The Sonic Hedgehog Pathway Mediates Carbamylated Erythropoietin-enhanced Proliferation and Differentiation of Adult Neural Progenitor Cells

Abstract

Carbamylated erythropoietin (CEPO), a well characterized erythropoietin (EPO) derivative, does not bind to the classical EPO receptor and does not stimulate erythropoiesis. Using neural progenitor cells derived from the subventricular zone of the adult mouse, we investigated the effect of CEPO on neurogenesis and the associated signaling pathways in vitro. We found that CEPO significantly increased neural progenitor cell proliferation and promoted neural progenitor cell differentiation into neurons, which was associated with up-regulation of Sonic hedgehog (Shh), its receptor ptc, and mammalian achaete-scute homolog 1 (Mash1), a pro-neuron basic helix-loop-helix protein transcription factor. Blockage of the Shh signaling pathway with a pharmacological inhibitor, cyclopamine, abolished the CEPO-induced neurogenesis. Attenuation of endogenous Mash1 expression by short-interfering RNA blocked CEPO-promoted neuronal differentiation. In addition, recombinant mouse Shh up-regulated Mash1 expression in neural progenitor cells. These results demonstrate that the Shh signaling pathway mediates CEPO-enhanced neurogenesis and Mash1 is a downstream target of the Shh signaling pathway that regulates CEPO-enhanced neuronal differentiation. Carbamylated erythropoietin (CEPO), a well characterized erythropoietin (EPO) derivative, does not bind to the classical EPO receptor and does not stimulate erythropoiesis. Using neural progenitor cells derived from the subventricular zone of the adult mouse, we investigated the effect of CEPO on neurogenesis and the associated signaling pathways in vitro. We found that CEPO significantly increased neural progenitor cell proliferation and promoted neural progenitor cell differentiation into neurons, which was associated with up-regulation of Sonic hedgehog (Shh), its receptor ptc, and mammalian achaete-scute homolog 1 (Mash1), a pro-neuron basic helix-loop-helix protein transcription factor. Blockage of the Shh signaling pathway with a pharmacological inhibitor, cyclopamine, abolished the CEPO-induced neurogenesis. Attenuation of endogenous Mash1 expression by short-interfering RNA blocked CEPO-promoted neuronal differentiation. In addition, recombinant mouse Shh up-regulated Mash1 expression in neural progenitor cells. These results demonstrate that the Shh signaling pathway mediates CEPO-enhanced neurogenesis and Mash1 is a downstream target of the Shh signaling pathway that regulates CEPO-enhanced neuronal differentiation. Ischemic stroke induces neurogenesis (1Jin K. Kim M. Lan J.Q. Mao X.O. Batteur S. Simon R.P. Greenberg D.A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 4710-4715Crossref PubMed Scopus (1009) Google Scholar, 2Zhang R.L. Kim Z.G. Zhang L. Chopp M. Neuroscience. 2001; 105: 33-41Crossref PubMed Scopus (534) Google Scholar, 3Parent J.M. Kim Z.S. Gong C. Derugin N. Ferriero D.M. Ann. Neurol. 2002; 52: 802-813Crossref PubMed Scopus (915) Google Scholar, 4Arvidsson A. Kim T. Kirik D. Kokaia Z. Lindvall O. Nat. Med. 2002; 8: 963-970Crossref PubMed Scopus (2416) Google Scholar). Erythropoietin (EPO) 2The abbreviations used are: EPO, erythropoietin; EPOR, EPO receptor; CEPO, carbamylated EPO; SVZ, subventricular zone; Shh, Sonic hedgehog; EGF, epidermal growth factor; bFGF, basic fibroblast growth factor; BrdUrd, bromodeoxyuridine; siRNA, short interfering RNA; RT-PCR, reverse transcription PCR; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; DAPI, 4′,6-diamidino-2-phenylindole. has neuroprotective effects for treatment of acute stroke via interaction with its receptor (EPOR) (5Brines M. Kim A. Kidney Int. 2006; 70: 246-250Abstract Full Text Full Text PDF PubMed Scopus (255) Google Scholar). Recent studies show that EPO increases neurogenesis in the subventricular zone (SVZ) of adult rodent brain under normal and stroke conditions via interaction with EPOR (6Wang L. Kim Z. Wang Y. Zhang R. Chopp M. Stroke. 2004; 35: 1732-1737Crossref PubMed Scopus (687) Google Scholar, 7Tsai P.T. Kim J.J. Kertesz N. Groszer M. Matter C. Gao J. Liu X. Wu H. Carmichael S.T. J. Neurosci. 2006; 26: 1269-1274Crossref PubMed Scopus (353) Google Scholar, 8Shingo T. Kim S.T. Shimazaki T. Weiss S. J. Neurosci. 2001; 21: 9733-9743Crossref PubMed Google Scholar). Systemic administration of EPO enhances stroke-induced neurogenesis (6Wang L. Kim Z. Wang Y. Zhang R. Chopp M. Stroke. 2004; 35: 1732-1737Crossref PubMed Scopus (687) Google Scholar). However, EPO also elevates hematocrit levels, which could lead to adverse effects on stroke recovery (6Wang L. Kim Z. Wang Y. Zhang R. Chopp M. Stroke. 2004; 35: 1732-1737Crossref PubMed Scopus (687) Google Scholar, 9Gassmann M. Kim K. Soliz J. Ogunshola O.O. Marti H.H. Hofer T. Grimm C. Heinicke I. Egli B. Adv. Exp. Med. Biol. 2003; 543: 323-330Crossref PubMed Scopus (105) Google Scholar). Carbamylated erythropoietin (CEPO), a non-erythropoietic derivative of EPO that does not bind to the classical EPOR (10Leist M. Kim P. Grasso G. Bianchi R. Villa P. Fratelli M. Savino C. Bianchi M. Nielsen J. Gerwien J. Kallunki P. Larsen A.K. Helboe L. Christensen S. Pedersen L.O. Nielsen M. Torup L. Sager T. Sfacteria A. Erbayraktar S. Erbayraktar Z. Gokmen N. Yilmaz O. CeramiHand C. Xie Q.W. Coleman T. Cerami A. Brines M. Science. 2004; 305: 239-242Crossref PubMed Scopus (710) Google Scholar), is neuroprotective for acute stroke but does not elevate hematocrit levels. The effect of CEPO on neurogenesis has not been investigated. Sonic hedgehog (Shh) is a member of the family of the hedgehog proteins known to exert important regulatory functions in patterning and growth in a large number of tissues during embryogenesis (11Ruiz i Altaba A. Kim P. Dahmane N. Nat. Rev. Cancer. 2002; 2: 361-372Crossref PubMed Scopus (651) Google Scholar, 12Goetz J.A. Kim L.M. Zeng X. Robbins D.J. BioEssays. 2002; 24: 157-165Crossref PubMed Scopus (52) Google Scholar, 13Roberts D.J. Kim R.L. Burke A.C. Nelson C.E. Morgan B.A. Tabin C. Development. 1995; 121: 3163-3174Crossref PubMed Google Scholar). In the mammalian brain, Shh plays an important role for the regulation of progenitor cell proliferation and differentiation (14Zhu G. Kim M.F. Zhao J. Yu Yung S. Kessler J.A. Dev. Biol. 1999; 215: 118-129Crossref PubMed Scopus (86) Google Scholar, 15Fu M. Kim V.C. Sham M.H. Pachnis V. Tam P.K. J. Cell Biol. 2004; 166: 673-684Crossref PubMed Scopus (150) Google Scholar, 16Lai K. Kim B.K. Gage F.H. Schaffer D.V. Nat. Neurosci. 2003; 6: 21-27Crossref PubMed Scopus (684) Google Scholar, 17Rowitch D.H. Kim B. Lee S.M. Flax J.D. Snyder E.Y. McMahon A.P. J. Neurosci. 1999; 19: 8954-8965Crossref PubMed Google Scholar). Shh binds to the transmembrane receptor protein patched (ptc), which, in the absence of Shh, exerts an inhibitory effect on the seven-transmembrane receptor smoothened (Smo) (18Gutierrez-Frias C. Kim R. Hernandez-Lopez C. Cejalvo T. Crompton T. Zapata A.G. Varas A. Vicente A. J. Immunol. 2004; 173: 5046-5053Crossref PubMed Scopus (44) Google Scholar, 19Ingham P.W. Kim A.P. Genes Dev. 2001; 15: 3059-3087Crossref PubMed Scopus (2504) Google Scholar). Binding of Shh to ptc blocks the inhibitory effect of ptc on Smo. Once activated, Smo induces a complex series of intracellular reactions that target the Gli family of transcription factors (11Ruiz i Altaba A. Kim P. Dahmane N. Nat. Rev. Cancer. 2002; 2: 361-372Crossref PubMed Scopus (651) Google Scholar). Gli1 is the principal effector of Shh signaling in neural progenitor cells (20Ahn S. Kim A.L. Nature. 2005; 437: 894-897Crossref PubMed Scopus (565) Google Scholar). In the present study, using neural progenitor cells derived from the SVZ of adult mice, we tested the hypothesis that CEPO via Shh signaling promotes proliferation and differentiation of neural progenitor cells. All experimental procedures were approved by the Institutional Animal Care and Use Committee of Henry Ford Hospital. Male mice (C57BL6/J, 6-8 weeks) were purchased from The Jackson Laboratory (Bar Harbor, ME). EPOR null mice (ΔEPOR mice, C57BL6 background) were provided by Dr. Constance Tom Noguchi at NIDDK, National Institutes of Health (44Chen Z.Y. Kim P. Prchal J.T. Noguchi C.T. J. Biol. Chem. 2007; 282: 25875-25883Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar). Neurosphere Culture—SVZ neural progenitor cells were dissociated from normal (n = 10) and ΔEPOR mice (n = 10) as previously reported (21Chiasson B.J. Kim V. Morshead C.M. van der Kooy D. J. Neurosci. 1999; 19: 4462-4471Crossref PubMed Google Scholar, 22Morshead C.M. Kim B.A. Craig C.G. McBurney M.W. Staines W.A. Morassutti D. Weiss S. van der Kooy D. Neuron. 1994; 13: 1071-1082Abstract Full Text PDF PubMed Scopus (1223) Google Scholar). The cells were plated at a density of 2 × 104 cells/ml in growth medium. Growth medium contains Dulbecco’s modified Eagle’s-F-12 medium (Invitrogen), 20 ng/ml of epidermal growth factor (EGF) (R&D Systems, Minneapolis, MN), and 20 ng/ml of basic fibroblast growth factor (bFGF) (R&D Systems). Dulbecco’s modified Eagle’s-F-12 medium contains l-glutamine (2 mm), glucose (0.6%), putrescine (9.6 μg/ml), insulin (0.025 mg/ml), progesterone (6.3 ng/ml), apo-transferrin (0.1 mg/ml), and sodium selenite (5.2 ng/ml). The generated neurospheres (primary sphere) were passaged by mechanical dissociation and reseeded as single cells at a density of 20 cells/μl in bFGF- and EGF-containing media (passage 1 cells). Passage 1 cells were processed for various experiments in the present study. These cells were cultured in reduced growth medium containing 10 ng/ml of EGF and 10 ng/ml of bFGF. To analyze the formation of secondary neurospheres, passage 1 neurospheres were collected and digested with 0.05% trypsin-EDTA (Invitrogen) for 5 min at 37 °C. They were then gently triturated with a fire-narrowed Pasteur pipette, spun down at 400 rpm for 3 min, resuspended in the reduced growth medium, and plated at 1 × 104 cells/ml in each well of a 24-well plate (Corning). The number of neurospheres was counted at 7 days in vitro. To analyze cell proliferation, bromodeoxyuridine (BrdUrd) (20 μg/ml), the thymidine analog that is incorporated into the DNA of dividing cells during S-phase was added into the reduced growth medium 18 h prior to termination of incubation. BrdUrd-positive cells were measured. To dynamically dividing neural progenitor was R.L. Kim Y. Wang Y. Y. Zhang Z.G. Chopp M. J. Neurosci. 2007; PubMed Scopus Google Scholar). neurospheres were in a with and 37 which was on the of a with a with was used for of with a in were at for a of 20 h using a and R.L. Kim Y. Wang Y. Y. Zhang Z.G. Chopp M. J. Neurosci. 2007; PubMed Scopus Google Scholar). To analyze of neural progenitor neurospheres were dissociated as single cells. These cells × 104 were plated in Dulbecco’s modified Eagle’s-F-12 medium containing but the growth factors for 7 medium was to as the differentiation medium. was with various to of neural progenitor cells. To signaling pathway is in the effects of CEPO on neural progenitor cells were in the of CEPO with of 5 progenitor cell proliferation and differentiation and of the pathway were measured. In addition, neural progenitor cells were with mouse Gli1 and with CEPO ng/ml). To the effects of CEPO on Mash1 neural progenitor cells were in the of CEPO ng/ml). Mash1 and protein were measured. To the role of Mash1 on CEPO-enhanced neuronal neural progenitor cells were with mouse Mash1 and with CEPO ng/ml). differentiation and Mash1 were measured. To the effect of Shh on neural progenitor cell proliferation, and Mash1 neural progenitor cells were in the of recombinant mouse Shh with and Mash1 were to the Mash1 in using target The were and the were by which of a and a DNA a RNA the mouse was for the of target and the All were at the with for of Gli1 was purchased from progenitor cells were using the the The of was to and protein were and h was using the L. Kim Z. Zhang R. Z. Chopp M. 2004; 15: PubMed Scopus Google Scholar). RNA was from neural progenitor cell using the RNA was on an using provided by the as 2 min at 10 min at and then of at and 1 min at °C. of the was by of dissociation single that a single DNA was during were on to that were was tested in and from experiments were used for of expression using the Kim 2001; PubMed Scopus Google Scholar). The for were using Shh Gli1 Mash1 and and of cultured cells were as previously R.L. Kim Z.G. Zhang L. Chopp M. Neuroscience. 2001; 105: 33-41Crossref PubMed Scopus (534) Google Scholar, R. Kim L. Zhang L. J. Z. Zhang Z. Chopp M. 2003; PubMed Scopus Google Scholar). The were used in the present mouse mouse mouse protein 2 mouse protein and mouse cells were in for min at were blocked with normal for min at The cells were then with the and with secondary were with The number of and cells and cell number were and the of each cell was were to L. Kim Z.G. Zhang R.L. Wang Y. Y. Chopp M. J. 2006; 26: PubMed Scopus Google Scholar). from neural progenitor cells were for 10 and at × for 20 in the of cell was using a protein of proteins were on the proteins were to and the were with the Shh patched Gli1 and Mash1 secondary were used by of results was by with The density was using an and of analyze cells were using a via the was using a in that of the and of L. Kim Z.G. Zhang R.L. Wang Y. Y. Chopp M. J. 2006; 26: PubMed Scopus Google Scholar). cells were and were by to each of by was The were as of was as CEPO and of the effect of CEPO on adult neural progenitor we a that has been used for the of neural progenitor cells L. Kim Z. Zhang R. Z. Chopp M. 2004; 15: PubMed Scopus Google Scholar, B.A. Kim S. Science. PubMed Scopus Google Scholar, R. Kim Z. Wang L. Wang Y. A. Zhang L. Morshead C. Chopp M. J. 2004; 24: PubMed Scopus Google Scholar, B.A. Kim R.L. Nat. 2005; 2: PubMed Scopus Google Scholar, Y. Kim S. T. M. Shimazaki T. Weiss S. R. H. J. Neurosci. PubMed Google Scholar). neural progenitor cells from the SVZ of the adult mouse were plated at a density of 20 cells/μl on the in the reduced growth medium ng/ml of and 10 ng/ml of cells 7 days in vitro. The of cells in neurospheres were and of neural progenitor cells. that of cells in neurospheres were an of and that of the cells single cells dissociated from neurospheres were reseeded on × 104 in medium the growth factors for 7 cells into a of neurons, a of and a of However, a of neurons, were not days in which is with studies R. Kim K. H. N. M. Y. T. Stroke. 2004; 35: PubMed Scopus Google Scholar). These that SVZ cells the of and of neural progenitor cells A. Kim H. PubMed Scopus Google Scholar). We then CEPO promotes neural progenitor cell SVZ cells were cultured in the reduced growth medium containing CEPO ng/ml). of single SVZ cells to CEPO and in a in the number and of neurospheres and with that in the and We the number of the secondary neurospheres in which the neurospheres were dissociated and reseeded at 1 × 104 cells/ml in the reduced growth medium. with the the number of the secondary neurospheres derived from the was significantly increased = = in treatment with CEPO significantly increased the number of BrdUrd-positive cells and and the number of and cells and that increases of number and of neurospheres from an of progenitor cells but not by an of cell In that cells that of the were dividing in the CEPO during a experimental dividing cells were in the at the results demonstrate that CEPO neural progenitor cell We the effect of CEPO on neural progenitor cell differentiation. cells dissociated from neurospheres were reseeded on × 104 and with CEPO for 7 days in differentiation medium the growth with CEPO and significantly increased the number of and and = in CEPO 10 ng/ml = in cells and the number of not with the number in the and The of cells could a of cell To the that CEPO as a factor for neural progenitor we the number of cells and found that the number of cells in the CEPO = was not significantly from the number in the = These that CEPO enhances neural progenitor cell differentiation into To CEPO enhances was by the and number of from cell In the cells a short However, cells complex neural progenitor cells were with CEPO that treatment with CEPO significantly increased the number and of and that CEPO promotes We then the effect of CEPO on SVZ cells from ΔEPOR with EPO significantly increased the number of cells in neural progenitor cells derived from mice = 2 = in but not the number of cells in ΔEPOR neural progenitor cells = 3 = in which is with studies (6Wang L. Kim Z. Wang Y. Zhang R. Chopp M. Stroke. 2004; 35: 1732-1737Crossref PubMed Scopus (687) Google Scholar, L. Kim Z.G. Zhang R.L. Wang Y. Y. Chopp M. J. 2006; 26: PubMed Scopus Google Scholar). However, treatment of ΔEPOR neural progenitor cells with CEPO significantly increased the number of cells = with the number in the = These results that EPOR is not for CEPO-enhanced neurogenesis. The the of CEPO on Cell and signaling pathway is for neurogenesis in the adult rodent brain (20Ahn S. Kim A.L. Nature. 2005; 437: 894-897Crossref PubMed Scopus (565) Google Scholar, V. Kim D.A. Dahmane N. P. Y. A. A. i Altaba A. Development. 2005; PubMed Scopus Google Scholar). To CEPO induces signaling we expression of Shh, its and and that neural progenitor cells Shh, ptc, and Gli1 and treatment with CEPO significantly up-regulated and However, in the absence of EGF bFGF, CEPO to of Shh in ptc in and Gli1 in that CEPO the Shh pathway in with EGF and bFGF. CEPO in the of 20 ng/ml EGF and 20 ng/ml not significantly the number of neurospheres = with the number in = These with that EGF at has a effect with Shh in the of proliferation of SVZ neural progenitor cells K. Kim B.K. Gage F.H. Schaffer D.V. Nat. Neurosci. 2003; 6: 21-27Crossref PubMed Scopus (684) Google Scholar). To Shh signaling is for CEPO to proliferation and differentiation of neural progenitor we neural progenitor cells with CEPO in the of the Shh of abolished the of CEPO to ptc and Gli1 expression but not CEPO-induced Shh expression and In addition, significantly reduced the of BrdUrd-positive and and and cells with the in the blocked CEPO-induced and These that the pathway regulates CEPO-enhanced neural progenitor cell proliferation and differentiation. To the effect of Shh on neural progenitor cell proliferation and we the number of and cells treatment with a of 5 ng/ml EGF, Shh has a effect on neural progenitor cells K. Kim B.K. Gage F.H. Schaffer D.V. Nat. Neurosci. 2003; 6: 21-27Crossref PubMed Scopus (684) Google Scholar). SVZ neural progenitor cells were cultured in the growth medium containing EGF with for 7 treatment to a of BrdUrd-positive cells from in the to In addition, treatment increased the number of cells in a with increases at a of ng/ml = for ng/ml and = for ng/ml 2 = in neural progenitor cells were cultured in the differentiation medium. These that Shh induces neural progenitor cell proliferation and which is with results R. Kim S. M. S. A. T. J.A. H. McMahon A.P. G. Neuron. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). CEPO Mash1 in in a a transcription promotes neuronal differentiation C.M. Kim R. O. S. C. J. M. A. J. 2004; PubMed Scopus Google Scholar). that cells were that cells neural progenitor cells To Mash1 is a target for CEPO-induced neuronal we Mash1 of neural progenitor cells with CEPO significantly increased Mash1 and and protein and at 1 and 7 days in with in the We then tested an in Mash1 regulates CEPO-promoted neuronal differentiation. We mouse Mash1 and that but not significantly Mash1 and protein in neural progenitor cells and h that the in endogenous Mash1 In addition, Mash1 and treatment with CEPO for h not significantly the number of cells in neural progenitor cells by with the number in neural progenitor cells by neural progenitor cells and Blockage of endogenous Mash1 by also significantly with CEPO and CEPO with and These that Mash1 mediates CEPO-promoted neuronal differentiation. In to CEPO increased in transcription factor L. Kim Z.G. Zhang R.L. Wang Y. Y. Chopp M. J. 2006; 26: PubMed Scopus Google Scholar). of Mash1 by Shh, and the pathway M. Kim O. L. van S. Dev. Biol. 2005; PubMed Scopus Google Scholar, L. Kim L. D.J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar, Kim S. J. J.J. M.F. Proc. Natl. Acad. Sci. U. S. A. 2002; PubMed Scopus Google Scholar, J.D. Kim N. J. T. G. Development. 2001; PubMed Google Scholar). that CEPO not of and in neural progenitor cells with in the the signaling pathway could a in CEPO-promoted neuronal differentiation. Shh is the CEPO and Mash1 then we that of Shh signaling Mash1 expression and CEPO-promoted neuronal differentiation. and that of neural progenitor cells with CEPO in the of significantly Mash1 and protein and cells and In addition, of the effector of Shh with significantly Mash1 expression in CEPO To Shh signaling is to Mash1 neural progenitor cells were with and for Mash1 with to a in Mash1 and Gli1 expression with the and that cells were These results that Shh signaling is to Mash1 expression in the absence of Shh as an in neuronal differentiation CEPO and The present that CEPO promotes adult neural progenitor cell proliferation and differentiation. Blockage of Shh with the Shh abolished the effect of CEPO on neural progenitor of Gli1 with of endogenous Mash1 with CEPO-promoted neuronal differentiation. These that CEPO enhances in neurogenesis which is by the Shh signaling pathway and that Mash1 is a downstream target of the Shh signaling pathway and regulates CEPO-enhanced neuronal differentiation. Using a in we show that the treatment of neural progenitor cells derived from the SVZ of adult mouse with CEPO increased the of cells and but not These demonstrate for the that in to its neuroprotective CEPO neural progenitor cell proliferation and promotes neural progenitor cell differentiation into The Shh signaling pathway neurogenesis in the adult brain K. Kim B.K. Gage F.H. Schaffer D.V. Nat. Neurosci. 2003; 6: 21-27Crossref PubMed Scopus (684) Google Scholar, V. Kim D.A. Dahmane N. P. Y. A. A. i Altaba A. Development. 2005; PubMed Scopus Google Scholar). Shh as a in with EGF to proliferation of neural cells in adult SVZ D. Kim P. J.M. A. M. J. Neurosci. 2002; PubMed Scopus Google Scholar). Shh increases neurogenesis in the SVZ, of Shh signaling in adult mice expression of Gli1 and SVZ cell proliferation K. Kim B.K. Gage F.H. Schaffer D.V. Nat. Neurosci. 2003; 6: 21-27Crossref PubMed Scopus (684) Google Scholar). Using mice, in which an is from and (20Ahn S. Kim A.L. Nature. 2005; 437: 894-897Crossref PubMed Scopus (565) Google in that Shh regulates neural and progenitor cells in the SVZ and the zone of adult we show that CEPO the Shh signaling pathway with of EGF and but CEPO on its is not to the Shh pathway The effects of CEPO on the Shh pathway as of neural progenitor cells with CEPO and cyclopamine, a pharmacological of the Shh abolished CEPO-induced Gli1 expression and in of CEPO-induced neural progenitor cell proliferation and neuronal differentiation. These that CEPO in with EGF and the Shh signaling pathway and the Shh pathway mediates the effect of CEPO on neural progenitor cells. transcription Mash1 and neural progenitor cell differentiation into L. Kim Z.G. Zhang R.L. Wang Y. Y. Chopp M. J. 2006; 26: PubMed Scopus Google Scholar, Kim L. P. D.J. J. Neurosci. PubMed Google Scholar). We and that 1 and neuronal differentiation T. Kim S.T. Shimazaki T. Weiss S. J. Neurosci. 2001; 21: 9733-9743Crossref PubMed Google Scholar, L. Kim Z.G. Zhang R.L. Wang Y. Y. Chopp M. J. 2006; 26: PubMed Scopus Google Scholar). The present that CEPO promoted neural progenitor cell differentiation into neurons, but not which was associated with up-regulation of Mash1 and of endogenous Mash1 with CEPO-promoted neuronal differentiation. In addition, of the Shh signaling pathway Gli1 and Mash1 we that CEPO the Shh signaling which Mash1 via of in neuronal differentiation. transcription factors Mash1 and downstream of the Shh signaling In to the Shh signaling and neurogenesis M. Kim O. L. van S. Dev. Biol. 2005; PubMed Scopus Google Scholar, L. Kim L. D.J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). studies on pathways on CEPO-induced neurogenesis. CEPO mediates in the by to a of the classical EPOR and the receptor (10Leist M. Kim P. Grasso G. Bianchi R. Villa P. Fratelli M. Savino C. Bianchi M. Nielsen J. Gerwien J. Kallunki P. Larsen A.K. Helboe L. Christensen S. Pedersen L.O. Nielsen M. Torup L. Sager T. Sfacteria A. Erbayraktar S. Erbayraktar Z. Gokmen N. Yilmaz O. CeramiHand C. Xie Q.W. Coleman T. Cerami A. Brines M. Science. 2004; 305: 239-242Crossref PubMed Scopus (710) Google Scholar, M. Kim G. Sfacteria A. P. Fratelli M. R. Xie Q.W. J. D. D. C. Coleman T. Cerami A. Proc. Natl. Acad. Sci. U. S. A. 2004; PubMed Scopus Google Scholar). from ΔEPOR mice that EPOR is not for the effect of CEPO on adult neural progenitor cells. studies of the of CEPO in neural progenitor cells In the present that the Shh signaling pathway mediates CEPO-enhanced neural progenitor cell proliferation and differentiation.