Title: Antiapoptotic Effects of Erythropoietin in Differentiated Neuroblastoma SH-SY5Y Cells Require Activation of Both the STAT5 and AKT Signaling Pathways
Abstract: The hematopoietic cytokine erythropoietin (Epo) prevents neuronal death during ischemic events in the brain and in neurodegenerative diseases, presumably through its antiapoptotic effects. To explore the role of different signaling pathways in Epo-mediated antiapoptotic effects in differentiated human neuroblastoma SH-SY5Y cells, we employed a prolactin receptor (PrlR)/erythropoietin receptor (EpoR) chimera system, in which binding of prolactin (Prl) to the extracellular domain activates EpoR signaling in the cytosol. On induction of apoptosis by staurosporine, Prl supports survival of the SH-SY5Y cells expressing the wild-type PrlR/EpoR chimera. In these cells Prl treatment strongly activates the STAT5, AKT, and MAPK signaling pathways and induces weak activation of the p65 NF-κB factor. Selective mutation of the eight tyrosine residues of the EpoR cytoplasmic domain results in impaired or absent activation of either STAT5 (mutation of Tyr343) or AKT (mutation of Tyr479) or both (mutation of all eight tyrosine residues). Most interestingly, Prl treatment does not prevent apoptosis in cells expressing mutant PrlR/EpoR chimeras in which either the STAT5 or the AKT signaling pathways are not activated. In contrast, ERK 1/2 is fully activated by all mutant PrlR/EpoR chimeras, comparable with the level seen with the wild-type PrlR/EpoR chimera, implying that activation of the MAPK signaling pathway per se is not sufficient for antiapoptotic activity. Therefore, the antiapoptotic effects of Epo in neuronal cells require the combinatorial activation of multiple signaling pathways, including STAT5, AKT, and potentially MAPK as well, in a manner similar to that observed in hematopoietic cells. The hematopoietic cytokine erythropoietin (Epo) prevents neuronal death during ischemic events in the brain and in neurodegenerative diseases, presumably through its antiapoptotic effects. To explore the role of different signaling pathways in Epo-mediated antiapoptotic effects in differentiated human neuroblastoma SH-SY5Y cells, we employed a prolactin receptor (PrlR)/erythropoietin receptor (EpoR) chimera system, in which binding of prolactin (Prl) to the extracellular domain activates EpoR signaling in the cytosol. On induction of apoptosis by staurosporine, Prl supports survival of the SH-SY5Y cells expressing the wild-type PrlR/EpoR chimera. In these cells Prl treatment strongly activates the STAT5, AKT, and MAPK signaling pathways and induces weak activation of the p65 NF-κB factor. Selective mutation of the eight tyrosine residues of the EpoR cytoplasmic domain results in impaired or absent activation of either STAT5 (mutation of Tyr343) or AKT (mutation of Tyr479) or both (mutation of all eight tyrosine residues). Most interestingly, Prl treatment does not prevent apoptosis in cells expressing mutant PrlR/EpoR chimeras in which either the STAT5 or the AKT signaling pathways are not activated. In contrast, ERK 1/2 is fully activated by all mutant PrlR/EpoR chimeras, comparable with the level seen with the wild-type PrlR/EpoR chimera, implying that activation of the MAPK signaling pathway per se is not sufficient for antiapoptotic activity. Therefore, the antiapoptotic effects of Epo in neuronal cells require the combinatorial activation of multiple signaling pathways, including STAT5, AKT, and potentially MAPK as well, in a manner similar to that observed in hematopoietic cells. The hematopoietic cytokine erythropoietin (Epo), 3The abbreviations used are: Epo, erythropoietin; EpoR, erythropoietin receptor; PrlR, prolactin receptor; Prl, prolactin; JAK2, Janus kinase 2; STAT5, signal transducer and activator of transcription 5; PI, phosphatidylinositol; MAPK, mitogen-activated protein kinase; PI(4,5)P2, phosphatidylinositol 4,5-bisphosphate; PI(3,4,5)P3, phosphatidylinositol 3,4,5-trisphosphate; GFP, green fluorescent protein; FACS, fluorescence-activated cell sorting; PBS, phosphate-buffered saline; BSA, bovine serum albumin; APC, allophycocyanin; 7-AAD, 7-aminoactinomycin D; p-NFH, phosphorylated Neurofilament-H; GFAP, glial fibrillary acidic protein; FACS, fluorescence-activated cell sorting; HA, hemagglutinin; TNF, tumor necrosis factor. a glycoprotein hormone produced primarily in the fetal liver and in the adult kidney, supports survival of erythroid progenitor cells and is essential for their proliferation and differentiation (1Lin C.S. Lim S.K. D'Agati V. Costantini F. Genes Dev. 1996; 10: 154-164Crossref PubMed Scopus (353) Google Scholar, 2Wu H. Liu X. Jaenisch R. Lodish H.F. Cell. 1995; 83: 59-67Abstract Full Text PDF PubMed Scopus (861) Google Scholar). Epo has been thought to act exclusively on erythroid progenitor cells. However, recent evidence indicates that Epo provides neuroprotective effects in the damaged brain during ischemic events and neurodegenerative diseases; the EpoR is expressed in several neuronal cell lines and in hippocampal and cortical neurons of rodent and human brains (3Juul S.E. Anderson D.K. Li Y. Christensen R.D. Pediatr. Res. 1998; 43: 40-49Crossref PubMed Scopus (342) Google Scholar, 4Marti H.H. Wenger R.H. Rivas L.A. Straumann U. Digicaylioglu M. Henn V. Yonekawa Y. Bauer C. Gassmann M. Eur. J. Neurosci. 1996; 8: 666-676Crossref PubMed Scopus (498) Google Scholar, 5Masuda S. Nagao M. Takahata K. Konishi Y. Gallyas F.J. Tabira T. Sasaki R. J. Biol. Chem. 1993; 268: 11208-11216Abstract Full Text PDF PubMed Google Scholar, 6Morishita E. Masuda S. Nagao M. Yasuda Y. Sasaki R. Neuroscience. 1997; 76: 105-116Crossref PubMed Scopus (628) Google Scholar). Expression of Epo also occurs in the brain and in in vitro cultured astrocytes and neurons (4Marti H.H. Wenger R.H. Rivas L.A. Straumann U. Digicaylioglu M. Henn V. Yonekawa Y. Bauer C. Gassmann M. Eur. J. Neurosci. 1996; 8: 666-676Crossref PubMed Scopus (498) Google Scholar, 7Bernaudin M. Marti H.H. Roussel S. Divoux D. Nouvelot A. MacKenzie E.T. Petit E. J. Cereb. Blood Flow Metab. 1999; 19: 646-651Crossref Scopus (679) Google Scholar, 8Bernaudin M. Bellail A. Marti H.H. Yvon A. Vivien D. Duchatelle I. Mackenzie E.T. Petit E. Glia. 2000; 30: 271-278Crossref PubMed Scopus (261) Google Scholar, 9Digicaylioglu M. Bichet S. Marti H.H. Wenger R.H. Rivas L.A. Bauer C. Gassmann M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3717-3720Crossref PubMed Scopus (435) Google Scholar, 10Konishi Y. Chui D.H. Hirose H. Kunishita T. Tabira T. Brain Res. 1993; 609: 29-35Crossref PubMed Scopus (249) Google Scholar). Together, these results suggest that Epo may function in the brain in a paracrine and/or autocrine fashion. Indeed, Epo reduces neuronal damage from ischemia in the brain of rodent models of stroke and also has a neuroprotective role against mechanical trauma, excitotoxins, neuroinflammation, and in an animal model of Parkinsonism (7Bernaudin M. Marti H.H. Roussel S. Divoux D. Nouvelot A. MacKenzie E.T. Petit E. J. Cereb. Blood Flow Metab. 1999; 19: 646-651Crossref Scopus (679) Google Scholar, 11Brines M.L. Ghezzi P. Keenan S. Agnello D. de Lanerolle N.C. Cerami C. Itri L.M. Cerami A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10526-10531Crossref PubMed Scopus (1289) Google Scholar, 12Digicaylioglu M. Lipton S. Nature. 2001; 412: 641-647Crossref PubMed Scopus (867) Google Scholar, 13Genc S. Kuralay F. Genc K. Akhisaroglu M. Fadiloglu S. Yorukoglu K. Fadiloglu M. Gure A. Neurosci. Lett. 2001; 298: 139-141Crossref PubMed Scopus (121) Google Scholar, 14Sadamoto Y. Igase K. Sakanaka M. Sato K. Otsuka H. Sakaki S. Masuda S. Sasaki R. Biochem. Biophys. Res. Commun. 1998; 253: 26-32Crossref PubMed Scopus (292) Google Scholar, 15Sakanaka M. Wen T.C. Matsuda S. Masuda S. Morishita E. Nagao M. Sasaki R. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 4635-4640Crossref PubMed Scopus (902) Google Scholar). Several studies show that Epo prevents apoptosis caused by cerebral ischemia in in vivo systems and by a variety of insults to in vitro cultured neuronal cell lines and primary cultured neurons (6Morishita E. Masuda S. Nagao M. Yasuda Y. Sasaki R. Neuroscience. 1997; 76: 105-116Crossref PubMed Scopus (628) Google Scholar, 12Digicaylioglu M. Lipton S. Nature. 2001; 412: 641-647Crossref PubMed Scopus (867) Google Scholar, 16Chong Z. Lin S. Kang J. Maises K. J. Neurosci. Res. 2003; 71: 659-669Crossref PubMed Scopus (155) Google Scholar, 17Kawakami M. Sekiguchi M. Sato K. Kozaki S. Takahashi M. J. Biol. Chem. 2001; 276: 39469-39475Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar, 18Ruscher K. Freyer D. Karsch M. Isaev N. Megow D. Sawitzki B. Priller J. Dirnagl U. Meisel A. J. Neurosci. 2002; 22: 10291-10301Crossref PubMed Google Scholar, 19Siren A.L. Fratelli M. Brines M. Goemans C. Casagrande S. Lewczuk P. Keenan S. Gleiter C. Pasquali C. Capobianco A. Mennini T. Heumann R. Cerami A. Ehrenreich H. Ghezzi P. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 4044-4049Crossref PubMed Scopus (927) Google Scholar, 20Wen T. Sadamoto Y. Tanaka J. Zhu P. Nakata K. Ma Y. Hata R. Sakanaka M. J. Neurosci. Res. 2002; 67: 795-803Crossref PubMed Scopus (192) Google Scholar). These results suggest that, like its role in erythroid progenitor cells, Epo supports survival of neuronal cells through antiapoptotic effects. Mice null for the EpoR (EpoR-/-) or Epo or Jak2 genes die at embryonic day 13 because of a deficiency in erythropoiesis that leads to severe anemia (1Lin C.S. Lim S.K. D'Agati V. Costantini F. Genes Dev. 1996; 10: 154-164Crossref PubMed Scopus (353) Google Scholar, 2Wu H. Liu X. Jaenisch R. Lodish H.F. Cell. 1995; 83: 59-67Abstract Full Text PDF PubMed Scopus (861) Google Scholar, 21Neubauer H. Cumano A. Muller M. Wu H. Huffstadt U. Pfeffer K. Cell. 1998; 93: 397-409Abstract Full Text Full Text PDF PubMed Scopus (682) Google Scholar, 22Parganas E. Wang D. Stravopodis D. Topham D.J. Marine J.C. Teglund S. Vanin E.F. Bodner S. Colamonici O.R. van Deursen J.M. Grosveld G. Ihle J.N. Cell. 1998; 93: 385-395Abstract Full Text Full Text PDF PubMed Scopus (912) Google Scholar). No gross morphological abnormalities were identified in the brain of these mice. Suzuki et al. (23Suzuki N. Ohneda O. Takahashi S. Higuchi M. Mukai H.Y. Nakahata T. Imagawa S. Yamamoto M. Blood. 2002; 100: 2279-2288Crossref PubMed Scopus (172) Google Scholar) generated a transgenically rescued EpoR-/- mouse by expression of the EpoR exclusively in the hematopoietic lineage using a GATA-1 minigene cassette. These mice are grossly normal despite the fact that they do not express EpoR in nonhematopoietic tissues, including the brain. This suggests that Epo signaling might not be necessary for normal brain development but could be required to respond to stresses in adults. In the hematopoietic system, the mechanisms by which the Epo signal is linked to gene transcription have been extensively investigated. The EpoR is a member of the type I cytokine receptor family that does not contain intrinsic tyrosine kinase activity (24Constantinescu S.N. Ghaffari S. Lodish H.F. Trends Endocrinol. Metab. 1999; 10: 18-23Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar). Instead, the signal is mediated by the Janus family protein-tyrosine kinase, JAK2. Once Epo binds to the EpoR, JAK2 associated with the EpoR cytoplasmic domain rapidly becomes activated by transphosphorylation. Subsequently, several tyrosine residues in the EpoR become phosphorylated and recruit multiple signaling molecules that contain Src homology 2 domains, including signal transducers and activators of transcription 5 (STAT5), phosphatidylinositol 3-kinase (PI 3-kinase), and phospholipase C-γ. One major EpoR intracellular signaling pathway involves STAT5. After binding to the activated EpoR, STAT5 becomes phosphorylated, dissociates from the EpoR, homodimerizes, and translocates to the nucleus, where it up-regulates expression of the antiapoptotic gene Bcl-X (25Damen J.E. Wakao H. Miyajima A. Krosl J. Humphries R.K. Cutler R.L. Krystal G. EMBO J. 1995; 14: 5557-5568Crossref PubMed Scopus (264) Google Scholar, 26Klingmuller U. Bergelson S. Hsiao J.G. Lodish H.F. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8324-8328Crossref PubMed Scopus (165) Google Scholar, 27Socolovsky M. Fallon A.E. Wang S. Brugnara C. Lodish H.F. Cell. 1999; 98: 181-191Abstract Full Text Full Text PDF PubMed Scopus (618) Google Scholar, 28Socolovsky M. Nam H.-S. Fleming M.D. Haase V.H. Brugnara C. Lodish H.F. Blood. 2001; 98: 3261-3273Crossref PubMed Scopus (581) Google Scholar). Socolovsky et al. (27Socolovsky M. Fallon A.E. Wang S. Brugnara C. Lodish H.F. Cell. 1999; 98: 181-191Abstract Full Text Full Text PDF PubMed Scopus (618) Google Scholar, 28Socolovsky M. Nam H.-S. Fleming M.D. Haase V.H. Brugnara C. Lodish H.F. Blood. 2001; 98: 3261-3273Crossref PubMed Scopus (581) Google Scholar) reported that mouse embryos and adults null for both Stat5a and Stat5b genes (Stat5a-/-5b-/-) are severely anemic and show increased levels of apoptosis in erythroid progenitors, demonstrating that STAT5 is one of the major antiapoptotic pathways activated by the EpoR. Activation of PI 3-kinase through recruitment to the activated EpoR results in conversion of the plasma membrane lipid phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) to phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3) (29Ghaffari S. Jagani Z. Kitidis C. Lodish H.F. Khosravi-Far R. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 6523-6528Crossref PubMed Scopus (126) Google Scholar, 30Kashii Y. Uchida M. Kirito K. Tanaka M. Nishijima K. Toshima M. Ando T. Koizumi K. Endoh T. Sawada K. Momoi M. Miura Y. Ozawa K. Komatsu N. Blood. 2000; 96: 941-949Crossref PubMed Google Scholar, 31Uddin S. Kottegoda S. Stigger D. Platanias L.C. Wickrema A. Biochem. Biophys. Res. Commun. 2000; 275: 16-19Crossref PubMed Scopus (84) Google Scholar, 32Cantley L.C. Science. 2002; 296: 1655-1657Crossref PubMed Scopus (4681) Google Scholar, 33Song G. Ouyang G. Bao S. J. Cell. Mol. Med. 2005; 9: 59-71Crossref PubMed Scopus (1551) Google Scholar). PI(3,4,5)P3 anchors the serine/threonine kinase AKT to the plasma membrane and induces a conformational change, which consequently allows phosphorylation of AKT by two other kinases. Activation of AKT requires phosphorylation of two regulatory residues, a threonine residue on the kinase domain and a serine residue on the C-terminal hydrophobic domain (Thr308 and Ser473 for AKT1; Thr309 and Ser474 for AKT2; and Thr305 and Ser472 for AKT3). Phosphorylation of the threonine residue, mediated by phosphoinositide-dependent kinase 1, is essential for AKT activation, whereas phosphorylation of the serine residue enhances the AKT activity about 10-fold. Several kinases, including phosphoinositide-dependent kinase 2, integrin-linked kinase, DNA-dependent protein kinase, protein kinase CβII, and the Rictor-mTOR complex, were suggested to phosphorylate the serine residue of AKT (32Cantley L.C. Science. 2002; 296: 1655-1657Crossref PubMed Scopus (4681) Google Scholar, 33Song G. Ouyang G. Bao S. J. Cell. Mol. Med. 2005; 9: 59-71Crossref PubMed Scopus (1551) Google Scholar, 34Sarbassov D.D. Guertin D.A. Ali S.M. Sabatini D.M. Science. 2005; 307: 1098-1101Crossref PubMed Scopus (5294) Google Scholar). However, which kinase is primarily responsible for the phosphorylation of the serine residue has not been clarified. Once activated, AKT phosphorylates and inactivates members of the Forkhead transcription factor family. Subsequently, this leads to reduced expression of several apoptotic proteins that are normally activated by the Forkhead transcription factors. In hematopoietic cells, the EpoR also activates other signaling pathways, including the phospholipase C-γ and Ras/MAPK pathways (24Constantinescu S.N. Ghaffari S. Lodish H.F. Trends Endocrinol. Metab. 1999; 10: 18-23Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar, 35Coolican S.A. Samuel D.S. Ewton D.Z. McWade F.J. Florini J.R. J. Biol. Chem. 1997; 272: 6653-6662Abstract Full Text Full Text PDF PubMed Scopus (555) Google Scholar, 36Gobert S. Duprez V. Lacombe C. Gisselbrecht S. Mayeux P. Eur. J. Biochem. 1995; 234: 75-83Crossref PubMed Scopus (59) Google Scholar, 37He T.C. Jiang N. Zhuang H. Wojchowski D.M. J. Biol. Chem. 1995; 270: 11055-11061Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 38Mason J.M. Beattie B.K. Liu Q. Dumont D.J. Barber D.L. J. Biol. Chem. 2000; 275: 4398-4406Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 39Ren H.Y. Komatsu N. Shimizu R. Okada K. Miura Y. J. Biol. Chem. 1994; 269: 19633-19638Abstract Full Text PDF PubMed Google Scholar, 40Tauchi T. Damen J.E. Toyama K. Feng G.S. Broxmeyer H.E. Krystal G. Blood. 1996; 87: 4495-4501Crossref PubMed Google Scholar, 41Gaffen S.L. Lai S.Y. Longmore G.D. Liu K.D. Goldsmith M.A. Blood. 1999; 94: 74-86Crossref PubMed Google Scholar, 42Haq R. Halupa A. Beattie B.K. Mason J.M. Zanke B.W. Barber D.L. J. Biol. Chem. 2002; 277: 17359-17366Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 43Tang T. Prasad K.S. Koury M.J. Brandt S.J. Biochem. J. 1999; 343: 615-620Crossref PubMed Google Scholar). These multiple pathways involved in Epo signaling provide the same output, namely antiapoptosis. Studies using EpoR mutants overexpressed in EpoR-/- erythroid progenitors showed that normal erythroid differentiation was fully supported when only some of these signal proteins were activated (44Wu H. Klingmuller U. Acurio A. Hsiao J.G. Lodish H.F. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1806-1810Crossref PubMed Scopus (144) Google Scholar), suggesting that these signaling pathways may play redundant roles. The detailed molecular mechanisms by which Epo exerts its neuroprotective effects have not been clarified. Recent papers have suggested that the antiapoptotic activity of Epo in neuronal cells is mediated by activation of the PI 3-kinase/AKT signaling pathways (16Chong Z. Lin S. Kang J. Maises K. J. Neurosci. Res. 2003; 71: 659-669Crossref PubMed Scopus (155) Google Scholar, 18Ruscher K. Freyer D. Karsch M. Isaev N. Megow D. Sawitzki B. Priller J. Dirnagl U. Meisel A. J. Neurosci. 2002; 22: 10291-10301Crossref PubMed Google Scholar, 45Chong Z.Z. Kang J.Q. Maiese K. Br. J. Pharmacol. 2003; 138: 1107-1118Crossref PubMed Scopus (203) Google Scholar); inactivation of the pro-apoptotic gene Bad (18Ruscher K. Freyer D. Karsch M. Isaev N. Megow D. Sawitzki B. Priller J. Dirnagl U. Meisel A. J. Neurosci. 2002; 22: 10291-10301Crossref PubMed Google Scholar); inhibition of glutamate secretion (17Kawakami M. Sekiguchi M. Sato K. Kozaki S. Takahashi M. J. Biol. Chem. 2001; 276: 39469-39475Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar); and activation of Bcl-XL (20Wen T. Sadamoto Y. Tanaka J. Zhu P. Nakata K. Ma Y. Hata R. Sakanaka M. J. Neurosci. Res. 2002; 67: 795-803Crossref PubMed Scopus (192) Google Scholar). Digicaylioglu and Lipton (12Digicaylioglu M. Lipton S. Nature. 2001; 412: 641-647Crossref PubMed Scopus (867) Google Scholar) suggested that activation of the EpoR in neurons triggers cross-talk between the JAK2 and NF-κB signaling pathways. Nevertheless, the contribution of each signaling pathway downstream of the EpoR to the neuroprotective activity of Epo in the brain is not clarified and may differ in different types of neurons. We demonstrate that activation of at least the STAT5 and AKT signaling pathways is essential for the antiapoptotic activity of Epo in neuronal cells. Cell Culture—SH-SY5Y cells were maintained in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum using collagen-coated plates (BD Biosciences). Differentiation was induced with 5 μm all-trans-retinoic acid (Sigma) in Neurobasal-A medium (Invitrogen) for 7 days. Generation of Ecotropic Receptor-overexpressing SH-SY5Y Cell Lines—SH-SY5Y cells were transfected with the plasmid pM5-EcoRneo that contains the murine ecotropic receptor cDNA and a neomycin resistance gene (46Baker B.W. Boettiger D. Spooncer E. Norton J.D. Nucleic Acids Res. 1992; 20: 5234Crossref PubMed Scopus (43) Google Scholar) using FuGENE (Roche Applied Science). Clones stably expressing the murine ecotropic receptor were isolated by drug selection using 1 mg/ml G418 (Invitrogen) for 2 weeks. Ten isolated clones were expanded and tested for growth rate, morphology, infection efficiency, differentiation by retinoic acid, and response to Epo upon induction of apoptosis. cDNA Constructs and Generation of Cells Expressing PrlR/EpoR Chimeras and the EpoR—PrlR/EpoR chimera constructs were made by standard subcloning methods. cDNAs encoding the wild-type and mutant EpoR intracellular domains were amplified by the PCR method using constructs described previously as templates (26Klingmuller U. Bergelson S. Hsiao J.G. Lodish H.F. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8324-8328Crossref PubMed Scopus (165) Google Scholar, 27Socolovsky M. Fallon A.E. Wang S. Brugnara C. Lodish H.F. Cell. 1999; 98: 181-191Abstract Full Text Full Text PDF PubMed Scopus (618) Google Scholar, 44Wu H. Klingmuller U. Acurio A. Hsiao J.G. Lodish H.F. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1806-1810Crossref PubMed Scopus (144) Google Scholar, 47Klingmuller U. Wu H. Hsiao J.G. Toker A. Duckworth B.C. Cantley L.C. Lodish H.F. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3016-3021Crossref PubMed Scopus (150) Google Scholar), fused with the cDNA of the extracellular domain of the PrlR as described (48Socolovsky M. Dusanter-Fourt I. Lodish H.F. J. Biol. Chem. 1997; 272: 14009-14012Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar), and subcloned into the pMSCVpuroIRES2-EGFP vector (49Marszalek J.R. Kitidis C. Dararutana A. Lodish H.F. J. Biol. Chem. 2004; 279: 23882-23891Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). All constructs were verified by sequencing. The N terminus of the extracellular domain of the PrlR/EpoR chimera contains the FLAG tag (DYKD-DDDK) as described (48Socolovsky M. Dusanter-Fourt I. Lodish H.F. J. Biol. Chem. 1997; 272: 14009-14012Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). The HA-tagged (YPYDVPDYA) murine EpoR expression construct was described previously (50Huang L.J. Constantinescu S.N. Lodish H.F. Mol. Cell. 2001; 8: 1327-1338Abstract Full Text Full Text PDF PubMed Scopus (234) Google Scholar). The HA-tagged human EpoR expression construct was provided by Dr. S. Constantinescu of the Ludwig Institute for Cancer Research, Brussels, Belgium. Retroviral supernatants were prepared by transient transfection of 5 μg of pMSCVpuroIRES2-EGFP or the PrlR/EpoR chimera expression construct or the HA-EpoR expression construct with 5 μg of pCL-Eco as described previously (49Marszalek J.R. Kitidis C. Dararutana A. Lodish H.F. J. Biol. Chem. 2004; 279: 23882-23891Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 51Naviaux R.K. Costanzi E. Haas M. Verma I.M. J. Virol. 1996; 70: 5701-5705Crossref PubMed Google Scholar). For retroviral infection, SH-SY5Y cells stably expressing the mouse ecotropic receptor (clone Eco-SH-SY5Y 7) were plated on 100-mm collagen-coated plates (BD Biosciences) and infected with retroviral supernatants in media with 4 μg/ml Polybrene (Sigma). Green fluorescent protein (GFP)-positive cells, indicating expression of PrlR/EpoR chimeras or the HA-EpoR, were isolated by fluorescence-activated cell sorting (FACS). For cells overexpressing PrlR/EpoR chimera, drug selection using 1 μg/ml puromycin was also applied. Once cells were sorted, integration of the correct PrlR/EpoR chimera cDNA was confirmed by genomic DNA PCR and sequencing. Surface Expression of FLAG-PrlR/EpoR Chimeras and HA-EpoR—Surface expression of PrlR/EpoR chimeras and the HA-EpoR was examined as described (50Huang L.J. Constantinescu S.N. Lodish H.F. Mol. Cell. 2001; 8: 1327-1338Abstract Full Text Full Text PDF PubMed Scopus (234) Google Scholar) with minor modifications. SH-SY5Y cells overexpressing PrlR/EpoR chimeras or the HA-EpoR were harvested with phosphate-buffered saline (PBS) containing 10 mm EDTA (10 mm EDTA/PBS) and incubated for 30 min in cold PBS containing 2% bovine serum albumin (BSA) and 4% donkey serum (Blocking buffer). Cells were then incubated for 1 h with mouse monoclonal anti-FLAG or anti-HA antibodies (Covance, 10 μg/ml in Blocking buffer), washed three times with PBS containing 0.2% BSA and 0.4% donkey serum (Washing buffer), and incubated with allophycocyanin (APC)-conjugated goat anti-mouse IgG secondary antibody (12.5 μg/ml in Blocking buffer; BD Biosciences) at 4 °C for 1 h. Cells were washed three times with Washing buffer and were analyzed by FACS. Median fluorescence intensities of APC for GFP-positive cells that correspond to the cells expressing PrlR/EpoR chimeras or the HA-EpoR were used for quantification. Apoptosis Analysis—Apoptosis was induced by treatment with 0.1 μm staurosporine (Calbiochem). For pretreatment, 30 pm Prl (National Hormone and Peptide Program) was added to cells 24 h prior to addition of staurosporine and removed when staurosporine was added. For concomitant treatment, 30 pm Prl was added at the same time as staurosporine. At 24 h after addition of staurosporine, cells were harvested by trypsin treatment. The cell population was then analyzed by FACS using APC-conjugated annexin V binding and 7-aminoactinomycin D (7-AAD) staining (BD Biosciences). For quantification of the "apoptosis proportion," both dead cell fraction and apoptotic cell fraction were calculated. Apoptosis induced by staurosporine was normalized by removing the background apoptosis detected in control differentiation medium. To facilitate comparisons, apoptosis induced by 0.1 μm staurosporine in each type of cells without Prl treatment was set equal to 1.0 (relative apoptosis proportion). For the EpoR-overexpressing cells, 25 pm Epo (Epogen®; Amgen) was used instead of Prl with the same procedures. For experiments involving LY294002 or wortmannin tests, 10 μm LY294002 (Calbiochem) or 100 nm wortmannin (Calbiochem) was added at the same time as Prl. All subsequent procedures were as described above. Immunoprecipitation and Western Blot Analysis—SH-SY5Y cell lysates were prepared as described (52Constantinescu S.N. Huang L.J. Nam H. Lodish H.F. Mol. Cell. 2001; 7: 377-385Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar) with minor modifications. In detail, SH-SY5Y cells differentiated for 7 days were growth factor-starved for 5 h, harvested with 10 mm EDTA/PBS, treated with Prl (or Epo for the EpoR-overexpressing cells) for 10 min, and lysed in Nonidet P-40 Lysis buffer (5 mm Tris, pH 7.4, 150 mm NaCl, 1% Nonidet P-40) with protease inhibitors (Roche Applied Science) and 1 mm sodium vanadate. The soluble fractions of the lysates were incubated with anti-STAT5 (Santa Cruz Biotechnology) or anti-AKT (Santa Cruz Biotechnology) or anti-ERK 1/2 (Cell Signaling Technology) antibodies. The immune complexes were recovered using protein-A-Sepharose beads and eluted with NuPAGE buffer (Invitrogen) containing 1% β-mercaptoethanol. Proteins from these immunoprecipitates were separated on NuPAGE gels (Invitrogen), transferred to nitrocellulose membranes (Schleicher & Schuell), and Western-blotted using anti-phospho-STAT5 (p-STAT5, Cell Signaling Technology) or anti-phosphoserine-AKT (p-Ser-AKT, Cell Signaling Technology) or anti-phospho-ERK 1/2 (p-ERK 1/2, Cell Signaling Technology) antibodies. For experiments in Fig. 7, anti-phosphothreonine-AKT (p-Thr-AKT, Cell Signaling Technology) was used instead of anti-p-Ser-AKT antibody. For quantification of protein loading, the membranes were stripped with 0.2 m Glycine buffer (pH 2.6) and reprobed with anti-STAT5 or anti-AKT or anti-ERK 1/2 antibodies. For cells overexpressing the EpoR, cells were treated with Epo instead of Prl, and subsequent procedures were as described above. To confirm expression of FLAG-tagged PrlR/EpoR chimeras and the exogenously expressed HA-tagged EpoR, SH-SY5Y cells overexpressing PrlR/EpoR chimeras or the HA-EpoRs were harvested with 10 mm EDTA/PBS and lysed in Nonidet P-40 lysis buffer with protease inhibitors. The lysates were analyzed by Western blotting using anti-FLAG or anti-HA antibodies (Covance). Nuclear Extract Preparation and Electrophoretic Mobility Shift Assay—SH-SY5Y cells differentiated for 7 days were growth factor-starved for 5 h, harvested with 10 mm EDTA/PBS, and treated with Prl (or Epo for the EpoR-overexpressing cells) for the times indicated in the figure legends. To isolate nuclear fractions, cells were resuspended in hypotonic buffer (10 mm HEPES, pH 7.9, 10 mm KCl, 0.1 mm EDTA, 0.1 mm EGTA, 1 mm Na3V2O4, 1 mm DTT, protease inhibitor (Roche Applied Science)), incubated for 15 min on ice, treated with Nonidet P-40 (0.5% final concentration), and mixed vigorously. The pellets were recovered by centrifugation. Nuclear extracts were made by resuspending the nuclear pellets in hypertonic buffer (20 mm HEPES, pH 7.9, 400 mm NaCl, 1 mm EDTA, 1 mm DTT, protease inhibitor (Roche Applied Science)). After 20 min of incubation on ice, the supernatants were collected by centrifugation and stored at -80 °C. As a positive control for NF-κB activation, nuclear extracts were made from SH-SY5Y cells treated with 50 ng/ml TNF-α (53Bui N.T. Livolsi A. Peyron J.F. Prehn J.H. J. Cell Biol. 2001; 152: 753-764Crossref PubMed Scopus (127) Google Scholar, 54Korner M. Tarantino N. Pleskoff O. Lee L.M. Debre P. J. Neurochem. 1994; 62: 1716-1726Crossref PubMed Scopus (30) Google Scholar) under the same procedures. The nuclear extracts were analyzed by Western blotting using anti-NF-κB p65 (Santa Cruz Biotechnology) or anti-TATA-binding protein (Santa Cruz Biotechnology) antibodies. For electrophoretic mobility shift assays, consensus NF-κB binding oligonucleotides (Santa Cruz Biotechnology) were labeled with 32P and used as a DNA probe. SH-SY5Y nuclear extracts were incubated with 0.25 ng of 32P-labeled oligonucle