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A Novel, Nerve Growth Factor-activated Pathway Involving Nitric Oxide, p53, and p21WAF1 Regulates Neuronal Differentiation of PC12 Cells

1997; Elsevier BV; Volume: 272; Issue: 38 Linguagem: Inglês

10.1074/jbc.272.38.24002

ISSN

1083-351X

Autores

Wojciech Poluha, Christopher M. Schonhoff, Kimberly S. Harrington, Mahesh B. Lachyankar, Nancy E. Crosbie, Dylan A. Bulseco, Alonzo H. Ross,

Tópico(s)

Cancer, Stress, Anesthesia, and Immune Response

Resumo

During development, neuronal differentiation is closely coupled with cessation of proliferation. We use nerve growth factor (NGF)-induced differentiation of PC12 pheochromocytoma cells as a model and find a novel signal transduction pathway that blocks cell proliferation. Treatment of PC12 cells with NGF leads to induction of nitric oxide synthase (NOS) (Peunova, N., and Enikolopov, G. (1995)Nature 375, 68–73). The resulting nitric oxide (NO) acts as a second messenger, activating the p21WAF1 promoter and inducing expression of p21WAF1 cyclin-dependent kinase inhibitor. NO activates the p21WAF1 promoter by p53-dependent and p53-independent mechanisms. Blocking production of NO with an inhibitor of NOS reduces accumulation of p53, activation of the p21WAF1 promoter, expression of neuronal markers, and neurite extension. To determine whether p21WAF1 is required for neurite extension, we prepared a PC12 line with an inducible p21WAF1 expression vector. Blocking NOS with an inhibitor decreases neurite extension, but induction of p21WAF1 with isopropyl-1-thio-β-d-galactopyranoside restored this response. Levels of p21WAF1 induced by isopropyl-1-thio-β-d-galactopyranoside were similar to those induced by NGF. Therefore, we have identified a signal transduction pathway that is activated by NGF; proceeds through NOS, p53, and p21WAF1 to block cell proliferation; and is required for neuronal differentiation by PC12 cells. During development, neuronal differentiation is closely coupled with cessation of proliferation. We use nerve growth factor (NGF)-induced differentiation of PC12 pheochromocytoma cells as a model and find a novel signal transduction pathway that blocks cell proliferation. Treatment of PC12 cells with NGF leads to induction of nitric oxide synthase (NOS) (Peunova, N., and Enikolopov, G. (1995)Nature 375, 68–73). The resulting nitric oxide (NO) acts as a second messenger, activating the p21WAF1 promoter and inducing expression of p21WAF1 cyclin-dependent kinase inhibitor. NO activates the p21WAF1 promoter by p53-dependent and p53-independent mechanisms. Blocking production of NO with an inhibitor of NOS reduces accumulation of p53, activation of the p21WAF1 promoter, expression of neuronal markers, and neurite extension. To determine whether p21WAF1 is required for neurite extension, we prepared a PC12 line with an inducible p21WAF1 expression vector. Blocking NOS with an inhibitor decreases neurite extension, but induction of p21WAF1 with isopropyl-1-thio-β-d-galactopyranoside restored this response. Levels of p21WAF1 induced by isopropyl-1-thio-β-d-galactopyranoside were similar to those induced by NGF. Therefore, we have identified a signal transduction pathway that is activated by NGF; proceeds through NOS, p53, and p21WAF1 to block cell proliferation; and is required for neuronal differentiation by PC12 cells. Neuronal differentiation is closely linked to cessation of cell proliferation, but how these states are connected remains a major unanswered question. Nerve growth factor (NGF) 1The abbreviations used are: NGF, nerve growth factor; NO, nitric oxide; NOS, nitric oxide synthase; nNOS, eNOS, iNOS, neuronal, endothelial, and inducible nitric oxide synthase, respectively; l-NAME,N-nitro-l-arginine methyl ester; IPTG, isopropyl 1-thio-β-d-galactopyranoside; nAChR, nicotinic acetylcholine receptor; MAP1B, microtubule-associated protein 1B; CDTA,trans-1,2-diaminocyclohexaneN,N,N′,N′-tetraacetic acid; Tricine,N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine. induces both cell cycle arrest and differentiation for PC12 pheochromocytoma cells (1Greene L.A. Tischler A.S. Proc. Natl. Acad. Sci. U. S. A. 1976; 73: 2424-2428Crossref PubMed Scopus (4861) Google Scholar). These effects are associated with induction of nitric oxide synthase (NOS) (2Hirsch D.B. Steiner J.P. Dawson T.M. Mammen A. Hayek E. Snyder S.H. Curr. Biol. 1993; 3: 749-754Abstract Full Text PDF PubMed Scopus (111) Google Scholar, 3Peunova N. Enikolopov G. Nature. 1995; 375: 68-73Crossref PubMed Scopus (475) Google Scholar), the p53 tumor suppressor (4Eizenberg O. Faber-Elman A. Gottlieb E. Oren M. Rotter V. Schwartz M. Mol. Cell. Biol. 1996; 16: 5178-5185Crossref PubMed Scopus (181) Google Scholar), and the p21WAF1cyclin-dependent kinase inhibitor (5Poluha W. Poluha D.K. Chang B. Crosbie N.E. Schonhoff C.M. Kilpatrick D.L. Ross A.H. Mol. Cell. Biol. 1996; 16: 1335-1341Crossref PubMed Scopus (225) Google Scholar, 6van Grunsven L.A. Billon N. Savatier P. Thomas A. Urdiales J.L. Rudkin B.B. Oncogene. 1996; 12: 1347-1356PubMed Google Scholar, 7Yan G.-z. Ziff E.B. J. Neurosci. 1995; 15: 6200-6212Crossref PubMed Google Scholar). However, the relationship among these signaling events, arrest, and differentiation is poorly understood. Nitric oxide (NO) is a regulatory molecule that influences many processes, perhaps including neuronal proliferation and differentiation. NO is synthesized from arginine by a family of three NOS proteins: neuronal NOS (nNOS), endothelial NOS (eNOS), and inducible NOS (iNOS) (8Knowles R.G. Moncada S. Biochem. J. 1994; 298: 249-258Crossref PubMed Scopus (2506) Google Scholar). In mature mammals, NO acts as a neurotransmitter, a regulator of blood pressure, and a toxin for killing pathogens. A role for NO in the developing nervous system is plausible because nNOS is absent from proliferating neuroblasts but is coexpressed with early markers of neuronal differentiation (9Dawson T.M. Snyder S.H. J. Neurosci. 1994; 14: 5147-5159Crossref PubMed Google Scholar, 10Bredt D.S. Snyder S.H. Neuron. 1994; 13: 301-313Abstract Full Text PDF PubMed Scopus (340) Google Scholar). InDrosophila, NOS is expressed in developing imaginal discs (11Kuzin B. Roberts I. Peunova N. Enikolopov G. Cell. 1996; 87: 639-649Abstract Full Text Full Text PDF PubMed Scopus (233) Google Scholar). Inhibition of NOS in larvae results in enhanced cell proliferation, and elevated NOS expression stunts development. In rats, NGF enhances expression of nNOS by cholinergic neurons of the basal forebrain (12Holtzman D.M. Lee S. Li Y.W. Chuacouzens J. Xia H.B. Bredt D.S. Mobley W.C. Neurochem. Res. 1996; 21: 861-868Crossref PubMed Scopus (34) Google Scholar). NGF treatment of PC12 cells induces expression of all three isoforms of NOS (2Hirsch D.B. Steiner J.P. Dawson T.M. Mammen A. Hayek E. Snyder S.H. Curr. Biol. 1993; 3: 749-754Abstract Full Text PDF PubMed Scopus (111) Google Scholar, 3Peunova N. Enikolopov G. Nature. 1995; 375: 68-73Crossref PubMed Scopus (475) Google Scholar), and inhibitors of NOS block NGF-induced cessation of proliferation and neurite extension for PC12 cells (3Peunova N. Enikolopov G. Nature. 1995; 375: 68-73Crossref PubMed Scopus (475) Google Scholar). Hence, NO acts as a regulator of cell proliferation which, in turn, influences process outgrowth. The tumor suppressor p53 may play a role in neural development, in addition to its well established function as an inducer of apoptosis or cell cycle arrest after certain types of cell stress, including DNA damage (13Ko L.J. Prives C. Genes Dev. 1996; 10: 1054-1072Crossref PubMed Scopus (2292) Google Scholar). Mice lacking functional p53 genes have an unusually large number of birth defects, such as failure of neural tube closure (14Armstrong J.F. Kaufman M.H. Harrison D.J. Clarke A.R. Curr. Biol. 1995; 5: 931-936Abstract Full Text Full Text PDF PubMed Scopus (389) Google Scholar). In some mouse embryos, the absence of p53 leads to exencephaly in which overgrowth of neural tissue in the fore- and midbrain leads to abnormalities in cranial development (15Sah V.P. Attardi L.D. Mulligan G.J. Williams B.O. Bronson R.T. Jacks T. Nat. Genet. 1995; 10: 175-179Crossref PubMed Scopus (497) Google Scholar). This overgrowth probably results from excessive cell proliferation rather than decreased cell death. Consistent with this role, a recent study found that p53 is required for NGF-induced neurite extension by PC12 cells (4Eizenberg O. Faber-Elman A. Gottlieb E. Oren M. Rotter V. Schwartz M. Mol. Cell. Biol. 1996; 16: 5178-5185Crossref PubMed Scopus (181) Google Scholar). p21WAF1 binds to and inhibits cyclin-dependent kinases and induces cell cycle arrest at G1/S (16El-Deiry W.S. Tokino T. Velculescu V.E. Levy D.B. Parsons R. Trent J.M. Lin D. Mercer W.E. Kinzler K.W. Vogelstein B. Cell. 1993; 75: 817-825Abstract Full Text PDF PubMed Scopus (7935) Google Scholar). p53 is a potent transcriptional activator for p21WAF1 (16El-Deiry W.S. Tokino T. Velculescu V.E. Levy D.B. Parsons R. Trent J.M. Lin D. Mercer W.E. Kinzler K.W. Vogelstein B. Cell. 1993; 75: 817-825Abstract Full Text PDF PubMed Scopus (7935) Google Scholar, 17El-Deiry W.S. Harper J.W. O'Connor P.M. Velculescu V.E. Canman C.E. Jackman J. Pietenpol J.A. Burrell M. Hill D.E. Wang Y. Wiman K.G. Mercer W.E. Kastan M.B. Kohn K.W. Elledge S.J. Kinzler K.W. Vogelstein B. Cancer Res. 1994; 54: 1169-1174PubMed Google Scholar), but expression of p21WAF1 also can be induced by p53-independent mechanisms (18Elbendary A. Berchuck A. Davis P. Havrilesky L. Bast R.C. Iglehart J.D. Marks J.R. Cell Growth Differ. 1994; 5: 1301-1307PubMed Google Scholar, 19Jiang H. Lin J. Su Z.-z. Collart F.R. Huberman E. Fisher P.B. Oncogene. 1994; 9: 3397-3406PubMed Google Scholar, 20Michieli P. Chedid M. Lin D. Pierce J.H. Mercer W.E. Givol D. Cancer Res. 1994; 54: 3391-3395PubMed Google Scholar, 21Sheikh M.S. Li X.-S. Chen J.-C. Shao Z.-M. Ordonez J.V. Fontana J.A. Oncogene. 1994; 9: 3407-3415PubMed Google Scholar, 22Steinman R.A. Hoffman B. Iro A. Guillouf C. Liebermann D.A. El-Houseini M.E. Oncogene. 1994; 9: 3389-3396PubMed Google Scholar). In vivo, expression of p21WAF1 is enhanced as myoblasts differentiate into muscle cells (23Halevy O. Novitch B.G. Spicer D.B. Skapek S.X. Rhee J. Hannon G.J. Beach D. Lassar A.B. Science. 1995; 267: 1018-1021Crossref PubMed Scopus (1092) Google Scholar, 24Skapek S.X. Rhee J. Spicer D.B. Lassar A.B. Science. 1995; 267: 1022-1024Crossref PubMed Scopus (460) Google Scholar, 25Parker S.B. Eichele G. Zhang P. Rawls A. Sands A.T. Bradley A. Olson E.N. Harper J.W. Elledge S.J. Science. 1995; 267: 1024-1027Crossref PubMed Scopus (1023) Google Scholar). p21WAF1 is not expressed in the mitotic germinal layer of the olfactory epithelium but is expressed by olfactory neurons (25Parker S.B. Eichele G. Zhang P. Rawls A. Sands A.T. Bradley A. Olson E.N. Harper J.W. Elledge S.J. Science. 1995; 267: 1024-1027Crossref PubMed Scopus (1023) Google Scholar). We (5Poluha W. Poluha D.K. Chang B. Crosbie N.E. Schonhoff C.M. Kilpatrick D.L. Ross A.H. Mol. Cell. Biol. 1996; 16: 1335-1341Crossref PubMed Scopus (225) Google Scholar) and others (6van Grunsven L.A. Billon N. Savatier P. Thomas A. Urdiales J.L. Rudkin B.B. Oncogene. 1996; 12: 1347-1356PubMed Google Scholar, 7Yan G.-z. Ziff E.B. J. Neurosci. 1995; 15: 6200-6212Crossref PubMed Google Scholar, 26Decker S.J. J. Biol. Chem. 1995; 270: 30841-30844Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar) have reported up-regulation of p21WAF1 expression in NGF-treated cell lines. We have demonstrated that p21WAF1 is required for survival of differentiating neuroblastoma cells (5Poluha W. Poluha D.K. Chang B. Crosbie N.E. Schonhoff C.M. Kilpatrick D.L. Ross A.H. Mol. Cell. Biol. 1996; 16: 1335-1341Crossref PubMed Scopus (225) Google Scholar), but van Grunsvenet al. (6van Grunsven L.A. Billon N. Savatier P. Thomas A. Urdiales J.L. Rudkin B.B. Oncogene. 1996; 12: 1347-1356PubMed Google Scholar) have suggested that after differentiation of PC12 cells, continued expression of p21WAF1 is not required to maintain their differentiated phenotype. Hence, it may be that p21WAF1 plays its most important role during differentiation. In this report, we provide the first evidence for a connection among these NGF-induced events. We find that NO activates the p21WAF1 promoter, resulting in expression of p21WAF1 protein. This link is partially dependent on p53, a potent transcriptional activator for p21WAF1 (16El-Deiry W.S. Tokino T. Velculescu V.E. Levy D.B. Parsons R. Trent J.M. Lin D. Mercer W.E. Kinzler K.W. Vogelstein B. Cell. 1993; 75: 817-825Abstract Full Text PDF PubMed Scopus (7935) Google Scholar). NO also is required for NGF-induced expression of two markers associated with neuronal differentiation. Using a PC12 line bearing an inducible expression vector for p21WAF1, we find that induction of recombinant p21WAF1 restores NGF-induced differentiation for cells treated with a NOS inhibitor. These data demonstrate a signal transduction pathway that is activated by NGF; proceeds through NOS, p53, and p21WAF1 to block cell proliferation; and is required for NGF-induced neuronal differentiation by PC12 cells. PC12 cells from Dr. David Kaplan (Montreal Neurological Institute) were maintained in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated horse serum, 5% heat-inactivated fetal bovine serum, and 100 μg/ml gentamycin at 37 °C under 5% CO2. These cells were found to be mycoplasma-free by the direct culture method (MYCOTRIM, Irvine Scientific, Santa Ana, CA). For PC12 cell differentiation, plastic dishes were treated overnight at 4 °C with 15 μg/ml rat tail collagen (Sigma) and 15 μg/ml poly-d-lysine (>300,000 Da, Sigma). The dishes were rinsed with distilled water, and then PC12 cells were plated in defined medium, as described (27Reinhold D.S. Neet K.E. J. Biol. Chem. 1989; 264: 3538-3544Abstract Full Text PDF PubMed Google Scholar), and treated with 100 ng/ml NGF (2.5 S, Bioproducts for Science, Indianapolis). After 3–4 days of treatment, cells with neurites at least five cell diameters long were scored as positive. N-Nitro-l-arginine methyl ester (l-NAME) competes with arginine for binding to all three isoforms of NOS and blocks enzymatic activity (8Knowles R.G. Moncada S. Biochem. J. 1994; 298: 249-258Crossref PubMed Scopus (2506) Google Scholar). In these studies, we used 20 mml-NAME and the same concentration of the inactive enantiomer, d-NAME, as a control. l-NAME used at this concentration is thought to be specific for NOS and with little effect on PC12 metabolism, growth rates, and NGF induction of immediate-early genes (3Peunova N. Enikolopov G. Nature. 1995; 375: 68-73Crossref PubMed Scopus (475) Google Scholar). We prepared a PC12 line bearing an inducible expression vector (LacSwitch, Stratagene, La Jolla, CA) for p21WAF1. In this system, mammalian cells are transfected with both a Lac-repressor-expressing vector and a lac-operator-containing p21WAF1vector. Expression of p21WAF1 occurs within 48 h after the addition of 25 mmisopropyl-1-thio-β-d-galactopyranoside (IPTG). The p21WAF1 plasmid for these experiments was prepared by J. Earheart and R. Pittman (University of Pennsylvania School of Medicine). In brief, the protein-encoding portion of the p21WAF1 cDNA was inserted into the pOPRSV1 plasmid (Stratagene) which includes a neomycin resistance gene. Expression of p21WAF1 is driven by a Rous sarcoma virus promoter but, in the absence of IPTG, is suppressed by adjoining lac operator sites. Plasmid p3′SS, which has the lac repressor gene under control of a cytomegalovirus early promoter, also was from Stratagene and was used without modification. PC12 cells were transfected with the p3′SS Lac repressor plasmid, using the cationic detergent Lipofectin (Life Technologies, Inc.). Plasmid DNA (6–7 μg/25-cm2 flask) was diluted in 100 μl of Dulbecco's modified Eagle's medium (serum-free). At the same time in a second tube, 20 μl of 1 mg/ml Lipofectin was diluted with 100 μl of Dulbecco's modified Eagle's medium. After a 30-min incubation at room temperature, the two solutions were combined and incubated for an additional 15 min at room temperature. This mix was then diluted with 2.5 ml of complete medium including serum, added to a 25-cm2 tissue culture flask containing about 3 × 106 cells, and incubated overnight at 37 °C. The resulting cells were selected for 14 days with medium containing 125 μg/ml hygromycin. This mass culture was derived from about 20 different hygromycin-resistant colonies. These cells were then transfected by the same procedure with the pOPRSV1 p21(WAF1) plasmid bearing the p21WAF1 cDNA and selected for 14 days with 100 μg/ml G418. The mass culture from the second transfection was derived from about 30 different G418-resistant colonies. The morphology and rates of proliferation of these cells were not obviously affected by the transfections. After treatment with NGF and NO drugs, cells were extracted as described (17El-Deiry W.S. Harper J.W. O'Connor P.M. Velculescu V.E. Canman C.E. Jackman J. Pietenpol J.A. Burrell M. Hill D.E. Wang Y. Wiman K.G. Mercer W.E. Kastan M.B. Kohn K.W. Elledge S.J. Kinzler K.W. Vogelstein B. Cancer Res. 1994; 54: 1169-1174PubMed Google Scholar). These samples (≈40 μg of protein/lane) were boiled under reducing conditions, subjected to electrophoresis on a 12.5% polyacrylamide gel, and electrotransferred to an Immobilon-P membrane (Millipore, Bedford, MA). Residual proteins in the gel were stained with Coomassie Blue to confirm even loading of the gel. The membrane was blocked for 1 h with 10% powdered milk in 0.2% Tween 20, Tris-buffered saline, and then incubated with 2 μg/ml anti-p53 monoclonal antibody Pab-1801 (Oncogene Science, Cambridge, MA), 1 μg/ml rabbit anti-p21WAF1 antibody C-19 (Santa Cruz Biotechnology, Santa Cruz, CA), or 35 μg/ml anti-nicotinic acetylcholine receptor (nAChR) monoclonal antibody mab35 (28Tzartos S.J. Rand D.E. Einarson B.L. Lindstrom J.M. J. Biol. Chem. 1981; 256: 8635-8645Abstract Full Text PDF PubMed Google Scholar). Immunoreactive proteins were detected with horseradish peroxidase-conjugated secondary antibodies (Amersham, Arlington Heights, IL) and a chemiluminescence reagent (29Matthews J.A. Batki A. Hynds C. Kricka L.J. Anal. Biochem. 1985; 151: 205-209Crossref PubMed Scopus (128) Google Scholar). The films were scanned with a Hewlett Packard ScanJet 3c. Brightness and contrast were adjusted with Photoshop, making the same adjustments for each band. Montages were assembled with CorelDraw, using only images from the same experiment and film. Cells were fixed for 10 min at room temperature with 4% paraformaldehyde in PBS and immunostained with 1 μg/ml of anti-p21WAF1 rabbit antibody C-19 and then a rhodamine-conjugated secondary antibody. The cells were then incubated with 1.0 mg/ml of NADPH, 0.25 mg/ml of nitro blue tetrazolium and 0.1 m Tris HCl for 4 h at 37 °C (30Mizukawa K. Vincent S.R. McGeer P.L. McGeer E.G. J. Comp. Neurol. 1989; 279: 281-311Crossref PubMed Scopus (243) Google Scholar). Samples were mounted in Citifluor (Ted Pella Inc., Reading, PA) and were viewed with a Zeiss Axioskop microscope and a 25× oil immersion objective lens, using bright field and fluorescence optics to detect diaphorase and p21WAF1, respectively. For detection of neuronal markers, cells were fixed for 10 min with 95% methanol, 5% acetic acid at −20 °C. The samples were stained with 35 μg/ml of monoclonal antibody mab35 against the α subunit of the nicotinic acetylcholine receptor (nAChR) (28Tzartos S.J. Rand D.E. Einarson B.L. Lindstrom J.M. J. Biol. Chem. 1981; 256: 8635-8645Abstract Full Text PDF PubMed Google Scholar) or ascites diluted 1:200 from hybridoma MAP1B-4 directed against microtubule-associated protein 1B (MAP1B) (31Bloom G.S. Luca F.C. Vallee R.B. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 5404-5408Crossref PubMed Scopus (175) Google Scholar, 32Luca F.C. Bloom G.S. Vallee R.B. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 1006-1010Crossref PubMed Scopus (54) Google Scholar). These antibodies were diluted with 0.3% bovine serum albumin, 0.2% Tween 20 in Tris-buffered saline. Both of these markers are expressed during development, although the time of initial onset is earlier for MAP1B (33Tohyama T. Lee V.M.-Y. Rorke L.B. Trojanowski J.Q. J. Comp. Neurol. 1991; 310: 285-299Crossref PubMed Scopus (87) Google Scholar). Micrographs were recorded with Kodak T-MAX 400 film and digitized with a Nikon Coolscan. Brightness and contrast were adjusted with Adobe Photoshop, and montages were assembled with CorelDraw. The figures were printed using a Kodak Colorease PS printer on Kodak Extatherm XLS paper. PC12 cells were treated for 60 h with or without NGF, l-NAME, ord-NAME. Then, using Lipofectin as described above, they were cotransfected with promoter constructs WWP-Luc or DM-Luc (16El-Deiry W.S. Tokino T. Velculescu V.E. Levy D.B. Parsons R. Trent J.M. Lin D. Mercer W.E. Kinzler K.W. Vogelstein B. Cell. 1993; 75: 817-825Abstract Full Text PDF PubMed Scopus (7935) Google Scholar) (6 μg/25-cm2 flask) and β-galactosidase plasmid pCH110 (6 μg/25-cm2 flask). After 16–20 h, cells were extracted for 15 min at room temperature with cell culture lysis reagent (Promega; 25 mm Tris phosphate, pH 7.8, 2 mmdithiothreitol, 2 mm CDTA, 10% glycerol, and 1% Triton X-100). These extracts were clarified by centrifugation, stored at −70 °C, and then used immediately after thawing. Mixtures of 20 μl of cell extract and 100 μl of luciferase assay reagent (20 mm Tricine, 1.07 mm(MgCO3)4Mg(OH)2·3H2O, 2 mm MgSO4, 0.1 mm EDTA, 33.3 mm dithiothreitol, 270 μm coenzyme A, 470 μm luciferin, 530 μm ATP, pH 7.8) were immediately placed in a model N Luminometer analyzer (Packard), and luciferase activities were measured. β-Galactosidase activities were measured using a kit from Promega. Assay 2× buffer (120 mm Na2HPO4, 80 mm NaH2PO4, 2 mmMgCl2, 100 mm β-mercaptoethanol, 1.33 mg/mlo-nitrophenyl-β-d-galactopyranoside) was mixed with an equal volume (100 μl) of cell extract. Samples were incubated overnight at 37 °C, and the reaction was stopped by the addition of 50 μl of 1 m Na2CO3. Optical densities at 420 nm were measured. Luciferase activities were normalized with β-galactosidase activities to eliminate any differences in transfection efficiencies. Using NGF-treated PC12 cells as a model system, we tested the relationship among several signaling events that occur during neuronal differentiation. We found that the NOS inhibitor, l-NAME, but not the inactive enantiomer d-NAME, inhibits NGF-induced neurite extension by 73 ± 15% (mean ± S.D.,n = 6), in agreement with Peunova and Enikolopov (3Peunova N. Enikolopov G. Nature. 1995; 375: 68-73Crossref PubMed Scopus (475) Google Scholar). We assayed expression of two markers associated with neuronal differentiation. As judged by immunofluorescence microscopy, MAP1B (34LoPresti P. Poluha W. Poluha D.K. Drinkwater E. Ross A.H. Cell Growth Differ. 1992; 3: 627-635PubMed Google Scholar,35Loeb D.M. Stephens R.M. Copeland T. Kaplan D.R. Greene L.A. J. Biol. Chem. 1994; 269: 8901-8910Abstract Full Text PDF PubMed Google Scholar) is up-regulated by NGF (Fig. 1). For this experiment, untreated PC12 cultures showed 12 ± 1% of cells positive for MAP1B, but NGF-treated cultures showed 87 ± 3% cells positive. l-NAME, but not d-NAME, reduced this NGF-induced increase in MAP1B expression (d-NAME + NGF-treated cells, 88 ± 4% positive; l-NAME + NGF-treated cells, 14 ± 2% positive). Averaging three experiments, l-NAME inhibited NGF induction of MAP1B-positive cells by 85 ± 10%. The pattern of expression for nAChR closely resembled that for MAP1B (36Henderson L.P. Gdovin M.J. Liu C. Gardner P.D. Maue R.A. J. Neurosci. 1994; 14: 1153-1163Crossref PubMed Google Scholar) (micrographs not shown). By Western blotting, we observed the same pattern of expression for nAChR (Fig. 2). Averaging three experiments, the relative intensities of the nAChR bands for control, NGF, NGF +d-NAME, NGF + l-NAME, and l-NAME were 1.0, 2.6 ± 0.2, 3.7 ± 1.6, 0.8 ± 0.4, and 0.8 ± 0.3, respectively. The anti-MAP1B antibody does not detect MAP1B protein by Western blotting. Hence, NO, like cell cycle arrest, is required for NGF-induced differentiation.Figure 2NGF-induced expression of nAChR is dependent on NO. PC12 cells were treated as in Fig. 1 and extracted for Western blotting.View Large Image Figure ViewerDownload Hi-res image Download (PPT) We then analyzed the role of NO in regulating expression of p53 and p21WAF1 (4Eizenberg O. Faber-Elman A. Gottlieb E. Oren M. Rotter V. Schwartz M. Mol. Cell. Biol. 1996; 16: 5178-5185Crossref PubMed Scopus (181) Google Scholar, 5Poluha W. Poluha D.K. Chang B. Crosbie N.E. Schonhoff C.M. Kilpatrick D.L. Ross A.H. Mol. Cell. 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Diaphorase staining allows visualization of NOS enzymatic activity, resulting in colored cells that can be detected by bright-field light microscopy (45Dawson T.M. Bredt D.S. Fotuhi M. Hwang P.M. Snyder S.H. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 7797-7801Crossref PubMed Scopus (1840) Google Scholar, 46Hope B.T. Michael G.J. Knigge K.M. Vincent S.R. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 2811-2814Crossref PubMed Scopus (1889) Google Scholar). This method is specific for NOS because other diaphorases are inactivated by fixation with paraformaldehyde. p21WAF1 was detected by immunofluorescence microscopy, using an anti-p21WAF1 antibody. For untreated cultures, few PC12 cells displayed diaphorase staining, p21WAF1immunoreactivity, or neurites (Fig. 5,A, C, and E, respectively). NGF treatment increased the percentages of cells with diaphorase activity (92%), p21WAF1 immunoreactivity (85%), and neurites (77%) (Fig. 5, B, D, and E, respectively). 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