Protein kinase Cbeta 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

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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 alpha -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 PKCbeta isozymes (PKCbeta I and PKCbeta II) by transducing cells with antisense cDNA of PKCbeta 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 PKCbeta 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 PKCbeta isozymes, whereas the PKCbeta -activated pathway is important in signaling heterologous PAR-1 desensitization in endothelial cells.

endothelium; protein kinase C isozymes; 12-O-tetradecanoylphorbol-13-acetate

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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 PKCbeta -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.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Materials. 12-O-tetradecanoylphorbol-13-acetate (TPA), staurosporine, histamine, hydrocortisone, and fibronectin were purchased from Sigma Chemical (St. Louis, MO). Human alpha -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.

PKCbeta I plasmid construction and transduction of HMEC. The 2.3-kilobase Xho I DNA fragment containing the entire cDNA for rat PKCbeta I was isolated from plasmid MT-PKCbeta I (a gift from Dr. John Knopf, Genetics Institute, Andover, MA). The antisense PKCbeta I cDNA fragment (PKCbeta 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 PKCbeta I cDNA was driven by immediate-early promoter of human cytomegalovirus. The pLNCX vector and pLNC-PKCbeta 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 PKCbeta (Transduction Labs, Lexington, KY) in TBST with 1% nonfat dry milk overnight at 4°C (this MAb recognizes PKCbeta I and PKCbeta 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
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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 alpha -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 alpha -thrombin. A: desensitization of PAR-1 to second thrombin challenge; after 5 min of treatment with 10 nM alpha -thrombin, cells were again challenged with 10 nM alpha -thrombin. B: thrombin-induced desensitization was reversible within 3 h; fura 2-preloaded cells were treated with 10 nM alpha -thrombin for 5 min, washed and incubated with fresh medium for 3 h, then rechallenged with 10 nM alpha -thrombin. C: desensitization of PAR-1 to thrombin receptor activation peptide (TRAP); HMEC were challenged with 10 nM alpha -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 alpha -thrombin, then cells were stimulated with 10 µM histamine (His). Values represent data from experiments repeated 4-5 times.

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 alpha -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 alpha -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 alpha -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 alpha -thrombin. All experiments were repeated 4-5 times.

Role of PKCbeta in mediating PAR-1 desensitization. We transduced HMEC with pLNCX vector alone (mock-infected, HMEC-mock) or vector containing PKCbeta I-antisense cDNA (HMEC-AS), as described previously (25). Western blot analysis showed that the expression of PKCbeta 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 PKCalpha , 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 PKCbeta 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 PKCbeta and PKCalpha 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 PKCbeta and PKCalpha are ~87,000.

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.

                              
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Table 1.   Comparison of thrombin-induced response

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 PKCbeta . 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 PKCbeta isozymes contribute to the TPA-induced heterologous desensitization of PAR-1 in endothelial cells.


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Fig. 4.   Effect of inhibition of PKCbeta expression on thrombin- and TPA-induced desensitization of PAR-1. A: alpha -thrombin-induced increase in [Ca2+]i measured in HMEC-AS. B: HMEC-AS were stimulated with 10 nM alpha -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 alpha -thrombin. D: 10 nM alpha -thrombin-induced increase in [Ca2+]i in HMEC-mock and HMEC-AS. Cells were incubated with indicated test components, then challenged with alpha -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.

PKCbeta 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 alpha -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.

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 alpha -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 alpha -thrombin. C: HMEC treated with TPA. Experiment was repeated 4 times, and results were similar.

To study the function of PKCbeta 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 PKCbeta 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 alpha -thrombin (10 nM for 5 min) and TPA (500 nM for 30 min), respectively. E and F: HMEC-AS treated with alpha -thrombin (10 nM for 5 min) and TPA (500 nM for 30 min), respectively. Data are representative of 3 separate experiments.

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

G protein-coupled receptors are subject to homologous and heterologous desensitization, as previously described for beta -adrenergic receptors (9). cAMP-dependent protein kinase A is involved in induction of heterologous desensitization of beta -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., alpha 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 PKCbeta 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 PKCbeta I and PKCbeta II isozymes in mediating heterologous desensitization, because endothelial cells predominantly express the Ca2+-dependent PKCalpha , PKCbeta I, and PKCbeta II variants (7, 25, 30; Lum and Malik unpublished observations). Moreover, we showed recently that PKCbeta 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 PKCbeta I cDNA in HMEC to inhibit the production of PKCbeta isozymes. Inasmuch as PKCbeta I and PKCbeta II are alternatively spliced gene products, expression of either antisense inhibits the expression of both isozymes (26). Immunoblots using the antibody recognizing PKCbeta I and PKCbeta II showed that expression of PKCbeta protein was markedly reduced in HMEC-AS without affecting PKCalpha (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 PKCbeta activation is critical in signaling heterologous desensitization of PAR-1. It is not clear why inhibition PKCbeta 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 PKCalpha 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 PKCbeta persisting in HMEC-AS that could account for the partial inhibition of heterologous desensitization.

In summary, PKCbeta isozymes provide an important regulatory signal mediating heterologous desensitization of PAR-1 in endothelial cells. These studies suggest modulation of the PKCbeta -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.

    REFERENCES
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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AJP Cell Physiol 274(2):C387-C395
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