Identification of functional domains of the human thrombopoietin receptor required for growth and differentiation of megakaryocytic cells.

To identify the functional domains of the human thrombopoietin (TPO) receptor essential for proliferation and megakaryocytic differentiation, we introduced human wild type c-mpl cDNA and deletion mutants of c-mpl cDNA into the human erythropoietin (EPO)-dependent cell line UT-7/EPO that does not express endogenous c-Mpl. TPO induced the proliferation and megakaryocytic differentiation of UT-7/EPO expressing wild type c-Mpl, as evidenced by increased levels of the CD41 antigen specific for cells of the megakaryocytic lineage and by changes in morphology. Mutational analysis of the cytoplasmic domain of c-Mpl identified four functional regions: (a) two C-terminal regions (amino acids 575-586 and 615-630) containing a domain essential for cell proliferation and megakaryocytic differentiation but not for DNA synthesis; (b) a region (amino acids 587-614) containing a negative domain for TPO-induced cell proliferation and megakaryocytic differentiation; and (c) a region (amino acids 565-574) including a box2 motif that is required for DNA synthesis. These deletion mutants will provide useful materials for analyzing the signals specific for TPO-induced proliferation and megakaryocytic differentiation.


To identify the functional domains of the human thrombopoietin (TPO) receptor essential for proliferation and megakaryocytic differentiation, we introduced human wild type c-mpl cDNA and deletion mutants of c-mpl cDNA into the human erythropoietin (EPO)-dependent cell line UT-7/EPO that does not express endogenous c-Mpl. TPO induced the proliferation and megakaryocytic differentiation of UT-7/EPO expressing wild type c-Mpl, as evidenced by increased levels of the CD41 antigen specific for cells of the megakaryocytic lineage and by changes in morphology. Mutational analysis of the cytoplasmic domain of c-Mpl identified four functional regions: (a) two C-terminal regions (amino acids 575-586 and 615-630) containing a domain essential for cell proliferation and megakaryocytic differentiation but not for DNA synthesis; (b) a region (amino acids 587-614) containing a negative domain for TPOinduced cell proliferation and megakaryocytic differentiation; and (c) a region (amino acids 565-574) including a box2 motif that is required for DNA synthesis. These deletion mutants will provide useful materials for analyzing the signals specific for TPO-induced proliferation and megakaryocytic differentiation.
Thrombopoietin (TPO) 1 supports the proliferation and differentiation of megakaryocyte progenitor cells as well as the differentiation of megakaryocytes. TPO exerts its action by binding to a specific cell surface receptor encoded by the protooncogene c-mpl (1)(2)(3)(4)(5). The c-mpl protooncogene was first identified as the cellular homolog of the viral oncogene v-mpl in the myeloproliferative leukemia virus (6). Based on homology with a member of the cytokine receptor superfamily, however, the c-mpl gene was predicted to encode a cytokine receptor (7)(8)(9). Experiments with an antisense oligomer against c-mpl and c-mpl-deficient mice revealed that the c-mpl gene encodes the receptor for TPO.
Like other members of the cytokine receptor superfamily, two regions of conserved sequences termed box1 and box2 have been identified in the intracellular domain of c-Mpl (8,10), and it was found that these motifs are essential for TPO-induced mitogenesis (11,12). In addition, transfection experiments with murine mutated c-mpl cDNA into UT-7 showed that the region distal to box2 is necessary for TPO-induced megakaryocytic differentiation (12). However, since c-mpl transcripts and c-Mpl proteins are detectable in UT-7 by reverse transcriptase polymerase chain reactions (RT-PCR) and Western blotting (8,13), the transfectants may respond to TPO through endogenous c-Mpl but not exogenous c-Mpl. Indeed, TPO supported the proliferation and megakaryocytic differentiation of UT-7/GM, a subline of UT-7 (14). 2 In this study we introduced human c-mpl cDNA into UT-7/EPO that does not express endogenous c-Mpl and generated in vitro models for cellular proliferation and megakaryocytic differentiation.

MATERIALS AND METHODS
Hematopoietic Growth Factors and Reagents-Recombinant human TPO and rabbit anti-human c-Mpl polyclonal antibody were 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 was provided by Sumitomo Pharmaceutical Company (Osaka, Japan). Human cDNAs of full-length c-mpl P (wild type) and c-mpl K (truncated) clones were provided by Dr. M. Okada (Eisai, Tsukuba, Japan) and Dr. S. Gisselbrecht (INSERM, Paris, France), respectively. A monoclonal antibody P2 (CD41, specific for platelet GPIIb-IIIa) was purchased from Immunotech (Marseilles, France).
Cell Culture-The UT-7 cell line was established from bone marrow cells obtained from a patient with acute megakaryocytic leukemia (15) and maintained in liquid culture with Iscove's modified Dulbecco's medium (IMDM; Life Technologies, Inc.) containing 10% fetal calf serum (FCS; HyClone Laboratories, Logan, UT) and 1 ng of granulocytemacrophage colony-stimulating factor/ml. UT-7/GM was isolated after long term culture of UT-7 cells and maintained like UT-7. 2 UT-7/EPO (16) and its transfectants were continuously maintained in IMDM containing 10% FCS and 1 unit of EPO/ml. The UT-7/TPO was maintained in IMDM containing 10% FCS and 10 ng of TPO/ml (14).
Preparation of c-mpl Deletion Mutants and Their Transfectants-To directionally delete the 3Ј-coding region of c-mpl P cDNA, the EcoRI cDNA fragment was inserted into the EcoRI site of pUC118. Plasmid pUC118, containing c-mpl cDNA, was digested with Sse8387I and XbaI, then the pUC118 fragment was digested exonuclease III at 25°C, and the reaction was stopped after various periods from 15 s to 5 min. The size and DNA sequence of the digested fragments were determined and inserted into pRcCMV. UT-7/EPO cells were transfected with pRcCMV containing full-length or various deletion mutants by conventional electroporation (250 V, 960 FD). These transfectants were selected as clones resistant to neomycin (1 mg/ml, Life Technologies, Inc.).
In Vitro Cell Proliferation Assay-DNA synthesis was measured by thymidine incorporation assay as follows. Briefly, cells were resuspended at a density of 1 ϫ 10 4 /0.1 ml in IMDM containing 5% FCS and incubated in 96-well culture-treated plates in the absence or presence of serial dilutions of TPO for 3 days. later the amount of radioactivity incorporated in cells was measured in a liquid scintillation counter. Viable cell numbers were assessed by trypan blue dye exclusion.
Analysis of Cell Surface Markers by Immunofluorescence-Cell-surface antigens were detected by immunofluorescence staining with P2 monoclonal or rabbit anti-human c-Mpl polyclonal antibodies. In brief, UT-7/EPO transfectant cells were incubated for 30 min at 4°C with the appropriately diluted antibody. After washing, the cells were incubated with fluorescein-labeled second antibody for 30 min at 4°C. After a second washing, signals were analyzed using a Becton Dickinson flow cytometer (FACScan; Becton Dickinson, Mountain View, CA), using 10,000 cells for each sample.
RT-PCR and Southern Blotting Analysis-Total cellular RNA was isolated from cells according to the methods of Chomczynski and Sacchi (17). RT-PCR was performed using oligonucleotide primers as follows. The c-mpl P forward 5Ј-AGACTGAGGCATGCCCTGTGG-3Ј (nucleotides 1567-1587) and reverse 5Ј-TGAAGGCTGCTGCCAATAGCT-3Ј (nucleotides 1908 -1888) primers amplified a 342-base pair fragment of the c-mpl P cDNA. The c-mpl K forward 5Ј-CCCACCTACCAAGGTC-CCTGGA-3Ј (nucleotides 1402-1422) and reverse 5Ј-TTAGAGTGTA-AGGAGCCGCGG-3Ј (nucleotides 1740 -1720) amplified a 339-base pair fragment of the c-mpl K cDNA. The ␤-actin forward 5Ј-TACCACTG-GCATCGTGATGGACT-3Ј and reverse 5Ј-TCCTTCTGCATCCTGTCG-GCAAT-3Ј amplified a 506-base pair fragment of the ␤-actin cDNA as a semiquantitative control. The cDNA was synthesized by reverse transcription using a commercial kit (Boehringer Mannheim, FRG). Total cellular RNA (1 g) was reverse transcribed using oligo(dT) primers followed by 35 PCR amplification cycles (94°C for 20 s, primer annealing at 56°C for 30 s, extension at 72°C for 40 s) in a Perkin-Elmer thermal cycler (GeneAmp PCR System 9600), and a final incubation at 60°C for 7 min. Amplification products were separated on 2% agarose-Tris-acetate-EDTA gels stained with ethidium bromide and photographed. The RT-PCR products were transferred to nylon membranes (Zeta-Probe; Bio-Rad) and incubated with c-mpl P or c-mpl K cDNA labeled with [␣-32 P]CTP by random priming. After an overnight incubation at 43°C in the presence of 50% formamide, blots were washed three times with 2 ϫ SSC, 0.5 ϫ SSC, 0.1 ϫ SSC, plus 0.1% SDS for 15 min each. The membranes were autoradiographed using Kodak XAR-5 film with an intensifying screen at Ϫ70°C.

RESULTS AND DISCUSSION
Endogenous c-Mpl Is Not Expressed in UT-7/EPO-We detected both P and K forms of c-mpl by Northern blotting in UT-7/GM and UT-7/TPO but not in UT-7 and UT-7/EPO cells (14). To detect small amounts of the mRNA, we performed RT-PCR using total cellular RNA from UT-7 and its sublines including UT-7/EPO. Ethidium bromide staining detected PCR products (both forms of c-mpl mRNA) in the samples from UT-7/GM, UT-7/TPO, and, to a lesser degree, from UT-7, but not from UT-7/EPO ( Fig. 1; left panel), indicating that UT-7/ EPO cells do not express endogenous c-Mpl. It was confirmed by Southern blotting of the PCR products with a 32 P-labeled c-mpl cDNA probe ( Fig. 1; right panel).
Preparation of UT-7/EPO Expressing Exogenous c-Mpl (UT-7/EPO-MplWT)-Based on these observations, we initially introduced full-length c-mpl P cDNA into the UT-7/EPO cells and examined whether exogenously expressed c-Mpl can transduce the signals for proliferation and megakaryocytic differentiation of the cells (Fig. 2A). We selected neomycin-resistant clones expressing high levels of c-Mpl on the surface of the cells by flow cytometry with a polyclonal antibody against the extracellular domain of c-Mpl (Fig. 2B) and designated them UT-7/EPO-MplWT.

Effect of TPO on the Proliferative Response of UT-7/EPO-MplWT-[ 3 H]
Thymidine incorporation assay revealed that the growth activity of TPO toward UT-7/EPO-MplWT in shortterm culture was almost similar to that of EPO (Fig. 3A). However, when the cell number was assessed over longer periods, the peak of growth was slightly reduced in the presence of TPO (Fig. 3B). UT-7/EPO-MplWT cells could be maintained in the presence of TPO alone for at least 3 months, whereas the parent cells could not (data not shown).
Effect of TPO on the Megakaryocytic Differentiation of UT-7/EPO-MplWT-We examined whether or not TPO induces megakaryocytic differentiation of UT-7/EPO-MplWT cells. As shown in Fig. 4, the mean intensity of GPIIb-IIIa antigens on the surface was increased after an incubation with TPO for 7 days. Consistent with this, some cells of TPO-stimulated UT-7/EPO-MplWT became much larger than EPO-stimulated UT-7/EPO-MplWT cells (Fig. 5). These observations indicated that c-Mpl protein expressed exogenously in UT-7/EPO can transduce signals for not only proliferation but also for megakaryocytic differentiation. Therefore, this system would be useful for identifying the functional domains of cytoplasmic c-Mpl.
Preparation of c-Mpl Deletion Mutants-To identify the regions of the receptor required for proliferation and megakaryocytic differentiation, a series of deletion mutants of the intracellular domain of c-Mpl were prepared as shown in Fig. 2A and introduced into the UT-7/EPO cells by electroporation. Stable transfectants were isolated after neomycin selection and designated UT-7/EPO-Mpl⌬5, UT-7/EPO-Mpl⌬21, UT-7/EPO-Mpl⌬49, UT-7/EPO-Mpl⌬61, UT-7/EPO-Mpl⌬box2, UT-7/EPO-Mpl⌬81, UT-7/EPO-Mpl⌬95, UT-7/EPO-Mpl⌬box1, and UT-7/ EPO-Mpl⌬120. We picked up a single colony from a methyl cellulose semisolid medium (18) and transferred it to liquid medium containing 1 unit of EPO/ml. In the following experiments, we analyzed at least three independent clones. FACS analysis using anti-c-Mpl antibody revealed that each clone expressed significant levels of exogenous c-Mpl on the surface of the cells (Fig. 2B).
To examine whether or not TPO-induced DNA synthesis leads to cell proliferation, we cultured the transfectants in the presence of TPO in liquid culture for several days. TPO alone sustained the survival and long-term proliferation of UT-7/ EPO-Mpl⌬5 and UT-7/EPO-Mpl⌬49, but not of the other transfectants ( Fig. 3B and data not shown). These results indicate that two C-terminal regions (amino acids 575-586 and 615-630) contain a domain essential for cell proliferation but not for DNA synthesis. This finding suggests that DNA synthesis does not always lead to cell proliferation. Alternatively, additional events besides DNA synthesis may be required to induce cell proliferation.
Effect of TPO on the Megakaryocytic Differentiation of Transfectants-To identify the functional domain(s) involved in megakaryocytic differentiation, we examined whether or not TPO can induce an increase in the intensity of the megakaryocytic markers in the transfectants. This was assessed by flow cytometry with an anti-GPIIb-IIIa antibody (CD41). When UT-7/EPO-Mpl⌬5 and UT-7/EPO-Mpl⌬49 cells were cultured with TPO (10 ng/ml) for 7 days, these cells increased the expression of CD41 antigens (Fig. 4). However, when others were cultured with TPO alone, most of the cells died within a few days.
To sustain the survival of these transfectants, we cultured the transfectants in medium containing both TPO (10 ng/ml) and EPO (1 unit/ml) for 7 days, then harvested the transfectant cells for flow cytometry with the CD41 antibody. The transfectant cells cultured with 1 unit/ml of EPO alone served as the negative control. The expression of CD41 antigens significantly EPO inhibited the TPO-induced increase in GPIIb-IIIa antigens on UT-7/EPO-Mpl⌬5 but not on UT-7/EPO-MplWT or UT-7/EPO-Mpl⌬49 (Fig. 4). The results were similar in other UT-7/EPO transfectants expressing c-Mpl⌬5 (data not shown). Porteu et al. (12) reported that although TPO did not induce the megakaryocytic differentiation of UT-7 cells expressing murine mutant c-Mpl with a deletion of 24 amino acids distal to the box2 region (residues 586 -609; corresponding to the human c-Mpl region at residues 604 -626 by homology), this effect could be restored by TPO plus EPO. These findings suggested that there is a physical association between the TPO and EPO receptors, as in the case for the EPO receptor and c-kit (19). However, in our system EPO did not promote the TPO-induced megakaryocytic differentiation of any transfectant including UT-7/EPO-Mpl⌬49 (lacking residues 587-635) and UT-7/EPO-Mpl⌬21 (lacking residues 615-635). Rather, EPO negatively acted on the TPO-induced megakaryocytic differentiation in UT-7/EPO-Mpl⌬5. Although this discrepancy may be due to a structural difference between human and murine c-Mpl, the possibility that endogenous c-Mpl expressed on UT-7 cells had affected the interaction of EPO and TPO signaling pathways cannot be completely excluded in their system (8,13).
We summarize the results in Table I. These transfectants could serve as a model system for analyzing TPO signals for cellular proliferation and megakaryocytic differentiation. These projects are in progress in our laboratory.  5. Changes in morphological appearance by TPO. UT-7/ EPO-MplWT cells were incubated with EPO (A; 1 unit/ml) or TPO (B; 10 ng/ml) for 1 week and then harvested for preparing cytospin smears. Some cells of TPO-stimulated UT-7/EPO-MplWT became much larger than EPO-stimulated UT-7/EPO-MplWT cells (original magnification, ϫ 40).