©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Thrombopoietin Induces Megakaryocyte Differentiation in Hematopoietic Progenitor FDC-P2 Cells (*)

(Received for publication, May 24, 1995; and in revised form, July 3, 1995)

Yuka Nagata (1)(§) Hiroshi Nagahisa (1) (2)(§) Yoko Aida (1) Keiichi Okutomi (1) Toshiro Nagasawa (2) Kazuo Todokoro (1)(¶)

From the  (1)Tsukuba Life Science Center, The Institute of Physical and Chemical Research (RIKEN), 3-1, Koyadai, Tsukuba, Ibaraki 305, Japan and the (2)Division of Hematology, Institute of Clinical Medicine, University of Tsukuba, 1-1, Tennoudai, Tsukuba, Ibaraki 305, Japan

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Thrombopoietin (Tpo) is a cytokine that specifically regulates megakaryocyte maturation and platelet production. Little is known about the molecular and cellular mechanism of the Tpo-induced megakaryocyte maturation process including polyploidization and platelet release. To study Tpo-induced megakaryocyte differentiation, a mouse cell line FD-TPO, which responds and grows with Tpo, was established from a interleukin-3-dependent hematopoietic progenitor cell line FDC-P2. The FD-TPO cells, expressing endogenous Tpo receptor, grew with Tpo in a dose-dependent manner. Further, Tpo stimulation dramatically induced expression of megakaryocyte/erythroid-specific transcription factors GATA-1 and NF-E2 in FD-TPO cells. Flow cytometry analysis demonstrated that expression of platelet-specific cell surface antigens including CD61 (GPIIIa) dramatically increased in Tpo-stimulated FD-TPO cells and that expression of myeloid-specific antigens, Gr-1 and Mac-1, decreased. Therefore, we concluded that the binding of Tpo to FD-TPO cells induces not only cell growth but also differentiation into mature megakaryocyte-like cells, and thus this cell line was found to be useful for the study of Tpo receptor-mediated growth and differentiation signals.


INTRODUCTION

Platelets are originally derived from pluripotent hematopoietic stem cells. The stem cells differentiate into committed megakaryocyte progenitors, the megakaryocytes mature by polyploidization and cytoplasmic maturation, and finally the matured megakaryocytes release a number of platelets. Studies on the regulatory mechanisms of this unique differentiation and maturation process have been hampered, since the specific factor regulating megakaryocyte and platelet formation had not been identified.

Souyri et al.(1) isolated and characterized a murine myeloproliferative leukemia virus and found that a v-oncogene (v-mpl) encodes for a cellular membrane protein, Mpl, which has considerable homology with the genes encoding the cytokine receptor superfamily. Methia et al. (2) then found that Mpl is selectively expressed in megakaryocytes and that antisense oligonucleotides of c-mpl specifically inhibit the formation of megakaryocyte colonies but not that of erythroid or granulocyte-macrophage colonies in vitro. Recently, the cognate ligand for the orphan receptor Mpl was identified as a specific cytokine, thrombopoietin (Tpo), (^1)which regulates the differentiation and maturation of megakaryocytes(1, 2, 3, 4, 5, 6, 7) .

Lineage-specific cytokines such as granulocyte-colony-stimulating factor, erythropoietin (Epo), and Tpo activate signals that induce cell maturation/differentiation, but little is known about these cytokine receptor-mediated differentiation-specific signaling pathways. It has been described that expression of erythroid/megakaryocyte-specific transcription factors GATA-1 and NF-E2 is induced when cells are committed to an erythroid or megakaryocyte lineage and that these factors transactivate the lineage-specific gene expression and determine the lineage specificity(8, 9, 10, 11) . It was also shown that GATA-1 activates Epo receptor gene expression and that the GATA-1 gene expression is amplified by Epo receptor-mediated signal, which in turn stimulates erythroid differentiation(12, 13) .

To study the molecular and cellular mechanisms of Tpo-induced megakaryocyte maturation and platelet formation, we established a Tpo-dependent mouse cell line FD-TPO, which can be also differentiated into megakaryocyte/platelet-like cells in response to Tpo. We found that the binding of Tpo to FD-TPO cells induces expression of megakaryocyte-specific transcription factors, which may in turn stimulate the expression of megakaryocyte/platelet-specific cell surface antigens.


MATERIALS AND METHODS

Reagents

Tpo cDNA isolated from mouse stromal cells MC3T3-G2/PA6, of which the sequence was confirmed to be identical with one previously reported (4) except that Ser is replaced by Pro, was inserted downstream of the SRalpha promoter in the expression vector pME18, which contained the neomycin-resistant gene with the simian virus 40 promoter. The plasmid was digested with ScaI and transfected into African green monkey kidney COS-7 cells, and the stable transfectants resistant to antibiotic G418 (1 mg/ml) were obtained. The conditioned media containing mouse Tpo were prepared by culturing the cells for 2.5 days without fetal calf serum. A mock transfected COS-7 cell supernatant was also prepared. Recombinant mouse IL-3 (1 10^6 units/mg) was purchased from Genzyme or prepared from conditioned medium of C127 cells expressing recombinant mouse IL-3.

Preparation of Antibodies

Anti-mouse platelet monoclonal antibody Pm1 was raised in Wistar rats using purified mouse platelets as immunogen. The hybridoma was derived from rat splenocytes fused with the murine myeloma P3X63Ag8U.1. Monoclonal antibody Pm1 was screened by specific binding to mouse pure platelets. The antibody was purified by protein G-Sepharose chromatography. Polyclonal rabbit antibody against mouse NF-E2 (C19) and rat monoclonal antibody against mouse GATA-1(N1) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Monoclonal anti-mouse CD61 (GPIIIa) antibody, anti-Mac-1 (CD11b) antibody, and anti-Gr-1 antibody were obtained from PharMingen (San Diego, CA).

Cell Culture

COS-7 transfectants expressing Tpo were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. IL-3-dependent mouse hematopoietic progenitor cell line FDC-P2 (14) was cultured in RPMI 1640 medium supplemented with 10% fetal calf serum and 500 units/ml mouse recombinant IL-3. The Tpo-dependent FD-TPO cells were established by culturing FDC-P2 cells in the presence of 10% COS-7 conditioned medium containing mouse recombinant Tpo without IL-3 for over 2 weeks, and after limiting dilution, the Tpo-dependent clonal cell line FD-TPO was maintained in the presence of 50 units/ml Tpo.

Cell Growth Assay

Cell proliferation was measured by a colorimetric assay using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(15) . Cells (1 10^5) in microtiter plates in 100 µl of culture medium in the presence or absence of various concentrations of recombinant mouse Tpo were cultured at 37 °C for 48 h. The Tpo concentration required for which the FD-TPO cells grew at one-half of the maximal growth rate was defined as 1 unit/ml.

Immunoblotting

The nuclear extracts (20 µg) were prepared by the modified method of Dignam et al.(16) , separated by 10% SDS-polyacrylamide gels and electrotransferred to ECL membrane (Amersham Corp.). The membrane was blocked in 5% bovine serum albumin in 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, and 0.5% Tween 20 (TBS-T) and incubated with anti-GATA-1 or anti-NF-E2 antibody for 2 h. After washing 3 times with TBS-T, the membrane was incubated with anti-mouse IgG-conjugated horseradish peroxidase antibody, and the antibody complexes were visualized by the enhanced chemiluminescence system (Amersham Corp.).

Flow Cytometry Analysis

Cell surface expressions of megakaryocyte/platelet-specific antigens and myeloid-specific antigens in Tpo-stimulated FD-TPO cells were analyzed using flow cytometry. FD-TPO cells cultured with Tpo or IL-3 (1 10^6 cells) were washed and resuspended in Dulbecco's phosphate-buffered saline containing 0.5% bovine serum albumin and 0.1% NaN(3). They were then incubated for 30 min at 4 °C with anti-megakaryocyte/platelet-specific rat monoclonal antibody Pm1 or anti-CD61 (GPIIIa) antibody, anti-mouse myeloid differentiation antigen Gr-1, or anti-mouse Mac-1 (CD11b) in a final volume of 50 µl. Cells were then washed and incubated with FITC-conjugated anti-rat IgG antibody for a further 30 min at 4 °C. Background fluorescence was determined by staining the cells directly with FITC-conjugated antibodies. All measurements were performed on a FACScan flow cytometer (Becton-Dickinson, San Jose, CA).


RESULTS

Establishment of a Tpo-dependent Mouse Cell Line

To study the molecular and cellular mechanism of Tpo-induced megakaryocyte growth and differentiation, we established a clonal cell line that responds and grows with Tpo. IL-3-dependent mouse hematopoietic progenitor cells FDC-P2 (14) were cultured with mouse recombinant Tpo, which was prepared from the conditioned medium of COS-7 cells expressing exogenous mouse Tpo, for over 2 weeks. Following the limiting dilution of the cells, a clonal cell line FD-TPO, which responds to both Tpo and IL-3, was established. FD-TPO cells responded and grew with recombinant mouse Tpo in a dose-dependent manner (Fig. 1A). These cells can be maintained for a long period in the presence of 50 units/ml Tpo. It was confirmed by reverse transcribed PCR analysis that endogenous c-mpl gene expression was induced in FD-TPO cells, but no c-mpl gene expression was observed in the parental FDC-P2 cells (Fig. 1B). Thus this cell line was found to be useful for further analyses of Tpo receptor-mediated signal transduction.


Figure 1: Establishment of a Tpo-dependent mouse cell line. A, Tpo-dependent cell growth. The FD-TPO cells were cultured in the presence of various amounts of mouse Tpo, and the cell proliferation was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. B, expression of Tpo receptor (Mpl). Reverse transcribed PCR analysis of c-mpl was performed with total RNA prepared from the parental FDC-P2 cells cultured with IL-3 (lane1) and the FD-TPO cells cultured with Tpo (lane2).



Induction of Lineage-specific Transcription Factors in Tpo-stimulated Cells

We examined whether or not FD-TPO cells can be differentiated into megakaryocyte/platelet-like cells by Tpo stimulation. The nuclear extracts prepared from the FD-TPO cells cultured with Tpo or IL-3, were immunoblotted with antibodies against megakaryocyte/erythroid-specific transcription factors GATA-1 and NF-E2. As shown in Fig. 2, the expressions of GATA-1 (p50) and NF-E2 (p45) were clearly observed in Tpo-stimulated cells, while neither GATA-1 nor NF-E2 was detected in the FD-TPO cells cultured with IL-3 and in the parental cells. The reverse transcribed PCR analysis also revealed that the transcriptional levels of GATA-1 and NF-E2 were significantly induced by Tpo stimulation, although low levels of these transcripts were detected in the FD-TPO cells cultured with IL-3 (data not shown). Thus, it was concluded that Tpo receptor-mediated signal transmits not only a growth signal but also a specific signal that induces the activation of the lineage-specific transcription factor GATA-1 and NF-E2 genes.


Figure 2: Induction of megakaryocyte/erythroid-specific transcription factors GATA-1 and NF-E2. Nuclear extracts of FD-TPO cells cultured with Tpo (+Tpo) or IL-3 (+IL3) were immunoblotted with anti-GATA-1 antibody (leftpanel) or with anti-NF-E2 antibody (rightpanel). The arrow-marked p50 and p45 indicate GATA-1 and NF-E2, respectively.



Enhanced Expression of Megakaryocyte/Platelet-specific Cell Surface Antigens in Tpo-stimulated FD-TPO Cells

Cell surface expressions of megakaryocyte/platelet-specific antigens in Tpo-stimulated FD-TPO cells were analyzed using flow cytometry. Cells were treated with anti-megakaryocyte/platelet-specific rat monoclonal antibody Pm1 or anti-CD61 (GPIIIa) antibody. Fig. 3, A and B, clearly shows that expression of both antigens significantly increased in FD-TPO cells cultured with Tpo (rightpanels) compared with the cells cultured with IL-3 (leftpanels). The molecule recognized by monoclonal Pm1 antibody has not been identified yet, but it can specifically immunostain the primary megakaryocytes and platelets and can also dramatically reduce the number of platelets in blood by the injection of antibody into mice (data not shown). Thus, the antigen recognized by Pm1 antibody is a specific cell surface molecule expressed in megakaryocytes and platelets. The GPIIIa is also known to be one of the megakaryocyte/platelet differentiation markers. Thus, these results suggested that Tpo activates the megakaryocyte-specific differentiation signal, which induces GATA-1 and NF-E2 gene expression and finally enhances the expression of megakaryocyte/platelet-specific cell surface antigens in FD-TPO cells.


Figure 3: Flow cytometry analysis of megakaryocyte/platelet-specific and myeloid-specific differentiation antigens in FD-TPO cells. FD-TPO cells cultured with Tpo (+Tpo; right panels) or IL-3 (+IL-3; left panels) were incubated with anti-CD61 (GPIIIa) antibody (A), anti-megakaryocyte/platelet-specific rat monoclonal antibody Pm1 (B), anti-Mac-1 antibody (C), or anti-Gr-1 antibody (D) and analyzed by a flow cytometer. Background fluorescence (brokenlines) was determined by staining the cells directly with FITC-conjugated antibodies.



We further examined the expression of myeloid-specific differentiation antigens, Gr-1 and Mac-1, in Tpo-stimulated cells. As shown in Fig. 3, C and D, both Gr-1 and Mac-1 antigens were expressed in low level in the FD-TPO cells cultured with IL-3 (leftpanels). However, the expression level of both antigens clearly decreased upon Tpo stimulation (Fig. 3, C and D, rightpanels). These results further confirmed that FD-TPO cells differentiated into megakaryocyte-like cells in response to Tpo stimulation and thus reduced the expression of myeloid-specific differentiation antigens.


DISCUSSION

The Tpo receptor-mediated signals activate the expression of lineage-specific transcription factor GATA-1 and NF-E2 genes and consequently induce expression of the megakaryocyte/platelet-specific cell surface antigens including GPIIIa and the Pm1-recognized antigen. These transcription factors, which were initially identified as erythroid-specific, are now known to be also expressed in megakaryocytes (9, 10) and induce expression of megakaryocyte-specific cell surface antigens GPIIIa/IIb and Ib(8) . However, this Tpo receptor-mediated differentiation mechanism is, at least in part, common to that of Epo. There must be Tpo-specific signaling cascades for determining the cell fate of megakaryocytes and for triggering the polyploidization and platelet release. It has to be resolved what signals activate the expression of GATA-1 and NF-E2 genes, whether or not specific STAT proteins and their associated molecules can determine cell specificity, cell function, and cell differentiation, what determines megakaryocyte specificity, and what is the difference between erythroid and megakaryocyte in differentiation signals and in transcription factors. A newly established FD-TPO cell line will be useful for further studies on these problems, and identification of cytoplasmic and nuclear signaling factors regulating cell differentiation is awaited.

The expression of megakaryocyte/platelet-specific antigens including CD61 and Pm1-recognized antigen was induced by Tpo stimulation. On the contrary, the expression of granulocyte- and macrophage-specific antigens including Gr-1 and Mac-1 decreased by Tpo stimulation. These results clearly indicate that FD-TPO cells cultured with Tpo differentiated into megakaryocyte-like cells. We also examined the possibility by flow cytometer that FD-TPO cells induce polyploidization and/or morphological change in response to Tpo. However, neither increase of DNA content nor morphological change (proplatelet formation, cytoplasmic process formation, or platelet release) was observed in FD-TPO cells cultured with Tpo. Therefore, we concluded that Tpo stimulation not only activates a growth signal but also induces a lineage-specific maturation/differentiation signal in the FD-TPO cells, and thus FD-TPO cells are very useful for the study of the molecular mechanism of the Tpo-induced megakaryocyte maturation process.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
The first two authors contributed equally to this work.

To whom correspondence should be addressed. Tel.: 81-298-36-9075; Fax: 81-298-36-9050; todokoro{at}rtcs1.riken.go.jp.

(^1)
The abbreviations used are: Tpo, thrombopoietin; Epo, erythropoietin; IL, interleukin; GP, glycoprotein; FITC, fluorescein isothiocyanate; PCR, polymerase chain reaction.


ACKNOWLEDGEMENTS

We thank T. Kanaya for tissue culture, S. H. Ha, T. Ohnuki, and T. Wada for helpful discussions, and Dr. T. Abe for encouragement.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.