TNF-α contributes to caspase-3 independent apoptosis in neuroblastoma cells: role of NFAT

Abstract

There is increasing evidence that soluble factors in inflammatory central nervous system diseases not only regulate the inflammatory process but also directly influence electrophysiological membrane properties of neurons and astrocytes. In this context, the cytokine TNF-α (tumor necrosis factor-α) has complex injury promoting, as well as protective, effects on neuronal viability. Up-regulated TNF-α expression has also been found in various neurodegenerative diseases such as cerebral malaria, AIDS dementia, Alzheimer's disease, multiple sclerosis, and stroke, suggesting a potential pathogenic role of TNF-α in these diseases as well. We used the neuroblastoma cells SK-N-MC. Transcriptional activity was measured using luciferase reporter gene assays by using lipofectin. We performed cotransfection experiments of NFAT (nuclear factor of activated T cells) promoter constructed with a dominant negative version of NFAT (dn-NFAT). Cell death was performed by MTT (3-(4,5-dimethylthiazol-2-yl)5,5-diphenyltetrazolium bromide) and TUNEL assays. NFAT translocation was confirmed by Western blot. Involvement of NFAT in cell death was assessed by using VIVIT. P53, Fas-L, caspase-3, and caspase-9 expressions were carried out by Western blot. The mechanisms involved in TNF-α-induced cell death were assessed by using microarray analysis. TNF-α causes neuronal cell death in the absence of glia. TNF-α treatment results in nuclear translocation of NFAT through activation of calcineurin in a Ca(2+) independent manner. We demonstrated the involvement of FasL/Fas, cytochrome c, and caspase-9 but the lack of caspase-3 activation. NB cell death was absolutely reverted in the presence of VIVIT, and partially diminished by anti-Fas treatment. These data demonstrate that TNF-α promotes FasL expression through NFAT activation in neuroblastoma cells and this event leads to increased apoptosis through independent caspase-3 activation.

Figure 1. TNF-α causes cell death of NB cells in the absence of glia.

A) SK-N-MC cells were treated with TNF-α (2, 5, 10, 20, and 40 ng/ml) and examined using the MTT assay after 24 h. Data are mean ± SD of three independent experiments performed in triplicate. Significant difference from controls: *p<0.01. B) Apoptosis determined by TUNEL. SK-N-MC cells were stained and TUNEL visualized under a fluorescence microscope with either 100 magnification. Bar: 50 µm.

Figure 2. Activation of NFAT activity in human NB cells by TNF-α.

A) NB cells were transfected with a NFAT reporter plasmid, and then stimulated by TNF-α at different doses. Luciferase activity was measured 16 h later. Results represent the mean ± SD of three independent experiments performed in triplicate. Significant difference from control: *p<0.01. B) NB cells were transfected with the NFAT reporter plasmid, and treated with TNF-α (20 ng/ml) in the presence of anti-TNF-α (10 µg/ml). Activity of NFAT-luc was measured after 16 h. Data are mean ± SD of three independent experiments performed in triplicate. Significant difference from stimulated cells: *p<0.01. C) SK-N-MC cells were transfected with the NFAT-luc reporter plasmid promoter construct plus an empty vector or an expression vector for a dominant negative (dn) of NFAT. Cells were treated with TNF-α (20 ng/ml) for 16 h and assayed for luciferase activity. Data are from three independent experiments, presented as mean ± SD. Significant difference between stimulated cells: **p = 0.05.

Figure 3. TNF-α induces Calcineurin Aα expression.

Top, representative immunoblots probed with antibodies against CaNα in NB cultures at different time set points after TNF-α treatment (20 ng/ml). The membrane was reprobed with α-tubulin antibody to confirm equal protein loading. Bottom, quantitation of CaNα levels was performed by densitometry. Values represent means ± SD of data from two independent experiments. Significant difference from control: *p<0.01.

Figure 4. Role of NFAT on TNF-α-mediated cell death.

A) Nuclear translocation of NFAT in TNF-α-stimulated SK-N-MC cells. Top, Western blot analysis of fractionated extracts from SK-N-MC cells incubated with TNF-α (20 ng/ml) for the indicated times. NFAT was detected with the anti-NFAT 672 antiserum. Gels shown are representative of 3 independent experiments. Antibodies directed against α-tubulin/Lamin B1 were used as a protein loading control. Each data point is the mean of three replications. Densitometric analysis was used to determine the level of cytoplasmic and nuclear lysates. B) Neuronal death induced by TNF-α is reverted by NFAT inhibition. SK-N-MC cells were untreated (control) or treated with TNF-α (20 ng/ml) alone or in combination with CsA (100 ng/ml) or VIVIT (2 µM) for 24 h before measuring cellular death by MTT. The results represent the means ± SD of three independent experiments performed in triplicate. Significant difference from TNF-α-stimulated cells: *p<0.01. DMSO 10% was used as a positive control of cell death. C) Apoptosis determined by TUNEL. SK-N-MC cells were stained with TUNEL and visualized under a fluorescence microscope with either 100 magnification. Bar: 50 µm. D) Human PBLs were incubated with TNF-α in the presence of VIVIT or PDTC. MTT activity was measured 24 h after. The results represent the means ± SD of three independent experiments performed in triplicate. DMSO 10% was used as a positive control of cell death.

Figure 5. FasL induction by TNF-α in SK-N-MC cells.

A) The level of FasL is increased in TNF-α treated NB cells. Protein lysates were prepared from NB cells 16 h after TNF-α stimulation. Top, lysates were analyzed by Western blot analysis by using antibody directed against FasL. Antibody directed against α-tubulin was used as a protein loading control. Bottom, densitometric analysis was used to determine the level of FasL expression in NB cells, with values normalized to tubulin levels. Error bars represent standard errors of the means. Significant difference from untreated cells: *p<0.01. B) SK-N-MC cells were pretreated with antiFas at the indicated doses before stimulation with TNF-α (20 ng/ml). Cell viability was determined using the MTT and LDH assays (C). The results represent the means ± SD of three independent experiments performed in triplicate. Significant difference from stimulated cells: **p = 0.01. D) Inhibition of TNF-α-induced apoptosis in SK-N-MC cells by the antagonistic peptide Kp7-6. SK-N-MC cells were stimulated with TNF-α in the presence or absence of Kp7-6 at the indicated doses. MTT activity was measured 24 h later. **p = 0.02.

Figure 6. The pathway was assembled using Ingenuity Pathways Analysis (Ingenuity Systems;www.ingenuity.com ), and it shows genes involved in both the extrinsic and intrinsic mechanisms of apoptosis.

Official gene symbols are used. Upregulation is represented by red, and downregulation is represented by green. Color intensity corresponds to the degree of differential regulation.

Figure 7. Effect of TNF-α on cytochrome c release, and cleavage of caspase-9 in SK-N-MC cells.

A) NB cells were incubated or not with TNF-α for the indicated times, and total extracts were immunoblotted with caspase-3 Ab. B) Caspase-3 was measured after 24 h of treatment by flow cytometry. Treatment with staurosporine 0.5-1 µM for 3 h was used as positive control. C) NB cells were incubated with TNF-α in the presence of CsA for 24 h. The mitochondria free cytosolic fractions were prepared for Western blot analysis as described under Experimental Procedures. α-tubulin was used as cytosolic marker and as control for protein loading. The graphs show the mean increase in cytochrome c/caspase-9 (D) activity levels in TNF-α treated cells compared to that of unstimulated controls. Porine antibody was used as a control to ensure proper fractionation and loading of mitochondrial pellet. Error bars represent standard errors of the means. Significant difference from stimulated cells: *p<0.01; **p = 0.01.

Figure 8. A proposed model for TNF-α-induced cell death in NB cells.