Neuronal differentiation and protection from nitric oxide-induced apoptosis require c-Jun-dependent expression of NCAM140

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.

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.

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.

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) β-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.

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.

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.

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, ×40). Rounded and shrunken cells are apoptotic. Cell death was quantitated by trypan blue exclusion and is presented in Fig. 5C.

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.

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.

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, ×40).