Thrombopoietin-induced polyploidization of bone marrow megakaryocytes is due to a unique regulatory mechanism in late mitosis.

Megakaryocytes undergo a unique differentiation program, becoming polyploid through repeated cycles of DNA synthesis without concomitant cell division. However, the mechanism underlying this polyploidization remains totally unknown. It has been postulated that polyploidization is due to a skipping of mitosis after each round of DNA replication. We carried out immunohistochemical studies on mouse bone marrow megakaryocytes during thrombopoietin- induced polyploidization and found that during this process megakaryocytes indeed enter mitosis and progress through normal prophase, prometaphase, metaphase, and up to anaphase A, but not to anaphase B, telophase, or cytokinesis. It was clearly observed that multiple spindle poles were formed as the polyploid megakaryocytes entered mitosis; the nuclear membrane broke down during prophase; the sister chromatids were aligned on a multifaced plate, and the centrosomes were symmetrically located on either side of each face of the plate at metaphase; and a set of sister chromatids moved into the multiple centrosomes during anaphase A. We further noted that the pair of spindle poles in anaphase were located in close proximity to each other, probably because of the lack of outward movement of spindle poles during anaphase B. Thus, the reassembling nuclear envelope may enclose all the sister chromatids in a single nucleus at anaphase and then skip telophase and cytokinesis. These observations clearly indicate that polyploidization of megakaryocytes is not simply due to a skipping of mitosis, and that the megakaryocytes must have a unique regulatory mechanism in anaphase, e.g., factors regulating anaphase such as microtubule motor proteins might be involved in this polyploidization process.

Regulation of APC activity by phosphorylation and regulatory factors.

Ubiquitin-dependent proteolysis of Cut2/Pds1 and Cyclin B is required for sister chromatid separation and exit from mitosis, respectively. Anaphase-promoting complex/cyclosome (APC) specifically ubiquitinates Cut2/Pds1 at metaphase-anaphase transition, and ubiquitinates Cyclin B in late mitosis and G1 phase. However, the exact regulatory mechanism of substrate-specific activation of mammalian APC with the right timing remains to be elucidated. We found that not only the binding of the activators Cdc20 and Cdh1 and the inhibitor Mad2 to APC, but also the phosphorylation of Cdc20 and Cdh1 by Cdc2-Cyclin B and that of APC by Polo-like kinase and cAMP-dependent protein kinase, regulate APC activity. The cooperation of the phosphorylation/dephosphorylation and the regulatory factors in regulation of APC activity may thus control the precise progression of mitosis.

Identification of human APC10/Doc1 as a subunit of anaphase promoting complex.

Anaphase-promoting complex or cyclosome (APC) is a ubiquitin ligase which specifically targets mitotic regulatory factors such as Pds1/Cut2 and cyclin B. Identification of the subunits of multiprotein complex APC in several species revealed the highly conserved composition of APC from yeast to human. It has been reported, however, that vertebrate APC is composed of at least eight subunits, APC1 to APC8, while budding yeast APC is constituted of at least 12 components, Apc1 to Apc13. It has not yet been clearly understood whether additional components found in budding yeast, Apc9 to Apc13, are actually composed of mammalian APC. Here we isolated and characterized human APC10/Doc1, and found that APC10/Doc1 binds to APC core subunits throughout the cell cycle. Further, it was found that APC10/Doc1 is localized in centrosomes and mitotic spindles throughout mitosis, while it is also localized in kinetochores from prophase to anaphase and in midbody in telophase and cytokinesis. These results strongly support the notion that human APC10/Doc1 may be one of the APC core subunits rather than the transiently associated regulatory factor.

Interleukin 3 and erythropoietin induce association of Vav with Tec kinase through Tec homology domain.

Although hematopoietic cytokine receptors lack tyrosine kinase domains, the binding of their ligands to the receptors induce rapid tyrosine phosphorylation of various cellular target proteins. The specific tyrosine kinases which phosphorylate these substrates, however, have not been identified, other than that JAK kinases which phosphorylate STAT proteins and the receptors. We found that the c-vav proto-oncogene product, Vav, is rapidly and transiently tyrosine-phosphorylated in response to erythropoietin and IL3 stimulations and that Tec kinase is also transiently activated by these cytokines. Immunoprecipitation experiments demonstrated that Tec kinase binds to Vav upon these cytokine stimulations and that Grb2 constitutively associates with Vav. In vitro binding assays showed that erythropoietin and IL3 stimulation induce the specific binding of Vav to Tec kinase through Tec homology domains. We therefore concluded that Tec kinase is one of the key enzymes in Epo and IL3 receptor-mediated signaling pathways and that Vav plays an important role in the cytokine receptor-mediated signal transduction.

Tec is involved in G protein-coupled receptor- and integrin-mediated signalings in human blood platelets.

Tec is a non-receptor type tyrosine kinase which is tyrosine phosphorylated and activated upon stimulation of hematopoietic cells with various cytokines. The role of Tec in G protein-coupled receptor- and integrin-mediated signalings has not been elucidated. We therefore investigated the regulation of Tec in human blood platelets. Tec was rapidly tyrosine phosphorylated in response to platelet agonists which activate G protein-coupled receptors such as thromboxane A2 analog (U46619), thrombin, and thrombin receptor activating peptide (TRAP). TRAP-induced phosphorylation in Tec was significantly reduced under the conditions which abrogate fibrinogen binding to GP IIb-IIIa and subsequent platelet aggregation. However, TRAP induced significant levels of the phosphorylation even under these conditions and also in thrombasthenic platelets which lack functional GP IIb-IIIa molecules, suggesting that activation of G-protein-coupled receptor causes the phosphorylation. To clarify whether integrin engagement by itself causes the phosphorylation in Tec, we examined the state of the phosphorylation in platelets activated by integrin engagement. Platelet adhesion to immobilized fibrinogen or collagen induced significant levels of the phosphorylation. Furthermore, Tec was translocated to cytoskeleton in response to TRAP in a manner dependent on platelet aggregation, suggesting that Tec can be a component of integrin-mediated signalings. These results collectively indicate that Tec is involved in G protein-coupled receptor- and integrin-mediated signalings in human blood platelets.

Characterization of erythropoietin receptor on erythropoietin-unresponsive mouse erythroleukemia cells.

A membrane receptor for erythropoietin was identified in various erythropoietin-unresponsive mouse erythroleukemia cells. Scatchard analyses of the binding of human 125I-labeled erythropoietin to T3C1-2-0, K-1, GM86 and 707 cells showed the presence of a single class of binding sites with apparent Kd values of 0.27-0.78 nM, which are slightly higher than those of erythropoietin-responsive cells. The number of binding sites varied from 110 to 930 per cell. Crosslinking of 125I-erythropoietin to its binding sites with disuccinimidyl suberate revealed the existence of a single binding protein with molecular mass of 63 kDa. No binding sites with higher molecular mass, as observed in erythropoietin-responsive cells, were detected, nor was any specific binding observed to the non-erythroid hematopoietic cell or to the human erythroleukemia cells examined.

Constitutive expression of exogenous c-myb gene causes maturation block in monocyte-macrophage differentiation.

A nuclear protooncogene c-myb has been hypothesized to play an important role in hematopoiesis, but little is known about the physiological function of the c-myb gene products. To study the role of c-myb gene expression in monocyte-macrophage differentiation and proliferation, we introduced exogenous c-myb gene into murine myelomonocytic leukemia WEHI-3B(D+) cells which can be induced to differentiate into mature monocytes with granulocyte-colony stimulation factor (G-CSF) and actinomycin D. Expression of the transfected gene was found to result in elevated levels of c-myb transcripts, which were not subject to normal down-regulation by differentiation induction. This constitutive expression of c-myb gene allowed the c-myb transfectants to differentiate into promonocytes with G-CSF and actinomycin D, but blocked further maturation from promonocytes to mature monocytes. It is concluded that normal down-regulation of c-myb gene expression during monocyte-macrophage differentiation is required for the maturation of promonocytes to mature monocytes.

Characterization of murine erythropoietin receptor genes.

We have isolated and characterized the murine genomic and complementary DNAs encoding erythropoietin (Epo) receptor from Epo-responsive and unresponsive mouse erythroleukemia cells. Two classes of Epo receptor cDNAs were isolated from Epo-responsive cells. One is a 55,000 Mr membrane-bound Epo receptor, and the other is a 29,000 Mr soluble Epo receptor lacking the transmembrane and cytoplasmic domains. As a result of alternative splicing, two insert sequences containing termination codons are produced, and the encoded polypeptide diverges four amino acids upstream from the transmembrane domain, adding 20 new amino acids before terminating. Amino acid sequence of the Epo receptor cDNA isolated from Epo-responsive cells was identical with that of Epo-unresponsive cells, indicating that Epo-responsiveness does not depend upon the primary structure of the Epo receptor (binding) protein. Analysis of 6.6 x 10(3) base-pairs (kb) genomic DNA segments covering complete Epo receptor gene and promoter regions revealed that potential regulatory elements (NF-E1, GF-1 or Eryf 1) for erythroid-specific and differentiation stage-specific gene expression are located in the promoter and 3' noncoding regions.

Regulation of megakaryocytopoiesis by thrombopoietin and stromal cells.

Thrombopoietin (Tpo) is a cytokine which stimulates megakaryocyte maturation. We found that Tpo is constitutively and ubiquitously expressed in all tissues examined, including bone marrow stromal cells, even in thrombocytopenia, thrombosis and steady-state condition in mice. Thus, platelet level in circulation is not regulated by Tpo gene expression. Furthermore, when the purified megakaryocytes were cocultured with the stromal cells, most of the megakaryocytes adhered to the stromal cells and remained unchanged, while free megakaryocytes induced proplatelet formation. Thus the stromal cells in bone marrow secrete Tpo and stimulate megakaryocytopoiesis, but the interaction of megakaryocytes with the stromal cells may suppress platelet formation. Study on signal transduction through Mp1 revealed that Tpo induces activation of JAK2 and Tyk2, which in turn activate STAT1, STAT3 and STAT5. Further, Tpo stimulates transcription factors GATA-1 and NF-E2, which induce differentiation markers, GPIIb/IIIa and Pm-1. In addition, Shc, Vav, Ras, Raf-1, MAPKK, MAPK and Pim-1 are also activated. Thus, Tpo activates a lineage-specific cascade as well as a specific JAK-STAT cascade and a common signaling cascade.

Immature megakaryocytes undergo apoptosis in the absence of thrombopoietin.

We examined withdrawal effects of recombinant mouse Tpo (rm-Tpo) on the apoptosis of mature and immature megakaryocytes in in vitro experiments. Apoptotic megakaryocytes were detected by double staining for acetylcholinesterase and by the TdT-mediated dUTP-biotin nick end labeling (TUNEL) method. When the purified mature megakaryocytes were cultured with or without rm-Tpo, the numbers of viable megakaryocytes, apoptotic megakaryocytes, and megakaryocytes with cytoplasmic processes were not significantly different between the two groups. In contrast, purified immature megakaryocytes underwent apoptosis when rm-Tpo was absent from the culture system. Murine bone marrow cells were cultured with rm-Tpo (50 U/mL) on days 1-7 to generate immature megakaryocytes and subsequently were cultured with different concentrations of rm-Tpo (0-50 U/mL) on days 8-14. The number of viable megakaryocytes was decreased and that of apoptotic megakaryocytes was increased by rm-Tpo in a dose-dependent manner. These results indicated a clear relation between the rm-Tpo level and the apoptosis of immature megakaryocytes.

Isolation and characterization of a genomic DDD mouse interleukin-3 gene.

A chromosomal DNA segment containing the entire gene for interleukin-3 (IL-3) was isolated from DDD mouse erythroleukemia cells, and its gene organization and nucleotide sequence were determined. Enhancer-like sequences in the second intron and G + C-rich sequence in the 5'-flanking region may play a role in the regulation of tissue-specific and inducible gene expression. Southern blot analyses revealed that constitutively IL-3-producing WEHI-3 cells have the rearranged IL-3 gene, and that genomic DNAs prepared from the adult and fetal mouse liver have the same organization of the IL-3 gene. No IL-3 gene transcript was detected in the mouse erythroleukemia cells by Northern blot analysis.

Isolation of a cDNA encoding a potential soluble receptor for human erythropoietin.

Analysis of human erythropoietin receptor-encoding cDNAs revealed the possible existence of a soluble receptor lacking the transmembrane and cytoplasmic domains.

Thrombopoietin induces activation of at least two distinct signaling pathways.

Thrombopoietin (Tpo) is a cytokine regulating megakaryocyte maturation and platelet formation. We studied Tpo-induced signal transduction, and found that Tpo induces phosphorylation of adapter molecules. Shc and Vav, and of serine/threonine kinases Raf-1 and mitogen-activated protein (MAP) kinases. Further, Tpo induced activation of Ras, MAP kinase kinase, MAP kinase and Pim-1. Taken together with other observations, we concluded that Tpo induces the activation of at least two distinct signaling pathways, a specific Tyk2-JAK2/STAT1-STAT3-STAT5 signaling cascade and a common Shc/Vav/Ras/Raf-1/MAP kinase kinase/MAP kinase signaling cascade.

Role of Flk-1 in mouse hematopoietic stem cells.

It was reported that human hematopoietic stem cells in bone marrow were restricted to the CD34(+)KDR(+) cell fraction. We found that expression levels of Flk-1, a mouse homologue of KDR, were low or undetectable in mouse Lin(-)c-Kit(+)Sca-1(+)CD34(low/-) cells as well as Hoechst33342(-) cells (side population), which have long-term reconstitution capacity. Furthermore, neither Flk-1(+)CD34(low/-) cells nor Flk-1(+)CD34(+) cells had long-term reconstitution capacity in mouse. Taken together with other observations using Flk-1-deficient mice, these results indicate that Flk-1 is essential for the development of hematopoietic stem cells in embryo but not for the function of hematopoietic stem cells in adult mouse bone marrow.

Cell-growth control by monomeric antigen: the cell surface expression of lysozyme-specific Ig V-domains fused to truncated Epo receptor.

Previously we have shown that the V(H) and V(L) fragments of an anti-hen egg lysozyme (HEL) antibody HyHEL-10 are weakly associated but can be driven together by antigen. By joining these antibody variable domains to the cytoplasmic portion of the murine erythropoietin receptor, we created a chimeric growth factor receptor that could be activated by HEL. After co-transfection with two plasmids encoding the respective chimeric receptors in IL-3 dependent murine pro-B Ba/F3 cells, a portion of the cells survived under antigen dependent stimulation without IL-3. These surviving cells all showed coexpression of the two chimeric receptor chains and demonstrated HEL dose-dependent growth stimulation without IL-3. When another IL-3 dependent cell line 32D was transfected with a variant of such chimeric receptor with a linker peptide (Gly-Ser-Gly) inserted between V(H)/V(L) and EpoR domains, an improved growth response was attained. These observations suggest the utility of heterodimeric Fv chimeric receptors in creating cells that respond to monomeric antigen.

Serum thrombopoietin level is not regulated by transcription but by the total counts of both megakaryocytes and platelets during thrombocytopenia and thrombocytosis.

Thrombopoietin (Tpo) regulates platelet production, but the mechanisms regulating the serum Tpo level and platelet count in circulation have been a subject of debate. Tpo was reported to be expressed mainly in liver and kidney, but we found that Tpo is expressed in all tissues examined: abundantly in liver, kidney, muscle, colon, brain and intestine, and moderately in bone marrow, spleen, lung, stomach, heart, thymus, ovary, and endothelial and leukemic cell lines. The levels of Tpo transcripts in major Tpo producing organs, liver and kidney, and in the platelet production sites bone marrow and spleen, were constant during acute thrombocytopenia induced by anti-platelet monoclonal antibody administration in mice, and during thrombocytosis induced by Tpo injection. Furthermore, we noticed that platelet count is not exactly inversely proportional to serum Tpo level. During acute thrombocytopenia, serum Tpo level transiently increased a few hours after antibody injection, and returned to the basal level just when matured megakaryocytes accumulated in bone marrow and spleen but the platelet count was still low. Matured megakaryocytes in bone marrow and spleen increased when the serum Tpo level decreased, and decreased when platelet count rebounded. Taken together with other observations, we propose here a modified version of Kuter and Rosenberg's theory, that is, Tpo is constitutively expressed in a variety of organs throughout the body, even in acute thrombocytopenia and thrombocytosis, and that the serum Tpo level is not regulated by Tpo gene expression nor only by platelet counts in circulation, but by the total counts of both megakaryocytes in bone marrow and spleen and of platelets in circulation.

Modulation of platelet activation in vitro by thrombopoietin.

Effect of human recombinant thrombopoietin (TPO) on platelet activation in vitro was studied. Although TPO itself did not cause platelet aggregation, it upregulated ADP-induced aggregation, especially the second wave of aggregation. This effect was dose-dependent for up to 5 ng/ml of TPO. When platelets were activated by epinephrine, collagen, or alpha-thrombin, similar effect was observed. However, TPO did not affect A23187- or PMA-induced aggregation, suggesting that TPO may have modulated the signal transduction pathway upstream of inositol 1,4,5-trisphosphate and diacylglycerol production. TPO also upregulated thrombin-induced alpha-granule secretion. To clarify the involvement of protein tyrosine phosphorylation, platelets were activated by TPO and/or suboptimal concentration of ADP, then tyrosine phosphorylation was detected by immunoblot analysis, using anti-phosphotyrosine monoclonal antibody. TPO by itself caused significant tyrosine phosphorylation of 146, 130, 122, 108, 97, 94, and 88 kDa proteins. Further, by using antibodies against signal transduction molecules for immunoprecipitation, we observed the significant tyrosine phosphorylation in Jak2 and Tyk2 molecules after TPO-stimulation. The results of the present experiment clearly indicate that TPO directly activated platelets and modulated intracellular signal transduction pathway.

Serum thrombopoietin (TPO) levels in patients with amegakaryocytic thrombocytopenia are much higher than those with immune thrombocytopenic purpura.

We assayed serum thrombopoietin (TPO) levels in amegakaryocytic thrombocytopenia (AMT) and immune thrombocytopenic purpura (ITP) patients by using a newly established enzyme-linked immunosorbent assay (ELISA). TPO levels in AMT patients were quite high (mean +/- SD = 13.7 +/- 11.2 fmoles/ml, n = 4), whereas those in ITP patients were only slightly higher (1.25 +/- 0.39, n = 12) than those of the healthy donors (0.55 +/- 0.2, n = 20). Furthermore, in ITP patients no correlation was observed between platelet counts and serum TPO levels (correlation coefficient = 0.14). We further assayed serum TPO levels sequentially during steroid treatment in patients with AMT and ITP. In one AMT patient serum TPO levels started to decrease in accordance with the increase of megakaryocyte counts, which preceded the increase in platelet counts. However, in ITP patients serum TPO levels did not change significantly throughout the course of the treatment despite the recovery of platelet counts. Based on these findings, we conclude that serum TPO levels may be regulated at least in part by megakaryocyte counts.