JOURNAL OF CELLULAR PHYSIOLOGY 147:157-165 (1991)
Regulatory Mechanisms for Thrombomodulin Expression in Human Umbilical Vein Endothelial Cells In Vitro KAZUNORI HIROKAWA AND NOBUO AOKI* First Department of Medicine, Tokyo Medical arid Dental University, Yushima, Bunkyo-ku, Tokyo 113, japan It has been reported that thrombomodulin (TM) expression in endothelial cells is modulated by various agents. We investigated cellular regulatory mechanisms for TM expression in human umbilical vein endothelial cells (HUVECs), incubated with agents, by measuring the time course changes in surface TM activity, total T M antigen in cell lysates, and T M mRNA levels. While dibutyryl CAMP (3 mM) increased TM mRNA levels in HUVECs and was followed by increased TM activity, dibutyryl cGMP had no effect on TM activity. Phorbol myristate acetate (PMA) induced rapid loss of surface TM activity (-8 h) and later increased TM mRNA levels between 4 h and 40 h (maximum at 24 h), resulting in biphasic effects on T M activity. Tumor necrosis factor or interleukin-1 p suppressed surface TM activity and TM mRNA levels. lnternalizationidegradation of TM in HUVECs incubated with PMA or cytokines was suggested by co-culture with chloroquine. The decrease in surface TM activity observed was not caused by the release of TM molecules from the cells into the conditioned media. These results suggest that T M activity in HUVECs is modulated by independent mechanisms involving cytoplasmic TM mRNA levels and internalizationidegradation of TM molecules. These regulatory mechanisms may involve protein kinase A and protein kinase Cdependent mechanisms but are independent of protein kinase G .
Thrombomodulin (TM) is a membrane glycoprotein expressed in endothelial cells (Esmon et al., 19821, syncytiotrophoblasts in placenta (Maruyama et al., 19851,megakaryocytic lineage cells including platelets (Suzuki et al., 1988),and even in mouse embryonic cells (Imada et al., 1987, 1990). TM functions mainly as a modulator of coagulation after formation of the thrombin-TM complex. Thrombin, bound to TM, loses its procoagulant activity and is accelerated to activate protein C several thousandfold (Esmon et al., 1982; Esmon, 1989; Dittman and Majerus, 1990). Activated protein C is a protease and with its cofactor protein S acts as an anti-coagulant by cleaving the activated coagulation factors V (Walker et al., 1979, 1980) and VIII (Fulcher et al., 1984). Qualitative and/or quantitative deficiency of protein C (Griffin et al., 1981) or protein S (Comp et al., 1984)was reported to be a causal factor of thrombotic tendency. Activated protein C injected into baboons protects from lethal injections of Escherichia coZi (Taylor et al., 1987) and purified (Kumada et al., 1988) or recombinant (Gomi et al., 1990) TM participates in defense against thrombininduced thrombosis in mice. Thus, the TM-protein C system is believed to be a physiologically important one which modulates coagulation. Furthermore, TM activity on endothelial cells is reduced by endotoxin (Moore et al., 19871, tumor necrosis factor (TNF) (Nawroth and Stern, 19861, and interleukin-1P (IL-lp) (Nawroth et al., 1986). Such agents also induce expression of the coagulation cofactor tissue factor on endothelial cells. G 1991 WILEY-LISS. INC.
This conversion of endothelial cell properties from anticoagulant to procoa ulant may relate to a hypercoagulable state followe by thrombosis and/or disseminated intravascular coagulation in sepsis, chronic inflammation, and cancer. Although thrombin-induced internalization of TM in endothelial cells was initially reported as a scavenging mechanism for thrombin by the cells (Maruyama and Majerus, 1985,19871,internalization was also involved in down-regulation of TM by TNF and endotoxin (Moore et al., 1987, 1989). Up-regulation of TM in HUVECs results from incubation with analogs of CAMP, forskolin, or active phorbol esters (Hirokawa and Aoki, 1990), which can activate protein kinase C (Castagna et al., 1982). However, precise cellular regulatory mechanisms of TM expression are not yet fully disclosed and several reports are conflicting with regard to internalization of TM induced by thrombin
%
*To whom reprint requestsicorrespondence should be addressed. Received July 17, 1990; accepted January 18, 1991. Abbreviations used: AT 111, antithrombin 111; BSA, bovine serum albumin; 4 p-PMA (PMA), 4P-phorbol 12-myristate 13-acetate; dBcAMP, dibutyryl CAMP; dBcGMP, dibutyryl cGMP; Dil-AcLDL, 1, 1‘-dioctadecyl-1-3,3, 3’, 3’-tetramethyl-indo-carbocyanine percholate; ECGS, endothelial cell growth supplement; HUVECs, human umbilical vein endothelial cells; IL-lp, interleukin-lp; IMDM, Iscove’s modified Dulbecco’s medium; TBS, Tris-buffered saline; TM, thrombomodulin; TNF, tumor necrosis factor.
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HIROKAWA AND AOKl
(Beretz et al., 1989) and effects of TNF or phorbol Wako Pure Chemical (Osaka, Japan). All other chemmyristate acetate (PMA) on transcription of the TM icals were reagent grade products. gene (Conway and Rosenberg, 1988; Scarpati and Cell culture Sadler, 1989; Hirokawa and Aoki, 1990). In addition, Human umbilical vein endothelial cells (HUVECs) PMA effects on surface TM activity are biphasic, with an initial reduction in TM activity, which can be were harvested from umbilical cord veins within 12 h abrogated by an inhibitor of protein kinase C, followed after delivery according to the method of Jaffe et al. by a significant enhancement of surface TM activity (1973aj. The cells were grown to confluence in IMDM (Hirokawa and Aoki, 1990). However, cellular mecha- supplemented with 20% FCS, 100 Uiml penicillinnisms of this biphasic effect are not clarified. In this streptomycin, 30 pgiml ECGS, 10 Uiml porcine mustudy, we measured the changes with time of HUVECs cosal heparin (Thornton et al., 1983)in 60 mm diameter surface TM activity, total TM antigen in cell lysates, gelatin-coated plastic dishes (Corning-Iwaki Glass, ToTM mRNA levels, and TM antigen released into the kyo, Japan) at 37"C, 5% C02. Cells were passed using conditioned media after incubation with various agents. trypsin-EDTA and grown to confluence in 48-well Our results suggest that PMA and cytokine-induced tissue culture cluster plates (Costar, Cambridge, MA) down-regulation of TM involves internalization and that had been previously coated with gelatin. All degradation of TM, and the biphasic effects of PMA experiments were performed on cells within three result from a distinct time course effect on internaliza- passages. The endothelial cell monolayer was characterized by the typical cobblestone morphology, presence tion of TM and cytoplasmic TM mRNA levels. of von Willebrand factor and Factor VIII complex by MATERIALS AND METHODS indirect immunofluorescence (Jaffe et al., 1973a,b), Materials uptake of Dil-Ac-LDL (Voyta et al., 1984), and detec4P-phorbol 12-myristate 13-acetate (PMA), chloro- tion of Weibel-Palade bodies by electron microscopy quine, sodium dodecyl sulfate (SDS),ethylene diamine (Wagner et al., 1982). tetraacetic acid (EDTA), ortho-phenylene diamine Assay of surface TM activity (OPD), porcine mucosal heparin, human a-thrombin (4000 NIH Uimg protein), and bovine serum albumin Surface thrombomodulin cofactor activity was deter(BSA) were purchased from Sigma (St. Louis, MO). mined using HUVEC monolayers (1.0-1.5 x lo5 cells/ Recombinant human interleukin-lp (IL-1p) was pur- well) in 48-well tissue culture plates. The cells were chased from Cistron Technology (Pine Brook, NJ). incubated with or without agents for the indicated Recombinant human tumor necrosis factor (TNFj was times at 37"C, 5% COz. After incubation, the treated purchased from Genzyme (Boston, MA). Iscove's modi- cells were washed three times with buffer A (150 mM fied Dulbecco's medium (IMDM), fetal calf serum NaC1, 2.5 mM CaCl,, 5 m iml BSA, 20 mM Tris-HC1, (FCS), penicillin-streptomycin, and 0.05% trypsin- pH 7.4) and then incubate for 30 min at 37°C in 75 p1 0.02% EDTA in phosphate buffered saline (trypsin- of the same buffer with 3.3 Uiml human thrombin and EDTA) were purchased from GIBCO (Grand Island, 10 pgiml human protein C. Activated protein C generNY). Collagenase (CLS 2) was purchased from Wor- ated in 50 pl of the reaction mixture was determined by thington Biochemical (Freehold, NJ). Endothelial cell measuring the cleavage of the chromogenic substrate, growth supplement (ECGS) was purchased from Col- 0.3 mM S-2366, in the presence of 6 Uiml AT I11 and 15 laborative Research (Belford,MA). Acetylated low den- U/ml heparin (Sala et al., 1984). A Vmax Kinetic sity lipoprotein labeled with l,l'-dioctadecyl-l-3,3,3',3'-Microplate Reader interfaced t o the Softmax software tetramethyl-indo-carbocyanine percholate (Dil-Ac-LDL) (Molecular Devices Corp., Palo Alto, CA) was used to was purchased from Biomedical Technologies (Stough- quantitate cleavage of S-2366 at 405 nm. ton, MA). Human protein C was purchased from AmerEndothelial cells in each well were counted by the ican Diagnostica (New York, NY). Human antithrom- method described by Drysdale et al. (1983). Briefly, the bin I11 (AT 111) was purchased from Green Cross cells were stained with 200 pliwell crystal violet (0.2% (Osaka, Japan). The chromogenic substrate, S-2366, in 2% ethanol) for 10 min at 37°C. Multiwell plates was purchased from Kabi Vitrum (Stockholm,Sweden). were rinsed with tap water and dried. Three hundred Dibutyryl CAMP (dBcAMP) was a gift from Daiichi microliters of 1% SDS was added to each well to Pharmaceutical (Tokyo, Japan). The nick-translation solubilize the stained cells. One hundred microliters of kit was purchased from Boehringer Mannheim (Ger- solubilized lysate from each well was transferred to many). The total RNA isolation kit was purchased from microtiter lates (Nunc, Denmark) and the absorbance Invitrogen (San Diego, CA). [(-u-~'PIdCTP (6,000 Cii of each we 1 was read at 550 nm. Cell numbers were mmol; 1 Ci = 37GBq) was purchased from New Re- calculated from a standard curve (OD 550 vs cell search Products (Boston, MA). The human TM gene number). Surface TM activity per lo5 cells was exwas isolated from a human leukocyte genomic library pressed as the percentage of the control kS.D. of four using a synthetic oligonucleotide. A 2.1 kb XhoI-NcoI determinations. Data were analyzed by the Student's fragment containing the coding region was isolated and t-test. subcloned into a pUC119 vector. This nucleotide seELISA for TM antigen in cell lysates quence determined by the dideoxy chain termination method was identical with that reported by Shirai et al. At the indicated times, medium was removed from (1988). A TthlllI-NheI fragment in the coding region the 48-well culture plates and quadruplicate cell monowas used as a hybridization probe. Complementary layers were extracted with 200 ~ 1 0 . 5 % Triton X-100 in DNA (cDNA) for human P-actin was purchased from phosphate buffered saline (PBS), pH 7.4. After 30 min,
f
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REGULATORY MECHANISMS FOR THROMBOMODULIN EXPRESSION IN HUVECS
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the cell monolayers were suspended with pasteur pi*** pettes. Cell lysates were stored at -20°C until assayed. Cell numbers were counted in another matched 48-well PMA ** I culture plate that had been equally treated with agents. Cell supernatants from the experimental wells r were also stored for measurement. Cell lysates were .+galso prepared from confluent HUVECs grown in 150 O 100cm2 tissue culture flasks. Protein in cell lysates was E measured by the method reported by Lowry et al. .-c (19511, using BSA as a standard. .-> Total TM antigen in cell lysates were measured by 9c 5 0 ELISA using anti-human TM monoclonal antibodies, u *** KA-2 and KA-4, whose characteristics were reported .-aC c previously (Kimura et al., 1989). Microtiter plates e coated with KA-2 at 60 pgiml in 0.05 M carbonate n O L , buffer were used for ELISA assays. After washing the ’ --- / 0 1 2 4 8 24 coated plates three times with Tris-buffered saline Incubation Time (h) (TBS) containing 0.05% Tween-20, aliquots of cell lysates diluted with TBS-Tween containing 5 mM Fig. 1. Time course of surface TM activity in HUVECs incubated CaClz (TBS-Tween-Ca2+)and 0.1% BSA were added to with dBcAMF’ or PMA. HUVECs grown in 48-well plates were the coated wells and incubated overnight at 37°C. The incubated with or without the indicated agents at 37°C in 5% CO,. At wells were then washed three times with the same the indicated times, surface TM activity was measured as described in buffer and 200 p1 of horseradish peroxidase-conjugated Materials and Methods. HUVECs were incubated with 3 mM dBcAMP ( 0 ) or M PMA (a). Surface TM activity per lo5 cells is expressed KA-4 were added to the wells and incubated for 4 h at as the percentage of the control ?S.D. of four determinations. Aster37°C. Horseradish peroxidase was conjugated to KA-4 isks represent significant differences from the unstimulated control according to the method of Nakane and Kawaoi (1974). cells (*P < 0.05, **P < 0.01, ***P < 0.001). After washing, 0.01% H,Oz and ortho-phenylendiamine (0.4 mgiml in 0.075 M citrate-sodium phosphate buffer, pH 5.0) were added to each well and incubated for 60 min at 37°C. The reaction was stopped by addition of 50 p1 4N H2S04to each well. The absorbance at 490 nm diographs, TM mRNA levels were normalized to the was measured using a Vmax Kinetic Microplate concentration of p-actin mRNA. Reader interfaced to Softmax software. Human placenRESULTS tal TM was used as a standard after isolation and The time course of surface TM cofactor activity purification by the previously described method in HUVECs incubated with dBcAMP or PMA (Kimura et al., 1989). As shown in Figure 1, PMA (lop8 M) effects were biphasic, with reduced TM activity occurring between Northern blotting 1-8 h, followed by a significant enhancement at 24 h Total cytoplasmic RNA was prepared from confluent (152 2 9.3%); 3 mM dBcAMP significantly enhanced HUVECs grown in 150 cmz tissue culture flasks using surface TM activity between 4 h (116 2 3.5%) and 8 h a total RNA isolation kit. Isolated RNA was heat- (124 ? 7.8%), followed by a slight reduction at 24 h. denatured and fractionated on formaldehyde-containing These results suggest that up-regulation of TM expresagarose gels. The gels were then stained with 1.0 pgiml sion involves protein kinase C or protein kinase A ethidium bromide for 30 min and washed with distilled dependent mechanisms consistent with previous obserwater four times for 30 min each. The RNA was then vations (Hirokawa and Aoki, 1990). transferred to a HyBond-N filter (Amersham InternaEffects of cyclic nucleotides on surface TM tional, Amersham, UK) by standard capillary blotting activity in HUVECs techniques in transfer buffer (Maniatis et al., 1982). The dose-response effects of cyclic nucleotide on TM The RNA was cross-linked to the nylon membrane by UV illumination. The probe employed for hybridization activity after 6 h incubation are shown in Figure 2. was a 32P-labelled450 bp DNA fragment corresponding Although dBcAMP enhanced surface TM activity sigto the human TM coding region, nick-translated to a nificantly between 3 and 10 mM, dBcGMP had no effect specific activity of > lo8 dpmipg. Nick-translation was up to 10 mM. These results suggest that CAMP-depenperformed using a nick-translation kit and [cx-~’PI- dent and cGMP-independent mechanisms are involved dCTP (6000 Ci/mmol). Prehybridizations and hybrid- in up-regulation of TM in HUVECs and eliminate the izations were performed at 42°C and washes were possibility that liberated free butyrate (Kooistra et al., performed at 60°C. Autoradiograms of Northern blots 1987) may affect surface TM activity. were prepared using Kodak X-OMAT AR film with an Effects of cytokines on the expression of intensifying screen, Lightning plus GK (Cronex, Duendothelial cell TM mRNA pont J a an), and exposed at -70°C. After washing with distilleIp water at 65°C for 30 min, the filter was dried The time-course effects of cytokines on the expression and rehybridized with 32P-labeled cDNA probe for of endothelial cell TM mRNA are shown in Figure 3. human p-actin. After densitometric analysis of autora- Northern blotting of TM mRNA demonstrated that 10
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HIROKAWA AND AOKI
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Fig. 2. Effects of cyclic nucleotides on surface TM activity in HUVECs. HUVECs grown in 48-well plates were incubated with or without 3 mM dBcAMP ( 0 ) or 3 mM dBcGMP (0)for 6 h at.37"C in a 5% GO, atmosphere. After incubation, surface TM activity was measured as described in Materials and Methods. Asterisks represent significant differences from the control (***P< 0.001).
U/ml TNF and 10 Uiml IL-1p induced a similar timedependent decrease in TM mRNA in HUVECs. These results agree with previous reports (Nawroth and Stern, 1986; Nawroth et al., 1986; Moore et al., 1989; Conway and Rosenberg, 1989; Hirokawa and Aoki, 1990). Effects of PMA and dBcAMP on the expression of endothelial cell TM mRNA As shown in F i e r e 4, lo-' M PMA and 3 mM dBcAMP irm~asedTM mRNA in HUVECs; lo-' M PMA increased TM mRNA in HUVECs between 4-40 h, with maximal effect at 24 h. The maximal increase induced by PMA varied from 300 to 1500%of control in several independent experiments (300, 5007 1100, 1500% of control), 3 mM dBcAMP also increased TM mRNA in HUVECs between 2-4 h with maximal effect at 2 h- In several independent experiments, BcAMP increased TM mRNA in HUVECs between 2-8 h and maximal effect was observed 150 to 1200% of control (150, 170, 1200 of control). Time course of surface TM activity and total TM antigen in HUVECs incubated with agents Although cytokines (10 Uiml IL-lp or TNF) decreased surface TM activity significantly between 4-48 h, they did not significantly decrease total TM antigen in cell lysates at 4 h, as shown in Figure 5. Several additional experiments confirmed this phenomena. PMA (lops M) decreased surface TM activity but not always total TM antigen in HUVECs after 4 h incubation (Hirokawa and Aoki, 1990). It was notable that PMA (lo-' M) increased total TM antigen in cell lysates at 24 h before surface TM activity increased at 48 h (Fig. 5C). Taken together, these results demonstrated that down-regulation of TM occurred initially
on the cell surface followed by loss of antigen in cell lysates and up-regulation occurred in reverse order. Analysis of conditioned media from these experiments for TM antigen by ELISA failed to show any detectable TM antigen. Effects of chloroquine on surface TM activity and total TM antigen in cell lysates The above observations raised questions concerning the fate of the down-regulated TM. Results shown in Figure 5 suggested that surface TM might be internalized and degraded intracellularly as reported previously. Hence, we investigated the effects of chloroquine, which inhibits lysozomal functions by increasing intralysosomal pH (Ohkuma and Poole, 19781, on surface TM activity and total TM antigen in HUVECs incubated with or without agents for 4 h. Two hundred micromole chloroquine did not affect surface TM activity in HUVECs incubated with or without agents, but increased total TM antigen in cell h a t e s as shown in Figure 6. These results i l s o demonstrate that incubation of HUVECs with PMA ( l o p 7M). TNF (10 Uiml), or IL-lp (10 U/ml) could decrease surface TM activity, but not total TM antigen in cell lysates. Thus, inhibition of lysosomal function by chloroquine resulted in decreased surface TM activity and increased total TM antigen in HUVECs incubated with PMA, TNF, or IL-lp. These results mean that such agents may induce internalization and degradation of TM in HUVECs. ~ e t ~ ~ e m eofn total t s TM antigen released into the COnditiOned media from HUVECs with agents Since analysis of conditioned media from HUVECs grown in 48-we11 plate by to show any detectable TM antigen, media from HUVECs grown in a 150 cm2 flask were analyzed. HUVECs were incubated with or without agents in 10 ml culture media for the indicated times. After incubation, the conditioned media and cell lysates were measured for TM antigen by ELISA as described in Materials and Methods. Compared with total TM antigen in cell lysates, only a small quantity of TM antigen was detected in the conditioned media as shown in Table 1. PMA (10-8 M, 24 h) or dBcAMp (3 mM, 6 h) increased total TM antigen in cell lysates up to 265% and 202%, respectively, whereas IL-1p (10 U/ml, 24 h) decreased TM antigen levels to 24% of control after normalization of TM antigen levels to protein content. A short incubation with PMA M, 6 h) did not affect total TM antigen levels significantly as observed previously (Scarpati and Sadler, 1989; Hirokawa and Aoki, 1990). In contrast to the drastic decrease in total TM antigen in cell lysates, TM antigen in the conditioned media from HUVECs incubated with IL-1p did not vary as much. These results suggested that down-regulated TM molecules are not released from the cell surface directly into conditioned media in a form detectable by our monoclonal antibodies. DISCUSSION TM expression in endothelial cells is regulated by various agents: endotoxin (Moore et al., 1987) and cytokines (Nawroth and Stern, 1986; Nawroth et al.,
REGULATORY MECHANISMS FOR THROMBOMODULIN EXPRESSION IN HUVECS
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Fig. 3. Effects of cytokines on the expression of endothelial cell TM mRNA. HUVECs were incubated with 10 Uiml TNF (upper panel)or 10 Uiml IL-lP (lower panel) for the indicated times. (A) Total RNA was isolated, electrophoresed, and transferred to a Hybond-N filter as described in Materials and Methods. The filter was hybridized with a
32P-labeled DNA probe. The positions of the ribosomal RNA size markers are indicated a t the left margin. (B) Autoradiographs were analyzed by densitometry, and values for TM were normalized to human p-actin. Normalized data is expressed as the percentage of TM mRNA relative to unstimulated control cells.
1987; Conway and Rosenberg, 1988) down-regulate TM; forskolin and CAMPanalogs up-regulate TM (Hirokawa and Aoki, 1990); active phorbol esters have biphasic effects such as down-regulation by short incubation (1-8 h) and up-regulation by long incubation
h) (Dittman et al., 1988; Scarpati and Sadler, 1989; Hirokawa and Aoki, 1990). As for cyclic nucleotides, up to 10 mM dBcGMP had no effect on TM expression by 6 h incubation while dBcAMP up-regulated it as shown in Figure 2. Inter(> 24
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HIROKAWA AND AOKI
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Fig. 4. Effects of PMA and dBcAMP on the expression of endothelial cell TM mRNA. HUVECs were incubated with M PMA (upper panel) or 3 mM dBcAMP (lower panel) for the indicated times. Northern blotting (A) and analysis of autoradiography by densitometry (B) were performed as described in Figure 3.
estingly, Mackie et al. (1986) reported that bovine endothelial cells contained a CAMP-dependent protein kinase (protein kinase A), three calcium-calmodulindependent protein kinases, protein kinase C, and tyrosine kinase, but no cGMP-dependent protein kinase (protein kinase G). They also found that the cells
contained numerous substrates for protein kinase A and protein kinase C. These results are consistent with the conclusion that TM expression is regulated by both protein kinase A and protein kinase C in human endothelial cells (Hirokawa and Aoki, 1990) and that dBcGMP had little effect on surface TM activity.
163
REGULATORY MECHANISMS FOR THROMBOMODULIN EXPRESSION IN HUVECS
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Fig. 5. Time course of surface TM activity and total TM antigen in HUVECs incubated with agents. HUVECs grown in 48-well plates were incubated with or without the indicated agents (A: IL-lP 10 Ulml, B: TNF 10 Ulml, C: PMA 10 *MI for indicated times. After incubation, surface TM activity was measured as described in Materials and Methods. In separate matched 48-well plates that had been equally treated with agents, medium was removed and quadruplicate
cell monolayers were extracted with 200 (*I 0.5% Triton X-100 in PBS. Total TM antigen in cell lysates were measured by ELISA methods a s described in Materials and Methods. Surface TM activity (upper panel) and TM antigen per lo5 cells (lower panel) are expressed as the percentage of the control z S I . of four determinations. Asterisks represent significant differences from the unstimulated control cells c*P < 0.05, **P i0.01, ***P < 0,001).
Active phorbol esters had biphasic effects on TM activity as described before, and both effects were related to the activation of protein kinase C (Hirokawa and Aoki, 1990). In addition, enhancement of TM activity by PMA was preceded by increased TM antigen in cell lysates (Fig. 5) resulting from increased TM mRNA levels (Fig. 4). This may reflect increased TM molecules in the Golgi-endoplasmiccomplex by de novo synthesis. TM activity in HLVECs incubated with PMA, thus, seems to vary as the result of two different mechanisms. Internalization and degradation of TM may be induced and is followed by increased TM expression due to PMA-induced up-regulation of TM mRNA. However, it may be possible that enhancement of TM activity by prolonged incubation with PMA results from down-regulation of protein kinase C, after activation of the enzyme, in cell membranes as reported by Pirotton et al. (1990) in bovine aortic endothelial cells. The time-course effect of cytokines on TM activity and total TM antigen in HUVECs as shown in Figure 5 suggested that down-regulation of TM has two serial events, since total TM antigen did not significantly decrease after a 4 h incubation with cytokines, whereas TM mRNA levels were remarkably decreased after 4 h incubation (Fig. 3). Effects of the cytokines may consist of internalizationidegradation of TM and decreasing TM mRNA levels. This decreasing effect on TM mRNA levels are different from the PMA effects and presum-
ably reflect mechanisms independent of protein kinase C activation. Internalization and degradation of TM by cytokines may be attributable in part to the activation of protein kinase C; however, a decreasing mechanism for cytoplasmic TM mRNA levels by cytokines should be further investigated. Results demonstrating that chloroquine increased total TM antigen in cell lysates of HUVECs incubated with PMA or cytokines (Fig. 6) suggested that inhibition of TM activity involved internalization and degradation of TM as reported by Moore et al. (1989). Internalization and degradation of TM was strongly suggested, since IL-10 decreased TM antigen in HUVECs and did not generate detectable TM antigen in the conditioned media (Table 1). Cellular regulatory mechanisms for TM expression in HUVECs incubated with agents was demonstrated to involve changes in TM mRNA levels (Figs. 3, 4). Dittman et al. (1988) reported that PMA had no effect on TM mRNA levels in mouse hemangioma cells and Scarpati and Sadler (1989) also reported that PMA or TNF had no effect on TM mRNA in HUVECs, although TNF decreased surface TM activity in HUVECs. Conway and Rosenberg (1988)reported that TNF decreased TM activity exclusively resulting from inhibition of transcription of TM gene, which is consistent with our results. Differing results among these reports may come from differences in experimental conditions, spe-
164
HIROKAWA AND AOKI
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Fig. 6. Effects of chloroquine on surface TM activity and total TM antigen in cell lysates. HUVECs grown in two 48-well plates were equally incubated with or without the indicated agents for 4 h. After incubation, surface TM activity (A) or total TM antigen in cell lysates (B) was measured in each plate as described in Materials and Methods and the legend to Figure 5. Asterisks represent significant differences from unstimulated control cells or between two groups (*P< 0.05, **P< 0.01, ***P< 0.001).
TABLE 1. Measurements of TM antigen released into the conditioned media from HUVECs incubated with agents'
Treatment Control PMA lo-* M 24 h PMA M6h dBcAMP 3 mM 6 h I L l S 10 U/ml24 h
Conditioned media TM antigen (ng/lO ml media)
Cell lysates TM antigen ( n g h g protein)
3.3 (100) 2.1 (64) 2.5 (76) 1.4 (42) 4.5 (1361
33.7 (100) 89.1 (265) 38.8 (115) 67.9 (202) 8.1 124)
'HUVECS grown in 150 cm' flasks were incubated with or without the indicated agents for indicated times. After incubation, media was removed and stored until assay at -20°C. Then HUVECs monolayers were extracted with 10 mlO.5%Triton X-100 in PBS. TAI antigen levels in conditioned media and cell lysates were measured by ELISA methods as described in Materials and Methods. Protein content of cell lysates was also measured by t h e h w r y method. Data expressed are the mean of duplicate determinations and the percentage of control levels is shown in parenthesis.
cies, normal cells and transformed cells, and number of passages. In addition, we demonstrated that IL-lP and TNF decreased TM mRNA levels similarly and that PMA or dBcAMP increased TM mRNA (Fig. 4). In conclusion, TM activity in HUVECs is modulated by independent mechanisms involving TM gene transcription and presumably intemalizatioddegradation of TM molecules. Each mechanism is stimulated and/or inhibited in HUVECs incubated with dBcAMP, PMA, IL-lp, and TNF. In addition, such regulatory mecha-
nisms involve protein kinase A- and protein kinase C-dependent mechanisms, but they are independent of protein kinase G. ACKNOWLEDGMENTS We thank Dr. S. Ohkawa and the nursery staff of Ohkawa Hospital (Matsudo) for collecting umbilical cords and placentae, Ms. M. Tanaka for her skillful technical assistance, Dr. N. Tanaka for performin the electron microscopy for Weibel-Palade bodies, an Mr. Y. Kita for his valuable suggestions on Northern blotting. We also gratefully thank Dr. D. J. Steams for discussions and critical reading of the manuscript. LITERATURE CITED
f
Beretz, A,, Freyssinet, J., Gauchy, J., Schmitt, D.A., Klein-Soyer, C., Edgell, C.S., and Cazenave, J. (1989) Stability of thrombinthrombomodulin complex on the surface of endothelial cells from human saphenus vein or from the cell line EA.hy 926. Biochem. J., 259:3540.
Castagna, M., Takai, Y., Kaibuchi, K., Sano, K., Kikkawa, U., and Nishizuka, Y. (1982) Direct activation of calcium-activated, phospholipid-dependent protein kinase by tumor-promoting phorbol esters. J. Biol. Chem., 257:7847-7851. Comp, P.C., Nixon, R.R., Cooper, M.R., and Esmon, C.T. (1984) Familial protein S deficiency is associated with recurrent thrombosis. J. Clin. Invest., 74.2082-2088. Conway, E.M., and Rosenberg, R.D. (1988) Tumor necrosis factor suppresses transcription of the thrombomodulin gene in endothelial cells. Mol. Cell. Biol., 8:5588-5592. Dittman, W.A., Kumada, T., Sadler, J.E., and Majerus, P.W. (1988)
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The structure and function of mouse thrombomodulin. Phorbol Maruyama, I., and Majerus, P.W. (1985) The turnover of thrombinmyristate acetate stimulates degradation and synthesis of thromthrombomodulin complex in cultured human umbilical vein endobomodulin without affecting mRNA levels in hemangioma cells. J . thelial cells and A549 lung cancer cells. Endocytosis and degradaBiol. Chem., 263:15815-15822, tion of thrombin. J. Biol. Chem., 260r15432-15438. Dittman, W.A., and Majerus, P.W. (1990) Structure and function of Maruyama, I. and Majerus, P.W. 11987)Protein C inhibits endocytosis thrombomodulin: A natural anticoagulant. Blood, 75:329336. of thrombin-thrombomodulin complexes in A549 lung cancer cells Drysdale, B-E., Zacharchuk, C.M., and Shin, H.S. (1983) Mechanism and human umbilical vein endothelial cells. Blood, 69:1481-1484. of macrophage-mediated cytotoxicity: Production of a soluble cyto- Moore, K.L., Andreoli, S.P., Esmon, N.L., Esmon, C.T., and Bang, toxic factor. J. Immunol., 131:2362-2367. N.U. (1987) Endotoxin enhances tissue factor and SuDoressesthromEsmon, N.L., Owen, W.G., and Esmon, C.T. (1982) Isolation of a bomodulin expression of human vascular endothelt6m in vitro. J. membrane-bound cofactor for thrombomodulin-catalyzed activation Clin. Invest., 79:124-130. of protein C. J. Biol. Chem., 257:859-864. Moore, K.L., Esmon. C.T.. and Esmon. N.L. (1989) Tumor necrosis Esmon, C.T. (1989) The role of protein C and thrombomodulin in the factor leads to the internalization and degradation of thrombomodregulation of blood coagulation. J. Biol. Chem., 264:47434746. ulin from the surface of bovine aortic endothelial cells in culture. Fulcher, C.A., Gardiner, J.E., Griffin, J.H., and Zimmerman, T.S. Blood, 73:159-165. (1984) Proteolytic inactivation of human factor VIII procoagulant Nakane, P.K., and Kawaoi, A. (1974) Peroxidase-labeled antibody. A orotein bv activated human orotein C and its analoev with factor V. new method of conjugation. J. Histochem. Cytochem., 22;10841091 Blood, 63":48&489. Gomi, K., Zushi, M., Honda, G., Kawahara, S., Matsuzaki, O., N&iih, P.P., and Stern, D.M. (1986) Modulation of endothelial cell hemostatic properties by tumor necrosis factor. J. Exp. Med., Kanabayashi, T., Yamamoto, S., Maruyama, I., and Suzuki, K. (1990) Antithrombotic effect of recombinant human thrombomodu163:740-745. Nawroth, P.P., Handley, D.A., Esmon, C.T., and Stern, D.M. (1986) lin on thrombin-induced thromboembolism in mice. Blood, 75:139& Interleukin 1induces endothelial cell Drocoadant while sumress1399. ing cell-surface anticoagulant activity. Pr"oc. Natl. Acad: Sci., Griffin, J.H., Evatt, B., Zimmerman, T.S., Kleiss, A.J., and Wideman, C. (1981) Deficiency of protein C in congenital thrombotic disease. U.S.A., 83:3460-3464. Ohkuma, S. and Poole. B. (1978) Fluorescence probe measurement of J. Clin. Invest., 68:1370-1373. the intralvsosomal DH in living cells and the Derturbation of DH bv Hirokawa, K. and Aoki, N. (1990) Up-regulation of thrombomodulin various agents. Prdc. Natl. Acid. Sc. U.S.A., -75:3327-3331. in human umbilical vein endothelial cells in vitro. J. Biochem., Pirotton, S., Robaye, B., Langneau, C., and Boeynaems, J-M. (1990) 108:839-845. Adenine nucleotides modulate phosphatidylcholine metabolism in Imada, M., Imada, S., Iwasaki, H., Kume, A,, Yamaguchi, H., and aortic endothelial cells. J. Cell. Physiol., 142:449457. Moore, E.E. (1987) Fetomodulin: Marker surface protein of fetal develoDment which is modulatable by cyclic AMP. Dev. Biol., Sala, N., Owen, W.G., and Collen, D. (1984) A functional assay of protein C in human plasma. Blood, 63:671-675. 122:483491. Imada, S., Yamaguchi, H., Nagumo, M., Katayanagi, S., Iwasaki, H. Scarpati, E.M. and Sadler, J.E. (1989) Regulation of endothelial cell coagulant properties. Modulation of tissue factor, plasminogen and Imada. M. (1990) Identification of fetomodulin, a surface activator inhibitors, and thrombomodulin by phorbol 12-myristate marker protein of fetal development, as thrombomodulin by gene 13-acetate and tumor necrosis factor. J. Biol. Chem., 264:20705cloning and functional assays. Dev. Biol., 14Or113-122. 20713. Jaffe. E.A.. Nachman. R.L., Becker, C.G., and Minick, C.R. (1973a) Culture of Shirai, T., Shiojiri, S., Ito, H., Yamamoto, S., Kusumoto, H., Deyash...~ .~~ ~- human endothelial cells derived from umbilical veins. iki, Y., Maruyama, I., and Suzuki, K. (1988) Gene structure of Identification~-bymorphologic and immunologic criteria. J. Clin. human thrombomodulin, a cofactor for thrombin-catalyzed activaInvest., 523745-2756. Jaffe, E.A., Hoyer, L.W., and Nachman, R.L. (197313) Synthesis of tion of protein C. J . Biochem., 103:281-285. antihemophilic factor antigen by cultured human endothelial cells. Suzuki, K., Nishioka, J., Hayashi, T., and Kosaka, Y. (1988) Functionally active thrombomodulin is present in human platelets. J. J. Clin. Invest., 522757-2764. Kimura. S.. Nagova. T.. and Aoki. N. (1989) Monoclonal antibodies to Biochem., 104:628-632. human thro~b'omcdulinwhose binding is calcium dependent. J. Taylor, F.B. Jr., Chang, A., Esmon, C.T., DAngelo, A,, ViganoBiochem., 105:47W83. DAngelo. S., and Blick, K.E. (1987) Protein C prevents the coaguKooistra, T., Van den Berg, J., Tons, A,, Platenburg, G., Rijken, D.C., lopathic and lethal effects of Escherichia coli infusion in the baboon. and Van den Berg, E. (1987) Butyrate stimulates tissue-type J. Clin. Invest., 79:91%925. plasminogen-activator synthesis in cultured human endothelial Thornton, S.C.. Mueller, S.N., and Levine, E.M. 11983) Human cells. Biochem. J. 247:605612. endothelial cells: Use of heparin in cloning and long-term serial Kumada. T.. Dittman. W.A., and Maierus. P.W. (1987) A role for cultivation. Science (Wash. DC), 222:623-625. thromhornndulin in the Dkhoeenesfs of thrombin-induced throm- Voyta, J.C., Via, D.P., Butterfield, C.E., and Zetter, B.R. (1984) ._.. . . ... boembolism in mice. Blobd, 71y728-833. Identification and isolation of endothelial cells based on their Lowry, O.H., Rosebrough, N.J., Farr, A.L., and Randall, R.J. (19511 increased uptake of acetylated-low densit.y lipoprotein. J . Cell Biol., Protein measurement with the folin phenol reagent. - J . Biol. Chem., 992034-2040. 193:265-275. Mackie, K., Lai, Y., Nairn, A.C., Greengard, P., Pitt, B.R., and Lazo, Wagner, D.D., Olmsted, J.B., and Marder, V.J. (1982) Immunolocalization of von Willebrand protein in Weibel-Palade bodies of human J.S. (1986) Protein phosphorylation in cultured endothelial cells. J. endothelial cells. J. Cell Biol., 95:355-360. Cell. Physiol., 128:367374. Maniatis, T.; Fritsch, E.F., and Sambrook, J. (1982) Molecular clon- Walker, F.J., Sexton, P.W., and Esmon, C.T. (1979) The inhibition of blood coagulation by activated protein C through the selective ing: A laboratory manual. Cold Spring Harbor Laboratory, Cold inactivation of activated factor V. Biochim. Biophys. Acta., Spring Harbor, NY. 571:333-342. Maruyama, I., Bell, C.E. and Majerus, P.W. (1985) Thrombomodulin is found on endothelium of arteries, veins, capillaries, and lymphat- Walker, F.J. (1980) Regulation of activated protein C by a new protein. A possible function for protein S.J . Biol. Chem., 2555521ics, and on syncytiotrophoblast of human placenta. J. Cell Biol., 101:363-371. 5524. "d
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Regulatory Mechanisms for Thrombomodulin Expression in Human Umbilical Vein Endothelial Cells In Vitro KAZUNORI HIROKAWA AND NOBUO AOKI* First Department of Medicine, Tokyo Medical arid Dental University, Yushima, Bunkyo-ku, Tokyo 113, japan It has been reported that thrombomodulin (TM) expression in endothelial cells is modulated by various agents. We investigated cellular regulatory mechanisms for TM expression in human umbilical vein endothelial cells (HUVECs), incubated with agents, by measuring the time course changes in surface TM activity, total T M antigen in cell lysates, and T M mRNA levels. While dibutyryl CAMP (3 mM) increased TM mRNA levels in HUVECs and was followed by increased TM activity, dibutyryl cGMP had no effect on TM activity. Phorbol myristate acetate (PMA) induced rapid loss of surface TM activity (-8 h) and later increased TM mRNA levels between 4 h and 40 h (maximum at 24 h), resulting in biphasic effects on T M activity. Tumor necrosis factor or interleukin-1 p suppressed surface TM activity and TM mRNA levels. lnternalizationidegradation of TM in HUVECs incubated with PMA or cytokines was suggested by co-culture with chloroquine. The decrease in surface TM activity observed was not caused by the release of TM molecules from the cells into the conditioned media. These results suggest that T M activity in HUVECs is modulated by independent mechanisms involving cytoplasmic TM mRNA levels and internalizationidegradation of TM molecules. These regulatory mechanisms may involve protein kinase A and protein kinase Cdependent mechanisms but are independent of protein kinase G .
Thrombomodulin (TM) is a membrane glycoprotein expressed in endothelial cells (Esmon et al., 19821, syncytiotrophoblasts in placenta (Maruyama et al., 19851,megakaryocytic lineage cells including platelets (Suzuki et al., 1988),and even in mouse embryonic cells (Imada et al., 1987, 1990). TM functions mainly as a modulator of coagulation after formation of the thrombin-TM complex. Thrombin, bound to TM, loses its procoagulant activity and is accelerated to activate protein C several thousandfold (Esmon et al., 1982; Esmon, 1989; Dittman and Majerus, 1990). Activated protein C is a protease and with its cofactor protein S acts as an anti-coagulant by cleaving the activated coagulation factors V (Walker et al., 1979, 1980) and VIII (Fulcher et al., 1984). Qualitative and/or quantitative deficiency of protein C (Griffin et al., 1981) or protein S (Comp et al., 1984)was reported to be a causal factor of thrombotic tendency. Activated protein C injected into baboons protects from lethal injections of Escherichia coZi (Taylor et al., 1987) and purified (Kumada et al., 1988) or recombinant (Gomi et al., 1990) TM participates in defense against thrombininduced thrombosis in mice. Thus, the TM-protein C system is believed to be a physiologically important one which modulates coagulation. Furthermore, TM activity on endothelial cells is reduced by endotoxin (Moore et al., 19871, tumor necrosis factor (TNF) (Nawroth and Stern, 19861, and interleukin-1P (IL-lp) (Nawroth et al., 1986). Such agents also induce expression of the coagulation cofactor tissue factor on endothelial cells. G 1991 WILEY-LISS. INC.
This conversion of endothelial cell properties from anticoagulant to procoa ulant may relate to a hypercoagulable state followe by thrombosis and/or disseminated intravascular coagulation in sepsis, chronic inflammation, and cancer. Although thrombin-induced internalization of TM in endothelial cells was initially reported as a scavenging mechanism for thrombin by the cells (Maruyama and Majerus, 1985,19871,internalization was also involved in down-regulation of TM by TNF and endotoxin (Moore et al., 1987, 1989). Up-regulation of TM in HUVECs results from incubation with analogs of CAMP, forskolin, or active phorbol esters (Hirokawa and Aoki, 1990), which can activate protein kinase C (Castagna et al., 1982). However, precise cellular regulatory mechanisms of TM expression are not yet fully disclosed and several reports are conflicting with regard to internalization of TM induced by thrombin
%
*To whom reprint requestsicorrespondence should be addressed. Received July 17, 1990; accepted January 18, 1991. Abbreviations used: AT 111, antithrombin 111; BSA, bovine serum albumin; 4 p-PMA (PMA), 4P-phorbol 12-myristate 13-acetate; dBcAMP, dibutyryl CAMP; dBcGMP, dibutyryl cGMP; Dil-AcLDL, 1, 1‘-dioctadecyl-1-3,3, 3’, 3’-tetramethyl-indo-carbocyanine percholate; ECGS, endothelial cell growth supplement; HUVECs, human umbilical vein endothelial cells; IL-lp, interleukin-lp; IMDM, Iscove’s modified Dulbecco’s medium; TBS, Tris-buffered saline; TM, thrombomodulin; TNF, tumor necrosis factor.
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HIROKAWA AND AOKl
(Beretz et al., 1989) and effects of TNF or phorbol Wako Pure Chemical (Osaka, Japan). All other chemmyristate acetate (PMA) on transcription of the TM icals were reagent grade products. gene (Conway and Rosenberg, 1988; Scarpati and Cell culture Sadler, 1989; Hirokawa and Aoki, 1990). In addition, Human umbilical vein endothelial cells (HUVECs) PMA effects on surface TM activity are biphasic, with an initial reduction in TM activity, which can be were harvested from umbilical cord veins within 12 h abrogated by an inhibitor of protein kinase C, followed after delivery according to the method of Jaffe et al. by a significant enhancement of surface TM activity (1973aj. The cells were grown to confluence in IMDM (Hirokawa and Aoki, 1990). However, cellular mecha- supplemented with 20% FCS, 100 Uiml penicillinnisms of this biphasic effect are not clarified. In this streptomycin, 30 pgiml ECGS, 10 Uiml porcine mustudy, we measured the changes with time of HUVECs cosal heparin (Thornton et al., 1983)in 60 mm diameter surface TM activity, total TM antigen in cell lysates, gelatin-coated plastic dishes (Corning-Iwaki Glass, ToTM mRNA levels, and TM antigen released into the kyo, Japan) at 37"C, 5% C02. Cells were passed using conditioned media after incubation with various agents. trypsin-EDTA and grown to confluence in 48-well Our results suggest that PMA and cytokine-induced tissue culture cluster plates (Costar, Cambridge, MA) down-regulation of TM involves internalization and that had been previously coated with gelatin. All degradation of TM, and the biphasic effects of PMA experiments were performed on cells within three result from a distinct time course effect on internaliza- passages. The endothelial cell monolayer was characterized by the typical cobblestone morphology, presence tion of TM and cytoplasmic TM mRNA levels. of von Willebrand factor and Factor VIII complex by MATERIALS AND METHODS indirect immunofluorescence (Jaffe et al., 1973a,b), Materials uptake of Dil-Ac-LDL (Voyta et al., 1984), and detec4P-phorbol 12-myristate 13-acetate (PMA), chloro- tion of Weibel-Palade bodies by electron microscopy quine, sodium dodecyl sulfate (SDS),ethylene diamine (Wagner et al., 1982). tetraacetic acid (EDTA), ortho-phenylene diamine Assay of surface TM activity (OPD), porcine mucosal heparin, human a-thrombin (4000 NIH Uimg protein), and bovine serum albumin Surface thrombomodulin cofactor activity was deter(BSA) were purchased from Sigma (St. Louis, MO). mined using HUVEC monolayers (1.0-1.5 x lo5 cells/ Recombinant human interleukin-lp (IL-1p) was pur- well) in 48-well tissue culture plates. The cells were chased from Cistron Technology (Pine Brook, NJ). incubated with or without agents for the indicated Recombinant human tumor necrosis factor (TNFj was times at 37"C, 5% COz. After incubation, the treated purchased from Genzyme (Boston, MA). Iscove's modi- cells were washed three times with buffer A (150 mM fied Dulbecco's medium (IMDM), fetal calf serum NaC1, 2.5 mM CaCl,, 5 m iml BSA, 20 mM Tris-HC1, (FCS), penicillin-streptomycin, and 0.05% trypsin- pH 7.4) and then incubate for 30 min at 37°C in 75 p1 0.02% EDTA in phosphate buffered saline (trypsin- of the same buffer with 3.3 Uiml human thrombin and EDTA) were purchased from GIBCO (Grand Island, 10 pgiml human protein C. Activated protein C generNY). Collagenase (CLS 2) was purchased from Wor- ated in 50 pl of the reaction mixture was determined by thington Biochemical (Freehold, NJ). Endothelial cell measuring the cleavage of the chromogenic substrate, growth supplement (ECGS) was purchased from Col- 0.3 mM S-2366, in the presence of 6 Uiml AT I11 and 15 laborative Research (Belford,MA). Acetylated low den- U/ml heparin (Sala et al., 1984). A Vmax Kinetic sity lipoprotein labeled with l,l'-dioctadecyl-l-3,3,3',3'-Microplate Reader interfaced t o the Softmax software tetramethyl-indo-carbocyanine percholate (Dil-Ac-LDL) (Molecular Devices Corp., Palo Alto, CA) was used to was purchased from Biomedical Technologies (Stough- quantitate cleavage of S-2366 at 405 nm. ton, MA). Human protein C was purchased from AmerEndothelial cells in each well were counted by the ican Diagnostica (New York, NY). Human antithrom- method described by Drysdale et al. (1983). Briefly, the bin I11 (AT 111) was purchased from Green Cross cells were stained with 200 pliwell crystal violet (0.2% (Osaka, Japan). The chromogenic substrate, S-2366, in 2% ethanol) for 10 min at 37°C. Multiwell plates was purchased from Kabi Vitrum (Stockholm,Sweden). were rinsed with tap water and dried. Three hundred Dibutyryl CAMP (dBcAMP) was a gift from Daiichi microliters of 1% SDS was added to each well to Pharmaceutical (Tokyo, Japan). The nick-translation solubilize the stained cells. One hundred microliters of kit was purchased from Boehringer Mannheim (Ger- solubilized lysate from each well was transferred to many). The total RNA isolation kit was purchased from microtiter lates (Nunc, Denmark) and the absorbance Invitrogen (San Diego, CA). [(-u-~'PIdCTP (6,000 Cii of each we 1 was read at 550 nm. Cell numbers were mmol; 1 Ci = 37GBq) was purchased from New Re- calculated from a standard curve (OD 550 vs cell search Products (Boston, MA). The human TM gene number). Surface TM activity per lo5 cells was exwas isolated from a human leukocyte genomic library pressed as the percentage of the control kS.D. of four using a synthetic oligonucleotide. A 2.1 kb XhoI-NcoI determinations. Data were analyzed by the Student's fragment containing the coding region was isolated and t-test. subcloned into a pUC119 vector. This nucleotide seELISA for TM antigen in cell lysates quence determined by the dideoxy chain termination method was identical with that reported by Shirai et al. At the indicated times, medium was removed from (1988). A TthlllI-NheI fragment in the coding region the 48-well culture plates and quadruplicate cell monowas used as a hybridization probe. Complementary layers were extracted with 200 ~ 1 0 . 5 % Triton X-100 in DNA (cDNA) for human P-actin was purchased from phosphate buffered saline (PBS), pH 7.4. After 30 min,
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REGULATORY MECHANISMS FOR THROMBOMODULIN EXPRESSION IN HUVECS
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the cell monolayers were suspended with pasteur pi*** pettes. Cell lysates were stored at -20°C until assayed. Cell numbers were counted in another matched 48-well PMA ** I culture plate that had been equally treated with agents. Cell supernatants from the experimental wells r were also stored for measurement. Cell lysates were .+galso prepared from confluent HUVECs grown in 150 O 100cm2 tissue culture flasks. Protein in cell lysates was E measured by the method reported by Lowry et al. .-c (19511, using BSA as a standard. .-> Total TM antigen in cell lysates were measured by 9c 5 0 ELISA using anti-human TM monoclonal antibodies, u *** KA-2 and KA-4, whose characteristics were reported .-aC c previously (Kimura et al., 1989). Microtiter plates e coated with KA-2 at 60 pgiml in 0.05 M carbonate n O L , buffer were used for ELISA assays. After washing the ’ --- / 0 1 2 4 8 24 coated plates three times with Tris-buffered saline Incubation Time (h) (TBS) containing 0.05% Tween-20, aliquots of cell lysates diluted with TBS-Tween containing 5 mM Fig. 1. Time course of surface TM activity in HUVECs incubated CaClz (TBS-Tween-Ca2+)and 0.1% BSA were added to with dBcAMF’ or PMA. HUVECs grown in 48-well plates were the coated wells and incubated overnight at 37°C. The incubated with or without the indicated agents at 37°C in 5% CO,. At wells were then washed three times with the same the indicated times, surface TM activity was measured as described in buffer and 200 p1 of horseradish peroxidase-conjugated Materials and Methods. HUVECs were incubated with 3 mM dBcAMP ( 0 ) or M PMA (a). Surface TM activity per lo5 cells is expressed KA-4 were added to the wells and incubated for 4 h at as the percentage of the control ?S.D. of four determinations. Aster37°C. Horseradish peroxidase was conjugated to KA-4 isks represent significant differences from the unstimulated control according to the method of Nakane and Kawaoi (1974). cells (*P < 0.05, **P < 0.01, ***P < 0.001). After washing, 0.01% H,Oz and ortho-phenylendiamine (0.4 mgiml in 0.075 M citrate-sodium phosphate buffer, pH 5.0) were added to each well and incubated for 60 min at 37°C. The reaction was stopped by addition of 50 p1 4N H2S04to each well. The absorbance at 490 nm diographs, TM mRNA levels were normalized to the was measured using a Vmax Kinetic Microplate concentration of p-actin mRNA. Reader interfaced to Softmax software. Human placenRESULTS tal TM was used as a standard after isolation and The time course of surface TM cofactor activity purification by the previously described method in HUVECs incubated with dBcAMP or PMA (Kimura et al., 1989). As shown in Figure 1, PMA (lop8 M) effects were biphasic, with reduced TM activity occurring between Northern blotting 1-8 h, followed by a significant enhancement at 24 h Total cytoplasmic RNA was prepared from confluent (152 2 9.3%); 3 mM dBcAMP significantly enhanced HUVECs grown in 150 cmz tissue culture flasks using surface TM activity between 4 h (116 2 3.5%) and 8 h a total RNA isolation kit. Isolated RNA was heat- (124 ? 7.8%), followed by a slight reduction at 24 h. denatured and fractionated on formaldehyde-containing These results suggest that up-regulation of TM expresagarose gels. The gels were then stained with 1.0 pgiml sion involves protein kinase C or protein kinase A ethidium bromide for 30 min and washed with distilled dependent mechanisms consistent with previous obserwater four times for 30 min each. The RNA was then vations (Hirokawa and Aoki, 1990). transferred to a HyBond-N filter (Amersham InternaEffects of cyclic nucleotides on surface TM tional, Amersham, UK) by standard capillary blotting activity in HUVECs techniques in transfer buffer (Maniatis et al., 1982). The dose-response effects of cyclic nucleotide on TM The RNA was cross-linked to the nylon membrane by UV illumination. The probe employed for hybridization activity after 6 h incubation are shown in Figure 2. was a 32P-labelled450 bp DNA fragment corresponding Although dBcAMP enhanced surface TM activity sigto the human TM coding region, nick-translated to a nificantly between 3 and 10 mM, dBcGMP had no effect specific activity of > lo8 dpmipg. Nick-translation was up to 10 mM. These results suggest that CAMP-depenperformed using a nick-translation kit and [cx-~’PI- dent and cGMP-independent mechanisms are involved dCTP (6000 Ci/mmol). Prehybridizations and hybrid- in up-regulation of TM in HUVECs and eliminate the izations were performed at 42°C and washes were possibility that liberated free butyrate (Kooistra et al., performed at 60°C. Autoradiograms of Northern blots 1987) may affect surface TM activity. were prepared using Kodak X-OMAT AR film with an Effects of cytokines on the expression of intensifying screen, Lightning plus GK (Cronex, Duendothelial cell TM mRNA pont J a an), and exposed at -70°C. After washing with distilleIp water at 65°C for 30 min, the filter was dried The time-course effects of cytokines on the expression and rehybridized with 32P-labeled cDNA probe for of endothelial cell TM mRNA are shown in Figure 3. human p-actin. After densitometric analysis of autora- Northern blotting of TM mRNA demonstrated that 10
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U/ml TNF and 10 Uiml IL-1p induced a similar timedependent decrease in TM mRNA in HUVECs. These results agree with previous reports (Nawroth and Stern, 1986; Nawroth et al., 1986; Moore et al., 1989; Conway and Rosenberg, 1989; Hirokawa and Aoki, 1990). Effects of PMA and dBcAMP on the expression of endothelial cell TM mRNA As shown in F i e r e 4, lo-' M PMA and 3 mM dBcAMP irm~asedTM mRNA in HUVECs; lo-' M PMA increased TM mRNA in HUVECs between 4-40 h, with maximal effect at 24 h. The maximal increase induced by PMA varied from 300 to 1500%of control in several independent experiments (300, 5007 1100, 1500% of control), 3 mM dBcAMP also increased TM mRNA in HUVECs between 2-4 h with maximal effect at 2 h- In several independent experiments, BcAMP increased TM mRNA in HUVECs between 2-8 h and maximal effect was observed 150 to 1200% of control (150, 170, 1200 of control). Time course of surface TM activity and total TM antigen in HUVECs incubated with agents Although cytokines (10 Uiml IL-lp or TNF) decreased surface TM activity significantly between 4-48 h, they did not significantly decrease total TM antigen in cell lysates at 4 h, as shown in Figure 5. Several additional experiments confirmed this phenomena. PMA (lops M) decreased surface TM activity but not always total TM antigen in HUVECs after 4 h incubation (Hirokawa and Aoki, 1990). It was notable that PMA (lo-' M) increased total TM antigen in cell lysates at 24 h before surface TM activity increased at 48 h (Fig. 5C). Taken together, these results demonstrated that down-regulation of TM occurred initially
on the cell surface followed by loss of antigen in cell lysates and up-regulation occurred in reverse order. Analysis of conditioned media from these experiments for TM antigen by ELISA failed to show any detectable TM antigen. Effects of chloroquine on surface TM activity and total TM antigen in cell lysates The above observations raised questions concerning the fate of the down-regulated TM. Results shown in Figure 5 suggested that surface TM might be internalized and degraded intracellularly as reported previously. Hence, we investigated the effects of chloroquine, which inhibits lysozomal functions by increasing intralysosomal pH (Ohkuma and Poole, 19781, on surface TM activity and total TM antigen in HUVECs incubated with or without agents for 4 h. Two hundred micromole chloroquine did not affect surface TM activity in HUVECs incubated with or without agents, but increased total TM antigen in cell h a t e s as shown in Figure 6. These results i l s o demonstrate that incubation of HUVECs with PMA ( l o p 7M). TNF (10 Uiml), or IL-lp (10 U/ml) could decrease surface TM activity, but not total TM antigen in cell lysates. Thus, inhibition of lysosomal function by chloroquine resulted in decreased surface TM activity and increased total TM antigen in HUVECs incubated with PMA, TNF, or IL-lp. These results mean that such agents may induce internalization and degradation of TM in HUVECs. ~ e t ~ ~ e m eofn total t s TM antigen released into the COnditiOned media from HUVECs with agents Since analysis of conditioned media from HUVECs grown in 48-we11 plate by to show any detectable TM antigen, media from HUVECs grown in a 150 cm2 flask were analyzed. HUVECs were incubated with or without agents in 10 ml culture media for the indicated times. After incubation, the conditioned media and cell lysates were measured for TM antigen by ELISA as described in Materials and Methods. Compared with total TM antigen in cell lysates, only a small quantity of TM antigen was detected in the conditioned media as shown in Table 1. PMA (10-8 M, 24 h) or dBcAMp (3 mM, 6 h) increased total TM antigen in cell lysates up to 265% and 202%, respectively, whereas IL-1p (10 U/ml, 24 h) decreased TM antigen levels to 24% of control after normalization of TM antigen levels to protein content. A short incubation with PMA M, 6 h) did not affect total TM antigen levels significantly as observed previously (Scarpati and Sadler, 1989; Hirokawa and Aoki, 1990). In contrast to the drastic decrease in total TM antigen in cell lysates, TM antigen in the conditioned media from HUVECs incubated with IL-1p did not vary as much. These results suggested that down-regulated TM molecules are not released from the cell surface directly into conditioned media in a form detectable by our monoclonal antibodies. DISCUSSION TM expression in endothelial cells is regulated by various agents: endotoxin (Moore et al., 1987) and cytokines (Nawroth and Stern, 1986; Nawroth et al.,
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28s -
%-
24h
-
TM
--e
I
c
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i20
I
0
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incubation Time (h)
+-
Actin
Fig. 3. Effects of cytokines on the expression of endothelial cell TM mRNA. HUVECs were incubated with 10 Uiml TNF (upper panel)or 10 Uiml IL-lP (lower panel) for the indicated times. (A) Total RNA was isolated, electrophoresed, and transferred to a Hybond-N filter as described in Materials and Methods. The filter was hybridized with a
32P-labeled DNA probe. The positions of the ribosomal RNA size markers are indicated a t the left margin. (B) Autoradiographs were analyzed by densitometry, and values for TM were normalized to human p-actin. Normalized data is expressed as the percentage of TM mRNA relative to unstimulated control cells.
1987; Conway and Rosenberg, 1988) down-regulate TM; forskolin and CAMPanalogs up-regulate TM (Hirokawa and Aoki, 1990); active phorbol esters have biphasic effects such as down-regulation by short incubation (1-8 h) and up-regulation by long incubation
h) (Dittman et al., 1988; Scarpati and Sadler, 1989; Hirokawa and Aoki, 1990). As for cyclic nucleotides, up to 10 mM dBcGMP had no effect on TM expression by 6 h incubation while dBcAMP up-regulated it as shown in Figure 2. Inter(> 24
162
HIROKAWA AND AOKI
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zas +
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0
-
10
20
30
Incubation Time (h)
Actin
Fig. 4. Effects of PMA and dBcAMP on the expression of endothelial cell TM mRNA. HUVECs were incubated with M PMA (upper panel) or 3 mM dBcAMP (lower panel) for the indicated times. Northern blotting (A) and analysis of autoradiography by densitometry (B) were performed as described in Figure 3.
estingly, Mackie et al. (1986) reported that bovine endothelial cells contained a CAMP-dependent protein kinase (protein kinase A), three calcium-calmodulindependent protein kinases, protein kinase C, and tyrosine kinase, but no cGMP-dependent protein kinase (protein kinase G). They also found that the cells
contained numerous substrates for protein kinase A and protein kinase C. These results are consistent with the conclusion that TM expression is regulated by both protein kinase A and protein kinase C in human endothelial cells (Hirokawa and Aoki, 1990) and that dBcGMP had little effect on surface TM activity.
163
REGULATORY MECHANISMS FOR THROMBOMODULIN EXPRESSION IN HUVECS
B
A
IL-1P 10U/rnfi
C
TNF 10U/mh
PMA 10-8M
Fig. 5. Time course of surface TM activity and total TM antigen in HUVECs incubated with agents. HUVECs grown in 48-well plates were incubated with or without the indicated agents (A: IL-lP 10 Ulml, B: TNF 10 Ulml, C: PMA 10 *MI for indicated times. After incubation, surface TM activity was measured as described in Materials and Methods. In separate matched 48-well plates that had been equally treated with agents, medium was removed and quadruplicate
cell monolayers were extracted with 200 (*I 0.5% Triton X-100 in PBS. Total TM antigen in cell lysates were measured by ELISA methods a s described in Materials and Methods. Surface TM activity (upper panel) and TM antigen per lo5 cells (lower panel) are expressed as the percentage of the control z S I . of four determinations. Asterisks represent significant differences from the unstimulated control cells c*P < 0.05, **P i0.01, ***P < 0,001).
Active phorbol esters had biphasic effects on TM activity as described before, and both effects were related to the activation of protein kinase C (Hirokawa and Aoki, 1990). In addition, enhancement of TM activity by PMA was preceded by increased TM antigen in cell lysates (Fig. 5) resulting from increased TM mRNA levels (Fig. 4). This may reflect increased TM molecules in the Golgi-endoplasmiccomplex by de novo synthesis. TM activity in HLVECs incubated with PMA, thus, seems to vary as the result of two different mechanisms. Internalization and degradation of TM may be induced and is followed by increased TM expression due to PMA-induced up-regulation of TM mRNA. However, it may be possible that enhancement of TM activity by prolonged incubation with PMA results from down-regulation of protein kinase C, after activation of the enzyme, in cell membranes as reported by Pirotton et al. (1990) in bovine aortic endothelial cells. The time-course effect of cytokines on TM activity and total TM antigen in HUVECs as shown in Figure 5 suggested that down-regulation of TM has two serial events, since total TM antigen did not significantly decrease after a 4 h incubation with cytokines, whereas TM mRNA levels were remarkably decreased after 4 h incubation (Fig. 3). Effects of the cytokines may consist of internalizationidegradation of TM and decreasing TM mRNA levels. This decreasing effect on TM mRNA levels are different from the PMA effects and presum-
ably reflect mechanisms independent of protein kinase C activation. Internalization and degradation of TM by cytokines may be attributable in part to the activation of protein kinase C; however, a decreasing mechanism for cytoplasmic TM mRNA levels by cytokines should be further investigated. Results demonstrating that chloroquine increased total TM antigen in cell lysates of HUVECs incubated with PMA or cytokines (Fig. 6) suggested that inhibition of TM activity involved internalization and degradation of TM as reported by Moore et al. (1989). Internalization and degradation of TM was strongly suggested, since IL-10 decreased TM antigen in HUVECs and did not generate detectable TM antigen in the conditioned media (Table 1). Cellular regulatory mechanisms for TM expression in HUVECs incubated with agents was demonstrated to involve changes in TM mRNA levels (Figs. 3, 4). Dittman et al. (1988) reported that PMA had no effect on TM mRNA levels in mouse hemangioma cells and Scarpati and Sadler (1989) also reported that PMA or TNF had no effect on TM mRNA in HUVECs, although TNF decreased surface TM activity in HUVECs. Conway and Rosenberg (1988)reported that TNF decreased TM activity exclusively resulting from inhibition of transcription of TM gene, which is consistent with our results. Differing results among these reports may come from differences in experimental conditions, spe-
164
HIROKAWA AND AOKI
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-
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-
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-
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Fig. 6. Effects of chloroquine on surface TM activity and total TM antigen in cell lysates. HUVECs grown in two 48-well plates were equally incubated with or without the indicated agents for 4 h. After incubation, surface TM activity (A) or total TM antigen in cell lysates (B) was measured in each plate as described in Materials and Methods and the legend to Figure 5. Asterisks represent significant differences from unstimulated control cells or between two groups (*P< 0.05, **P< 0.01, ***P< 0.001).
TABLE 1. Measurements of TM antigen released into the conditioned media from HUVECs incubated with agents'
Treatment Control PMA lo-* M 24 h PMA M6h dBcAMP 3 mM 6 h I L l S 10 U/ml24 h
Conditioned media TM antigen (ng/lO ml media)
Cell lysates TM antigen ( n g h g protein)
3.3 (100) 2.1 (64) 2.5 (76) 1.4 (42) 4.5 (1361
33.7 (100) 89.1 (265) 38.8 (115) 67.9 (202) 8.1 124)
'HUVECS grown in 150 cm' flasks were incubated with or without the indicated agents for indicated times. After incubation, media was removed and stored until assay at -20°C. Then HUVECs monolayers were extracted with 10 mlO.5%Triton X-100 in PBS. TAI antigen levels in conditioned media and cell lysates were measured by ELISA methods as described in Materials and Methods. Protein content of cell lysates was also measured by t h e h w r y method. Data expressed are the mean of duplicate determinations and the percentage of control levels is shown in parenthesis.
cies, normal cells and transformed cells, and number of passages. In addition, we demonstrated that IL-lP and TNF decreased TM mRNA levels similarly and that PMA or dBcAMP increased TM mRNA (Fig. 4). In conclusion, TM activity in HUVECs is modulated by independent mechanisms involving TM gene transcription and presumably intemalizatioddegradation of TM molecules. Each mechanism is stimulated and/or inhibited in HUVECs incubated with dBcAMP, PMA, IL-lp, and TNF. In addition, such regulatory mecha-
nisms involve protein kinase A- and protein kinase C-dependent mechanisms, but they are independent of protein kinase G. ACKNOWLEDGMENTS We thank Dr. S. Ohkawa and the nursery staff of Ohkawa Hospital (Matsudo) for collecting umbilical cords and placentae, Ms. M. Tanaka for her skillful technical assistance, Dr. N. Tanaka for performin the electron microscopy for Weibel-Palade bodies, an Mr. Y. Kita for his valuable suggestions on Northern blotting. We also gratefully thank Dr. D. J. Steams for discussions and critical reading of the manuscript. LITERATURE CITED
f
Beretz, A,, Freyssinet, J., Gauchy, J., Schmitt, D.A., Klein-Soyer, C., Edgell, C.S., and Cazenave, J. (1989) Stability of thrombinthrombomodulin complex on the surface of endothelial cells from human saphenus vein or from the cell line EA.hy 926. Biochem. J., 259:3540.
Castagna, M., Takai, Y., Kaibuchi, K., Sano, K., Kikkawa, U., and Nishizuka, Y. (1982) Direct activation of calcium-activated, phospholipid-dependent protein kinase by tumor-promoting phorbol esters. J. Biol. Chem., 257:7847-7851. Comp, P.C., Nixon, R.R., Cooper, M.R., and Esmon, C.T. (1984) Familial protein S deficiency is associated with recurrent thrombosis. J. Clin. Invest., 74.2082-2088. Conway, E.M., and Rosenberg, R.D. (1988) Tumor necrosis factor suppresses transcription of the thrombomodulin gene in endothelial cells. Mol. Cell. Biol., 8:5588-5592. Dittman, W.A., Kumada, T., Sadler, J.E., and Majerus, P.W. (1988)
REGULATORY MECHANISMS FOR THROMBOMODULIN EXPRESSION IN HUVECS
165
The structure and function of mouse thrombomodulin. Phorbol Maruyama, I., and Majerus, P.W. (1985) The turnover of thrombinmyristate acetate stimulates degradation and synthesis of thromthrombomodulin complex in cultured human umbilical vein endobomodulin without affecting mRNA levels in hemangioma cells. J . thelial cells and A549 lung cancer cells. Endocytosis and degradaBiol. Chem., 263:15815-15822, tion of thrombin. J. Biol. Chem., 260r15432-15438. Dittman, W.A., and Majerus, P.W. (1990) Structure and function of Maruyama, I. and Majerus, P.W. 11987)Protein C inhibits endocytosis thrombomodulin: A natural anticoagulant. Blood, 75:329336. of thrombin-thrombomodulin complexes in A549 lung cancer cells Drysdale, B-E., Zacharchuk, C.M., and Shin, H.S. (1983) Mechanism and human umbilical vein endothelial cells. Blood, 69:1481-1484. of macrophage-mediated cytotoxicity: Production of a soluble cyto- Moore, K.L., Andreoli, S.P., Esmon, N.L., Esmon, C.T., and Bang, toxic factor. J. Immunol., 131:2362-2367. N.U. (1987) Endotoxin enhances tissue factor and SuDoressesthromEsmon, N.L., Owen, W.G., and Esmon, C.T. (1982) Isolation of a bomodulin expression of human vascular endothelt6m in vitro. J. membrane-bound cofactor for thrombomodulin-catalyzed activation Clin. Invest., 79:124-130. of protein C. J. Biol. Chem., 257:859-864. Moore, K.L., Esmon. C.T.. and Esmon. N.L. (1989) Tumor necrosis Esmon, C.T. (1989) The role of protein C and thrombomodulin in the factor leads to the internalization and degradation of thrombomodregulation of blood coagulation. J. Biol. Chem., 264:47434746. ulin from the surface of bovine aortic endothelial cells in culture. Fulcher, C.A., Gardiner, J.E., Griffin, J.H., and Zimmerman, T.S. Blood, 73:159-165. (1984) Proteolytic inactivation of human factor VIII procoagulant Nakane, P.K., and Kawaoi, A. (1974) Peroxidase-labeled antibody. A orotein bv activated human orotein C and its analoev with factor V. new method of conjugation. J. Histochem. Cytochem., 22;10841091 Blood, 63":48&489. Gomi, K., Zushi, M., Honda, G., Kawahara, S., Matsuzaki, O., N&iih, P.P., and Stern, D.M. (1986) Modulation of endothelial cell hemostatic properties by tumor necrosis factor. J. Exp. Med., Kanabayashi, T., Yamamoto, S., Maruyama, I., and Suzuki, K. (1990) Antithrombotic effect of recombinant human thrombomodu163:740-745. Nawroth, P.P., Handley, D.A., Esmon, C.T., and Stern, D.M. (1986) lin on thrombin-induced thromboembolism in mice. Blood, 75:139& Interleukin 1induces endothelial cell Drocoadant while sumress1399. ing cell-surface anticoagulant activity. Pr"oc. Natl. Acad: Sci., Griffin, J.H., Evatt, B., Zimmerman, T.S., Kleiss, A.J., and Wideman, C. (1981) Deficiency of protein C in congenital thrombotic disease. U.S.A., 83:3460-3464. Ohkuma, S. and Poole. B. (1978) Fluorescence probe measurement of J. Clin. Invest., 68:1370-1373. the intralvsosomal DH in living cells and the Derturbation of DH bv Hirokawa, K. and Aoki, N. (1990) Up-regulation of thrombomodulin various agents. Prdc. Natl. Acid. Sc. U.S.A., -75:3327-3331. in human umbilical vein endothelial cells in vitro. J. Biochem., Pirotton, S., Robaye, B., Langneau, C., and Boeynaems, J-M. (1990) 108:839-845. Adenine nucleotides modulate phosphatidylcholine metabolism in Imada, M., Imada, S., Iwasaki, H., Kume, A,, Yamaguchi, H., and aortic endothelial cells. J. Cell. Physiol., 142:449457. Moore, E.E. (1987) Fetomodulin: Marker surface protein of fetal develoDment which is modulatable by cyclic AMP. Dev. Biol., Sala, N., Owen, W.G., and Collen, D. (1984) A functional assay of protein C in human plasma. Blood, 63:671-675. 122:483491. Imada, S., Yamaguchi, H., Nagumo, M., Katayanagi, S., Iwasaki, H. Scarpati, E.M. and Sadler, J.E. (1989) Regulation of endothelial cell coagulant properties. Modulation of tissue factor, plasminogen and Imada. M. (1990) Identification of fetomodulin, a surface activator inhibitors, and thrombomodulin by phorbol 12-myristate marker protein of fetal development, as thrombomodulin by gene 13-acetate and tumor necrosis factor. J. Biol. Chem., 264:20705cloning and functional assays. Dev. Biol., 14Or113-122. 20713. Jaffe. E.A.. Nachman. R.L., Becker, C.G., and Minick, C.R. (1973a) Culture of Shirai, T., Shiojiri, S., Ito, H., Yamamoto, S., Kusumoto, H., Deyash...~ .~~ ~- human endothelial cells derived from umbilical veins. iki, Y., Maruyama, I., and Suzuki, K. (1988) Gene structure of Identification~-bymorphologic and immunologic criteria. J. Clin. human thrombomodulin, a cofactor for thrombin-catalyzed activaInvest., 523745-2756. Jaffe, E.A., Hoyer, L.W., and Nachman, R.L. (197313) Synthesis of tion of protein C. J . Biochem., 103:281-285. antihemophilic factor antigen by cultured human endothelial cells. Suzuki, K., Nishioka, J., Hayashi, T., and Kosaka, Y. (1988) Functionally active thrombomodulin is present in human platelets. J. J. Clin. Invest., 522757-2764. Kimura. S.. Nagova. T.. and Aoki. N. (1989) Monoclonal antibodies to Biochem., 104:628-632. human thro~b'omcdulinwhose binding is calcium dependent. J. Taylor, F.B. Jr., Chang, A., Esmon, C.T., DAngelo, A,, ViganoBiochem., 105:47W83. DAngelo. S., and Blick, K.E. (1987) Protein C prevents the coaguKooistra, T., Van den Berg, J., Tons, A,, Platenburg, G., Rijken, D.C., lopathic and lethal effects of Escherichia coli infusion in the baboon. and Van den Berg, E. (1987) Butyrate stimulates tissue-type J. Clin. Invest., 79:91%925. plasminogen-activator synthesis in cultured human endothelial Thornton, S.C.. Mueller, S.N., and Levine, E.M. 11983) Human cells. Biochem. J. 247:605612. endothelial cells: Use of heparin in cloning and long-term serial Kumada. T.. Dittman. W.A., and Maierus. P.W. (1987) A role for cultivation. Science (Wash. DC), 222:623-625. thromhornndulin in the Dkhoeenesfs of thrombin-induced throm- Voyta, J.C., Via, D.P., Butterfield, C.E., and Zetter, B.R. (1984) ._.. . . ... boembolism in mice. Blobd, 71y728-833. Identification and isolation of endothelial cells based on their Lowry, O.H., Rosebrough, N.J., Farr, A.L., and Randall, R.J. (19511 increased uptake of acetylated-low densit.y lipoprotein. J . Cell Biol., Protein measurement with the folin phenol reagent. - J . Biol. Chem., 992034-2040. 193:265-275. Mackie, K., Lai, Y., Nairn, A.C., Greengard, P., Pitt, B.R., and Lazo, Wagner, D.D., Olmsted, J.B., and Marder, V.J. (1982) Immunolocalization of von Willebrand protein in Weibel-Palade bodies of human J.S. (1986) Protein phosphorylation in cultured endothelial cells. J. endothelial cells. J. Cell Biol., 95:355-360. Cell. Physiol., 128:367374. Maniatis, T.; Fritsch, E.F., and Sambrook, J. (1982) Molecular clon- Walker, F.J., Sexton, P.W., and Esmon, C.T. (1979) The inhibition of blood coagulation by activated protein C through the selective ing: A laboratory manual. Cold Spring Harbor Laboratory, Cold inactivation of activated factor V. Biochim. Biophys. Acta., Spring Harbor, NY. 571:333-342. Maruyama, I., Bell, C.E. and Majerus, P.W. (1985) Thrombomodulin is found on endothelium of arteries, veins, capillaries, and lymphat- Walker, F.J. (1980) Regulation of activated protein C by a new protein. A possible function for protein S.J . Biol. Chem., 2555521ics, and on syncytiotrophoblast of human placenta. J. Cell Biol., 101:363-371. 5524. "d
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