Stem Cells, Vol. 16, No. 1, 1-6,
January 1998
© 1998 AlphaMed Press
Hematopoietic Deficiencies in c-mpl and TPO Knockout Mice
Maximilien Murone,
David A. Carpenter,
Frederic J. de Sauvage
The Department of Molecular Oncology, Genentech, Inc., South San Francisco, California, USA
Key Words. Thrombopoietin • c-mpl • Platelets • Stem cells • Thrombocytopenia • Hematopoiesis
Dr. Frederic J. de Sauvage, Genentech, Inc., One DNA Way, South San Francisco, CA 94080, USA.
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Abstract
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Thrombopoietin (TPO) is the primary regulator of megakaryocyte (Meg) and platelet production. Its receptor, c-mpl, is a member of the cytokine receptor superfamily. Major insight into the physiological role of this receptor/ligand pair came from the study of mice carrying disrupted alleles of these two genes. Both TPO and c-mpl knockout mice are viable, but have a 90% reduction in platelet counts. Their thrombocytopenia is caused by a reduction in progenitor cell numbers and a decrease in Meg ploidy. However, the Megs and platelets produced in the absence of TPO or c-mpl appear morphologically and functionally normal indicating that, in vivo, the main role of TPO is to control their numbers, rather than their maturation. In addition to its effect on the Meg lineage, TPO also affects hematopoietic stem cells as measured by a reduction of the repopulating capacity of bone marrow cells from c-mpl-deficient mice. Finally, analysis of these gene targeted mice provided substantial evidence to a model where the circulating TPO level is directly regulated by the platelet mass through binding to c-mpl receptors present at the platelet surface. This elegant feedback mechanism allows a tight regulation of the amount of TPO available to stimulate megakaryocytopoiesis.
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Introduction
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Thrombocytopenia, or lack of platelets, is a significant medical problem today. Currently the only method to alleviate thrombocytopenia is platelet transfusion [1]. Megakaryocytopoiesis, as well as platelet formation, are key events of hemostasis, and the understanding of the mechanisms underlying megakaryocyte (Meg) differentiation from pluripotent stem cells is consequently of major medical interest. Little was known of the factors regulating Meg proliferation and maturation until the cloning and characterization of the c-mpl receptor [2] and its ligand thrombopoietin (TPO) [3-6].
The model that preceded the cloning of TPO was that different cytokines (or sets of cytokines) were regulating Meg proliferation and differentiation [7]. It is now well established that the key factor controlling both is TPO [3-5, 8]. Other cytokines are able to induce Meg expansion, including interleukin 1 (IL-1), IL-11, kit ligand (KL), erythropoietin (EPO), IL-3, IL-6 and GM-CSF [7], but their contribution is moderate with respect to the effect of TPO [9]. The cloning of TPO may be critical to alleviate thrombocytopenia in the clinic, but also allows for a larger scope of in vitro Meg study, as this cell type is fairly rare in the hematopoietic milieu. One of the tools available to determine the function of cytokines and cytokine receptors in hematopoiesis is the use of transgenic and knockout mouse technology. As in vitro studies have implicated TPO and its cognate receptor, c-mpl, in multiple aspects of megakaryocytopoiesis, it was of great interest to analyze their physiological role by generating mice carrying disrupted alleles of the TPO and c-mpl genes. This review will focus on the analysis of the phenotype of these two strains of knockout mice.
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Mice Lacking TPO or c-mpl Are Thrombocytopenic
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Human c-mpl is a 635 amino acid, cell surface receptor most homologous to the EPO and IL-3 receptors [2]. Its extracellular domain contains the typical cysteines and WSXWS motifs conserved in most members of the cytokine receptor superfamily. The intracellular domain contains two regions of conserved homology, designated box 1 and box 2 [10]. The myeloproliferative leukemia virus is a murine retrovirus containing a truncated c-mpl gene where the extracellular domain has been replaced by the envelope protein. Mice infected with myeloproliferative leukemia virus exhibit dramatic proliferation of several hematopoietic lineages [11], and in vitro infection of bone marrow cells leads to immortalized factor independent cell lines of various hematopoietic lineages [12]. To determine the role of c-mpl in vivo, two separate groups generated c-mpl null mice, disrupting the gene in embryonic stem (ES) cells by the insertion of a targeted neomycin resistance marker [13, 14]. C-mpl–/– mice are normal in appearance but have thrombocytopenia with 100% penetrance [13]. Both Meg and platelet counts are reduced by about 90% in c-mpl–/– mice [13, 14], while all other blood lineages are normal in number and appearance ( Table 1). Interestingly, the mean platelet volume is increased from an average of 4.7 µm3 in wild-type mice to 6.25 µm3 in c-mpl–/– mice [13]. These results indicate that c-mpl is key to the regulation of the Meg lineage and is likely to be the receptor for a cytokine involved in the regulation of platelet production.
By the time these mice were available, the c-mpl ligand had been purified, cloned and shown to have a pattern of activity consistent with being TPO. The human TPO gene encodes a 353 amino acid protein with a 21 amino acid signal sequence, which is cleaved to produce a 332 amino acid mature protein. The overall structure of TPO can be divided into two parts: the amino terminus of the protein, which is homologous to EPO, and a highly glycosylated carboxyl terminus region which shows no homology to any known protein. A truncated form of TPO lacking the carboxyl terminus is as active as the full length molecule in vitro, indicating that this domain is sufficient for receptor binding and activation [3]. The carboxyl half of the protein contains 6 N-linked and 18 O-linked glycosylation sites and is rich in the amino acids proline, serine and threonine [15]. By analogy with EPO, it has been suggested that glycosylation of the molecule is responsible for the long half-life of the protein in vivo. This hypothesis is supported by the observation that a truncated form of TPO lacking the carboxyl terminus has a half-life of 1.5 h, while the full length glycosylated TPO has a half-life of 18-24 h [16]. To generate TPO knockout mice, 23 amino acids of the region with EPO homology were replaced in a mouse genomic clone by the neomycin resistance marker. The resulting targeting vector was used to electroporate ES cells, and clones resistant to neomycin were screened for homologous recombination at the TPO locus. Litters resulting from interbreeding TPO+/– mice produced viable TPO–/– mice in a Mendelian ratio, indicating that TPO is not essential for embryogenesis and development [17]. Adult mice carrying disrupted alleles of the TPO gene showed no apparent physical abnormalities, but analysis of their blood revealed an ~90% reduction in platelet counts with respect to wild-type mice ( Table 2). Blood cell counts for all other blood cell types were normal. As previously seen in c-mpl–/– mice, the volume of the remaining platelets in TPO–/– mice was significantly increased (8.7 µm3 versus 4.7 µm3 in TPO+/+ mice). Analysis of the bone marrow and spleen of TPO–/– mice revealed that the number of Megs was also drastically reduced (again, approximately 10% of littermate control). Measurement of Meg ploidy in TPO–/– mice versus wild-type mice indicated a decrease in mean Meg ploidy [17]. Interestingly, TPO seems to work in a gene dosage dependent manner as TPO+/– mice have platelet and Meg counts intermediate between wild-type and knockout ( Table 2). Taken together, these data demonstrate that TPO is the primary physiological regulator of Meg and platelet production. Furthermore, the similarity in phenotype of TPO and c-mpl deficient mice strongly supports TPO being the only ligand for c-mpl and conversely that c-mpl is the only functional receptor for TPO.
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Effect of TPO on Progenitor Cells
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The ability of cytokines to expand progenitor cells ex vivo is of major clinical and scientific interest. Investigators have been looking for a cytokine or combination of cytokines that will yield a large expansion of progenitor cells, while keeping those cells pluripotent. These cells could be directly transplanted or would be an ideal target for further modification by gene therapy. Soon after its discovery, it became apparent that the effects of TPO would not be restricted to the megakaryocytic lineage but would be broader than anticipated. In vitro, TPO not only supports the growth of Meg progenitors [18] but also enhances the proliferation of erythroid progenitors [19, 20]. Administration of recombinant TPO leads to a significant expansion of colony forming units (CFU-)megakaryocyte (MEG), BFU-E, and CFU-granulocyte-macrophage (CFU-GM) in both normal and myelosuppressed mice [21]. The physiological relevance of effects of TPO on progenitor cells was again established using gene targeted mice. In vitro colony assays using bone marrow from TPO–/– or c-mpl–/– mice in semisolid media show a reduction in CFU-MEG, consistent with the role of this receptor/ligand in megakaryocytopoiesis. Interestingly, total hematopoietic progenitor cell numbers were reduced in both types of knockout mice. Reductions ranging from 70% to over 90% were observed in multipotential CFU-Mixture, CFU-GM, and especially BFU-E progenitors [14, 22]. This reduction in hematopoietic progenitors is reversible as injections of TPO into TPO–/– mice and normal mice show dramatic increases in bone marrow, spleen and peripheral blood progenitors [22]. The increase in circulating progenitors also suggests that TPO may be useful as a mobilizing agent. Although the circulating white and red blood cell counts of the TPO–/– and c-mpl–/– are normal and do not reflect the multilineage effect of TPO, it has been shown that TPO can shorten the duration of the anemia and leukopenia in some murine preclinical models of myelosuppressive therapy [23].
In vitro studies have shown that TPO may actually directly support the proliferation of human [24, 25] and murine [26, 27] hematopoietic stem cells (HSC). The direct effect of TPO at such an early stage is consistent with the expression profile of its receptor. C-mpl transcripts can be identified in human CD34+CD38– cells [28]. Similarly, in murine fetal liver the AA4+Sca+ population is highly enriched for progenitor cells, and fluorescence-activated cell sorter analysis has indicated that approximately 50% of this population is positive for c-mpl expression [29]. When these AA4+Sca+ cells are cocultured on stromal cells with TPO as a single factor, there is a significant increase in proliferation of hematopoietic progenitor cells as compared with stroma alone [29]. When analyzed in a competitive repopulation assay, all the stem cell activity segregates with the AA4+Sca+mpl+ cell population compared to AA4+Sca+mpl– cells [30]. One of the most convincing demonstrations of the direct effect of TPO on HSCs comes from the analysis of the c-mpl knockout mice by competitive repopulation assays. LinloSca+ cells derived from the bone marrow of c-mpl-deficient mice contained significantly fewer repopulation units compared to the cells isolated from wild-type animals [30].
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TPO Is Not Required for the Production of Normal Platelets
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The analysis of TPO and c-mpl-deficient mice summarized above demonstrates that TPO controls platelet production by affecting the proliferation and/or apoptosis of progenitor cells as well as Meg ploidy. However, even with their reduced platelet counts, neither TPO nor c-mpl knockout mice bleed spontaneously. In a tail clip assay, their bleeding times were prolonged only three- to fourfold compared to wild-type mice [31], suggesting that the remaining platelets are functional and still able to form a thrombus. Indeed, structural and functional analysis demonstrated that the platelets from knockout mice were similar to the ones from wild-type mice. Ultrastructural analysis by electron microscopy performed on platelets and Megs from c-mpl–/– and TPO–/– mice did not reveal any significant cytological differences when compared to wild-type [31]. Functionally, platelets derived from knockout mice responded to agonists such as adenosine diphosphate by upregulating fibrinogen binding sites as efficiently as normal platelets, and their adhesion to extracellular matrix proteins was normal. These data suggest that, in vivo, the TPO/c-mpl system is not required for the formation of normal Megs and platelets. This is in contrast to in vitro data where TPO seems to be required for the production of normal Megs and platelets [9, 32], indicating that it may be difficult to reconstitute the exact hematopoietic microenvironment in vitro. Multiple cytokines with megakaryocytopoietic activity have been shown to work in c-mpl or TPO-deficient mice and may compensate for the maturational role of TPO in vivo but may not be able to fulfill its proliferative function [22]. Alternatively, once a stem cell has been committed stochastically to a hematopoietic lineage, a program of intracellular signaling and activation of transcription factors may have been initiated and will lead the cell to proper and complete differentiation. In this scheme, the role of cytokines such as TPO is to regulate the number of cells reaching maturation by controlling survival and proliferation.
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Regulation of TPO Production
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TPO and c-mpl deficient mice have proven to be a useful model for studying the regulation of TPO production. The serum of c-mpl–/– mice, like that of other thrombocytopenic animals, contains high levels of TPO [13, 33-35]. To explain the inverse relationship between platelet count and circulating TPO level, it was tempting to speculate that a sensing mechanism was responsible for detecting lower platelet counts and stimulating TPO transcription in a similar way that EPO transcription is regulated in response to anemia and low oxygen pressure [36-38].
The TPO gene is expressed in both human and mouse as a 1.8 kb mRNA. Northern blot analysis indicates the presence of the transcript in the kidney, the liver, and, to a lesser extent, in the spleen and bone marrow [3, 39-42]. However, when the amount of TPO mRNA was analyzed in the liver and kidney of c-mpl–/– mice, no differences could be measured with respect to wild-type mice [43]. Similar results were obtained in mice when acute thrombocytopenia was induced by injection of an antimouse platelet serum or by irradiation [40, 41]. These data indicate that, in these models, TPO transcription in the kidney and liver is not modulated in response to platelet counts. These results are consistent with the gene dosage effect observed in TPO heterozygous mice which further suggests that TPO production is not regulated by the platelet mass [17].
In an alternative model, the level of circulating TPO could be regulated by the platelet mass itself which could act as a sink and directly control the amount of circulating TPO available to reach the bone marrow and stimulate the production of additional platelets [44-46]. Again, the c-mpl–/– mice provided a tool to explore this model further. Platelets from c-mpl–/– mice are not able to bind TPO compared to wild-type platelets which bind, internalize and degrade TPO. Consequently, pharmacokinetics studies showed that the half-life of 125I-TPO was increased in c-mpl–/– mice since their platelets are not capable of removing the circulating TPO [43]. Injection of purified normal platelets into c-mpl–/– mice restored normal levels of serum TPO [43]. Similarly, in vitro, TPO activity can be depleted from serum in a dose-dependent manner by incubation with different concentrations of platelets [40, 46]. Taken together, these results indicate that TPO plasma levels are regulated directly by platelets through binding to the c-mpl receptor present at their surface.
However, another mouse model for thrombocytopenia, the NF-E2–/– mouse, seemed at first to conflict with the model. These mice, which lack the transcription factor p45 NF-E2, have virtually no circulating platelets, but increased numbers of Megs. Most of them die at or soon after birth from severe bleeding [47]. Surprisingly, normal levels of serum TPO were found in these mice [47, 48]. Recent studies showed that radiolabled TPO injected into NF-E2-deficient mice is cleared from the serum through c-mpl receptors found on Megs and on large numbers of Meg fragments present in the spleen, suggesting that TPO levels can not only be regulated by mature platelets, but also by Megs themselves [48]. This model may reflect what has been observed in patients affected by idiopathic thrombocytopenic purpura (ITP), a chronic thrombocytopenic syndrome characterized by increased platelet destruction but with normal platelet production and Meg mass. Similarly to the NF-E2–/– mouse, patients with ITP do not have increased levels of circulating TPO [49].
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Summary
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In summary, TPO and c-mpl gene targeted mice have proved to be invaluable tools to study the physiological contribution of this cytokine to Meg and platelet production. Although TPO has both proliferative and maturation properties, its major role appears to be the control of platelet numbers while other cytokines may be able to compensate for its maturation role. Beside its expected effect on the megakaryocytic lineage, these mice have also demonstrated the direct effect of TPO on progenitor cells from other lineages and on HSCs. Finally, this model has helped to clarify the mechanisms by which the circulating levels of TPO are regulated by the platelet mass.
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accepted for publication September 25, 1997.
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Top
Abstract
Introduction
