Itaru Matsumura

Transcriptional regulation of the cyclin D1 promoter by STAT5: its involvement in cytokine‐dependent growth of hematopoietic cells

STAT5 is a member of a family of transcription factors that participate in the signal transduction pathways of many hormones and cytokines. Although STAT5 is suggested to play a crucial role in the biological effects of cytokines, its downstream target(s) associated with cell growth control is largely unknown. In a human interleukin‐3 (IL‐3)‐dependent cell line F‐36P‐mpl, the induced expression of dominant‐negative (dn)‐STAT5 and of dn‐ras led to inhibition of IL‐3‐dependent cell growth, accompanying the reduced expression of cyclin D1 mRNA. Also, both constitutively active forms of STAT5A (1*6‐STAT5A) and ras (H‐rasG12V) enabled F‐36P‐mpl cells to proliferate without added growth factors. In NIH 3T3 cells, 1*6‐STAT5A and H‐rasG12V individually and cooperatively transactivated the cyclin D1 promoter in luciferase assays. Both dn‐STAT5 and dn‐ras suppressed IL‐3‐induced cyclin D1 promoter activities in F‐36P‐mpl cells. Using a series of mutant cyclin D1 promoters, 1*6‐STAT5A was found to transactivate the cyclin D1 promoter through the potential STAT‐binding sequence at −481 bp. In electrophoretic mobility shift assays, STAT5 bound to the element in response to IL‐3. Furthermore, the inhibitory effect of dn‐STAT5 on IL‐3‐dependent growth was restored by expression of cyclin D1. Thus STAT5, in addition to ras signaling, appears to mediate transcriptional regulation of cyclin D1, thereby contributing to cytokine‐dependent growth of hematopoietic cells.

E2F1 and c-Myc Potentiate Apoptosis through Inhibition of NF-κB Activity that Facilitates MnSOD-Mediated ROS Elimination

Overexpression of c-Myc or E2F1 sensitizes host cells to various types of apoptosis. Here, we found that overexpressed c-Myc or E2F1 induces accumulation of reactive oxygen species (ROS) and thereby enhances serum-deprived apoptosis in NIH3T3 and Saos-2. During serum deprivation, MnSOD mRNA was induced by NF-kappaB in mock-transfected NIH3T3, while this induction was inhibited in NIH3T3 overexpressing c-Myc or E2F1. In these clones, E2F1 inhibited NF-kappaB activity by binding to its subunit p65 in competition with a heterodimeric partner p50. In addition to overexpressed E2F1, endogenous E2F1 released from Rb was also found to inhibit NF-kappaB activity in a cell cycle-dependent manner by using E2F1(+/+) and E2F1(-/-) murine embryonic fibroblasts. These results indicate that E2F1 promotes apoptosis by inhibiting NF-kappaB activity.

Thrombopoietin-induced differentiation of a human megakaryoblastic leukemia cell line, CMK, involves transcriptional activation of p21(WAF1/Cip1) by STAT5.

Although thrombopoietin (TPO) is known to play a fundamental role in both megakaryopoiesis and thrombopoiesis, the molecular mechanism of TPO-induced megakaryocytic differentiation is not known. In a human megakaryoblastic leukemia cell line, CMK, that showed some degree of megakaryocytic differentiation after culture with TPO, the cyclin-dependent kinase (Cdk) inhibitor p21(WAF1/Cip1), but not p27(Kip1), p16(INK4A), p15(INK4B), or p18(INK4C), was found to be upregulated in an immediately early response to TPO. The expression of p21 was found to be sustained over a period of 5 days by treatment with TPO in large polyploid cells that developed in response to TPO, but not in small undifferentiated cells, indicating a close correlation between the ligand-induced differentiation and p21 induction in CMK cells. To examine potential roles of Cdk inhibitors in megakaryocytic differentiation, CMK cells were transfected with the p21, p27, or p16 gene, together with a marker gene, beta-galactosidase, and were cultured with medium alone for 5 days. The ectopic expression of p21 or p27 but not of p16 led to induction of megakaryocytic differentiation of CMK cells. Overexpression of the N-terminal domain (amino acids [aa] 1 to 75) of p21 was sufficient to induce megakaryocytic differentiation, whereas that of the C-terminal domain (aa 76 to 164) had little or no effect on morphological features. Furthermore, we found that although TPO induced tyrosine phosphorylation of both STAT3 and STAT5 in CMK cells, only STAT5 showed binding activities to potential STAT-binding sites that locate in the promoter region of p21 gene (p21-SIE sites), thereby leading to transactivation of p21. These results suggested that p21 induction, possibly mediated through activated STAT5, could play an important role in TPO-induced megakaryocytic differentiation.

E2F1 and c-Myc in cell growth and death.

Cell cycle machinery controls not only cell growth but also cell survival and death. For example, overexpression of c-Myc or E2F1, which are involved in G1/S transition, causes apoptosis under certain conditions. Furthermore, endogenous E2F1 also participates in apoptosis, as evidenced by the defect of apoptosis in E2F1-deficient mice. Candidate molecules that mediate c-Myc- and E2F1-enhanced apoptosis include p14/p19ARF, ornithine decarboxylase and lactate degydrogenase-A (for c-Myc) as well as p14/p19ARF, p73, Apaf-1 and caspase-3 (for E2F1). c-Myc also activates the CD95/Fas-FADD-mediated death signal. c-Myc and E2F1 inhibit NF-kB activities induced by TNFa or reactive oxygen species. Therefore, c-Myc and E2F1 regulate cell growth and death not only by inducing transcription but also by modulating signal transduction pathways.

Identification of a cytokine-induced antiapoptotic molecule anamorsin essential for definitive hematopoiesis.

Many growth factors and cytokines prevent apoptosis. Using an expression cloning method, we identified a novel antiapoptotic molecule named Anamorsin, which does not show any homology to known apoptosis regulatory molecules such as Bcl-2 family, caspase family, or signal transduction molecules. The expression of Anamorsin was completely dependent on stimulation with growth factors such as interleukin 3, stem cell factor, and thrombopoietin in factor-dependent hematopoietic cell lines, and forced expression of Anamorsin conferred resistance to apoptosis caused by growth factor deprivation in vitro. Furthermore, Anamorsin was found to act as an antiapoptotic molecule in vivo because Anamorsin−/− mice die in late gestation due to defective definitive hematopoiesis in the fetal liver (FL). Although the number of hematopoietic stem/progenitor cells in the FL did not decrease in these mice, myeloid, and particularly erythroid colony formation in response to cytokines, was severely disrupted. Also, Anamorsin−/− erythroid cells initiated apoptosis during terminal maturation. As for the mechanism of Anamorsin-mediated cell survival, a microarray analysis revealed that the expression of Bcl-xL and Jak2 was severely impaired in the FL of Anamorsin−/− mice. Thus, Anamorsin is considered to be a necessary molecule for hematopoiesis that mediates antiapoptotic effects of various cytokines.

Involvement of Prolonged Ras Activation in Thrombopoietin-Induced Megakaryocytic Differentiation of a Human Factor-Dependent Hematopoietic Cell Line

ABSTRACT Thrombopoietin (TPO) is a hematopoietic growth factor that plays fundamental roles is both megakaryopoiesis and thrombopoiesis through binding to its receptor, c-mpl. Although TPO has been shown to activate various types of intracellular signaling molecules, such as the Janus family of protein tyrosine kinases, signal transducers and activators of transcription (STATs), and ras, the precise mechanisms underlying TPO-induced proliferation and differentiation remain unknown. In an effort to clarify the mechanisms of TPO-induced proliferation and differentiation, c-mpl was introduced into F-36P, a human interleukin-3 (IL-3)-dependent erythroleukemia cell line, and the effects of TPO on the c-mpl-transfected F-36P (F-36P-mpl) cells were investigated. F-36P-mpl cells were found to proliferate and differentiate at a high rate into mature megakaryocytes in response to TPO. Dominant-negative (dn) forms of STAT1, STAT3, STAT5, and ras were inducibly expressed in F-36P-mpl cells, and their effects on TPO-induced proliferation and megakaryocytic differentiation were analyzed. Among these dn molecules, both dn ras and dn STAT5 reduced TPO- or IL-3-induced proliferation of F-36P-mpl cells by ∼30%, and only dn ras could inhibit TPO-induced megakaryocytic differentiation. In accord with this result, overexpression of activated ras (H-rasG12V) for 5 days led to megakaryocytic differentiation of F-36P-mpl cells. In a time course analysis on H-rasG12V-induced differentiation, activation of the ras pathway for 24 to 28 h was required and sufficient to induce megakaryocytic differentiation. Consistent with this result, the treatment of F-36P-mpl cells with TPO was able to induce prolonged activation of ras for more than 24 h, whereas IL-3 had only a transient effect. These results suggest that prolonged ras activation may be involved in TPO-induced megakaryocytic differentiation.

Suppressor of cytokine signalling-1 gene silencing in acute myeloid leukaemia and human haematopoietic cell lines.

The aim of this study was to investigate whether the suppressor of cytokine signalling (SOCS)‐1 can act as a tumour suppressor when functioning as a negative regulator of the Janus family tyrosine kinases (JAKs), which have been reported to play important roles in leukaemogenesis. For this purpose, we carried out molecular analysis of the SOCS‐1 gene in human acute myeloid leukaemia (AML) and human haematopoietic cell lines. Sequencing alterations in the coding region were found in two of 90 primary AML samples and one of 17 cell lines. Hypermethylation of the SOCS‐1 gene was also observed in 72% of primary cases and 52% of cell lines and aberrant methylation strongly correlated with reduced expression. Transfection of SOCS‐1 into Jurkat cells harbouring the mutation and methylation suppressed cell growth at a low serum concentration. These findings indicate that SOCS‐1 is frequently silenced in haematopoietic malignancies, mainly as a result of hypermethylation, and suggest that SOCS‐1 may be able to function as a tumour suppressor.

Cell Cycle Regulation in Hematopoietic Stem/Progenitor Cells

Hematopoietic stem cells (HSCs) are characterized by pluripotentiality and a capacity for self-renewal. In order to both maintain a supply of mature blood cells and not to exhaust HSCs throughout the lifespan of the organism, most HSCs remain quiescent and only a limited number enter the cell cycle. In HSCs, the cell cycle is crucially regulated by external factors such as cytokines and interactions with stromal cells and the extracellular matrix (ECM) in the bone marrow (BM) microenvironment. In addition, intrinsic transcription factors expressed in HSCs, including c-Myb, GATA-2, HOX family proteins, and Bmi-1, also control their growth through their effect on gene transcription. In terms of the particular roles in regulation of the cell-cycle, p21WAF1 (p21) and p27KIP1 (p27) were shown to maintain the quiescence of HSCs and of progenitor cells,

Full Oncogenic Activities of v-Src Are Mediated by Multiple Signaling Pathways Ras AS AN ESSENTIAL MEDIATOR FOR CELL SURVIVAL

Tyrosine kinase oncoproteins cause simultaneous activation of multiple intracellular signaling pathways. However, the precise mechanisms by which individual pathways induce oncogenesis are not well understood. We have investigated the roles of individual signaling pathways in v-Src-dependent cell growth and survival by inhibiting one particular pathway. v-Src induced constitutive activation of signal transducers and activators of transcription 3 (STAT3), phosphatidylinositol 3-kinase, and Ras in murine Ba/F3 cells and led to factor-independent proliferation. Dominant-negative mutants of STAT3 (STAT3D) and phosphatidylinositol 3-kinase (Δp85) inhibited v-Src-dependent growth by ∼60 and ∼40%, respectively. Moreover, dominant-negative Ras (N17) induced severe apoptosis, which was accompanied by down-regulation of Bcl-2 and activation of caspase-3. Although cells overexpressing Bcl-2 or caspase-3 inhibitors remained viable even when N17 was expressed, the growth was reduced by ∼85%. During N17- and STAT3D-induced growth suppression, expression of cyclin D2,cyclin D3, c-myc, and c-fos was suppressed by N17, whereas that of cyclin D2,cyclin E, and c-myc was suppressed by STAT3D. Thus, v-Src-activated Ras and STAT3 are involved in distinct but partly overlapping transcriptional regulation of cell cycle regulatory molecules. These results suggest that the full oncogenic activity of v-Src requires simultaneous activation of multiple signalings, in which Ras is particularly required for survival.

Japanese epidemiological survey with consensus statement on Japanese guidelines for treatment of iron overload in bone marrow failure syndromes

Many patients with bone marrow failure syndromes need frequent transfusions of red blood cells, and most of them eventually suffer from organ dysfunction induced by excessively accumulated iron. The only way to treat transfusion-induced iron overload is iron chelating therapy. However, most patients have not been treated effectively because daily/continuous administration of deferoxamine is difficult for outpatients. Recently, a novel oral iron chelator, deferasirox, has been developed, and introduction of the drug may help many patients benefit from iron chelation therapy. In this review, we will discuss the current status of iron overload in transfusion-dependent patients, and the development of Japanese guidelines for the treatment of iron overload in Japan, which were established by the National Research Group on Idiopathic Bone Marrow Failure Syndromes in Japan.