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INTRODUCTION |
Lead toxicity has been identified as the most important global
environmental health hazard because of its prevalence in the environment and its potential to cause long lasting learning deficits and behavioral abnormalities particularly in children (1-5). Acute
neonatal exposure leads to cerebral-microvascular pathology including
blood-brain barrier dysfunction, cerebellar hemorrhage, and cerebral
edema (6-8). The mechanisms leading to these diverse manifestations of
lead toxicity are mostly unknown.
The biochemical and molecular mechanism(s) of action of lead
neurotoxicity have not yet been fully elucidated. Several studies have
shown, however, that calcium-dependent events are potential intracellular targets of lead (9, 10). Lead is reported to alter a
number of calcium-mediated cellular processes including calcium
channels and second messenger systems. Lead is a potent blocker of
calcium channels, activates calmodulin with higher affinity than
calcium (Ca2+), and most importantly picomolar
concentrations can substitute for Ca2+ in activating
protein kinase C (PKC)1
(11-13). PKC is a phospholipid-dependent
diacylglycerol-activated serine/threonine protein kinase consisting of
a multigene family of closely related, but distinct, isoenzymes (14,
15). Activation of PKC by lead results in the induction of the
immediate-early-response genes c-fos, c-jun, and
erg-1 (16, 17). Homodimerization (Jun family proteins) and
heterodimerization (Jun and Fos family members) of these early response
gene proteins form the activator protein-1 complex (AP-1) that mediates
its subsequent effects on gene transcription (18-20). Recent studies
using rat PC12 pheochromocytoma cells show that lead increases AP-1 DNA
binding activity via a PKC-dependent pathway (21) and
NF-
B activity through the activation of the MAP kinase family of
kinases (22). Given the ability of lead to interfere with several
signal transduction pathways and transcription factors, it is likely
that lead alters gene expression in its target cells and thereby
interferes with multiple cellular events in the developing brain (14,
23).
Experimental evidence points to multiple cellular targets including
neurons, astroglia, and the microvasculature, at which lead may act in
the developing brain (24). Astroglia are particularly important as the
signals leading to expression of the blood-brain barrier phenotype
appear to originate in the astrocytes and depend upon intimate
astroglial-endothelial interactions (25-27). Lead has been shown to
increase the permeability of the blood-brain barrier, the function of
which is regulated by perivascular astrocytes (6, 26, 27). Earlier
studies from this laboratory have shown that lead inhibits
astroglia-induced microvessel formation in vitro (12).
Astrocytes, therefore, appear particularly vulnerable to the toxic
effects of lead (28, 29).
In this study, we have examined the effects of lead on (a)
differential gene expression using cDNA expression microarrays and
(b) the activity of transcription factors and related signal responsive kinases involved in the intracellular signal transduction leading to gene expression in immortalized human fetal astrocyte (SV-FHA) cultures. Our results indicate that among the genes induced by
lead, vascular endothelial growth factor (VEGF) known also as vascular
permeability factor is one of the most sensitive. Subsequent
experiments determine the relative contributions of PKC, AP-1, and
hypoxia-inducible factor 1 (HIF-1) regulatory pathways on lead- induced
VEGF expression.
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EXPERIMENTAL PROCEDURES |
Materials--
Dulbecco's modified Eagle's medium with 4.5 g/liter glucose and L-glutamine (Mediatech Cellgro); fetal
bovine serum (Gemini Bio-Products, Inc.); lead acetate, phorbol
12-myristate 13-acetate (PMA), dimethyl sulfoxide (Me2SO),
dithiothreitol, sodium acetate, EDTA, salmon sperm DNA (Sigma);
GF-109203, protease inhibitor mixture set I (Calbiochem); luciferase
assay kit (Promega); poly(dI-dC) (Amersham Pharmacia Biotech); VEGF
monoclonal antibody (Oncogene); Opti-MEM I, T4 polynucleotide kinase,
and gentamycin (Life Technologies, Inc.); AP-1 consensus
oligonucleotide (Santa Cruz Biotechnology) were purchased for the
study. HIF-1 consensus sequences were custom synthesized by the DNA
Synthesis Core Facility, School of Hygiene and Public Health, Johns
Hopkins University. Random primed DNA labeling kit (Roche Molecular
Biochemicals), [
-32P]ATP (specific activity, 3000 Ci/mmol; NEN Life Science Products), and [
-32P]dCTP
(specific activity, 3000 Ci/mmol) (Amersham Pharmacia Biotech) were
purchased for probe labeling.
Cell Culture and Treatments--
Immortalized human fetal
astrocyte (SV-FHA) cells were kindly provided by Dr. Stanimirovic
(Institute of Biological Sciences, National Research Council of Canada)
and cultured in medium containing Dulbecco's modified Eagle's medium
with 4.5 g/liter glucose and L-glutamine, 10% fetal bovine
serum, and 50 µg/ml gentamycin (30). Cells were grown to 80-90%
confluency prior to treatment with lead acetate, sodium acetate, and
PMA. Lead acetate and sodium acetate were dissolved in
sterile-distilled water and PMA in Me2SO, and directly
added to the complete medium. Final concentration of Me2SO
in the medium was 0.006% (v/v) and equal volume was used as control
for PMA-exposed cells. To study the effects on VEGF expression, cells
were exposed to 10 µM lead acetate for 24 h or 100 nM PMA for 6 h in complete medium unless otherwise
mentioned. To study the effects on transcription factors AP-1 and
HIF-1, cells were exposed to 10 µM lead acetate for 3 and
4 h, respectively, and to 100 nM PMA for 1 h
prior to isolation of nuclear proteins.
cDNA Expression Microarray Assay--
SV-FHAs, grown in
10-cm culture dishes to 80-90% confluency, were exposed to either 10 µM lead acetate or 10 µM sodium acetate as
control. Total cellular RNA was extracted from the cells 24 h
later using RNeasy isolation kit (Qiagen) according to manufacturer's protocol. Poly(A)+ RNA was isolated from total cellular RNA
samples and reverse transcribed in the presence of
[32P]dATP to generate radiolabeled cDNA probe, and
purified onto Chromaspin-200 DEPC H2O column chromatography
according to the Atlas Pure RNA labeling protocol
(CLONTECH). Neuroarray cDNA microarrays (CLONTECH) were hybridized with
32P-labeled cDNA probes from each experimental
condition according to the manufacturer's instructions. Briefly, the
membranes were soaked in deionized H2O and then blocked
with sheared salmon testes DNA in ExpressHyb solution at 68 °C for
30 min. Five µl of Cot-1 DNA was added to the labeled
cDNA probe and heat denatured for 2 min followed by chilling on ice
for 2 min. The cDNA probes were then added to ExpressHyb solution
and the microarray blots were hybridized overnight with continuous
agitation at 68 °C. The membranes were washed with Wash Solution 1 and Wash Solution 2 (supplied with kit) for 30 min each at 68 °C,
and finally with 2 × SSC for 5 min at room temperature. The blots
were exposed to a phosphorimaging screen at room temperature for
overnight and the array image was visualized by phosphorimaging using
the Bio-Image analyzer BAS 2500 (Fujifilm). Hybridized dot intensities
on the microarrays were quantified using CLONTECH
AtlasImage software. Normalization of the data was performed by
dividing the intensity for each gene on a blot by the average intensity
of all of the genes on that blot.
Northern Blot Analysis--
Total cellular RNA was isolated from
cells using an RNeasy isolation kit (Qiagen Inc.) as above. Northern
blot analysis was performed according to the method of Sambrook and
co-workers (31) with minor modifications as described previously (32).
The cDNA probe for human VEGF (KpnI-SpeI
restriction fragment/582 base pairs) was labeled with
[32P]dCTP using a random primed DNA labeling kit (Roche
Molecular Biochemicals) according to the manufacturer's instructions.
Heat-denatured probe was hybridized to the membrane at 42 °C
overnight (16-18 h) in 10 ml of hybridization buffer (50% formamide,
5 × SSE, 2.5 × Denhardt's solution, 0.5% SDS, and
0.2 mg/ml salmon testes DNA). The blots were washed as follows:
three times in 0.1% SDS in 1 × SSC for 20 min at room
temperature; and two times in the same buffer for 30 min each at
65 °C. Radioactivities corresponding to mRNA signals for VEGF
were quantified by phosphorimaging using the Bio-Image analyzer BAS
2500 (Fujifilm). Membranes were stripped of probe by washing in 1 × SSC containing 0.1% SDS at 85-90 °C for 10 min and then
rehybridized with cDNA probe to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Results are expressed relative to GAPDH.
Preparation of Nuclear Extracts--
Nuclear extracts were
prepared according to the method of Stein and co-workers (33). Briefly,
cells were washed with ice-cold PBS twice and harvested by scraping in
1 ml of cold PBS. The harvested cells were centrifuged at 14,000 × g for 5 min. The cell pellets were resuspended in 100 µl of lysis buffer (10 mM HEPES, pH 7.9, 60 mM KCl, 1 mM EDTA, 1 mM
dithiothreitol, 1 × protease inhibitor mixture, and 0.5% Nonidet
P-40) and incubated on ice for 5 min. Cell lysates were centrifuged at
1,200 × g for 5 min at 4 °C. The nuclei recovered
in the pellet were washed with 100 µl of lysis buffer without Nonidet
P-40 and centrifuged at 1,200 × g for 5 min at
4 °C. The pellet was suspended in 100 µl of nuclear suspension
buffer (250 mM Tris, pH 7.8, 60 mM KCl, 1 mM dithiothreitol, and 1 × protease inhibitor
mixture) and lysed by three cycles of quick freezing in liquid nitrogen
and thawing at 37 °C. The nuclear lysates were centrifuged at
14,000 × g for 10 min at 4 °C and the supernatants
were stored at 
70 °C until use for the electrophoretic mobility
shift assays.
Electrophoretic Mobility Shift Assay--
Electrophoretic
mobility shift assay (gel-shift) was carried out to analyze the DNA
binding activity of AP-1 and HIF-1 according to Ying and co-workers
(34) with some modifications. Briefly, AP-1 consensus double-stranded
oligonucleotides (5'-CGCTTGATGACTCAGCCGGAA-3') were
purchased from Santa Cruz Biotechnology, Inc. The sense
(5'-GCCCTACGTGCTGTCTCA-3') and antisense (5'-TGAGACAGCACGTAGGGC-3')
HIF-1 binding sequences were synthesized by the DNA Synthesis Core
Facility of Johns Hopkins University. The AP-1 consensus
oligonucleotide (5 pmol) was end-labeled with
[-32P]ATP (3000 Ci/mmol; NEN Life Science Products
Inc.) using T4 polynucleotide kinase (Life Technologies, Inc.) and then
purified through Sephadex G-25 (Roche Molecular Biochemicals). To
generate double stranded HIF-1 consensus oligonucleotide probes, the
sense oligonucleotides underwent 5' end labeling with
[-32P]ATP (NEN Life Science Products Inc.) and T4
polynucleotide kinase (Life Technologies, Inc.). The labeled sense
strands were then annealed to a 10-fold excess of antisense strands by
heating to 85 °C for 10 min, allowed to cool down to room
temperature over a period of 2-3 h, and then purified through Sephadex
G-25.
Nuclear extracts (10-15 µg) were incubated on ice for 20 min
with 5 µl of a reaction buffer containing 1 µg of poly(dI-dC) (Amersham Pharmacia Biotech), 50 mM Tris-HCl (pH 7.5), 500 mM NaCl, 20% glycerol, 5 mM EDTA, 5 mM dithiothreitol in a total volume of 24 µl. One
microliter of respective 32P-labeled (25,000-30,000 cpm)
oligonucleotide probe was added to the reaction mixture and incubated
further at room temperature for 20 min. For competition studies,
100-fold excess of unlabeled oligonucleotide was added to the reaction
mixture during the incubation on ice before the addition of labeled
probe. DNA-protein complexes were resolved by polyacrylamide gel
electrophoresis using 6% nondenaturing gel at 180 V for 3 h in
0.25 × TBE (45 mM Tris borate and 1 mM EDTA). Gels were vacuum dried and specific bands were quantified by
phosphorimaging using the Bio-Imaging analyzer BAS 2500 (Fujifilm).
Immunoblot Analysis--
To evaluate VEGF protein levels, cells
were exposed to lead acetate (10 µM) or sodium acetate
for 24 h. Cells were washed with ice-cold PBS, harvested by
scraping in 1 ml of cold PBS and centrifuged at 14,000 × g for 5 min. Three hundred µl of RIPA buffer (1 × PBS, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS) containing
freshly added protease inhibitor mixture (Calbiochem) were added to
each tube and sonicated for 10 s, centrifuged at 14,000 × g for 5 min and the supernatants were stored at 70 °C. One hundred µg of cell extracts were diluted to a total volume of 1 ml using 10 mM Tris (pH 7.4) and 100 mM NaCl;
30 µl of equilibrated heparin-agarose beads (Sigma) were added and
the mixture rocked at 4 °C overnight as described by Ferrara and
Henzel (35). The beads were pelleted at 5,000 × g for
1 min, washed in 400 mM NaCl and 20 mM Tris (pH
7.4), and recentrifuged at 5,000 × g for 1 min; the
supernatant was removed and discarded. Next, 20 µl of 2 × loading buffer (20% glycerol, 100 mM Tris-HCl (pH 6.8),
4% SDS, and 0.2% bromphenol blue) were added and the slurry was
heated at 100 °C for 10 min. SDS-polyacrylamide gel electrophoresis
and immunoblotting were performed according to the method of Laemmli (36) with modifications. Samples (20 µl ) were loaded onto a 4-20%
gradient Tris glycine pre-cast gel (Novex) together with a full range
Rainbow recombinant protein molecular weight marker (Amersham Pharmacia
Biotech) and run at 150 V for 1 h. Proteins were transferred
electrophoretically onto a nitrocellulose membrane at constant current
of 50 mA for 1 h. After blocking with 5% nonfat dried milk in
1 × Tris-buffered saline (100 mM Tris-HCl, 0.9% NaCl, pH 7.4) and 0.1% Tween-20 (TBS-T) overnight at 4 °C, the nitrocellulose membranes were incubated with VEGF monoclonal antibody (Oncogene) at 1:1000 dilution in 1 × TBS containing 0.1% bovine serum albumin for 1 h at room temperature. After washing 3 times with 1 × TBST for 10 min each, the membranes were incubated with horseradish peroxidase-conjugated protein A/G (Pierce) at 1:10,000 dilution for 1 h. Horseradish peroxidase reaction product was then
visualized by enhanced chemiluminescence using an ECL Western blotting
detection kit (Amersham Pharmacia Biotech) and the digitized images
were quantified by densitometry (Molecular Dynamics).
Transient Expression Assays--
The involvement of specific PKC
isoforms was examined using dominant-negative (DN) mutants of PKC-
kindly provided by Dr. Albert Descoteaux (37) and PKC-
kindly
provided by Dr. P. M. Blumberg (38). These catalytically inactive
mutants compete with the corresponding endogenous isoforms (37). Cells
were transfected with 20 µg of either control vector DNA (pCIN-4) or the dominant-negative expression vectors DN-PKC- or DN-PKC- by
electroporation (240 V and 1050 microfarads) in a volume of 500 µl of
cell suspension and replated. The involvement of AP-1 was examined by
using an AP-1 luciferase reporter kindly supplied by Dr. Joseph
Bressler (21) and a jun dominant-negative (TAM-67) plasmid
expression vector kindly supplied by Dr. Michael Birrer (39). The
jun mutant (TAM-67) lacks the transactivation domain while
retaining full DNA binding capacity and inhibits AP-1 function by
homodimerizing and binding to the DNA at the AP-1 sites as well as by
forming heterodimers with wild type c-Fos to produce inactive
heterodimers (39). Four µg of AP-1 luciferase reporter plasmid DNA
along with either 20 µg of TAM-67 or control (CMV) plasmid DNA were
used for electroporation as indicated above. Cells were allowed to
recover for 24 h in a 5% CO2, 95% air
incubator at 37 °C. The cells were given fresh medium
containing 10% serum and either lead acetate (10 µM) or sodium acetate (10 µM) as
control and incubated for an additional 24 h. Alternatively, cells
received PMA (100 nM) or Me2SO (0.06% v/v) as
control and were incubated for an additional 6 h.
The involvement of HIF-1 was examined by using a VEGF-luciferase
reporter construct (pGL-MAP11wt) and a dominant-negative form of
HIF-1 (HIF-1DN) plasmid expression vector (pCEP/HIF-1DN) kindly supplied by Dr. Greg Semenza (40). The pGL-MAP11wt
construct includes the 5'-flanking region of human VEGF promoter
containing the HIF-1 consensus DNA-binding site cloned to an SV40
promoter-luciferase transcription unit (40). The pCEP/HIF-1DN
construct encodes a form of HIF-1 lacking both the amino-terminal
basic domain required for DNA-binding and the carboxyl-terminal
transactivation domain. HIF-1DN heterodimerizes with endogenous
HIF-1
, generating biologically inactive heterodimers that are unable
to bind DNA. Cells were co-transfected with 4 µg of pGL-MAP11wt
plasmid DNA along with either 20 µg of pCEP/HIF-1DN or control
(pCEP4) plasmid DNA by electroporation as mentioned above. The total
amount of plasmid DNA was adjusted to 24 µg. Twenty-four hours later
cells were given fresh complete medium containing either lead acetate (10 µM) or sodium acetate (10 µM) as
control and incubated for 24 h. In addition, plates containing
transfected cells were incubated in 1% O2 for 24 h at
37 °C. Cells were harvested and cell extracts were prepared using
reporter lysis buffer (Promega). Luminescence was measured in 20 µl
of cell extract using a luciferase assay system kit (Promega). Activity
was expressed as relative light units/mg of cellular protein as
determined by the Bradford method (41).
To investigate the role of AP-1 and HIF-1 in lead-induced VEGF
expression, cells were electroporated with either 20 µg of TAM-67 or
control (CMV) plasmid DNA and 20 µg of pCEP/HIF-1DN or control
(pCEP4) control plasmid DNA, respectively. Transfected cells were
exposed to either lead or sodium acetate as indicated above. Total
cellular RNA was extracted and VEGF and GAPDH levels were measured by
Northern blot analysis as described above.
Statistical Analysis--
Comparisons involving multiple groups
were done by ANOVA followed by Bonferroni/Dunn post-hoc test.
Comparison between any two groups was done by two-tailed Student's
t test. An overall level of significance of 0.01 was used to
determine differences.
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RESULTS |
Lead Induces Differential Gene Expression in
SV-FHAs--
cDNA expression microarray analyses were performed to
identify lead-sensitive genes in SV-FHA cells. Poly(A)+ RNA
was isolated from SV-FHAs exposed to either lead acetate (10 µM) or sodium acetate as control for 24 h and used
for cDNA expression microarray analysis. The cDNA microarrays
used contain 588 selected genes of which 131 were consistently
expressed in the SV-FHAs. Among these expressed genes, 12% appeared
induced following exposure to lead. Of these induced genes one of the most lead-sensitive was VEGF (Fig. 1,
A and B). Subsequent quantification of spot
intensities using CLONTECH AtlasImage software
revealed that lead induced VEGF mRNA 3.0-fold (p < 0.005) compared with sodium-treated controls (Fig. 1C). No
significant difference in VEGF expression levels was observed between
sodium-treated and untreated controls (not shown).
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Fig. 1.
cDNA expression microarrays analysis of
differential gene expression by lead in SV-FHAs. Cells grown in
normal serum (80-90% confluency) were exposed to either lead acetate
(10 µM) or sodium acetate (10 µM) as
control (n = 4). Poly(A)+ RNA was isolated,
and reverse transcribed to cDNA in the presence of
[32P]dATP as described under "Experimental
Procedures." cDNA samples from each experimental condition were
hybridized to 8 independent cDNA Neuroarrays
(CLONTECH). A, gene expression values
were visualized by phosphorimaging and are depicted in a scatter plot.
A best fit line was generated by least squares analysis. VEGF is one of
only several genes that are differentially regulated by lead acetate
treatment. B, VEGF gene expression was identified with
CLONTECH AtlasImage software based on analysis of
three replicate blots from cells treated with sodium acetate
(left column) or lead acetate (right column).
Each gene is represented by a pair of cDNA on each blot.
C, quantification of spot intensities shows significant
up-regulation of VEGF mRNA in SV-FHAs exposed to lead relative to
that of sodium or no treatment group (not shown). *, p < 0.005.
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Lead Induces VEGF mRNA and Protein Levels in
SV-FHAs--
Northern blot analysis of total cellular RNA confirmed
that lead induced VEGF mRNA levels in SV-FHAs compared with cells
treated identically with sodium acetate. Induction of VEGF mRNA in
SV-FHAs by lead was concentration and time-dependent, with
maximal induction observed at 10 µM lead following
24 h of treatment (Fig. 2,
A and B). Quantification of VEGF hybridization by
phosphorimaging revealed that lead induced VEGF expression 3-fold
(312 ± 25% versus 100 ± 9%; p < 0.001) compared with sodium-treated controls (Fig. 2, A
and B) consistent with the magnitude of induction found in the microarray analysis. The effect of lead on VEGF protein levels was
examined. Cells were exposed to 10 µM lead acetate or
sodium acetate as control for 24 h and cell extracts were
subjected to immunoblot analysis using anti-human VEGF monoclonal
antibody. An intense VEGF immunoreactive protein band at ~46 kDa
(Fig. 3) was detected. Densitometric
quantification showed that VEGF protein levels were increased ~2-fold
(188 ± 20% versus 100 ± 16%; p < 0.01) by lead (Fig. 3).
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Fig. 2.
Induction of VEGF by lead is concentration-
and time-dependent in SV-FHAs. SV-FHA cells were
exposed to different concentrations (0.1-50 µM) of lead
acetate for 24 h (A) or to 10 µM lead
acetate for 4-48 h (B). Total cellular RNA (20 µg/lane)
was subjected to Northern analysis using a 582-base pair
32P-labeled human VEGF cDNA probe. Blots were stripped
and rehybridized with a probe to GAPDH. Specific hybridization was
quantified by phosphorimaging and normalized to GAPDH. VEGF induction
was maximum in response to 10 µM lead following 24 h
of exposure. Data show mean ± S.E. (n = 6) of
percent induction relative to sodium-treated controls. *,
p < 0.001.
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Fig. 3.
Lead induces VEGF protein levels in
SV-FHAs. Cells were exposed either to lead acetate (10 µM) or sodium acetate (10 µM) as control
for 24 h. One hundred micrograms of total cellular extract were
incubated with heparin-agarose beads and the heparin binding fraction
subjected to immunoblot analysis using an anti-human VEGF monoclonal
antibody and horseradish peroxidase-conjugated Protein A/G as described
under "Experimental Procedures." Blots were exposed to ECL-film and
the 46-kDa VEGF-immunoreactive protein band was quantified by
densitometry. Values are shown as percent induction (mean ± S.E.;
n = 6) relative to sodium-treated controls set as
100%. *, p < 0.01. Blots shown are from
representative experiments.
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Involvement of PKC in VEGF Induction by Lead--
The signal
transduction mechanism involved in the induction of VEGF mRNA by
lead was examined. Since, lead is a potent activator of PKC in other
cell types, the PKC dependence of lead-induced VEGF expression in
SV-FHAs was examined. In addition, PMA, a selective activator of PKC,
was used as a positive control for this experiment. Treatment of
SV-FHAs with lead acetate (10 µM) for 24 h and PMA (100 nM) for 6 h induced VEGF mRNA expression
>2-fold (241 ± 28% versus 100 ± 7%;
p < 0.001) and ~2-fold (193 ± 14%
versus 100 ± 5%; p < 0.001),
respectively (Fig. 4, A and
B). Pretreatment of SV-FHAs with GF-109203 (2 µM), a selective cell-permeable PKC inhibitor, for 30 min
completely inhibited the induction of VEGF mRNA observed following
lead and PMA treatment (Fig. 4, A and B).
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Fig. 4.
Lead induction of VEGF mRNA is
PKC-dependent. SV-FHAs cells were pretreated with PKC
inhibitor GF-109203 (2 µM) for 30 min before exposure to
either lead acetate (10 µM) or sodium acetate (10 µM) as control for 24 h, or before exposure to PMA
(100 nM) or Me2SO (0.006% v/v) as control for
6 h. Northern blot analysis of total cellular RNA for VEGF
relative to GAPDH mRNA was performed as described under
"Experimental Procedures." Pretreatment with GF-109203 completely
inhibited VEGF induction by lead (A) or PMA (B).
Values represent mean ± S.E. (n = 6-10) relative
to controls (set at 100%). **, p < 0.001; *,
p < 0.01. Blots shown are from representative
experiments.
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To further evaluate the PKC dependence of VEGF induction by lead, we
used the dominant negative of the conventional PKC isoform PKC- and
the novel isoform PKC-. Cells were transfected with control vector
(pCIN-4) or with either DN-PKC- or DN-PKC- expression vectors.
Twenty-four hours later, cells were exposed to either lead acetate (10 µM) or sodium acetate (10 µM) as control
for an additional 24 h, or alternatively to either PMA (100 nM) or Me2SO (0.06% v/v) as control for an
additional 6 h. Northern hybridization of total cellular RNA
showed >2-fold induction of VEGF mRNA (212 ± 21%
versus 100 ± 6%; p < 0.001) in
control- transfected cells exposed to lead acetate and ~1.7-fold
induction (168 ± 13% versus 100 ± 8%;
p < 0.001) in response to PMA when compared with the respective controls (Fig. 5, A
and B). The induction of VEGF by lead and PMA was completely
inhibited by DN-PKC- (p < 0.001) (Fig. 5,
A and B). In contrast, DN-PKC- had no effect
on the induction of VEGF by lead (199 ± 18%) or PMA (149 ± 10%) compared with respective control-transfected cells.
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Fig. 5.
Induction of VEGF by lead is
nPKC--dependent. SV-FHA cells were
transfected with 20 µg of either control vector or dominant-negative
PKC- (DN-PKC-) expression vector as described under
"Experimental Procedures." Twenty-four hours later, cells were
exposed to either lead acetate (10 µM) or sodium acetate
(10 µM) for an additional 24 h (A), or to
PMA (100 nM) or Me2SO (0.006% v/v) as control
for an additional 6 h (B). Northern blot analysis of
total cellular RNA (20 µg/lane) was performed using a 582 base pairs
32P-labeled human VEGF cDNA probe followed by a GAPDH
probe as described under "Experimental Procedures." Specific
hybridization was quantified by phosphorimaging and normalized to
GAPDH. VEGF induction was completely inhibited in cells transfected
with DN-PKC-. Values represent mean ± S.E. (n = 6-8) relative to controls set at 100%. *, p < 0.001. Blots shown are from representative experiments.
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Involvement of AP-1 in Lead-induced VEGF Expression--
To
evaluate further the downstream signaling involved in the mechanisms of
lead's action, we examined the effects of lead on AP-1 complex in
SV-FHAs. Nuclear proteins (15 µg) isolated from SV-FHAs exposed to
lead acetate (10 µM) for 3 h and PMA (100 nM) for 1 h were subjected to electrophoretic mobility
shift assay using 32P-labeled AP-1 consensus
oligonucleotide probes. Quantification by phosphorimaging revealed a
2-fold increase in AP-1 specific consensus DNA binding activity
(210 ± 19% versus 100 ± 17%; p < 0.01) in lead-exposed cells relative to sodium treated controls (Fig. 6A). PMA stimulated a
similar induction of AP-1 DNA binding activity (238 ± 2%
versus 100 ± 5%; p < 0.01) (Fig.
6A). The AP-1 specific band disappeared when 100-fold excess
of unlabeled AP-1 oligonucleotide was added to the reaction mixture
(data not shown).
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Fig. 6.
Lead activates AP-1 DNA binding activity and
AP-1- dependent reporter gene expression. A, nuclear
protein was isolated from SV-FHAs exposed to either lead acetate (10 µM) or sodium acetate as control for 3 h, or to
either PMA (100 nM) or Me2SO (0.006% v/v) as
control for 1 h. Electrophoretic mobility shift assays (15 µg of
protein/lane) were performed using 32P-labeled AP-1
consensus oligonucleotide probes as described under "Experimental
Procedures." AP-1 DNA binding activity increased >2-fold in cells
exposed to either lead or PMA. B, cells were co-transfected
with 4 µg of AP-1/luciferase reporter constructs along with either 20 µg of the c-jun dominant-negative TAM-67 or control (CMV)
plasmid DNA. After 24 h, cells were exposed to either lead acetate
(10 µM) or sodium acetate as control for an additional
18-20 h. Luciferase expression was quantified in 20 µl of cell
extracts by measuring luminescence normalized to total protein as
described under "Experimental Procedures." Data represents
mean ± S.E. relative to sodium-treated controls (set at 100%).
*, p < 0.01. Blots shown are from representative
experiments.
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To determine whether lead specifically activates AP-1-dependent
transcriptional activity, the ability of lead to induce AP-1-dependent luciferase reporter gene expression was examined. Cells were
transfected with either 4 µg of control CMV-500 plasmid expression
vector or with AP-1/luciferase reporter constructs and 24 h later
exposed to lead acetate (10 µM) or sodium acetate as
control for 18-20 h. Lead increased the luciferase activity of cell
extracts >2-fold (221 ± 8% versus 100 ± 3%;
p < 0.001) (Fig. 6B). To further evaluate the AP-1 dependence of AP-1/luciferase induction by lead, cells were
co-transfected with a jun dominant negative (TAM-67)
expression vector or with control (CMV) vectors. TAM-67 lacks the
transactivation domain of Jun protein and inhibits AP-1 function by
binding to the consensus AP-1 sites as inactive homodimers or
heterodimers. AP-1/luciferase induction by lead was inhibited by 72%
(p < 0.001) in the TAM-67 co-transfected cells (Fig.
6B).
Since three AP-1 recognition sites are present in the VEGF 5'-flanking
region (42), the direct involvement of AP-1 in lead-induced VEGF
expression in SV-FHAs was examined. Cells were transfected with either
control vector (CMV) or TAM-67 expression vector and 24 h later
exposed to either lead acetate (10 µM) or sodium acetate (10 µM) as control for an additional 24 h. Northern
hybridization of total cellular RNA showed >2-fold induction of VEGF
mRNA (236 ± 9% versus 100 ± 2%;
p < 0.001) in control CMV vector-transfected cells
exposed to lead acetate compared with those exposed to sodium acetate.
TAM-67 inhibited lead-induced VEGF mRNA expression by ~86%
(p < 0.001) (Fig.
7).
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Fig. 7.
Induction of VEGF by lead is
AP-1-dependent. Cells were transfected with either 20 µg of
control (CMV) or c-jun dominant-negative TAM-67 expression
vectors and 24 h later exposed to either lead acetate (10 µM) or sodium acetate (10 µM) as control
for an additional 24 h. Total cellular RNA (20 µg/lane) was
subjected to Northern analysis using a 582-base pair
32P-labeled human VEGF cDNA probe followed by a GAPDH probe
as described under "Experimental Procedures." Specific
hybridization was quantified by phosphorimaging and normalized to
GAPDH. Expression of VEGF mRNA was significantly inhibited in cells
transfected with TAM-67. Data represents mean ± S.E.
(n = 6-8) relative to sodium-treated controls (set at
100%). *, p < 0.001. Blots shown are from
representative experiments.
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Effects of Lead on HIF-1 Activation--
Since the transcription
factor HIF-1 is a potent activator of VEGF transcription, the effects
of lead on HIF-1 DNA binding activity were also examined. Nuclear
proteins isolated from SV-FHAs exposed to either lead acetate (0.1-50
µM) or sodium acetate for 0.5-6 h were subjected to
electrophoretic mobility shift assay using 32P-labeled
oligonucleotide probes containing HIF-1 binding consensus sequences.
Quantification of shifted bands by phosphorimaging showed a small but
reproducible and significant increase in HIF-1 DNA binding activity in
response to lead exposure. The HIF-1 specific band disappeared when
140-fold excess of unlabeled oligonucleotide probe was added to the
reaction mixture (not shown). The induction of HIF-1 activity was
concentration and time-dependent, with maximum induction occurring at 10 µM lead exposure for
4 h relative to the control (196 ± 28%
versus 100 ± 7%; p < 0.001) (Fig.
8, A and B).
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Fig. 8.
Effects of lead on the HIF-1 DNA binding
activity. A, cells were exposed to either varying
concentrations of lead acetate (0.1-50 µM) or sodium
acetate as control for 4 h. Nuclear proteins (15 µg/lane) were
analyzed by electrophoretic mobility shift assay using
32P-labeled oligonucleotide probes containing HIF-1
binding consensus sequences as described under "Experimental
Procedures." B, cells were exposed to either lead
acetate (10 µM) or sodium acetate as control for 0.5-6
h. HIF-1 DNA binding activity was determined as above. Maximum activity
was observed at 10 µM concentration following 4 h of
exposure. Data represent mean ± S.E. relative to controls (set at
100%). *, p < 0.01. Blots shown are from
representative experiments.
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Because the VEGF induction by lead was found to be PKC- dependent, we
examined the role of PKC in HIF-1 induction using the specific PKC
activator PMA and the selective PKC inhibitor GF-109203. Electrophoretic mobility shift assay showed that PMA (100 nM) induced HIF-1 binding activity ~2-fold (196 ± 34% versus 100 ± 12%; p = 0.01) with
maximum activity occurring at 1 h of exposure. Cells were
pretreated with GF-109203 (2 µM) for 30 min prior to lead
(10 µM) or PMA (100 nM) exposure for 4 and
1 h, respectively. Lead induction of HIF-1 DNA binding activity
was inhibited in cells pretreated with the GF-109203 both in the case
of lead and PMA (Fig. 9, A
and B).
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Fig. 9.
Involvement of PKC in HIF-1 induction
by lead. Cells were pretreated with GF-109203 (2 µM)
for 30 min prior to treatment with either lead acetate (10 µM) or sodium acetate for 4 h (A), or
with either PMA (100 nM) or Me2SO (0.006% v/v)
as control for 1 h (B). Nuclear proteins were analyzed
by electrophoretic mobility shift assay. Induction of HIF-1 DNA binding
activity by either lead or PMA was inhibited in cells pretreated with
the GF-109203. Data represents mean ± S.E. relative to controls
(set at 100%). *, p < 0.01; **, p < 0.001. Blots shown are from representative experiments.
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Involvement of HIF-1 in Transcriptional Activation of VEGF--
To
determine whether the increase in HIF-1 DNA binding activity in
lead-treated cells reflects a change in HIF-1 regulated transcriptional
events, cells were co-transfected with 4 µg of a VEGF-luciferase
expression vector (p-GL/MAP11wt) along with either 20 µg of a
HIF-1 dominant-negative expression vector (pCEP/HIF-1DN) or its
control pCEP4 plasmid DNA. The total amount of plasmid DNA transfected
was held constant at 24 µg. The VEGF/luciferase reporter construct
includes the 5'-flanking region of human VEGF promoter containing the
HIF-1 consensus DNA-binding site (40). The pCEP/HIF-1DN construct
encodes a form of HIF-1 lacking both the amino-terminal basic domain
required for DNA binding and the carboxyl-terminal transactivation
domain. HIF-1DN heterodimerizes with HIF-1 generating
biologically inactive heterodimers and inhibits HIF-1-regulated
reporter gene expression. Twenty-four hours after transfection, cells
were exposed to either lead acetate (10 µM) or sodium
acetate for an additional 18-20 h. There was no significant difference
in luciferase activity in cells exposed to lead or sodium acetate (Fig.
10A). As a positive control,
transfected cells were exposed to hypoxia (1% O2 at
37 °C) for 24 h. Hypoxia increased luciferase activity >8-fold
(867 ± 40% versus 100 ± 6%; p < 0.001) relative to normoxic cells co-transfected with p-GL/MAP11wt
and pCEP4 plasmid DNA. In the presence of pCEP/HIF-1DN, hypoxia-induced luciferase activity was inhibited by 60%
(p < 0.001) (Fig. 10A) demonstrating that
pCEP/HIF-1DN effectively inhibits HIF-1-dependent reporter gene
expression under these experimental conditions.
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Fig. 10.
Lead does not induce HIF-1-dependent
transcriptional activity. A, cells were co-transfected
with 4 µg of a VEGF-luciferase reporter construct (pGL-MAP11wt) along
with either 20 µg of a HIF-1 dominant-negative expression vector
(pCEP/HIF-1DN) or its control plasmid DNA (pCEP4). The pGL-MAP11wt
construct includes the 5'-flanking region of the human VEGF promoter
containing the HIF-1 consensus DNA-binding site as described under
"Experimental Procedures." Total amount of transfected plasmid DNA
was held constant at 24 µg. Twenty-four hours after transfection,
cells were exposed either to lead acetate (10 µM) or
sodium acetate (10 µM) as control, or to hypoxia (1%
O2 at 37 °C) as positive control for additional 24 h. Luciferase expression was quantified in 20 µl of cell extracts by
measuring luminescence normalized to total proteins.
Hypoxia-induced luciferase expression is inhibited by HIF-1
dominant-negative. B, cells were transfected with
20 µg of either pCEP/HIF-1DN or control plasmid vector
pCEP4, and 24 h later exposed to either 10 µM lead
acetate or sodium acetate as control for additional 24 h. Total
cellular RNA (20 µg /lane) was subjected to Northern analysis.
Specific VEGF hybridization was quantified by phosphorimaging and
normalized to GAPDH as described under "Experimental Procedures."
Northern data revealed no inhibition of lead-induced VEGF expression by
HIF-1 dominant-negative. Data represents mean ± S.E.
(n = 6-8) relative to controls (set at 100%). *,
p < 0.001. Blots shown are from representative
experiments.
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The potential role of HIF-1 in the transcriptional activation of VEGF
by lead was similarly examined using the dominant-negative HIF-1DN.
SV-FHAs were transfected with either the control pCEP4 vector DNA or
pCEP/HIF-1DN (20 µg) expression vectors. Twenty-four hours later,
cells were exposed to lead acetate (10 µM) or sodium acetate as control for an additional 24 h. Northern hybridization of total RNA revealed 3-fold induction of VEGF mRNA by lead
(306 ± 22% versus 99 ± 4%; p < 0.001 relative to control) in cells transfected with control pCEP4
plasmid. pCEP/HIF-1DN had no effect on the induction of VEGF
mRNA by lead (Fig. 10B).
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DISCUSSION |
Lead is a widespread environmental toxicant whose developmental
neurotoxicity remains a major medical issue. There is growing evidence
that lead can directly alter cellular physiology at multiple levels
which include interference with ion channels and activation of second
messengers, in particular calcium-dependent messengers, that ultimately affect transcription factors and gene expression. This
report demonstrates that lead alters the expression of human fetal
astrocyte genes and defines the second messenger and transcription factors involved in the induction of one of these genes, VEGF/vascular permeability factor. VEGF induction was observed in this study in
response to low micromolar total lead in 10% serum-containing medium,
a condition in which almost all lead will be chelated by serum
proteins. Actual measurements of free-lead concentrations in
serum-containing cell culture medium by Audesirk and co-workers (43),
predicts that the free lead levels under our experimental conditions
were in the nanomolar range, a concentration that is relevant to human
toxicity (5, 16, 24). To our knowledge this is the first application of
cDNA microarray analysis to questions of lead toxicity and the
first detailed dissection of the effect of lead on cell signaling and
transcription factor function in cells derived from the human central
nervous system.
It has been established previously in rodent cells and purified protein
preparations that lead alters a number of diverse calcium-sensitive
processes. These include inhibition of calcium-channels, activation of
calmodulin, and activation of PKC (11, 13, 44). Of these, PKC appears
to be particularly relevant. The PKC family comprises more than 10 isoforms categorized into three structurally related groups:
conventional PKCs (cPKCs: , , and ) are regulated by
calcium and diacylglycerol or phorbol ester; novel PKCs (nPKCs: 
,
, 
, and ) are sensitive to diacylglycerol and phorbol ester but are calcium-independent; and atypical PKCs (aPKCs: 
, µ, and 
) are insensitive to calcium, diacylglycerol, and phorbol ester (45). Activation of both calcium-dependent and
calcium-independent PKC isoforms by lead has been described in rodent
and bovine cells such as glial, endothelial, and PC12 pheochromocytoma
cell lines (12, 46, 47). It has been proposed that lead alters PKC function by interacting with a high affinity site within the
NH2-terminal regulatory domain either at the calcium
activation site or cysteine-rich zinc-finger-like binding site (11, 15,
48). A role for PKC in the induction of VEGF by lead described in this
report is supported by complementary approaches. The PKC activator PMA
induced VEGF mRNA levels in the human fetal astrocyte cell line
and, furthermore, both lead and PMA induction of VEGF were blocked by
the selective PKC inhibitor GF-109203. Since GF-109203 inhibits both
calcium-dependent and calcium-independent PKC isoforms, the
specific isoforms involved in the VEGF response were further examined
using dominant-negative mutants of representative
calcium-dependent conventional PKC- and
calcium-independent novel PKC- isoforms. Such catalytically inactive
dominant-negative molecules act by competing with the corresponding
endogenous isoforms (37). The selective involvement of PKC-, but not
the PKC-, in VEGF induction by lead and PMA was directly
demonstrated through its inhibition by DN-PKC-. These results
clearly implicate a specific role of PKC- in this lead response in
SV-FHAs. Similarly, PKC- has been found to play a role in the
induction of immediate early gene expression in the rat PC12 cells
(47). The potential involvement of other PKC isoforms in response of
human astrocytes to lead is currently under investigation.
PKC activation induces the transcription of immediate early response
genes such as c-jun and c-fos that comprise the
AP-1 transcription factor through either Jun homodimerization or
Jun/Fos heterodimerization (18-20). The present study used multiple
approaches to show that lead alters AP-1 activity in SV-FHAs. Increased
AP-1 DNA binding activity in nuclear proteins isolated from
lead-treated cells was shown by electrophoretic mobility shift assay
similar to that reported in the rat PC12 cell line (21). The functional relevance of this in vitro assay is demonstrated by the
ability of lead to induce AP-1-dependent reporter gene
expression in transiently transfected SV-FHA cells and by the
inhibition of this lead-induced transcriptional response by the
c-jun dominant-negative TAM-67 (39). Finally, the
physiological relevance of this lead-induced AP-1 activation in SV-FHAs
was examined. Given the presence of AP-1 consensus binding sites in the
5'-flanking region of VEGF (40, 42), we investigated the role of AP-1
in VEGF expression in response to lead. The involvement of AP-1 in the
lead-mediated induction of VEGF mRNA was directly demonstrated
through its inhibition by TAM-67. These results clearly demonstrate the
requirement for AP-1 transcription factors for lead-induced VEGF expression.
Due to the prominent role of HIF-1 in the regulation of VEGF
expression, a similar multifaceted approach was taken to examine the
effects of lead on HIF-1 and its potential role in VEGF induction by
lead. HIF-1 is a heterodimeric transcription factor consisting of
HIF-1 and HIF-1 subunits and is essential for the activation of
genes mediating responses to hypoxia (40, 49). HIF-1 mediated transcriptional activity depends upon both the post-translational stabilization of HIF-1, probably through selective protection from
ubiquitin-dependent proteolysis, and the increased activity of HIF-1 transactivating domains which may together exert
synergistic effects (50). Hypoxia sensing mechanisms upstream to HIF-1
activation are not well defined in astrocytic cells but may mirror a
mechanism partially characterized in type I cells of the carotid body
and in PC12 cells that involve increased cytoplasmic calcium from calcium channel activation, PKC, and heme metabolism. The ability of
lead to alter calcium-mediated processes (9, 10), to activate PKC
(11-13), and to interfere with heme biosynthesis (51) suggested a
potential interface with HIF-1 function. While our gel-shift assay
results indicate that exposing cells to either lead or the PKC-agonist
PMA enhances HIF-1 binding to its DNA consensus sequence ~2-fold, no
downstream HIF-1-dependent VEGF-specific transcriptional events could be identified using two complementary methods. Neither lead nor PMA enhanced reporter gene expression under transcriptional control of HIF-1 binding sequences derived from the 5'-flanking region
of the human VEGF gene in intact SV-FHAs. In addition, VEGF induction
by lead was not affected by the HIF-1 dominant-negative under
conditions that inhibited a much stronger hypoxia-induced VEGF
response. These disparate results might be explained by a consideration
of the different magnitudes of HIF-1 induction seen in response to lead
and hypoxia. The magnitude of increased HIF-1 binding activity in
nuclear proteins derived from lead-treated cells while statistically
significant was small and possibly inadequate to generate a
transcriptional response. Alternatively, lead may generate HIF-1
heterodimers comprised of -like Per-ARNT-Sim (PAS) family members
such as neural specific nPAS-2 (52) that are less effective in
transcriptional activation than heterodimers induced by hypoxia
(53). The possibility that lead might modulate HIF-1-dependent hypoxic responses has not been addressed.
VEGF is a dimeric secretory protein containing an amino terminus
secretory sequence (42, 54) and exerts its action via high-affinity
binding to phosphotyrosine kinase receptors Flt-1 and Flk-1 (55, 56).
Compelling evidence indicates that VEGF is a fundamental regulator of
physiological and pathological angiogenesis (35, 54). VEGF is crucially
involved in mediating vascular changes following neuronal injury (57).
Overexpression of VEGF can contribute to progression of several
disorders such as intracerebral hemorrhage (58), development of brain
edema (59), and disruption of blood-brain barrier, pathological
processes seen in acute lead toxicity (6, 7). In addition neurons have
recently been found to express high-affinity VEGF receptors and thereby
might be influenced directly to VEGF disregulation (60).
In conclusion, this is the first evidence that lead induces the
expression of a growth factor (i.e. VEGF). This is also the first dissection of the second messenger pathways and transcription factors that mediate the effects of lead on gene expression in human
central nervous system cells. The potential roles of VEGF and other
astrocytic gene products in the spectrum of the vascular and cognitive
symptoms associated with lead toxicity warrants further investigation.
§¶,
,
TOP
ABSTRACT
INTRODUCTION
