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From the Department of Cellular Physiological Chemistry, Graduate School, Tokyo Medical and Dental University, Yushima, Bunkyo-ku, Tokyo, Japan.
Correspondence to Dr I. Morita, Department of Cellular Physiological Chemistry, Graduate School, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo, 113-8549 Japan. E-mail morita.cell@tmd.ac.jp
Abstract
Objective— Cyclooxygenase-1 (COX-1), but not COX-2, is expressed in human platelets, and thromboxane A2 (TXA2) produced via COX-1 induces platelet aggregation. The objectives of this study were to investigate the expression of COX-1 and COX-2 during platelet differentiation and to determine whether these enzymes are involved in the differentiation.
Methods and Results— CD34+ progenitor cells isolated from human cord blood were cultured with thrombopoietin and c-kit ligand. The cells differentiated into megakaryocytes (CD34-/CD41+) after 8 days of culture and into platelets (CD41+/prodium iodide-) after 14 days of culture. The CD34+cells expressed a trace of COX-1 gene and no COX-2 gene. On day 5, COX-2 gene expression was observed and continued throughout the remainder of the culture. COX-1 gene expression increased after 8 days of culture. The treatment of this liquid culture with indomethacin, a dual inhibitor of COX-1 and COX-2, and NS-398, a COX-2–specific inhibitor, suppressed megakaryocyte differentiation. In contrast, at a dose of 10-7 M, mofezolac, which is a highly selective inhibitor of COX-1, did not affect differentiation. NS-398–induced suppression of megakaryocyte differentiation was partly abrogated by stable analogues of TXA2.
Conclusions— We report here that COX-2 and COX-1 are constitutively expressed in megakaryocytes, and TXA2 produced by COX-2 plays an important role in megakaryocytopoiesis.
Key Words: megakaryocytopoiesis ? cyclooxygenase-1 ? cyclooxygenase-2 ? platelets ? thromboxane A2
Introduction
Cyclooxygenase-1 (COX-1) and COX-2 are enzymes that convert arachidonic acid to prostaglandin H2. The biological roles and properties of COX-1 are different from those of COX-2. It was originally thought that the function of COX-1 was involved in physiological phenomena, whereas that of COX-2 was involved in various pathologies.1 One of the main roles of COX-1 is the production of thromboxane A2 (TXA2) in activated platelets.1 Because platelets are anucleated cells, COX-1 content is a consequence of gene expression in precursor cells known as megakaryocytes. However, a recent study showed that human megakaryocytes in bone marrow express COX-2 proteins.2 The expression and function of COX isozymes and prostanoids in megakaryocytes are largely unknown.
Megakaryocytes originate from pluripotent stem cells through a differentiation process that involves stem cell commitment, nuclear polyploidization, and cytoplasmic maturation leading to the production of platelets.3 The initial stage of megakaryocyte development involves sequential proliferation of CD34+ hematopoietic stem cells into proliferating megakaryoblasts and then into bipotent erythromegakaryocytic progenitor cells.4 The second phase involves nuclear polyploidization, increase in cell size, formation of a demarcation membrane system in the cytoplasm, and expression of lineage-specific cell surface markers.5 The terminal differentiation process involves shedding of proplatelet fragments that become functional platelets.6
Proliferation and maturation of megakaryocyte precursors are regulated by several cytokines. Thrombopoietin (TPO) plays a major role in forming megakaryocytes and in producing platelets.4 Stem cell factor, IL-3, IL-6, IL-11, and basic fibroblast growth factor also contribute to megakaryocyte formation.7,8 Several leukemic cell lines such as HEL,9 Dami,10 CMK,11 Meg-01,12 UT-7,13 M-07e,14 EST-IU,15 MKPL-1,16 and LAMA-8417 have been established to study megakaryocyte differentiation. These cell lines were not suitable for physiological studies of megakaryocyte terminal differentiation. Recently, we developed a human primary culture system in which human hematopoietic stem cells differentiate into megakaryocytes after treatment with several cytokine cocktails.18
Prostaglandin E2 (PGE2) regulates osteoclast differentiation through elevation of cAMP levels19 and 15-deoxy-;12,14 prostaglandin J2 regulates adipocyte differentiation through activation of peroxisome proliferator-activating receptors.20 It has been reported that PGE2 supports T-cell differentiation.21
In the present study, we have shown the expression patterns of COX-1 and COX-2 in a liquid culture system for megakaryocyte terminal differentiation from hematopoietic stem cells into proplatelet formation. We have also demonstrated that inhibition of COX-2 activity suppresses megakaryocytopoiesis and a TXA2 analogue partially abrogated this suppression.
Methods
Purification and Culture of CD34+ Cells
Umbilical cord blood (CB) samples from normal full-term newborn infants were obtained from Tokyo Metropolitan Bokutou Hospital after informed consents were obtained from the mothers. CB samples were diluted 2-fold with phosphate-buffered saline (PBS) and separated by centrifugation (800g, 20 minutes) on Ficoll-Paque (density=1.077 g/mL; Pharmacia Biotech AB, Uppsala, Sweden) to obtain mononuclear cell preparations. CD34+ progenitor cells were purified from these preparations using a Dynal CD34 Progenitor Cell Selection System (Dynal AS, Oslo, Norway). First, anti-CD34 antibody-conjugated beads bound to the progenitor cells to allow separation from other cells in the preparation. Then, the progenitor cells were detached from the antibody-coated beads. Flow cytometric analysis of purified cell preparations using a phycoerythrin-conjugated anti-CD34 monoclonal antibody (clone BIRMA-K3; DAKO, Glostrup, Denmark) showed that >95% of the selected cells were positive for CD34. These cells were cultured in X-vivo 20 medium (BioWhittaker, Walkersville, Md) containing 50 ng/mL TPO (PeproTech EC, London, UK) and 40 ng/mL c-kit ligand (KL) (Biosource, Camarillo, Calif) at an initial density of approximate 1x105 cells/mL. Cultures were maintained at 37°C in humidified 5% CO2 atmosphere.
RNA Preparation and Semi-quantification of mRNA
Total RNA was extracted from cells using Trizol Reagent (GIBCO BRL, Gaithersburg, Md).22 cDNA was synthesized from 2 μg of total RNA using the SUPER SCRIPT First-Strand Synthesis System (GIBCO BRL) according to the manufacturer’s protocol, and reaction product was submitted to PCR amplification using a Gene Amp PCR System 9600 (Perkin Elmer, Emeryville, Calif). The primers (CLONTECH Laboratories) for detection of COX-1, COX-2, thromboxane receptor (TP) and hypoxanthine guanine phosphoribosyltransferase (HPRT) were as follows: COX-1: 5'-TGCC-CAGCTCCTGGCCCGCCGCTT-3' and 5'-GTGCATCAACACA-GGCGCCTCTTC-3'23; COX-2: 5'-TTCAAATGAGATTG-TGGGAAAATTGCT-3' and 5'-AGATCATCTCTGCCTGAGT-ATCTT-3';24 TP: 5'-CTCCTCATCTACTTGCGCGT-3' and 5'-CAGGGTCAAAGAGCATGCAA-3';25 and HPRT: 5'-GGCG-TCGTGATTAGTGATGATGAACC-3' and 5'-CTTGCGACC-TTGACCATCTTTGGA-3'.
PCR reaction for HPRT and COX-1 was repeated for 30 cycles, and each cycle included denaturation at 95°C for 1 minute, annealing at 55°C for 1 minute, and primer extension at 72°C for 1 minute. PCR reaction for COX-2 and TP was repeated for 36 cycles, and each cycle included denaturation at 95°C for 1 minute, annealing at 58°C for 1 minute, and primer extension at 72°C for 1 minute. The PCR products were electrophoresed through a 1.2% agarose gel and visualized by staining the gel with ethidium bromide.
COX Enzyme Assay
Cells from day 14 of culture (3x106 cells/0.5 mL) were incubated with 10 μmol/L [1-14C]arachidonic acid for 10 minutes at 37°C. The reaction mixture was then acidified (pH 3.0) and extracted with 2 mL of ethyl acetate. The resulting organic phase was evaporated to dryness and the residue was applied to thin-layer chromatographic plates. The plates were developed with a solvent system of isooctane/ethyl acetate/water/acetic acid (50:110:100:20, by volume). Distribution of radioactivity on the plate was detected by BAS 2000 imaging analyzer (Fuji X, Tokyo, Japan).
Immunocytochemistry of COX-1 and COX-2
Cells in X-vivo 20 medium were seeded on 48-well plates at a density of 1x105 cells/mL and incubated for 14 days at 37°C. The medium was removed, and the cells were fixed in PBS–2% formaldehyde for 30 minutes at room temperature. After two washings with PBS, the cells were permeabilized in PBS buffer containing 1% fetal bovine serum (FBS) and 0.5% saponin for 15 minutes. The cells were subsequently incubated with the primary antibodies (anti-COX-1 and anti-COX-2; Oxford Biomedical Research, Oxford, Mich) diluted 1:20 in PBS with 1% FBS for 60 minutes at room temperature. Samples were washed with PBS containing 1% FBS, then incubated for 60 minutes at room temperature with the secondary antibodies (fluorescein isothiocyanate -conjugated goat anti-mouse IgG and FITC-conjugated goat anti-rabbit IgG) diluted 1:40 in PBS with 1% FBS. The samples were washed with PBS containing 1% FBS and then rinsed with PBS. For negative control staining, the same procedure as described was performed, but without the primary antibody. Fluorescence confocal microscopy (FV300/FLUOVIEW, Olympus, Japan) was used with an argon laser as the excitation source. A 40x objective and laser-power setting of 30 milliwatts were used for detection of subcellular COX-1 and COX-2 after immunocytochemical staining.
Determination of Megakaryocyte and Platelet Number in Culture by Flow Cytometry
On day 14, cultured cells were collected and rinsed with PBS, then centrifuged at 1000g for 10 minutes and fixed with 2% formaldehyde for 60 minutes. After the cells were washed twice with 0.1% BSA-PBS, they were incubated with a 1:100 dilution of FITC-conjugated mAb against human CD41 antibody (clone 5B12; DAKO, Glostrup, Denmark) and 15 μg/mL propidium iodide (PI) (Calbiochem, San Diego, Calif). Cell-associated immunofluorescence was analyzed, and cells were sorted by FACScan and a Vantage flow cytometer using Cell Quest software (Becton Dickinson, San Jose, Calif). Cells that were doubly positive for CD41 and PI (CD41+/PI+) on FACScan were counted as megakaryocytes, and the number of CD41-positive and PI-negative (CD41+/PI-) cells (excluding small debris or microparticles) were counted as proplatelets. The identification of megakaryocytes and platelets was performed by electron microscopy using forward scatter as an indicator of cell size and the expression of CD42b proteins.18 The identification of proplatelets was also performed by electron microscopy using as a reference the same distribution of forward scatter as platelets isolated from human peripheral blood and aggregated by thrombin.
Statistics
Data are expressed as mean±SEM. The statistical significance of differences of the means was determined by one-way analysis of variance (P<0.05).
Results
Expression of COX-1 and COX-2 in Human Megakaryocytes and Platelets Differentiated From CD34+ Cells
CD41 was substantially expressed at day 8 after induction by TPO and KL. The percent of CD41+ cells obtained was 3.7% (background level), 74.1%, 82.6%, and 88.9% on day 3, 8, 13, and 18, respectively. In this system, we first examined the profile of PG production in megakaryocytes. As shown in Figure 1, TXB2 was mainly converted from arachidonic acid (Figure 1). Next, we determined the expression levels of COX-1 and COX-2 mRNA during megakaryocyte differentiation (Figure 1). As expected, the expression of COX-1 mRNA was slightly detected on days 3 and 5, and it gradually increased with culture age. The expression of COX-2 mRNA was detected on day 5, and it was constitutively expressed throughout the remainder of the culture.
Figure 1. Time-dependent expression of COX-1 and COX-2 mRNA during human megakaryocytopoiesis and thrombocytopoiesis and TXB2 production in differentiated megakaryocytes. CD34+ cells were purified from human umbilical cord blood and cultured for 14 days with 50 ng/mL TPO and 40 ng/mL c-kit ligand. Cells from day 15 of culture (3x106 cells/0.5 mL) were incubated with 10 μmol/L [1-14C] arachidonic acid for 10 minutes at 37°C. The products were separated by TLC as described in Methods. The mRNA was purified at different stages of megakaryocytopoiesis (days 3, 5, 8, 11, and 15) and reverse-transcribed in the presence of random hexamers. Total cDNAs were then amplified using a set of oligonucleotides specific for COX-1 and COX-2 mRNA. HPRT was used as a control in the PCR.
To determine what kind of cells expressed COX proteins during megakaryocytopoiesis, cells on various culture days were stained with anti-COX-1 and anti-COX-2 antibodies. On day 3, neither COX-1 proteins nor COX-2 proteins were detected in any cells; on day 5, traces of COX-1 proteins and large amounts of COX-2 proteins were seen in megakaryocytes. As previously mentioned,18 morphological identification of these cells was performed on day 15. Some cells had abundant cytoplasm and multinucleated cells. Using an electron microscope, the cells were characterized as mature megakaryocytes. The other cells displayed very small size without nuclei, and using propidium iodide staining and electron microscope, the cells were characterized as platelets. As shown in Figure 2, COX-1 proteins were detected in platelets that displayed small size without nuclei, but not in megakaryocytes that displayed large size with multinuclei, whereas COX-2 proteins were still detected in megakaryocytes but not in platelets on day 15. Microscopic observation revealed that COX-2 proteins were localized in the cytoplasm of megakaryocytes (Figure 2, inset).
Figure 2. Time-dependent expression of COX-1 and COX-2 proteins during megakaryocytopoiesis in humans. At several stages of megakaryocytopoiesis, the cells were fixed and stained by antibodies against COX-1 or COX-2 and FITC-conjugated secondary antibody. Insert represents the cytoplasm localization of COX-2 at higher magnification.
Effects of COX Inhibitors on Megakaryocytopoiesis
Because COX-2 is constitutively expressed in megakaryocytes, we next examined whether COX-2-induced PG synthesis was involved in megakaryocyte differentiation and platelet formation. The treatment of this culture with indomethacin or NS-398 (a specific inhibitor of COX-226) caused a decrease in the number of CD41+/PI+ cells on day 14 (Figure 3). The number of platelets (CD41+/PI-) also decreased, but treatment in the late stage (days10 to 14) failed to suppress platelet formation (control: 14.10±0.16x105 cells; NS-398: 14.91±0.10x 105 cells; no significant difference). In contrast, mofezolac, a specific inhibitor of COX-1,27,28 did not affect the megakaryocyte differentiation and platelet formation (Figure 4). These data indicate that COX-2 is involved in megakaryocyte formation.
Figure 3. Dose-dependent effect of indomethacin and NS-398 on megakaryocytopoiesis and thrombocytopoiesis. CD34+cells were purified from human umbilical cord blood and cultured for 14 days with 50 ng/mL TPO and 40 ng/mL c-kit ligand. Indomethacin (IND) or NS-398 at the indicated doses was added throughout the 14 days of culture. Columns and vertical bars represent mean±SEM of triplicate samples.*P<0.05 versus control.
Figure 4. Dose-dependent effect of mofezolac on megakaryocytopoiesis and thrombocytopoiesis. CD34+ cells were purified from human umbilical cord blood and cultured for 14 days with 50 ng/mL TPO and 40 ng/mL c-kit ligand. NS-398 (10-7M) added throughout the 14 days of culture. Mofezolac at the indicated doses was added throughout the 14 days of culture. Columns and vertical bars represent mean±SEM of triplicate samples. **P<0.01 versus control.
Involvement of TXA2 in Megakaryocytopoiesis
It is known that megakaryocytes and platelets produce TXA2 and PGD2,29,30 and the main product in megakaryocytes that differentiated in our system was TXA2 (Figure 1). Therefore, we next investigated whether PGs produced by COX-2 affect megakaryocytopoiesis. The inhibition of megakaryocytopoiesis by NS-398 was specifically abrogated by the simultaneous addition of TXA2-receptor agonists, U44069 (3 μmol/L), U44619 (1 mmol/L), and I-BOP (10 μmol/L) (Figure 5a). However, the simultaneous addition of PGD2 did not abrogate the suppression (data not shown). To assess the involvement of TXA2 on megakaryocytopoiesis, we examined the gene expressions of thromboxane synthase (TXS) and TXA2 receptors during megakaryocyte differentiation. TXS was detected during megakaryocytopoiesis (Figure 6). During our cultures, we detected gene expressions of TP and TP? (Figure 6). Finally, the treatment with U51605, an inhibitor of TXS, caused suppression of megakaryocytopoiesis and thrombocytopoiesis, as shown in Figure 5b. By treatment with U51605, TXB2 secretion was completely suppressed but PGE2 secretion was increased (day-5 control culture: TXB2=280 pg/mL, PGE2=not detectable; the treatment of NS-398, TXB2, and PGE2=not detectable; and the treatment with US51605: TXB2=not detectable, PGE2=270 pg/mL, respectively) However, the treatment with PGE2 in this system did not affect the megakaryocyte formation (data not shown).
Figure 5. Involvement of TXA2 in megakaryocytopoiesis. a, CD34+cells were purified from human umbilical cord blood and cultured for 14 days with 50 ng/mL TPO and 40 ng/mL c-kit ligand. NS-398 (10-7M), U44069 (3 μM), U44619 (1 mmol/L), and I-BOP (10 μmol/L) were added throughout the 14 days of culture. Columns and vertical bars represent mean±SEM of triplicate samples. **P<0.01 versus control. #P<0.01 versus NS-398 alone. b, CD34+cells were purified from human umbilical cord blood and cultured for 14 days with 50 ng/mL TPO and 40 ng/mL c-kit ligand, then U51605 (10 μmol/L) was added throughout the 14 days of culture. Columns and vertical bars represent mean±SEM of triplicate samples. **P<0.01 versus control.
Figure 6. Time-dependent expression of TXS, TP, and TP? mRNA during human megakaryocytopoiesis. At several stages of megakaryocytopoiesis, mRNA were purified and reverse-transcribed in the presence of oligo dT. The total cDNAs were then amplified using a set of oligonucleotides specific for TXS, TP, and TP?. Ethidium bromide staining of PCR products yielded 2 bands. The larger and the smaller band in each reaction correspond to TP and TP? mRNA, respectively.
Discussion
It is widely recognized that TXA2 produced by COX-1 in platelets stimulates aggregation. However, Weber and Zimmermann31 have reported that COX-2 mRNA and proteins were expressed in human platelets, and the extent of COX-2 expression might be an important factor in aspirin resistance. These findings suggest the possibility that megakaryocytes express COX-2 and COX-1. In a related report, when CMK and MEG-01 leukemia cell lines were differentiated to megakaryocytes by phorbol-12-myristate acetate, COX-2 was expressed. However, in that system, COX-2 mRNA significantly increased with only 3 hours of phorbol-12-myristate acetate treatment, and the level decreased rapidly,29,30 so it was not clearly demonstrated whether COX-2 was expressed constitutively in megakaryocytes. Here, we have reported that megakaryocytes differentiated from human cord blood expressed COX-2 as well as COX-1. The expression of COX-2 mRNA during megakaryocytopoiesis was shown by RT-PCR, and COX-2 protein was expressed in megakaryocytes, as demonstrated by immunostaining. These results were supported by the study in which megakaryocytes from bone marrow biopsies and those derived from thrombopoietin-treated CD34(+) hemopoietic progenitor cells in culture were positive for COX-1 and COX-2.2 In our culture system, the extraction of RNA and immunostaining were performed on the indicated day before medium change; however, in those experiments after medium change, the expression of COX-2 did not vary (data not shown), indicating that COX-2 may be expressed constitutively. This is not surprising because constitutive expression of COX-2 has been observed in several tissues and cells such as rat brain,32 maculadensa of the kidney,33 tracheal epithelium,34 pancreatic islets,35 and hepatic stellate cells.36 Thus, expression of COX-2 in many specialized cell types appears to be differentially sensitive to stimuli that regulate the unique physiological activities of each tissue.
During megakaryocytopoiesis, TXS mRNA, TP mRNA, and TP? mRNA were also detected by RT-PCR. A single gene encodes the human TP, of which there are 2 splice variants, TP and TP?. The mRNA for both splice variants have been demonstrated in platelets, but the protein of TP? was not detected in human platelets.25
In our experiments, mRNA for TP and TP? were expressed during megakaryocytopoiesis. Although we did not determine the protein expression, TX agonist or TXS inhibitor affected megakaryocytopoiesis in our system, indicating that TPs are functionally active.
Lorenz et al37 have demonstrated that COX-2–deficient mice had markedly reduced numbers of erythroid and myeloid colony-forming cells in the recovery phase after treatment with 5-fluorouracil. They showed that the platelet number on day 8 after 5-fluorouracil treatment was also clearly reduced in COX-2–deficient mouse (COX-2-/+=7.5x105/μL versus COX-2-/-=2.1x105/μL).
However, the number of platelets did not change in COX-2–deficient mouse cells without 5-fluorouracil treatment. These results suggest that COX-2 is involved in megakaryocytopoiesis, but other factors may compensate for COX-2 deficiencies in development. In our experiments, the treatment of the late phase of culture with NS-398 did not affect the number of platelets. These data were supported by a previous report in which inhibition of TXS by aspirin after 10 days of culture has no effect on platelet production.38
Despite the inhibition of platelet aggregation by classic NSAIDs (inhibition activity: COX-1>COX-2), a COX-1–specific inhibitor, mofezolac, failed to suppress megakaryocytopoiesis (Figure 4). These data are supported by clinical data that classic NSAID therapy does not cause thrombocytopenia and by the lack of reports about decreased platelet numbers in COX-1–deficient mouse cells. In contrast, a recent study has demonstrated that in a patient with moderate thrombocytopenia, the protein level of COX-1 decreased dramatically but that reduced levels of COX-1 protein is posttranscriptional.39 From this information, therefore, it is difficult to infer that COX-1 is involved in megakaryocytopoiesis.
It is recognized that NSAIDs such as aspirin possess antioxidant properties that could contribute importantly to their cardiovascular beneficial effects. For example, Wu et al have demonstrated that long-term aspirin treatment in vivo markedly reduced the production of vascular superoxide anion by decreasing the NAD(P)H oxidase activity.40 It was also reported that NAD(P)H oxidase-dependent superoxide anion was involved in platelet recruitment.41 Although these reports suggest that the inhibition of superoxide anion by NSAIDs suppresses platelet activation, there has been no report that superoxide anion is involved in megakaryocytopoiesis.
We have no data that can explain the relationship between aspirin-resistance and COX-2 expression in platelets. Eikelboom42 reported that aspirin-resistant TX biosynthesis increased the risk of cardiovascular events in a high-risk population. Aspirin-resistant TX biosynthesis will mean a COX-2–dependent TX synthesis in platelets or other cells. Cipollone et al43 have speculated that in unstable angina episodes of aspirin-insensitive TXA2 biosynthesis reflect extraplatelet source, but Weber et al31 have shown that COX-2 in platelets was a possible factor in aspirin resistance. In our study, we have shown that COX-2 and COX-1 existed in terminal differentiated megakaryocytes and demonstrated that COX-2, but not COX-1, were able to convert endogenous arachidonic acid to prostaglandins.44 Therefore, if demarcation membrane system was abnormal in the patients with cardiovascular events, then it may induce a formation of COX-2–dependent/aspirin-resistant platelets.
In summary, this study indicates that COX-2 and COX-1 are constitutively expressed in megakaryocytes, and TXA2 produced by COX-2 plays an important role in megakaryocytopoiesis. The results of large clinical trials treated with COX-2 specific inhibitors have recently raised some concerns regarding the cardiovascular safety.45,46 This problem is still disputed, but the data in the present study will support that COX-2 selective inhibitors were not associated with an increased risk of cardiovascular thrombotic events.
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