Norio Komatsu

A Truncated Erythropoietin Receptor That Fails to Prevent Programmed Cell Death of Erythroid Cells

A form of the human erythropoietin receptor (EPOR) was identified in which the cytoplasmic region is truncated by alternative splicing. The truncated form of the receptor (EPOR-T) is the most prevalent form of EPOR in early-stage erythroid progenitor cells, but the full-length EPOR (EPOR-F) becomes the most prevalent form in late-stage progenitors. EPOR-T can transduce a mitogenic signal. However, cells transfected with EPOR-T are more prone to programmed cell death than those expressing EPOR-F. EPOR-F may transduce a signal to prevent programmed cell death that is independent of the mitogenic signal, and alternative splicing of the EPOR gene may have an important role in erythropoiesis.

Activation of the thrombopoietin receptor by mutant calreticulin in CALR-mutant myeloproliferative neoplasms.

Recurrent somatic mutations of calreticulin (CALR) have been identified in patients harboring myeloproliferative neoplasms; however, their role in tumorigenesis remains elusive. Here, we found that the expression of mutant but not wild-type CALR induces the thrombopoietin (TPO)-independent growth of UT-7/TPO cells. We demonstrated that c-MPL, the TPO receptor, is required for this cytokine-independent growth of UT-7/TPO cells. Mutant CALR preferentially associates with c-MPL that is bound to Janus kinase 2 (JAK2) over the wild-type protein. Furthermore, we demonstrated that the mutant-specific carboxyl terminus portion of CALR interferes with the P-domain of CALR to allow the N-domain to interact with c-MPL, providing an explanation for the gain-of-function property of mutant CALR. We showed that mutant CALR induces the phosphorylation of JAK2 and its downstream signaling molecules in UT-7/TPO cells and that this induction was blocked by JAK2 inhibitor treatment. Finally, we demonstrated that c-MPL is required for TPO-independent megakaryopoiesis in induced pluripotent stem cell-derived hematopoietic stem cells harboring the CALR mutation. These findings imply that mutant CALR activates the JAK2 downstream pathway via its association with c-MPL. Considering these results, we propose that mutant CALR promotes myeloproliferative neoplasm development by activating c-MPL and its downstream pathway.

Characterization of stage progression in chronic myeloid leukemia by DNA microarray with purified hematopoietic stem cells.

Chronic myeloid leukemia (CML) is characterized by the clonal expansion of hematopoietic stem cells (HSCs). Without effective treatment, individuals in the indolent, chronic phase (CP) of CML undergo blast crisis (BC), the prognosis for which is poor. It is therefore important to clarify the mechanism underlying stage progression in CML. DNA microarray is a versatile tool for such a purpose. However, simple comparison of bone marrow mononuclear cells from individuals at different disease stages is likely to result in the identification of pseudo-positive genes whose change in expression only reflects the different proportions of leukemic blasts in bone marrow. We have therefore compared with DNA microarray the expression profiles of 3456 genes in the purified HSC-like fractions that had been isolated from 13 CML patients and healthy volunteers. Interestingly, expression of the gene for PIASy, a potential inhibitor of STAT (signal transducer and activator of transcription) proteins, was down-regulated in association with stage progression in CML. Furthermore, forced expression of PIASy has induced apoptosis in a CML cell line. These data suggest that microarray analysis with background-matched samples is an efficient approach to identify molecular events underlying the stage progression in CML.

A Distinct Function of STAT Proteins in Erythropoietin Signal Transduction

The Janus kinase (JAK)-signal transducers and activators of transcription (STAT) pathway is an important signaling pathway of interferons and cytokines. We examined the activation of STAT proteins induced by interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), or erythropoietin (EPO) using the human leukemia cell line, UT-7, which requires these cytokines for growth. IL-3, GM-CSF, and EPO induced DNA-binding activity to the oligonucleotides corresponding to the sis-inducible elements (SIE) of c-fos, in addition to the β-casein promoter (β-CAP), SIE- and β-CAP-binding proteins were identical to Stat1α and Stat3 complex and to Stat5 protein, respectively. This indicates that IL-3, GM-CSF, and EPO commonly activated Stat1α, Stat3, and Stat5 proteins in UT-7. However, EPO hardly activated Stat1α and Stat3 in UT-7/GM, which is a subline of UT-7 that grows slightly in response to EPO. Transfection studies revealed that UT-7/GM cells constitutively expressing Stat1α, but not Stat3, can grow as well in response to EPO as GM-CSF, suggesting that Stat1α is involved in the EPO-induced proliferation of UT-7. Thus, although Stat1α, Stat3, and Stat5 proteins are activated by GM-CSF, IL-3, and EPO, our data suggest that each STAT protein has a distinctive role in the actions of cytokines.

Construction of ELISA system to quantify human ST2 protein in sera of patients.

The human ST2 gene can be specifically induced by growth stimulation in fibroblastic cells, and can also be induced by antigen stimulation in Th2 cells. The gene encodes a soluble secreted protein, ST2, and a transmembrane protein, ST2L, which are closely related to the interleukin-1 receptor. To gain insight into the biological roles of the ST2 gene, three monoclonal antibodies (MAbs) against human ST2 gene products were obtained. To obtain these antibodies, immunization was carried out using two different immunogens: purified soluble human ST2 protein (hST2), and COS7 cells, which express the extracellular portion of human ST2L. 2A5 and FB9 MAbs were derived from the immunization with soluble hST2, and HB12 was derived from the COS7 cell immunization. All three antibodies were shown to detect native forms of the human ST2 gene products by immunoprecipitation, flow cytometry, and enzyme-linked immunosorbent assay (ELISA). In the competitive ELISA using biotinylated and nonlabelled MAbs, neither FB9 nor HB12 affected the binding of 2A5 to ST2 gene products. Based on this result, we constructed a sandwich ELISA system using 2A5 and FB9 to measure the concentration of soluble hST2 in sera. The ELISA, combined with the flow cytometry using these antibodies, will be a useful tool for elucidating the functions of human ST2 gene products in individuals.

Activation of extracellular signal-regulated kinases ERK1 and ERK2 induces Bcl-xL up-regulation via inhibition of caspase activities in erythropoietin signaling.

Erythropoietin (EPO) can rescue erythroid cells from apoptosis during erythroid development, leading to red cell production. However, the detailed mechanism of how EPO protects erythroid cells from apoptosis is still open to question. To address this problem, we used a human EPO‐dependent leukemia cell line UT‐7/EPO and normal erythroid progenitor cells. After deprivation of EPO, UT‐7/EPO cells underwent apoptosis, accompanied by down‐regulation of the Bcl‐xL protein. In addition, the cleaved products of caspase‐3, p11 and p21, and a few cleaved forms of inhibitor of caspase‐activated DNase (ICAD) were detected in these cells. When the cells were pre‐treated with the pancaspase inhibitor Z‐VAD‐FMK, the ratio of apoptotic cells was significantly reduced, suggesting that EPO protects the UT‐7/EPO cells from apoptosis via inhibition of caspase activities. When an MEK 1/2 inhibitor U0126 inhibited activities of extracellular signal‐regulated kinases (ERKs), the expression of Bcl‐xL protein was down‐regulated and subsequently apoptosis was induced. Interestingly, Z‐VAD‐FMK blocked U0126‐induced down‐regulation of Bcl‐xL protein and apoptosis, strongly suggesting that Bcl‐xL expression is regulated by caspases which lies downstream of ERK activation pathway in EPO signaling. Importantly, these findings were also observed in normal erythroid progenitor cells. In conclusion, the activation of ERKs by EPO up‐regulates Bcl‐xL expression via inhibition of caspase activities, resulting in the protection of erythroid cells from apoptosis.

The production of tissue inhibitors of metalloproteinases (TIMPs) in megakaryopoiesis: possible role of platelet‐ and megakaryocyte‐derived TIMPs in bone marrow fibrosis

We quantified tissue inhibitor of metalloproteinase (TIMP)‐1 and TIMP‐2 in serum and plasma in normal control subjects and patients with a low or high platelet count, using one‐step sandwich enzyme immunoassays. The serum levels of TIMP‐1 and TIMP‐2 were 101.1 ± 13.3 ng/ml, and 82.7 ± 26.3 ng/ml, respectively, in normal subjects. In patients with an elevated platelet count, such as in essential thrombocytosis, polycythaemia vera, and myelofibrosis, serum levels of TIMP‐1 and TIMP‐2 were 351.6 ± 200.9 ng/ml and 148.9 ± 84.0 ng/ml, respectively. Serum levels of TIMP‐1 and TIMP‐2 in patients with a low platelet count, such as in aplastic anaemia and idiopathic thrombocytopenic purpura, were 57.2 ± 25.8 ng/ml and 19.7 ± 7.68 ng/ml, respectively. The serum level of TIMP‐1 was significantly correlated with the platelet count in all subjects. The correlation between the serum level of TIMP‐2 and the platelet count was not as strong. The level of TIMP‐1 in platelet‐depleted plasma was not correlated with the platelet count.

FOXO3A as a key molecule for all-trans retinoic acid–induced granulocytic differentiation and apoptosis in acute promyelocytic leukemia

All-trans retinoic acid (ATRA) induces granulocytic differentiation and apoptosis in acute promyelocytic leukemia (APL) cells, although the detailed mechanisms are not fully understood. We investigated ATRA-induced cellular responses mediated by the transcription factor FOXO3A in APL cells. FOXO3A was constitutively phosphorylated and localized in the cytoplasm in both APL-derived NB4 cells and primary APL cells. Upon treating the cells with ATRA, FOXO3A phosphorylation was reduced and FOXO3A translocated into the nucleus. In addition, the expression of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), a target molecule for FOXO3A, was increased at the transcriptional and protein levels. As expected, transfection of a short hairpin RNA (shRNA) oligonucleotide specific for FOXO3A significantly inhibited ATRA-induced granulocytic differentiation and apoptosis in NB4 cells. In NB4-derived ATRA-resistant NB4/RA cells, neither FOXO3A nuclear localization nor subsequent TRAIL induction was observed after ATRA treatment. Furthermore, forced expression of active FOXO3A in the nucleus induced TRAIL production and apoptosis in NB4/RA cells. We conclude that activation of FOXO3A is an essential event for ATRA-induced cellular responses in NB4 cells. FOXO3A is a promising target for therapeutic approaches to overcome ATRA resistance in APL.

Active FKHRL1 overcomes imatinib resistance in chronic myelogenous leukemia‐derived cell lines via the production of tumor necrosis factor‐related apoptosis‐inducing ligand

FKHRL1 (also called FOXO3a) is a member of the Forkhead Box, class O (FOXO) subfamily of forkhead transcription factors and functions downstream of Bcr–Abl tyrosine kinase as a phosphorylated inactive form in chronic myelogenous leukemia (CML). The Bcr–Abl tyrosine kinase inhibitor imatinib induces cell cycle arrest and subsequent apoptosis via the conversion of FKHRL1 from the phosphorylated inactive form to the dephosphorylated active form in CML‐derived cell lines. In the present study, we examined whether active FKHRL1 can overcome resistance to imatinib. To this end, we generated a 4‐hydroxytamoxifen‐inducible active FKHRL1 (FKHRL1‐TM; a triple mutant of FKHRL1 in which all three Akt phosphorylation sites have been mutated)–estrogen receptor fusion protein expression system in CML‐derived imatinib‐resistant cell lines. 4‐Hydroxytamoxifen inhibited cell growth and cell cycle progression, and subsequently induced apoptosis, accompanied by upregulation of tumor necrosis factor‐related apoptosis‐inducing ligand (TRAIL). Thus, active FKHRL1 antagonized deregulated proliferation and induced apoptosis in these cell lines. In addition, imatinib‐resistant cells underwent apoptosis after transfection with full‐length TRAIL cDNA. Collectively, our results suggest that active FKHRL1 can overcome imatinib resistance in CML cells, in part via TRAIL production. (Cancer Sci 2007; 98: 1949–1958)

Megakaryocytopoiesis in vitro of patients with essential thrombocythaemia: effect of plasma and serum on megakaryocytic colony formation

Summary. TO clarify the mechanism of increased numbers of megakaryocytes in patients with essential thrombocythaemia (ET), we studied in vitro megakaryocytopoiesis in ET and other myeloproliferative disorders, using a megakaryocyte colony assay in methylcellulose containing plasma or serum and medium conditioned by phytohaemagglutinin (PHA) stimulated leucocytes (PHA‐LCM). Megakaryocytic colony formation was supported well by heparinized or citrated plasma and citrated serum which was harvested after clot formation of citrated plasma. Whole serum was inhibitory for megakaryocytic colony growth. The addition of platelet releasates and partially purified platelet derived growth factor (PDGF) resulted in a decrease in the number of megakaryocytic colonies. These findings suggested that platelet‐derived factor(s) in serum was inhibitory to megakaryocytic colony formation.