THROMBOSIS RESEARCH 59; 841-850,199O 0049-3848/90 $3.00 + .OO Printed in the USA. Copyright (c) 1990 Pergamon Press plc. All rights reserved.

MEMBRANE CYCLIC AMP INCREASES THROMBOMODULIN EXPRESSION ON SURFACE OF CULTURED HUMAN UMBILICAL VEIN ENDOTHELIAL CELLS Hidemi

Ishii,

Keiichiro and

Department of Sciences,

(Received

Clinical Teikyo

26.3.1990;

Kizaki, Hiroyuki Uchiyama, Mutsuyoshi Kazama

Faculty Biochemistry, University, Sagamiko, Kanagawa 199-01, Japan.

accepted

in revised form 4.7.1990

Shuichi

Horie

of Pharmaceutical Tsukui-gun,

by Editor H. Yamazaki)

ABSTRACT AMP (Bt,cAMP; final concentration lDibutyryl-cyclic 5 mfl) or beraprost sodium (synthetic prostacyclin, 100 enhanced the expression of thrombomodulin (TM; an nM) factor of endothelial cells) on the anticoagulant surface of cultured human umbilical vein membrane endothelial cells up to 1.4 times over the control 9 hrs after the treatment, while the within expression below the control level at 12 hrs and fell thereafter. 8-Bromo-CAMP (final concentration l-5 or mM) 3-isobutyl-1-methylxanthine (IBMX; an inhibitor of phosphodiesterase; final concentration 10-1000 _eM) enhanced the expression of TM on the cell surface at 12 hrs after the treatment. The enhancement of TM expression caused by BtcAMP was inhibited by incubation with phorbol 12-myris z ate 13-acetate. These results that suggest CAMP stimulates expression of TM in the endothelial cells.

INTRODUCTION Thrombin Key

words :

acts

as

a procoagulant

thrombomodulin, PMA , endothelial

CAMP, cells

841

factor beraprost

through

conversion sodium,

of IBMX,

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fibrinogen to fibrin and activation of Factors V, VIII and XIII, though it also acts as an anticoagulant factor through the activation of protein C (1,2). A key cofactor of protein C activation by thrombin is thrombomodulin (TM), which is located on the membrane surface of endothelial cells (1.3). The protein C activated by TM-thrombin complex inactivates Factors Va and VIIIa (1) and plasminogen activator inhibitor (4). Elevation of TM activity on the membrane surface is considered to promote the anticoagulation pathway of the blood coagulation cascade and depression of the activity favors the procoagulation pathway. Thus, blood coagulation at the cell surface is considered to be controlled by the change of TM expression. Exposure of endothelial cells to endotoxin (5) and tumor necrosis factor (TNF)(G) & vitro results in a sustained fall in the TM activity and antigen on the cell surface. Also an exposure of the cells to phorbol 12-myristate 13-acetate, which activates protein kinase the TM activity (7). These results suggested that C, decreases those treatments of the cells enhance degradation of the protein.. However, the regulatory mechanisms of TM expression on the cell surface have not been established yet. Cyclic AMP is an intracellular messenger produced in response to extracellular signals. The present study was undertaken to evaluate the effect of cyclic AMP on TM expression on the endothelial cell surface.

Materials: Reagents were purchased from Wako Pure Chemical Industries, Osaka, Japan, unless otherwise indicated. Dibutyryl 8-bromo-CAMP and IBMX were purchased from cyclic AMP (BtpcAMP), was purchased Sigma (St. Louis, MO, USA). Boc-Leu-Ser-Thr-Arg-MCA Thrombin purified from from Peptide Institute Inc., Osaka, Japan. human plasma was provided by Green Cross Co. Ltd., Osaka, Japan. Beraprost sodium (TRK-100) was donated by Toray Co. Ltd., Tokyo, anti-human TM IgGs and Purified human TM, mouse monoclonal Japan. the horseradish peroxidase (HRP)-labeled anti-TM IgGs were prepared according to the reported methods (8). Protein C was Suzuki et al. (9). isolated from human plasma according to Human umbilical vein endothelial cells were Cell culture: (10) and were grown to harvested by the method of Jaffe et al. confluence in Dulbecco’s minimum essential medium (D’MEM, Flow Scotland) supplemented with 10% heatLaboratories, Irvine, inactivated fetal calf serum (FCS, Hyclone Laboratories, Logan, 37’C 72 U/ml penicillin and 50 ug/ml streptomycin at UT), an atmosphere consisting of 95% air and 5% CO? (CO, incubaunder The cultured cells were identified as endothellal cells by tor). observation of typical monolayer phase-contrast microscopic growth and by the detection of von Willebrand factor produced in von Willebrand factor was determined by using a von the medium. Willebrand reagent (Behring Werke, Marburg, West Germany). After the cells were routinely subcultured by reaching confluence trypsinization with 0[05% trypsin solution.

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The confluent cells in the second Incubation procedure: 2.0 x lo4 subculture on a 96-well microplate (A/S Nunc, cells/well) were washed once with D’MEM and then incubated in required time in the CO, D’MEM containing test samples for the the cultured medium was incubator. After the incubation, collected and the monolayer cells were used for the assay of TM on the cell surface and the total antigen activity and antigen in the cells. Total TM antigen in endothelial cell monolayers was Assays: extracted with 200 ,el of 50 mM Tris-HCl containing 0.15 M NaCl, X-100 and 1 mM benzamidine hydrochloride, pH 7.5, 0.5% Triton for 30 min at 4-C. The TM antigen levels in the cell extract and the conditioned media were measured by enzyme immunoassay using monoclonal anti-TM IgGs (TMmAb 20, 2 and 11) as previously described (8). Purified placental TM was used as a standard. TM antigen level on the cell surface was measured by a modification of the previous method (8). Each culture well containing the confluent cells was washed with D’MEM and then 0.2 the HRP-labeled second antibodies (TMmAb 2 and 11; ml of 50ng/ml) in 10 mM phosphate, 0.1 M NaCl, 0.2% BSA and 0.5% sucrose, pH 7.0, (PBS/BSA/sucrose) was placed in each well. After incubation for 1 hr at 37’C in the CO 2 incubator, the plate was washed 4 times with PBS/BSA/sucrose. The HRP activity of the antibodies bound on the cell was measured according to the method for assay of total TM antigen in cells (8). Specific binding of the HRP-labeled antibodies on the cells was obtained by subtraction of the binding of the labeled antibodies measured in the presence of an excess of non-labeled antibodies (5 fig/ml) from the binding measured without the non-labeled antibodies. Results are given as relative expression (%) of TM on the cell surface compared with the untreated control. The- surface TM activity was measured by a modification of the previous method (8). The culture wells containing the confluent cells were washed once with 20 mM Tris-HCl containing 0.1 M NaCl and 5 mg/ml bovine serum albumin (PH 7.5, buffer A). Thrombin (final concentration, 2 NIH U/ml) and human protein C (final concentration, 50 fig/ml) in 0.1 ml of buffer A containing 1 mM CaClpwere added to each well. The cells were incubated for 90 min at 37’C in the CO incubator. A solution of antithrombinIII (final concentra t ion, 2 U/ml) and heparin (final concentration, 8 U/ml) in 0.1 ml of 50 mM Tris-HCl, 0.1 M NaCl and 1 mM CaCl, (pH 8.5, buffer B) was then added to block thrombin activity. Controls containing thrombin and protein C in the absence of cells were treated similarly. A mixture of 0.2 ml of conditioned medium and 0.25 ml of 50 /IM Boc-Leu-Ser-ThrArg-MCA in buffer B was incubated in a test tube for 10 min at 37°C. The reaction was terminated by adding 0.5 ml of 20% acetic acid. 7-Amino-4-methylcoumarin (AMC) liberated was then measured by using a spectrofluorophotometer with excitation at 380 nm and emission at 460 nm, using a standard curve constructed with standard AMC. RESULTS

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al

Incubation

time

(hrs)

Fig.1. Effects of dibutyryl CAMP on total TM antigen level in cultured human endothelial cells and on the TM antigen level and activity on the cell surface. T.he confluent endothelial cells (2 x104 cells) in each well were washed once with D’MEM and then incubated with several concentrations of dibutyryl CAMP in D’MEM for various times. At the indicated times, total TM antigen levels in the cells (A) and on the cell surface (B) and TM activity on the cell surface (C) were measured. 0 control; 0 1 mM BtcAMP; A 3 mM Bt,cAMP; ?? , 5 rnk Bt,cAMP . Each point ind?cates the mein + S.D. of 4 wells. Effect of Bt CT essio on endothelial ‘r he total TM antigen cultured human level in cells umbilical vein endothelial cells (Fig. 1.A) and the antigen level 1.B) and cofactor activity (Fig. 1 ,C) of TM on the cell (Fig. surface were elevated during 9 hrs after treatment with 1 to 5 The elevations of these parameters were dosemM Bt*cAMP. dependent with respect to Bt CAMP. At 6 hrs after the treatment total! TM antigen level in the cells and with 5 mM Bt CAMP, the 1 eve1 and the cofactor activity on the surface were the antigen 1.4 and 1.4 times the control levels, increased to 1.5, the levels of these parameters began to respectively. However, and were below the control values at 24 hrs decrease after 9 hrs 3 and 5 mM BtecAMP. The degree of after the treatment with depression of TM in the cells was also dose-dependent w,ith three parameters behaved in parallel respect to BtzcAMP. All with each other after the treatment. beraprost sodium, a synthetic prostacyclin, The influence of on the expression of TM antigen on the cell surface was compared The level on the cell surface with that of BtzcAMP (Fig. 2). that of the untreated control at 3 hrs increased to 1.2 times The antigen level, after treatment with 100 nM beraprost. returned to the control level at 9 hrs and was below the however, control level at 12 hrs after the treatment.

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r

150

0

12

6

Incubation

time

18

24

(hrs)

Changes in TM antigen level on the membrane surface of Fig.2. endothelial cells following treatment with beraprost sodium or The confluent endothelial cells (2 x104 cells) in dibutyryl-CAMP. incubated with 5 mM each well were washed with D’MEM and then ( 0 ) or 100 nM beraprost sodium ( A ) for various dibutyryl-CAMP times. At the indicated times, TM antigen levels on the cell , control. Each point indicates the surface were measured. 0 mean + S.D. of 4 wells. The behavior treatment.

was

closely

similar

to

that

in

the

case

of

Bt*cAMP

The TM Influence of 8-bromo-CAMP and IBMX on TM expression expression on the cell surface was elevated gradually until 12 hrs after the 8-bromo-CAMP treatment and then increased markedly. Treatment with 5 mM 8-bromo-CAMP increased the antigen level to 1.2 times the control level at 12 hrs and to 1.9 times the control at 24 hr (Fig. 3). The elevation was also dose-dependent. IBMX, a phosphodiesterase inhibitor, also enhanced the TM antigen level on the cell surface (Fig. 4). The enhancement of TM expression caused by IBMX was not observed within 9 hrs after the treatment, but the expression was markedly enhanced after 12 hrs in a dose-dependent manner at doses above 10 ,uM. The level was 1.8 times that of the control at 24 hrs after 1 mM IBMX treatment. Reduction of the TM antigen level to below the control level was not found within 24 hrs after treatment with either compound. Inhibition The behavior outcome

treatment

by PMA of the enhancement of TM expression bv Btw effects of phorbol 12-myristate 13-acetate (PMA) on the of the TM antigen level on the cell surface and on the of BtcAMP treatment were investigated (Fig. 5). A single with 0.5 or 1 ,eM PMA reduced the TM antigen level on

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200 f

I 0

I

I

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I

6

12

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24

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(hrs)

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(hrs 1

Fig.3. LeftElevation of TM antigen level on the membrane surface of cultured endothelial cells by treatment of 8-bromoCAMP. The confluent cells were washed once with D’MEM and then incubated with 0 mM ( 0 1, 1 mM ( 0 1, 3 mM ( A ) or 5 mM ( ?? ) 8-bromo-CAMP for various times. TM antigen level on the cell surface was measured at the indicated times. Each point indicates the mean + S.D. of 4 wells. Fig.4. RightElevation of TM antigen level on the membrane surface of cultured endothelial cells by treatment with IBMX. The incubated confluent cells were washed once with D’MEM and then with 0 IIM ( 0 1, 10 ,uM ( ?? 1, 100 ,KM ( A ) or 1000 .cM IBMX ( m 1 TM antigen level on the membrane surface was for various times. measured at the indicated times. Each point indicates the mean + S.D. of 4 wells. timeand dose-dependently. The reduction of the cell surface The elevation of the TM for 24 hrs. antigen level was maintained inhibited by level caused by 5 mM Bt,cAMP was antigen The increase of simultaneous incubation with 0.5 or 1 BM PMA. about 1.5 times over the control at 3 hrs after antigen level of 5 mM BtzcAMP treatment was suppressed to the control level by of TM antigen level observed at 24 hrs 1 ,uM PMA. The reduction after the BtcAMP treatment was slightly accelerated by the incubation wit i PMA. DISCUSSION We have demonstrated here that CAMP increases TM expression Two patterns of increase in on the membrane of endothelial cells. treatment with TM expression were found. Within 6 hrs after Bt2cAMP, beraprost (a synthetic prostacyclin which increases

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0

1

1

6

12

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1

time

847

I

24 18 (hrs)

Fig.5. Inhibiting effect of PMA on elevation of TM antigen level on the endothelial cell surface caused by Bt,cAMP treatment. The were washed once with D’MEM and then confluent cells incubated with 5 mM Bt,cAMP I 0 1 containing 0.5 ,uM ( A ) or 1.0 fiM ) or 1.0 ,oM ( ? ? ) PMA for 0.5 DLM ( A ( ?? 1 PMA or with various times. TM antigen level on the membrane surface was Each point indicates the mean of measured at the indicated times. 3 wells. or 8-bromo-CAMP, intracellular CAMP (11)) the TM levels were increased 1.4 times over the untreated control, and 8-bromo-CAMP or IBMX increased the level by more than 1.8 times over the control after 12 hrs. It has been reported that TM expression is regulated by the processes of transcription (121, translation (13) and degradation (6) of the protein and that the half life (t1/2) of TM is 20 hrs in mouse hemangioma cells (7). Based on the times required for the first increases in the TM level after the treatments with these agents, it is postulated that intracellular influences CAMP level translational and posttranslational regulations of the protein through shifting the TM mRNA from the untranslated pool into the polysomes and/or causes a depression of TM degradation. It has been reported that degradation of Fe-r receptor on the membrane of human monoblast cell line U937 is also depressed by Bt -CAMP treatment (14). 2 Furthermore, a decrease in the TM expression after TNF treatment is already apparent at 6 hrs after the treatment, and it has been suggested that the decrease is due to suppression of a translational or posttranslational mechanism in TM biosynthesis On the other hand, the marked increase in the expression (13). found at 12 hrs after the treatment with 8-bromo-CAMP or IBMX may be due to stimulation of TM biosynthesis accompanied with acceleration of transcription of the TM gene or decrease in the degradation of the corresponding mRNA.

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or beraprost may reflect degradation of intracellular CAMP by activation of CAMP phosphodiesterase. Bt -CAMP incorporated in the cells and CAMP produced in the cells +I y beraprost treatment are degraded by the phosphodiesterase, the activity of which in cells is enhanced by Bt2-CAMP treatment (15-1’7). 8-Bromo-CAMP incorporated in the cells is not degraded in the cells and CAMP produced in the cells is accumulated following IBMX treatment. These results suggest again that intracellular CAMP level is important for the regulation of TM expression of endothelial cells. Furthermore, no TM antigen was detected in the conditioned media in any of the present experiments. This finding indicates that the reduction of TM antigen on the cell surface observed at 12 hrs after the BtcAMP or beraprost treatment is not due to the release of TM into the medium. It was suggested that suppression of TM expression on the cell surface caused by PMA treatment was due to down-regulation through phosphorylation of TM by protein kinase C (7). Suppression of TM expression caused by PMA treatment was confirmed in the present experiment, and simultaneous treatment with PMA and BtzcAMP inhibited the elevation of TM expression observed in the case of treatment with Bt,cAMP alone. Thus, these data indicate that TM expression on the cell surface is controlled not only by CAMP level but also by phosphorylation of TM through protein kinase C. Enhancement of the TM expression on the endothelial cell surface might play a role in reducing thrombotic events through activation of protein C. Experimental manipulation to enhance net TM expression on the endothelial cell surface has not previously been achieved, but in the present paper, we have demonstrated that the expression of TM on endothelial cell membrane is finding may provide an important clue to enhanced by CAMP. This elucidate the regulation mechanism of TM expression.

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ESMON,C.T., and and suppresses endothelium in

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G.MOORE.K.L., ESMON,C.T., and ESMON,N.L. Tumor necrosis factor leads to the internalization and degradation of thrombomodulin from the surface of bovine aortic endothelial cells culture. Blood 73, 159-165, 1989, --_ --

in

7.DITTMAN,W.A., KUMADA,T., SADLER,J.E., and MAJERUS,P.W. The structure function of thrombomodulin: Phorbol and mouse myristate acetate stimulates degradation and synthesis of thrombomodulin hemangioma without affecting mRNA levels in cells. J. Biol. Chem . 233. 15815-15822, 1988. 8.ISHII.H.. NAKANO,M., TSUBOUCHI,J., UCHIYAMA,H., ISHIKAWA,T., HIRAISHI,S., TAHARA,C., MIYAJIMA.Y., and KAZAMA,M. Establishment of enzyme immunoassay of human thrombomodulin in plasma and urine using monoclonal antibodies. Thromb_._Haemost. 63, X57-162, 1990. S.SUZUKI,K., Inactivation protein C.

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HATTORI,M., and as suppressors of Biochem. Pharmac. _.-

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in fibroblasts by intracellular adenosin monophosphate. Ps. 459-462, 1972.

Natl.

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of cyclic U.S.A. .s_9*

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Cyclic AMP increases thrombomodulin expression on membrane surface of cultured human umbilical vein endothelial cells.

Dibutyryl-cyclic AMP (Bt2cAMP; final concentration 1-5 mM) or beraprost sodium (synthetic prostacyclin, 100 nM) enhanced the expression of thrombomodu...
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