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Molecular and Cellular Biology, August 2002, p. 5357-5366, Vol. 22, No. 15
0270-7306/02/$04.00+0     DOI: 10.1128/MCB.22.15.5357-5366.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Neuronal Differentiation and Protection from Nitric Oxide-Induced Apoptosis Require c-Jun-Dependent Expression of NCAM140

Zhiwei Feng, Lei Li, Poh Yong Ng, and Alan G. Porter*

Institute of Molecular and Cell Biology, Singapore 117609, Republic of Singapore

Received 26 November 2001/ Returned for modification 13 February 2002/ Accepted 17 April 2002


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ABSTRACT
 
c-Jun, a crucial component of the dimeric transcription factor activating protein 1 (AP-1), can regulate apoptosis induced by oxidative stress and has been implicated in neuronal differentiation, but the mechanisms are largely unknown. We found that specific inhibition of transcription or stable transfection with cDNA encoding dominant-negative c-Jun sensitized SH-SY5Y neuroblastoma cells (TAM-67 cells) to apoptosis induced by the nitric oxide (NO) donor sodium nitroprusside or SIN-1. TAM-67 cells also became refractory to nerve growth factor (NGF)-induced neuronal differentiation. Dominant-negative c-Jun abolished expression of a 140-kDa neural cell adhesion molecule (NCAM140) and dramatically enhanced the expression of NCAM180 in TAM-67 cells. Inhibition of c-Jun in TAM-67 cells also resulted in a corresponding decrease in the amount of NCAM140 mRNA and an increase in the amount of NCAM180 mRNA. Reexpression of NCAM140 in TAM-67 cells restored NGF-induced neuronal differentiation and resistance to NO-induced apoptosis. Our results show that c-Jun/AP-1, through up-regulation of NCAM140, plays an important role in both NGF-induced neuronal differentiation and resistance to apoptosis induced by NO in neuroblastoma cells. As NCAM140 and NCAM180 are translated from differentially spliced mRNAs transcribed from the same gene, alternative splicing of NCAM pre-mRNA (and consequently the synthesis of the smaller NCAM140 species) appears to be regulated by c-Jun/AP-1.


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INTRODUCTION
 
Excessive excitatory neurotransmission induced by glutamate may cause oxidative stress, which contributes to neurodegenerative disorders, stroke, trauma, and seizures (15, 56). Overstimulation of glutamate receptors generates nitric oxide (NO) and reactive oxygen species, leading to free radical-induced neuronal cell apoptotic or necrotic death (2, 34). NO is enigmatic. As an effector of apoptosis, it kills a variety of cell types, including neuronal cells, through a p53-dependent mitochondrial pathway, though it can also protect various cells from death induced by other toxic stimuli (9). NO has recently received more attention because of its ability to indirectly stimulate mRNA transcription, which leads to a plethora of functions, including neuronal damage (7).

The activating protein 1 (AP-1) family of transcription factors includes homodimers and heterodimers of Jun (c-Jun, JunB, JunD), Fos (c-Fos, FosB, Fra1, Fra2), activating transcription factor (ATF2, ATF3/LRF1, B-ATF), and Jun dimerization partners (JDP-1 and JDP-2) (50). The various dimers can associate with specific AP-1 binding sites in DNA to activate or repress mRNA transcription. AP-1 family members can either promote or block apoptosis. Thus, c-Jun counteracts cell death after DNA damage and during liver development (9, 35, 44) and AP-1 protects macrophages from excess NO (58). It is unclear whether c-Jun is pro- or antiapoptotic in UV-induced apoptosis (49, 60), but c-Jun is proapoptotic in various neuronal models, for example, nerve growth factor (NGF) withdrawal (20, 24, 25). Remarkably, c-Jun can even be proapoptotic or antiapoptotic depending on the differentiation status of the neuronal cell (37).

Oxidative stress caused by glutamate receptor activation induces AP-1 activity, which may play a regulatory role in neuronal damage (55). At a further level of complexity, NO can either activate or repress AP-1 in neuronal cells, which probably reflects the amount and strength of signal, cell type differences, and the combinatorial diversity of AP-1 heterodimers (7, 23, 53). It is not, however, known if NO-provoked activation or repression of AP-1 in neurons is pro- or antiapoptotic, and the target genes regulated by NO and AP-1 in various neuronal cell populations remain to be identified.

In the nervous system, the cell death, differentiation, and regeneration machineries may share some components and c-Fos/c-Jun (AP-1) may be one such component (25). c-Jun is involved in differentiation in a number of systems, for example, in hematopoietic cells (38). In neuronal cells, c-Jun is required for neurite regeneration (19) and is involved in Ras- and NGF-induced PC12 neural cell differentiation (36, 37, 47). Germ line mutations in the c-Jun gene are embryonic lethal, but there are no obvious neural abnormalities, at least not up to midgestation (26). However, mice lacking ATF-2 or c-Fos have severe defects in the development of the central nervous system (30, 45). The mechanism of action and target genes of c-Jun, c-Fos, or ATF-2 in AP-1 complexes in neuronal development and differentiation are yet to be identified.

Neural cell adhesion molecule (NCAM) belongs to the immunoglobulin superfamily, and three major isoforms may be generated from a single gene by alternative splicing (18, 54). Family members, acting as either receptors or substrates on the cell surface, may be involved in the regulation of neuronal differentiation, neurite guidance, and branching (1, 16, 59). NCAM may also control neuronal survival, as inhibition of NCAM by antibody cross-linking induces neuronal apoptosis (3).

Here we report the functions of c-Jun/AP-1 in NGF-induced neuronal differentiation and apoptosis induced by chemical NO donors in a human neuroblastoma cell line. As the NCAM gene has an AP-1 site in its promoter (13), we investigated NCAM as a potentially important AP-1 target gene in the regulation of neuronal survival and differentiation.


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MATERIALS AND METHODS
 
Materials. The human neuroblastoma cell line SH-SY5Y (40) was obtained from Eva Feldman (University of Michigan) and maintained in Dulbecco's modified medium containing 10% fetal bovine serum, 100 U of penicillin/ml, and 100 µg of streptomycin/ml. The substrate that ensures cell plating is presumed to be extracellular matrix proteins, either exogenous or derived from the bovine serum. SIN-1 was purchased from Calbiochem. Sodium nitroprusside (SNP), 1-(5-isoquinolinesulfonyl)-2-methylpiperizine (H-7), mouse NGF, and aphidicolin were obtained from Sigma Co. The polyclonal antibody to c-Jun was from Oncogene Science. The annexin V-fluorescein isothiocyanate apoptosis detection kit, polyclonal antibody to caspase-3, and monoclonal antibody to poly(ADP-ribose) polymerase (PARP) were from Pharmingen. The monoclonal antibody to Bcl-2 was from Transduction Laboratories Inc., and the antibody to NCAM was purchased from Chemicon International Inc. Poly(dI-dC) was obtained from Amersham Pharmacia Biotech. The AP-1-specific oligonucleotide (5'-CGC TTG ATG ACT CAG CCG GAA-3') was obtained from Santa Cruz Biotechnology. The plasmid encoding dominant-negative c-Jun (TAM-67) was provided by R. Jaggi (University of Bern, Bern, Switzerland). The pIREShyg vector and hygromycin were bought from Clontech, and LipofectAMINE and the preamplification system were from Life Technologies, Inc. The complete protease inhibitor cocktail and cell proliferation reagent WST-1 were purchased from Roche. The luciferase assay kit and AP-1 reporter plasmid were purchased from Stratagene. The RNeasy MiniKit was bought from Qiagen.

Cell growth, viability, and neuronal differentiation. To generate stable cell lines, the TAM-67 plasmid (8) was cotransfected in SH-SY5Y cells in a 10:1 mixture with pIREShyg by using LipofectAMINE following the user's manual. The stable transformed cells were selected and maintained in medium with 100 ng of hygromycin/ml. The plasmid encoding the 140-kDa NCAM (NCAM140) fused to c-myc was transfected, and stable cell lines were selected with Zeosin (100 µg/ml). To measure cell death, cells (3 x 105/ml) were plated in 96-well plates and treated with SNP, SIN-1, or H-7 for up to 16 h. In some experiments, actinomycin D was added together with the SNP to measure the effects of inhibiting transcription on SNP toxicity. Cell death was measured by trypan blue uptake or crystal violet staining (28), or in a few experiments, loss of mitochondrial function was measured with WST-1 reagent. TAM-67 and vector control cells were plated at 105 cells/ml in 6-cm-diameter plates, and the cells were counted every 24 h for 5 days to determine the growth rates. For neuronal differentiation, the method was modified from the method described in reference 29. In brief, the cells were treated with NGF (1 µg/ml) for 8 to 10 days and aphidicolin (5 µg/ml) was added to the culture medium from day 2 onwards. After 8 to 10 days of differentiation, the cells were photographed under a light microscope.

Preparation of total RNA and cytoplasmic and nuclear proteins and RT PCR. SH-SY5Y cells stably transformed with TAM-67 and the control plasmid vector were harvested after treatment and washed in ice-cold phosphate-buffered saline. For cytoplasmic proteins, the cell pellets were suspended in ice-cold lysis buffer (100 mM NaCl, 20 mM Tris-HCl [pH 8.0], 1% Nonidet P-40, and complete protease inhibitor cocktail) and incubated on ice for 30 min. The samples were centrifuged, and supernatants were collected as cytoplasmic proteins. To isolate nuclear proteins, cells were homogenized in a Dounce homogenizer in buffer A (20 mM HEPES [pH 7.9], 1.5 mM MgCl2, 10 mM KCl, 0.5 mM dithiothreitol, and complete protease inhibitor cocktail). After centrifugation at 600 x g, the pellets containing nuclei were resuspended in buffer C (20 mM HEPES [pH 7.9], 1.5 mM MgCl2, 420 mM KCl, 1 mM EDTA, 25% glycerol, 0.5 mM dithiothreitol, and complete protease inhibitor cocktail) and incubated at 4°C for 20 min. The samples were centrifuged at 100,000 x g for 20 min at 4°C, and the supernatants were collected as nuclear proteins (20). Total RNA isolation was done following the protocol included in the RNeasy kit. cDNA was synthesized following the manufacturer's protocol for the SuperScript preamplification system. Reverse transcriptase PCR (RT PCR) was performed with primers homologous to the sequence of the 1st exon (ATG CTG CAA ACT AAG GAT CTC ATC TGG ACT TTG) and the 19th exon (TGC TTT GCT CTC GTT CTC CTT TGT CTG TGT G) for NCAM140 and primers homologous to the 18th exon (GCC TGC CGA CAC CAC AGC CAC) and the 19th exon (TGC TTT GCT CTC GTT CTC CTT TGT CTG TGT G) for NCAM180.

Electrophoretic mobility shift assay. Reactions were conducted in a total volume of 20 µl (21). Typically, 5 µl of nuclear extracts (5 µg of protein) was added to the reaction buffer containing 100 mM KCl, 10% glycerol, 0.2 mM EDTA, 0.5 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 1 µg of leupeptin/ml, 20 mM HEPES (pH 7.9), and 2 µg of poly(dI-dC). The mixture was incubated on ice for 20 min, and 105 cpm (3,000 Ci/mmol) of [{gamma}-32P]ATP-labeled oligonucleotide probe with an AP-1 site (5'-CGC TTG ATG ACT CAG CCG GAA-3') was then added. In the antibody supershift experiment, 1 µg of c-Jun antibody was added with nuclear proteins and the reactions were incubated for a further 20 min at 24°C. Samples were electrophoresed in 5% polyacrylamide gels in Tris-glycine buffer (pH 8.5) for 3 h at 4°C. The gels were dried and autoradiographed with intensifying screens at -88°C.

Annexin staining and immunoblotting. Cells were treated with SNP for 16 h, and the cells were stained with annexin V-fluorescein isothiocyanate following the manufacturer's instructions and then analyzed by flow cytometry. For Western blot analysis, cells were lysed in a buffer containing complete protease inhibitor cocktail. After centrifugation, 30 to 50 µg of soluble cytoplasmic proteins was electrophoresed in 6 to 12% polyacrylamide gels and transferred to nitrocellulose membranes. Immunoblotting was carried out with antibodies in phosphate-buffered saline with 0.2% Tween 20 (Sigma) and 5% bovine serum albumin (Sigma). After washing, the membrane was probed with horseradish peroxidase-conjugated donkey antiserum to rabbit or mouse immunoglobulin G (Amersham Pharmacia Biotech) and developed by the enhanced chemiluminescence method (Amersham Pharmacia Biotech).

Reporter assay. Both 0.5 µg of ß-galactosidase and 1.5 µg of AP-1 reporter plasmids were cotransfected in vector control and TAM-67 cells in 24-well plates. Forty hours later, cells were treated with 32 nM 12-O-tetradecanoylphorbol-13-acetate (TPA) for 6 h. Medium was removed, and cells were washed with phosphate-buffered saline. Cells were removed with 300 µl of lysis buffer and were frozen at -88°C for 20 min. After thawing at 37°C, cells were centrifuged. Twenty microliters of supernatant was added to 100 µl of luciferase substrate and incubated for 30 min at 37°C. The reaction was stopped by adding 500 µl of 1 M sodium carbonate, and luciferase activity was measured with the TD-20e luminometer. An aliquot of the same sample was used to determine ß-galactosidase activity for normalizing luciferase activity.


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RESULTS
 
NO donors kill SH-SY5Y cells by apoptosis. The generation of NO in cells can be mimicked with chemical donors of NO (9). Various neuronal cell lines, including SH-SY5Y, are susceptible to apoptosis induced by different NO donors (22, 39, 57). NO donors SNP and SIN-1 (at 0.5 to 2 mM) release NO intracellularly, which induces apoptosis of neuroblastoma cells in part via oxidative stress. SH-SY5Y cells treated with 1.5 to 2.0 mM SNP were cultured various times for up to 16 h, and cell death was measured by trypan blue uptake or crystal violet staining (28). Cell death was measurable 8 h after SNP treatment, and over half the cells were dead by 16 h (Fig. 1A). Prolonged SNP treatment did not further increase the number of dead cells (data not shown).



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  		FIG. 1. Apoptotic cell death induced by SNP in SH-SY5Y cells. (A) SH-SY5Y cells were incubated with 2 mM SNP for the indicated times, and cell death was quantitated using crystal violet staining. SD, standard deviation. (B) Western immunoblot analysis of the proteolytic activation of caspase 3. SH-SY5Y cells were incubated with 2 mM SNP for the indicated times. (C) Western immunoblot analysis of the proteolytic cleavage of PARP. SH-SY5Y cells were treated with 2 mM SNP for the indicated times.

To verify that the mode of cell death was apoptosis, DNA fragmentation assays (not shown), Western blot analysis of caspase 3 activation, and PARP cleavage assays were performed. Correlating with the onset of cell death, the cleavage products of caspase 3 and PARP were first detected 8 h after SNP addition and PARP was completely cleaved at 16 h (Fig. 1B and C). As DNA fragmentation, caspase 3, and PARP are known markers of apoptosis (43), these results confirm that under our experimental conditions, SNP-treated SH-SY5Y cells died by apoptosis.

Dominant-negative c-Jun sensitizes SH-SY5Y cells to NO-induced apoptosis. It is well established that NO regulates gene expression, but it is unclear how this might influence the susceptibility of neuronal cells to NO-induced apoptosis (7). Inhibition of transcription in SH-SY5Y cells by nontoxic doses of actinomycin D sensitized the cells to NO-induced cell death (Fig. 2), suggesting that new or continuous gene transcription is required for protection of these cells from NO toxicity.



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  		FIG. 2. Actinomycin D (ActD) sensitizes SH-SY5Y neuroblastoma cells to SNP-induced apoptosis. SH-SY5Y cells were left untreated or treated with SNP at 1.5 or 2 mM in the absence or presence of actinomycin D at 0.05 and 0.1 µg/ml. Cell death was quantitated using crystal violet staining. SD, standard deviation.

NO can induce or repress AP-1 in neuronal cells (7), but it is not known how the regulation of AP-1 activity by NO relates to neuronal death and survival (55). To determine if NO donors induce AP-1 DNA binding activity, SH-SY5Y cells were treated with SNP and cell extracts were probed with an AP-1-specific oligonucleotide. SNP clearly induced AP-1 DNA binding activity, which was first detected at 2 h and peaked at 8 h after SNP addition (Fig. 3A, left panel). Maximal activation of AP-1 (Fig. 3A, left panel) preceded apoptosis (Fig. 1A), suggesting that AP-1 might regulate NO toxicity in SH-SY5Y cells. To determine if c-Jun was present in this AP-1 complex, cell extracts from the 8-h time point were incubated with the AP-1-specific oligonucleotide in the presence or absence of a c-Jun antibody. Figure 3A (right panel) shows that AP-1 binding (lane 2) was completely blocked by the c-Jun antibody (lane 1), demonstrating that c-Jun is a crucial component of the complex induced by SNP.



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  		FIG. 3. Dominant-negative c-Jun blocks induction of AP-1 activity in TAM-67 cells. (A) DNA band shift analyses in native polyacrylamide gels were performed using an oligonucleotide containing AP-1 sites. Left panel, nuclear extracts of SH-SY5Y cells were prepared at the indicated times after 2 mM SNP treatment. Right panel, supershift analysis using c-Jun antibodies. Nuclear extracts were prepared from SH-SY5Y cells treated with SNP for 8 h. Lane 1, 1 µg of polyclonal antibody against c-Jun was added before the AP-1-specific oligonucleotide was added. Lane 2, no c-Jun antibody was added. (B) Western blot analysis of c-Jun expression in SH-SY5Y vector control cells (lane 1) and dominant-negative c-Jun in TAM-67 cells (lane 2). (C) ß-Galactosidase and AP-1 reporter plasmids were cotransfected into SH-SY5Y vector control and TAM-67 cells. Lysates were prepared from cells treated for 6 h with 32 nM TPA or left untreated. AP-1 activity was measured by luciferase assay. Vec con, vector control. SD, standard deviation.

To clarify the function of AP-1 in NO-induced apoptosis, we generated stable SH-SY5Y cell lines expressing TAM-67, a dominant-negative mutant c-Jun protein. TAM-67 has 104 amino acids deleted from the transactivation domain of c-Jun and displays no transcriptional activity, but it can dimerize efficiently with Fos, Jun, and ATF-2 family members and bind to the AP-1 consensus DNA sequence, thereby antagonizing endogenous AP-1 (8). Western blot analysis confirmed the expression of the truncated dominant-negative c-Jun protein in TAM-67 cells but not in the cells transformed with the vector control plasmid (Fig. 3B). Next, SH-SY5Y vector control and TAM-67 cells were transiently transfected with a plasmid containing AP-1 sites linked to a luciferase reporter gene and treated with SNP. No luciferase activity was detected in repeated experiments (data not shown). A significant 10-fold stimulation of luciferase activity was, however, observed in vector control cells (but not TAM-67 cells) treated with TPA, a known inducer of AP-1 activity (Fig. 3C). This shows that AP-1 activity is blocked as expected in SH-SY5Y cells expressing dominant-negative c-Jun and suggests that NO or its by-product(s) quenches luciferase activity, thereby preventing a direct evaluation of NO-induced AP-1 transcriptional activity.

Since AP-1 is potentially oncogenic and plays a role in cell proliferation (50), the inhibition of its activity may affect SH-SY5Y cell growth. There were no differences in growth rates of vector control and TAM-67 cells over 4 days, showing that blocking c-Jun/AP-1 does not appear to affect cell proliferation (data not shown). However, in six independent experiments, TAM-67 cells were markedly more sensitive to apoptosis induced by different concentrations of SNP compared with vector control SH-SY5Y cells (Fig. 4A). Similar results were obtained with two other independent clones expressing dominant-negative c-Jun (data not shown). To independently verify the difference in sensitivities of vector control and TAM-67 cells to SNP, we quantitated the externalization of phosphatidylinositol by annexin V staining using flow cytometry. In contrast to vector control cells, there was appreciable annexin V staining of TAM-67 cells (Fig. 4B), which again shows that suppression of AP-1 activity sensitizes SH-SY5Y cells to cell death induced by SNP.



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  		FIG. 4. Dominant-negative c-Jun sensitizes SH-SY5Y cells to NO-induced apoptosis. (A) Vector control cells and TAM-67 cells were treated with the indicated concentrations of SNP for 16 h, and cell death was quantitated using crystal violet staining. SD, standard deviation. (B) Cell death was determined by flow cytometry using annexin V staining to detect externalized phosphatidylserine. Vector control cells and TAM-67 cells were left untreated or treated with 1.5 mM SNP for 16 h. M1 marks the positions of the annexin V-stained cells. (C) Vector control cells and TAM-67 cells were treated with the indicated concentrations of the NO donor SIN-1 for 16 h, and cell death was quantitated using crystal violet staining.

Similar results were obtained with an alternative NO donor, SIN-1, which also kills SH-SY5Y cells by apoptosis (39). Figure 4C shows that the SIN-1-induced death of TAM-67 cells was markedly higher than that of vector control cells, confirming that blocking AP-1 function sensitizes SH-SY5Y cells to NO toxicity induced by different NO donors.

NCAM140 is regulated by c-Jun/AP-1 and protects SH-SY5Y cells from apoptosis induced by NO and the protein kinase inhibitor H-7. There are several AP-1 target genes which have been implicated in the resistance of neuronal cells to apoptosis, including NCAM (3, 13). There are three major species of NCAM based on their molecular masses: 120-, 140-, and 180-kDa NCAM (16). In untreated SH-SY5Y cells, a polyclonal antibody to NCAM detected NCAM140 whereas NCAM120 and NCAM180 were not detectable (Fig. 5A, left panel). In complete contrast, NCAM140 was virtually absent in TAM-67 cells and, strikingly, there was a high level of NCAM180 in TAM-67 cells (Fig. 5A, left panel). Similar results were obtained with two other independent clones expressing dominant-negative c-Jun (data not shown). Whereas NCAM140 levels declined sharply after NO donor treatment, the levels of NCAM180 showed an increase (Fig. 5B).



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  		FIG. 5. NCAM140 protects SH-SY5Y cells from SNP-induced apoptosis. (A) Analysis of expression of NCAM140 and NCAM180 in vector control SH-SY5Y cells or TAM-67 cells. Left panel, Western blot analysis; right panel, RT PCR analysis. (B) Western blot analysis of expression of NCAM140 and NCAM180 in vector control, TAM-67, and TAM-67/NCAM140 cells treated with 2 mM SNP for the indicated times. (C) Cell death assays of SNP-treated vector control cells, TAM-67 cells, and two clones of TAM-67/NCAM140 cells (TAM-67/NCAM140-1 and TAM-67/NCAM140-2). Cells were either left untreated or treated with the indicated concentrations of SNP for 16 h. Cell death was measured using trypan blue exclusion. SD, standard deviation.

To test whether the change in the NCAM expression pattern in TAM-67 cells was due to altered transcriptional regulation, RT PCR was performed with RNAs from vector control and TAM-67 cells with PCR primers specific for either NCAM140 mRNA or NCAM180 mRNA. Consistent with the protein results, the level of NCAM140 mRNA in TAM-67 cells was much lower than that in vector control cells; conversely, the amount of NCAM180 mRNA in TAM-67 cells was far higher than that in vector control cells (Fig. 5A, right panel). Thus, the dramatically different yields of NCAM140 and NCAM180 are reflected in the respective mRNA levels.

To determine whether the change in the NCAM expression pattern in TAM-67 cells increases their vulnerability to SNP-induced apoptosis, a cDNA encoding NCAM140 was stably transfected into TAM-67 cells. NCAM140 was expressed in several independent clones (designated TAM-67/NCAM140), although very surprisingly, NCAM180 was not expressed (Fig. 5B). Two independent clones of TAM-67/NCAM140 cells showed a marked resistance to SNP-induced apoptosis, compared with that in TAM-67 cells, which was similar to that in vector control cells (Fig. 5C), suggesting that NCAM140 plays a protective role in SH-SY5Y cells subjected to NO stress. In support of this, microscopic examination revealed that NO-induced apoptosis was dramatically lower in TAM-67/NCAM140 and vector control cells than in TAM-67 cells (Fig. 6).



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  		FIG. 6. NCAM140 rescues TAM-67 cells from SNP-induced apoptosis. Vector control SH-SY5Y cells, TAM-67 cells, and two clones of TAM-67/NCAM140 cells were treated with 1.5 mM SNP for 16 h. Cells were photographed (magnification, x40). Rounded and shrunken cells are apoptotic. Cell death was quantitated by trypan blue exclusion and is presented in Fig. 5C.

We finally investigated whether constitutive expression of NCAM140 also protects TAM-67 cells from apoptosis induced by other oxidative stressors and different classes of apoptosis-inducing agents. The protein kinase inhibitor H-7 induces apoptosis in SH-SY5Y cells at concentrations ranging from 20 to 100 µM (46). Similar to the results with SNP and SIN-1, TAM-67 cells were more sensitive to H-7 than parental SH-SY5Y cells (Fig. 7A). Moreover, two independent clones of TAM-67/NCAM140 were significantly more resistant to H-7-induced apoptosis than TAM-67 cells, exhibiting sensitivities comparable to parental SH-SY5Y cells (Fig. 7B). Although SH-SY5Y cells are sensitive to UV light, staurosporine, and hydrogen peroxide, we consistently found no significant differences in the sensitivity of SH-SY5Y, TAM-67, and TAM-67/NCAM140 cells to these agents (data not shown). Thus, NCAM140 appears to afford TAM-67 cells significant protection from apoptosis induced by some, but not all, classes of apoptosis inducers.



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  		FIG. 7. NCAM140 counteracts H-7-induced apoptosis. (A) Vector control cells and TAM-67 cells were either left untreated or treated with the indicated micromolar concentrations of the protein kinase inhibitor H-7 for 16 h, and cell death was quantitated using trypan blue exclusion. SD, standard deviation. (B) Cell death assays of H-7-treated vector control cells, TAM-67 cells, and two clones of TAM-67/NCAM140 cells (TAM-67/NCAM140-1 and TAM-67/NCAM140-2). Cells were either left untreated or treated with the indicated micromolar concentrations of H-7 for 16 h. Cell death was measured using trypan blue exclusion.

NCAM140 and c-Jun/AP-1 are required for NGF-induced neuronal differentiation of SH-SY5Y cells. In the nervous system, different species of AP-1 are activated during neuronal regeneration and differentiation (19, 25) but their precise involvement in these processes is not established. The SH-SY5Y neuroblastoma cell line represents a well-characterized model of neuronal differentiation (40). SH-SY5Y cells can be terminally differentiated into neurons with NGF (29), but it is not known if NGF induces AP-1 and whether AP-1 in turn regulates neuronal differentiation. Electrophoretic mobility shift assays showed that AP-1 DNA binding activity was indeed induced in SH-SY5Y cells 4 to 8 h after NGF stimulation (Fig. 8A), long before neuronal differentiation was evident. Vector control SH-SY5Y cells treated with NGF for 4 days showed obvious neurite extensions and after 8 to 10 days showed classical neuronal features, such as shrinkage of the cell body and a typical network of neurite connections (Fig. 9). In contrast, few if any TAM-67 cells differentiated into neurons under the same conditions (Fig. 9). Identical results were obtained with several independent TAM-67 clones (data not shown).



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  		FIG. 8. NCAM140 is required for NGF-induced Bcl-2 up-regulation in SH-SY5Y cells. (A) NGF (1 µg/ml) activates AP-1 DNA binding activity in SH-SY5Y cells. Nuclear extracts were prepared at the indicated times after NGF treatment. DNA band shift analyses were performed using an oligonucleotide containing AP-1 sites, and the AP-1 complexes were analyzed in 4% native polyacrylamide gels. (B and C) Western blot analyses of Bcl-2 expression (B) or NCAM expression (C) in vector control SH-SY5Y, TAM-67, and TAM-67/NCAM140 cells. Cytoplasmic proteins were prepared at the indicated numbers of days after NGF treatment.



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  		FIG. 9. NCAM140 is required for NGF-induced neuronal differentiation of SH-SY5Y cells. All cells were treated with 1 µg of NGF/ml plus aphidicolin for 8 days and then photographed (magnification, x40).

There are many reports that the antiapoptotic protein Bcl-2 is elevated during differentiation of neuronal cells, including SH-SY5Y, and it has been observed that Bcl-2 regulates neurite outgrowth and neuronal differentiation in at least some cell types (21, 27, 31). Bcl-2 up-regulation is generally considered one biochemical marker of neuronal differentiation (21). Western blot analysis demonstrated that Bcl-2 was steadily up-regulated at day 4 through day 8 following treatment of SH-SY5Y cells with NGF (Fig. 8B). Under the same conditions, Bcl-2 was not up-regulated by NGF in TAM-67 cells and had disappeared by 8 days (Fig. 8B). Similar results were obtained with two other independent clones. Together, these results show that c-Jun/AP-1 is required for NGF-induced neuronal differentiation as judged by both morphological and biochemical criteria.

NCAM plays an important role in axonal growth (59). Cell surface NCAM binding inhibits proliferation of neurons and promotes their differentiation (1), and NCAM is down-regulated during terminal neuronal differentiation (41). Does the switch from NCAM140 to NCAM180 in TAM-67 cells (in which c-Jun/AP-1 is blocked) account for the failure of these cells to differentiate? Figure 8C shows that NCAM140 and NCAM180 were up-regulated in vector control SH-SY5Y cells by 4 days after NGF treatment but that both had dramatically declined by day 8. In NGF-treated TAM-67 cells, there was no obvious expression and elevation of NCAM140, but the high expression of NCAM180 was maintained from day 0 until day 4 though it was virtually undetectable at day 8 after NGF addition (Fig. 8C). To determine whether the expression and induction of NCAM140 plays any role in neuronal differentiation, TAM-67/NCAM140 stable cell lines were incubated with NGF for up to 8 days. NCAM140 expression increased from day 0 to day 4 after NGF treatment, as it did for the vector control cells (Fig. 8C). These TAM-67/NCAM140 cells (Fig. 9) underwent neuronal differentiation that was indistinguishable from that in vector control SH-SY5Y cells (Fig. 9). Interestingly, the levels of Bcl-2 (the biochemical marker of neuronal differentiation) were also clearly restored and even increased by NGF by day 8 in TAM-67/NCAM140 cells (Fig. 8B) whereas NCAM180 was undetectable at all times (Fig. 8C). Similar results were obtained with two other independent clones. Thus, NCAM140 (positively regulated by c-Jun/AP-1) is the NCAM species that is required for NGF-induced neuroblastoma cell differentiation.


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DISCUSSION
 
Oxidative stress, including NO toxicity, can activate AP-1 and kill neuronal cells, but so far the relevant functions and target genes of AP-1 have not been elucidated (7, 55). In particular, it is unclear whether AP-1 family members facilitate cell death or, conversely, are part of a protective response that attempts to counteract neuronal apoptosis. Our data suggest that c-Jun/AP-1 plays a protective role by regulating the expression of one or more specific target genes. An antiapoptotic role for as-yet-uncharacterized AP-1 transcription factors has also been found in NO-induced macrophage apoptosis, UV-induced apoptosis of fibroblasts, and okadaic acid-induced cell death of PC12 neuronal cells (19, 44, 58, 60). Thus, AP-1 factors may play a more general role in counteracting diverse external stresses (35).

Which are the AP-1 target genes in SH-SY5Y cells that prevent NO-induced apoptosis? We found no evidence that AP-1 counteracts NO-induced apoptosis merely by its ability to regulate cell proliferation because the growth rates of TAM-67 and SH-SY5Y cells were indistinguishable. Rather, our data strongly suggest that NCAM140 (but not NCAM180) is at least partly responsible for counteracting NO-induced apoptosis of SH-SY5Y cells. The reason why NCAM180 was present in TAM-67 cells but disappeared upon overexpression of NCAM140 is unknown, but it might conceivably be due to a negative feedback mechanism. The protective and differentiating effects of NCAM140 and the disappearance of NCAM180 in NCAM140-overexpressing cells were essentially the same in several independent TAM-67 clones, which argues against the possibility that nonspecific genetic mutations arose during cell selection and influenced NCAM synthesis.

AP-1 DNA binding activity was steadily induced in SH-SY5Y cells from about 2 to 8 h after the addition of SNP, yet NCAM140 and NCAM180 were differentially expressed in SH-SY5Y and TAM-67 cells even in the absence of SNP treatment. This suggests that basal AP-1 activity, undetectable by DNA band shift analysis, rather than inducible AP-1, is responsible for the differential expression of NCAM140 and NCAM180. The significance of SNP-inducible AP-1 remains to be understood, but it is possible that other as-yet-unidentified SNP-inducible genes contribute to AP-1-mediated neuroblastoma cell survival. Our study provides the first direct evidence that a specific NCAM species (NCAM140) can prevent oxidative stress-induced apoptosis of neuronal cells, although the induction of apoptosis by NCAM antibody cross-linking had already hinted that NCAM can regulate neuronal cell viability (3).

c-Jun/AP-1 controls neuronal proliferation, survival, and regeneration, but the role(s) of members of the AP-1 transcription factor complex in neuronal differentiation is unclear (25). Mice lacking c-Jun die while in the embryonic stage, and so the possible involvement of c-Jun in neuronal development cannot be fully explored (26). However, c-Fos- and ATF-2-null mice have severe neural abnormalities, suggesting a role for AP-1 family members in neuronal development and differentiation (30, 45). c-Jun/AP-1 appears to regulate neurite outgrowth (19, 36), but further studies were required to establish whether c-Jun/AP-1 is also required for terminal neuronal differentiation. NGF-treated SH-SY5Y cells undergo terminal differentiation into cells that show a typical neuronal phenotype (29). We employed morphological changes and Bcl-2 up-regulation as markers of neuronal differentiation in these cells (21, 41). We found that NGF promotes early activation of AP-1 after 4 to 8 h, which is well before the onset of NGF-induced neuronal differentiation. Our results show that c-Jun/AP-1-regulated expression of NCAM140 is essential for promoting neuronal differentiation according to the morphological and biochemical criteria mentioned above.

What is the molecular basis for the dual functions of NCAM140 in neuronal differentiation and protection from stress-induced apoptosis? NCAM140 might mediate these functions by homophilic or heterophilic interactions at the cell surface, by a unique signal transduction pathway, or by dual signaling pathways emanating from complexes of NCAMs with fibroblast growth factor receptors (FGFR) (51). We will consider mainly the latter two possibilities.

NCAM is a member of the immunoglobulin superfamily that promotes axonal growth, fasciculation, and cell adhesion (16, 59). Alternative splicing of pre-mRNA encoded by a single NCAM gene generates three major isoforms, two of which—the NCAM140 and the NCAM180 species—have extracellular, transmembrane, and cytoplasmic domains (16, 54). NCAM140 is identical to NCAM180 in the extracellular immunoglobulin-like domain but lacks a 261-amino-acid insert in the cytoplasmic domain that results from the splicing out of exon 18 (4, 17, 18). There is evidence that NCAM140 promotes neurite outgrowth in migrating growth cones and may be more potent in this respect than NCAM180 (17, 33). Little is known about intracellular signaling mediated by the individual NCAM cytoplasmic domains. NCAM140 (but not NCAM180) binds the nonreceptor tyrosine kinase p59fyn and the focal adhesion kinase p125fak; subsequently, both kinases are phosphorylated (5). Conversely, NCAM180, but not NCAM140, binds cytosolic spectrin and reduces the lateral mobility of NCAM in the plasma membrane (5, 42). NCAM140-dependent neurite outgrowth is impaired in fyn-minus neurons, suggesting that fyn is involved in neuronal differentiation (6). There is some evidence that NCAM140 signaling involves the Ras-mitogen-activated protein (MAP) kinase pathway via extracellular-signal-regulated protein kinase 1 (ERK1) and ERK2 activation, perhaps involving fyn (16, 33, 48). Thus, the abolition of NCAM140 expression in our TAM-67 cells might prevent a signaling pathway required for neurite outgrowth that cannot be substituted by NCAM180.

A role for NCAM140 signaling in the suppression of NO-induced apoptosis is harder to understand, as NCAM140 has not previously been shown to be involved in cell survival per se. However, it is well established that p125fak can mediate death protection in a variety of cell types (10). Especially interesting is the finding that p125fak is upstream of a phosphatidylinositol 3'-kinase survival pathway in the hydrogen peroxide-induced apoptosis of a glioblastoma cell line (52). It is also relevant that the MAP kinases ERK1 and ERK2 are selectively phosphorylated after NCAM140 stimulation (48) because ERK kinases have been implicated in resistance to apoptosis in neuronal cells (55, 61). Future investigations are required to determine whether p125fak, phosphatidylinositol 3'-kinase(s), and the MAP kinases are important in NCAM140-mediated suppression of NO-induced apoptosis.

There is considerable evidence that neurite outgrowth stimulated by NCAM molecules is dependent on the tyrosine kinase activity of the FGFR in neurons (51). NCAM-stimulated neurite outgrowth was shown to involve both a Ras-MAP kinase pathway and FGFR-dependent signaling via phospolipase C{gamma} and protein kinase C (32). In pancreatic tumor cells, surface NCAM binds to and stimulates the FGFR-4 tyrosine kinase, which activates many signaling molecules including phospolipase C{gamma} and protein kinase C, culminating in the induction of ß1-integrin-dependent cell matrix adhesion (11). It is quite possible that activation of similar NCAM/FGFR complexes also occurs in neuronal cells and contributes to neuronal differentiation via ß1-integrin-dependent cell matrix contacts. Because FGF signaling and cell adhesion can both independently promote cell viability, we speculate that NCAM and FGFR might also cooperate to ensure neuronal cell survival. It will be interesting to know if the NCAM140 isoform is involved in FGFR-dependent signaling in neuronal cell survival and differentiation.

The genetic regulation of the various isoforms of NCAM is poorly understood. We demonstrated that suppression of c-Jun/AP-1 in TAM-67 cells switched the synthesis of NCAM140 to NCAM180 at both the protein and mRNA levels. As alternative splicing of pre-mRNA encoded by a single NCAM gene generates NCAM140 and NCAM180, it is quite possible that c-Jun/AP-1 controls the production or activity of a splicing factor(s) that is required to generate NCAM140 mRNA. There are at least seven SR proteins which regulate alternative splicing (12), and it is noteworthy that SR proteins modulate NCAM pre-mRNA splicing in an in vitro system (14). Conceivably, c-Jun/AP-1 might regulate SR proteins that in turn determine the relative abundance of NCAM140 and NCAM180 in neuronal cells. This would provide a novel mechanism whereby c-Jun/AP-1 regulates NCAM140, both of which are required for NGF-induced neuronal differentiation and resistance to apoptosis induced by NO donors in SH-SY5Y neuroblastoma cells.


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ACKNOWLEDGMENTS
 
We thank E. Feldman, University of Michigan, for SH-SY5Y cells; R. Jaggi, University of Bern, Bern, Switzerland, for the plasmid encoding the dominant-negative c-Jun (TAM-67); Christiane Volbracht and Hannes Hentze for helpful comments; and Hannes Hentze for help with the artwork.

This work was generously supported by the Institute of Molecular and Cell Biology, Singapore.

A.G.P. is an adjunct staff member of the Department of Surgery, National University of Singapore.


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FOOTNOTES
 
* Corresponding author. Mailing address: Institute of Molecular & Cell Biology, 30 Medical Dr., Singapore 117609, Republic of Singapore. Phone: 6874-3761. Fax: 6779-1117. E-mail: mcbagp{at}imcb.nus.edu.sg. Back


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Molecular and Cellular Biology, August 2002, p. 5357-5366, Vol. 22, No. 15
0270-7306/02/$04.00+0     DOI: 10.1128/MCB.22.15.5357-5366.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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