Cholinergic stimulation of AP-1 and NF kappa B transcription factors is differentially sensitive to oxidative stress in SH-SY5Y neuroblastoma: relationship to phosphoinositide hydrolysis

Abstract

Oxidative stress appears to contribute to neuronal dysfunction in a number of neurodegenerative conditions, notably including Alzheimer's disease, in which cholinergic receptor-linked signal transduction activity is severely impaired. To test whether oxidative stress could contribute to deficits in cholinergic signaling, responses to carbachol were measured in human neuroblastoma SH-SY5Y cells exposed to H2O2. DNA binding activities of two transcription factors that are respondent to oxidative conditions, AP-1 and NF kappa B, were measured in nuclear extracts. H2O2 and carbachol individually induced dose- and time-dependent increases in AP-1 and NF kappa B. In contrast, when given together, H2O2 concentration dependently (30-300 microM) inhibited the increase after carbachol in AP-1. Carbachol's stimulation of NF kappa B was not inhibited except with a high concentration (300 microM) of H2O2, which was associated with impaired activation of protein kinase C. Lower concentrations of H2O2 (30-300 microM) inhibited carbachol-induced [3H]phosphoinositide hydrolysis, and this inhibition correlated (r = 0.95) with the inhibition of carbachol-induced AP-1. Activation [3H]phosphoinositide hydrolysis by the calcium ionophore ionomycin was unaffected by H2O2, indicating that phospholipase C and phosphoinositides were impervious to this treatment. In contrast, activation with NaF of G-proteins coupled to phospholipase C was concentration dependently inhibited by H2O2, indicating impaired G-protein function. These effects of H2O2 are similar to signaling impairments reported in Alzheimer's disease brain, which involve deficits in receptor- and G-protein-stimulated phosphoinositide hydrolysis, but not phospholipase C activity. Thus, these findings indicate that oxidative stress may contribute to impaired phosphoinositide signaling in neurological disorders in which oxidative stress occurs, and that oxidative stress can differentially influence transcription factors activated by cholinergic stimulation.

Fig. 1.

Concentration-dependent effects of H₂O₂ on AP-1 (A) and NFκB (B) DNA binding activities. SH-SY5Y cells were exposed to 30–300 μm H₂O₂ for 2 hr. Cells were harvested, nuclear extracts were prepared, and AP-1 and NFκB DNA binding activities were measured by EMSA as described in Materials and Methods. Two NFκB bands were resolved and are designated as UPPER and LOWER to indicate the slower and faster mobilities, respectively. Values are given as the percent of controls that were not exposed to H₂O₂. Mean ± SEM. n = 6–7. *p < 0.05 compared with control values (ANOVA with a post hoc Dunnett multiple comparisons test).

Fig. 2.

Carbachol-stimulated AP-1 and NFκB DNA binding activities. SH-SY5Y cells were treated without (Control) or with 1 mm carbachol for 1 hr, followed by EMSA measurements of AP-1 (A) and NFκB (B) in nuclear extracts. Arrows indicate the single AP-1 band and the two NFκB bands. Where indicated, reaction mixtures contained 100-fold excess unlabeled AP-1 or NFκB oligonucleotides. Supershifted (SS) bands are indicated in samples that were incubated with antibodies to the Fos family of proteins, p65 or p50. ns, Nonspecific.

Fig. 3.

Carbachol concentration-dependent (A–D) and time-dependent (E) activation of AP-1 (A, C) and NFκB (B, D). SH-SY5Y cells were treated with 3 × 10⁻⁷ to 10⁻³mcarbachol (A–D) for 1 hr (n = 3–4) or with 10⁻³m carbachol (E) for 0.25–20 hr (n = 3–4). Cells were harvested, and AP-1 and NFκB DNA binding activities were measured in nuclear extracts as described in Materials and Methods. Both bands of NFκB were measured together to calculate overall stimulation caused by carbachol. Values are given as the percent of untreated cells (controls). Mean ± SEM. *p < 0.05 compared with control values (no carbachol) (ANOVA with a post hocDunnett multiple comparisons test).

Fig. 4.

Modulation by H₂O₂ of carbachol-stimulated AP-1 (A, B) and NFκB (B–D) DNA binding. SH-SY5Y cells were incubated with the indicated concentration (30–300 μm) of H₂O₂ for 1 hr followed by addition of 1 mm carbachol for an additional hour. Cells were harvested, and AP-1 and NFκB DNA binding activities were measured as described in Materials and Methods. Values in A, C, and D are given as the percent of controls that were not exposed to H₂O₂, or, in B, as the percent of values obtained with carbachol in the absence of H₂O₂. Mean ± SEM (n = 3–7). *p < 0.05 compared with carbachol stimulation in the absence of H₂O₂ (ANOVA with a post hoc Dunnett multiple comparisons test).

Fig. 5.

Time dependence of the inhibition by H₂O₂ of carbachol-stimulated AP-1. SH-SY5Y cells were incubated with 150 μmH₂O₂ for 0–2 hr before the addition of 1 mm carbachol. After 1 hr exposure to carbachol, AP-1 DNA binding activity was measured in nuclear extracts as described in Materials and Methods. Values are given as the percent of AP-1 in untreated cells. Mean ± SEM. *p < 0.05 compared with control values for basal or with carbachol alone (no H₂O₂) (ANOVA).

Fig. 6.

MTT reduction in SH-SY5Y cells. Cells were incubated with 50, 100, or 300 μmH₂O₂ for 15, 30, 60, or 120 min followed by measurement of MTT reduction as described in Materials and Methods. Data are expressed as the percent of controls that were not exposed to H₂O₂. Mean ± SEM (n = 4).

Fig. 7.

Time-dependent activation of AP-1 (A) and NFκB (B, C) by PMA. SH-SY5Y cells were incubated with 1 μm PMA, and AP-1 and NFκB DNA binding activities were measured in nuclear extracts as described in Materials and Methods. Values are given as the percent of controls that were not exposed to PMA. Mean ± SEM (n = 4).

Fig. 8.

Modulation by H₂O₂ of PMA-stimulated AP-1 (A) and NFκB (B) DNA binding. SH-SY5Y cells were incubated for 1 hr with 30–300 μm H₂O₂ followed by incubation for 2 hr with 1 μm PMA, and AP-1 and NFκB DNA binding were measured in nuclear extracts as described in Materials and Methods. Values are given as the percent of those obtained with PMA in the absence of H₂O₂ (as shown in Fig. 6). Mean ± SEM (n = 5). *p < 0.05 compared with cells treated with PMA alone (no H₂O₂) (ANOVA).

Fig. 9.

PMA-induced translocation of protein kinase C-α.A, SH-SY5Y cells were incubated with 0.1 μm PMA for 5, 10, 15, 20, and 30 min, membrane (M) and cytosolic (C) fractions were prepared, and protein kinase C-α was measured by immunoblotting as described in Materials and Methods. B, The immunoblots of membrane and cytosol protein kinase C-α were quantitated by densitometry, and data were calculated as percentage of maximal protein kinase C-α (30 min PMA treatment for the membrane fraction and no PMA treatment for the cytosol fraction).n = 3 for both membrane and cytosol protein kinase C-α using samples from three individual experiments.

Fig. 10.

Inhibition by H₂O₂ of PMA-induced translocation of protein kinase C-α. SH-SY5Y cells were incubated with 300 μm H₂O₂ for 45 min followed by the addition of 0.1 μm PMA. After 15 min, protein kinase C-α was measured in the membrane (M) and cytosol (C) fractions. Data were calculated as the percent of maximal protein kinase C (30 min PMA treatment for the membrane fraction and no PMA treatment for the cytosol fraction). Mean ± SEM (n = 3). *p < 0.05 compared with cells not exposed to H₂O₂ (paired t test).

Fig. 11.

Inhibition by H₂O₂ of [³H]phosphoinositide hydrolysis. SH-SY5Y cells were prelabeled with [³H]inositol for 48 hr, resuspended in assay buffer, incubated for 10 min with the indicated concentration of H₂O₂ followed by the addition of 1 mm carbachol (A), 20 mm NaF (plus 10 μm AlCl₃) (B), or 50 μm ionomycin (C). After an additional incubation for 30 min, [³H]inositol monophosphate was measured as described in Materials and Methods. Values with each stimulant were corrected for basal [³H]inositol monophosphate production in each experiment, which are shown withsquares. D, [³H]phosphoinositide hydrolysis was calculated as the percent of control values obtained from cells exposed to each stimulant in the absence of H₂O₂. Mean ± SEM (n = 8–9). *p < 0.05 compared with agonist stimulation in the absence of H₂O₂(ANOVA with a post hoc Bonferroni test).