Enhanced expression of thrombomodulin by intracellular cyclic AMP-increasing agents in two human megakaryoblastic leukemia cell lines

THROMBOSIS RESEARCH 58; 615624,199O 0049-3848/90 $3.00 + .OOPrinted in the USA. Copyright(c) 1990 Pergamon Press plc. All rights reserved.

ENHANCED EXPRESSION OF THROMBOMODULIN BY INTRACELLULAR CYCLIC AMP-INCREASING AGENTS IN TWO HUMAN MEGAKARYOBLASTIC LEUKEMIA CELL LINES

Takahiko Ito’, Michinori Ogura’, Yoshihisa Morishita’, Junki Takamatsu’, Ikuro Maruyamd, Shuji Yamamoto? Kohei Ogawaq and Hidehiko Saito’ *the First Department of Internal Medicine, Nagoya University School of Medicine, Nagoya 466; Qhird Department of Internal Medicine, Kagoshima University School of Medicine, Kagoshima, Kanoshima 890: sBio-Science Laboratory. Life Science Research Laboratories, Asahi Chemical Industry, Fuji, Shizuoka 416, Japan. (Received 13.2.1990; accepted in revised form 30.3.1990 by Editor H. Yamazaki)

ABSTRACT

Functionally active thrombomodulin (TM) was expressed in human megakaryoblastic leukemia (MEG-01s) cells. We examined the effect of agents that increased the intracellular concentration of CAMP on the expression of TM by these cells. N6,02dibutyryl CAMP (dbcAMP) markedly enhanced TM antigen, activity, and mRNA level in MEG-01s cells. Other agents, &bromo-CAMP (8BrcAMP), forskolin, and prostaglandin El were also effective for the enhancement. Moreover, similar enhancement of TM by these agents was also observed in another human leukemia cell line, HEL, which has megakaryocytic markers. In contrast to the marked enhancement of TM expression by these agents, the expression of the other megakaryocytic markers including platelet glycoproteins IIMlIa, Ib, von Willebmnd factor and D-thromboglobulin was not stimulated in MEG-01s or HEL cells. These results suggest that expression of TM is rather specifically regulated by CAMP in human megakaryocytes.

INTRODUCTION Thrombomodulin (TM) is a cell surface glycoprotein initially found on vascular endothelial cells( 1,2) that acts as a cofactor for thrombin-catalyzed activation of protein C, a vitamin Kdependent serine protease zymogen. Activated protein C is a potent anticoagulant that selectively inactivates the coagulation cofactors Va (3) and V& (4). Therefore, TM has been recognized as an important regulator for in vivo coagulation. This cofactor is widely distributed in tissue on the luminal surface of vasucular endothelium (5). Recently, we found that TM is also expressed on the surface of human megakaryocytes, platelets, and a human megakaryoblastic cell line (MEG-O 1) (6). The molecular weight and the specific activity of the cofactor activity of the TM of human platelets have been shown to be identical to those of placenta TM (7). Key Words Thrombomodulin,

Protein C, Cyclic AMP, Megakaryocytes 615

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The expression of TM by endothelial cells appears to be influenced by various stimuli. Endotoxin (8), interleukin 1 (9), tumor necrosis factor (lo), and phorbol diester (11) have been shown to down-regulate the expression of surface TM. On the other hand, the up-regulating mechanism of this protein has not been elucidated in endothelial cells, or in megakaryocytes. In the course of our studies on the differentiation induction of human megakaryoblastic cell lines by various agents, we incidentally found that intracellular cAMP-elevating agents markedly and specifically enhance TM expression in a human megakaryoblastic cell line, MEG-O 1s. Similar results were also obtained in an erythroblastic cell line, HEL which co-expresses numerous megakaryocytic markers (12). Our results suggest that the expression of TM in megakaryocytes is regulated by intracellular CAMP. MATERIALS AND METHODS Cells and Cell Culture: The establishment and some properties of a human megakaryoblastic leukemia cell line, MEG-O 1, and of the subline MEG-O 1s were previously described ( 13,14,15). MEG-O 1s cells seem to have more immature characteristics than the original MEG-O 1 cells; the former cells grow in single suspension without attachment to the culture dish, and the expression of platelet glycoprotein GP Rb’lIla is weaker than that in MEG-01 cells (13,15). HEL was obtained from the American Tissue Culture Collection (Rockville, MD). These cells were passaged in RPM1 1640 medium (GIBCO, Grand Island, NY) supplemented with 10% heat-inactivated fetal calf serum (FCS, Flow Laboratories, Stamnore, NSW, Australia), penicillin G (100 U/ml) and streptomycin (50 l&ml). Before addition of various agents, cells were resuspended at 2 x 105/ml in the complete serum-free medium, COSMEDIUM-001 (Cosmo Bio, Tokyo), in which MEG01s and HEL cells could be maintained. For isolation of cellular RNAs, RPM1 1640 medium containing 1 % BSA was used instead of COSMEDIUM-001. 12-O-tetradecanoylphorbol- 13acetate (TPA), fotskolin and PGEt were dissolved in dimethyl sulfoae (DMSO) and added to the cell culture such that the final DMSO concentration did not exceed 0.1 %. This concentration of DMSO did not affect the growth or differentiation of the cells. Other reagents were dissolved in dist.HzO. All reagents added to the culture were obtained from Sigma Chemical Co., St Louis. The morphology of the cells was examined in cytocentrifuged preparations stained with MayGriinwald-Giemsa. Viable cell counts were determined by using trypan blue dye exclusion. Assav of Thrombomodulin (TM) Activitv: Human protein C was purified as described (3) with a slight modification. Eluate from a heparin-Sepharose (Pharmacia AB, Uppsala, Sweden) column was further purified by immunoaffinity chromatography on a column of Ca2+-dependent antiprotein C monoclonal antibody HPC-3-immobilized Sepharose that was generously provided by Dr. Matsuda, Jichi Medical School, Tochigi-Ken, Japan (16). Cell surface activity of TM was assayed as cofactor activity towards thrombin-catalyzed activation of protein C, as described (7). Cells (2 x 106) were washed three times with buffer A (20 mM Hepes, pH 7.4, 145 mM NaCl, 1 mM KCI, 1 mM MgS04, 1 mM CaC12) , then suspended in 100 1.11 of buffer A containing purified protein C (80 ps/ml). Ten ul of human thrombin (Sigma Chemical Co, 4000 NIH Unit/mg protein, 0.2 NM U/ml final concentration), was added, and the mixture was incubated for 60 min at 37 “C. In the absence of cells, activation of protein C was not detected with this concentmtion of thrombin. MEG-01s and HEL cells were >95% viable by trypan blue dye exclusion after the incubation. Cells were pelleted, and the reaction was terminated by the addition of 10 pl of 100 U,fnlantithrombinIlI(Behringwerke, Marburg&ahn, F. R. Germany) and 10 pl of 10 U/ml heparin to 100 p1 of the resultant supematant. Substrate solution (480 ul) containing 0.1 mM of fluorogenie substrate Boc-Leu-Ser-Thr-Arg-MCA (17) (Peptide Institute Co., Osaka), 25 mM Tris-HCI, pH 8.0, 0.15 M NaCI, 2 mM CaC12was added, then further incubated for 10 min at 37 “C. After termination of the reaction by adding 50 % acetic acid, fluorescence of the released aminomethylcoumarin (AMC) was monitored with a fluorospectrometer (model FP-770, Japan Spectroscopic Co, Tokyo) at 380 mu (excitation) and 440 nm (emisssion). TM activity was quantitated by subtracting the amount of AMC in parallel assays in the absence of thrombin, and expressed as pmol AMC formed/tube(600 ul)/min. Without thrombin, amidolytic activity of 0.1-0.3 pmol AMC/tube/min were detected in preparations from both MEG-01s and HEL cells. This activity was dependent on the cell number but was undetectable in the absence of protein C, therefore,

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unknown proteolytic activity for protein C seems to be present in these cells. In addtion, this activity was not affected by the reagents added to the culture in the present study. Northern Blot Analvsis: Total cellular RNAs were extracted by using guanidine thiocyanate procedure from MEG-01s cells that had been treated with 3 mM dbcAMP. Alliquots (10 pg) of RNAs were electrophoresed on formaldehyde denatured 1% agarose gel and transferred to a nitrocellulose filter (nitroplus 2000, Micron Separations Inc., Westboro, MA). The SmaI-KpnI fragment (approximately 1.3 kb) of TM cDNA (18) was random-labelled with [a-32P]dCTP (Amersham International plc, Buckinghamshire, England), using the Klenow fragment in a commercial kit (Amersham). Hybridization was carried out using the labelled fragment ( 19). The filter was washed with 0.1 x standard saline citrate (SSC), 0.1 % SDS, at 42 “C and was exposed to Kodak X-OMAT AR film at -80 “C for 3 days. Immunofluorescence Studies: Cell surface and cytoplasmic antigens were detected with an indirect immunofluorescence technique as previously described (15). Preparations were viewed in a Olympus fluorescence microscope, and further analyzed by flow cytometry with EPICS Profile (Coulter Electronics, Inc., Hialeath, FL). Anti-human TM monoclonal antibody (MoAb) was produced as previously described (20). Anti-platelet GPIIMUa MoAb, HPL-3 (21,22), was provided by Dr K. Furukawa, Department of Medicine, Nagoya University Branch Hospital. Anti-platelet GPIb MoAb (AN5 1) and anti-von Willebrand factor (vWf) MoAb were obtained from Dakopatts, Glostrup, Denmark. Rabbit Antihuman D-thromboglobulin (B-TG) polyclonal antibody (PoAb) was obtained from Amersham International plc. Normal mouse serum and normal rabbit serum were used as negative controls of primary antibody for MoAb and PoAb, respectively. RESULTS Immunofluorescence analysis using anti-TM monoclonal antibody revealed that exposure of MEG-01s cells to 3 mh4 N6,02-dibutyryl CAMP (dbcAMP) for 48 hr markedly increased the amount of TM antigen (Fig. 1). Under the condition in which 4 % of cells treated with control serum showed a positive background, the population of cells reacted with the antibody was increased from 30 % to 90 %. In the presence of 10 pg/ml cycloheximide, an inhibitor of protein

Fluorescent

Intensity

(log)

F&J Effect of dbcAMP on the expression of TM antigen in MEG-O 1s cells. MEG-O 1s cell were cultured with ( ) or without ( -) 3 mM dbcAMP, or with 3 mM dbcAMP in the presence of 10 ps/rnl cycloheximide ( - - -) for 48 hr. TM antigen on 10000 cells were then analyzed by flow cytometry. Normal mouse semm was used as negative control for primary antibody (.- - .- - --) . The data are presented as histograms, log10 relative fluorescence intensity (x axis; arbitrary units) versus number of cells (y axis; till scale=400).

618

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0 0

.Ol

.l

dbcAhlP

1

10

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I*-,--,‘-,-.,-.,..,

0

12

24

( mM )

36

48

60

72

hr

Fin.2: Dose and time dependent increase in TM activity of MEG-O 1s cells treated with dbcAMP. MEG-01s cells were cultured for 48 hr with various concentrations of dbcAMP (a), or were cultured with 3 mM dbcAMP for various times (b). synthesis, no increase of the antigen was observed following the addition of dbcAMP. This concentration of cycloheximide did not affect the viability of the cells. In the original MEG-01 cells, the poulation of cells which reacted with the antibody was increased from 10 % to 30 %. Since more antigen was found to be expressed in MEG-01s than in MEG-O 1, MEG-01s cells were used in the following study. To measure more quantitatively the amount of TM molecule, and to know whether the expressed TM was functional, we assayed TM activity in the cells. The cofactor activity of MEG-01s cells for thrombin-catalyzed activation of protein C also increased in both dose and time dependent manner after treatment with dbcAMP (Fig.Za and 2b). The increased activity could be detected after 48 hr of treatment with a dose of 0.03 mM or higher of dbcAMP. When 3 mM dbcAMP was dbcAMP

(-1

(4

I

hr

2

71

3

t-1

612247

I

/

F&J Effect of dbcAMP on TM mRNA levels in MEG-01s cells. MEG-01s cells were incubated with or without 3 mM dbcAMP for indicated times, and total RNAs extracted were analyzed by Northern blot.

619

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used, the activity increased within 2 hr, and reached a maximum at 48 hr, approximately 26fold over the control cells. To determine whether the enhanced TM expression is due to the increased TM mKNA, preparations of mRNA from MEG-01s cells were tested in Nothem blot hybridization experiments with a labeled TM cDNA. Time-dependent accumulation of thrombomodulin mRNA after stimulation with dbcAMP was detected (Fig.3). The accumulations were detectable within 2 hr, and peak levels were not reached until 24 hr. These results indicate that intracellular CAMP regulates transcription of TM gene in MEG-O 1s cells. Dibutyryl CAMP may have enhanced expression of TM of MEG-01s cells by mechanisms unrelated to cyclic nucleotides. To exclude these alternative possibilities, other nucleotide analogs and agents believed to raise intracelullar levels of CAMP were tested and compared to the results of dbcAMP treatment (Fig.4). Treatment with another cell-permeant cyclic nucleotide, 8-bromoCAMP (8BrcAMP), also enhanced the expression of TM, about 18-fold with a dose of 3 mM, but was less effective than with the same dose of dbcAMP. Prostaglandin Et (PGEJ is known to increase CAMP by stimulating adenylate cyclase coupled with G-protein in platelets. After exposure of 100 nM PGEt, a 4-fold increase in TM was detected. Higher concentrations of PGE, failed to induce further increase and saturation of the receptors above this concentration was suggested (data not shown). A direct activator of adenylate cyclase, forskolin, also increased the expression in a concentration-dependent manner. Approximately a lo-fold increase was observed with 30 uM forskolin. A small increase of TM was found after treatment with 100 uM isobutylmethylxanthine (IBMX), a phosphodiesterase inhibitor. When combined with low concentration of forskolin, IBMX synergistically enhanced the expression of TM. The addition of 3 mM dbcGMP also increased TM, while such an increase was not detected with the same concentration of 8BrcGMP. Since treatment with 1 mM sodium butymte affected the expression, intracellular butyrate, produced by hydrolysis of dbcAMP or dbcGMP, was suggested to have another effects on TM expression in MEG-01s cells. We previously reported that 12-Q-tetradecanoylphorbol- 13-acetate (TPA) induced differentiation of MEG-01s cells (16). TPA also enhanced the expression of TM, but was less effective in the enhancement than CAMP analogs, although used with a concentration (100 nM) enough for the cell differentiation. We further investigated the effect of intracellular CAMP-increasing agents on the expression of TM using another cell line, HEL. Before treatment, no TM antigen or activity was detected in HEL cells (Fig.5 and Table 1). When the cells were treated with dbcAMP, 8BrcAMP, forskolin, or PGE 1, the expression of TM was significantly enhanced, as was observed in MEG-O 1s cells.

Control dbcAMP 0.3 mM dbzAMP 3 mM BBrcAMP3 mM POE1 100 nM Forskolin 30 pM Fonkolin 1 )IM IBMX 1M) PM in 1 MM+ IBMX 100 pM dtcGMP 3mM BBrcGMP3 mM Sodium bUylate 1 mM TPA 100 nM 0

5 Thrombomodulin

10 Activity

15

20

(pmol AMC/min)

&4: Effect of various agents upon TM expression in MEG-01s cells. After MEG-01s cells were treated with various agents for 48 hr, TM activity was assayed. The results are the mean + S.D. of three to five different experiments.

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BY CYCLIC AMP

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Control dbcAMP 3mM 8BrcAMP 3 mM Forskolin 30 pM PGEl 100 nM TPA 100 nM 0

1

3

2

Thrombomodulin

Activity

(pmol AMC/min)

j&J: Effect of various agents upon TM expression in HEL cells. HEL cells were treated with various agents for 48 hr, and TM activity was assayed. Data represent mean + S.D. of three different experiments.

To exclude any effects of serum, all these experiments were carried out in the complete serumfree medium. However, the same experiments using RPM1 1640 with 10 % FCS gave similar results, indicating that serum had no additional effects in our experiments. While 8BrcAMP and forskolin considerably enhanced the expression of TM, these agents had little effects on the morphology of MEG-O 1s and HEL cells for 4 days. In some MEG-O 1s cells, slight convolution of nuclei was observed, however, multiplication of nuclei was not evident. Neither cytoplasmic granules nor protrusions were not increased. Moreover, immunofluorescence study demonstrated that the expression of otherplatelet specific proteins including GP IlMlIa, GP Ib, vWf and D-TG was not increased after exposure to 8BrcAMP or forskolin in MEG-01s or HEL cells (Table 1). TABLE 1 Specific Enhancement of Thrombomodulin by AMP-Increasing

MEG-01s Control 8BrcAMP (3 mM) Forskolin (30 pM) HEL Control 8BrcAMP (3 mM) Forskolin (30 PM)

Agents in MEG-O 1s and HEL

TM

GPllb’IUa

GPIb

vWF

D-TG

25+5 87*4 76+7

18*3 15&4 17*5

<4 <4 <4

35*3 36+2 33+4

28*4 30a2 25*3

<4 8+2 15i2

67*4 68*3 69*5

<4 <4 <4

<4 <4 <4

<4 <4 <4

Cell surface antigens (TM, GP IIMIIa and GP Ib) were analyzed by flow cytometry as shown in Fig. 1. Cytoplasmic antigens (vWf and D-TG) were examined with fluorescent microscope. Values (percentage of positive cells) are the means f S.D. of three determinations.

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DISCUSSION

Thrombomodulin (TM) plays a key role in the protein C anticoagulant pathway. Activated protein C complexes with protein S, another vitamin K-dependent protein, to interact with platelets or endothelial surface membrane (1,2). We previously reported that MEG-01 cells, as well as human megakaryocytes, synthesize protein S ( 15), and the presence of this protein in platelets was also reported (23). Although the role of TM and protein S in megakaryocytes or platelets remains to be investigated, they may participate in the local control of the protein C anticoagulant pathway. In this report, we have demonstrated that intracellular CAMP-increasing agents enhance the expression of cell surface TM in human megakaryoblastic leukemia cell lines by inducing transcription of TM mRNA. Cyclic AMP-dependent gene transcription has been shown in many eukaryotic genes, and some CAMP-responsive elements have been analyzed (24). Among these elements, Activator Protein-2 (AP-2)-binding elements has shown to be involved in the initiation of the gene transcription after treatment of phorbol diester, as well as CAMP (25). Nucleotide sequence of human TM was recently determined (26). It is of our interest that the 5’ flanking region of the gene contains a AP-2 binding consensus sequence (23), TCCCCCAC, at the nucleotide -576 residue. Since both CAMP analogs and a phorbol diester TPA increased the expression of TM in MEG-01s and HEL cells, this signal transduction may be mediated by AP-2. Whether AP-2 binds to the region in these cells remains to be determined. Cyclic AMP analogs have been reported to induce cell maturation in some cell lines. It was shown that PGEr and cholera toxin induced the formation of chemotactic receptor without morphological maturation in I-IL-60 cells (27). Dibutyryl CAMP was found to induce most potently the receptor formation followed by morphological maturation, but 8BrcAMP was almost inactive in the receptor induction or morphological change (27). On the contrary, another study demonstrated that 8BrcAMP and chorela toxin increased nitroblue tetrazolium (NBT) reductive capacity with morphological maturation beyond the promyelocyte stage (28). In MEG-01s cells and HEL cells, both 8BrcAMP and forskolin, which were potent inducers of TM expression, did not increase megakaryocytic markers including GP IlMlIa, GP Ib, vWF and g-TG. Moreover, morphological maturation was not significant (figure not shown). Although phorbol diester TPA also increased TM (Fig.4, 5), this enhancement was accompanied by increase of other markers and morphological maturation as previously reported (16,29). Therefore, our results suggest that CAMP analogs rather specifically induce the expression of thrombomodulin in these cells, and that the induction was not associated with overall cell differentiation as in the case of TPA. It has been shown that the initial mediator of the signal transduction after exposure to TPA is protein kinase C (30). In MEG-01 cells, TPA induced a protracted translocation of protein kinase C from the cytoplasm to the plasma membrane which was followed by the down-regulation of the enzyme (31). The signal after the activation of protein kinase C may diverse in the cells and seems to enhance the transcription of many genes. It is diflicult to decide whether TM gene expression in normal megakaryocytes is regulated by CAMP, since normal human megakaryocytes are hard to isolate in significant amounts. Although both MEG-01s and HEL are derived from neoplastic cells, these cell lines provide useful model systems for studying TM gene expression because of the differentiated properties of megakaryocytes. In this regards, it should be pointed out that some studies on TM have been performed in another neoplastic cells (11, 20). Our results raise the possibility that intracellular CAMP-increasing agents such as prostaglandins increase TM in megakaryocytes and, hence, in platelets. Therefore, these agents may exert another anti-thrombotic effect in addition to their well known inhibitory effects on platelet aggregation. Moreover, our findings obtained with two distinct cell lines hint that a similar mechanism may also exist in other cell types, including endothelial cells. After this work was completed, Imada et al. reported that fetomodulin (32), a cell surface glycoprotein in murine fibroblasts which responded to CAMP derivatives, might be identical with TM (33).

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ACKNOWLEDGEMENTS Part of this work was presented at The 3 1st Annual Meeting of the AMERICAN SOCIETY OF HEMATOLOGY held on December l-5, 1989, in Atlanta, GA, and published as an abstract (34). This work was supported in part by grants-in-aid for Scientific Research from the Ministry of Education, Science and Culture, grants from the Ministry of Health and Welfare of Japan, and the Aichi Blood Disease Research Juridical Foundation. REFERENCES 1. ESMON, C.T. The regulation of natural anticoagulant pathways. Science235, 1987. 2. ESMON C.T. The roles of protein C and thrombomodulin coagulation. JBiol Chem, 264,4743-4746, 1989.

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