 |
 Previous Article | Next Article 
Volume 16, Number 19,
Issue of October 1, 1996
pp. 5914-5922
Copyright ©1996 Society for Neuroscience
Cholinergic Stimulation of AP-1 and NF B Transcription Factors
Is Differentially Sensitive to Oxidative Stress in SH-SY5Y
Neuroblastoma: Relationship to Phosphoinositide Hydrolysis
Xiaohua Li,
Ling Song, and
Richard S. Jope
Department of Psychiatry and Behavioral Neurobiology, University of
Alabama at Birmingham, Birmingham, Alabama 35294-0017
 ABSTRACT
- INTRODUCTION
- MATERIALS AND METHODS
- RESULTS
- DISCUSSION
- FOOTNOTES
- REFERENCES
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 B, were measured in nuclear extracts.
H2O2 and carbachol individually induced dose-
and time-dependent increases in AP-1 and NFB. In contrast, when
given together, H2O2 concentration dependently
(30-300 µM) inhibited the increase after carbachol in
AP-1. Carbachol's stimulation of NFB was not inhibited except with
a high concentration (300 µM) of
H2O2, which was associated with impaired
activation of protein kinase C. Lower concentrations of
H2O2 (30-300 µM) inhibited
carbachol-induced [3H]phosphoinositide hydrolysis, and
this inhibition correlated (r = 0.95) with the
inhibition of carbachol-induced AP-1. Activation of
[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.
Key words:
oxidative stress;
transcription factors;
AP-1;
NFB;
phosphoinositide;
cholinergic signaling
INTRODUCTION
Oxidative stress appears to be one of the primary
factors contributing to neuronal dysfunction in a number of
debilitating conditions, potentially including Alzheimer's disease,
aging, and epilepsy (Halliwell, 1992 ; Coyle and Puttfarcken, 1993;
Shigenaga et al., 1994). Activation of two transcription factors, AP-1
and NFB, represents cellular signaling processes that are
particularly responsive, or susceptible, to oxidative stress (Abate et
al., 1990; Staal et al., 1990). Thus, it has been reported that
exposure of a variety of cell types to oxidants induces increases in
both AP-1 and NFB DNA binding, as measured by the electrophoretic
mobility shift assay (EMSA) (for review, see Karin and Smeal, 1992;
Siebenlist et al., 1994). Therefore, activation of these two
transcription factors comprises potentially important signaling
pathways for mediation of cellular responses to oxidative stress, which
can range from adaptive mechanisms, such as the induction of
antioxidant enzymes, to terminal signals leading to cell death.
Activation of AP-1 and NFB in many cases constitutes a downstream
consequence of signaling pathways that activate protein kinase C (for
review, see Karin and Smeal, 1992; Siebenlist et al., 1994). For
example, the phosphoinositide signal transduction system, in which
receptors coupled to the G-proteins Gq and G11 stimulate phospholipase
C to hydrolyze phosphoinositides, activates protein kinase C subsequent
to diacylglycerol production from the cleaved phosphoinositides
(Fisher, 1995). Protein kinase C activates both AP-1, by altering the
phosphorylation state of the Jun and Fos immediate early gene proteins,
and NFB, by phosphorylating the inhibitory protein IkB and reducing
its interactions with the transcription factor proteins (Karin and
Smeal, 1992; Finco and Baldwin, 1995). Thus, in many cells agents that
activate phosphoinositide hydrolysis have been shown to stimulate
protein kinase C and to increase AP-1 and NFB DNA binding
activities as measured by the EMSA with nuclear extracts. Both
the AP-1 and NFB transcription factors are composed of protein
dimers. Members of the Jun and Fos protein families interact to form a
heterogeneous mixture of AP-1 dimers, which are usually detected as a
single band using the EMSA. NFB consists of dimers of two families
of proteins, exemplified by p50 and p65 (RelA), which are often
resolved into two or more bands by the EMSA.
The purpose of this investigation was to test whether oxidative stress
modulated the activation of AP-1 and NFB transcription factors
induced by stimulation of the phosphoinositide signal transduction
system in neuronal cells. Focus was placed on identifying the
consequences of oxidative stress on stimulation by carbachol of
cholinergic muscarinic receptor-associated responses. This is of
special interest because oxidative stress has been linked to neuronal
dysfunction in Alzheimer's disease (Behl et al., 1994; Friedlich and
Butcher, 1994; Hensley et al., 1994), in which there is a severe
impairment of cholinergic activity that includes a large deficit in
carbachol-induced phosphoinositide signaling (Greenwood et al., 1995)
(for review, see Jope, 1996). Thus, the goal was to determine whether
there could be a direct link between deficits in cholinergic
receptor-induced signaling and oxidative stress in Alzheimer's
disease. To test the possibility that oxidative stress may directly
impair responses to cholinergic stimulation, cultured human
neuroblastoma SH-SY5Y cells, which have been used widely to study
muscarinic receptor function (Fisher, 1995), were exposed to
H2O2 to induce oxidative stress,
and the effects on signaling processes leading to activation of the
AP-1 and NFB transcription factors were examined.
MATERIALS AND METHODS
Cell culture. Human neuroblastoma SH-SY5Y cells
(kindly provided by Dr. S. K. Fisher, University of Michigan) were
grown in 100 mm culture dishes (Corning, Corning, NY) in RPMI medium
(Cellgro, Herndon, VA) containing 5% fetal clone II (Hyclone, Logan,
UT), 10% horse serum, 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin. Cells were plated at a
density of 105 cells per dish and were harvested ~48-72
hr later, after the treatments described in Results.
MTT assay. The method of Hansen et al. (1989) was used to
measure MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium
bromide) reduction. Cells (7 × 104 cells/well) were
grown in 24-well plates for 48 hr, H2O2 (50, 100, or 300 µM) was added, and after 15, 30, 60, or 120 min MTT (1 mg/ml) was added. After 20 min incubation at 37°C, lysis
buffer was added (10% SDS, 25% dimethylformamide, pH 4.7). After
incubation overnight at 37°C, absorbance at 570 nm was measured in
duplicate with a microtiter plate reader. Data are expressed as the
percent of values obtained with cells not exposed to
H2O2.
EMSA. To prepare nuclear extracts, cells were washed two
times with PBS, and 4 ml of lysis buffer (10 mM Tris, pH
7.4, 3 mM MgCl2, 10 mM NaCl, 0.5%
NP40) was added. Lysed cells were centrifuged at 4000 × g for 5 min at 4°C. The pellet was resuspended in 30 µl
of buffer containing 20 mM HEPES, pH 7.9, 20% glycerol,
0.3 M NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 1 mM dithiothreitol (DTT), 0.1 mM  -glycerophosphate, 0.05 mM vanadate, 1 mM phenylmethylsulfonyl fluoride, and 1 µg/ml each of
pepstatin A, leupeptin, and aprotinin. After extraction on ice for 30 min, the samples were centrifuged at 16,000 × g for 15 min at 4°C. The supernatant containing nuclear proteins was
transferred to a microfuge tube, an aliquot was removed for
determination of the protein concentration (Bradford, 1976), and
samples were stored at  80°C.
EMSAs were performed using a double-stranded 15 base pair
oligonucleotide (5 -CTAGGGGGACTTTCC-3) containing the NFB consensus
sequence or an 18 base pair oligonucleotide (5-CTAGTGATGAGTCAGCCG-3)
containing the AP-1 consensus sequence, which was radiolabeled as
described previously (Unlap and Jope, 1995). For the binding reaction,
the nuclear protein extract (2 µg for AP-1; 10 µg for NFB) was
incubated in a total volume of 20 µl in binding buffer containing 20 mM HEPES, pH 7.9, 4% glycerol, 1 mM
MgCl2, 50 mM KCl, 0.5 mM DTT, 1 µg poly (dI-dC), and ~10,000 cpm radiolabeled DNA for 30 min at
4°C. Where indicated, reactions included 100-fold excess unlabeled
AP-1 or NFB oligonucleotides, and supershift analyses were performed
with antibodies to the Fos family of proteins (kindly provided by Dr.
M. J. Iadarola, National Institutes of Health), p65 (Rockland,
Gilbertsville, PA), or p50 (Rockland). DNA-protein complexes were
resolved on a preelectrophoresed 6% nondenaturing polyacrylamide gel
in 0.25 × TBE (22.3 mM Tris, 22.3 mM
boric acid, and 0.5 mM EDTA) at 4°C for 1.5 hr at 150 V. Subsequently, the gel was dried under vacuum and exposed to film. The
amount of DNA-protein complex present was analyzed using a Phosphor
Imager (Molecular Dynamics, Sunnyvale, CA), and specific bands were
identified by displacement of radioactivity with excess unlabeled
oligonucleotides and supershift analyses. Data from treated cells were
compared with controls using ANOVA.
Protein kinase C- translocation. SH-SY5Y cells were
treated with phorbol 12-myristate 13-acetate (PMA) as indicated in
Results, followed by two washes with ice-cold PBS. Cells were harvested
in TE buffer (10 mM Tris-Cl, pH 7.4, and 1 mM
EDTA), sonicated on ice for 15 sec, and centrifuged at 16,000 × g for 30 min at 4°C. The pellets were washed with TE
buffer, resuspended by sonication, and used as the membrane fractions.
The supernatants were centrifuged at 16,000 × g for 30 min at 4°C, and the resultant supernatants were used as the cytosol
fractions. Protein concentrations of both fractions were measured using
the Bradford protein assay (1976), and aliquots were solubilized in
Laemmli sample buffer (Laemmli, 1970) by boiling for 2 min. Proteins
(25 µg) from the membrane and cytosol fractions were resolved in 9%
SDS-polyacrylamide gels, transferred to nitrocellulose membranes, and
probed with an antibody to protein kinase C- (Life Technologies,
Gaithersburg, MD). Immunoreactive bands were analyzed by densitometry,
and statistical significance was determined using the paired Student's
t test.
Phosphoinositide hydrolysis. SH-SY5Y cells in 100 mm culture
dishes were prelabeled with 75 µCi/ml [3H]inositol
(American Radiolabeled Chemicals, St. Louis, MO) for 2 d at 37°C
in RPMI medium containing 5% fetal clone II, 10% horse serum, 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin. Labeled cells were washed and suspended in
Krebs'-bicarbonate-HEPES buffer (30 mM HEPES, pH 7.4, 122 mM NaCl, 3.6 mM NaHCO3, 1.2 mM MgCl2, 5 mM KCl, 1.3 mM CaCl2, 11 mM glucose) and washed
twice. Suspended cells (105 cells in 0.5 ml) were incubated
at 37°C for 10 min with or without the addition of
H2O2 followed by incubation with 1 mM carbachol, 20 mM NaF (with 10 µM AlCl3), or 50 µM ionomycin
(with 2.2 mM CaCl2) for 30 min. The reaction
was stopped by adding 1.7 ml of CHCl3:MeOH:12N HCl
(1:2:0.01). Inositol monophosphate, inositol, and lipids were
fractionated as described previously (Jope and Li, 1989). Radioactivity
was measured in each fraction, and data were analyzed using ANOVA with
a post hoc Bonferroni test.
RESULTS
Exposure of SH-SY5Y neuroblastoma cells to 30-300
µM H2O2 for 2 hr caused
concentration-dependent changes in AP-1 and NFB DNA binding, which
resulted in bell-shaped curves (Fig. 1). AP-1 was
stimulated by low concentrations of H2O2 to a
maximum of ~250% of control with 100 µM
H2O2, followed by a concentration-dependent
diminution in the stimulation of AP-1 to a minimum of 170% of control
with 300 µM H2O2. Two bands of
NFB DNA binding activity were apparent in the EMSA, designated as
upper and lower to indicate the bands with slower and faster
mobilities, respectively. NFB DNA binding activity responded to
H2O2 in a bell-shaped, concentration-dependent
manner similar to that observed with AP-1. Low concentrations of
H2O2 increased NFB to maximums of ~350 and
200% of control for the lower and upper NFB bands, respectively,
achieved with 100 µM H2O2,
followed by decreased stimulation with higher concentrations of
H2O2 and reduction to below control levels of
the upper band with 300 µM
H2O2.
Fig. 1.
Concentration-dependent effects of
H2O2 on AP-1 (A) and NFB
(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
NFB DNA binding activities were measured by EMSA as described in
Materials and Methods. Two NFB 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).
[View Larger Version of this Image (21K GIF file)]
Treatment of SH-SY5Y cells with carbachol increased AP-1 and NFB DNA
binding activities (Fig. 2). AP-1 was detected as a
single band in the EMSA, and it was virtually eliminated by inclusion
of excess unlabeled AP-1 oligimer in the binding reaction. Incubation
with an antibody to the Fos family of proteins supershifted the AP-1
band to one with slower mobility. NFB was detected as two major
bands that were increased after treatment of the cells with carbachol,
and the top one sometimes resolved into a doublet. These bands were
reduced in the presence of excess unlabeled NFB oligimer. Incubation
of nuclear extracts with an antibody to p65 reduced the intensity of
both NFB bands and produced a supershifted band with slower
mobility, and anti-p50 reduced the intensity of the lower NFB band
and two supershifted bands appeared.
Fig. 2.
Carbachol-stimulated AP-1 and NFB 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 NFB (B)
in nuclear extracts. Arrows indicate the single AP-1
band and the two NFB bands. Where indicated, reaction mixtures
contained 100-fold excess unlabeled AP-1 or NFB oligonucleotides.
Supershifted (SS) bands are indicated in samples that
were incubated with antibodies to the Fos family of proteins, p65 or
p50. ns, Nonspecific.
[View Larger Version of this Image (51K GIF file)]
Carbachol induced concentration-dependent and time-dependent induction
of AP-1 and NFB DNA binding activities in SH-SY5Y cells (Fig.
3). AP-1 was increased maximally to 1000% of control by
a 2 hr treatment with 30 µM carbachol with an
EC50 of ~2 µM carbachol. NFB DNA binding
(total of both bands) reached a maximum increase that was almost 300%
of control with 300 µM carbachol, and the
EC50 of carbachol was ~20 µM. Carbachol (1 mM) induced rapid increases in both transcription factors,
which reached peak levels after 2 hr of treatment. Afterward, AP-1
decreased relatively steadily over time, whereas NFB consistently
rebounded after an initial decrease, followed by a decline at 20 hr.
Fig. 3.
Carbachol concentration-dependent
(A-D) and time-dependent (E) activation
of AP-1 (A, C) and NFB
(B, D). SH-SY5Y cells were treated with
3 × 10 7 to 103 M
carbachol (A-D) for 1 hr (n = 3-4)
or with 103 M carbachol (E)
for 0.25-20 hr (n = 3-4). Cells were harvested,
and AP-1 and NFB DNA binding activities were measured in nuclear
extracts as described in Materials and Methods. Both bands of NFB
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 hoc
Dunnett multiple comparisons test).
[View Larger Version of this Image (36K GIF file)]
Although carbachol and H2O2 each individually
stimulated AP-1 DNA binding activity, treatment with
H2O2 inhibited carbachol-induced AP-1 (Fig.
4A) concentration dependently. The
responses to carbachol in the presence of the lower
H2O2 concentrations were especially remarkable
because H2O2 alone stimulated AP-1 to values up
to 250% of control (with 100 µM
H2O2), whereas the stimulation by carbachol of
AP-1 was concentration dependently decreased by
H2O2, with an almost 50% reduction in the
response to carbachol attained with 100 µM
H2O2. This represents a conservative estimate
of the H2O2-induced inhibition of the response
to carbachol because a portion of the AP-1 stimulation in the presence
of H2O2 plus carbachol is contributed by
H2O2. Higher concentrations of
H2O2 further decreased carbachol-stimulated
AP-1, and 300 µM H2O2 eliminated
stimulation by carbachol. These inhibitory effects of pretreatment with
H2O2 on the response to carbachol are shown in
Figure 3B, where values are expressed as the percent of the
response to carbachol in the absence of
H2O2.
Fig. 4.
Modulation by H2O2 of
carbachol-stimulated AP-1 (A, B) and
NFB (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 NFB 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).
[View Larger Version of this Image (33K GIF file)]
The interactions of carbachol and H2O2 on the
stimulation of NFB were quite distinct from those on AP-1 (Fig. 4).
For the lower NFB band, the effects of carbachol and
H2O2 were approximately additive of the
individual responses. Thus, H2O2 alone
maximally increased NFB (lower band) by 350% at a concentration of
100 µM, and 100 µM
H2O2 increased the response to carbachol by
>300%. Only at the highest concentration of
H2O2 (300 µM) was there clear
inhibition of the stimulation by carbachol. The response of the upper
band of NFB to H2O2 plus carbachol was more
difficult to discern because of the relatively small stimulation
produced by each agent individually. There appeared to be a slight
inhibitory effect of H2O2 on the response to
carbachol because the individual responses were less than additive, but
this was not clearly evident except with the highest concentrations of
H2O2.
Examination of the pretreatment time-dependence of the inhibition by
150 µM H2O2 of carbachol-induced
AP-1 revealed that it was rapid and reversible. The data in Figure
5 show that the greatest inhibition of
carbachol-stimulated AP-1 occurred when cells were exposed to
H2O2 immediately before the addition of
carbachol. Preincubation with H2O2 lessened its
inhibitory effect in a time-dependent manner, with clear attenuation of
the inhibition occurring with a 2 hr H2O2
preincubation before addition of carbachol. Concurrently, there was a
time-dependent increase in AP-1 induced by H2O2
in the absence of carbachol.
Fig. 5.
Time dependence of the inhibition by
H2O2 of carbachol-stimulated AP-1. SH-SY5Y
cells were incubated with 150 µM
H2O2 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).
[View Larger Version of this Image (23K GIF file)]
To measure the magnitude of the altered oxidation state caused by
H2O2, the concentration- and time-dependent
effects of H2O2 on SH-SY5Y cells were assessed
using the MTT assay, which measures cellular redox changes resulting
from impaired mitochondrial enzyme function. Exposure of cells to 50 µM H2O2 resulted in a 10%
decrease in MTT reduction, and decreases of ~20 and 30% were
obtained after incubation with 100 and 300 µM
H2O2, respectively (Fig. 6).
Fig. 6.
MTT reduction in SH-SY5Y cells. Cells were
incubated with 50, 100, or 300 µM
H2O2 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).
[View Larger Version of this Image (20K GIF file)]
To test whether H2O2 could impair the
stimulation of AP-1 or NFB DNA binding by reducing the activation of
protein kinase C, two strategies were employed using PMA to directly
activate protein kinase C. First, the effects of
H2O2 on AP-1 and NFB stimulated by PMA were
measured, and second, the effect of H2O2 on
PMA-induced membrane translocation of protein kinase C- was
measured. PMA (1 µM) induced time-dependent increases in
each of the transcription factors, with AP-1 being activated to the
greatest extent and the upper NFB band the least (Fig.
7). Only at a concentration of 300 µM did
H2O2 consistently inhibit PMA-induced
activation of AP-1 or NFB (Fig. 8).
Fig. 7.
Time-dependent activation of AP-1
(A) and NFB (B, C) by PMA. SH-SY5Y
cells were incubated with 1 µM PMA, and AP-1 and NFB
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).
[View Larger Version of this Image (19K GIF file)]
Fig. 8.
Modulation by H2O2 of
PMA-stimulated AP-1 (A) and NFB (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 NFB 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).
[View Larger Version of this Image (18K GIF file)]
In the second series of experiments designed to measure the
interactions of H2O2 and protein kinase C,
measurements were made of the modulation by
H2O2 of the PMA-induced translocation to the
membrane of protein kinase C-, the major subtype expressed in these
cells. PMA (0.1 µM) induced a time-dependent
translocation of protein kinase C- from the cytosol to the membrane
(Fig. 9). The PMA-induced translocation of protein
kinase C- was inhibited by only 40% with 300 µM
H2O2 (Fig. 10). These findings
indicate that inhibition of carbachol-stimulated AP-1 activation by low
concentrations of H2O2 was unlikely to be
attributable to impaired activation of protein kinase C. However,
because 300 µM H2O2 inhibited
PMA-induced protein kinase C activation, this mechanism likely
contributes to the inhibition by 300 µM
H2O2 of AP-1 and NFB activation induced by
carbachol and by PMA.
Fig. 9.
PMA-induced translocation of protein kinase C-.
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- was measured by immunoblotting
as described in Materials and Methods. B, The
immunoblots of membrane and cytosol protein kinase C- were
quantitated by densitometry, and data were calculated as percentage of
maximal protein kinase C- (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- using samples from three individual experiments.
[View Larger Version of this Image (20K GIF file)]
Fig. 10.
Inhibition by H2O2 of
PMA-induced translocation of protein kinase C-. 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- 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).
[View Larger Version of this Image (41K GIF file)]
To determine whether H2O2 modulated signaling
induced by carbachol at a site upstream from protein kinase C
activation, carbachol-induced phosphoinositide hydrolysis was measured.
SH-SY5Y cells were prelabeled with [3H]inositol, and
carbachol-induced [3H]phosphoinositide hydrolysis was
measured with or without a 10 min preexposure to 30-500
µM H2O2. Figure
11A shows that
H2O2 inhibited carbachol-induced
phosphoinositide hydrolysis concentration dependently. There was a
significant correlation between the H2O2
concentration-dependent inhibition of carbachol-stimulated AP-1 and
[3H]phosphoinositide hydrolysis (r = 0.95; p < 0.001).
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 with
squares. 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).
[View Larger Version of this Image (27K GIF file)]
To identify the site of the phosphoinositide signal transduction system
that was susceptible to inhibition by H2O2,
[3H]phosphoinositide hydrolysis was measured using NaF to
activate G-proteins coupled to phospholipase C (Jope, 1988) or using
the calcium ionophore ionomycin to directly activate phospholipase C
(Fisher et al., 1989). H2O2 treatment inhibited
NaF-induced [3H]phosphoinositide hydrolysis (Fig.
11B) with a concentration dependence similar to that
observed with carbachol stimulation, except at the higher
concentrations of H2O2 where the response to
NaF was inhibited less than was carbachol stimulation (Fig.
11D). Incubation of SH-SY5Y cells with
H2O2 did not alter the level of Gq/11 in
cell membranes detected by quantitative immunoblots (data not shown),
indicating that the function, rather than the level, of Gq/11 was
impaired by H2O2. Ionomycin-stimulated
[3H]phosphoinositide hydrolysis was unaffected by
H2O2 (Fig. 11C). Thus,
calcium-activated phospholipase C and phosphoinositide substrates were
impervious to treatment with H2O2, whereas
G-protein activation was impaired.
DISCUSSION
The primary objective of this study was to determine whether
oxidative stress influenced cholinergic muscarinic receptor-induced
signaling processes in neuronal cells. Oxidative stress caused by
H2O2 was found to markedly impair
carbachol-induced AP-1 DNA binding, an effect that was apparently
attributable to impaired activation of the phosphoinositide signal
transduction system, whereas carbachol-induced NFB DNA binding was
resistant to oxidative stress except at the highest concentration of
H2O2 tested (300 µM), where
inhibition was associated with reduced activation of protein kinase C. Thus, not only was neuronal carbachol-induced signaling found to be
susceptible to inhibition by oxidative stress, but two downstream
responses to carbachol, activation of AP-1 and NFB DNA binding, were
revealed to be differentially sensitive to impairment by
H2O2.
It was interesting to find that, whereas individually, carbachol and
H2O2 each increased AP-1 DNA binding in SH-SY5Y
cells, coincident exposure to both agents impaired this response.
Oxidative stress has been reported many times previously, and in a wide
variety of cell types, to increase AP-1, as well as NFB DNA binding
(for review, see Karin and Smeal, 1992; Siebenlist et al., 1994). These
effects of oxidative stress, in this case caused by
H2O2, also were observed in SH-SY5Y cells, with
100 µM H2O2 causing a maximal
stimulation (to 250% of basal) of AP-1. Thus, it is highly unlikely
that inhibition by H2O2 of carbachol-induced
AP-1 was attributable to a direct inhibitory effect of
H2O2 on AP-1 constituent proteins or DNA
binding (because H2O2 alone was stimulatory);
it is more likely that a site upstream in the cholinergic signaling
cascade was impaired by H2O2. Inhibition of
protein kinase C activation may have contributed to the maximal
impairment of AP-1 that was induced by 300 µM
H2O2, because this treatment reduced
PMA-induced AP-1 activation and protein kinase C translocation.
(Unfortunately, carbachol-induced translocation of protein kinase C to
the membrane was not detectable in these cells.) However, the
inhibitory effects of lower concentrations of
H2O2 on carbachol-induced AP-1 indicated that
an earlier response to carbachol, before protein kinase C activation,
was susceptible to inhibition by H2O2.
Measurements of phosphoinositide hydrolysis stimulated by carbachol
revealed a severe inhibition with low concentrations of
H2O2, and the magnitude of the inhibition
correlated closely (r = 0.95) with the
H2O2 concentration-dependent inhibition of
carbachol-induced AP-1. Thus, it appears most likely that diminution of
carbachol-stimulated AP-1 activation caused by oxidative stress induced
by H2O2 primarily resulted from inhibition of
carbachol-induced phosphoinositide hydrolysis.
The site of action of H2O2 that accounted for
the inhibition of phosphoinositide hydrolysis appeared not to involve
changes in phospholipase C or the inositol phospholipids, because
ionomycin-induced phospholipase C activation was unaltered by
H2O2. However, because G-protein-activated (by
NaF), as well as carbachol-stimulated, phosphoinositide hydrolysis was
impaired by H2O2, it appears that the G-protein
is a likely target of the inhibitory actions of
H2O2. Further studies will be required to
identify the mechanism by which H2O2 impairs
G-protein function. However, it is known that blocking sulfhydryl
groups on the G-protein -subunit impairs its activation of
phospholipase C (Jope et al., 1987), so this represents one likely
candidate site for the inhibitory action of
H2O2. Because phosphoinositide hydrolysis
stimulated by direct activation of G-proteins was inhibited by
H2O2, it is unlikely that there is selectivity
for muscarinic responses in this modulatory effect, but responses to
other receptors remain to be investigated.
Surprisingly, carbachol-stimulated NFB was impervious to the
inhibitory effect of low concentrations of H2O2
on phosphoinositide hydrolysis, which impaired the stimulation of AP-1.
This suggested that different signaling pathways contributed to NFB
and AP-1 activation induced by carbachol, a proposal also supported by
the 10-fold difference in the carbachol EC50 for
stimulation of NFB (20 µM) and AP-1 (2 µM). The mechanism of the resilience of carbachol-induced
NFB to H2O2 remains to be identified.
However, the sensitivity of G-protein-activated phosphoinositide
hydrolysis to inhibition by H2O2 may provide a
clue to this problem. Because both the G-protein -subunits and the
 -dimers constitute signaling elements, we speculate that
bifurcation of signaling at the level of the G-protein subunits may
account for the differential responses of NFB and AP-1 to activation
by carbachol and inhibition by H2O2. Thus, the
subunit activating phosphoinositide hydrolysis that leads to AP-1
activation was susceptible to inhibition by
H2O2, whereas the other signaling subunit
leading to activation of NFB remained unaffected except at the
highest concentration of H2O2. Although the
G-protein -subunit classically has been identified as the primary
activator of phospholipase C, Clapham and colleagues (Stehno-Bittel et
al., 1995) recently raised the intriguing possibility that the
-dimer fulfills this role. Thus, it remains to be determined
which activated G-protein subunits are coupled to activation of NFB
and AP-1 after stimulation with carbachol and whether these are
differentially susceptible to inhibition by oxidative stress.
The observations that carbachol caused a relatively prolonged
activation of NFB and AP-1 and that the effects of
H2O2 persisted well beyond its lifetime
demonstrate the prolonged signaling potential of these agents.
Activation of NFB is often a transient effect, but recent reports
have identified conditions in which activation is more extended (for
review, see Finco and Baldwin, 1995). Thompson et al. (1995) provided
evidence that the release of NFB from IB mediates transient
responses, whereas prolonged activation of NFB is associated
additionally with release from the inhibitory action of IB.
Similarly, stimulation of the levels of Fos-related antigens (e.g., Fra
1, Fra 2) has been associated with prolonged activation of AP-1
compared with more transient responses attributable to increased levels
of c-Fos (Pennypacker et al., 1995; Hyman and Nestler, 1996).
Regulation of the duration of activation of NFB and AP-1, and their
eventual roles as mediators of apoptosis or cell survival, may depend
on the selectivity of the inhibitory and constitutive proteins that are
affected by stimulatory agents, an issue remaining to be addressed in
identifying the responses to carbachol and to
H2O2 in neuronal cells.
The inhibition by oxidative stress induced with
H2O2 on cholinergic muscarinic
receptor-stimulated signaling reported here is remarkably similar to
impaired cholinergic responses observed in Alzheimer's disease brain
in a number of studies. Notably, cholinergic agonist-induced
phosphoinositide hydrolysis is severely impaired in Alzheimer's
disease, and impaired G-protein function has been identified as a
primary site causing this deficient phosphoinositide signaling (for
review, see Jope, 1996). These impairments correspond remarkably
closely to the H2O2-induced inhibition of
cholinergic receptor- and G-protein-stimulated phosphoinositide
hydrolysis in SH-SY5Y cells. Moreover, A peptides, which contribute
to amyloid plaque formation in Alzheimer's disease, have been shown to
induce neurotoxicity via oxidative stress mechanisms, including
increasing the concentration of H2O2 (Behl et
al., 1994; Hensley et al., 1994). Thus, impairments in signaling
induced by H2O2 in SH-SY5Y cells may model some
of the mechanisms contributing to neuronal dysfunction in Alzheimer's
disease and other neurological disorders in which oxidative stress
occurs.
FOOTNOTES
Received April 23, 1996; revised July 1, 1996; accepted July 8, 1996.
This work was supported by National Institutes of Health Grant
AG06569.
Correspondence should be addressed to Dr. Richard S. Jope, Department
of Psychiatry and Behavioral Neurobiology, Sparks Center 1057, University of Alabama at Birmingham, Birmingham, AL
35294-0017.
REFERENCES
-
Abate C,
Patel L,
Rauscher FJ,
Curran T
(1990)
Redox
regulation of fos and jun DNA-binding activity in vitro.
Science
249:1157-1161 .
[Abstract/Free Full Text]
-
Behl C,
Davis JB,
Lesley R,
Schubert D
(1994)
Hydrogen
peroxide mediates amyloid protein toxicity.
Cell
77:817-827 .
[Web of Science][Medline]
-
Bradford MM
(1976)
A rapid and sensitive method for the
quantitation of microgram quantities of protein utilizing the principle
of protein dye binding.
Anal Biochem
72:248-254 .
[Web of Science][Medline]
-
Coyle JT,
Puttfarcken P
(1993)
Oxidative stress, glutamate,
and neurodegenerative disorders.
Science
262:689-695 .
[Abstract/Free Full Text]
-
Finco TS,
Baldwin AS
(1995)
Mechanistic aspects of
NF-B regulation: the emerging role of phosphorylation and
proteolysis.
Immunity
3:263-272 .
[Web of Science][Medline]
-
Fisher SK
(1995)
Homologous and heterologous regulation of
receptor-stimulated phosphoinositide hydrolysis.
Eur J Pharmacol
288:231-250 .
[Web of Science][Medline]
-
Fisher SK,
Domask LM,
Roland RM
(1989)
Muscarinic receptor
regulation of cytoplasmic Ca2+ concentrations in human
SK-N-SH neuroblastoma cells: Ca2+ requirements for
phospholipase C activation.
Mol Pharmacol
35:195-204 .
[Abstract]
-
Friedlich AL,
Butcher LL
(1994)
Involvement of free oxygen
radicals in -amyloidosis: an hypothesis.
Neurobiol Aging
15:443-455 .
[Web of Science][Medline]
-
Greenwood AF,
Powers RE,
Jope RS
(1995)
Phosphoinositide
hydrolysis, Gq, phospholipase C, and protein kinase C in post mortem
human brain: effects of post mortem interval, subject age, and
Alzheimer's disease.
Neuroscience
69:125-138 .
[Web of Science][Medline]
-
Halliwell B
(1992)
Reactive oxygen species and the central
nervous system.
J Neurochem
59:1609-1623 .
[Web of Science][Medline]
-
Hansen MB,
Nielsen SE,
Berg K
(1989)
Reexamination and
further development of a precise and rapid dye method for measuring
cell growth/cell kill.
J Immunol Methods
119:203-210 .
[Web of Science][Medline]
-
Hensley K,
Carney JM,
Mattson MP,
Aksenova M,
Harris M,
Wu JF,
Floyd R,
Butterfield DA
(1994)
A model for -amyloid aggregation and
neurotoxicity based on free radical generation by the peptide:
relevance to Alzheimer's disease.
Proc Natl Acad Sci USA
91:3270-3274 .
[Abstract/Free Full Text]
-
Hyman SE,
Nestler EJ
(1996)
Initiation and adaptation: a
paradigm for understanding psychotropic drug action.
Am J Psychiatry
153:151-162 .
[Abstract/Free Full Text]
-
Jope RS
(1988)
Modulation of phosphoinositide hydrolysis by
NaF and aluminum in rat cortical slices.
J Neurochem
51:1731-1736 .
[Web of Science][Medline]
-
Jope RS (1996) Cholinergic muscarinic receptor signaling by
the phosphoinositide signal transduction system in Alzheimer's
disease. Alzheimer's Dis Rev 1:2-14.
-
Jope RS,
Li X
(1989)
Inhibition of inositol phospholipid
synthesis and norepinephrine-stimulated hydrolysis in rat brain slices
by excitatory amino acids.
Biochem Pharmacol
38:589-596 .
[Web of Science][Medline]
-
Jope RS,
Casebolt TL,
Johnson GVW
(1987)
Modulation of
carbachol-stimulated inositol phospholipid hydrolysis in rat cerebral
cortex.
Neurochem Res
12:693-700 .
[Web of Science][Medline]
-
Karin M,
Smeal T
(1992)
Control of transcription factors by
signal transduction pathways: the beginning of the end.
Trends Biochem Sci
17:418-422 .
[Web of Science][Medline]
-
Laemmli UK
(1970)
Cleavage of structural proteins during the
assembly of the head of bacteriophage T4.
Nature
227:680-685 .
[Medline]
-
Pennypacker KR,
Hong J-S,
McMillian MK
(1995)
Implications of
prolonged expression of Fos-related antigens.
Trends Pharmacol Sci
16:317-321 .
[Medline]
-
Shigenaga MK,
Hagen TM,
Ames BN
(1994)
Oxidative damage and
mitochondrial decay in aging.
Proc Natl Acad Sci USA
91:10771-10778 .
[Abstract/Free Full Text]
-
Siebenlist U,
Franzoso G,
Brown K
(1994)
Structure,
regulation and function of NFB.
Annu Rev Cell Biol
10:405-455 .
[Web of Science]
-
Staal FJT,
Roederer M,
Herzenberg LA,
Herzenberg LA
(1990)
Intracellular thiols regulate activation of nuclear
factor B and transcription of human immunodeficiency virus.
Proc Natl Acad Sci USA
87:9943-9947.
[Abstract/Free Full Text]
-
Stehno-Bittel L,
Krapivinsky G,
Krapivinsky L,
Perez-Terzic C,
Clapham DE
(1995)
The G-protein subunit transduces the
muscarinic receptor signal for Ca2+ release in
Xenopus oocytes.
J Biol Chem
270:30068-30074 .
[Abstract/Free Full Text]
-
Thompson JE,
Phillips RJ,
Erdjument-Bromage H,
Tempst P,
Ghosh S
(1995)
IB- regulates the persistent response in a
biphasic activation of NF-B.
Cell
80:573-582 .
[Web of Science][Medline]
-
Unlap T,
Jope RS
(1995)
Diurnal variation in kainate-induced
AP-1 activation in rat brain: influence of glucocorticoids.
Mol Brain Res
28:193-200 .
[Medline]
This article has been cited by other articles:
|
|
|
|
 
J. Yuan, A. Lugea, L. Zheng, I. Gukovsky, M. Edderkaoui, E. Rozengurt, and S. J. Pandol
Protein kinase D1 mediates NF-{kappa}B activation induced by cholecystokinin and cholinergic signaling in pancreatic acinar cells
Am J Physiol Gastrointest Liver Physiol,
December 1, 2008;
295(6):
G1190 - G1201.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S.-Y. Kim, M. Seo, Y. Kim, Y.-I. Lee, J.-M. Oh, E.-A. Cho, J.-S. Kang, and Y.-S. Juhnn
Stimulatory Heterotrimeric GTP-binding Protein Inhibits Hydrogen Peroxide-induced Apoptosis by Repressing BAK Induction in SH-SY5Y Human Neuroblastoma Cells
J. Biol. Chem.,
January 18, 2008;
283(3):
1350 - 1361.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. Choi, J. H. Kim, E.-J. Roh, M.-J. Ko, J.-E. Jung, and H.-J. Kim
Nuclear Factor-{kappa}B Activated by Capacitative Ca2+ Entry Enhances Muscarinic Receptor-mediated Soluble Amyloid Precursor Protein (sAPP{alpha}) Release in SH-SY5Y Cells
J. Biol. Chem.,
May 5, 2006;
281(18):
12722 - 12728.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
N. G. Bazan and W. J. Lukiw
Cyclooxygenase-2 and Presenilin-1 Gene Expression Induced by Interleukin-1beta and Amyloid beta 42 Peptide Is Potentiated by Hypoxia in Primary Human Neural Cells
J. Biol. Chem.,
August 9, 2002;
277(33):
30359 - 30367.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. O. R. Borges, M. L. C. Abreu, C. S. Porto, and M. C. W. Avellar
Characterization of Muscarinic Acetylcholine Receptor in Rat Sertoli Cells
Endocrinology,
November 1, 2001;
142(11):
4701 - 4710.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
L. Song, P. De Sarno, and R. S. Jope
Muscarinic Receptor Stimulation Increases Regulators of G-protein Signaling 2 mRNA Levels through a Protein Kinase C-dependent Mechanism
J. Biol. Chem.,
October 15, 1999;
274(42):
29689 - 29693.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
G. M. Denning, M. A. Railsback, G. T. Rasmussen, C. D. Cox, and B. E. Britigan
Pseudomonas pyocyanine alters calcium signaling in human airway epithelial cells
Am J Physiol Lung Cell Mol Physiol,
June 1, 1998;
274(6):
L893 - L900.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. Zhang, M. Lesort, R. P. Guttmann, and G. V. W. Johnson
Modulation of the in Situ Activity of Tissue Transglutaminase by Calcium and GTP
J. Biol. Chem.,
January 23, 1998;
273(4):
2288 - 2295.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. M. Auerbach and M. Segal
Peroxide Modulation of Slow Onset Potentiation in Rat Hippocampus
J. Neurosci.,
November 15, 1997;
17(22):
8695 - 8701.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
W. A. Pedersen, Q. Guo, B. K. Hartman, and M. P. Mattson
Nerve Growth Factor-independent Reduction in Choline Acetyltransferase Activity in PC12 Cells Expressing Mutant Presenilin-1
J. Biol. Chem.,
September 5, 1997;
272(36):
22397 - 22400.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
