
Pennsylvania, Philadelphia, PA). This antibody recognizes cleaved and
intact PAR-1 (6, 15, 37). Cells were grown on glass coverslips coated
with fibronectin. After treatments, cells were fixed in 1%
paraformaldehyde in PBS, pH 7.4, for 15 min at room temperature. Cells
were blocked with 1% bovine serum albumin in PBS for 30 min at room
temperature. Cells were subsequently incubated for 1 h with MAb ATAP2
diluted 1:100 in PBS containing 0.2% bovine serum albumin. After three
washes with 50 mM Tris · HCl, pH 7.6, cells were
incubated with rhodamine-conjugated goat anti-mouse IgG diluted 1:40 in
50 mM Tris · HCl, pH 7.6, containing 5% nonfat dry
milk for 1 h at room temperature. In some experiments, cells were
permeabilized with 0.2% Nonidet P-40 for 30 min at room temperature after they were fixed. Coverslips were mounted in mounting solution, and immunofluorescence microscopy was performed using standard epifluorescence optics (19).
Cytosolic
Ca2+
determination.
The increase in cytosolic Ca2+
concentration
([Ca2+]i)
was measured in cells grown on 25-mm-diameter glass coverslips using
our method (20). After treatment, cells were loaded with 20 µM fura 2-acetyoxymethyl ester for 1 h at 25°C, washed twice in Hanks' balanced salt solution with 20 mM
N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, pH 7.4, and placed in a Sykes-Moore perfusion chamber, which was
set on a Nikon Diaphot microscope stage equipped for fura 2 fluorescence measurement. An optically isolated group of two to four
cells was excited with alternate wavelengths of 340 and 380 nm, and
emission was collected at 510 nm. Fluorescence intensity was measured
at 10 points/s. Background counted at the beginning of each experiment
was subtracted automatically during data collection. At the end of each
experiment, 10 µM ionomycin was added to obtain fluorescence of
Ca2+-saturated fura 2 and 0.1 M
EDTA was added to obtain fluorescence of free fura 2.
Statistics.
Statistical analysis was performed by two-tailed Student's
t-test. Significance was accepted at
P < 0.05.
 |
RESULTS |
Thrombin produces homologous desensitization of PAR-1.
Stimulating HMEC preloaded with fura 2 with 10 nM thrombin increased
[Ca2+]i.
After the initial thrombin exposure, cells failed to respond to a
second thrombin stimulation (Fig.
1A).
To study reversibility of desensitization of PAR-1, we first treated
cells with thrombin and tested the response to thrombin several hours
later. Homologous desensitization of PAR-1 was reversible after 3 h
(Fig. 1B). Thrombin pretreatment
also prevented the effect of TRAP in increasing
[Ca2+]i
(Fig. 1C). We also pretreated cells
with TRAP, then stimulated them with thrombin or TRAP. TRAP
pretreatment abolished the effect of a second stimulation by thrombin
or TRAP (data not shown). However, thrombin pretreatment did not
influence the histamine response, which showed the characteristic
increase in
[Ca2+]i
(Fig. 1D). Pretreatment of cells
with staurosporine, an inhibitor of PKC activation, did not prevent
homologous desensitization of PAR-1 (data not shown).

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Fig. 1.
Homologous desensitization of proteolytically activated thrombin
receptor 1 (PAR-1) by -thrombin. Human dermal microvascular
endothelial cells (HMEC) were loaded with 20 µM fura 2-acetoxymethyl
ester for 1 h at 25°C, and change in cytosolic
Ca2+ concentration
([Ca2+]i)
was measured in response to 10 nM -thrombin.
A: desensitization of PAR-1 to second
thrombin challenge; after 5 min of treatment with 10 nM -thrombin,
cells were again challenged with 10 nM -thrombin.
B: thrombin-induced desensitization
was reversible within 3 h; fura 2-preloaded cells were treated with 10 nM -thrombin for 5 min, washed and incubated with fresh medium for 3 h, then rechallenged with 10 nM -thrombin.
C: desensitization of PAR-1 to
thrombin receptor activation peptide (TRAP); HMEC were challenged with
10 nM -thrombin for 5 min, then challenged with 10 µM TRAP.
Inset: untreated HMEC response to 10 µM TRAP. D: thrombin-exposed cells
remain sensitive to histamine; HMEC were treated with 10 nM
-thrombin, then cells were stimulated with 10 µM histamine (His).
Values represent data from experiments repeated 4-5 times.
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Phorbol ester treatment prevents thrombin-induced activation of
PAR-1.
We incubated endothelial cells with different concentrations of TPA for
30 min and studied the thrombin-induced rise in
[Ca2+]i.
Effects of TPA on thrombin-induced PAR-1 activation were concentration dependent. After cells were treated with 100 nM TPA, 10 nM thrombin caused a rise in
[Ca2+]i
similar to that in control cells. However, the thrombin-induced rise in
[Ca2+]i
was inhibited by ~50% after 30 min of treatment with 250 nM TPA, and
the response was fully inhibited by treatment with 500 nM TPA (Fig.
2A). We
also treated endothelial cells with TPA for different time intervals
and studied the activation of PAR-1. TPA treatment caused a
time-dependent decrease in the thrombin-induced increases in
[Ca2+]i;
maximum inhibition was observed in cells treated with TPA for 30 min
(Fig. 2B). To study the
reversibility of the TPA effect, we preincubated HMEC with 500 nM TPA
for 30 min, washed the cells, and incubated them without TPA for up to
24 h. Thrombin did not elicit a response at 3 and 8 h after TPA
treatment, whereas the response to thrombin was restored by 24 h after
TPA treatment (Fig. 2C). TPA
treatment inhibited the increase in
[Ca2+]i
in response to thrombin as well as histamine (Fig.
2D), indicating that PKC activation
with TPA induced heterologous PAR-1 desensitization. Pretreatment of
cells with 100 nM staurosporine prevented the TPA-induced inhibition of
the
[Ca2+]i
signal (Fig. 2E).

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Fig. 2.
Effect of 12-O-tetradecanoylphorbol-13-acetate (TPA) on
thrombin-induced increase in
[Ca2+]i.
A: desensitization to thrombin was
dependent on TPA concentration; HMEC were pretreated with different
concentrations of TPA for 30 min, then challenged with 10 nM
-thrombin. B: desensitization to
thrombin after TPA exposure was time dependent; HMEC were preincubated
with 500 nM TPA for 15 or 30 min, then challenged with 10 nM
-thrombin. C: reversibility of
TPA-induced desensitization of PAR-1; HMEC were treated with 500 nM TPA
for 30 min, then washed and incubated with fresh medium for up to 24 h;
cells were stimulated with 10 nM -thrombin to measure rise in
[Ca2+]i
at these times. D: TPA exposure also
desensitized cells to histamine (His); HMEC were treated with 500 nM
TPA for 30 min, then stimulated with 10 µM histamine.
Inset: histamine response in cells not
treated with TPA. E: staurosporine
prevented desensitization of thrombin response; HMEC were preincubated
with 100 nM staurosporine for 10 min, then with 500 nM TPA for 30 min;
after these treatments, cells were stimulated with 10 nM -thrombin.
All experiments were repeated 4-5 times.
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Role of PKC
in mediating PAR-1 desensitization.
We transduced HMEC with pLNCX vector alone (mock-infected, HMEC-mock)
or vector containing
PKC
I-antisense cDNA (HMEC-AS), as described previously (25). Western blot analysis showed that the
expression of PKC
was significantly reduced in HMEC-AS compared with
HMEC (Fig. 3). We did not observe any
difference between control HMEC and HMEC-mock (data not shown). The
protein level of PKC
, another
Ca2+-dependent PKC isoform
expressed in endothelial cells, did not change in HMEC-AS (Fig. 3).

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Fig. 3.
Immunoblot of protein kinase C (PKC) isoforms in HMEC and HMEC
containing PKC I antisense DNA
(HMEC-AS). Lane 1, HMEC;
lane 2, HMEC-AS. Total cell proteins
(60 µg/lane) were separated on SDS-PAGE and transferred to
nitrocellulose membrane strips, and strips were blotted with PKC and
PKC monoclonal antibodies. Experimental details are described in
MATERIALS AND METHODS. Molecular
weights of proteins were determined using known molecular weight marker
proteins. Molecular weights of PKC and PKC are ~87,000.
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We studied thrombin-induced increases in
[Ca2+]i
in control HMEC, HMEC-mock, and HMEC-AS. We compared the fura 2 fluorescence ratio during the basal period, thrombin-induced increases
in peak, and decay time in these cells (Table
1). HMEC-mock did not differ significantly
from control HMEC. Although HMEC-AS showed small increases in fura 2 fluorescence and decay times compared with control HMEC, HMEC-AS did
not differ significantly from HMEC-mock (Table 1). Therefore, we used
HMEC-mock as controls for HMEC-AS.
Thrombin (10 nM) increased
[Ca2+]i
in HMEC-AS to levels similar to HMEC-mock (Fig.
4A, Table
1). After 5 min of treatment with 10 nM thrombin, 10 µM TRAP failed
to increase
[Ca2+]i
in HMEC-AS (Fig. 4B,) indicating that
thrombin-induced desensitization of PAR-1 was independent of PKC
.
TPA treatment caused ~90% inhibition of the thrombin-induced
increase in
[Ca2+]i
in HMEC-mock but not in HMEC-AS (Fig.
4C). Staurosporine pretreatment fully prevented this inhibition (Fig.
4D). TPA treatment of HMEC-AS restored ~60% of the inhibition of the thrombin-induced increase in
[Ca2+]i
(Fig. 4, C and
D), indicating that PKC
isozymes
contribute to the TPA-induced heterologous desensitization of PAR-1 in
endothelial cells.

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Fig. 4.
Effect of inhibition of PKC expression on thrombin- and TPA-induced
desensitization of PAR-1. A:
-thrombin-induced increase in
[Ca2+]i
measured in HMEC-AS. B: HMEC-AS were
stimulated with 10 nM -thrombin for 5 min, then challenged with 10 µM TRAP. C: mock-infected HMEC
(HMEC-mock) and HMEC-AS were incubated with 500 nM TPA for 30 min, then
challenged with 10 nM -thrombin. D:
10 nM -thrombin-induced increase in
[Ca2+]i
in HMEC-mock and HMEC-AS. Cells were incubated with indicated test
components, then challenged with -thrombin. Staurosporine (100 nM)
was incubated with cells for 10 min. Cells were treated with 500 nM TPA
for 30 min. y-Axis, difference (mean ± SE) between peak and basal fura 2 fluorescence ratios.
Significantly different from untreated HMEC-mock:
* P < 0.05;
** P < 0.001.
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PKC
regulates cell surface PAR-1 expression.
We carried out immunofluorescence staining using MAb against PAR-1 (see
MATERIALS AND METHODS) to study the
cell surface PAR-1 expression. In control HMEC, PAR-1 was uniformly
distributed on the cell surface (Fig.
5A).
After HMEC were treated with 10 nM thrombin for 5 min, surface
expression of PAR-1 was greatly decreased, as shown by decreased
staining of PAR-1 (Fig. 5B).
Staurosporine (100 nM) treatment itself had no effect on cell surface
PAR-1 expression (Fig. 5C).
Staurosporine pretreatment also did not prevent a thrombin-induced
decrease in surface expression of PAR-1 (Fig.
5D). Cell surface expression of
PAR-1 was restored within 3 h after thrombin treatment (Fig.
5E).

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Fig. 5.
Effects of -thrombin and TPA on cell surface expression of PAR-1 in
HMEC. Cells were treated with 500 nM TPA for 30 min or 10 nM thrombin
for 5 min, then fixed with 1% paraformaldehyde in PBS for 15 min.
Cells were washed and incubated with monoclonal antibody ATAP2 for 1 h.
After this incubation period, cells were washed and incubated with
rhodamine-conjugated goat anti-mouse IgG. Experimental details are
described in MATERIALS AND METHODS.
A: cell surface PAR-1 in HMEC control
without treatment. B: loss of cell
surface PAR-1 in thrombin-treated HMEC.
C: HMEC incubated with 100 nM
staurosporine for 10 min. D:
persistent loss of cell surface PAR-1 in HMEC incubated with 100 nM
staurosporine for 10 min, then treated with 10 nM thrombin.
E: return of cell surface PAR-1 in
cells treated with 10 nM thrombin, then washed and incubated in fresh
medium for 3 h. F: loss of cell
surface PAR-1 in HMEC treated with 500 nM TPA for 30 min.
G: persistent cell surface PAR-1 in
HMEC treated with 100 nM staurosporine for 10 min before TPA treatment.
H: return of cell surface PAR-1 after
TPA treatment in HMEC that were washed and incubated in fresh medium
for 24 h. Data are representative of 5 separate experiments. Bar, 15 µm.
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Cell surface expression of PAR-1 in HMEC decreased after treatment with
500 nM TPA for 30 min (Fig. 5F).
However, pretreating cells with 100 nM staurosporine for 10 min
prevented the TPA-induced loss of cell surface expression of PAR-1
(Fig. 5G). Cell surface PAR-1
expression was fully restored 24 h after TPA treatment (Fig. 5H).
Cells were permeabilized and stained with anti-PAR-1 MAb to study
internalization of PAR-1 induced by thrombin and TPA. In control
permeabilized cells, anti-PAR-1 MAb staining was uniform (Fig.
6A). In
cells treated with thrombin or TPA, intracellular fluorescence was
increased (Fig. 6, B and
C), suggesting internalization of
PAR-1.

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Fig. 6.
Thrombin or TPA treatment induced internalization of PAR-1 in HMEC.
Cells were exposed to 10 nM -thrombin for 5 min or 500 nM TPA for 30 min and then fixed. Fixed cells were permeabilized and stained with
monoclonal antibody ATAP2. Experimental details are described in
MATERIALS AND METHODS.
A: control HMEC.
B: HMEC exposed to -thrombin.
C: HMEC treated with TPA. Experiment
was repeated 4 times, and results were similar.
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To study the function of PKC
in decreasing PAR-1 expression on the
cell surface, we carried out studies using HMEC-mock and HMEC-AS. In
untreated HMEC-mock (Fig.
7A) and
HMEC-AS (Fig. 7B), expression of
PAR-1 on the cell surface was similar to control HMEC. Thrombin (10 nM)
treatment for 5 min decreased cell surface expression of PAR-1 in
HMEC-mock and HMEC-AS (Fig. 7, C and
D). However, treatment with 500 nM
TPA reduced the surface expression of PAR-1 in HMEC-mock (Fig.
7E), but it did not affect PAR-1
surface expression in HMEC-AS (Fig.
7F), indicating the importance of PKC
in regulating the loss of cell surface PAR-1 induced by TPA but
not by thrombin.

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Fig. 7.
Effect of thrombin and TPA on cell surface expression of PAR-1 in
HMEC-mock and HMEC-AS. Expression of PAR-1 on cell surface was detected
by immunofluorescence. Experimental details are described in Fig. 5
legend and MATERIALS AND METHODS.
Untreated HMEC-mock (A) and
untreated HMEC-AS (B) show cell
surface expression of PAR-1 similar to control HMEC (Fig.
5A).
C and
D: HMEC-mock treated with -thrombin
(10 nM for 5 min) and TPA (500 nM for 30 min), respectively.
E and
F: HMEC-AS treated with -thrombin
(10 nM for 5 min) and TPA (500 nM for 30 min), respectively. Data are
representative of 3 separate experiments.
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 |
DISCUSSION |
G protein-coupled receptors are subject to homologous and heterologous
desensitization, as previously described for
-adrenergic receptors
(9). cAMP-dependent protein kinase A is involved in induction of
heterologous desensitization of
-adrenergic receptors (8, 12, 18,
29). The activation of PKC has been proposed to mediate heterologous
receptor desensitization in the case of receptors coupled to
phospholipase C activation (e.g.,
1B-adrenergic, neurokinin-2,
and C5a receptors) (2, 3, 10). PKC activation may also be involved in
induction of heterologous desensitization of the thrombin receptor,
PAR-1 (5). Moreover, Mirza et al. (24) recently showed heterologous
desensitization of PAR-1 in human umbilical vein endothelial cells by
the activation of proteinase-activated receptor-2 (PAR-2). They showed
that stimulation of human umbilical vein endothelial cells with
PAR-2-activating peptide (SLIGRL) caused internalization of PAR-1 and
also attenuated the thrombin-mediated increase in
[Ca2+]i
(24). Furthermore, it has been proposed that PAR-1 internalization occurring secondary to PKC-induced phosphorylation of the receptor's COOH terminal regulates PAR-1 desensitization (37).
In the present study we investigated the function of PKC
isozymes
[which are key
Ca2+-activated PKC isoforms in
endothelial cells (7, 25, 30)] in the mediation of PAR-1
desensitization. Desensitization of PAR-1 was evaluated by measuring
changes in
[Ca2+]i
in response to thrombin. We observed that heterologous desensitization of PAR-1 induced by TPA was abolished by pretreatment of endothelial cells with the PKC inhibitor staurosporine, whereas this did not prevent homologous desensitization induced by thrombin. These data
suggest that heterologous desensitization is PKC dependent.
We compared cell surface expression of PAR-1 after thrombin or TPA
challenge to explain the observed differences in homologous and
heterologous desensitization of PAR-1. Immunofluorescence showed that
thrombin and TPA challenges decreased cell surface expression of PAR-1
(Fig. 5). The TPA-induced desensitization of PAR-1 was reversible,
inasmuch as resensitization to thrombin was evident within 24 h after
TPA treatment. This time course is consistent with the time required
for de novo synthesis of PAR-1 (5). Thrombin-induced desensitization of
PAR-1 was also reversible; however, the recovery period in this case
was 3 h. The relatively rapid recovery may be due to translocation of
the intracellular PAR-1 to the cell surface, which is capable of
restoring receptor function within this time (13). The time course of alterations in cell surface PAR-1 expression is consistent with kinetics of desensitization induced by TPA and thrombin. Inasmuch as
the TPA-induced loss of cell surface PAR-1 was abolished by staurosporine (unlike the thrombin-induced loss of cell surface PAR-1),
these results further suggest that heterologous PAR-1 desensitization
was secondary to PKC activation.
We studied the role of PKC
I and
PKC
II isozymes in mediating
heterologous desensitization, because endothelial cells predominantly express the Ca2+-dependent PKC
,
PKC
I, and
PKC
II variants (7, 25, 30; Lum
and Malik unpublished observations). Moreover, we showed recently that
PKC
I overexpression in HMEC
augments the TPA-induced increase in endothelial permeability (25),
suggesting the importance of this isoform in regulating endothelial
function. We used the strategy of introducing the antisense of
PKC
I cDNA in HMEC to inhibit
the production of PKC
isozymes. Inasmuch as
PKC
I and PKC
II are alternatively spliced
gene products, expression of either antisense inhibits the expression
of both isozymes (26). Immunoblots using the antibody recognizing
PKC
I and
PKC
II showed that expression of
PKC
protein was markedly reduced in HMEC-AS without affecting PKC
(Fig. 3). We observed that the thrombin-induced increase in
[Ca2+]i
in HMEC-AS was similar to control HMEC, and thrombin remained capable
of inducing homologous desensitization. This finding that thrombin-induced PAR-1 desensitization was PKC independent is consistent with the involvement of GRKs in mediating thrombin-induced homologous desensitization of PAR-1 (16, 28). In contrast, heterologous
desensitization induced by TPA was severely impaired in HMEC-AS (i.e.,
the
[Ca2+]i
signal in response to thrombin was restored to ~60% of its normal
value), indicating that PKC
activation is critical in signaling
heterologous desensitization of PAR-1. It is not clear why inhibition
PKC
production did not fully prevent heterologous desensitization of PAR-1, as was the case with
staurosporine. One possibility is that other PKC isozymes such as
PKC
can also contribute to the mechanism of heterologous
desensitization. Inasmuch as staurosporine prevents activation of these
isozymes, this may explain its effect in preventing the desensitization
response. Another possibility is that there may be a residual amount of PKC
persisting in HMEC-AS that could account for the partial inhibition of heterologous desensitization.
In summary, PKC
isozymes provide an important regulatory signal
mediating heterologous desensitization of PAR-1 in endothelial cells.
These studies suggest modulation of the PKC
-activated signaling
pathway as a means of regulating endothelial cell surface PAR-1
activity.
 |
ACKNOWLEDGEMENTS |
This work was supported by National Heart, Lung, and Blood
Institute Grants HL-27016 and HL-45638 and by the American Heart and
Lung Associations (Chicago).
 |
FOOTNOTES |
Address for reprint requests: C. Tiruppathi, Dept. of Pharmacology (M/C
868), University of Illinois, 835 S. Wolcott Ave., Chicago, IL
60612-7343.
Received 15 October 1996; accepted in final form 16 October 1997.
 |
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