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Thrombopoietin Regulates Bcl-xL Gene Expression through Stat5 and Phosphatidylinositol 3-Kinase Activation Pathways*

Open AccessPublished:December 27, 2001DOI:https://doi.org/10.1074/jbc.M109824200
      Thrombopoietin (TPO), an essential factor for megakaryopoiesis and thrombopoiesis, works as a survival factor for megakaryocytic lineage cells. However, little is known about the molecular mechanism in detail. We show here that TPO supports the survival of TPO-dependent leukemia cell line UT-7/TPO and normal megakaryocytic progenitors via the induction of Bcl-xL, an anti-apoptotic member of the Bcl-2 family. We further analyzed the signal transduction pathways required for TPO-induced Bcl-xL gene expression. A reporter assay with various lengths of Bcl-x gene promoter revealed that both Stat- and nuclear factor κB-binding sites are prerequisites for TPO-induced promoter activity. Consistent with these results, TPO induced the binding of Stat5 and subunits of nuclear factor κB, p50, and c-Rel to the Bcl-x gene promoter. AG490, a specific inhibitor for Jak2, and LY294002, a specific inhibitor for phosphatidylinositol (PI) 3-kinase, reduced the protein level of Bcl-xL in UT-7/TPO cells, accompanied by an increase in the ratio of apoptotic cells. Interestingly, LY294002 enhanced the TPO-induced DNA binding activity of Stat5 without affecting the Jak2 activation and tyrosine phosphorylation of Stat5. Concomitantly, confocal microscopy revealed that LY294002 clearly inhibited the nuclear export of Stat5, suggesting that PI 3-kinase regulates the subcellular localization of Stat5. Taken together, our results suggest that both Jak-Stat and PI 3-kinase activation pathways regulate the TPO-induced survival of megakaryocytic cells via Bcl-xL gene expression. In addition, our data suggest possible cross-talk between these two signaling pathways.
      TPO
      thrombopoietin
      PI
      phosphatidylinositol
      IL
      interleukin
      PBS
      phosphate-buffered saline
      EPO
      erythropoietin
      GSK-3
      glycogen synthase kinase-3
      NF-κB
      nuclear factor-κB
      FCS
      fetal calf serum
      EMSA
      electrophoretic mobility shift assay
      Thrombopoietin (TPO)1 is the major regulator of megakaryopoiesis and thrombopoiesis, leading to platelet production (
      • Kaushansky K.
      • Broudy V.C.
      • Lin N.
      • Jorgensen M.J.
      • McCarty J.
      • Fox N.
      • Zucker-Franklin D.
      • Lofton-Day C.
      ,
      • Kaushansky K.
      ). One of the most important functions of TPO is to support the survival of megakaryocytic lineage cells. Withdrawal of TPO causes the apoptosis of both TPO-responsive cell lines (
      • Ritchie A.
      • Vadhan-Raj S.
      • Broxmeyer H.
      ) and in immature megakaryocytic progenitor cells (
      • Osada M.
      • Komeno T.
      • Todokoro K.
      • Takizawa M.
      • Kojima H.
      • Suzukawa K.
      • Ninomiya H.
      • Abe T.
      • Nagasawa T.
      ). An animal model study demonstrated that disruption of the TPO gene or its receptor c-mpl gene causes severe thrombocytopenia (
      • Gurney A.
      • Carver-Moore K.
      • de Sauvage F.J.
      • Moore M.W.
      ,
      • de Sauvage F.
      • Carver-Moore K.
      • Luoh S.M.
      • Ryan A.
      • Dowd M.
      • Eaton D.L.
      • Moore M.W.
      ). Furthermore, point mutations of c-mpl gene were found in some patients with congenital amegakaryocytic progenitor cells (
      • Gurney A.
      • Carver-Moore K.
      • de Sauvage F.J.
      • Moore M.W.
      ,
      • de Sauvage F.
      • Carver-Moore K.
      • Luoh S.M.
      • Ryan A.
      • Dowd M.
      • Eaton D.L.
      • Moore M.W.
      ). Although it is well recognized that TPO is essential for the survival of megakaryocytic lineage cells, the precise molecular mechanism of how TPO prevents megakaryocytic progenitor cells from apoptosis is still open to question.
      Bcl-2 family proteins play key roles in the control of programmed cell death by hematopoietic cytokines (
      • Gross A.
      • McDonnell J.M.
      • Korsmeyer S.J.
      ). First, hematopoietic cytokines induce the expression of anti-apoptotic members of these proteins, such as Bcl-2 and Bcl-xL. These anti-apoptotic Bcl-2 proteins protect apoptosis through the inhibition of cytochrome c release from the mitochondria (
      • Gross A.
      • McDonnell J.M.
      • Korsmeyer S.J.
      ). Second, hematopoietic cytokines inhibit apoptosis through phosphorylation of BAD, a pro-apoptotic member of the Bcl-2 family. Phosphorylation of BAD causes altered intracellular distribution and leads to loss of pro-apoptotic function of this protein (
      • del Peso L.
      • Gonzalez-Garcia M.
      • Page C.
      • Herrera R.
      • Nunez G.
      ). Suppression of pro-apoptotic members of this family, known as BH3-only proteins, is another important function of cytokines to protect hematopoietic cells from apoptosis (
      • Sanz C.
      • Benito A.
      • Inohara N.
      • Ekhterae D.
      • Nunez G.
      • Fernandez-Luna J.L.
      ,
      • Shinjyo T.
      • Kuribara R.
      • Inukai T.
      • Hosoi H.
      • Kinoshita T.
      • Miyajima A.
      • Houghton P.J.
      • Look A.T.
      • Ozawa K.
      • Inaba T.
      ). Thus, although all of these mechanisms are important for cytokine-mediated anti-apoptotic effects, it appears that Bcl-xL plays central roles as a downstream molecule of hematopoietic cytokine signaling. Several lines of evidence support this notion. Bcl-xL is expressed in both CD34+/CD38− multipotent hematopoietic stem cells (
      • Park J.
      • Bernstein I.
      • Hockenbery D.
      ,
      • Peters R.
      • Leyvraz S.
      • Perey L.
      ) and committed progenitors having erythroid (
      • Silva M.
      • Grillot D.
      • Benito A.
      • Richard C.
      • Nunez G.
      • Fernandez-Luna J.
      ,
      • Gregoli P.
      • Bondurant M.
      ) or monocytic lineage properties (
      • Sanz C.
      • Benito A.
      • Silva M.
      • Albella B.
      • Richard C.
      • Segovia J.C.
      • Insunza A.
      • Bueren J.A.
      • Fernandez L.J.
      ). Genetic elimination of Bcl-xL genes causes severe deficiency of hematopoietic cells resulting from the increased ratio of apoptotic cells (
      • Motoyama N.
      • Wang F.
      • Roth K.A.
      • Sawa H.
      • Nakayama K.
      • Nakayama K.
      • Negishi I.
      • Senju S.
      • Zhang Q.
      • Fujii S.
      • Loh D.Y.
      ).
      Bcl-xL expression is mainly regulated by hematopoietic cytokines through the activation of intracellular signal transduction pathways (
      • Grad J.M.
      • Zeng X.R.
      • Boise L.H.
      ). Although a variety of signal transduction molecules are activated by hematopoietic cytokines, recent studies have revealed that Jak-Stat and PI 3-kinase activation pathways mainly regulate Bcl-xL gene expression. In the erythropoietin (EPO)-dependent cell line HCD-57, EPO induced the binding of Stat5 to the consensus Stat-binding sites on the Bcl-x gene promoter (
      • Silva M.
      • Benito A.
      • Sanz C.
      • Prosper F.
      • Ekhterae D.
      • Nunez G.
      • Fernandez-Luna J.L.
      ). Stat5 is also required for IL-3-dependent survival of Baf-3 cells (
      • Dumon S.
      • Santos S.C.
      • Debierre-Grockiego F.
      • Gouilleux-Gruart V.
      • Cocault L.
      • Boucheron C.
      • Mollat P.
      • Gisselbrecht S.
      • Gouilleux F.
      ). In contrast, IL-15 induced the binding of Stat6 to the Bcl-xgene promoter in mast cells (
      • Masuda A.
      • Matsuguchi T.
      • Yamaki K.
      • Hayakawa T.
      • Yoshikai Y.
      ). In addition to these in vitro studies, Stat5a and Stat5b knockout mice also demonstrated that Stat5 is required for Bcl-xL expression in erythroid lineage cells (
      • Socolovsky M.
      • Fallon A.
      • Wang S.
      • Brugnara C.
      • Lodish H.
      ). Another important molecule for Bcl-xL regulation is PI 3-kinase. PI 3-kinase is a key molecule for controlling apoptosis in a variety of cell type. PI 3-kinase prevents cells from apoptosis through a variety of mechanisms. Recently, some reports have shown that PI 3-kinase is involved in the regulation of Bcl-xL expression. Leverrier et al. (
      • Leverrier Y.
      • Thomas J.
      • Mathieu A.L.
      • Low W.
      • Blanquier B.
      • Marvel J.
      ) reported that PI 3-kinase is required for IL-3- and insulin-like growth factor-1-induced Bcl-xL expression in Baf3 cells. Like these cytokines, TPO activates both Jak-Stat and PI 3-kinase pathways in TPO-dependent cell lines and primary megakaryocytic cells (
      • Drachman J.G.
      • Sabath D.F.
      • Fox N.E.
      • Kaushansky K.
      ,
      • Miyakawa Y.
      • Rojnuckarin P.
      • Habib T.
      • Kaushansky K.
      ). Furthermore, a recent study has indicated that megakaryocytic progenitor cells express Bcl-xL (
      • Sanz C.
      • Benet I.
      • Richard C.
      • Badia B.
      • Andreu E.J.
      • Prosper F.
      • Fernandez-Luna J.L.
      ). Taken together, we hypothesized that TPO supports the survival of megakaryocytic progenitor cells through Jak-Stat- and/or PI 3-kinase-dependent Bcl-xL expression.
      In this study, we examined the role of Bcl-xL in TPO-dependent cell survival. Furthermore, we tried to clarify the role of both Jak-Stat and PI 3-kinase activation pathways in the regulation of Bcl-xL expression and subsequent apoptosis. We show here that Bcl-xL expression is dependent on TPO in TPO-dependent human leukemic cell line UT-7/TPO and megakaryocytic cells. We also show that TPO regulates Bcl-xLgene expression through Jak2-dependent activation of Stat5 and PI 3-kinase-dependent activation of NF-κB. Finally, we show that subcellular localization of Stat5 is in part regulated by PI 3-kinase activity.

      DISCUSSION

      In this study, we focused our attention on elucidating the precise mechanisms through which TPO maintains the survival of megakaryocytic progenitor cells. We showed that the Bcl-xL protein, an important member of the Bcl-2 family, is involved in TPO-dependent cell survival. We also showed that two distinct signal transduction pathways, Jak2-Stat5 and PI 3-kinase activation pathways, are required for TPO-induced transcriptional regulation of the Bcl-xL gene expression. Finally, we showed that subcellular localization of Stat5 is in part dependent on PI 3-kinase activity.
      Bcl-xL works as an essential anti-apoptotic factor in various types of cells including hematopoietic cells (
      • Gross A.
      • McDonnell J.M.
      • Korsmeyer S.J.
      ). It was previously reported that primitive hematopoietic progenitor cells express Bcl-xL and that Bcl-xL is required for the long term survival of the hematopoietic stem cells (
      • Park J.
      • Bernstein I.
      • Hockenbery D.
      ,
      • Peters R.
      • Leyvraz S.
      • Perey L.
      ). In addition, it was reported that Bcl-xL is required for the differentiation and maturation of committed hematopoietic progenitor cells. For example, Bcl-xL has crucial roles in the development of erythroid lineage cells (
      • Silva M.
      • Grillot D.
      • Benito A.
      • Richard C.
      • Nunez G.
      • Fernandez-Luna J.
      ,
      • Gregoli P.
      • Bondurant M.
      ,
      • Gregory T.
      • Yu C.
      • Ma A.
      • Orkin S.
      • Blobel G.
      • Weiss M.
      ). Monocytic differentiation of myeloid lineage progenitor cells also requires Bcl-xL expression (
      • Sanz C.
      • Benito A.
      • Silva M.
      • Albella B.
      • Richard C.
      • Segovia J.C.
      • Insunza A.
      • Bueren J.A.
      • Fernandez L.J.
      ). Furthermore, Bcl-xL is also expressed in normal megakaryocytic progenitor cells (
      • Sanz C.
      • Benet I.
      • Richard C.
      • Badia B.
      • Andreu E.J.
      • Prosper F.
      • Fernandez-Luna J.L.
      ). In this study, we showed that Bcl-xL is inducibly expressed by TPO in TPO-responsive human leukemic cell line UT-7/TPO and normal human megakaryocytes. We also showed that Bcl-2, another important anti-apoptotic Bcl-2 family member (
      • Gross A.
      • McDonnell J.M.
      • Korsmeyer S.J.
      ), is detectable in UT-7/TPO cells and normal megakaryocytic cells. However, TPO deprivation did not affect Bcl-2 expression. Furthermore, TPO stimulation did not induce Bcl-2 expression. Although we could not completely exclude the possibility that Bcl-2 has some anti-apoptotic function in megakaryocytic cells, our data strongly suggested that Bcl-xL expression is a prerequisite for cell survival in megakaryocytic lineage cells.
      The expression of Bcl-xL in hematopoietic cells is mainly regulated by hematopoietic cytokines at the transcriptional level (
      • Grad J.M.
      • Zeng X.R.
      • Boise L.H.
      ). Consistent with this notion, the regulatory region of the Bcl-x gene contains several cytokine-responsive elements, including Stat-binding sites and NF-κB binding sites (
      • Grad J.M.
      • Zeng X.R.
      • Boise L.H.
      ,
      • Grillot D.A.
      • Gonzalez G.M.
      • Ekhterae D.
      • Duan L.
      • Inohara N.
      • Ohta S.
      • Seldin M.F.
      • Nunez G.
      ). Among them, Stat proteins are a unique family of proteins that dually function as signal transducers and transcription factors. It is well known that Stat proteins are activated by a variety of cytokines and control the growth, survival, or differentiation of hematopoietic cells (
      • Coffer P.
      • Koenderman L.
      • de Groot R.P.
      ,
      • Smithgall T.
      • Briggs S.D.
      • Schreiner S.
      • Lerner E.C.
      • Cheng H.
      • Wilson M.B.
      ). Several studies have reported that Bcl-xL is one of the most important target genes of Stat proteins; however, the activation pattern of Stat proteins involved in Bcl-xL gene control is different in each cytokine. In EPO-dependent murine cell line HCD-57 cells, Stat5 regulates the Bcl-xL gene in response to EPO (
      • Silva M.
      • Benito A.
      • Sanz C.
      • Prosper F.
      • Ekhterae D.
      • Nunez G.
      • Fernandez-Luna J.L.
      ). Stat5 is also involved in IL-3-induced Bcl-xL gene expression (
      • Dumon S.
      • Santos S.C.
      • Debierre-Grockiego F.
      • Gouilleux-Gruart V.
      • Cocault L.
      • Boucheron C.
      • Mollat P.
      • Gisselbrecht S.
      • Gouilleux F.
      ). In contrast, LIF up-regurates Bcl-xL gene expression through Stat1 activation in cardiocytes (
      • Fujio Y.
      • Kunisada K.
      • Hirota H.
      • Yamauchi T.K.
      • Kishimoto T.
      ). Furthermore, Stat 3 plays a crucial role in IL-6-dependentBcl-xL gene expression in myeloma cell line U-266 (
      • Catlett-Falcone R.
      • Landowski T.H.
      • Oshiro M.M.
      • Turkson J.
      • Levitzki A.
      • Savino R.
      • Ciliberto G.
      • Moscinski L.
      • Fernandez-Luna J.L.
      • Nunez G.
      • Dalton W.S.
      • Jove R.
      ). Stat6 also has the ability to regulate the Bcl-xL gene in IL-15 signaling (
      • Masuda A.
      • Matsuguchi T.
      • Yamaki K.
      • Hayakawa T.
      • Yoshikai Y.
      ). TPO activates Stat3 and Stat5 in normal megakaryocytes (
      • Drachman J.G.
      • Sabath D.F.
      • Fox N.E.
      • Kaushansky K.
      ). In addition, we previously reported that TPO activates Stat3 and Sta5 in UT-7/TPO cells (
      • Komatsu N.
      • Kirito K.
      ). In this study, we found that Stat5 but not Stat3 binds to the putative Stat-binding site on the Bcl-x gene promoter. Furthermore, a dominant negative mutant of Stat5 but not Stat3 clearly inhibited the TPO-inducedBcl-x gene promoter activity. These results indicate that Stat5 has the capacity to control the Bcl-x gene in TPO signaling. Previously, we showed that Stat3 activation is correlated with TPO-induced cellular proliferation (
      • Komatsu N.
      • Kirito K.
      ). Transgenic mice with a dominant negative Stat3 using the GATA-1 gene promoter regulatory region revealed a delayed recovery of the number of megakaryocytes and of platelet production after 5-fluorouracil-induced thrombocytopenia.
      K. Kirito, M. Osawa, H. Morita, R. Shimizu, M. Yamamoto, A. Oda, H. Fujita, M. Tanaka, K. Nakajima, Y. Miura, K. Ozawa, and N. Komatsu, Blood, in press.
      However, we did not observe any significant increase in the ratio of apoptotic cells in the megakaryocytic lineage cells of these mice.
      K. Kirito, M. Osawa, H. Morita, R. Shimizu, M. Yamamoto, A. Oda, H. Fujita, M. Tanaka, K. Nakajima, Y. Miura, K. Ozawa, and N. Komatsu, unpublished observation.
      Together with these observations, it is hypothesized that Stat3 and Stat5 have distinct functions in TPO signaling; Stat3 regulates cell growth and Stat5 works as an anti-apoptotic effector. It would be attractive to analyze the effects of genetic elimination of Stat5a and/or Stat5b on megakaryopoiesis, especially on the survival of megakaryocytic cells.
      In addition to Stat proteins, NF-κB plays an important role in gene regulation of Bcl-xL in many types of cells. There are some putative NF-κB-binding sites on the regulatory region of theBcl-x gene as illustrated in Fig. 3 (
      • Grad J.M.
      • Zeng X.R.
      • Boise L.H.
      ,
      • Grillot D.A.
      • Gonzalez G.M.
      • Ekhterae D.
      • Duan L.
      • Inohara N.
      • Ohta S.
      • Seldin M.F.
      • Nunez G.
      ). Lee et al. (
      • Lee H.H.
      • Dadgostar H.
      • Cheng Q.
      • Shu J.
      • Cheng G.
      ) reported that the NF-κB-binding sites (III) 77 bp and 62 bp upstream of the transcription start sites are required for CD40-mediated Bcl-xL expression in B cells. In contrast, Glasgoet al. (
      • Glasgow J.N.
      • Wood T.
      • Perez-Polo J.R.
      ) reported that other NF-κB-binding sites are located in the region of about 847 bp (II) and 967 bp (I) upstream of the transcription start sites, and that this region is crucial forBcl-xL gene expression in pheochromocytoma cell line PC12 cells. In this study, we showed that deletion of NF-κB binding sites I and II did not affect the activity of the Bcl-x gene promoter (Fig. 3). However, deletion of the region containing NF-κB binding site III and Stat-binding site reduced the Bcl-xLpromoter activity. Based on these findings, we concluded that proximal (III) but not distal NF-κB binding sites (I and II) are essential for TPO-induced Bcl-xL expression. Indeed, we found that TPO enhanced the binding of NF-κB complex on this proximal NF-κB binding site I. The NF-κB family of proteins includes p50, p52, p65, c-Rel, and Rel-B (
      • Siebenlist U.
      • Franzoso G.
      • Brown K.
      ,
      • Thanos D.
      • Maniatis T.
      ). Among them, we showed that p50 and c-Rel subunits bind to theBcl-xL gene promoter. This is consistent with a previous report that p50 and c-Rel can bind to the NF-κB binding sites on theBcl-xL gene promoter (
      • Chen C.
      • Edelstein L.C.
      • Gelinas C.
      ). In addition, we showed that normal megakaryocytes express these two subunits (Fig. 7 C). p50 and c-Rel are also expressed in CD34-positive hematopoietic cells and are required for the survival of these cells (
      • Pyatt D.W.
      • Stillman W.S.
      • Yang Y.
      • Gross S.
      • Zheng J.H.
      • Irons R.D.
      ). In contrast, committed erythroid progenitors (BFU-E) mainly express p50, p52, and p65 (
      • Zhang M.Y.
      • Sun S.C.
      • Bell L.
      • Miller B.A.
      ). Therefore, the combination of NF-κB subunits expressed in hematopoietic cells depends on cell lineage.
      In UT-7/TPO cells, LY294002 reduced the TPO-induced DNA binding activity of NF-κB, indicating that TPO-induced activation of NF-κB is dependent on PI 3-kinase activity. Recent studies have revealed that PI 3-kinase induced DNA binding activity of NF-κB through Akt activation and subsequent IκB phosphorylation (
      • Ozes O.N.
      • Mayo L.D.
      • Gustin J.A.
      • Pfeffer S.R.
      • Pfeffer L.M.
      • Donner D.B.
      ,
      • Romashkova J.A.
      • Makarov S.S.
      ). We previously showed that TPO activated Akt in a PI 3-kinase-dependent manner (
      • Tanaka M.
      • Kirito K.
      • Kashii Y.
      • Uchida M.
      • Watanabe T.
      • Endo H.
      • Endoh T.
      • Sawada K.
      • Ozawa K.
      • Komatsu N.
      ). Taken together, the DNA binding activity of NF-κB may be enhanced through TPO-induced PI 3-kinase-Akt activation.
      It is noteworthy that there was a large discrepancy between the Bcl-xL level and the DNA binding activity of NF-κB. The expression level of Bcl-xL proteins was not down-regulated by treatment with 50 μg/ml LY294002, at which dosage the DNA binding activity of NF-κB was drastically suppressed in UT-7/TPO cells. A possible explanation for this discrepancy is that decreased NF-κB activity was compensated for Stat5, resulting in the maintenance of the Bcl-xL level. Surprisingly, LY294002 clearly enhanced the DNA binding activity of Stat5. This finding suggested that PI 3-kinase is one of the important upstream modulators of Stat proteins. However, the LY294002 treatment did not affect the Jak2 activation or tyrosine phosphorylation of Stat5. Interestingly, we found that nuclear localization of Stat5 was enhanced by LY294002 treatment. The nuclear transport mechanism of Stat5 has not been well described, and it is unclear how PI 3-kinase regulates Stat5 distribution. Recent work by Ginger et al. (
      • Ginger R.S.
      • Dalton E.C.
      • Ryves W.J.
      • Fukuzawa M.
      • Williams J.G.
      • Harwood A.J.
      ) may provide an explanation for this mechanism. They demonstrated that Gsk-A, aDictyostelium homolog of glycogen synthase kinase-3 (GSK-3) regulates the nuclear translocation of Dictyostelium Stat proteins, Dd-STATa; serine phosphorylation by GskA promoted the nuclear export of Dd-STATa. Importantly, mammalian Stat5B contains potential GSK-3 phosphorylation sites in the N-terminal region. Recently, we reported that TPO activates GSK-3 in a PI 3-kinase- and Akt-dependent manner in UT-7/TPO cells (
      • Tanaka M.
      • Kirito K.
      • Kashii Y.
      • Uchida M.
      • Watanabe T.
      • Endo H.
      • Endoh T.
      • Sawada K.
      • Ozawa K.
      • Komatsu N.
      ). Collectively, PI 3-kinase may regulate the function of Stat5 through the activation of GSK-3 and subsequent changes in subcellular localization of Stat5. If so, Stat5 can work as a rescue unit to maintain Bcl-xL under an emergency condition in which PI 3-kinase function is abrogated.
      We showed here that TPO supports the survival of megakaryocytic cells, in part mediated through the induction of the anti-apoptotic protein, Bcl-xL. In addition, we showed that Bcl-xL gene expression is mainly regulated by the Jak2-Stat5 and PI 3-kinase activation pathways in TPO signaling. Surprisingly, we found that the subcellular distribution of Stat5 is in part regulated by PI 3-kinase activity. This suggested that there is a possible cross-talk in Bcl-xL gene regulation between the Jak-Stat and PI 3-kinase activation pathways. Although the concept is speculative at present, the cross-talk between them may contribute to the redundancy of TPO signaling to support the survival of megakaryocytic lineage cells.

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