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