Mie Uchida

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.

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.

A Functional Role of Mitogen-Activated Protein Kinases, Erk1 and Erk2, in the Differentiation of a Human Leukemia Cell Line, UT-7/GM: A Possible Key Factor for Cell Fate Determination Toward Erythroid and Megakaryocytic Lineages

The mitogen-activated protein (MAP) kinase cascade is a key regulator of mammalian cell proliferation and differentiation. In this study, we examined the roles of 2 members of the MAP kinase family, extracellular signal-regulated kinase 1 (Erk1) and Erk2, in erythropoietin (EPO)-induced erythroid differentiation and thrombopoietin (TPO)-induced megakaryocytic differentiation. UT-7/GM was used as a model system because this cell line is an erythroid/megakaryocytic bipotent cell line that can be induced to differentiate into the erythroid and megakaryocytic lineages by EPO and TPO, respectively. The kinetics of activation of Erk1 and Erk2 were examined during erythroid and megakaryocytic differentiation of UT-7/GM cells. EPO induced a transient activation of these kinases, peaking after 1 minute of stimulation and then declining quickly almost to the basal level. In contrast, TPO-induced activation of the kinases peaked at 10 minutes and persisted for up to 60 minutes, similar to the activation by granulocyte-macrophage colony-stimulating factor. The percentage of EPO-induced hemoglobin-positive cells was elevated by the addition of PD98059, a specific inhibitor of MEK1 (MAP kinase/ERK kinase 1). In contrast, PD98059 clearly reduced the amount of glycoprotein IIb/IIIa antigens induced by TPO on UT-7/GM cells. Thus, inactivation of Erk1 and Erk2 kinases promoted EPO-induced erythroid differentiation and suppressed TPO-induced megakaryocytic differentiation of UT-7/GM cells. In conclusion, the activation of Erk1 and Erk2 kinases may be a critical event in the determination of cell fate and the differentiation processes of the erythroid and megakaryocytic lineages.

Erythropoietin Overcomes Imatinib-Induced Apoptosis and Induces Erythroid Differentiation in TF-1/bcr-abl Cells

Targeting BCR‐ABL tyrosine kinase by treatment with the selective inhibitor imatinib (formerly STI571, Gleevec) has proved to be highly efficient for inhibiting leukemic growth in vitro. In addition, in clinical trials, imatinib has produced high response rates in patients with chronic myeloid leukemia (CML) in chronic phase and blastic crisis. However, episodes of severe cytopenia were also frequently observed, leading to discontinuation of therapy in some cases. Therefore, it is important to examine whether administration of cytokines overcomes the adverse effects of imatinib in in vitro systems. In this study, we examine the effects of granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) and erythropoietin (EPO) on TF‐1/bcr‐abl (which was generated by transduction of a bcr‐abl fusion gene into the TF‐1 cell line) as a model system for CML with blastic crisis. Imatinib induced apoptosis in TF‐1/bcr‐abl cells but not in the parental TF‐1 cells. However, GM‐CSF, a survival factor of the parental TF‐1 cells, protected TF‐1/bcr‐abl cells from imatinib‐induced apoptosis in a dose‐dependent manner. Concomitantly, constitutive phosphorylation of Stat5 and FKHRL1 was significantly inhibited by imatinib, and the inhibition was canceled by the addition of GM‐CSF, accompanied by upregulation of Bcl‐xL and downregulation of p27/Kip1. In addition, although untreated TF‐1/bcr‐abl cells had lost responsiveness to both GM‐CSF and EPO and showed autonomous growth, GM‐CSF enhanced phosphorylation of Stat5 and FKHRL1 in these cells. Importantly, imatinib‐treated TF‐1/bcr‐abl cells differentiated into hemoglobin‐positive cells in the presence of EPO, as in the case for the parental TF‐1 cells. Taken together, imatinib‐treated CML cells may differentiate into mature cells in the presence of differentiation‐inducing cytokines such as EPO.

Activation of FKHRL1 Plays an Important Role in Protecting Erythroid Cells from Erythropoietin Deprivation-Induced Apoptosis in a Human Erythropoietin-Dependent Leukemia Cell Line, UT-7/EPO

FKHRL1 is one of the human homologues of DAF-16, which is concerned with longevity inCaenorhabditis elegans. Previously, we demonstrated that FKHRL1 functions downstream of Akt in erythropoietin (EPO) signaling and that it is directly phosphorylated by activated Akt. Because phosphorylated FKHRL1 loses its transcriptional activity and translocates into the cytoplasm, FKHRL1 appears to be nonfunctional in the presence of EPO. Conversely, EPO deprivation leads to FKHRL1 dephosphorylation and its translocation into the nucleus, suggesting that FKHRL1 becomes active as a transcription factor in the absence of EPO. On the basis of these findings, we hypothesized, by analogy withC elegans, that erythroid cells possess self-defense machinery against life-threatening surroundings. We prepared a dominant-negative mutant of FKHRL1 (FKHRL1-DN) lacking the transactivation domain and prepared FKHRL1 small interfering RNA (siRNA), and we used constructs to transfect a human EPO-dependent cell line, UT-7/EPO. In the parental cells, 24-hour EPO deprivation induced transient cell cycle arrest without apoptosis. On the other hand, stable transfectants expressing FKHRL1-DN or FKHRL1 siRNA underwent rapid apoptosis after EPO deprivation in the UT-7/EPO cells. In conclusion, FKHRL1 activation plays an important role in the extension of survival of erythroid cells after EPO deprivation. This phenomenon appears to correspond to dauer formation in C elegans. Thus, the mechanism of lifespan extension may be broadly conserved fromC elegans to humans.