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Erythropoietin-dependent erythropoiesis: New insights and questions
Blood Cells, Molecules, and Diseases 36 (2006) 232 – 238 www.elsevier.com/locate/ybcmd
Erythropoietin-dependent erythropoiesis: New insights and questions Don M. Wojchowski a,⁎, Madhu P. Menon a,b , Pradeep Sathyanarayana a , Jing Fang a , Vinit Karur a , Estelle Houde a , William Kapelle a , Oleg Bogachev a a
Maine Medical Center Research Institute and Program in Stem Cell Biology and Regenerative Medicine, ME 04074-7205, USA b Molecular Medicine Program, The Pennsylvania State University, University Park, PA 16802, USA Submitted 11 January 2006 (Communicated by J. Hoffman, M.D., 11 January 2006)
Abstract Committed erythroid progenitor cells require exposure to erythropoietin (Epo) for their survival and for their quantitatively regulated transition to red blood cells. With regard to Epo signal transduction mechanisms, much has been learned from analyses in cell line models, fetal liver or spleenderived primary erythroblasts and human CD34pos progenitor cells from cord blood or mobilized bone marrow. Presently, we have developed an ex vivo system that efficiently supports the expansion and development of murine adult bone-marrow-derived erythroid progenitor cells. This system is outlined together with its demonstrated utility in studying (for the first time) the signaling capacities of two knocked-in phosphotyrosine-deficient Epo receptor alleles (EpoR-H and EpoR-HM). Ways in which these studies advance an understanding of core Epo signal transduction events are outlined. Also introduced are two new putative negative regulators of Epo-dependent erythropoiesis, DYRK3 and DAPK2 kinases. © 2006 Elsevier Inc. All rights reserved. Keywords: Erythropoiesis; Epo; Epo receptor signal transduction; Stat5
Introduction Factors that regulate the survival of maturing erythroblasts are clinically relevant to the anemia of chemotherapy and chronic disease (e.g., renal disease, myelodysplasia, multiple myeloma) [1–3]. Erythropoietin (Epo) is one key erythropoietic factor that exerts its effects via a single transmembrane receptor (EpoR) and an at least partially defined set of positively acting signal transduction pathways [4]. Jak2, as pre-assembled with the Epo receptor (EpoR), acts upstream as an essential Epoactivated Janus kinase to mediate the phosphorylation of eight conserved phosphotyrosine sites within the EpoR's distal cytoplasmic domain [4,5]. Among these sites, EpoR PY343 binds Stat5 [6], while PY479 binds p85/PI3-kinase [7]. Subsequently, Stat5 has been argued to stimulate Bcl-x gene expression [8], while PI3-kinase stimulates AKT-associated survival pathways [9] and inhibits pro-apoptotic activities of the forkhead transcription factor Fox03a [10]. ⁎ Corresponding author. PI, Program in Stem and Progenitor Cell Biology, Maine Medical Center Research Institute, 81 Research Drive, Scarborough, ME 04074-7205, USA. E-mail address: [email protected] (D.M. Wojchowski). 1079-9796/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.bcmd.2006.01.007
Despite the apparent complexity (and conservation) of EpoR PY signaling scaffolds, mice expressing a minimal PY-null EpoR allele (EpoR-HM) or a related allele with a singularly restored PY343 site (EpoR-H allele) somewhat unexpectedly support steady-state erythropoiesis [11]. This outcome at least suggests that EpoR PY479 and PY343 sites, and coupled pathways, may be non-essential. At the recent Red Cell Club meeting (Chicago, 2005), work from our laboratory was presented which revealed first that an EpoR-PY343-Stat5 signaling axis, while dispensable for steady-state erythropoiesis, is critical for red cell formation during anemia (i.e., stress erythropoiesis). Using a novel ex vivo system [12], we further have been able to quantitatively assess (for the first time) signal transduction events which are activated by these minimal knocked-in EpoR alleles in developmentally staged bonemarrow-derived erythroblasts. This paper summarizes these findings and also introduces two new apparent negative regulators of late erythropoiesis: DYRK3, an erythroidrestricted YAK family dual-specificity kinase [13], and DAPK2, a pro-apoptotic S/T kinase [14]. Speculation also is provided on ways that the above factors might act to coordinately control red cell production under normal and anemic conditions.
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An optimized ex vivo system for murine adult bone-marrow-derived (PRO) erythroblast development Ontogenetically, erythropoiesis is supported by several tissues. This includes primitive erythropoiesis in yolk sac and subsequent transitions to fetal liver and to bone marrow at a perinatal stage [15,16]. In addition, spleen can be induced by anemia to generate red cells [17–20], and this appears to involve a BMP4-dependent expansion of a unique progenitor pool [21]. In fetal liver and induced spleen, erythroid progenitor cells are wellrepresented [22,23], and this makes these systems convenient for cell biological and biochemical investigations, especially in murine models. Most clinical instances of defective or deregulated erythropoiesis, however, involve the bone marrow compartment [24–27]. In addition, increasing evidence exists to indicate that significant differences may exist in erythropoietic
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regulation in bone marrow as compared to fetal liver or spleen [28,29]. Recently, we have worked to develop an efficient expansion and ex vivo developmental system for primary bone-marrow-derived murine (pro)erythroblasts (see Fig. 1). Among several base media tested, serum-free StemPro-34 proved to most efficiently support murine bone-marrowderived (pro)erythroblast expansion when supplemented with SCF (100 ng/mL), Epo (2.5 U/mL), h-transferrin (75 μg/mL), dexamethasone (1 μM), beta-estradiol (1 μM), BSA (0.5%), 2mercaptoethanol (0.1 mM) and L-glutamine (1.5 mM). In particular, at 3.5 days of culture, KitposCD71high erythroblasts routinely were represented at ≥50%. The balance of cells typically was comprised of Kit neg CD71high erythroblasts (∼30%), CD71 high Ter119 pos erythroblasts (∼10%) and ≤10% non-erythroid Linpos cells.
Fig. 1. System for the ex vivo expansion and development of adult murine bone-marrow-derived erythroblasts. (A) Outlined are steps involved in the initial expansion and subsequent isolation of stage-specific progenitor cells. In brief, bone marrow cells were prepared and were cultured in SP34-EX media for 3.5 days with partial media changes on days 1 and 2. For progenitor cell isolation, Linpos cells were first were depleted. KitposCD71high and KitnegCD71high progenitor cells then were isolated via MACS. To generate KitnegCD71lowTer119pos erythroblasts, isolated KitnegCD71high cells were transferred to a serum-free BSA, insulin and transferrin (BIT) medium containing Epo at 1 U/mL. (B) Diagrammed are the various discrete stages of developing erythroid progenitor cells generated via the above procedure.
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Via a step-wise procedure of depleting Linpos cells (EasySep) and Kit-selection (MACS), discrete populations of relatively homogeneous (and developmentally staged) Kitpos CD71high and KitnegCD71high erythroblasts were generated in useful numbers (approximately 6 × 106 and 4 × 106 cells, respectively, per mouse). Each population proved to be highly Epo-responsive, especially the KitposCD71high cohort. In addition, when shifted to a minimal differentiation medium necessarily containing BSA, insulin and transferrin (BIT medium) plus Epo, KitnegCD71high progenitors efficiently developed over a 24- to 48-h interval to Ter119pos low-FALS late-stage erythroblasts, up
to 50% of which continued with the process of enucleation to form reticulocytes. Overall, this system provides new opportunities to investigate regulators of the development of early CFUe-like progenitors as derived from the bone marrow compartment of wild-type and variously genetically altered mouse models. Core positive effectors of Epo receptor signal transduction As introduced above, the abilities of minimal PY-deficient EpoR alleles to support steady-state erythropoiesis (PY-null
Fig. 2. Core positive effectors of Epo receptor signal transduction. (A) Outlined for the wild-type Epo receptor (wt-EpoR) are core positive signal transduction pathways including Epo-dependent Jak2 activation; Jak2-mediated EpoR PY site phosphorylation; PY343 coupling to Stat5; PY479 coupling to p85/PI3-kinase; and Jak2dependent (PY site independent) activation of Mek1,2/ERK1,2. (B) Diagrammed are two PY-deficient knocked-in Epo receptor alleles: EpoR-H, which retains a single PY343 Stat5 binding site, and EpoR-HM in which this site is mutated to F343 (PY-null allele). Retained major positive signal transduction pathways also are outlined.
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EpoR-HM allele) and stress erythropoiesis (PY343-retaining EpoR-H allele) raise basic questions concerning the nature of core EpoR signal transduction events that support erythroblast growth and survival. We recently have assessed basic signaling capacities of these EpoR forms and the wt-EpoR in primary bone-marrow-derived erythroblasts prepared using the above ex vivo expansion system (see Fig. 2) [12]. The wt-EpoR proved to efficiently activate ERK1,2, AKT, p70S6K, JNK1,2 and Stat5 (but not Stat1 or 3) and detectably stimulate p60Src. For EpoR-H and EpoR-HM, AKT and p70S6K activation was markedly diminished and JNK1,2 stimulation was nominal. For EpoR-H (but not EpoR-HM), Stat5 was activated at essentially wild-type efficiency. Finally, EpoR-HM not only remained coupled to ERK1,2 but substantially hyper-stimulated this response pathway. Steady-state erythropoiesis, especially as supported via EpoR-HM, therefore may involve primarily a core EpoR/ Jak2/MEK1,2/ERK1,2 signaling axis (and/or might somehow laterally engage additional effectors, e.g., via adaptors such as Gab2 or IRS2) [30,31]. In contrast, based on an observed requirement for EpoRPY343/Stat5 signaling (as supported by EpoR-H), stress erythropoiesis is proposed to depend further upon key positively acting Stat5 target genes [32]. In the present system, EpoR-PY343/Stat5 target genes proved to include the pleiotropic cytokine oncostatin-M [32], and Pim-1 as an anti-apoptotic kinase which acts in parallel with AKT and mTOR [33]. Bcl-xl, however, was not observed to be induced at significant levels by any studied EpoR allele [8,32]. Bioactivities of minimal EPO receptor alleles We also have further investigated the bioactivities of EpoRHM and EpoR-H alleles both in vivo and ex vivo. Despite its
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ability to support steady-state erythropoiesis, and as indicated above, the PY-null allele EpoR-HM failed to efficiently support stress erythropoiesis in several tested contexts. This included poor correction of anemia due to red cell hemolysis or to 5fluorouracil depletion of progenitor cell pools. In addition, EpoR-HM progenitor cells failed to efficiently repopulate the erythron during bone marrow transplantation. In response to Epo dosing, EpoR-HM mice also displayed multi-fold defects in reticulocyte production. In each context, however, stress erythropoiesis was efficiently rescued upon the selective restoration of a single PY-343 Stat5 binding site in EpoR-H [12,32]. These findings interestingly distinguish between requirements for Epo action during steady state vs. stress erythropoiesis at the level of EpoR signal transduction (see Fig. 3). They also point to critical roles in the latter process for key positively acting Stat5 target genes. In ex vivo analyses of erythroblasts expressing the EpoR-H allele, outcomes in general were consistent with in vivo analyses. EpoR-H erythroblasts possessed near wild-type growth, survival and differentiation properties. EpoR-HM erythroblasts, in contrast, proved to have markedly more compromised Epo-dependent survival and proliferative capacities than suggested by in vivo data (e.g., ∼30-fold deficit in Epo-dependent 3HdT incorporation) [32]. These defects were not reversed by increased Epo dosing, and levels of Epo expression in EpoR-HM mice were not greatly increased [32]. Together, these findings raise the prospect that erythropoiesis in EpoR-HM mice may be supported by a distinct and as yet unidentified erythropoietic factor. EpoR-HM erythroblasts also faltered in their ability to differentiate to Ter119pos low-FALS late-stage erythroblasts. Interestingly, this specific defect was essentially corrected upon U0126 inhibition of MEK1,2/ ERK1,2 signaling (as otherwise hyper-activated via EpoR-
Fig. 3. Summarized bioactivities of minimal EpoR-H and EpoR-HM alleles. For EpoR-HM, note deficiencies in the abilities to support stress erythropoiesis in vivo and erythroblast growth and survival ex vivo. For both EpoR-HM and EpoR-H, note the limited capacities to protect against TRAIL-induced apoptosis.
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HM, see above). This suggests a requirement for a downmodulation in ERK1,2 signaling in parallel with a commitment to erythrocyte maturation [12,34]. In pilot experiments, sensitivity of EpoR-H, EpoR-HM and wt-EpoR erythroblasts to TRAIL also was initially investigated. TRAIL (TNF-related apoptosis inducing ligand) is a type II membrane-bound TNF orthologue that can induce apoptosis in cell lines and primary tumor cells [35]. Furthermore, upregulation of TRAIL and its receptors in bone marrow cells from patients with aplastic anemia and myelodysplatic syndrome (MDS) has been reported [25,36]. TRAIL, in fact, proved to induce apoptosis in Kit pos CD71high bone-marrow-derived erythroblasts (as assessed by FITC-annexin V staining). EpoR-HM erythroblasts also were damaged by TRAIL, but this effect was masked by high-level apoptosis in the absence of TRAIL. Unexpectedly, EpoR-H erythroblasts were observed in these preliminary studies to likewise be hyper-sensitive to TRAIL-induced apoptosis. This requires further study but at present suggests that a C-terminal functional motif in the EpoR distal cytoplasmic domain may selectively mediate resistance to TRAIL-induced erythroblast death.
DYRK3 and DAPK2 as new putative negative regulators of erythropoiesis Our development of the above model systems also provides unique opportunities for transcriptome analyses of Epodependent erythropoiesis. We have begun to undertake this task and in initial analyses have followed-up on two perhaps under-studied kinases that have proven (in analyses to date) to be expressed predominantly in erythroid cells, DYRK3 and DAPK2. DYRK3 is a dual-specificity kinase which is related in its catalytic domains to DYRK1 or minibrain kinase, a regulator of neuronal cell development [37]. DYRK3, however, lacks an NLS and possesses unique N- and C-terminal domains [37]. In one approach to discovering DYRK3's functional roles, we have generated and initially characterized DYRK3−/− mice. In the absence of DYRK3, steady-state erythropoiesis (and hematopoiesis) is largely unperturbed. During stress erythropoiesis, however (e.g., 5-fluorouracil-induced anemia), DYRK3−/− mice exhibit significantly increased reticulocyte production and appear to be partially protected against anemia. DYRK3 therefore may place an upper limit on red cell
Fig. 4. Overall model for Epo receptor propagated erythroblast growth and survival signals, including putative negative modulation by DYRK3 and DAPK2 kinases.
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production during stress erythropoiesis. DYRK3 action mechanisms (as well as the potential clinical utility of DYRK3 inhibitors) are receiving active consideration. DAPK2 is a S/T CAM kinase and an orthologue of the tumor-suppressor DAPK-1. DAPK2, however, is less widely expressed and in initial studies was observed to be expressed at elevated levels in maturing erythroblasts [14]. In cell line studies, DAPK2 has been shown to exert pro-apoptotic effects [38] and may also affect autophagy [39]. In a UT7epo cell line model, we further have observed sensitization to apoptosis due to DAPK2 ectopic expression and increased survival due to siRNA knockdown [40]. These initial findings provide impetus for the development of both gain- and loss-of-function mouse models to better establish the significance of DAPK2 regulation of late erythropoiesis. In Fig. 4, a schematic summary of possibly integrated actions of EpoR, DYRK3 and DAPK2 is outlined. Acknowledgments Supported by NIH grants (to DMW) R01HL44491, R01DK59472 and P20RR18789. References [1] M. Boogaerts, M. Mittelman, P. Vaupel, Beyond anaemia management: evolving role of erythropoietin therapy in neurological disorders, multiple myeloma and tumour hypoxia models, Oncology 69 (Suppl. 2) (2005) 22–30. [2] A. List, S. Kurtin, D.J. Roe, A. Buresh, D. Mahadevan, D. Fuchs, L. Rimsza, R. Heaton, R. Knight, J.B. Zeldis, Efficacy of lenalidomide in myelodysplastic syndromes, N. Engl. J. Med. 352 (6) (2005) 549–557. [3] C. Lacombe, Resistance to erythropoietin, N. Engl. J. Med. 334 (10) (1996) 660–662. [4] T.D. Richmond, M. Chohan, D.L. Barber, Turning cells red: signal transduction mediated by erythropoietin, Trends Cell. Biol. 15 (3) (2005) 146–155. [5] L.I. Zon, Blood 100 (2002) A58. [6] J.E. Damen, H. Wakao, A. Miyajima, J. Krosl, R.K. Humphries, R.L. Cutler, G. Krystal, Tyrosine 343 in the erythropoietin receptor positively regulates erythropoietin-induced cell proliferation and Stat5 activation, EMBO J. 14 (22) (1995) 5557–5568. [7] U. Klingmuller, H. Wu, J.G. Hsiao, A. Toker, B.C. Duckworth, L.C. Cantley, H.F. Lodish, Identification of a novel pathway important for proliferation and differentiation of primary erythroid progenitors, Proc. Natl. Acad. Sci. U. S. A. 94 (7) (1997) 3016–3021. [8] M. Socolovsky, A.E. Fallon, S. Wang, C. Brugnara, H.F. Lodish, Fetal anemia and apoptosis of red cell progenitors in Stat5a−/−5b−/− mice: a direct role for Stat5 in Bcl-X(L) induction, Cell 98 (2) (1999) 181–191. [9] D. Bouscary, F. Pene, Y.E. Claessens, O. Muller, S. Chretien, M. FontenayRoupie, S. Gisselbrecht, P. Mayeux, C. Lacombe, Critical role for PI 3kinase in the control of erythropoietin-induced erythroid progenitor proliferation, Blood 101 (9) (2003) 3436–3443. [10] Y. Kashii, M. Uchida, K. Kirito, M. Tanaka, K. Nishijima, M. Toshima, T. Ando, K. Koizumi, T. Endoh, K. Sawada, et al., A member of Forkhead family transcription factor, FKHRL1, is one of the downstream molecules of phosphatidylinositol 3-kinase-Akt activation pathway in erythropoietin signal transduction, Blood 96 (3) (2000) 941–949. [11] H. Zang, K. Sato, H. Nakajima, C. McKay, P.A. Ney, J.N. Ihle, The distal region and receptor tyrosines of the Epo receptor are non-essential for in vivo erythropoiesis, EMBO J. 20 (12) (2001) 3156–3166. [12] M.P. Menon, J. Fang, D.M. Wojchowski, Core erythropoietin receptor signals for late erythroblast development, Blood (2005).
237
[13] J.N. Geiger, G.T. Knudsen, L. Panek, A.K. Pandit, M.D. Yoder, K.A. Lord, C.L. Creasy, B.M. Burns, P. Gaines, S.B. Dillon, mDYRK3 kinase is expressed selectively in late erythroid progenitor cells and attenuates colony forming unit erythroid development, Blood 97 (4) (2001) 901–910. [14] M. Menon, B. Vincent, A. Breggia, D. Wojchowski, Death associated protein kinase 2 (DAPK2): a novel Epo modulated apoptotic regulator of late erythropoiesis, Blood 104 (11) (2004) abstract #582. [15] K.E. McGrath, J. Palis, Hematopoiesis in the yolk sac: more than meets the eye, Exp. Hematol. 33 (9) (2005) 1021–1028. [16] J. Palis, G.B. Segel, Developmental biology of erythropoiesis, Blood Rev. 12 (2) (1998) 106–114. [17] D. Orlic, J.M. Wu, R.D. Carmichael, F. Quaini, M. Kobylack, A.S. Gordon, Increased erythropoiesis and 2′5′—A polymerase activity in the marrow and spleen of phenylhydrazine-injected rats, Exp. Hematol. 10 (5) (1982) 478–485. [18] V.C. Broudy, N.L. Lin, G.V. Priestley, K. Nocka, N.S. Wolf, Interaction of stem cell factor and its receptor c-kit mediates lodgment and acute expansion of hematopoietic cells in the murine spleen, Blood 88 (1) (1996) 75–81. [19] L.C. Ou, D. Kim, W.M. Layton Jr., R.P. Smith, Splenic erythropoiesis in polycythemic response of the rat to high-altitude exposure, J. Appl. Physiol. 48 (5) (1980) 857–861. [20] A.M. Vannucchi, F. Paoletti, S. Linari, C. Cellai, R. Caporale, P.R. Ferrini, M. Sanchez, G. Migliaccio, A.R. Migliaccio, Identification and characterization of a bipotent (erythroid and megakaryocytic) cell precursor from the spleen of phenylhydrazine-treated mice, Blood 95 (8) (2000) 2559–2568. [21] L.E. Lenox, J.M. Perry, R.F. Paulson, BMP4 and Madh5 regulate the erythroid response to acute anemia, Blood 105 (7) (2005) 2741–2748. [22] O. Ohneda, N. Yanai, M. Obinata, Microenvironment created by stromal cells is essential for a rapid expansion of erythroid cells in mouse fetal liver, Development 110 (2) (1990) 379–384. [23] K.M. Moritz, G.B. Lim, E.M. Wintour, Developmental regulation of erythropoietin and erythropoiesis, Am. J. Physiol. 273 (6 Pt 2) (1997) R1829–R1844. [24] J.J. Brazil, P. Gupta, Constitutive expression of the Fas receptor and its ligand in adult human bone marrow: a regulatory feedback loop for the homeostatic control of hematopoiesis, Blood Cells Mol. Dis. 29 (1) (2002) 94–103. [25] T. Kakagianni, N.C. Giannakoulas, E. Thanopoulou, A. Galani, S. Michalopoulou, A. Kouraklis-Symeonidis, N.C. Zoumbos, A probable role for trail-induced apoptosis in the pathogenesis of marrow failure Implications from an in vitro model and from marrow of aplastic anemia patients, Leuk. Res. (in press) (Electronic publication ahead of print). [26] S. Nakao, X. Feng, C. Sugimori, Immune pathophysiology of aplastic anemia, Int. J. Hematol. 82 (3) (2005) 196–200. [27] P. Gupta, G.A. Niehans, S.C. LeRoy, K. Gupta, V.A. Morrison, C. Schultz, D.J. Knapp, R.A. Kratzke, Fas ligand expression in the bone marrow in myelodysplastic syndromes correlates with FAB subtype and anemia, and predicts survival, Leukemia 13 (1) (1999) 44–53. [28] M.O. Muench, R. Namikawa, Disparate regulation of human fetal erythropoiesis by the microenvironments of the liver and bone marrow, Blood Cells Mol. Dis. 27 (2) (2001) 377–390. [29] M. Tanaka, Y. Hirabayashi, T. Sekiguchi, T. Inoue, M. Katsuki, A. Miyajima, Targeted disruption of oncostatin M receptor results in altered hematopoiesis, Blood 102 (9) (2003) 3154–3162. [30] A. Wickrema, S. Uddin, A. Sharma, F. Chen, Y. Alsayed, S. Ahmad, S.T. Sawyer, G. Krystal, T. Yi, K. Nishada, et al., Engagement of Gab1 and Gab2 in erythropoietin signaling, J. Biol. Chem. 274 (35) (1999) 24469–24474. [31] F. Verdier, S. Chretien, C. Billat, S. Gisselbrecht, C. Lacombe, P. Mayeux, Erythropoietin induces the tyrosine phosphorylation of insulin receptor substrate-2. An alternate pathway for erythropoietin-induced phosphatidylinositol 3-kinase activation, J. Biol. Chem. 272 (42) (1997) 26173–26178. [32] M.P. Menon, V. Karur, O. Bogacheva, O. Bogachev, B. Cuetara, D.M. Wojchowski, Signals for stress erythropoiesis are integrated via an erythropoietin receptor phosphotyrosine-PY343-Stat5 axis. Journal of Clinical Investigation (in press).
238
D.M. Wojchowski et al. / Blood Cells, Molecules, and Diseases 36 (2006) 232–238
[33] P.S. Hammerman, C.J. Fox, M.J. Birnbaum, C.B. Thompson, The Pim and Akt oncogenes are independent regulators of hematopoietic cell growth and survival, Blood 105 (11) (2005) 4477–4483. [34] J. Zhang, H.F. Lodish, Constitutive activation of the MEK/ERK pathway mediates all effects of oncogenic H-ras expression in primary erythroid progenitors, Blood 104 (6) (2004) 1679–1687. [35] J.L. Spivak, The anaemia of cancer: death by a thousand cuts, Nat. Rev., Cancer 5 (7) (2005) 543–555. [36] D. Campioni, P. Secchiero, F. Corallini, E. Melloni, S. Capitani, F. Lanza, G. Zauli, Evidence for a role of TNF-related apoptosis-inducing ligand (TRAIL) in the anemia of myelodysplastic syndromes, Am. J. Pathol. 166 (2) (2005) 557–563. [37] D. Zhang, K. Li, C.L. Erickson-Miller, M. Weiss, D.M. Wojchowski,
DYRK gene structure and erythroid-restricted features of DYRK3 gene expression, Genomics 85 (1) (2005) 117–130. [38] T. Kawai, F. Nomura, K. Hoshino, N.G. Copeland, D.J. Gilbert, N.A. Jenkins, S. Akira, Death-associated protein kinase 2 is a new calcium/ calmodulin-dependent protein kinase that signals apoptosis through its catalytic activity, Oncogene 18 (23) (1999) 3471–3480. [39] B. Inbal, S. Bialik, I. Sabanay, G. Shani, A. Kimchi, DAP kinase and DRP1 mediate membrane blebbing and the formation of autophagic vesicles during programmed cell death, J. Cell Biol. 157 (3) (2002) 455–468. [40] J. Fang, M.P. Menon, M. Tschan, B. Torbett, D.M. Wojchowski, Deathassociated protein kinase-2 (DAPK2) potentiates erythroid progenitor cell apoptosis in response to cisplatin, and cytokine withdrawal, American Society Of Hematology-Annual Meeting, 2005, Abstracts Dec 2005.
Erythropoietin-dependent erythropoiesis: New insights and questions Don M. Wojchowski a,⁎, Madhu P. Menon a,b , Pradeep Sathyanarayana a , Jing Fang a , Vinit Karur a , Estelle Houde a , William Kapelle a , Oleg Bogachev a a
Maine Medical Center Research Institute and Program in Stem Cell Biology and Regenerative Medicine, ME 04074-7205, USA b Molecular Medicine Program, The Pennsylvania State University, University Park, PA 16802, USA Submitted 11 January 2006 (Communicated by J. Hoffman, M.D., 11 January 2006)
Abstract Committed erythroid progenitor cells require exposure to erythropoietin (Epo) for their survival and for their quantitatively regulated transition to red blood cells. With regard to Epo signal transduction mechanisms, much has been learned from analyses in cell line models, fetal liver or spleenderived primary erythroblasts and human CD34pos progenitor cells from cord blood or mobilized bone marrow. Presently, we have developed an ex vivo system that efficiently supports the expansion and development of murine adult bone-marrow-derived erythroid progenitor cells. This system is outlined together with its demonstrated utility in studying (for the first time) the signaling capacities of two knocked-in phosphotyrosine-deficient Epo receptor alleles (EpoR-H and EpoR-HM). Ways in which these studies advance an understanding of core Epo signal transduction events are outlined. Also introduced are two new putative negative regulators of Epo-dependent erythropoiesis, DYRK3 and DAPK2 kinases. © 2006 Elsevier Inc. All rights reserved. Keywords: Erythropoiesis; Epo; Epo receptor signal transduction; Stat5
Introduction Factors that regulate the survival of maturing erythroblasts are clinically relevant to the anemia of chemotherapy and chronic disease (e.g., renal disease, myelodysplasia, multiple myeloma) [1–3]. Erythropoietin (Epo) is one key erythropoietic factor that exerts its effects via a single transmembrane receptor (EpoR) and an at least partially defined set of positively acting signal transduction pathways [4]. Jak2, as pre-assembled with the Epo receptor (EpoR), acts upstream as an essential Epoactivated Janus kinase to mediate the phosphorylation of eight conserved phosphotyrosine sites within the EpoR's distal cytoplasmic domain [4,5]. Among these sites, EpoR PY343 binds Stat5 [6], while PY479 binds p85/PI3-kinase [7]. Subsequently, Stat5 has been argued to stimulate Bcl-x gene expression [8], while PI3-kinase stimulates AKT-associated survival pathways [9] and inhibits pro-apoptotic activities of the forkhead transcription factor Fox03a [10]. ⁎ Corresponding author. PI, Program in Stem and Progenitor Cell Biology, Maine Medical Center Research Institute, 81 Research Drive, Scarborough, ME 04074-7205, USA. E-mail address: [email protected] (D.M. Wojchowski). 1079-9796/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.bcmd.2006.01.007
Despite the apparent complexity (and conservation) of EpoR PY signaling scaffolds, mice expressing a minimal PY-null EpoR allele (EpoR-HM) or a related allele with a singularly restored PY343 site (EpoR-H allele) somewhat unexpectedly support steady-state erythropoiesis [11]. This outcome at least suggests that EpoR PY479 and PY343 sites, and coupled pathways, may be non-essential. At the recent Red Cell Club meeting (Chicago, 2005), work from our laboratory was presented which revealed first that an EpoR-PY343-Stat5 signaling axis, while dispensable for steady-state erythropoiesis, is critical for red cell formation during anemia (i.e., stress erythropoiesis). Using a novel ex vivo system [12], we further have been able to quantitatively assess (for the first time) signal transduction events which are activated by these minimal knocked-in EpoR alleles in developmentally staged bonemarrow-derived erythroblasts. This paper summarizes these findings and also introduces two new apparent negative regulators of late erythropoiesis: DYRK3, an erythroidrestricted YAK family dual-specificity kinase [13], and DAPK2, a pro-apoptotic S/T kinase [14]. Speculation also is provided on ways that the above factors might act to coordinately control red cell production under normal and anemic conditions.
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An optimized ex vivo system for murine adult bone-marrow-derived (PRO) erythroblast development Ontogenetically, erythropoiesis is supported by several tissues. This includes primitive erythropoiesis in yolk sac and subsequent transitions to fetal liver and to bone marrow at a perinatal stage [15,16]. In addition, spleen can be induced by anemia to generate red cells [17–20], and this appears to involve a BMP4-dependent expansion of a unique progenitor pool [21]. In fetal liver and induced spleen, erythroid progenitor cells are wellrepresented [22,23], and this makes these systems convenient for cell biological and biochemical investigations, especially in murine models. Most clinical instances of defective or deregulated erythropoiesis, however, involve the bone marrow compartment [24–27]. In addition, increasing evidence exists to indicate that significant differences may exist in erythropoietic
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regulation in bone marrow as compared to fetal liver or spleen [28,29]. Recently, we have worked to develop an efficient expansion and ex vivo developmental system for primary bone-marrow-derived murine (pro)erythroblasts (see Fig. 1). Among several base media tested, serum-free StemPro-34 proved to most efficiently support murine bone-marrowderived (pro)erythroblast expansion when supplemented with SCF (100 ng/mL), Epo (2.5 U/mL), h-transferrin (75 μg/mL), dexamethasone (1 μM), beta-estradiol (1 μM), BSA (0.5%), 2mercaptoethanol (0.1 mM) and L-glutamine (1.5 mM). In particular, at 3.5 days of culture, KitposCD71high erythroblasts routinely were represented at ≥50%. The balance of cells typically was comprised of Kit neg CD71high erythroblasts (∼30%), CD71 high Ter119 pos erythroblasts (∼10%) and ≤10% non-erythroid Linpos cells.
Fig. 1. System for the ex vivo expansion and development of adult murine bone-marrow-derived erythroblasts. (A) Outlined are steps involved in the initial expansion and subsequent isolation of stage-specific progenitor cells. In brief, bone marrow cells were prepared and were cultured in SP34-EX media for 3.5 days with partial media changes on days 1 and 2. For progenitor cell isolation, Linpos cells were first were depleted. KitposCD71high and KitnegCD71high progenitor cells then were isolated via MACS. To generate KitnegCD71lowTer119pos erythroblasts, isolated KitnegCD71high cells were transferred to a serum-free BSA, insulin and transferrin (BIT) medium containing Epo at 1 U/mL. (B) Diagrammed are the various discrete stages of developing erythroid progenitor cells generated via the above procedure.
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Via a step-wise procedure of depleting Linpos cells (EasySep) and Kit-selection (MACS), discrete populations of relatively homogeneous (and developmentally staged) Kitpos CD71high and KitnegCD71high erythroblasts were generated in useful numbers (approximately 6 × 106 and 4 × 106 cells, respectively, per mouse). Each population proved to be highly Epo-responsive, especially the KitposCD71high cohort. In addition, when shifted to a minimal differentiation medium necessarily containing BSA, insulin and transferrin (BIT medium) plus Epo, KitnegCD71high progenitors efficiently developed over a 24- to 48-h interval to Ter119pos low-FALS late-stage erythroblasts, up
to 50% of which continued with the process of enucleation to form reticulocytes. Overall, this system provides new opportunities to investigate regulators of the development of early CFUe-like progenitors as derived from the bone marrow compartment of wild-type and variously genetically altered mouse models. Core positive effectors of Epo receptor signal transduction As introduced above, the abilities of minimal PY-deficient EpoR alleles to support steady-state erythropoiesis (PY-null
Fig. 2. Core positive effectors of Epo receptor signal transduction. (A) Outlined for the wild-type Epo receptor (wt-EpoR) are core positive signal transduction pathways including Epo-dependent Jak2 activation; Jak2-mediated EpoR PY site phosphorylation; PY343 coupling to Stat5; PY479 coupling to p85/PI3-kinase; and Jak2dependent (PY site independent) activation of Mek1,2/ERK1,2. (B) Diagrammed are two PY-deficient knocked-in Epo receptor alleles: EpoR-H, which retains a single PY343 Stat5 binding site, and EpoR-HM in which this site is mutated to F343 (PY-null allele). Retained major positive signal transduction pathways also are outlined.
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EpoR-HM allele) and stress erythropoiesis (PY343-retaining EpoR-H allele) raise basic questions concerning the nature of core EpoR signal transduction events that support erythroblast growth and survival. We recently have assessed basic signaling capacities of these EpoR forms and the wt-EpoR in primary bone-marrow-derived erythroblasts prepared using the above ex vivo expansion system (see Fig. 2) [12]. The wt-EpoR proved to efficiently activate ERK1,2, AKT, p70S6K, JNK1,2 and Stat5 (but not Stat1 or 3) and detectably stimulate p60Src. For EpoR-H and EpoR-HM, AKT and p70S6K activation was markedly diminished and JNK1,2 stimulation was nominal. For EpoR-H (but not EpoR-HM), Stat5 was activated at essentially wild-type efficiency. Finally, EpoR-HM not only remained coupled to ERK1,2 but substantially hyper-stimulated this response pathway. Steady-state erythropoiesis, especially as supported via EpoR-HM, therefore may involve primarily a core EpoR/ Jak2/MEK1,2/ERK1,2 signaling axis (and/or might somehow laterally engage additional effectors, e.g., via adaptors such as Gab2 or IRS2) [30,31]. In contrast, based on an observed requirement for EpoRPY343/Stat5 signaling (as supported by EpoR-H), stress erythropoiesis is proposed to depend further upon key positively acting Stat5 target genes [32]. In the present system, EpoR-PY343/Stat5 target genes proved to include the pleiotropic cytokine oncostatin-M [32], and Pim-1 as an anti-apoptotic kinase which acts in parallel with AKT and mTOR [33]. Bcl-xl, however, was not observed to be induced at significant levels by any studied EpoR allele [8,32]. Bioactivities of minimal EPO receptor alleles We also have further investigated the bioactivities of EpoRHM and EpoR-H alleles both in vivo and ex vivo. Despite its
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ability to support steady-state erythropoiesis, and as indicated above, the PY-null allele EpoR-HM failed to efficiently support stress erythropoiesis in several tested contexts. This included poor correction of anemia due to red cell hemolysis or to 5fluorouracil depletion of progenitor cell pools. In addition, EpoR-HM progenitor cells failed to efficiently repopulate the erythron during bone marrow transplantation. In response to Epo dosing, EpoR-HM mice also displayed multi-fold defects in reticulocyte production. In each context, however, stress erythropoiesis was efficiently rescued upon the selective restoration of a single PY-343 Stat5 binding site in EpoR-H [12,32]. These findings interestingly distinguish between requirements for Epo action during steady state vs. stress erythropoiesis at the level of EpoR signal transduction (see Fig. 3). They also point to critical roles in the latter process for key positively acting Stat5 target genes. In ex vivo analyses of erythroblasts expressing the EpoR-H allele, outcomes in general were consistent with in vivo analyses. EpoR-H erythroblasts possessed near wild-type growth, survival and differentiation properties. EpoR-HM erythroblasts, in contrast, proved to have markedly more compromised Epo-dependent survival and proliferative capacities than suggested by in vivo data (e.g., ∼30-fold deficit in Epo-dependent 3HdT incorporation) [32]. These defects were not reversed by increased Epo dosing, and levels of Epo expression in EpoR-HM mice were not greatly increased [32]. Together, these findings raise the prospect that erythropoiesis in EpoR-HM mice may be supported by a distinct and as yet unidentified erythropoietic factor. EpoR-HM erythroblasts also faltered in their ability to differentiate to Ter119pos low-FALS late-stage erythroblasts. Interestingly, this specific defect was essentially corrected upon U0126 inhibition of MEK1,2/ ERK1,2 signaling (as otherwise hyper-activated via EpoR-
Fig. 3. Summarized bioactivities of minimal EpoR-H and EpoR-HM alleles. For EpoR-HM, note deficiencies in the abilities to support stress erythropoiesis in vivo and erythroblast growth and survival ex vivo. For both EpoR-HM and EpoR-H, note the limited capacities to protect against TRAIL-induced apoptosis.
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HM, see above). This suggests a requirement for a downmodulation in ERK1,2 signaling in parallel with a commitment to erythrocyte maturation [12,34]. In pilot experiments, sensitivity of EpoR-H, EpoR-HM and wt-EpoR erythroblasts to TRAIL also was initially investigated. TRAIL (TNF-related apoptosis inducing ligand) is a type II membrane-bound TNF orthologue that can induce apoptosis in cell lines and primary tumor cells [35]. Furthermore, upregulation of TRAIL and its receptors in bone marrow cells from patients with aplastic anemia and myelodysplatic syndrome (MDS) has been reported [25,36]. TRAIL, in fact, proved to induce apoptosis in Kit pos CD71high bone-marrow-derived erythroblasts (as assessed by FITC-annexin V staining). EpoR-HM erythroblasts also were damaged by TRAIL, but this effect was masked by high-level apoptosis in the absence of TRAIL. Unexpectedly, EpoR-H erythroblasts were observed in these preliminary studies to likewise be hyper-sensitive to TRAIL-induced apoptosis. This requires further study but at present suggests that a C-terminal functional motif in the EpoR distal cytoplasmic domain may selectively mediate resistance to TRAIL-induced erythroblast death.
DYRK3 and DAPK2 as new putative negative regulators of erythropoiesis Our development of the above model systems also provides unique opportunities for transcriptome analyses of Epodependent erythropoiesis. We have begun to undertake this task and in initial analyses have followed-up on two perhaps under-studied kinases that have proven (in analyses to date) to be expressed predominantly in erythroid cells, DYRK3 and DAPK2. DYRK3 is a dual-specificity kinase which is related in its catalytic domains to DYRK1 or minibrain kinase, a regulator of neuronal cell development [37]. DYRK3, however, lacks an NLS and possesses unique N- and C-terminal domains [37]. In one approach to discovering DYRK3's functional roles, we have generated and initially characterized DYRK3−/− mice. In the absence of DYRK3, steady-state erythropoiesis (and hematopoiesis) is largely unperturbed. During stress erythropoiesis, however (e.g., 5-fluorouracil-induced anemia), DYRK3−/− mice exhibit significantly increased reticulocyte production and appear to be partially protected against anemia. DYRK3 therefore may place an upper limit on red cell
Fig. 4. Overall model for Epo receptor propagated erythroblast growth and survival signals, including putative negative modulation by DYRK3 and DAPK2 kinases.
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production during stress erythropoiesis. DYRK3 action mechanisms (as well as the potential clinical utility of DYRK3 inhibitors) are receiving active consideration. DAPK2 is a S/T CAM kinase and an orthologue of the tumor-suppressor DAPK-1. DAPK2, however, is less widely expressed and in initial studies was observed to be expressed at elevated levels in maturing erythroblasts [14]. In cell line studies, DAPK2 has been shown to exert pro-apoptotic effects [38] and may also affect autophagy [39]. In a UT7epo cell line model, we further have observed sensitization to apoptosis due to DAPK2 ectopic expression and increased survival due to siRNA knockdown [40]. These initial findings provide impetus for the development of both gain- and loss-of-function mouse models to better establish the significance of DAPK2 regulation of late erythropoiesis. In Fig. 4, a schematic summary of possibly integrated actions of EpoR, DYRK3 and DAPK2 is outlined. Acknowledgments Supported by NIH grants (to DMW) R01HL44491, R01DK59472 and P20RR18789. References [1] M. Boogaerts, M. Mittelman, P. Vaupel, Beyond anaemia management: evolving role of erythropoietin therapy in neurological disorders, multiple myeloma and tumour hypoxia models, Oncology 69 (Suppl. 2) (2005) 22–30. [2] A. List, S. Kurtin, D.J. Roe, A. Buresh, D. Mahadevan, D. Fuchs, L. Rimsza, R. Heaton, R. Knight, J.B. Zeldis, Efficacy of lenalidomide in myelodysplastic syndromes, N. Engl. J. Med. 352 (6) (2005) 549–557. [3] C. Lacombe, Resistance to erythropoietin, N. Engl. J. Med. 334 (10) (1996) 660–662. [4] T.D. Richmond, M. Chohan, D.L. Barber, Turning cells red: signal transduction mediated by erythropoietin, Trends Cell. Biol. 15 (3) (2005) 146–155. [5] L.I. Zon, Blood 100 (2002) A58. [6] J.E. Damen, H. Wakao, A. Miyajima, J. Krosl, R.K. Humphries, R.L. Cutler, G. Krystal, Tyrosine 343 in the erythropoietin receptor positively regulates erythropoietin-induced cell proliferation and Stat5 activation, EMBO J. 14 (22) (1995) 5557–5568. [7] U. Klingmuller, H. Wu, J.G. Hsiao, A. Toker, B.C. Duckworth, L.C. Cantley, H.F. Lodish, Identification of a novel pathway important for proliferation and differentiation of primary erythroid progenitors, Proc. Natl. Acad. Sci. U. S. A. 94 (7) (1997) 3016–3021. [8] M. Socolovsky, A.E. Fallon, S. Wang, C. Brugnara, H.F. Lodish, Fetal anemia and apoptosis of red cell progenitors in Stat5a−/−5b−/− mice: a direct role for Stat5 in Bcl-X(L) induction, Cell 98 (2) (1999) 181–191. [9] D. Bouscary, F. Pene, Y.E. Claessens, O. Muller, S. Chretien, M. FontenayRoupie, S. Gisselbrecht, P. Mayeux, C. Lacombe, Critical role for PI 3kinase in the control of erythropoietin-induced erythroid progenitor proliferation, Blood 101 (9) (2003) 3436–3443. [10] Y. Kashii, M. Uchida, K. Kirito, M. Tanaka, K. Nishijima, M. Toshima, T. Ando, K. Koizumi, T. Endoh, K. Sawada, et al., A member of Forkhead family transcription factor, FKHRL1, is one of the downstream molecules of phosphatidylinositol 3-kinase-Akt activation pathway in erythropoietin signal transduction, Blood 96 (3) (2000) 941–949. [11] H. Zang, K. Sato, H. Nakajima, C. McKay, P.A. Ney, J.N. Ihle, The distal region and receptor tyrosines of the Epo receptor are non-essential for in vivo erythropoiesis, EMBO J. 20 (12) (2001) 3156–3166. [12] M.P. Menon, J. Fang, D.M. Wojchowski, Core erythropoietin receptor signals for late erythroblast development, Blood (2005).
237
[13] J.N. Geiger, G.T. Knudsen, L. Panek, A.K. Pandit, M.D. Yoder, K.A. Lord, C.L. Creasy, B.M. Burns, P. Gaines, S.B. Dillon, mDYRK3 kinase is expressed selectively in late erythroid progenitor cells and attenuates colony forming unit erythroid development, Blood 97 (4) (2001) 901–910. [14] M. Menon, B. Vincent, A. Breggia, D. Wojchowski, Death associated protein kinase 2 (DAPK2): a novel Epo modulated apoptotic regulator of late erythropoiesis, Blood 104 (11) (2004) abstract #582. [15] K.E. McGrath, J. Palis, Hematopoiesis in the yolk sac: more than meets the eye, Exp. Hematol. 33 (9) (2005) 1021–1028. [16] J. Palis, G.B. Segel, Developmental biology of erythropoiesis, Blood Rev. 12 (2) (1998) 106–114. [17] D. Orlic, J.M. Wu, R.D. Carmichael, F. Quaini, M. Kobylack, A.S. Gordon, Increased erythropoiesis and 2′5′—A polymerase activity in the marrow and spleen of phenylhydrazine-injected rats, Exp. Hematol. 10 (5) (1982) 478–485. [18] V.C. Broudy, N.L. Lin, G.V. Priestley, K. Nocka, N.S. Wolf, Interaction of stem cell factor and its receptor c-kit mediates lodgment and acute expansion of hematopoietic cells in the murine spleen, Blood 88 (1) (1996) 75–81. [19] L.C. Ou, D. Kim, W.M. Layton Jr., R.P. Smith, Splenic erythropoiesis in polycythemic response of the rat to high-altitude exposure, J. Appl. Physiol. 48 (5) (1980) 857–861. [20] A.M. Vannucchi, F. Paoletti, S. Linari, C. Cellai, R. Caporale, P.R. Ferrini, M. Sanchez, G. Migliaccio, A.R. Migliaccio, Identification and characterization of a bipotent (erythroid and megakaryocytic) cell precursor from the spleen of phenylhydrazine-treated mice, Blood 95 (8) (2000) 2559–2568. [21] L.E. Lenox, J.M. Perry, R.F. Paulson, BMP4 and Madh5 regulate the erythroid response to acute anemia, Blood 105 (7) (2005) 2741–2748. [22] O. Ohneda, N. Yanai, M. Obinata, Microenvironment created by stromal cells is essential for a rapid expansion of erythroid cells in mouse fetal liver, Development 110 (2) (1990) 379–384. [23] K.M. Moritz, G.B. Lim, E.M. Wintour, Developmental regulation of erythropoietin and erythropoiesis, Am. J. Physiol. 273 (6 Pt 2) (1997) R1829–R1844. [24] J.J. Brazil, P. Gupta, Constitutive expression of the Fas receptor and its ligand in adult human bone marrow: a regulatory feedback loop for the homeostatic control of hematopoiesis, Blood Cells Mol. Dis. 29 (1) (2002) 94–103. [25] T. Kakagianni, N.C. Giannakoulas, E. Thanopoulou, A. Galani, S. Michalopoulou, A. Kouraklis-Symeonidis, N.C. Zoumbos, A probable role for trail-induced apoptosis in the pathogenesis of marrow failure Implications from an in vitro model and from marrow of aplastic anemia patients, Leuk. Res. (in press) (Electronic publication ahead of print). [26] S. Nakao, X. Feng, C. Sugimori, Immune pathophysiology of aplastic anemia, Int. J. Hematol. 82 (3) (2005) 196–200. [27] P. Gupta, G.A. Niehans, S.C. LeRoy, K. Gupta, V.A. Morrison, C. Schultz, D.J. Knapp, R.A. Kratzke, Fas ligand expression in the bone marrow in myelodysplastic syndromes correlates with FAB subtype and anemia, and predicts survival, Leukemia 13 (1) (1999) 44–53. [28] M.O. Muench, R. Namikawa, Disparate regulation of human fetal erythropoiesis by the microenvironments of the liver and bone marrow, Blood Cells Mol. Dis. 27 (2) (2001) 377–390. [29] M. Tanaka, Y. Hirabayashi, T. Sekiguchi, T. Inoue, M. Katsuki, A. Miyajima, Targeted disruption of oncostatin M receptor results in altered hematopoiesis, Blood 102 (9) (2003) 3154–3162. [30] A. Wickrema, S. Uddin, A. Sharma, F. Chen, Y. Alsayed, S. Ahmad, S.T. Sawyer, G. Krystal, T. Yi, K. Nishada, et al., Engagement of Gab1 and Gab2 in erythropoietin signaling, J. Biol. Chem. 274 (35) (1999) 24469–24474. [31] F. Verdier, S. Chretien, C. Billat, S. Gisselbrecht, C. Lacombe, P. Mayeux, Erythropoietin induces the tyrosine phosphorylation of insulin receptor substrate-2. An alternate pathway for erythropoietin-induced phosphatidylinositol 3-kinase activation, J. Biol. Chem. 272 (42) (1997) 26173–26178. [32] M.P. Menon, V. Karur, O. Bogacheva, O. Bogachev, B. Cuetara, D.M. Wojchowski, Signals for stress erythropoiesis are integrated via an erythropoietin receptor phosphotyrosine-PY343-Stat5 axis. Journal of Clinical Investigation (in press).
238
D.M. Wojchowski et al. / Blood Cells, Molecules, and Diseases 36 (2006) 232–238
[33] P.S. Hammerman, C.J. Fox, M.J. Birnbaum, C.B. Thompson, The Pim and Akt oncogenes are independent regulators of hematopoietic cell growth and survival, Blood 105 (11) (2005) 4477–4483. [34] J. Zhang, H.F. Lodish, Constitutive activation of the MEK/ERK pathway mediates all effects of oncogenic H-ras expression in primary erythroid progenitors, Blood 104 (6) (2004) 1679–1687. [35] J.L. Spivak, The anaemia of cancer: death by a thousand cuts, Nat. Rev., Cancer 5 (7) (2005) 543–555. [36] D. Campioni, P. Secchiero, F. Corallini, E. Melloni, S. Capitani, F. Lanza, G. Zauli, Evidence for a role of TNF-related apoptosis-inducing ligand (TRAIL) in the anemia of myelodysplastic syndromes, Am. J. Pathol. 166 (2) (2005) 557–563. [37] D. Zhang, K. Li, C.L. Erickson-Miller, M. Weiss, D.M. Wojchowski,
DYRK gene structure and erythroid-restricted features of DYRK3 gene expression, Genomics 85 (1) (2005) 117–130. [38] T. Kawai, F. Nomura, K. Hoshino, N.G. Copeland, D.J. Gilbert, N.A. Jenkins, S. Akira, Death-associated protein kinase 2 is a new calcium/ calmodulin-dependent protein kinase that signals apoptosis through its catalytic activity, Oncogene 18 (23) (1999) 3471–3480. [39] B. Inbal, S. Bialik, I. Sabanay, G. Shani, A. Kimchi, DAP kinase and DRP1 mediate membrane blebbing and the formation of autophagic vesicles during programmed cell death, J. Cell Biol. 157 (3) (2002) 455–468. [40] J. Fang, M.P. Menon, M. Tschan, B. Torbett, D.M. Wojchowski, Deathassociated protein kinase-2 (DAPK2) potentiates erythroid progenitor cell apoptosis in response to cisplatin, and cytokine withdrawal, American Society Of Hematology-Annual Meeting, 2005, Abstracts Dec 2005.