Abstract

Plasminogen activator inhibitor-1 (PAI-1) is present in the platelet -granule and is released on activation. However, there is some debate as to whether the megakaryocyte and platelet synthesize PAI-1, take it up from plasma, or both. We examined the expression of PAI-1 in differentiating megakaryocytic progenitor cells (UT-7) and in CD34+/CD41 cells from cord blood. UT-7 cells differentiated with thrombopoietin (TPO) resembled megakaryocytes (UT-7/TPO) with respect to morphology, ploidy, and the expression of glycoprotein IIb-IIIa. PAI-1 messenger RNA (mRNA) expression was upregulated and PAI-1 protein synthesized in the UT-7/TPO cells accumulated in the cytoplasm without being released spontaneously. In contrast, erythropoietin (EPO)-stimulated UT-7 cells (UT-7/EPO) did not express PAI-1 mRNA after stimulation with TPO because they do not have endogenous c-Mpl. After cotransfection with human wild-typec-mpl, the cells (UT-7/EPO-MPL) responded to phorbol 12-myristate 13-acetate (PMA), tumor necrosis factor- (TNF-), and interleukin-1β (IL-1β) with enhanced PAI-1 mRNA expression within 24 to 48 hours. However, induction of PAI-1 mRNA in UT-7/EPO-MPL cells by TPO required at least 14-days stimulation. UT-7/EPO cells expressing c-Mpl changed their morphology and the other characteristics similar to the UT-7/TPO cells. TPO also differentiated human cord blood CD34+/CD41 cells to CD34/CD41+ cells, generated morphologically mature megakaryocytes, and induced the expression of PAI-1 mRNA. These results suggest that both PAI-1 mRNA and de novo PAI-1 protein synthesis is induced after differentiation of immature progenitor cells into megakaryocytes by TPO.

PLATELETS MAY AFFECT clot lysis by a variety of mechanisms. Platelets contain factor XIII, which, on activation, cross-links fibrin monomer and α2-plasmin inhibitor (α2-PI), thereby rendering it more resistant to digestion by plasmin.1,2 Activated platelets also secrete plasminogen activator inhibitor-1 (PAI-1) and α2-PI, which inhibit plasmin formation and activity, respectively.3,4 PAI-1 is a member of serine protease inhibitor super family, and is the major physiological inhibitor of fibrinolysis. Platelets are the main reservoir of PAI-1, with approximately 85% of circulating PAI-1 contained within platelet α-granules.5 It has been reported that platelet PAI-1 exists predominantly in a latent or inactive form,6,7 suggesting that its effect on fibrinolysis may be limited. However, Fay et al reported that PAI-1–deficient platelets inhibited tissue-type plasminogen activator (t-PA)–mediated clot lysis to a substantially lesser extent than normal platelets.8 Positive PAI-1 immunostaining of human platelets and megakaryocytes has also been shown.9However, PAI-1 messenger RNA (mRNA) was neither detected by Northern blot analysis of human platelet RNA, nor amplified from reverse-transcribed human platelet RNA.10 Therefore we questioned whether a relationship may exist between thrombopoietin (TPO)-induced megakaryocytic differentiation from progenitor cells and PAI-1 synthesis.

TPO, the recently isolated and cloned ligand for the cytokine receptor Mpl,11-15 is a hematopoietic growth factor that regulates platelet production. TPO stimulates the proliferation of megakaryocyte progenitor cells, promotes megakaryocyte terminal differentiation, and is essential for the production and maintenance of normal levels of thrombopoiesis.16-19 In the past, the study of megakaryocytic differentiation was limited because of the rarity of megakaryocytes in normal bone marrow, the poorly defined cell population, and inadequate assay methods. The UT-7 cell line was established from bone marrow cells of a patient with acute megakaryoblastic leukemia.20 In particular, recently isolated UT-7/TPO cells have an absolute dependence on TPO and show mature megakaryocytic features.21 In contrast, the UT-7/erythropoietin (EPO) cell line shows erythroid development without TPO dependency due to a lack of endogenous c-Mpl expression.22 Therefore, comparison of these cell lines will be useful for evaluating the PAI-1 response during megakaryocyte development induced by TPO.

In the present study, we used these UT-7 cell lines and their transfectants with or without transfection with c-mplcomplementary DNA (cDNA) as a model system and show expression of PAI-1 mRNA and de novo synthesis of PAI-1 protein with TPO-dependent differentiation. We also confirm these results using CD34+progenitor cells isolated from human cord blood.

MATERIALS AND METHODS

Hematopoietic growth factors and reagents.

Recombinant human TPO was provided by the Kirin Brewery Co, Ltd (Gumma, Japan). Recombinant human EPO was a gift from the Life Science Research Institute of Snow Brand Milk Company (Tochigi, Japan). Recombinant human granulocyte-macrophage colony stimulating factor (GM-CSF) was provided by Sumitomo Pharmaceutical Company (Osaka, Japan). Human cDNA clones for full-length c-mpl P (wild type) andc-mpl K (truncated) were kindly provided by Dr M. Okada (Eisai, Tsukuba, Japan) and Dr S. Gisselbrecht (INSERM, Paris, France), respectively.

Cell culture.

The original UT-7 cell line (UT-7/OR) was established from bone marrow cells obtained from a patient with acute megakaryocytic leukemia20 and maintained in liquid culture with Iscove’s modified Dulbecco’s medium (IMDM; GIBCO Laboratories, Grand Island, NY) containing 10% fetal calf serum (FCS; Hyclone Laboratories, Logan, UT) and 1 ng/mL of GM-CSF. UT-7/GM was isolated after long-term culture of UT-7 cells and maintained as described for the UT-7 cells.23 The UT-7/EPO cell line, which is a subclone of UT-7, was maintained continuously in IMDM containing 10% FCS and 1 U/mL of EPO.22 The UT-7/TPO cell line was maintained in IMDM containing 10% FCS and 10 ng/mL of TPO.21 Primary human umbilical cord-derived endothelial cells (HUVEC) were harvested from human umbilical cord veins treated with 0.1% collagenase as described elsewhere,24 and grown on fibronectin-precoated culture plate in Medium-199 (GIBCO Laboratories), containing 15% FCS, 2 mmol/L of glutamine, 15 mmol/L of HEPES, 100 μg/mL of heparin, and 60 μg/mL of endothelial cell growth supplement (Equitech-Bio Inc, Ingram, TX).

Reverse-transcriptase polymerase chain reactions (RT-PCR) and Southern blotting analysis.

Total RNA was isolated from cells according to the methods of Chomczynski and Sacchi.25 RT-PCR was performed using oligonucleotide primers as follows. The PAI-1 forward 5′-GAACAAGGATGAGATCAGCACC-3′ (nucleotides 402-423) and reverse 5′-ACTATGACAGCTGTGGATGAGG-3′ (nucleotides 1151-1172) primers; GP-Ibα forward 5-AAGCTGGAGAAGCTCAGTCTGG-3′ (nucleotides 535-556) and reverse 5′-CTCCTTAGTGGATTCTTGTGTTGG-3′ (nucleotides 1072-1095); PF-4 forward 5′-GCTGAAGCTGAAGAAGATGGG-3′ (nucleotides 98-108) and reverse 5′-TAGCAAATGCACACACGTAGG-3′ (nucleotides 324-344); urokinase-type plasminogen activator (u-PA) forward 5′-GATCTGATGCTCTTCAGCTGG-3′ (nucleotides 389-409) and reverse 5′-CTGCTCCGGATAGAGATAGTCG-3′ (nucleotides 1038-1059); Protease-activated receptor 1 (PAR-1) forward 5′-TGTCTGTGTCAGCAGCATAAGC-3′ (nucleotides 1292-1313) and reverse 5′-CTTGGAATAACACCGTCATCTCG-3′ (nucleotides 1688-1710); PAR-3 forward 5′-GGTAACATGTGGACTGGTGTGG-3′ (nucleotides 777-798) and reverse 5′-AATGGAGCTCCTTGCACTATGC-3′ (nucleotides 1334-1355);c-mpl P forward 5′-CAAGGCTTCTTCTACCACAGC-3′ (nucleotides 934-954) and reverse 5′-TCAGTCTCCTGTAGTGTGCAGG-3′ (nucleotides 1552-1573); glyceraldehyde-3-phosphate dehydrogenase (GAPDH) forward 5′-CCCATGGCAAATTCCATGGCA-3′ (nucleotides 215-235) and reverse 5′-GGTGGACCTGACCTGCCGTCTAGA-3′ (nucleotides 759-782). Each first-strand cDNA was synthesized by RT using a commercial kit (Roche Molecular System, Branchburg, NJ). Total cellular RNA (1 μg) was RT using oligo-dT16 primers, and amplified by PCR (94°C for 60 seconds, primer annealing at 56°C for 60 seconds, extension at 72°C for 60 seconds) for 30 cycles in a Program Temp Control System PC-700 (Astec, Tokyo, Japan), and a final incubation at 72°C for 10 minutes. Amplification products were separated on 1.5% agarose gels stained with ethidium bromide and photographed. The RT-PCR products were transferred to nylon membranes and incubated with PAI-1 and c-mpl P cDNA labeled with32P-αCTP by the random-priming method. After an overnight incubation at 65°C in 5× SSPE (150 mmol/mL NaCl, 10 mmol/mL sodium phosphate, pH 7.4, containing 1 mmol/mL EDTA) with 5× Denhardt’s solution, 20 μg/mL of nonhomologous salmon sperm DNA, and 0.5% (wt/vol) sodium dodecyl sulfate (SDS), the blots were washed three times with 2× SSC (150 mmol/mL NaCl, 15 mmol/mL sodium citrate, pH 7.0), 0.5× SSC, 0.1× SSC, plus 0.1% SDS for 15 minutes each. The membranes were autoradiographed using Kodak XAR-5 film (Eastman Kodak Co, Rochester, NY) with an intensifying screen at −80°C.

Preparation of c-mpl transfectant.

A c-Mpl expression vector was generated by ligation of full-lengthc-mpl cDNA into the pRc cytomegalovirus (CMV) mammalian expression vector. Electroporation was used for stable transfection of the plasmid into UT-7/EPO cells, as described previously.26In brief, 20 μg of pRcCMV containing c-mpl cDNA was introduced into 1 × 107 UT-7/EPO cells resuspended in 0.25 mL of RPMI 1640 medium containing 10% fetal bovine solution (FBS) by electropulse at 250V, 960 μFD. Transfected cells were seeded at 3 to 5 × 107 cells/mL in IMDM medium containing 10% FBS and 1 U/mL of EPO and neomycin- (Life Technologies Inc, Grand Island, NY) resistant clones were selected.

Metabolic labeling and immunoprecipitation.

UT-7/OR, UT-7/TPO cells, and HUVEC (1 × 107 cells/mL) were metabolically labeled for 15 minutes with 250 μCi/mL35S-methionine (EXPRE35S35S; Du Pont Co, Wilmington, DE) in 2.5 mL of methionine-free medium, as previously described.27 The cells were further incubated in serum-free medium supplemented with unlabeled methionine, and were harvested at various time intervals. Culture media was separated from cell pellets by centrifugation, and stored at −80°C immediately. The cells were washed with phosphate-buffered saline (PBS) and lysed on ice in lysis buffer composed of 20 mmol/L Tris-HCl (pH 7.4), 135 mmol/L NaCl, 20% glycerol, 1% NP-40, 1 mmol/L phenylmethylsulfonylfluoride (PMSF), 15 μg/mL aprotinin, and 2 mmol/L sodium orthovanadate. The culture media and cell lysates were precleared with protein A-Sepharose (Pharmacia Biotech), and incubated at 4°C for 1 hour with shaking with mouse antihuman PAI-1 monoclonal antibodies (MoAb) (JTI-3 and JTI-4) and nonimmune mouse immunoglobulin G (IgG) (Cappel ICN Pharmaceuticals Inc, Aurora, OH) bound to protein A-Sepharose beads.28 The immunoprecipitates were washed to remove unbound proteins and the bound proteins were eluted from the Sepharose by heating at 100°C for 5 minutes and then subjected to electrophoresis on 10% SDS-polyacrylamide gels.29 The gels were dried and exposed to radiograph film for autoradiography at −80°C.

Separation of human CD34-positive cells and megakaryocytic colony formation.

Human cord blood was obtained with informed consent from women who underwent normal vaginal delivery, and approximately 50 mL of cord blood was collected. To deplete adherent cells, cord blood cells were incubated with silica beads (KAC-2, Japan Antibody Institute, Gumma, Japan) at 37°C for 30 minutes. The cells were separated as the interface mononuclear FH cells by centrifugation (400g, 30 minutes at 25°C) over Ficoll Hypaque (FH; 1.077 g/cm3; Pharmacia Biotech, Uppsala, Sweden). The nonadherent mononuclear cells were adjusted to 5 × 107 cells/mL in PBS without Ca2+ and Mg2+, and CD34+ progenitor cells were isolated using a magnetic cell sorting system (Dynal CD34 progenitor cell selection system; Dynal, Oslo, Norway) according to the manufacturer’s instructions. CD34+-enriched cells were plated at 1 × 106 cells/mL in megakaryocyte culture medium containing 20% heparinized plasma (from the human cord blood) in 24-well tissue culture plates (Costar Corp, Cambridge, MA) as described previously.30-32 They were cultured at 37°C with 5% CO2 for 12 days.

Flow cytometry.

Cell-surface antigens were detected by immunofluorescence using fluorescein isothiocyanate (FITC; Becton Dickinson, Mountain View, CA) conjugated mouse antihuman CD34 and phycoerythrin (PE)-conjugated antihuman CD41 MoAb. In brief, UT-7 cells or isolated cord blood CD34+ progenitor cells were incubated for 30 minutes at 4°C with the appropriately diluted antibodies. After washing, cell-bound fluorescence on 5,000 to 10,000 cells/sample was determined with a flow cytometer (FACScan; Becton Dickinson).

RESULTS

Expression of GPIb-α, PF-4, u-PA, and PAI-1 in UT-7 megakaryocytic cell lines.

We examined the expression of GPIb-α, PF-4, and u-PA in the UT-7 cell lines by means of RT-PCR. The GPIb-α transcript was detected in the original UT-7 cell lines and in UT-7/TPO cells, but was undetectable in UT-7/EPO or UT-7/GM cells. PF-4, a protein specific to megakaryocytes and platelets, was detected solely in UT-7/TPO cells, and was entirely absent in UT-7/OR, UT-7/EPO, or UT-7/GM cells. In contrast, the u-PA mRNA was scarcely detectable UT-7/TPO cells as well as in platelets (data not shown). These results and previous studies21suggest that the UT-7/TPO cells have mature megakaryocyte characteristics with respect to morphology, ploidy, and the expression of megakaryocyte-specific proteins. Thus, we examined the effect of differentiation into megakaryocytes on PAI-1 expression using these cell lines. As shown in Fig 1, the PAI-1 expression level was detected in UT-7/OR and UT-7/GM cells, with abundant expression in the UT-7/TPO cells, but the transcript was scarcely detected in UT-7/EPO cells.

Fig. 1.

Expression of PAI-1 mRNAs in UT-7 cell lines. Total cellular RNAs were extracted from UT-7 cell lines (UT-7/OR, UT-7/EPO, UT-7/GM, and UT-7/TPO) and expressions of PAI-1 mRNA were evaluated by RT-PCR (see Materials and Methods). The RT-PCR products were resolved by agarose gel electrophoresis, and the bands were transferred to a membrane and hybridized to 32P-labeled PAI-1 or β-actin cDNA probes.

Fig. 1.

Expression of PAI-1 mRNAs in UT-7 cell lines. Total cellular RNAs were extracted from UT-7 cell lines (UT-7/OR, UT-7/EPO, UT-7/GM, and UT-7/TPO) and expressions of PAI-1 mRNA were evaluated by RT-PCR (see Materials and Methods). The RT-PCR products were resolved by agarose gel electrophoresis, and the bands were transferred to a membrane and hybridized to 32P-labeled PAI-1 or β-actin cDNA probes.

Pulse-chase analysis of PAI-1 protein in UT-7/TPO cells.

We evaluated the synthesis of PAI-1 and its secretion from UT-7/OR, UT-7/TPO cells, and HUVECs by pulse-chase analysis. The cells were pulse-labeled with [35S]-methionine for 15 minutes and were chased with cell lysate and culture medium for various periods. As shown in Fig 2, PAI-1 protein was not synthesized in UT-7/OR cells. In contrast, the protein produced for 15 minutes was apparent in the UT-7/TPO cell lysate and did not appear in the culture medium. Although UT-7/TPO cells express protease-activated receptor-1 (PAR-1) and PAR-3, a recently identified thrombin receptor of platelets and megakaryocytes,33addition of thrombin to the pulse-labeled cells had no effect on the release of PAI-1 protein (data not shown).

Fig. 2.

Pulse-chase analysis of PAI-1 in UT-7/OR, UT-7/TPO, and HUVEC. UT-7/OR, UT-7/TPO, and HUVEC were incubated for 60 minutes in methionine-free medium and then pulse labeled with35S-methionine for 15 minutes, followed by a chase with excess of unlabeled methionine for the indicated period. Cells and media were harvested at appropriate intervals, and PAI-1 was immunoprecipitated from each sample with mouse antihuman PAI-1 MoAb (JTI-3 and JTI-4) or normal mouse IgG. Subsequently, they were subjected to SDS-polyacrylamide gel electrophoresis (10% separating gels) and autoradiography.

Fig. 2.

Pulse-chase analysis of PAI-1 in UT-7/OR, UT-7/TPO, and HUVEC. UT-7/OR, UT-7/TPO, and HUVEC were incubated for 60 minutes in methionine-free medium and then pulse labeled with35S-methionine for 15 minutes, followed by a chase with excess of unlabeled methionine for the indicated period. Cells and media were harvested at appropriate intervals, and PAI-1 was immunoprecipitated from each sample with mouse antihuman PAI-1 MoAb (JTI-3 and JTI-4) or normal mouse IgG. Subsequently, they were subjected to SDS-polyacrylamide gel electrophoresis (10% separating gels) and autoradiography.

The effect of TPO and other ligands on PAI-1 mRNA expression.

Because UT-7/EPO cells are committed to the erythroid lineage,22 and do not express endogenous c-Mpl, we introduced full-length c-mpl cDNA into the UT-7/EPO cells (Fig3A). We selected neomycin-resistant clones expressing high levels of c-Mpl on the surface of the cells as determined by flow cytometry with a polyclonal antibody against the extracellular domain of c-Mpl. These cells were designated as UT-7/EPO-MPL cells. Using UT-7/OR, UT-7/TPO, UT-7/EPO, and UT-7/EPO-MPL cells, we examined the effect of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, phorbol 12-myristate 13-acetate (PMA), and TPO on the expression of PAI-1 mRNA. TNF-α and IL-1β induced the expression of PAI-1 mRNA within 48 hours in all cell lines (Fig 4). Incubation with 10 nmol/mL PMA resulted in a more-rapid increase in the PAI-1 mRNA expression. Although the expressions of GP-Ibα and PF-4 mRNA were also upregulated by PMA for 24 hours, UT-7/EPO-MPL cells did not obviously change their morphology, except for mild adhesion to the plastic dishes and extension of pseudopadia (data not shown). In contrast, TPO did not increase PAI-1 mRNA expression until day 7 (Fig3B). The effect of TPO on the growth of UT-7/EPO-MPL cells was similar to that of EPO when incubated for several days. However, over a long-term culture of 14 to 28 days, the TPO-stimulated UT-7/EPO-MPL cells gradually enlarged and became nearly identical with UT-7/TPO cells. In parallel with these morphological changes, PAI-1 transcript was increased and visible by RT-PCR on day 14.

Fig. 3.

Effect of TPO on the expression of PAI-1 mRNA in UT-7/EPO transfected with c-Mpl (UT-7/EPO-MPL). (A) Total cellular RNAs extracted from UT-7/EPO, UT-7/EPO transfected with c-Mpl (UT-7/EPO-MPL), and UT-7/TPO were RT and amplified by PCR. The RT-PCR products were resolved by agarose gel electrophoresis, and the bands were transferred to a membrane and hybridized to32P-labeled c-mpl P cDNA probes. (B) UT-7/EPO-MPL cells were cultured in the presence of 1 U/mL EPO or 10 ng/mL TPO. The cells were harvested at the indicated time points (0 to 28 days) and subjected to RT-PCR analysis for PAI-1 and GAPDH mRNA expression. PAI-1 RT-PCR products were analyzed on 2% agarose gels and transferred to membranes and hybridized to 32P-labeled PAI-1 cDNA probes.

Fig. 3.

Effect of TPO on the expression of PAI-1 mRNA in UT-7/EPO transfected with c-Mpl (UT-7/EPO-MPL). (A) Total cellular RNAs extracted from UT-7/EPO, UT-7/EPO transfected with c-Mpl (UT-7/EPO-MPL), and UT-7/TPO were RT and amplified by PCR. The RT-PCR products were resolved by agarose gel electrophoresis, and the bands were transferred to a membrane and hybridized to32P-labeled c-mpl P cDNA probes. (B) UT-7/EPO-MPL cells were cultured in the presence of 1 U/mL EPO or 10 ng/mL TPO. The cells were harvested at the indicated time points (0 to 28 days) and subjected to RT-PCR analysis for PAI-1 and GAPDH mRNA expression. PAI-1 RT-PCR products were analyzed on 2% agarose gels and transferred to membranes and hybridized to 32P-labeled PAI-1 cDNA probes.

Fig. 4.

Effect of TNF-, IL-1β, and PMA on the expression of PAI-1 mRNA in UT-7 cell lines. At the initiation of (A) UT-7/OR, (B) UT-7/TPO, (C) UT-7/EPO, and (D) UT-7/EPO-MPL culture, any one of 10 ng/mL TNF-, 10 ng/mL IL-1β, and 10 nmole/mL PMA was added to the culture. The cells were harvested at the indicated times (0, 24, and 48 hours) and examined by RT-PCR followed by Southern blot analysis. Each value (mean ± SD, n = 3) shows the ratio of PAI-1 expression after 24 and 48 hours of culture versus the respective control (0 hour) expression.

Fig. 4.

Effect of TNF-, IL-1β, and PMA on the expression of PAI-1 mRNA in UT-7 cell lines. At the initiation of (A) UT-7/OR, (B) UT-7/TPO, (C) UT-7/EPO, and (D) UT-7/EPO-MPL culture, any one of 10 ng/mL TNF-, 10 ng/mL IL-1β, and 10 nmole/mL PMA was added to the culture. The cells were harvested at the indicated times (0, 24, and 48 hours) and examined by RT-PCR followed by Southern blot analysis. Each value (mean ± SD, n = 3) shows the ratio of PAI-1 expression after 24 and 48 hours of culture versus the respective control (0 hour) expression.

Expression of PAI-1 during short-term liquid culture of CD34+ cord blood cells.

To verify the parallel increase of PAI-1 mRNA expression with apparent morphological change to a more megakaryocytic-like cell, we examined the expression of c-mpl P and PAI-1 mRNA transcripts in the presence of TPO in a short-term liquid culture of cord blood. CD34+ cells were isolated from normal human cord blood using a magnetic cell sorting system and cultured for 6 to 12 days in a megakaryocyte culture medium as previously described.31,32,34 As shown in the upper panel of Fig 5, cells with morphologic features of immature megakaryocytes, including basophilic cytoplasm and budding, began to appear after 3 days of culture. Mature megakaryocytes-like features with polyploid nuclei were observed after 6 days. Total cellular RNA was isolated on days 1, 3, and 6, and the expression of c-mpl P and PAI-1 mRNA was examined by semiquantitative RT-PCR analysis. On day 1, CD34+ cells expressed neither c-mpl P nor PAI-1 mRNA transcript just after isolation, whereas GAPDH mRNA, which was used as an internal control, was readily detectable (Fig 5, lower panel, day 1). The expression of c-mpl P was observed after 3 days of culture, which is apparently preceded by polyploidization. In contrast, PAI-1 mRNA transcript was not present on day 3, but was apparent only after 6 days of culture in parallel with the appearance of mature megakaryocytes. This chronological order of PAI-1 expression is consistent with the findings obtained with the UT-7 cell lines.

DISCUSSION

Platelets can modify fibrinolysis through several mechanisms. Platelet arterial thrombi are more resistant to lysis by plasminogen activators than platelet-poor thrombi.35 Several studies suggest that PAI-1, which inhibits fibrinolysis by binding irreversibly to the active site of t-PA and u-PA, is a major determinant of the resistance of platelet-rich clots to lysis by t-PA.36,37 Activation of platelets results in release of PAI-1 from the α-granules. In this study, we have investigated the mechanism of production of PAI-1 during the process of megakaryocyte differentiation. Among the UT-7 cell lines, the UT-7/OR cells have some properties of megakaryocytes, including polyploidy and positive staining of platelet peroxidase.20 In contrast, UT-7/EPO cells have progressed further in erythroid development than the parent UT-7 cells,22 and UT-7/GM cells are of the erythroid-megakaryocytic bipotential lineage, because they have the capacity to differentiate into erythroid or megakaryocytic lineages by treatment with EPO and TPO, respectively.23Morphologically, UT-7/TPO cells have mature megakaryocytic characteristics, such as a developed demarcation membrane in the cytoplasm and a multinucleated appearance with a high level of DNA content.21 In addition, UT-7/TPO cells contain high levels of GP-Ibα, PF4 and PAI-1 mRNA (Fig 1). Therefore, we initially hypothesized that PAI-1 expression might be closely related to megakaryocyte differentiation induced by TPO. In contrast, u-PA mRNA transcript was decreased in the UT-7/TPO cells and not detected in platelets. Because immature cells and many cell lines originated from cancer cells are apt to have both PAI-1 and u-PA mRNAs,38,39 this result may also support the differentiation of UT-7, developed from megakaryoblastic leukemia, into a more megakaryocytic cell line, UT-7/TPO.

PAI-1 protein was synthesized de novo in UT-7/TPO cells as shown by the [35S]-methionine–labeled pulse-chase analysis (Fig 2). Interestingly, unlike HUVECs and other cell lines,40UT-7/TPO cells did not secrete PAI-1 immediately after synthesis (Fig2). This may represent a storage pool of PAI-1, although whether or not it is eventually secreted awaits further experimentation. Furthermore, PAI-1 was not secreted after thrombin stimulation, although UT-7/TPO cells express both PAR-1 and PAR-3 protease receptors capable of binding thrombin (data not shown). This indicates that the signal transduction system for these receptors probably differ between megakaryocytes and platelets.

To clarify the mechanism of TPO-dependent induction, we compared the induction of PAI-1 by other agents such as PMA, IL-1β, and TNF-α. Analyses of the mechanism involved in megakaryocytic differentiation and the expression of megakaryocytic genes were performed with PMA-induced human megakaryocytic cell line models.41Hill40 and Konkle42 showed that PMA-induced PAI-1 mRNA expression and the accumulation of PAI-1 protein in Dami cells and CHRF-288 cell lines, respectively. However, because PMA is a chemical agent and not a physiological regulator, its action on megakaryoblastic cell lines may not always mimic normal megakaryocytopoiesis. Because c-Mpl specifically regulates megakaryocytopoiesis and thrombopoiesis through activation by its ligand TPO,18 we forced c-Mpl expression in UT-7/EPO cells, which do not express endogenous c-Mpl, and studied the effect of TPO stimulation on the transfected cells (UT-7/EPO-MPL). UT-7/EPO-MPL cells not only depended on EPO for growth and survival, but they also acquired the ability to proliferate and differentiate in the presence of TPO. PAI-1 mRNA expression was induced by PMA in UT-7/EPO-MPL cells within 12 to 48 hours (Fig 4). In addition, the proinflammatory cytokines, IL-1β and TNF-α, which are known to enhance PAI-1 production through increased transcription rate, also induced PAI-1 mRNA in UT-7/EPO-MPL for 48 hours without inducing apparent morphological changes. Furthermore, the comparisons of PAI-1 mRNA expression between UT-7/OR and UT-7/TPO or UT-7/EPO and UT-7/EPO-MPL show that upregulations of PAI-1 production by these cytokines are independent of c-Mpl presentation. In contrast, PAI-1 mRNA transcripts were detectable in UT-7/EPO-MPL cells only after 14 days stimulation with TPO, a time when these cells changed morphology to mature megakaryocytes-like feature (Fig 3). Our data suggest that megakaryocytes response to TPO through c-Mpl is essential for their maturation, differentiation, and constitutive expression of PAI-1 mRNA. However, because PAI-1 message is not detected in platelets but the protein is,9,10 it is also possible that mRNA turnover might contribute in part to the regulation of PAI-1 message level.43 Recently, PU.1/Spi-1, an Ets-related transcription factor, was found to be selectively induced by TPO and it increased the transcription activity of megakaryocyte-related gene promoters, whereas PMA did not.44 Because the PAI-1 gene promoter contains several Ets-binding core sequences GGAA (GGAA −621, −460, −396, and −61) and a predicted GATA-binding site (GATA −426),45 these elements could be responsible for the TPO action.

In normal cord blood CD34/CD41+ colonies induced by TPO, c-mpl mRNA expression was induced before the detection of PAI-1 mRNA transcript and PAI-1 mRNA expression peaked at 6 days (Fig 5), and reached a plateau on day 12 (data not shown). Although PAI-1 mRNA appeared faster in these colonies than in the UT-7/EPO-MPL cells, a difference in the maturation stage between these two groups may be a contributing factor. Thus, the expression of PAI-1 was first shown at the transcriptional level both in leukemic and normal megakaryocytes.

Fig. 5.

Expression of PAI-1 and c-Mpl mRNA during short-term culture of normal human cord blood CD34+ progenitor cells with TPO. The CD34+ cells were isolated from human cord blood using antihuman CD34 MoAb bound to magnetic microspheres and cultured in the presence of 10 ng/mL TPO. The cells were harvested at the indicated time points and subjected to morphologic examination on Wright-Giemsa staining cytospin slides (upper panel) and RT-PCR analysis for PAI-1, c-mpl P and GAPDH mRNA expression (lower panel). Amplified products were analyzed on 2% agarose gels followed by ethidium bromide staining. M, molecular size marker.

Fig. 5.

Expression of PAI-1 and c-Mpl mRNA during short-term culture of normal human cord blood CD34+ progenitor cells with TPO. The CD34+ cells were isolated from human cord blood using antihuman CD34 MoAb bound to magnetic microspheres and cultured in the presence of 10 ng/mL TPO. The cells were harvested at the indicated time points and subjected to morphologic examination on Wright-Giemsa staining cytospin slides (upper panel) and RT-PCR analysis for PAI-1, c-mpl P and GAPDH mRNA expression (lower panel). Amplified products were analyzed on 2% agarose gels followed by ethidium bromide staining. M, molecular size marker.

In summary, we report here that PAI-1 mRNA is expressed in megakaryocytes and is accompanied by de novo PAI-1 protein synthesis. This endogenous PAI-1 synthesis may be closely related to TPO-dependent megakaryocyte maturation.

ACKNOWLEDGMENT

We thank Dr Hiroshi Tomizuka for helpful discussion regarding the flow cytometry analyses of cultured cells.

Supported in part by a Grant-in-Aid for Scientific Research, No. 09470234 from the Ministry of Education, Science, Sports, and Culture of Japan, by a grant from Nippon Foundation, and by a grant from Jichi Medical School Young Investigator Award.

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. section 1734 solely to indicate this fact.

REFERENCES

REFERENCES
1
Reed
GL
Matsueda
GR
Haber
E
Platelet factor XIII increases the fibrinolytic resistance of platelet-rich clots by accelerating the crosslinking of α2-antiplasmin to fibrin.
Thromb Haemostat
68
1992
315
2
Rubens
FD
Perry
DW
Hatton
MWC
Bishop
PD
Packham
MA
Kinlough-Rathbone
RL
Platelet accumulation on fibrin-coated polyethylene: Role of platelet activation and factor XIII.
Thromb Haemostat
73
1995
850
3
Stringer
HAR
van Swieten
P
Heijnen
HFG
Sixma
JJ
Pannekoek
H
Plasminogen activator inhibitor-1 released from activated platelets plays a key role in thrombolysis resistance.
Arterioscler Thromb
14
1994
1452
4
Robbie
LA
Booth
NA
Croll
AM
Bennett
B
The roles of α2-antiplasmin and plasminogen activator inhibitor 1 (PAI-1) in the inhibition of clot lysis.
Thromb Haemostat
70
1993
301
5
Kruithof
EKO
Nicolosa
G
Bachmann
F
Plasminogen activator inhibitor 1: Development of a radioimmunoassay and observations on its plasma concentration during venous occlusion and after platelet aggregation.
Blood
70
1987
1645
6
Hekman
CM
Loskutoff
DJ
Endothelial cells produce a latent inhibitor of plasminogen activators that can be activated by denaturants.
J Biol Chem
260
1985
11581
7
Kunitada
S
FitzGerald
GA
Fitzgerald
DJ
Inhibition of clot lysis and decreased binding of tissue-type plasminogen activator as a consequence of clot retraction.
Blood
79
1992
1420
8
Fay
WP
Eitzman
DT
Shapiro
AD
Madison
EL
Ginsburg
D
Platelets inhibit fibrinolysis in vitro by both plasminogen activator inhibitor-1 dependent and independent mechanisms.
Blood
83
1994
351
9
Simpson
AJ
Booth
NA
Moore
NR
Bennett
B
Distribution of plasminogen activator inhibitor (PAI-1) in tissues.
J Clin Pathol
44
1991
139
10
Pérez
GN
Nelles
L
Deckmyn
H
Vermylen
J
PAI-1 is not synthesized in human platelets.
Thromb Haemost
65
1991
719
11
Lok
S
Kaushansky
K
Holly
RD
Kuijper
JL
Lofton-Day
CE
Oort
PJ
Grant
FJ
Heipel
MD
Burkhead
SK
Kramer
JM
Bell
LA
Sprecher
CA
Blumberg
H
Jonson
R
Prunkard
D
Ching
AFT
Mathewes
SL
Bailey
MC
Forstrom
JW
Buddle
MM
Osborn
SG
Evans
SJ
Sheppard
PO
Presnell
SR
O’hara
PJ
Hagen
FS
Roth
GJ
Foster
DC
Cloning and expression of murine thrombopoietin cDNA and stimulation of platelet production in vivo.
Nature
369
1994
565
12
de Sauvage
FJ
Hass
PE
Spencer
SD
Malloy
BE
Gurney
AL
Spencer
SA
Darbonne
WC
Henzel
WJ
Wong
SC
Kuang
W
Oles
KJ
Hultgren
B
Solberg Jr
LA
Goeddel
DV
Eaton
DL
Stimulation of megakaryocytopoiesis and thrombopoiesis by the c-Mpl ligand.
Nature
369
1994
533
13
Kaushansky
K
Lok
S
Holly
RD
Broudy
VC
Lin
N
Bailey
MC
Forstrom
JW
Buddle
MM
Oort
PJ
Hagen
FS
Roth
GJ
Papayannopoulou
T
Foster
DC
Promotion of megakaryocyte progenitor expansion and differentiation by the c-Mpl ligand thrombopoetin.
Nature
369
1994
568
14
Wendling
F
Maraskovsky
E
Debili
N
Florindo
C
Teepe
M
Titeux
M
Methia
N
Breton-Gorius
J
Cosman
D
Vainchenker
W
c-Mpl ligand is a humoral regulator of megakaryocytopoiesis.
Nature
369
1994
571
15
Bartley
TD
Bogenberger
J
Hunt
P
Li
YS
Lu
HS
Martin
F
Chang
MS
Samal
B
Nichol
JL
Swift
S
Johnson
MJ
Hsu
RY
Parker
VP
Suggs
S
Skrine
JD
Merewether
LA
Clongston
C
Hsu
E
Hokom
MM
Hornkohl
A
Choi
E
Pangelinan
M
Sun
Y
Mar
V
McNinch
J
Simonet
L
Jacobsen
F
Xie
C
Shutter
J
Chute
H
Basu
R
Selander
L
Trollinger
D
Sieu
L
Padilla
D
Trail
G
Elliott
G
Izumi
R
Covey
T
Crouse
J
Gracia
A
Xu
W
Del Castillo
J
Biron
J
Cole
S
Hu
MCT
Pacifici
R
Ponting
I
Saris
C
Wen
D
Yung
YP
Lin
H
Bosselman
RA
Identification and cloning of a megakaryocyte growth and development factor that is a ligand for the cytokine receptor Mpl.
Cell
77
1994
1117
16
Debili
N
Wendling
F
Katz
A
Guichard
J
Breton-Gorius
J
Hunt
P
Vainchenker
W
The Mpl-ligand or thrombopoietin or megakaryocyte growth and differentiative factor has both direct proliferative and differentiative activities on human megakaryocyte progenitors.
Blood
86
1995
2516
17
Kaushansky
K
Broudy
VC
Lin
N
Jorgensen
MJ
McCarty
J
Fox
N
Zucker-Franklin
D
Lofton-Day
C
Thrombopoietin, the Mpl ligand, is essential for full megakaryocyte development.
Proc Natl Acad Sci USA
92
1995
3234
18
Gurney
AL
Carver-Moore
K
de Sauvage
FJ
Moore
MW
Thrombocytopenia in c-mpl-deficient mice.
Science
265
1994
1445
19
de Sauvage
FJ
Carver-Moore
K
Luoh
S
Ryan
A
Dowd
M
Eaton
DL
Moore
MW
Physiological regulation of early and late stages of megakaryocytopoiesis by thrombopoietin.
J Exp Med
183
1996
651
20
Komatsu
N
Nakauchi
H
Miwa
A
Ishihara
T
Eguchi
M
Moroi
M
Okada
M
Sato
Y
Wada
H
Yawata
Y
Suda
T
Miura
Y
Establishment and characterization of a human leukemic cell line with megakaryocytic features: Dependency on granulocyte-macrophage colony-stimulating factor, interleukin 3, or erythropoietin for growth and survival.
Cancer Res
51
1991
341
21
Komatsu
N
Kunitama
M
Yamada
M
Hagiwara
T
Kato
T
Miyazaki
H
Eguchi
M
Yamamoto
M
Miura
Y
Establishment and characterization of the thrombopoietin-dependent megakaryocytic cell line, UT-7/TPO.
Blood
87
1996
4552
22
Komatsu
N
Yamamoto
M
Fujita
H
Miwa
A
Hatake
K
Endo
T
Okano
H
Katsube
T
Fukumaki
Y
Sassa
S
Miura
Y
Establishment and characterization of an erythropoietin-dependent subline, UT-7/Epo, derived from human leukemic cell line, UT-7.
Blood
82
1993
456
23
Komatsu
N
Kirito
K
Shimizu
R
Kunitama
M
Yamada
M
Uchida
M
Takatoku
M
Eguchi
M
Miura
Y
In vitro development of erythroid and megakaryocytic cells from a UT-7 subline, UT-7/GM.
Blood
89
1997
4021
24
Masuyama
J
Minato
N
Kano
S
Mechanisms of lymphocyte adhesion to human vascular endothelial cells in culture.
J Clin Invest
77
1986
1596
25
Chomczynski
P
Sacchi
N
Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.
Anal Biochem
162
1987
156
26
Takatoku
M
Kametaka
M
Shimizu
R
Miura
M
Komatsu
N
Identification of functional domains of the human thrombopoietin receptor required for growth and differentiation of megakaryocytic cells.
J Biol Chem
272
1997
7259
27
Harlow
E
Lane
D
Antibodies.
1988
Cold Spring Harbor Laboratory
New York, NY
28
Sakata
Y
Murakami
T
Noro
A
Mori
K
Matsuda
M
The specific activity of plasminogen activator inhibitor-1 in disseminated intravascular coagulation with acute promyelocytic leukemia.
Blood
77
1991
1949
29
Laemlli
UK
Cleavage of structural proteins during the assembly of the head of bacteriophage T4.
Nature
227
1970
680
30
Kikuchi
J
Furukawa
Y
Iwase
S
Terui
Y
Nakamura
M
Kitagawa
S
Kitagawa
M
Komatsu
N
Miura
Y
Polyploidization and functional maturation are two distinct processes during megakaryocytic differentiation: Involvement of cylin-dependent kinase inhibitor p21 in polyploidization.
Blood
89
1997
3980
31
Debili
N
Issaad
C
Massé
J
Guichard
J
Katz
A
Breton-Gorius
J
Vainchenker
W
Expression of CD34 and platelet glycoproteins during human megakaryocytic differentiation.
Blood
80
1992
3022
32
Choi
ES
Nichol
JL
Hokom
MM
Hornkohl
AC
Hunt
P
Platelets generated in vitro from proplatelet-displaying human megakaryocytes are functional.
Blood
85
1995
402
33
Ishihara
H
Connolly
AJ
Zeng
D
Kahn
ML
Zheng
YW
Timmons
C
Tram
T
Coughlin
SR
Protease-activated receptor 3 is a second thrombin receptor in humans.
Nature
386
1997
502
34
Sasaki
Y
Takahashi
T
Tanaka
I
Nakamura
K
Okubo
Y
Nakagawa
O
Narumiya
S
Nakao
K
Expression of prostacyclin receptor in human megakaryocytes.
Blood
90
1997
1039
35
Jang
I
Gold
HK
Ziskind
AA
Fallon
JT
Holt
RE
Leinbach
RC
May
JW
Collen
D
Differential sensitivity of erythrocyte-rich and platelet-rich arterial thrombi to lysis with recombinant tissue-type plasminogen activator.
Circulation
79
1989
920
36
Levi
M
Biemond
BJ
van Zonneveld
A
Cate
JW
Pannekoek
H
Inhibition of plasminogen activator inhibitor-1 acitivity results in promotion of endogenous thrombolysis and inhibition of thrombus extension in models of experimental thrombosis.
Circulation
85
1992
305
37
Farrehi
PM
Ozaki
CK
Carmeliet
P
Fay
WP
Regulation of arterial thrombolysis by plasminogen activator inhibitor-1 in mice.
Circulation
97
1998
1002
38
de Vries
TJ
Quax
PH
Denijn
M
Verrijp
KN
Verheijen
JH
Verspaget
HW
Weidle
UH
Ruiter
DJ
van Muijen
GN
Plasminogen activators, their inhibitors, and urokinase receptor emerge in late stages of melanocytic tumor progression.
Am J Pathol
144
1994
70
39
Lund
LR
Romer
J
Ronne
E
Ellis
V
Blasi
F
Dano
K
Urokinase-receptor biosynthesis, mRNA level and gene transcription are increased by transforming growth factor beta 1 in human A549 lung carcinoma cells.
EMBO J
10
1991
3399
40
Hill
SA
Shaughnessy
SG
Joshua
P
Ribau
J
Austin
RC
Podor
TJ
Differential mechanisms targeting type 1 plasminogen activator inhibitor and vitronectin into the storage granules of a human megakaryocytic cell line.
Blood
87
1996
5061
41
Lemarchandel
V
Ghysdael
J
Mignotte
V
Rahuel
C
Romeo
PH
GATA and Ets cis-acting sequences mediate megakaryocyte-specific expression.
Mol Cell Biol
13
1993
668
42
Konkle
BA
Schick
PK
He
X
Liu
RJ
Mazur
EM
Plasminogen activator inhibitor-1 mRNA is expressed in platelets and megakaryocytes and the megakaryoblastic cell line CHRF-288.
Arterioscler Thromb
13
1993
669
43
Sachs
AB
Messenger RNA degradation in eukaryotes.
Cell
74
1993
413
44
Doubeikovski
A
Uzan
G
Dougeikovski
Z
Prandini
M
Porteu
F
Gisselbrecht
S
Dusanter-Fourt
I
Thrombopoietin-induced expression of the glycoprotein IIb gene involves the transcription factor PU.1/Spi-1 in UT7-Mpl Cells.
J Biol Chem
272
1997
24300
45
Riccio
A
Lund
LR
Sartorio
R
Lania
A
Andreasen
PA
Danø
K
Blasi
F
The regulatory region of the human plasminogen activator inhibitor type-1 gene.
Nucleic Acids Res
16
1988
2805