Interferon γ Induces Neurite Outgrowth by Up-regulation of p35 Neuron-specific Cyclin-dependent Kinase 5 Activator via Activation of ERK1/2 Pathway

Interferon gamma (IFN-γ) is a cytokine predominantly involved in antiproliferative and antiviral responses, immune surveillance, and tumor suppression. However, it has been shown that IFN-γ is also involved in central nervous system development. Here we studied the underlying mechanism for IFN-γ-induced neuronal differentiation using the human neuroblastoma Paju cell line. Our results indicate that IFN-γ treatment led to neurite outgrowth followed by growth arrest in the G1 phase of the cell cycle. IFN-γ induced ERK1/2 phosphorylation and subsequently the transcription factor early gene response 1, which in turn up-regulated p35 expression and increased cyclin-dependent kinase 5 (Cdk5) activity. IFN-γ-induced neurite outgrowth was abolished by the treatment of MEK1/2 kinase inhibitors, such as U0126 and PD98059, which inhibit the ERK1/2 activation and subsequently prevent the up-regulation of p35 expression and Cdk5 activity. In agreement with the role of p35-Cdk5 in neuronal differentiation, small interfering RNA targeting Cdk5 abrogate the IFN-γ-induced neurite outgrowth. Together, these results demonstrate for the first time that IFN-γ-triggered neuronal differentiation mediated through the up-regulation of p35-associated Cdk5 depends on the activation of the ERK1/2 pathway. Therefore, the present study suggests that IFN-γ is not only involved in tumorigenicity but also involved in neurogenesis by regulating cell proliferation and differentiation. Interferon gamma (IFN-γ) is a cytokine predominantly involved in antiproliferative and antiviral responses, immune surveillance, and tumor suppression. However, it has been shown that IFN-γ is also involved in central nervous system development. Here we studied the underlying mechanism for IFN-γ-induced neuronal differentiation using the human neuroblastoma Paju cell line. Our results indicate that IFN-γ treatment led to neurite outgrowth followed by growth arrest in the G1 phase of the cell cycle. IFN-γ induced ERK1/2 phosphorylation and subsequently the transcription factor early gene response 1, which in turn up-regulated p35 expression and increased cyclin-dependent kinase 5 (Cdk5) activity. IFN-γ-induced neurite outgrowth was abolished by the treatment of MEK1/2 kinase inhibitors, such as U0126 and PD98059, which inhibit the ERK1/2 activation and subsequently prevent the up-regulation of p35 expression and Cdk5 activity. In agreement with the role of p35-Cdk5 in neuronal differentiation, small interfering RNA targeting Cdk5 abrogate the IFN-γ-induced neurite outgrowth. Together, these results demonstrate for the first time that IFN-γ-triggered neuronal differentiation mediated through the up-regulation of p35-associated Cdk5 depends on the activation of the ERK1/2 pathway. Therefore, the present study suggests that IFN-γ is not only involved in tumorigenicity but also involved in neurogenesis by regulating cell proliferation and differentiation. Interferon gamma (IFN-γ) 1The abbreviations used are: IFN, interferon; Cdk5, cyclin-dependent kinase 5; dn-Cdk5, dominant-negative Cdk5; Egr1, early gene response 1; ERK, extracellular signal-regulated kinase; mAb, monoclonal antibody; siRNA, small interfering RNA; TNF, tumor necrosis factor; TRAIL, TNFα-related apoptosis-inducing ligand; NGF, nerve growth factor; HRP, horseradish peroxidase; PI3K, phosphatidylinositol 3-kinase; MOPS, 4-morpholinepropanesulfonic acid; PBS, phosphate-buffered saline; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase. is a cytokine with pleiotropic actions that is profoundly involved in antiproliferative and antiviral responses, immune surveillance, and tumor suppression (1.Stark G.R. Kerr I.M. Williams B.R. Silverman R.H. Schreiber R.D. Annu. Rev. Biochem. 1998; 67: 227-264Crossref PubMed Scopus (3380) Google Scholar). However, it has also been reported that IFN-γ plays a role in embryonic neural differentiation and neuronal survival. For example, IFN-γ enhances nerve growth factor (NGF)-induced neurite outgrowth (2.Improta T. Salvatore A.M. Di Luzio A. Romeo G. Coccia E.M. Calissano P. Exp. Cell Res. 1988; 179: 1-9Crossref PubMed Scopus (42) Google Scholar), increases neuronal numbers in embryonic cortical and hippocampal cultures (3.Jonakait G.M. Wei R. Sheng Z.L. Hart R.P. Ni L. Neuron. 1994; 12: 1149-1159Abstract Full Text PDF PubMed Scopus (61) Google Scholar), and promotes cholinergic differentiation of septal nucleus and basal forebrain neurons (4.Barish M.E. Mansdorf N.B. Raissdana S.S. Dev. Biol. 1991; 144: 412-423Crossref PubMed Scopus (57) Google Scholar). IFN-γ also appears to affect the differentiation of neural stem cells (5.Wong G. Goldshmit Y. Turnley A.M. Exp. Neurol. 2004; 187: 171-177Crossref PubMed Scopus (162) Google Scholar). While in combination with tumor necrosis factor (TNF) superfamily death ligands, such as TNFα, Fas ligand/CD95 ligand, and TNF-related apoptosis-inducing ligand (TRAIL), IFN-γ can induce apoptosis in both human brain cells and neuroblastoma cells (6.Saas P. Boucraut J. Quiquerez A.L. Schnuriger V. Perrin G. Desplat-Jego S. Bernard D. Walker P.R. Dietrich P.Y. J. Immunol. 1999; 162: 2326-2333PubMed Google Scholar, 7.Fulda S. Debatin K.M. Oncogene. 2002; 21: 2295-2308Crossref PubMed Scopus (231) Google Scholar, 8.Pouly S. Becher B. Blain M. Antel J.P. J. Neuropathol. Exp. Neurol. 2000; 59: 280-286Crossref PubMed Scopus (107) Google Scholar). However, the potentiation mechanisms of IFN-γ on death receptor-mediated apoptosis are not fully understood, and no study has yet illustrated the molecular mechanisms of IFN-γ-induced neuronal differentiation. To elucidate the effects of IFN-γ on cell survival and death and to further discern the signaling pathways that are responsible for IFN-γ-induced neuronal differentiation, we conducted a series of experiments using Paju cells, a neuroblastoma cell line that was established from human neural crest-derived tumor (9.Zhang K.Z. Westberg J.A. Holtta E. Andersson L.C. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 4504-4508Crossref PubMed Scopus (132) Google Scholar). As observed in some other neuroblastomas (10.Encinas M. Iglesias M. Liu Y. Wang H. Muhaisen A. Cena V. Gallego C. Comella J.X. J. Neurochem. 2000; 75: 991-1003Crossref PubMed Scopus (584) Google Scholar, 11.Kaplan D.R. Matsumoto K. Lucarelli E. Thiele C.J. Neuron. 1993; 11: 321-331Abstract Full Text PDF PubMed Scopus (313) Google Scholar), Paju cells also can be differentiated into neuronal phenotype by extracellular stimuli (12.Westberg J.A. Zhang K.Z. Andersson L.C. FASEB J. 1999; 13: 1621-1626Crossref PubMed Scopus (23) Google Scholar). Cyclin-dependent kinase 5 (Cdk5) is a small serine/threonine kinase that has been implicated in neuronal migration, differentiation, and survival during central nervous system development. Cdk5 kinase activity has been shown to be dependent upon p35, a neuron-specific Cdk5 activator (13.Tsai L.H. Delalle I. Caviness Jr., V.S. Chae T. Harlow E. Nature. 1994; 371: 419-423Crossref PubMed Scopus (810) Google Scholar). Expression of p35 and Cdk5 kinase activity have been shown to increase during in vitro neuronal differentiation, and mutant mice lacking either Cdk5 or p35 exhibit defective neuronal migration and cortical lamination in the developing mouse brain (14.Ohshima T. Ward J.M. Huh C.G. Longenecker G. Veeranna, Pant H.C. Brady R.O. Martin L.J. Kulkarni A.B. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 11173-11178Crossref PubMed Scopus (808) Google Scholar, 15.Chae T. Kwon Y.T. Bronson R. Dikkes P. Li E. Tsai L.H. Neuron. 1997; 18: 29-42Abstract Full Text Full Text PDF PubMed Scopus (664) Google Scholar). Increased p35 expression and Cdk5 activity are essential for NGF-induced neurite outgrowth in rat pheochromocytoma PC12 cells and are also required for Fas-mediated neuronal differentiation in vivo (16.Harada T. Morooka T. Ogawa S. Nishida E. Nat. Cell Biol. 2001; 3: 453-459Crossref PubMed Scopus (281) Google Scholar, 17.Desbarats J. Birge R.B. Mimouni-Rongy M. Weinstein D.E. Palerme J.S. Newell M.K. Nat. Cell Biol. 2003; 5: 118-125Crossref PubMed Scopus (268) Google Scholar). We found that treatment with IFN-γ also resulted in neuritic outgrowth of Paju cells and generated nearly pure populations of human neuron-like cells, which provided a model to explore the molecular mechanisms of IFN-γ-induced neuronal differentiation. Treatment of Paju cells with IFN-γ significantly increased the expression of p35 and Cdk5 activity through the activation of the extracellular-signal regulated kinase1/2 (ERK1/2) and the early gene response 1 (Egr1) pathway. In addition, co-treatment with Fas monoclonal antibody (mAb), but not TRAIL, enhanced IFN-γ-induced neuronal differentiation. In this study, we demonstrated that the signaling pathway initiated by IFN-γ occurred through the ERK-Egr1-p35-Cdk5 pathway. Antibodies and Materials—Primary monoclonal antibodies included anti-human Fas mAb (CH-11) and caspase-8 (Medical and Biological Laboratories, Nagoya, Japan), and DNA fragmentation factor 45 (DFF45; StressGen, Victoria, BC, Canada). Primary polyclonal rabbit antibodies included anti-human caspase-3 and ERK1/2 (StressGen), p-ERK and Egr1 (Cell Signaling Technology, Beverly, MA), p35, Cdk5, and actin (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Secondary antibodies included HRP-conjugated goat anti-mouse IgG2b and IgG1 antibodies (Southern Biotech, Birmingham, AL), and HRP-conjugated goat anti-rabbit antibody (Jackson ImmunoResearch Laboratories, West Grove, PA). Recombinant human TRAIL (amino acids 114–281) was obtained from PeproTech, Inc. (Rocky Hill, NJ). RPMI 1640 medium was purchased from Invitrogen, and [γ-32P]ATP (3,000 Ci/mmol) was purchased from PerkinElmer Life Sciences. PD98059, U0126, and LY294002 were purchased from New England Biolabs. Small interfering RNA (siRNA) against Cdk5 was purchased from Upstate Biotechnology (Dharmacon). Protein quantification reagents were obtained from Bio-Rad Laboratories, and enhanced chemiluminescence reagents for Western blot analysis were purchased from Amersham Biosciences. Protease inhibitor mixtures, IFN-γ, roscovitine, and other chemicals were obtained from Sigma. Wild-type and dominant-negative Cdk5 cDNAs in pcDNA3.1 vector were kindly provided by Dr. Li-Huei Tsai (Harvard Medical School, Cambridge, MA). Cell Culture—Paju, a human neural crest-derived tumor cell line, was a gift from Dr. L. Andersson (Haartman Institute, University of Helsinki, Helsinki, Finland). The cells were grown in RPMI 1640 medium supplemented with penicillin (20 units/ml), streptomycin (20 μg/ml), and 10% (v/v) heat-inactivated fetal bovine serum. Cells were maintained at 37 °C in a saturated humidity atmosphere containing 95% air and 5% CO2. Death Receptor Analysis—Expression of Fas, DR4, and DR5 receptors was measured by flow cytometry. Briefly, 0.1 μg of phycoerythrin-conjugated anti-human Fas, DR4, and DR5 (mouse IgG1) or mouse IgG1 (negative control) was added to 1 × 106 cells in 200 μl of PBS containing 2% fetal bovine serum and 0.02% sodium azide. Cells were incubated for 1 h in the dark at 4 °C, washed with PBS, and suspended in 500 μl of PBS. Samples were analyzed using a BD Biosciences FACScan (Mountain View, CA), and the data were processed using Cell Quest software (BD Biosciences). Cell Viability, Apoptosis, and Cell Cycle Analysis—To examine cell viability, apoptotic death, and cell cycle progression, cells (15 × 103/cm2) were plated in 6-well plates or 100-mm dishes, and treatments were started at h Cells were at time and cells were using the C. C. J. Biol. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). The of cells in was also measured with in plates G.M. 2004; PubMed Scopus Google Scholar). cells were analyzed by flow for 1997; PubMed Scopus Google Scholar). Cell cycle analysis was by the DNA with Briefly, cells grown in a were by washed with PBS, and by in and in at 4 The washed was using a containing 200 and for at °C in the The cell cycle was analyzed with a flow using software Cell and were in in PBS at for washed with PBS containing and for with PBS containing bovine serum and The cells were incubated with monoclonal rabbit anti-human in PBS containing bovine serum and washed in PBS with and incubated at for with anti-rabbit monoclonal antibody in PBS with the cells were washed with PBS and with The cells were observed with a Paju cell differentiation was by for neurite outgrowth. Cells or with a of the of the cell were as Western grown in a were in (20 10% 1 and inhibitor at × for at 4 °C, were of protein from were by and to The were with in (20 500 and and incubated at 4 °C with the antibody in the The were washed in and incubated with the antibodies in anti-mouse and anti-mouse or anti-rabbit to the antibody The were washed and by of and cells were grown in 6-well plates at a of × The cells were with 5 μg of or dominant-negative Cdk5 using to the of targeting Cdk5 containing was using from to the Cells were to for h and were as Cdk5 were in containing sodium 1 1 and inhibitor The were at × for For Cdk5 the was incubated with and for 4 h at 4 The Cdk5 were in with 1 1 and μg of and 5 of [γ-32P]ATP were for at °C, the was by μl of the on that were washed with followed by with was measured with a In some in vitro Cdk5 kinase activity was also analyzed by Western Paju Cells to Death study demonstrated that IFN-γ some neuroblastoma cells to death receptor-mediated apoptosis S. Debatin K.M. Oncogene. 2002; 21: 2295-2308Crossref PubMed Scopus (231) Google Scholar). To IFN-γ enhances apoptosis induced by Fas and TRAIL, we first analyzed the expression of death receptors in Paju analysis that Paju cells cell Fas and DR5 but to expression of the cells to IFN-γ for h significantly increased Fas no increase in and DR5 expression was we Fas and TRAIL can induce apoptosis in Paju As we reported C. C. J. Biol. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar, S. I. M. M. M.E. J. PubMed Scopus Google Scholar), cells are to apoptosis induced by Fas mAb and TRAIL However, cultures of Paju cells not of apoptosis treatment with Fas mAb or TRAIL In the or of caspase-8 expression is with the of neuroblastoma cells to death receptor-mediated has been that IFN-γ cells to apoptosis by the expression of In of we to examine IFN-γ can Paju cells to Western blot analysis that of caspase-8 expression in Paju cells and of the cells to IFN-γ resulted in increase of caspase-8 at this expression was for at h the treatment However, no of caspase-8 were The expression of of and DNA fragmentation factor 45 was not the treatment with no of the caspase-3 and DNA fragmentation factor 45 were the To further IFN-γ Paju cells to death cell death was measured treatment with IFN-γ in combination with either Fas mAb or Cells were with IFN-γ for h the of Fas mAb or TRAIL, and cell was by the h Fas mAb or TRAIL either or in combination with IFN-γ to induce apoptosis in Paju cells The activation of caspase-8 and was also analyzed by Western Treatment with IFN-γ or in combination with these increased caspase-8 expression However, these treatments to of caspase-8 and results indicate that IFN-γ is of caspase-8 expression but not to induce that a of be involved in the of neuroblastoma cells to IFN-γ in Paju treatment with IFN-γ induced neuronal differentiation in Paju cells as by neurite outgrowth. treatment with IFN-γ, neurite to on 1 The increases in and of the were 5 IFN-γ were by the neuritic and the cells differentiated into nearly pure populations of human neuron-like that these neuron-like cells the of neurons In to IFN-γ, Fas mAb TRAIL treatment enhanced the neurite outgrowth in Paju To IFN-γ treatment the from proliferation to neuronal differentiation, Paju cells were to IFN-γ for of and cell proliferation was by in cell IFN-γ treatment significantly cell and this was on 1 Treatment with Fas mAb also cell but was Fas growth In treatment with TRAIL no effects on cell The actions of IFN-γ and Fas mAb on cell proliferation were also with cell To IFN-γ mediated of neuronal differentiation from the of IFN-γ on cell cycle of Paju cells was from flow with that cell cycle arrest occurred at G1 these results indicate that IFN-γ-induced neuronal differentiation is initiated in the G1 phase of the cell and differentiation as shown by neurite outgrowth a and IFN-γ-induced p35-associated Cdk5 has been shown to be essential for NGF-induced neurite outgrowth in PC12 cells and neuronal differentiation in (16.Harada T. Morooka T. Ogawa S. Nishida E. Nat. Cell Biol. 2001; 3: 453-459Crossref PubMed Scopus (281) Google Scholar, 17.Desbarats J. Birge R.B. Mimouni-Rongy M. Weinstein D.E. Palerme J.S. Newell M.K. Nat. Cell Biol. 2003; 5: 118-125Crossref PubMed Scopus (268) Google Scholar). We plays a role in IFN-γ-induced neuronal differentiation of Paju We first the expression of p35 and Cdk5 in Paju treatment with IFN-γ, the of p35 increased at 1 h and at 5 h of IFN-γ treatment The of p35 and to basal on 1 IFN-γ However, the of Cdk5 not significantly the IFN-γ we the of Cdk5 activity IFN-γ was with and the activity of Cdk5 was using as a The activity of Cdk5 increased IFN-γ and this the of is in agreement with that Cdk5 activity is dependent on p35 T. Kwon Y.T. Bronson R. Dikkes P. Li E. Tsai L.H. Neuron. 1997; 18: 29-42Abstract Full Text Full Text PDF PubMed Scopus (664) Google Scholar, T. Morooka T. Ogawa S. Nishida E. Nat. Cell Biol. 2001; 3: 453-459Crossref PubMed Scopus (281) Google Scholar, M. H. Kwon Y.T. Tsai L.H. Dev. 1996; PubMed Scopus Google Scholar). We studied Cdk5 is involved in cell differentiation. series of experiments were we the of dominant-negative Cdk5 gene into Paju cells IFN-γ-induced neurite outgrowth. Treatment of the Paju cells with IFN-γ at h Cdk5 gene resulted in increase in neurite IFN-γ to induce neurite outgrowth in cells and In the of we studied of Cdk5 expression can cell differentiation. Cdk5 expression was using targeting Cdk5 of significantly Cdk5 protein expression and kinase activity IFN-γ-induced neurite outgrowth was also Cdk5 expression was using In addition, we treatment of IFN-γ in combination with either Fas mAb or TRAIL can cell differentiation. from this study that co-treatment with Fas mAb, but not TRAIL, enhanced IFN-γ-induced cell differentiation as by the of neurite Western analysis also p35 expression was the treatment and Fas IFN-γ not Treatment with Fas mAb or TRAIL not increase in p35 these treatments also not neurite outgrowth. Therefore, we that increased p35-Cdk5 kinase activity is required for IFN-γ-induced neuronal differentiation in Paju Signaling in IFN-γ-induced a study, activation of ERK1/2 was shown to be and for NGF-induced neurite up-regulation of p35 by ERK1/2 activation is essential of this pathway (16.Harada T. Morooka T. Ogawa S. Nishida E. Nat. Cell Biol. 2001; 3: 453-459Crossref PubMed Scopus (281) Google Scholar). it has also been shown that up-regulation of p35 is mediated by transcription factor To ERK1/2 activation also IFN-γ-induced cell differentiation in Paju cells, we IFN-γ treatment in the present system resulted in the activation of ERK1/2 and Egr1 pathway. ERK1/2 are through phosphorylation by kinase A. PubMed Scopus Google Scholar). Western blot that IFN-γ phosphorylation of ERK1/2 at and at 1 h was in Paju cells The phosphorylation of ERK1/2 to at 5 it was the Egr1 not be in the However, a increase was observed 1 h IFN-γ a of Egr1 was also at and 5 h treatment We studied ERK1/2 activation mediated IFN-γ-induced neuronal differentiation in Paju cells using a inhibitor of kinase 1 U0126 a inhibitor of and a inhibitor of mitogen-activated protein kinase. The Paju cells were with IFN-γ for 5 h in the or of and the cells were for Western blot analysis Treatment with U0126 or ERK1/2 phosphorylation and p35 expression treatment not have a on the suppression of ERK1/2 phosphorylation or p35 expression The in the p35 upon of ERK1/2 activity with the in Cdk5 activity in Paju cells treatment with ERK1/2 significantly the neurite in Paju cells We also Cdk5 pathway H. Y. S. Zhang L. Kulkarni A.B. Pant H.C. J. Biol. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). activation through phosphorylation K. A. D.R. Full Text PDF PubMed Scopus Google Scholar). In Paju cells, activation was either with or IFN-γ treatment as in phosphorylation Treatment with a in a and with a of LY294002 to prevent IFN-γ-induced neurite outgrowth and p35 up-regulation these results that the signaling pathway is required for neuronal differentiation induced by IFN-γ in Paju superfamily death and and IFN-γ have been shown to be involved in apoptotic death of the brain cells during and central nervous system J. P. G. B. R. B. J. J. 2002; PubMed Google Scholar, J.A. C.G. J.M. R.P. Exp. Neurol. 2001; PubMed Scopus Google Scholar). it has been shown that these also a role in neurogenesis (5.Wong G. Goldshmit Y. Turnley A.M. Exp. Neurol. 2004; 187: 171-177Crossref PubMed Scopus (162) Google Scholar, S. Exp. Neurol. 1997; PubMed Scopus Google Scholar, A. 2002; 67: PubMed Scopus Google Scholar). Fas and TRAIL have to induce these also cell proliferation and differentiation C. A.M. J. 2003; PubMed Scopus Google Scholar, Nat. Rev. Immunol. 2003; 3: PubMed Scopus Google Scholar), which that the role of Fas and TRAIL in the central nervous system is the of Here we have demonstrated the effects of these on apoptosis in Paju Treatment of Paju cells with Fas mAb or TRAIL to induce As shown as as in S. E. B. Debatin K.M. Oncogene. 2001; PubMed Scopus Google Scholar, M.E. M. L.H. U. Thiele C.J. Res. 2003; Google Scholar), the of Fas and TRAIL to induce apoptosis be to the expression of caspase-8 in Paju cells IFN-γ has been shown to death expression and increase caspase-8 expression the to death receptor-mediated apoptosis S. Debatin K.M. Oncogene. 2002; 21: 2295-2308Crossref PubMed Scopus (231) Google Scholar). IFN-γ increased Fas not and expression and caspase-8 expression in Paju cells 1, a and the of Paju cells to apoptosis was not by co-treatment of IFN-γ in combination with either Fas mAb or TRAIL 1, and apoptosis to be cell in the nervous and mechanisms be involved in the of Paju cells to apoptotic In of the mechanism of Paju cells, we found that treatment with IFN-γ induced the cells to into a neuronal has been shown that the cells are to apoptosis the cells A. V. M. C. S. A. J. Biochem. 2001; PubMed Scopus Google Scholar, A. A. Res. Google Scholar), of the activation of ERK, and Paju cells activation of during IFN-γ and treatment with the inhibitor LY294002 in a Therefore, Paju cells were to which be the of activation of pathway. we cell cycle cell cycle arrest is required for neuronal differentiation, which is regulated by such as and that inhibit phosphorylation of the protein Full Text PDF PubMed Scopus Google Scholar). IFN-γ treatment led to growth arrest in the G1 phase of the cell by a in proliferation be with of the neurite outgrowth results are in agreement with that in cell the of cells to is in the G1 phase of the cell cycle and that the from proliferation to differentiation is at cell cycle L. Dev. 2001; 11: PubMed Scopus Google Scholar). We subsequently the pathway responsible for the IFN-γ-induced neuronal differentiation in Paju from the present study indicate that treatment with IFN-γ up-regulated p35 the neuron-specific activator for Cdk5, and p35-associated Cdk5 activation is required and for the IFN-γ-induced neuronal differentiation in Paju As shown in of Cdk5 activity by either of or dominant-negative vector IFN-γ-induced neurite outgrowth. We further studied pathway is for IFN-γ-induced from 4 demonstrated that treatment of U0126 or not only p35 expression and Cdk5 activity but also IFN-γ-induced neurite ERK1/2 activation resulted in the of Egr1, a of a transcription factor that to the of p35 and p35 expression (16.Harada T. Morooka T. Ogawa S. Nishida E. Nat. Cell Biol. 2001; 3: 453-459Crossref PubMed Scopus (281) Google Scholar). the present results that p35-Cdk5 activity is implicated in neurogenesis but from the role of In the activation of and up-regulation of p35 have been observed in NGF-induced neuronal differentiation in PC12 cells and also in neuronal differentiation in vivo (16.Harada T. Morooka T. Ogawa S. Nishida E. Nat. Cell Biol. 2001; 3: 453-459Crossref PubMed Scopus (281) Google Scholar, 17.Desbarats J. Birge R.B. Mimouni-Rongy M. Weinstein D.E. Palerme J.S. Newell M.K. Nat. Cell Biol. 2003; 5: 118-125Crossref PubMed Scopus (268) Google Scholar, P. Veeranna, M. P. Kulkarni A.B. Pant H.C. J. Biol. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). are also up-regulated during neuronal and Therefore, these with the present the that IFN-γ-induced p35-Cdk5 activity be implicated in neural in the nervous We L. and Dr. for and

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