Characterization of transcriptional regulation of neurogranin by nitric oxide and the role of neurogranin in SNP-induced cell death: implication of neurogranin in an increased neuronal susceptibility to oxidative stress

Abstract

Neurogranin (Ng), a calmodulin (CaM)-binding protein kinase C (PKC) substrate, regulates the availability of Ca(2+)/CaM complex and modulates the homeostasis of intracellular calcium in neurons. Previous work showed Ng oxidation by NO donor induces increase in [Ca(2+)](i). The current study demonstrated that the gene transcription of Ng could be up-regulated by various nitric oxide (NO) donors via a NO-soluble guanylyl cyclase (sGC)-mediated pathway. Furthermore, ectopic expression of neuronal nitric oxide synthase (nNOS) in human embryonic kidney 293 cells (HEK 293) exhibited a nNOS-concentration-dependent biphasic regulatory effect on Ng gene transcription. One of the NO donors, sodium nitroprusside (SNP), however, induced cell death of neuroblastoma Neuro-2a cells. The potency of SNP-induced cell death was shown to be higher in Neuro-2a cells expressing recombinant Ng, as compared with Neuro-2a control cells without Ng expression in cell viability and apoptosis assays. Single-cell fluorescence imaging and site-directed mutagenesis studies suggest that Ng promotes SNP-induced cell death through an amplification of calcium-mediated signaling, which requires the interaction between CaM and IQ motif of Ng. Increased neuronal susceptibility rendered by Ng in response to pathophysiological NO production is suggested to be involved in the selective vulnerability of neurons to oxidative insults in the CNS.

Figure 1

Analysis of Ng promoter activity upon NO donor stimulation in HEK 293 cells and Ng mRNA abundance in primary cortical neurons. (a) Ng promoter activity was measured in HEK 293 cells stimulated with various kinds of NO donors (indicated below each bar) at 200 μM concentration for 24 h. (b) Relative Ng mRNA abundance in 12-day cultured cortical neurons with NOR-3 treatment for 24 h was assessed by real-time PCR. PCR results were represented as relative folds of mRNA level over non-treated group (arbitrarily assigned as 1 fold). (c) Ng promoter activities were measured in HEK 293 cells with stimulation of ODQ alone or NOR-3 in the presence or absence of ODQ. Data are represented as means ± SEM (n=3) and tested by Student's t-test. ** p < 0.01, *** p < 0.001 (compared with the control group).

Figure 2

nNOS exhibits a dose-dependent biphasic effect on Ng promoter activity. (a) Ng promoter activities were measured in HEK 293 cells with cotransfection of indicated concentrations of pcDNA3.1-nNOS. The total amount of plasmid transfected was normalized with pcDNA3.1 vector. Data are shown as means ± SEM (n=6) and processed with one-way ANOVA followed by post-hoc analyses of Student's t-test. *** p< 0.001 (compared with the control pcDNA3.1 vector group). (b) Representative nNOS protein expression levels in HEK 293 cells cotransfected with pcDNA3.1 vector in lane 1, 3 μg, or pcDNA3.1-nNOS plasmid in: lane 2, 0.5 μg; lane 3, 1 μg; lane 4, 2 μg and lane 5, 3 μg. Aliquots of extracted protein were used either for luciferase measurement as shown in panel (a), or for Western blotting using nNOS antibody as shown in this panel. Western blotting for α-tubulin was used as a loading control.

Figure 3

SNP suppresses Ng promoter activity in the presence of Ng protein. (a) Ng promoter activities were measured in Neuro-2a and clone1 cells with stimulation of indicated concentrations of 4 h SNP treatment. Data were processed by one-way ANOVA and represented as means ± SEM (n=3). *** p < 0.001 (compared with clone1 control). (b) Ng promoter activities were measured at various time points in indicated cell lines with either PBS or 200 μM SNP treatment. PBS or SNP were applied to cultures 20 h post-transfection and incubated for 4 h followed by replacing with fresh medium. Data are shown as means ± SEM (n= 6).

Figure 4

Ng promotes Neuro-2a cell death induced by prolonged SNP or A23187 stimulation. (a) Cell viability of Neuro-2a and clone1 cells was determined by MTT assay after 12 h stimulation with various concentrations of SNP. (b) Detection of DNA fragmentation induced by prolonged SNP stimulation (12 h) in Neuro-2a and clone1 cells. Lanes 1, 3, 5, 7 correspond to Neuro-2a cells treated with 0.2, 2, 20, 200 μM SNP, respectively. Lanes 2, 4, 6, 8 correspond to clone1 cells treated with 0.2, 2, 20, 200 μM SNP, respectively. Lane 9, 1 kb DNA ladder. (c) Cell viability of Neuro-2a and clone1 cells were determined by MTT assay after 24 h stimulation with various concentrations of A23187. For panel a and c, the cell viability was calculated by the following equation: MTT OD value of sample/MTT OD value of control (cell treated with PBS). Data are shown as means ± SEM (n=6).

Figure 5

A functional IQ motif in Ng is required for promoting intracellular calcium increase and NO-induced cell death. (a) Following transfection with different forms of Ng cDNA constructed into pIRES2-EGFP vector, Neuro-2a cells were treated with 200 μM SNP for 12 h. Cell viability was measured by MTT reduction assay. Data were normalized by densitometric scanning of film from Western blotting against EGFP. Data were represented as % of the control (normalized MTT reading from Neuro-2a cells transfected with pIRES2-EGFP vector). Data shown are means ± SEM (n=6). *** p < 0.001 (compared with the vector control by Student's t-test). Transient expression of variations of Ng in Neuro-2a cells were resolved on 10% to 20% gradient non-reduced PAGE and subjected to Western blotting against Ng antibodies. Lane 1, transfected with pIRES2-EGFP vector; 2, transfected with WT Ng cDNA; 3, transfected with I33Q Ng cDNA; 4, transfected with Cys mutant Ng cDNA. (b) Following transfections, Neuro-2a cells were loaded with x-rhod-1, and successfully transfected cells were identified by a green fluorescence filter and chosen as region of interest (ROI). Fluorescence changes were monitored using a filter for x-rhod-1. Representative normalized (F/F₀) fluorescence changes versus time are shown in the line plot. Arrow indicates the SNP application time. The bar chart shows averaged peak fluorescence changes from various transfections indicated in response to 200 μM SNP. Data shown are means ± SEM (n=9 for all transfections). ***p < 0.001 obtained from Student's t test (compared with the data from pIRES2-EGFP transfection).

Figure 6

A hypothetic role of Ng in NMDA receptor-mediated neuronal death. The activation of NMDA receptor by excitatory presynaptic signal cause Ca²⁺ transient in neuron which can be amplified by the 'mass action' of Ng. The overacting Ca²⁺ transients induce neuronal death by itself or cause the dissociation of CaM from Ng and form more Ca²⁺/CaM complex which sustains the NMDA-mediated activation of nNOS which implicates dephophorylation of nNOS by calcineurin. This sustained over-activation of nNOS then produce overdose NO which induces neuronal apoptosis.