Regulation of megakaryocytopoiesis and platelet production: Lessons from animal models

REVIEW AITIOLE Regulation of megakaryocytopoiesis and platelet production: Lessons from animal models FREDERIC J. DE SAUVAGE, JEAN-LUC VILLEVAL, and RAMESH A. SHIVDASANI SOUTH SAN FRANCISCO, CALIFORNIA, AND BOSTON, MASSACHUSETTS

Abbreviation: Tpo = thrombopoietin

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istorically, the study of megakaryocyte differentiation has been hampered by the rarity of this cell in hematopoietic tissues, the difficulties associated with isolating and handling sufficient numbers of cells, the absence of adequate differentiation in most established cell lines, and the lack of a specific growth factor. The discovery of thrombopoietin (Tpo) in 1994 was followed by significant and rapid progress in the understanding of megakaryocyte biology and platelet production. Because platelet production is a dynamic process that is influenced by many factors in vivo, some of the most valuable insights have emerged from animal studies. Here we review knockout and transgenic mouse models of Tpo, its receptor c-Mpl, and hematopoietic-restricted transcription factors that have contributed these insights. TPO AND C-MPL DEFICIENCIES IN VIVO

The proto-oncogene c-Mpl is a member of the cytokine receptor superfamily with sequence similarity to the erythropoietin and granulocyte colony-stimulating factor receptors.12 Expression of c-Mpl in normal mice is largely restricted to hematopoietie tissues-primitive hematopoietic progenitors, megakaryocytes,

From the Departmentof MolecularOncology,GenentechInc., South San Francisco;and the Departmentof AdultOncologyand Medicine, Dana-FarberCancerInstitute and HarvardMedical School,Boston. Submittedfor publicationJune 10, 1997;revisionsubmittedAug. 12, 1997; acceptedSept. 4, 1997. Reprintrequests: FredericJ. de Sanvage,PhD, Departmentof Molecular Oncology,GenentechInc., 460 Pt. San BrunoBlvd., SouthSan Francisco, CA 94080. J Lab Clin Med 1998; 131:496-401. CopyrightO 1998by Mosby,Inc. 0022-2143/98 $5.00 + 0 511189724 496

and platelets2,3--suggesting that c-Mpl and its ligand may be specific regulators of megakaryocytopoiesis. 3 Indeed, knockout mice deficient in c-Mpl 4 reveal platelet counts that are >85% reduced relative to littermate controls, with 100% penetrance and without significant difference in red blood cell, total leukocyte, neutrophil, and eosinophil counts. Size and cellularity of lymphoid organs, cell type ratios, and maturation markers of B and T lymphocytes in bone marrow, spleen, and thymus are also within normal limits. A decrease in bone marrow megakaryocytes that is directly proportional to the degree of thrombocytopenia suggests that the absence of c-Mpl function results in a highly specific loss of both megakaryocytes and platelets in vivo and points to the c-Mpl ligand as the major regulator of megakaryocyte and platelet production in vivo. The ligand for c-Mpl is a cytokine with a unique structure; the amino terminus is homologous to erythropoietin, while the C-terminal glycosylated domain is unrelated to any known protein (reviewed in reference 5). As observed in c-Mpl -/- mic e, disruption of the Tpo gene, by replacement of part of the erythropoietin homology domain with a neomycin resistance cassette, also leads to a 90% drop in the platelet count. 6 In contrast to c-Mpl heterozygous mice, which do not differ from the wild type, a significant alteration in platelet counts (67% of wild-type) is observed in Tpo +/- mice. Histopathology of the hematopoietic tissues in both c-Mpl -/- and Tpo -/- mice reveals a substantial loss (- 10% of control) of megakaryocytes, while Tpo +/mice have intermediate (60% of control) megakaryocyte levels. Micrometric measurements in c-Mpl -/mice indicate that the mutant megakaryocytes are ~20% smaller than normal cells in overall as well as in nucleus size. Ploidy analysis of bone marrow megakary-

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ocytes from both mutant mouse strains further reveals reduced DNA content relative to normal. 6,7 Analysis of megakaryocyte progenitor cells from Tpo -/-, c-Mpl -/- and normal mice in serum-containing semisolid media indicates a greatly reduced number of megakaryocyte progenitor cells in both knockout mice, consistent with a primary role for this ligand-receptor pair in megakaryocyte development.6, 8 Remarkably, granulocyte-macrophage, erythroid, and multipotential progenitors are also decreased in Tpo -/- and c-Mpl -/mice, 8 suggesting that Tpo exerts additional effects on an early common progenitor cell rather than restricted effects on the megakaryocyte lineage. This is consistent with the observation that Tpo enhances not only megakaryocyte colony growth but also the proliferation of erythroid progenitors in vitro and in vivo. 9,10 Furthermore, the involvement of Tpo and c-Mpl in early aspects of hematopoiesis correlates with the detection of c-Mpl expression in the AA4+Sca ÷ subpopulation of murine hematopoietic cells. 11 This effect of Tpo on primitive hematopoietic progenitors was recently confirmed by a competitive repopulation assay in which Linl°Sca+ cells purified from normal mice demonstrated a greater repopulation capacity than the same cell subpopulation isolated from c-Mpl -/- mice. 46 In summary, the Tpo/c-Mpl ligand-receptor combination regulates aspects of multipotential hematopoietic development as well as of thrombocytopoiesis in particular. Interestingly, although Tpo- and c-Mpl-deficient mice have reduced platelet counts, they do not bleed spontaneously, and both megakaryocytes and platelets from these knockout mice have a normal ultrastructure. 47 In both instances, platelets are functionally normal, as measured by the up-regulation of fibrinogen binding in response to agonist stimulation or the ability to attach to extracellular matrix proteins. These results suggest that in vivo Tpo is not required for the formation of normal megakaryocytes and platelets but is necessary to stimulate their production in optimal numbers. The residual thrombocytopoiesis in these knockout mice raises the question of which cytokines, if any, function to compensate for the in vivo absence of Tpo or c-Mpl function; this question remains under active investigation. ANIMAL MODELS OF TPO OVEREXPRESSION

Early clinical trials of Tpo in patients with advanced malignancies show accelerated platelet recovery, with little toxicity, after myelosupressive therapy. 12-14Additionally, the therapeutic use of Tpo may extend to patients with familial or idiopathic thrombocytopenias, where the side effects of its long-term administration are unknown. The further possibility that Tpo may be involved in human disease, notably through overexpres-

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sion in essential thrombocythemia or as a growth factor for leukemic cells, has led investigators to use a variety of experimental approaches to study animals subjected to chronic Tpo overexpression. Long-term exposure to Tpo in mice has been accomplished by using retroviral gene transfer in hematopoietic stem cells. In one model,15J 6 mice followed for more than 15 months have the same life expectancy as controls but develop chronic thrombocytosis (platelet levels increased 5 times over controls), mild anemia, and megakaryocytosis in the bone marrow, spleen, liver, and lymph nodes. Progenitor cell numbers from all myeloid lineages, including pluripotent cells, are decreased in the marrow but greatly increased in the spleen and peripheral blood. Remarkably, normal marrow elements are almost completely replaced over time by new bone trabeculae, large reticulin fibers, and numerous megakaryocytes; osteosclerosis appears to result from increased matrix deposition. These features are likely caused by an indirect effect, such as increased plasma levels of platelet-derived growth factor and transforming growth factor-[3, mediators known to be released by megakaryocytes. Using a similar approach of retroviral gene transfer, we have observed a more dramatic syndrome. 17 In addition to developing megakaryocyte hyperplasia, splenomegaly, extramedullary hematopoiesis, severe myelofibrosis, and osteosclerosis, all of the mice died (median survival -7 months) from a myeloproliferative disorder characterized by two successive phases. During the first 2 months, thrombocytosis was accompanied by leukocytosis and severe anemia; except for erythrocyte colony-forming units, which were slightly decreased, the total number of progenitors from all lineages was increased. Two months after transplantation, platelet and nucleated blood cell numbers, as well as marrow and spleen progenitors, began a progressive decline, culminating in death from severe pancytopenia. Splenic fibrosis was prominent, and 2 mice died with a large number of circulating blast cells, which is suggestive of leukemic transformation. Plasma Tpo activity remained elevated throughout the study period (0.3 to 1.2 gg/ml). Subcutaneous 18 or intravenous 19 injections of adenoviral vectors containing the human Tpo cDNA have also been undertaken. In immunocompetent mice, transient (2 weeks) elevation of the platelet count 18 has been accompanied by megakaryocyte hyperplasia without myelofibrosis or osteosclerosis. In other studies, thrombocytopenia later developed in mice because of anti-Tpo antibodies19; further investigation is needed to know whether this autoimmune disease is a model for some human thrombocytopenias. In immunodeficient mice, sustained high levels of plasma Tpo lead to

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leukocytosis, anemia, myelofibrosis, and osteosclerosis.19 Finally, transgenic mice expressing human Tpo under the control of a liver-specific apolipoprotein E promoter 20 can maintain serum levels of human Tpo up to 3 ng/ml and fourfold higher platelet counts throughout their lives. These mice show megakaryocytosis in the marrow, spleen, and liver; anemia; and an increase in circulating myeloid and lymphoid cells without evidence of myelofibrosis. Taken together, these studies suggest that the severity of the clinical syndrome with Tpo overexpression is related to the level of plasma Tpo. Transgenic mice with more than a 300-fold elevation in plasma Tpo levels reveal fourfold to fivefold the level of thrombocytosis without significant additional pathology. 2° In contrast, when Tpo is transduced with a retrovirus in hematopoietic cells, depending on the serum concentration achieved, mice develop either severe myelofibrosis and osteosclerosis without premature death or a myeloproliferative disease akin to human idiopathic myelofibrosis, with lethal evolution to pancytopenia or blast transformation. Although additional studies are required to establish the mechanisms leading to fibrosis and osteosclerosis, we speculate that megakaryocyte hyperplasia in response to Tpo results in locally increased production of potent stimuli for these processes. These models may further our understanding of the pathophysiology of this and related diseases. THE ROLE OF HEMATOPOIETIC TRANSCRIPTION FACTORS IN MEGAKARYOCYTE DEVELOPMENT

Current concepts postulate that hematopoietic cytokines promote growth and survival of committed progenitors whose differentiation potential is realized through transcriptional mechanisms. However, the precise relationships between transcription factors and secreted cytokines in orchestrating hematopoiesis is poorly understood. Although the available evidence suggests that Tpo controls aspects of megakaryocyte growth as well as platelet differentiation, targeted gene disruption of two hematopoietic-specific transcription factors has revealed critical transcriptional pathways in megakaryocyte maturation. The intersections between these pathways and intracellular signaling by Tpo through c-Mpl represents an important area for future investigations. Tissue-specific, high-level, developmentally regulated expression of the globin genes in maturing red blood cells is controlled by a cluster of cis elements located at considerable distance from the structural genes and collectively known as the locus control regions. 21,22 Within these locus control regions, distinct aspects of globin gene expression map to a remarkably small num-

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ber of DNA sequence motifs, including GATA and an extended AP-l-related GCTGAGTCA site designated NF-E2 (reviewed in reference 23). The predominant transcription factors interacting with these cis elements in erythroid cells are the zinc-finger protein GATA124,25 and the basic leucine zipper heterodimer NFE2, 26,27 respectively. Both GATA-1 and the hematopoietic-restricted 45 kd subunit of NF-E2 (p45) are coexpressed in erythroid cells and megakaryocytes. Mice lacking p45 NF-E2 develop to term without apparent anemia but exhibit a severe hemorrhagic diathesis in the neonatal period that results in death of the majority of animals, 2s whereas surviving mutant mice reveal a compensated anemia with dyserythropoiesis throughout their lives, 29 the predominant hematologic abnormality being a virtually complete absence of circulating platelets. This abnormality can be traced to an arrest in late maturation of megakaryocytes, which are present in large numbers but reveal a significantly expanded cytoplasm with few platelet-specific granules and a highly disorganized system of demarcation membranes. There is no interference with endomitosis and Tpo signaling through c-Mpl is intact, at least with respect to megakaryocyte proliferation in vivo. Megakaryocyte biology has been more difficult to study in mice lacking GATA-1 because of early embryonic lethality as a result of arrested erythroid maturation and severe anemia. 3°,31 Recently, gene targeting within upstream cis elements of the murine X-linked GATA-1 locus created a hypomorphic mutation for the erythroid lineage that permits survival of some mice with milder, compensated anemia32; an additional consequence of this mutation is complete, selective attenuation of GATA-1 expression within megakaryocytes.33 Mice with this genetic alteration resemble Tpo and cMpl knockout mice in that platelet counts are about 15% of normal and the few platelets are unusually large. However, megakaryocyte numbers are vastly increased, reflecting a greatly increased proliferative capacity of megakaryocyte progenitors, and the ultrastructure of the mature megakaryocytes reveals striking nuclear and cytoplasmic abnormalities. The nucleus is hyperlobulated with increased numbers of nucleoli, whereas the cytoplasm is disproportionately small and underdeveloped for the degree of nuclear maturation. Both demarcation membranes and platelet-specific granules are decreased, and there is abundant endoplasmic reticulum, much of it within an expanded, organelle-poor peripheral zone. In summary, megakaryocytes lacking GATA-1 exhibit excessive proliferation and arrested cytoplasmic maturation, which results in thrombocytopenia without evident hemorrhage. Several aspects of the megakaryocyte abnormalities in the absence of NF-E2 or GATA-1 merit discussion.

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First, these two knockout mouse models represent the sum of transcription factors with known megakaryocyte and platelet functions in vivo. Although proteins of the Ets family have been implicated in the regulation of some megakaryocyte-specific genes in vitro, 34,35 their role in megakaryocyte differentiation or platelet formation in vivo is unknown. Similarly, the importance of other transcription factors known to be expressed at high levels in mature megakaryocytes, including cMyb, c-Myc, GATA-2, and SCL/Tal-1, remains uncertain. Second, it is remarkable that critical megakaryocyte functions are fulfilled by transcription factors that are also important for erythroid-specific gene expression. This overlap highlights the relationship between these hematopoietic cell lineages derived from a common precursor. 3637 Finally, there are important similarities and differences between the megakaryocyte abnormalities in these two gene knockouts. Absence of either transcription factor leads to low platelet counts (which differ in degree) and arrested megakaryocyte cytoplasmic maturation with the shared feature of decreased numbers of platelet-specific granules. However, in contrast to GATA-1 deficiency, absence of p45 NF-E2 does not lead to hyperproliferation or accumulation of immature megakaryocyte precursors. Perhaps most importantly with respect to mechanisms of thrombocytopoiesis, the cytoplasmic abnormalities in the two varieties of knockout megakaryocytes are vastly different.2s, 33 Taken together, these findings imply that GATA- 1 and NF-E2 play essential roles in distinct transcriptional pathways that culminate in platelet differentiation within the megakaryocyte cytoplasm. Because the critical target genes of both transcription factors are unknown, it is unclear to what extent these transcriptional pathways overlap. INSIGHTS INTO REGULATION OF SERUM TPO LEVELS FROM MOUSE MODELS

Analysis of some of the above knockout mice has provided valuable insights into the understanding of how serum Tpo levels may be regulated in vivo. As in other thrombocytopenias, c-Mpl-deficient mice, representing the first mouse model of chronic thrombocytopenia, have a dramatic elevation in circulating Tpo levels 4 without having a detectable increase in Tpo mRNA levels in any organ. 38-4° Consistent with the suggested absence of transcriptional up-regulation of the Tpo gene in thrombocytopenic animals, Tpo +/- mice have a platelet level intermediate between wild-type and Tpo-/- mice. 6 This gene dosage effect is particularly suggestive that Tpo production p e r s e is regulated independently of the platelet mass. According to one model, 41-43 Tpo is released into the circulation at a constant rate and its serum concentra-

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tion is regulated by the platelets themselves; experiments in c-Mpl-deficient mice are consistent with such a mechanism. Predictably, and in contrast to normal platelets, those from c-Mpl -/- mice do not bind, internalize, or degrade Tpo. 38 Thus the absence of Tpo receptors in c-Mpl-/- mice leads to reduced clearance of Tpo from the plasma and an increase in its half-life. The injection of platelets purified from normal donors reconstitutes normal platelet levels in c-Mpl-/- or artificially thrombocytopenic mice38,39 and rapidly decreases the plasma level of Tpo to that found in normal mice. This supports the aforementioned model by demonstrating that Tpo receptors present on platelets are sufficient to achieve rapid removal of the high amount of Tpo circulating in c-Mpl -/- mice. Remarkably, serum Tpo levels are not increased in p45 NF-E2 knockout mice despite a profound decrease in circulating platelet numbers.28, 44 Following the fate of radiolabeled Tpo injected into these animals indicates that it is rapidly cleared by megakaryocyte fragments found, for the most part, in the spleen and by intact megakaryocytes in the spleen and bone marrow. This situation is probably akin to that encountered in patients with immune thrombocytopenic purpura, where Tpo levels may be low despite low platelet numbers because the excess megakaryocytes serve as a source of c-Mpl to clear free Tpo from the circulation. 45 Indeed, Tpo binding to tissue megakaryocytes has been demonstrated by several investigators and is probably one component of the regulation of serum Tpo levels in vivo. CONCLUSIONS

The animal models summarized in this review contribute greatly to our understanding of megakaryocyte development and platelet production. Although they establish Tpo and c-Mpl as the major regulators of megakaryocyte growth and differentiation, residual platelet production and the absence of bleeding in Tpo -/- and c-Mpl -/- mice raise the possibility that other ligand-receptor pairs may also provide some of these functions in vivo. Several of the animal models discussed here further suggest that Tpo can influence hematopoietic progenitors of other lineages directly along with the development of myelofibrosis osteosclerosis, perhaps indirectly. Knockout mice have provided invaluable insights into how lineage-restricted transcription factors may establish programs of gene expression associated with terminal megakaryocyte maturation, and they implicate two "erythroid" transcription factors, GATA-1 and NF-E2, in this process. Finally, a combination of animal models has served to clarify the mechanisms by which the serum concentration of Tpo is regulated. These and other animal mod-

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els w i l l c o n t i n u e to s e r v e as a rich s o u r c e o f insights into m e g a k a r y o c y t e and platelet biology. REFERENCES

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